COMPREHENSIVE ANALYTICAL CHEMISTRY VOLUME
51
ADVISORY BOARD Joseph A. Caruso University of Cincinnati, Cincinnati, OH, USA Hendrik Emons Joint Research Centre, Geel, Belgium Gary Hieftje Indiana University, Bloomington, IN, USA Kiyokatsu Jinno Toyohashi University of Technology, Toyohashi, Japan Uwe Karst University of Mu¨nster, Mu¨nster, Germany Gyo¨rgy Marko-Varga AstraZeneca, Lund, Sweden Janusz Pawliszyn University of Waterloo, Waterloo, Ont., Canada Susan Richardson US Environmental Protection Agency, Athens, GA, USA
Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK
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Wilson & Wilson’s
COMPREHENSIVE ANALYTICAL CHEMISTRY FOOD CONTAMINANTS AND RESIDUE ANALYSIS
VOLUME
51 Edited by ´ YOLANDA PICO Laboratori de Bromatologia i Toxicologia Facultat de Farmacia Universitat de Valencia Av. Vicent Andre´s Estelle´s s/n. 46100 Burjassot Valencia, Spain
Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo
Wilson & Wilson’s
COMPREHENSIVE ANALYTICAL CHEMISTRY
Edited by ´ D. BARCELO Research Professor Department of Environmental Chemistry IIQAB-CSIC Jordi Girona 18-26 08034 Barcelona Spain
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CONTENT S
Contributors to Volume 51 Volumes in the Series Preface Series Editor’s Preface 1. Challenges in Chemical Food Contaminants and Residue Analysis
xiii xix xxiii xxv
1
Michel W.F. Nielen and Hans J.P. Marvin 1. Introduction 2. Emerging Contaminants 3. Masked Contaminants 4. Unknown Bioactive Contaminants 5. Emerging Technologies 6. Validation and QA/QC Challenges 7. Conclusions and Future Trends Acknowledgements References
2. International Regulations on Food Contaminants and Residues
2 7 14 15 18 22 24 25 25
29
Ioannis S. Arvanitoyannis 1. Introduction to EU and USA Legislation 2. Topics/Categories Covered under EU Legislation 3. Topics/Categories Covered under USA Legislation References
3. Guidelines on Quality Implementation for Analytical Methods
29 31 63 73
77
Sergio Caroli 1. Introduction 2. The Role of Quality 3. Conclusions References
77 78 86 87
vii
viii
Contents
4. Immunochemical and Receptor Technologies: The Role of Immunoassay, Immunoaffinity Chromatography, Immunosensors and Molecularly Imprinted Polymeric Sensors
91
Marinel la Farre´, Elena Martı´nez and Damia` Barcelo´ 1. Introduction 2. Definitions and Concepts 3. Immunoassay Formats 4. Immunoassay Development 5. Immunochemical Technologies 6. Biomimics: Molecularly Imprinted Polymers (MIPs) 7. Commercial Instrumentation and Future Perspectives References
5. Advanced Sample Preparation Techniques for the Analysis of Food Contaminants and Residues
92 93 95 96 101 113 122 125
131
Pat Sandra, Frank David and Gerd Vanhoenacker 1. Introduction 2. Sample Preparation Methods for VOCs 3. Sample Preparation Methods for SVOCs and NVOCs 4. Fractionation and Clean-Up 5. Evaporation 6. Derivatization 7. Conclusion References
6. Recent Developments in Chromatographic Techniques
131 133 138 162 170 170 171 172
175
Katerina Mastovska 1. Introduction 2. Fast Chromatographic Separation 3. Two-Dimensional Chromatographic Separation 4. Overcoming Adverse Matrix Effects 5. Future Trends References
7. New Approaches in Mass Spectrometry
175 176 188 191 196 197
201
Yolanda Pico´ 1. Introduction 2. High Resolution and Accurate Mass Analysers 3. Tandem Mass Spectrometry 4. High Resolution Mass Spectrometers in Tandem 5. Conclusions and Future Trends References
201 204 211 219 227 228
Contents
8. Capillary-Based Separation Techniques
ix
231
Peter Viberg 1. 2. 3. 4. 5. 6.
Introduction Miniaturization and the Capillary Format Capillary Liquid Chromatography Capillary Electroseparations Detection Techniques in Capillary-Based Separations Automation and On-Line/In-Capillary Coupled Solid-Phase Extraction in Capillary Electrophoresis 7. Applications in Determination of Food Contaminants Using Capillary Electrophoresis References
9. Pesticide Residues
231 232 233 235 250 251 252 253
257
Anna Sannino 1. Introduction 2. Physical and Chemical Properties 3. Health Effects 4. Analytical Methods 5. Instrumental Determination 6. LC-MS-MS Applications 7. Conclusions and Future Trends References
10. Veterinary Drug Residues
257 258 259 272 278 295 301 302
307
Sherri B. Turnipseed and Wendy C. Andersen 1. Introduction 2. Physical and Chemical Properties Affecting Analytical Methodology 3. Human Health Effects from Veterinary Drug Residues 4. Analytical Methods 5. Occurrence in Food 6. Future Trends References
307 313 315 317 328 329 332
11. Analytical Strategies to Control the Illegal Use of Banned Growth Promoters in Meat Producing Animals
339
Bruno Le Bizec, Jean-Philippe Antignac and Gaud Pinel 1. Introduction 2. Chemical Properties, Health effects 3. Analytical Methods 4. Occurrence in Foods 5. Future Trends References
339 340 342 357 358 358
x
Contents
12. Mycotoxins
363
Carlo Brera, Barbara De Santis, Francesca Debegnach and Marina Miraglia 1. Introduction 2. Classes of Mycotoxins 3. Health Effects 4. Sampling 5. Analytical Methods 6. Occurrence in Food and Feed 7. Decontamination Procedures 8. Future Needs Acknowledgement References
13. Phycotoxins
364 366 376 383 385 402 416 418 419 419
429
Kevin J. James, Daniel O’Driscoll, Javier Garcı´a Ferna´ndez and Ambrose Furey 1. Introduction 2. Seafood-Poisoning Toxins 3. Paralytic Shellfish Poisoning (PSP) Toxins 4. Diarrhetic Shellfish Poisoning (DSP) Toxins 5. Azaspiracid Poisoning (AZP) Toxins 6. Amnesic Shellfish Poisoning (ASP) Toxins 7. Yessotoxins (YTXs) 8. Neurotoxin Shellfish Poisoning (NSP) Toxins References
14. Persistent Organochlorine Pollutants, Dioxins and Polychlorinated Biphenyls
429 430 430 432 439 442 445 448 452
457
Marie-Louise Scippo, Gauthier Eppe, Claude Saegerman, Georges Scholl, Edwin De Pauw, Guy Maghuin-Rogister and Jean-Franc- ois Focant 1. Introduction 2. Physical and Chemical Properties and Use of POPs 3. Health Effects 4. Analytical Methods 5. Occurrence in Food 6. Future Trends References
15. Brominated Flame Retardants as Food Contaminants
457 460 462 467 495 496 498
507
Adrian Covaci, Stefan Voorspoels, Kyle D’Silva, Janice Huwe and Stuart Harrad 1. 2. 3. 4.
Introduction Analytical Methods Occurrence in Food Regulatory/Safety Aspects and On-Going Monitoring Programmes
507 512 542 560
Contents
5. Future Trends in the BFR Research in Food Acknowledgement References
16. Metals
xi
562 563 563
571
Clinio Locatelli 1. Introduction 2. Analytical Procedure for Metal Determination 3. Metals of Interest 4. Directive for Trace Metals in Food 5. Conclusions and Future Trends References
17. Polycyclic Aromatic Hydrocarbons
571 573 585 589 589 590
599
Katsumi Tamakawa 1. Introduction 2. Physical and Chemical Properties 3. Health Effects 4. Analytical Methods 5. Occurrences of PAHs in Food 6. Future Trend Acknowledgements References
18. Methods for the Determination of N-Nitroso Compounds in Food and Biological Fluids
599 601 602 611 632 642 644 644
653
Sidney S. Mirvish 1. Introduction 2. Physical and Chemical Properties of NOC 3. Health Effects 4. Analytical Methods 5. Occurrence of NOC in Foods 6. Conclusions and Future Trends Acknowledgements References
19. Heterocyclic Amines
654 656 657 659 676 679 680 680
685
Mark G. Knize and James S. Felton 1. 2. 3. 4. 5. 6. 7.
Introduction Physical and Chemical Properties Health Effects Formation of Heterocyclic Amines Food Sample Analysis Occurrence in Food Exposure Assessment
685 686 687 688 689 697 700
xii
Contents
8. Regulations and Future Trends Acknowledgements References
20. Acrylamide, Chloropropanols and Chloropropanol Esters, Furan
701 701 701
705
Richard H. Stadler and Till Goldmann 1. Acrylamide 2. Chloropropanols and Chloroesters 3. Furan 4. Conclusion and Future Trends References
21. Food Contact Materials
705 714 722 728 728
733
Catherine Simoneau 1. Introduction 2. Physical and Chemical Properties 3. Analytical Methods 4. Health Effects 5. Occurrence in Foods 6. Future Trends References
22. Non-Target Multi-Component Analytical Surveillance of Food Contact Materials
733 736 741 765 768 768 771
775
Leon Coulier, Sander Koster, Bas Muilwijk, Leo van Stee, Ruud Peters, Esther Zondervan-van den Beuken, Rinus Rijk, Monique Rennen, Winfried Leeman, Geert Houben and William D. van Dongen 1. Introduction 2. Legislation on Food Contact Materials 3. Non-Target Analysis 4. Examples of Non-Target Analysis from the Recent Literature 5. Safety Assessment of Migrants from FCM 6. Conclusions and Future Trends References
List of Abbreviations Subject Index
776 776 778 782 785 791 792
795 807
CONTRIBUTORS TO VOLUME 51
Wendy C. Andersen US FDA, Animal Drugs Research Center, Denver Fed Ctr, Lakewood, CO 80225, USA Jean-Philippe Antignac LABERCA-Ecole Nationale Ve´te´rinaire de Nantes, Route de Gachet, BP 50707, F-44307 Nantes Cedex 3, France Ioannis S. Arvanitoyannis University of Thessaly, School of Agricultural Sciences, Fytokou Str., 38446 Nea Ionia Magnesias, Volos, Greece Damia` Barcelo´ Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18, 08034 Barcelona, Spain Carlo Brera Istituto Superiore di Sanita`, Centro Nazionale per la Qualita` degli Alimenti e per i Rischi Alimentari, Reparto OGM e Xenobiotici di origine fungina, Viale Regina Elena, 299, 00161 Rome, Italy Sergio Caroli Instituto Superiore di Sanita, Viale Regina Elena, 299, 00151 Rome, Italy Leon Coulier Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Adrian Covaci Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium and Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium Frank David Research Institute for Chromatography, Kennedypark 26, B-8500 Kortrijk, Belgium
xiii
xiv
Contributors to Volume 51
Francesca Debegnach Istituto Superiore di Sanita`, Centro Nazionale per la Qualita` degli Alimenti e per i Rischi Alimentari, Reparto OGM e Xenobiotici di origine fungina, Viale Regina Elena, 299, 00161 Rome, Italy Edwin De Pauw Department of Chemistry, Laboratory of Mass Spectrometry, CART (Centre of analysis of residues in trace), University of Liege, Alle´e de la Chimie, 6, Bat B6c, Sart Tilman, B-4000 Liege, Belgium Barbara De Santis Istituto Superiore di Sanita`, Centro Nazionale per la Qualita` degli Alimenti e per i Rischi Alimentari, Reparto OGM e Xenobiotici di origine fungina, Viale Regina Elena, 299, 00161 Rome, Italy Kyle D’Silva Waters Corporation, Atlas Park, Simonsway, Manchester, M22 5PP, UK Gauthier Eppe Department of Chemistry, Laboratory of Mass Spectrometry, CART (Centre of analysis of residues in trace), University of Liege, Alle´e de la Chimie, 6, Bat B6c, Sart Tilman, B-4000 Liege, Belgium Marinel la Farre´ Environmental Chemistry, Spain
IIQAB-CSIC,
Jordi
Girona
18,
08034
Barcelona,
James S. Felton Chemistry, Materials, and Life Sciences Directorate, Lawrence Livermore National Laboratory, University of California, Livermore, California, USA Jean-Franc- ois Focant Department of Chemistry, Laboratory of Mass Spectrometry, CART (Centre of analysis of residues in trace), University of Liege, Alle´e de la Chimie, 6, Bat B6c, Sart Tilman, B-4000 Liege, Belgium Ambrose Furey PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Javier Garcı´a Ferna´ndez PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Till Goldmann Nestle´ Research Centre, P.O. Box 44, CH 1000 Lausanne 26, Switzerland
Contributors to Volume 51
xv
Stuart Harrad Division of Environmental Health and Risk Management, Public Health Building, School of Geography, Earth, and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK Geert Houben Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Janice Huwe Biosciences Research Laboratory, USDA-ARS, P.O. Box 5674, University Station, Fargo, North Dakota 58105, USA Kevin J. James PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Mark G. Knize Chemistry, Materials, and Life Sciences Directorate, Lawrence Livermore National Laboratory, University of California, Livermore, California, USA Sander Koster Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Bruno Le Bizec LABERCA-Ecole Nationale Ve´te´rinaire de Nantes, Route de Gachet, BP 50707, F-44307 Nantes Cedex 3, France Winfried Leeman Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Clinio Locatelli Department of Chemistry ‘‘G. Ciamician’’, University of Bologna, Via F. Selmi 2, I-40126 Bologna, Italy and CIRSA (Centro Interdipartimentale di Ricerca per le Scienze Ambientali), Laboratory of Environmental Analytical Chemistry, University of Bologna, Via S. Alberto 163, I-48100 Ravenna, Italy Guy Maghuin-Rogister Department of Food Sciences, Laboratory of Food Analysis, CART (Centre of analysis of residues in traces), University of Liege, Boulevard de Colonster, 20, Bat B43-bis, Sart Tilman, B-4000 Liege, Belgium Elena Martı´nez Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18, 08034 Barcelona, Spain
xvi
Contributors to Volume 51
Hans J.P. Marvin RIKILT-Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands Katerina Mastovska USDA, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA Marina Miraglia Istituto Superiore di Sanita`, Centro Nazionale per la Qualita` degli Alimenti e per i Rischi Alimentari, Reparto OGM e Xenobiotici di origine fungina, Viale Regina Elena, 299, 00161 Rome, Italy Sidney S. Mirvish Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68198-6802, USA Bas Muilwijk Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Michel W.F. Nielen RIKILT-Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands and Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, The Netherlands Daniel O’Driscoll PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Cork Institute of Technology, Bishopstown, Cork, Ireland Ruud Peters Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Yolanda Pico´ Laboratori de Bromatologia i Toxicologia, Facultat de Farmacia, Universitat de Valencia, Av. Vicent Andre´s Estelle´s s/n, 46100 Burjassot, Valencia, Spain Gaud Pinel LABERCA-Ecole Nationale Ve´te´rinaire de Nantes, Route de Gachet, BP 50707, F-44307 Nantes Cedex 3, France Monique Rennen Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Rinus Rijk Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands
Contributors to Volume 51
xvii
Claude Saegerman Department of Infectious and Parasitic Diseases, Epidemiology and Risk analysis applied to Veterinary Sciences, University of Liege, Boulevard de Colonster, 20, Bat B42, Sart Tilman, B-4000 Liege, Belgium Pat Sandra Department of Organic Chemistry, University of Ghent, Krijgslaan 281-S4bis, B-9000 Ghent, Belgium Anna Sannino Stazione Sperimentale Conserve Alimentari, Food Safety Department, Viale Tanara, 31/A, 43100 Parma, Italy Georges Scholl Department of Chemistry, Laboratory of Mass Spectrometry, CART (Centre of analysis of residues in trace), University of Liege, Alle´e de la Chimie, 6, Bat B6c, Sart Tilman, B-4000 Liege, Belgium Marie-Louise Scippo Department of Food Sciences, Laboratory of Food Analysis, CART (Centre of analysis of residues in traces), University of Liege, Boulevard de Colonster, 20, Bat B43-bis, Sart Tilman, B-4000 Liege, Belgium Catherine Simoneau Community Reference Laboratory for Food Contact Materials, European Commission, DG-Joint Research Centre, Institute for Health and Consumer Protection, Unit Physical and Chemical Exposure, T.P. 260, Ispra Va 21020, Italy Richard H. Stadler Quality Management Department, Nestle´ Product Technology Centre Orbe, CH 1350 Orbe, Switzerland Katsumi Tamakawa Sendai City Institute of Public Health, 2-5-10, Oroshimachi-higashi, Wakabayashi ward, Sendai city, Miyagi prefecture, 984-0002, Japan Sherry B. Turnipseed US FDA, Animal Drugs Research Center, Denver Fed Ctr, Lakewood, CO 80225, USA William D. van Dongen Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Leo van Stee Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands Gerd Vanhoenacker Research Institute for Chromatography, Kennedypark 26, B-8500 Kortrijk, Belgium
xviii
Contributors to Volume 51
Peter Viberg Technical Analytical Chemistry, Lund University, Getingeva¨gen 60, PO Box 124, SE-22100 Lund, Sweden Stefan Voorspoels European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium Esther Zondervan-van den Beuken Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands
VOLUMES IN THE SERIES
Vol. 1A
Vol. 1B Vol. 1C Vol. 2A
Vol. 2B
Vol. 2C
Vol. 2D Vol. 3
Vol. 4
Vol. 5
Vol. 6 Vol. 7 Vol. 8
Vol. 9
Analytical Processes Gas Analysis Inorganic Qualitative Analysis Organic Qualitative Analysis Inorganic Gravimetric Analysis Inorganic Titrimetric Analysis Organic Quantitative Analysis Analytical Chemistry of the Elements Electrochemical Analysis Electrodeposition Potentiometric Titrations Conductometric Titrations High-Frequency Titrations Liquid Chromatography in Columns Gas Chromatography Ion Exchangers Distillation Paper and Thin Layer Chromatography Radiochemical Methods Nuclear Magnetic Resonance and Electron Spin Resonance Methods X-ray Spectrometry Couiometric Analysis Elemental Analysis with Minute Sample Standards and Standardization Separation by Liquid Amalgams Vacuum Fusion Analysis of Gases in Metals Electroanalysis in Molten Salts Instrumentation for Spectroscopy Atomic Absorption and Fluorescence Spectroscopy Diffuse Reflectane Spectroscopy Emission Spectroscopy Analytical Microwave Spectroscopy Analytical Applications of Electron Microscopy Analytical Infrared Spectroscopy Thermal Methods in Analytical Chemistry Substoichiometric Analytical Methods Enzyme Electrodes in Analytical Chemistry Molecular Fluorescence Spectroscopy Photometric Titrations Analytical Applications of Interferometry Ultraviolet Photoelectron and Photoion Spectroscopy Auger Electron Spectroscopy Plasma Excitation in Spectrochemical Analysis
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Volumes in the Series
Vol. 10 Vol. 11 Vol. 12
Vol. 13
Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. Vol. Vol. Vol. Vol.
19 20 21 22 23
Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.
24 25 26 27 28 29 30 31 32 33 34
Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.
35 36 37 38 39 40 41 42 43
Vol. 44 Vol. 45 Vol. 46
Organic Spot Tests Analysis The History of Analytical Chemistry The Application of Mathematical Statistics in Analytical Chemistry Mass Spectrometry Ion Selective Electrodes Thermal Analysis Part A. Simultaneous Thermoanalytical Examination by Means of the Derivatograph Part B. Biochemical and Clinical Application of Thermometric and Thermal Analysis Part C. Emanation Thermal Analysis and other Radiometric Emanation Methods Part D. Thermophysical Properties of Solids Part E. Pulse Method of Measuring Thermophysical Parameters Analysis of Complex Hydrocarbons Part A. Separation Methods Part B. Group Analysis and Detailed Analysis Ion-Exchangers in Analytical Chemistry Methods of Organic Analysis Chemical Microscopy Thermomicroscopy of Organic Compounds Gas and Liquid Analysers Kinetic Methods in Chemical Analysis Application of Computers in Analytical Chemistry Analytical Visible and Ultra-violet Spectrometry Photometric Methods in Inorganic Trace Analysis New Developments in Conductometric and Oscillometric Analysis Titrimetric Analysis in Organic Solvents Analytical and Biomedical Applications of Ion-Selective Field-Effect Transistors Energy Dispersive X-ray Fluorescence Analysis Preconcentration of Trace Elements Radionuclide X-ray Fluorecence Analysis Voltammetry Analysis of Substances in the Gaseous Phase Chemiluminescence Immunoassay Spectrochemical Trace Analysis for Metals and Metalloids Surfactants in Analytical Chemistry Environmental Analytical Chemistry Elemental Speciation – New Approaches for Trace Element Analysis Discrete Sample Introduction Techniques for Inductively Coupled Plasma Mass Spectrometry Modern Fourier Transform Infrared Spectroscopy Chemical Test Methods of Analysis Sampling and Sample Preparation for Field and Laboratory Countercurrent Chromatography: The Support-Free Liquid Stationary Phase Integrated Analytical Systems Analysis and Fate of Surfactants in the Aquatic Environment Sample Preparation for Trace Element Analysis Non-destructive Microanalysis of Cultural Heritage Materials Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues Biosensors and Modern Biospecific Analytical Techniques Analysis and Detection by Capillary Electrophoresis Proteomics and Peptidomics: New Technology Platforms Elucidating Biology
Volumes in the Series
Vol. Vol. Vol. Vol.
47 48 49 50
Modern Instrumental Analysis Passive Sampling Techniques in Environmental Monitoring Electrochemical (Bio) Sensor Analysis Analysis, Fate and Removal of Pharmaceuticals in the Water Cycle
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PREFACE
All consumers have the right to expect and demand good-quality and safe food at affordable prices. This right was recognized by the participants at the United Nations Conference on Food and Agriculture, held in the United States (US) in 1943, which laid the foundation for the creation of Food and Agriculture Organization (FAO). Because of this, food safety and consumer protection are topics of highest priority. In the same way, producing a food supply safe and of good quality is a prerequisite to successful domestic and international food trade, and a key to sustainable development of national agricultural resources. Chemical analysis is a vital component of every quality management system. Determination of organic contaminants in food needs to continually refine and explore new technologies to enhance sensitivity, selectivity, separation, interpretation, and adaptability of methodology. Though a well-established field in many senses, new methods, instruments, and modifications are decisive to analytical chemists and instrumentalists who are challenged with the identification and quantitation of new and already recognized xenobiotics of health concern. This book treats different aspects of the analysis of contaminants and residues in food, and highlights some current concerns facing this field. The content is initiated by an overview on food safety, the objectives and importance of determining contaminants and residues in food, and the problems and challenges associated to these analyses. This is followed by full details of relevant European and United States regulations. Topics, such as conventional chromatographic methods, accommodating clean up, and preparing substances for further instrumental analysis, are encompassed with new analytical techniques that have significantly been developed over the past few years, like solid phase microextraction, liquid chromatography-mass spectrometry, immunoassays, and biosensors. A wide range of toxic contaminants and residues, from pesticides to mycotoxins or dioxins are examined. This book can be a practical resource that offers ideas on how to choose the most effective techniques for determining these compounds. The book contains 22 chapters written by different experts in each field that cover the emerging topics in food contaminants and residue analysis, and it is organized into three sections: the first one covers the regulatory framework including validation and quality assurance of analytical methods, the second treats the latest developments in analytical techniques, and the third provides information about the determination of specific contaminants and residues in food. Logically structured and with numerous examples Food Contaminants and Residue Analysis is a valuable tool for food analysis as a reference and trained xxiii
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Preface
guide. The book is addressed to serve as a general reference for post-graduate students, as well as a practical reference guide for a wide range of experts: biologists, biochemists, microbiologists, food chemists, toxicologists, chemists, agronomists, hygienists, and everybody who need to use the analytical techniques for evaluating food safety. Each chapter contains enough references to the literature to help as an effective resource for more detailed information. Many people have collaborated in this project, directly or indirectly, and I would like to acknowledge my debts to them. Prof. Damia` Barcelo´ for the trust put in me to develop this work and for his help and support through this time; the people from the editorial staff of Elsevier (Andrew Gent, Joan Annuels, Anne Russum) for their willingness to solve any problem related to the production of the book and their patience; and finally and foremost, the authors (the edition of this book would not have been possible without their cooperation) and the readership (you are the ones who will make the book an integral part of food contaminants and residue research). I hope the book lives up to the expectations. Yolanda Pico´
S ER I E S E D I TOR ’ S P R E F A CE
Food safety has become a key issue in our society, especially now that much of the food we consume comes from other parts of the world. The public in general is increasingly concerned about the quality of the products they consume every day and to meet that need, more information is being made available in the media. Various cases have become headline news, such as the dioxin problem in Belgium, antibiotics in shrimps imported from Asia to Europe, and the high levels of persistent organic pollutants detected in European salmon as compared to the American salmon. These cases have alerted the authorities and the public in general that more efforts are needed on food control. In this respect this volume edited by my old friend and colleague, Yolanda Pico´, is timely and I am delighted I was able to convince her to collate such a book for the Comprehensive Analytical Chemistry series. This is only the second book on the topic of food residues in the series. The first, collated by another old friend and colleague, AR Fernandez Alba, was entitled Pesticide Residues published as Vol. 43 in the series. I am sure that food safety will be the subject of further volumes in the future. This book contains 19 chapters; the first three are devoted to general aspects of food analysis. Chapters 4 to 8 cover sample preparation techniques, chromatographic-mass spectrometric methods, capillary electrophoresis and immunochemical methods. Chapters 9 to 19 report on analytical applications of a broad range of contaminants, including pesticides, antibiotics, growth promoters, mycotoxins, phycotoxins, persistent organic pollutants brominated flame retardants and heterocyclic amines. The content is comprehensive and covers most of the problems encountered in food residue analysis, so will act as a useful reference for those who are new to the field and/or expert food laboratories. The book discusses most aspects of food contaminant and residue analysis and I expect it will become a key reference for food residue specialists. Finally I would like to thank not only the editor for compiling such a worldclass book but also all the authors for their contributions. D. Barcelo´ Department of Environmental Chemistry, IIQAB-CSIC Barcelona, Spain
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CHAPT ER
1 Challenges in Chemical Food Contaminants and Residue Analysis Michel W.F. Nielen and Hans J.P. Marvin
Contents
1. Introduction 1.1 The food chain: A global issue 1.2 Issues according to the Rapid Alert System for Food and Feed 1.3 Issues according to the feed and food industry 1.4 The consumer perception 2. Emerging Contaminants 2.1 Brominated flame retardants 2.2 Perfluorinated organic substances 2.3 Endocrine disruptors 2.4 Metal speciation 2.5 Alkaloid biotoxins 2.6 Peptide and protein growth promoters 2.7 Genetically modified organisms (GMO) 2.8 Nanoparticles 3. Masked Contaminants 4. Unknown Bioactive Contaminants 4.1 Bioactivity-directed identification of emerging unknown contaminants 4.2 Metabolomics approach to the identification of emerging unknown contaminants 4.3 In silico prediction approach to the identification of unknowns 5. Emerging Technologies 5.1 Omics 5.2 Bioassays 5.3 (Bio)nanotechnology 5.4 Ambient mass spectrometry 6. Validation and QA/QC Challenges 6.1 Biochemical or biological rapid screening assays 6.2 Comprehensive instrumental analysis methods
Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00001-9
2 3 4 6 6 7 8 8 9 10 10 11 12 13 14 15 15 17 17 18 18 19 20 20 22 22 23
r 2008 Elsevier B.V. All rights reserved.
1
2
Michel W.F. Nielen and Hans J.P. Marvin
7. Conclusions and Future Trends Acknowledgements References
24 25 25
1. INTRODUCTION Nowadays food is produced and distributed in a global market leading to stringent legislation and regulation for food quality and safety in order to protect consumers and ensure fair trade. Despite these efforts, food safety incidents occasionally occur and originate from both microbial and chemical contamination. Pesticide and veterinary drug residues, endocrine disruptors, food additives and packaging materials, environmental contaminants (including dioxins and heavy metals) and contaminants of natural origin (including mycotoxins and marine toxins) are of particular concern. As a consequence of the introduction of food commodities containing ingredients produced by modern biotechnology and resulting legal requirements of safety and labeling, a strong additional demand for adequate methods of analysis has occurred. Risk analysis provides a framework for regulatory authorities to protect the consumer from potential food safety hazards and is performed in an iterative manner by food safety managers (regulatory authorities), risk assessors (scientists) and stakeholders (i.e., consumers, industry, non-governmental organizations). The assessment of food safety is a scientific exercise performed by scientists and consists of hazard identification, hazard characterization, exposure assessment and risk characterization. An important prerequisite for performing risk assessment adequately is the presence of data generated by reliable and fit-for-purpose analytical methods to estimate the level of exposure and intake of the consumer to contaminants and residues. Hence, the accuracy of risk assessment will benefit from the availability of comprehensive quantitative monitoring and consumption data. However, cost and time considerations of food safety managers (in regulatory institutions and industry) favour the development and implementation of inexpensive and rapid screening methods having a limited scope and providing qualitative or semiquantitative ‘‘on-off’’ data only. Global food production practices and the changing climate showed that new unexpected food safety hazards and risks may appear in the food and feed production chain stressing the need for analytical tools capable of early warning for such emerging risks. Some of these potential food safety hazards and methodologies capable of detecting known and unknown emerging contaminants are discussed and related challenges defined. It is argued that monitoring programmes should anticipate these new conspicuous threats. In this context a key role is proposed for bioactivity-based screening concepts and bioactivity-directed identification tools. The development of both rapid screening methods and comprehensive tools covering as many contaminants as possible including emerging and even unknown contaminants is justified by the different needs from food safety
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stakeholders. The rapid screening developments are facing the challenge of multiplex detection in order to extend their scope, the comprehensive methods on the other hand are facing major challenges in generic sample preparation and advanced data evaluation.
1.1 The food chain: A global issue The food chain as schematically represented by Figure 1 is rather complex and many factors worldwide play a role in the final issue of food quality and safety. Raw materials for feed and food production come from all over the world with very different local climate, harvesting and storage conditions, all having an impact on the occurrence of microbiological and chemical contaminants such as mycotoxins, pesticide residues, environmental pollution and packaging migrants. The feed producer will mix different raw materials according to its specifications and add additives such as stabilizers but in some cases also medication. Medicated feed production can cause drug residues in nonmedicated feed produced at the same facility due to carry-over. Next, the feed is transported to the first consumer level, i.e., the farm animals. The increased awareness of animal welfare might put a stronger demand on feed-related riskbenefit issues. At the farm stage again additives, but also pesticides, veterinary drugs or even illegal hormones might be applied, which residues and/or metabolites can again build up in the food chain. The food industry produces food products and/or food ingredients but they also yield a waste stream which is at least partly recycled and used as feed ingredient. Packaging into smaller
Raw material
Retailers
Feed
Farmers +animals
Industry, processing
Figure 1 Simplified representation of the food chain.
Consumers
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pieces might increase the migration issue of chemicals from the packaging into the food commodity. Following transport the final products come to the retailers where a storage issue might influence the final contamination load. Products being beyond the storage limit date might be recycled and end up in the feed stream again. Finally the consumer buys food that must be stored and prepared for cooking, actions known for their potential introduction of contaminants if hygiene guidelines are not followed. During cooking, food processing contaminants, such as acrylamide, heterocyclic amines and polycyclic hydrocarbons, are introduced but some of the contaminants present might be degraded (or bioactivated) so the real load of dietary intake of contaminants is not so easy to determine. The occurrence of residues from intentionally added chemicals somewhere in the food chain can in theory be avoided, but very much depends on the attitude and behaviour of the actors in the food production chain. Retrospective studies on recent food safety incidents have shown that the human factor (unawareness, fraudulent and illegal actions) plays an important role in the development of food safety incidents [1]. Globalization of food trade, changing climate conditions and agricultural practices, changing food consumption patterns and environmental pollution are all drivers of food safety risks and should be taken into account in systems aimed at identifying emerging food safety hazards and risks. Control of food safety standards, monitoring of contaminants and knowledge about the fate of food contaminants through the entire food chain is needed thus requiring the availability of analysis methods dedicated to the different parts and their actors within the chain.
1.2 Issues according to the Rapid Alert System for Food and Feed The Rapid Alert System for Food and Feed (RASFF) is primarily a tool for exchange of information between food and feed authorities in the European Union (EU) member states in cases where a risk to human health has been identified and measures have been taken, such as withholding, recalling, seizure or rejection of the products concerned. The European Commission (EC) publishes weekly overviews of RASFF alert and information notifications on its website, and the summarizing annual reports provide an overview of the numbers of notifications and the categories of food products and hazards that they pertained to [2]. These annual reports also highlight conspicuous developments within the particular year. Kleter et al. [3] have explored the utility of notifications filed through RASFF to identify emerging trends in food safety issues. To this end RASFF information and alert notifications published in the four-year period of July 2003–June 2007 amounting to a total of 11,403 notifications were divided into categories and analysed. The breakdown per hazard category is given in Figure 2. The major categories included chemical (44%), mycotoxins (29%) and microbiological hazards (17%), which together accounted for the majority of the notifications (90%). Within the chemical hazard category, contaminants in products from seafood (30%) and spices and condiments (15%) were the most commonly reported. The most frequently reported are allergens (e.g., sulfite and
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Hazards reported through RASFF July 2003 - June 2007 (12,641 hazards)
Chemical Mycotoxin Microbiological Others, including fraud, biological hazards, labeling, chemical hazards, quality, hygiene, defective packaging and others
Figure 2 Breakdown of food chain hazards according to Kleter et al. [3].
histamine), heavy metals (e.g., cadmium, mercury and lead), veterinary antibiotics (e.g., the nitrofurans; furazolidone and nitrofurazone, as well as chloramphenicol), dyes (e.g., Sudan 1 and 4) and pesticides (e.g., dimethoate, isophenfos-methyl and omethoate). Aflatoxins account for the majority (93%) of mycotoxins and are mainly (84%) found in nuts, of which (54%) have been imported from Iran. Microbial contaminants include moulds, viruses and bacteria. Bacteria species were most frequently reported of which Salmonella and its subspecies were the most numerous (57% of the reports) followed by Listeria monocytogenes (16%). It should be noted that the number of reports in the RASFF not necessarily reflects the extent of a specific food safety problem because the nature of the RASFF system implicitly yields a multiplication of a specific finding. Based on warnings from the EU member states authorities in other countries will check suspicious lots which will give an additional RASFF notification. Secondly, cost and time considerations limit the scope of survey and monitoring programs, hence many potential food safety hazards will not be monitored at all or at best be accidentally picked up. The application of more generic screening methods such as bioassays in routine monitoring programs may circumvent this problem and increase the chance of finding new (re)emerging food safety hazards. In general, EC regulators and legislators require the availability of fit-for-purpose analysis methods having a comprehensive contaminant scope in order to provide the data for risk assessment, the establishment of maximum residue limits and the development and execution of monitoring plans. Harmonization of validated methods and interpretation of results is a prerequisite in order to allow their use in data banks and to avoid internal and external trade disputes.
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1.3 Issues according to the feed and food industry According to the General Food Law [4], the producers are responsible for the safety of food and feed they produce, storage, transport, selling (or eventually dispose), tracking and tracing. They should not place unsafe food or feed on the market, and should have a full traceability system for rapid withholding and recalling in case of alerts. Last but not least, unsafe situations should be prevented using critical control points hazard analyses (HACCP) and good manufacturing practices. Because of this responsibility and associated demand to maintain their core product integrity the need of the industry for rapid and inexpensive screening methods has never been greater. It is obvious that unloading an incoming ship or truck carrying raw materials cannot wait for the results from a full comprehensive contaminant analysis; a first screening result should be available in minutes, not days. As a compromise a limited number of key expected contaminants will be rapidly tested by, for example, dipstick-like screening assays, and other contaminant parameters will be checked less frequently. Ideally a rapid screening test would allow the detection of all relevant contaminants and still be inexpensive. But reality is far from that, putting a strong demand for international cooperation on the development of rapid multi-detection methods for chemical contaminants in the food chain.
1.4 The consumer perception Generally food consumers prefer high quality and intrinsically safe food for a low price, but they are also aware of food-related health and safety concerns. Following the request of the European Food Safety Authority (EFSA) and the EC DG-SANCO a special Eurobarometer was carried out in 2005 aiming at an assessment of how people in the EU perceive risk focusing particularly on food safety [5]: 42% of the EU citizens thought that their health could be damaged by the food they eat or by other consumer goods. When they were asked to freely associate the food-related problems they mentioned most often food poisoning (16%), immediately followed by chemicals including pesticides and toxic substances (14%) and obesity (13%). However, following a reminder of possible risks they expressed widespread concerns having on top of their ‘‘worry-scale’’ pesticide residues in fruit, vegetables or cereals and residues such as antibiotics or hormones in meat. In the mid-range environmental pollutants like mercury or dioxins were considered. Surprisingly EU citizens are less concerned about their personal factors such as food hygiene and food preparation (including preparation induced chemical risks). Asked about regulatory consumer protection 62% agreed that food safety laws are strict, despite some reservations regarding their enforcement. Concerning information sources consumers, physicians and scientists were trusted most while economic actors such as farmers, manufacturers and retailers are the least trusted. According to the same Eurobarometer consumers when they go for shopping indeed go for high quality (42%) and low pricing (40%). Although being based on almost 25,000 interviews (approximately 1,000 per member state) these data should be considered as an
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estimation only. Classical chemical residues and contaminant issues were mentioned but, for example, natural contaminants such as toxins were obviously not considered and/or unknown by the interviewers and the respondents. Moreover consumers might show irrational behaviour in answering questions: they tend to underestimate the safety aspect related to their own lifestyle with respect to food hygiene during storage and cooking and to overestimate the external risk of chemical residues. Recently Verbeke et al. [6] discussed why consumers behave as they do with respect to food safety and risk information. Some irrational and inconsistent behaviours were explained based on the nature of the risk and individual psychological processes. Improvement of traceability and labeling, segmented communication approaches and public involvement in risk management decision making might contribute to restore consumer confidence and reduce the gap between risk perception of consumers and experts. Also the consumer expectations regarding food safety and quality require the availability of fit-for-purpose analysis methods having a comprehensive contaminant scope. Harmonization of methods and interpretation of results is again a prerequisite: consumers tend to trust scientists but they will get much more worried when scientists provide contradictory information!
2. EMERGING CONTAMINANTS An emerging risk is an issue that in the future may pose a risk to human health, to animals or the environment [7]. In Europe, regulation (EC) No 178/2002 includes the responsibility of identification of such emerging risks [4]. The indication of an emerging risk may relate to a significant exposure to a hazard not recognized earlier or to a new/increased exposure to a known hazard. Some current and potential future emerging risk issues are listed in Table 1.
Table 1
Some current and potential future emerging risks in the food chain
Abbreviation
Description
EFSA opinion
BFR PFOS ER Metals
Brominated flame retardants Perfluorinated organic substances Endocrine disruptors Arsenic, cadmium, lead, mercury, methylmercury, organotin Pyrrolizidine and ergot toxins Abuse of peptide and protein growth promoters Gene modification of plants and gene doping of animals Nanoparticles
Yes Requested No Yes
Alkaloids Proteins GMO Nano
Yes No Yes No
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2.1 Brominated flame retardants Brominated Flame Retardants (BFRs) such as polybrominated diphenylethers (PBDEs) and hexabromocyclododecane isomers (HBCD) are widespread occurring in the environment and belong to the group of persistent organic pollutants (POPs): BFRs show persistence in the environment like their polychlorinated biphenyls (PCBs) and dioxins/dibenzofurans (PCDDs/PCDFs) analogues. BFRs are suspects for various toxic effects, including endocrine disruption which might be enhanced following in vivo hydroxylation. The EFSA has drafted an opinion on PCBs [8] and advice on relevant compounds in the BFR group. It can be expected that BFRs occur in the food chain accompanied by several other POPs in different concentrations (PCBs, PCDD/PCDF, PAH, chlorinated pesticides). The complex mixture thus obtained requires analysis methods having a huge separation and identification power. Comprehensive gas chromatography combined with time-of-flight mass spectrometry (GC GC/TOFMS) is an expensive but comprehensive solution to the analysis of BFRs and related substances in the food chain [9,10]. Contrary to conventional techniques, implementation of a GC GC/TOFMS multi-analyte/multi-class strategy in food safety control seems feasible, provided that an adequate solution is found for the automated data interpretation challenge related to the very large fourdimensional datasets. Apart from that, the development of bioactivity-based screening approaches remains challenging since they can not only provide an additional screening tool, but even disclose the presence of unknown bioactive pollutants. The DR-CALUXs, an aryl hydrocarbon receptor-based transcription activation bioassay, is being used for dioxin screening in several laboratories [11]. Also a surface plasmon resonance (SPR) biosensor assay for measurement of the thyroid disrupting activity of hydroxylated halogenated aromatic pollutants has been described based on binding with specific human transport proteins [12]. A real comprehensive exposure assessment would integrate the data from these entirely different but highly complementary approaches.
2.2 Perfluorinated organic substances Perfluorinated compounds (PFCs) or perfluorinated organic substances (PFOS) are used in a wide variety of industrial applications [13]. As a consequence these compounds show a global distribution in the environment [14]. They have been detected not only in environmental samples and fish but also in human blood and liver, and in several wildlife species [15]. PFOS show persistence in the environment and some of them have been related to different carcinogenic actions, for example, perfluorooctanoic acid has been identified as a potent hepatocarcinogen in rodents [16]. Meanwhile PFOS have been recognized by the EFSA and concentration levels, contamination pathways and toxicological potency should be assessed in the food chain. So far most of the analysis methods are based on liquid chromatography coupled to mass spectrometry or tandem mass spectrometry approaches (LC/MS,
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LC/MS/MS) preceded by solid-phase extraction. A key issue is the avoidance of contamination during sampling, storage and analysis: PFOS are everywhere inside the laboratory and its instruments. The specific chemical and physical properties of PFOS hinder the development of rapid screening methods: it is for example unlikely that antibodies can be raised successfully against the PFOS family, no biorecognition-based methods have been reported yet. Still many challenges remain even for the LC/MS approach: the development of simplified sample preparation protocols and harmonization of methods are key issues anyway.
2.3 Endocrine disruptors Many contaminants occurring in the food chain can be considered as endocrine disruptors: certain pesticides, POPs and metabolites thereof, phytoestrogens (present in fruit and vegetables and soy products), hormones like estradiol endogenously present in cow’s milk and eggs, and residues of illegally applied hormones. Several synthetic endocrine disruptors are actually POPs and suspect for carcinogenity; information related to phytoestrogens is rather contradictory in this respect: both protection and promotion of cancers is claimed in literature [17]. The main classes of phytoestrogens are isoflavones, lignans, coumestans and natural stilbenes, and show structural similarities with potent estrogens. Their consumption by healthy adults may be without risk but the problem might be totally different when exposure occurs at critical stages of development, i.e., at foetal and prepubertal children. Soy-milk-based baby-food is especially relevant to check for adverse effects of phytoestrogens [18]. Cow’s milk on the other hand should be checked for both estradiol and phytoestrogens. For an adequate risk assessment it is crucial to know how much phytoestrogens (or endocrine disruptors in general) are added to the ‘‘diet’’ of vulnerable consumers. Apart from endocrine disruptor analyses in the diet a clear insight into the endogenous estrogen background levels is needed. Recently, new data were presented using sensitive gas chromatography high-resolution mass spectrometry (GC/HRMS) down to the 2 ng/L level in plasma samples [19]. It was shown that the endogenous levels in prepubertal children are much lower than previously thought based on less specific immunoassays; as a result the diet-contribution to the total exposure becomes much more critical and relevant. Within the scope of the EU project BioCop (phyto)estrogen levels have been assessed both in soy and cow’s milk products [20]. Several bioactivity-based approaches are feasible for the screening of endocrine disruptors in the food chain. Apart from the SPR biosensor assay based on binding with specific human transport proteins already mentioned [12], robust transcription activation bioassays are available for estrogens [21]. The performance of the latest generation based on recombinant yeast cells fulfilled all validation and ISO 17025 accreditation requirements and the results obtained compared very well with GC/MS data [22]. Also a highly challenging transcriptomics approach is being explored within BioCop [20]. An MCF7 cell line is exposed to sample extracts. Next the messenger RNA (mRNA) is extracted
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from the cells and the cDNA is hybridized on a microarray carrying 47,000 human transcripts. Up and down regulation of specific transcripts was observed which will allow the design of dedicated microarrays having a limited number of transcripts for endocrine disruptor fingerprinting. A major challenge will be to ensure compatibility of real sample extracts with the cells, overall robustness and validation issues.
2.4 Metal speciation The total content of the classical heavy metals, lead, cadmium and mercury in foodstuffs and animal feeding stuff, is regulated by EU maximum levels. However, for some trace elements their speciation is very important in terms of food and feed safety and information on the total content only does not give adequate information for correct toxicological risk assessment. For example, in the case of arsenic, the inorganic forms are the most toxic while for mercury, methylmercury is the most toxic form. Seafood is the major dietary source for arsenic and mercury in the European population [23]. For speciation analysis of trace elements the use of either LC or GC with inductively coupled plasma MS (GC- and LC/ICPMS) is currently the state-of-the-art [24,25]. These techniques have been known for a couple of decades but their use is not routine yet, probably due to rather expensive instrumentation, the need for skilled personnel and the lack of standardization, thus clearly defining some of the urgent challenges. Another issue is the lack of rapid and simple field sensors for speciation analysis which address at least partly the toxicity of the specific metal forms. In the past, different microbial biosensors were developed and evaluated in aqueous model systems [26,27]. These transcription activation biosensors contain a reporter gene under metal responsive element(s). Once the cell and the responsive element detect a metal/metalloid the responsive element on the DNA will switch on the transcriptional and translational machinery producing firefly luciferase leading to light emission directly responding to the concentration. These metal biosensors determine intrinsically the bioavailable fraction of the metal, giving an estimation of the toxic potential and complementing the chemical methods where total concentration of the metals is determined. The ability of this type of biosensors to work in real food and feed extracts is a major challenge that still needs to be explored. Some people are hesitating to work with such genetically modified bacterial cell biosensors. To overcome these hesitations luminescent cells immobilised onto a fibre dipstick might be applied to metal and metal speciation analysis [28].
2.5 Alkaloid biotoxins The EFSA is very interested in alkaloid biotoxins and has requested scientific opinions for ergot alkaloids (EA) in food, and for tropane and pyrrolizidine alkaloids (TA and PA) in feed; an opinion on EA in feed has been completed showing that there is a lack of data on EA patterns in feed materials and on toxic effects [29]. In particular ergotamine and ergocristine are of concern. PA are
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widespread and can be found in many plant genera and therefore also in feed, food and herbs, jacobine and lycopsamine being the most abundant. TA, such as atropine and scopolamine, are mainly found in feed as contaminants from Datura species. Plants producing TA have expanded dramatically in parts of Europe and problems are emerging. Data on the sensitivity of animal species towards the alkaloids are incomplete and do not allow the establishment of tolerance levels for individual alkaloids and mixtures thereof; nevertheless the data available so far indicate that adverse effects may occur in animals. The very limited and often incomplete data on tissue distribution and residual concentrations in edible tissues, milk and eggs do not allow an estimation of carry-over rates to food for human consumption. Data on human exposure and sensitivity towards the alkaloids are very incomplete and do not allow the establishment of tolerance levels for individual alkaloids and mixtures thereof. No harmonized methods are available yet, although different suggestions ranging from ELISA to thin layer chromatography (TLC) and LC/MS/MS can be found in literature [30]. A general issue in the determination of these alkaloids is a lack of reliable analytical standards, (certified) reference materials, proficiency schemes and a lack of harmonized regulation.
2.6 Peptide and protein growth promoters The use of growth promoters in food producing animals has been banned in many countries since 1988 [31]. Thanks to harmonization efforts most of the EU member states are capable of detecting steroids and b-agonists at the required level, although large differences in specific analyte coverage still exist. Hormone criminality is believed to be linked with sports doping and to occur via international networks. As in sports doping it can be predicted that the abuse will shift from classical growth promoters such as steroids and b-agonists to peptides and proteins when the veterinary control of the former becomes more effective. Bovine and porcine somatotropin (bST and pST), the equivalents of human growth hormone, are 22 kDa proteins and commercially available as recombinant preparations. They are important endocrine factors influencing metabolic and somatogenic processes including growth, immune function, reproduction and lactation. Their species specificity implies low toxicity in humans, apart from potential rare allergic reactions. Major concerns are the observed increased levels of insulin-like growth factor (IGF-1) in milk [32], which are connected to the incidence of cancer [33]. Detection via instrumental LC/MS/MS approaches is feasible [34]. Recently a rapid SPR biosensor immunoassay has been proposed to detect somatotropins in illegal preparations [35]. However, routine sampling and analysis of somatotropins and associated proteins in blood, milk and other relevant matrices is still a major challenge due to the complexity of the endogenous regulation (Figure 3), protein binding and the pulsatile secretion by the pituitary gland. As a result the blood levels show a high intra- and interindividual variability. In view of this it is rather unlikely that measurement of a single parameter can provide a validated tool for the enforcement of the ban on somatotropin and
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Figure 3 Endogenous regulation along the somatotropin (growth hormone) axis.
related growth promoters. A huge challenge is envisaged that an assembly of parameters has to be measured followed by appropriate statistics in order to validate its diagnostic value versus untreated control populations.
2.7 Genetically modified organisms (GMO) Within the framework of safety assessment of food produced by means of modern genetic engineering the comparative analysis of the genetic modified (GM) crop with its unmodified genetic counterpart is an important part [36]. Two approaches are followed to detect intentional or unintentional alteration of the chemical composition of the GMO, i.e., a targeted approach (i.e., investigating defined constituents) and an untargeted approach using profiling technologies to analyse differences in RNA, proteins and metabolites [37]. Targeted analysis of single compounds with focus on important nutrients and critical toxicants has been successfully applied to demonstrate the safety of GM foods. Some criticisms have been expressed on the targeted approach as being biased and focused on known compounds and expected results [38]. This may become increasingly a problem in the second generation of GM crops where the introduction of completely new biosynthetic pathways or modifications in key enzymes in the primary or secondary structure may result in unexpected changes. Non-targeted approaches using profiling technologies based on DNA (e.g., microarray methods), proteins (two-dimensional gel electrophoresis followed by MSand/ or metabolites (using e.g., nuclear magnetic resonance (NMR), GC/MS and LC/ MS) are nowadays explored for their potential within the food safety assessment of GM food crops.
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In the EU the release of GMO and GMO-derived ingredients into the environment, and the marketing of GMO-derived food and animal feed is regulated within various specific directives and regulations. The EU regulations 1829/2003 and 1830/2003 concern the premarket safety assessment and the traceability and labeling of food and feed products derived from authorized GMOs establish that food or feed materials containing GMO-derived ingredients above the set threshold of 0.9% must be labeled as such. Implementation and enforcement of this regulation is generally performed by PCR-based methods using GMO-specific DNA probes. These methods must meet a number of agreed quality requirements. On the other hand, the presence of unauthorized GMO is not allowed at all in food and feed. In these cases, qualitative methods, such as DNA microarray methods may in the near future become very useful since these methods allow the detection of many different GMOs and GMO elements in one analysis, albeit their quantitative performance is limited [39]. Major challenges in GMO detection are quantitative aspects, validation and harmonization. Knowledge about the measurement uncertainty at the level of 0.9% is crucial already, and once a minimum required performance level (MRPL) has been assigned to the ban on unauthorized GMO that challenge will become immense.
2.8 Nanoparticles Application of nanoparticles — particles with one or more dimensions at the nanoscale — in food, medicine and agricultural products is booming and many nanobased products are already on the market [40]. Inherent to their size and surface-to-volume ratio nanoparticles often show a high chemical reactivity. The quantum-size and Coulomb-charging effects of nanoparticles may yield particles with exceptionally electric conductivity or resistance, high capacity for storing or transferring heat or changed solubility properties. Within food technology nanoparticles are used in food conservation, dietary supplements, food additives, packaging materials, functional foods and intelligent food. Some typical examples of application in the food area can be found: bakeries in Western Australia have incorporated nanocapsules containing omega-3 fatty acids in bread, the capsules will open only in the acidic environment of the stomach; the company NutraLeaset utilizes micelles with a diameter of 30 nm to deliver lycopenes, beta-carotene, lutein, phytosterols, CoQ10 and omega-3 fatty acids; Unilever is developing low fat ice creams by decreasing the size of the emulsion particles and The Oilfresh Corporation marketed a new nanoceramic product that prevents the oxidation and agglomeration of fats in deep fryers. It is envisaged that within agricultural practices nanoparticles can be used for precision farming, meaning that autonomous nanosensors are applied for realtime monitoring and early warning of plant health issues, and for controlled release of pesticides and herbicides encapsulated in nanomicelles. Some of these developments already come into reality are: Syngenta is using nanoemulsions in its pesticide products and also marketing the microencapsulated product Gutbusters that releases its content in the alkaline environment of the insect stomach; and a growth-promoting product PrimoMaxxs is used to strengthen
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the physical structure of turfgrass. The unique properties of nanoparticles make them attractive in the applications mentioned previously but also impose new unforeseen risks, hence making an evaluation of the appropriateness of the current risk assessment protocols and methods necessary. The appropriateness of risk assessment methodologies currently in place to deal with the new properties of the nanoparticles is being addressed by different authorities. Current methods for the identification of nanoparticle hazards are probably adequate but the methods for characterization of the hazard (i.e., establishment of toxicological dose-response relationships) and subsequent exposure assessment need to be adapted. Improvement of current assessment methodology should consider the following aspects: (i) physical parameters such as particle size, size distribution, surface charge, (ii) agglomeration and disagglomeration properties in different environments, (iii) impurities within, and adsorbed species onto the surface and (iv) biological processes involving nanoparticles, including translocation, cellular uptake and toxicological mechanisms. The analytical challenge for the required monitoring of nanoparticles in the food chain is immense. A vast array of analytical techniques is typically used for the characterization of the physical properties of manufactured nanoparticles: the mean size and its distribution is measured by techniques like photon correlation spectroscopy (PCS), laser diffractometry (LD), light scattering (LS), differential mobility analysis, TOFMS and microscopy, while electrophoresis is typically used to determine the particle charge density [41,42]. The crystalline structure of a nanosuspension can be assessed by differential scanning calorimetry (DSC) [41], polarized optical microscopy and scanning electron microscopy [43]. Electron spectroscopy for chemical analysis (ESCA), X-ray photoelectron spectroscopy (XPS), secondary ions mass spectroscopy (SIMS) and matrix assisted laser desorption ionization MALDI TOFMS are used for surface structure and chemical composition analysis of nanoparticles [42]. It should be stressed that so far the key issue of isolation and sample preparation of nanoparticles from food or feed samples has been hardly addressed. Definitely it will be very difficult to maintain the integrity of the nanoparticle during sample preparation and the subsequent analysis. Apart from that, many of the characterization tools employed and cited previously will not be easily implemented within a routine food control environment.
3. MASKED CONTAMINANTS A masked contaminant is defined as a contaminant present in such a form that it will escape from rapid screening or instrumental analysis and remain undetected. A contaminant might be conjugated with carbohydrates, sulfates, amino acids, fatty acids or bound to proteins or nucleic acids in the sample of interest. Rapid screening tests such as immuno or receptor assays are based on molecular recognition and will fail recognition when the binding sites of the molecule become less accessible due to modification or steric hindrance elsewhere in the molecule. Chromatographic and mass spectrometric methods
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will fail as well due to retention time shifts and changes in m/z. A typical example is the occurrence of glycosylated Fusarium mycotoxins in wheat and maize, as recently shown for deoxynivalenol (DON) and zearalenone by Berthiller et al. [44,45]. In addition to that it was shown that the ratio between free and glycosylated mycotoxins can change during fermentation processes, for example, in beer breweries [46]. It can be assumed that all biotoxins are vulnerable to some degree of conjugation and escape at least partly from detection, unless appropriate modifications to sample preparation schemes are being implemented. Risk assessments and intake data of biotoxins should be reconsidered taking this phenomenon into account, but first of all appropriate analytical methods should become available. Another example is residues of the nitrofuran antibiotics, the use of which has been prohibited within the EU because of their potentially harmful effects on human health. Former analysis of the parent drugs turned out to be completely inappropriate since they do not persist in edible tissues due to rapid metabolization. The nitrofuran metabolites on the other hand form persisting protein-bound residues which will remain undetected. Conneely et al. [47] described a method for the determination of the total content of nitrofuran metabolites in tissues incorporating an acidic hydrolysis combined with a nitrobenzaldehyde derivatization step. The method was validated for porcine liver at the 5-ng/g level. Subsequent application to poultry had a global impact: high percentages of non-compliant samples were detected and an MRPL was assessed by the EC for harmonization of residue control in importing and exporting countries.
4. UNKNOWN BIOACTIVE CONTAMINANTS Unknown bioactive contaminants might emerge in the food chain due to climate changes, illegal production methods such as the application of designer steroids and b-agonists in cattle fattening or even because of an act of terrorism. Irrespective of the origin, they will remain undetected and escape from control as long as contaminant monitoring is restricted to a defined list of target substances. At least three different approaches can be distinguished for the detection and mass spectrometric identification of unknown contaminants, a bioactivitydirected, a metabolomics-like and an in silico prediction approach.
4.1 Bioactivity-directed identification of emerging unknown contaminants Ideally, the sample should be extracted and purified in such a way to maintain all relevant contaminant classes of interest, the first challenge being clearly in the field of generic sample preparation development. The sample extract is analysed for bioactivity using a suitable assay (whole cell-, receptor-, immuno-, microbiological inhibition assay, etc.). When the sample has been screened suspect in one of these assays and the cause cannot be identified using the
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Sample pretreatment and clean-up
Gradient Liquid Chromatography
Optional on-line (Q)TOF MS(/MS) Flow split Bioactivity screening Bioassay plate
Collection plate
Identification off-line UPLC-(Q)TOFMS(/MS) GC-TOFMS
Figure 4 Generic set-up for bioactivity-directed identification of emerging unknown contaminants.
prescribed confirmatory analysis method targeted for the presence of known contaminants, then possibly an emerging unknown contaminant is present in the sample. Next the unknown can be identified using the generic experimental setup shown in Figure 4. In short, the suspect sample is fractionated by gradient LC and narrow fractions are collected in duplicate using a parallel 96-well plate setup. One plate is re-analysed for bioactivity using the original bioassay yielding a bioactivity chromatogram or ‘‘biogram’’. The duplicate well number(s) of the suspect positions in the biogram are subjected to full-scan LC/TOFMS or GC/TOFMS techniques in order to perform chemical identification of the bioactive unknown, preferably using accurate mass measurement and isotope fitting which allows elemental composition assessment of the molecular ion and its collision-induced dissociation fragments. This approach has been demonstrated for cell-, immuno- and SPR biosensor assays and applied successfully to the identification of b-agonists in feed, hormones in urine and antibiotics in chicken muscle [48–50]. A key issue in this approach is the biocompatibility between the LC fraction and the subsequent bioactivity measurement. In this respect we have good experience with wells filled with a mixture of 2 mL dimethylsulfoxide (DMSO) and 50 mL water as a keeper solvent prior to the fractionation. The
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water–acetonitrile eluate from the LC gradient yields a large number of filled wells which are simply evaporated overnight in a fume hood, leaving the substances of interest in a tiny volume of water/DMSO. Of course, an LC fractionation is also directly compatible with atmospheric pressure ionization MS. That option requires only one well plate for fraction collection and bioactivity assessment and the suspect well number is then simply correlated with the retention time in the parallel MS system [51].
4.2 Metabolomics approach to the identification of emerging unknown contaminants Another sophisticated means of identification of unknown contaminants is based on a metabolomics-like approach. Again the sample should be extracted and purified in such a way to maintain all relevant contaminant classes of interest. Next the sample, still being a highly complex mixture, is analysed by a highresolution chromatographic technique such as ultra performance liquid chromatography (UPLC), GC or even comprehensive GC GC, combined with a sensitive full-scan MS technique such as TOF, ion trap or FT Orbitrap. The data from the sample replicates are aligned in the retention time domain and compared with the data from a set of reference sample replicates. Finally uni- or multi-variate statistics are applied in order to assess the significant differences between the suspect sample and the regular reference situation. By using appropriate data analysis software contaminants could be retrieved automatically from an oily preparation, drinking water and grass samples [52]. Successful application of this approach requires the availability of a clean, stable and highly reproducible chromatography system, including reproducibility in solvent and column impurities. Moreover, the reference situation is crucial, requiring a more or less reproducible sample matrix background. For homogeneous samples such as drinking water this can be relatively easily achieved but an adequate reference for inherently inhomogeneous samples having a fluctuating composition such as feed will be very difficult to obtain. Last but not least, intelligent data analysis software is required which can automatically correct for small changes in retention time and/or mass accuracy and is capable of keeping the underlying raw data accessible for retrospective analysis, reprocessing and evaluation in its original software format. Note that a GC GC/TOFMS analysis creates a challenging four-dimensional dataset. Ignoring all these factors will yield many irrelevant data from system impurities, bulk composition changes, etc., and probably not identify the unknown emerging contaminant.
4.3 In silico prediction approach to the identification of unknowns Starting from a chemical core structure of a specific contaminant or contaminant class and having knowledge about reactivity at specific atom positions in that core structure, one can predict in silico the chemical structure of unknown contaminants, metabolites and emerging risks. On the basis of such a structure
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physicochemical parameters such as solubility, polarity, acidity can be estimated as well, thus providing a basis for the prediction of chromatographic and mass spectrometric behaviour of the unknown. Finally, GC- or LC/MS techniques can be used to perform a targeted search on the presence of the predicted unknown in samples of interest. Recently we demonstrated the feasibility of this approach by a UPLC/TOFMS search for in silico predicted metabolites and designer modifications of glucocorticosteroids in urine [53]. Even the potential modifications can be automated: we modified a commercially available software package for metabolite finding in such a way that all kinds of chemical modifications were automatically searched for in the TOFMS dataset yielding a listing of both known compounds already present in a user library and unexpected options caused by a combination of different reactions relative to the core structure, all supported by elemental compositions thanks to the accurate mass measurement.
5. EMERGING TECHNOLOGIES 5.1 Omics Genomics, proteomics and metabolomics (‘‘omics’’) are extremely valuable tools in studying biological processes, in bio- and disease marker discovery, in drug discovery, in nutrition and toxicology. Generally, cells, plants or animals are exposed and divided into a treated and an untreated group. Following the experiment both groups are analysed at the mRNA, the protein or the metabolite level using DNA microarrays, 2D-gel electrophoresis plus MALDI/TOFMS or LC/MS, NMR plus GC- or LC/MS, respectively. Next the differential regulation of thousands of targets is assessed using appropriate statistics. Usually both experimental groups are well-defined and identical, except for the treatment, and the dose of exposure is relatively high as compared to levels normally encountered in food contaminant and residue analysis. On the other hand, biomarkers thus obtained might be used for the development of dedicated screening assays based on PCR, tailored DNA microarrays, receptor or immunoassays. Assuming that such screening assays will be developed at least two scenarios can be distinguished: (i) exposure of a standard cell system or organism to the food sample extract of interest followed by isolation of mRNA, proteins or metabolites from the cells and analysis using the developed dedicated screening assay and (ii) isolation of mRNA, proteins or metabolites directly from biofluids in the food sample, usually restricted to farm animals and crops. A major challenge would be to obtain, purify and maintain the integrity of a representative isolate. But most important of all will be the validation of the biomarker targets versus their natural background variability in food, feed and biofluid matrices: real-life is quite different from standard cells or organisms! The on-going European project on new technologies to screen multiple chemical contaminants in food, acronym BioCop, has taken this challenge and is studying both transcriptomics and proteomics for chemical food contaminant analysis [20]. An MCF-7 standard cell line is used and exposed with food sample extracts for
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phytoestrogen and mycotoxin analysis, next the extracted mRNA from the cells is analysed by tailored DNA microarrays. In the proteomics topic of BioCop a multiplex SPR biosensor immunoassay is being developed to analyse protein biomarkers in blood of bovines for the screening of steroid abuse in cattle fattening.
5.2 Bioassays The acceptance and application of whole-cell bioassays in routine chemical food contaminant analysis is progressing slowly but steadily. Typical examples are the DR-CALUXs bioassay and hormone reporter gene bioassay. The principle of such assays is illustrated in Figure 5: Genetically modified cells are exposed to the sample extract containing the contaminant(s). The cells are modified in such a way that they express the receptor protein of interest, in this example the aryl hydrocarbon (Ah) receptor which binds to dioxins and PCBs. Upon binding the receptor–ligand complex travels into the cell nucleus and binds onto the modified DNA at specific responsive elements which act as a switch for the activation of a gene encoding for the production of a marker protein such as luciferase or green fluorescent protein (GFP); as a result the cells will generate light upon exposure, even at trace levels [54]. Cell assays can be performed in parallel in 96-well plates and require hardly any reagents. Only the suspect samples will require confirmatory analysis using expensive instrumental analysis methods such as GC/HRMS in the case of dioxins. A key issue in the application to food and feed samples is the stability of the cells. In general, mammalian cells seem to be more vulnerable to cell toxicity caused by matrix components and require a more stringent sample clean-up in contaminant and residue analysis. Yeast cells on the other hand are inherently more robust, allowing application to
Figure 5 Schematic representation of the mechanism of the DR-CALUXs. Reproduced and kindly provided by T.F.H. Bovee [54].
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residue analysis of estrogens in feed and urine samples following a simple solidphase extraction step [54].
5.3 (Bio)nanotechnology The nanosciences intend to deliver radical new products and processes at nanoscale dimensions by manipulation of surfaces and macromolecular assemblies. In 2005, Bruce et al. [55] reviewed the role of nanotechnology in food analysis but no real-life application of (bio)nanotechnology in chemical food contaminant analysis was presented. Since then the situation is not so much different. However, it should be noted that any SPR biosensor instrument already used in chemical food contaminant analysis is actually an example of bionanotechnology since the biointeraction is measured within a distance of 150 nanometre of the functionalized surface and the flow cells of the microfluidic system are typically in the order of some tens of nanolitres. For example, by applying SPR, Farre´ et al. [56] showed part-per-trillion sensitivity for the pesticide atrazine in natural water samples and Haasnoot et al. [57] showed the SPR determination of sulfonamide antibiotics in broiler serum and plasma as a prediction tool for residue levels in edible tissues. Another option is the use of localized surface plasmons (LSPs) sustained by functionalized silver or gold nanoparticles: a highly specific, label-less immunosensor has been constructed for the residue analysis of the anabolic steroid stanozolol down to the nanomolar range [58]. The nanoscale format of SPR can also be coupled successfully to nanoLC/TOFMS as demonstrated for the residue analysis of the antibiotic enrofloxacin in chicken muscle [59]. Gold nanoparticles can also be applied to enhance the performance of immunochromatographic strip tests (‘‘dipsticks’’) as shown by Tanaka et al. [60]. A special application of nanoparticles in the detection of chemical food contaminants will be the use of quantum dots (QDs). QDs are semiconducting crystals of a few nanometres and have unique photophysical properties such as size-tunable luminescence spectra, high quantum yields, broad absorption and narrow emission wavelengths. As a result they will be increasingly used as fluorescent labels in, for example, immunoassays [61], also in chemical food contaminant and residue analysis. Apart from detection, nanomaterials might be used in sample preparation as well. Zhao et al. [62] used carbon nanotubes as a solid-phase extraction adsorbent for the GC/MS/MS analysis of barbiturate drug residues in pork at sub-ppb levels.
5.4 Ambient mass spectrometry In 2004 a novel ambient ionization technology, called Desorption ElectroSpray Ionization (DESI), was proposed by the Cooks group at Purdue University [63,64]. For the first time, surfaces can be probed and analysed by MS under real native ambient conditions, without any sample preparation or matrix addition. The typical lay-out of a DESI source is shown in Figure 6. In short, a pneumatically assisted electrospray is used to produce charged microdroplets
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Solvent HV N2
electrospray desorbed ions
et inl of S M
Sample
moving sample stage in air
Figure 6 Schematic view of a DESI source.
and gas-phase solvent ions that are directed onto the sample at a surface. The ionization process closely resembles conventional ESI-MS; however, the sample is not present in the solvent nor ionized during the electrospray process, and is therefore less vulnerable to ionization suppression caused by the presence of salts and other interfering matrix components. Instead, ionization of the sample molecules takes place at or above the surface as a result of proton transfer, electron transfer, or ion transfer by gas-phase ions [65]. Reagents can be added to the electrospray solvent in order to tune the selectivity and the sensitivity of the DESI process [66]. That versatility is a great advantage over the direct analysis in real time (DART) ionization alternative in which helium or nitrogen is used as a reaction gas [67]. Depending on the capabilities of the MS, targeted (selected ion mode), untargeted (full-scan mode) or accurate mass analysis is possible, yielding for example, new biomarkers from differential metabolomics with the aid of principal component analysis (PCA) [68]. The ion transfer line to the atmospheric inlet of the MS can be extended and integrated with the electrospray nozzle, thus creating a probe for in situ sensing of any native or cut surface. Widely variable substrates such as paper, metal, Teflon, human skin, animal organs such as pancreas, liver and brain, plant parts such as stems, seeds, flowers and roots, glass, leather, bricks, polymers (Nylon, latex, PDMS) and silica gel have been sampled successfully. Measurements take on average 3 s but high-throughput analysis (3 samples/s) with a moving belt has also been described [69]. Automated chemical imaging of surfaces is possible. Depending on the dimensions of the electrospray tip and the distance to the surface, spot sizes will vary from 2 2 mm down to 50 50 mm. In terms of analytes, the technique is more versatile than standard ESI-MS, as also highly non-polar molecules such as carotenoids, steroids, terpenes and even volatiles can be analysed. The sensitivity of the technique lies in the low picogram or high
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femtogram range. DESI-MS is also believed to be quantitative but we have some reservations in that respect. So far, the applicability of DESI technology has been demonstrated in forensics (detection of explosives, of drug residues on banknotes, Ecstasy), pharmaceutical [69–71], plant sciences (in vivo analysis of plant alkaloids) [72], and clinical analysis, diagnosis of cancerous tissue [73] and metabolomics [68]. The option of direct surface analysis of plant alkaloids, pesticide residues and natural toxins will be very attractive for chemical food contaminant analysis. For complex, mixtures the combination of TLC and DESI can be considered, as recently demonstrated for alkaloid dietary supplements [74].
6. VALIDATION AND QA/QC CHALLENGES The validation requirements for contaminants and residue analysis in the food chain are laid down in different international regulations. Some contaminant plus sample matrix groups are using the concept of normalized and harmonized reference methods, in other situations the requirements are limited to method validation and data interpretation issues. In any case, the number of available certified reference materials and interlaboratory studies is far too low, mainly due to financial limitations and other priorities. Two issues are particularly challenging: (i) the validation of biochemical or biological rapid screening methods and (ii) the validation of generic comprehensive instrumental methods.
6.1 Biochemical or biological rapid screening assays Validations for classical instrumental chemical analysis methods are relatively straightforward, but the introduction of (multiplex) biochemical or biological rapid screening assays requires a new fit-for-purpose approach. Typically the data output is generating qualitative or semi-quantitative results, while the scope of bioactive analytes covered might be rather diverse in terms of physicochemical parameters. As a pragmatic solution we applied the following validation procedure to the validation of a whole-cell estrogen bioassay: first, five different bioactive estrogen representatives were selected ranging from polar to apolar chemistries assuming that both known and unknown estrogens would have physicochemical characteristics within that range. Then, representative sample matrices, in this case calf urine and three different types of feed matrix, and acceptable spiking levels were defined based on current legislation and/or expected sensitivity of the bioassay for that particular compound. For each sample matrix both unspiked and samples spiked with the representative individual estrogen were analysed. By doing so the unspiked samples were always screened compliant while the spiked samples were always screened suspect non-compliant at the levels chosen, allowing validation as a qualitative screening method and assessment of CCa and CCb values according to 2002/657/EC; even ISO 17025 accreditations could be obtained [75,76]. Moreover,
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Challenges in Chemical Food Contaminants and Residue Analysis
chem + 1 ng/ml 17bE2 urine + 1 ng/ml 17bE2 blank chem blank urine
1000
response units
800
600
400
200
CCα
0
-200
July 2003
# control samples
May 2005
Figure 7 Long-term performance of a validated estrogen bioassay for blank and estradiolcontaining control samples [22].
as shown in Figure 7 routine operation of the validated bioassay showed that the non-compliant control sample was always screened above the CCa decision limit and declared non-compliant while the compliant blank was only screened two times false non-compliant over a two-year period, despite the inherent signal variability of such a bioassay [22]. In other words, even a biological assay can provide consistent results as an on/off screening tool in residue and contaminant control.
6.2 Comprehensive instrumental analysis methods Thanks to the introduction of highly sensitive ion trap and TOF mass analysers in GC/MS and LC/MS full-scan positive and negative ion data of the residues and contaminants in a particular sample of interest can be easily obtained, provided that the analytes are effectively extracted and survived the clean-up step, the injection, chromatographic separation and ionization. Then the sky is the limit and hundreds of residues and contaminants can be searched for. QA/ QC considerations will require the co-analysis of control samples, and then particularly the preparation of a non-compliant control sample having a known concentration of all analytes of interest will be a huge task. The co-analysis of such a control sample will be crucial for the assessment of false compliant results, especially because in the end automated raw data processing and evaluation will be the only option to handle the screening for hundreds of residues and contaminants. It should be noted that state-of-the-art raw data processing and evaluation software will function quite well in academic standards but most often fail in real samples at residue or contaminant level.
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Considering validation of the total analysis including data processing it might be questioned whether the immense efforts of a full validation for all these hundreds of analytes in all sample matrices of interest is really required. In analogy with Section 6.1 a set of a limited number of representatives might be chosen for an initial in-house validation covering a range of physicochemical analyte characteristics and/or contaminant (sub)groups. Ideally, the choice of these representatives should be validated by performing at least a within-day replicate analysis experiment of blanks and control samples containing all analytes of interest. Additional contaminants of interest might be added to the control sample during routine operation thus providing a continuous extension of the scope of the method.
7. CONCLUSIONS AND FUTURE TRENDS The current use of routine monitoring systems for a limited number of chemical food contaminants and residues in food industry and food control laboratories does not mean in any way that the challenges we are facing are less, they simply evolved under the influence of globalization, new food technologies, climate change, etc. Not surprisingly different stakeholders have different wishes for improvements in chemical food contaminant and residue analysis: in industry priority will be at the implementation of rapid and inexpensive methods having a scope for those substances which are of paramount importance to their specific core product integrity. Recognized challenges are further increase of speed and reduction of costs, and extension of the scope. Despite their primary responsibility for food safety as laid down in the General Food Law, it cannot be reasonably expected from the food industry that they take care of any emerging known or unknown risk, which might pop up somewhere in the food chain. On the other hand, some of the emerging issues raised in this chapter will cause a problem sooner or later, requiring the availability of analysis methods having a comprehensive scope in order to provide an early warning to the food safety authorities and generate the data for risk assessment. We believe that bioactivity-based screening methods and full-scan accurate mass spectrometric identification methods are highly complementary technologies and both essential in this respect. It is recognized that still major efforts should be put to generic sample preparation and automated data processing and evaluation before such comprehensive tools can be routinely applied in a real-life environment. Current EU monitoring plans are restricted to a limited list of target contaminants and residues in a limited number of samples. Also current validation requirements are typically developed for target analysis. The advocated more comprehensive scope of analysis does not fit easily into such policies. It is recommended that at least a part of the associated efforts and resources is moved towards the actual application of comprehensive methods for known and unknown emerging food contaminants.
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ACKNOWLEDGEMENTS Partners in EU-framework projects SAFEFOODS, BioCop (www.biocop.org) and CONffIDENCE (www.confidence.org), and colleagues at RIKILT-Institute of Food Safety are acknowledged for their stimulating contributions.
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CHAPT ER
2 International Regulations on Food Contaminants and Residues Ioannis S. Arvanitoyannis
Contents
1. Introduction to EU and USA Legislation 2. Topics/Categories Covered under EU Legislation 2.1 Food safety 2.2 Food quality 2.3 Water quality 3. Topics/Categories Covered under USA Legislation 3.1 Food safety 3.2 Technological aspects 3.3 Food quality 3.4 Water protection and management References
29 31 31 51 60 63 63 65 68 71 73
1. INTRODUCTION TO EU AND USA LEGISLATION Over the past 15 years a series of food safety crises (Bovine Spongiform Encephalopathy (BSE), dioxins, high pesticide and antibiotic content in several foods, high nitrates content, presence of coliforms in drinking water, usage of Sudan Red 1 and formation of acrylamide among others) occurred within the frame of European Union (EU) thus resulting in great losses of human lives and capital. These crises made EU citizens more alert but also increased considerably the EU legislative task in an attempt to undertake preventive instead of corrective measures. One of the top priority areas was dealing with the safety and hygiene directives. In practical terms, the requirements on primary producers amount, in the main, to fairly basic hygiene procedures. Primary producers must ensure that hazards are acceptably controlled and respect other existing legislation. Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00002-0
r 2008 Elsevier B.V. All rights reserved.
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Primary producers will not be required to apply Hazard Analysis and Critical Control Point (HACCP)-based procedures. Primary producers will have to be registered with Competent Authorities, although existing forms of registration may be used for this purpose. Stakeholders are being consulted on the form the controls will take to ensure they are practicable and can be addressed in Good Practice Guides to be initiated by industry with support from other stakeholders. For most people in the EU access to potable and clean water in quite abundant quantities is taken for granted. Most people do not realise, however, that all human activities put a burden on water quality and quantity. All polluted water, whether polluted by households, industry or agriculture, returns back, one way or another, to the environment and may cause damage to human health or the environment (http://www.food.gov.uk/science/surveillance). In August 199, Food Quality Protection Act was signed in US — a comprehensive overhaul of laws that regulate pesticides in food. The new law establishes a single, strong health-based standard by using the best science available, and, for the first time, provides Americans with the ‘‘right-to-know’’ about health risks from pesticides. In August 1996, it was signed into law the Safe Water Drinking Act of 1996, strengthening protections to ensure that American families have clean, safe tap water to drink. On 2 December 1997, the Food and Drugs Administration (FDA) approved irradiation of meat products for controlling disease-causing microorganisms. Disease causing microorganisms that can be controlled by irradiation include the Escherichia coli O157:H7 and Salmonella species (http://www.hhs.gov/news/press/2000pres/20000316a.html). The Food Safety and Inspection Service (FSIS) regulates all raw beef, pork, lamb, chicken and turkey, as well as processed meat and poultry products, including hams, sausage, soups, stews, pizzas and frozen dinners (any product that contains 2 percent or more cooked poultry or 3 percent or more raw meat). Under the Federal Meat Inspection Act, the Poultry Products Inspection Act and the Egg Products Inspection Act, FSIS inspects all meat, poultry and eggs prepared for distribution in commerce, including imported products. FSIS develops and improves analytical procedures for detecting microbiological and chemical adulterants and infectious and toxic agents in meat and poultry products (http://www.usda.gov/news/pubs/97arp/arp4.htm). With 27.4 million pounds of ready-to-eat poultry products recalled due to fears of Listeria. Since January 2000, all federally inspected meat and poultry slaughtering and processing plants operate under the HACCP system to ensure meat safety. Schumer’s legislation will give the United State Department of Agriculture (USDA) the authority needed to establish and enforce microbiological standards. These standards will include specific pathogens like Listeria, Salmonella, E. coli and others identified as threats (http://schumer.senate.gov/SchumerWebsite/pressroom/ press_releases/PR01257.html). Food contamination refers to the presence in food of harmful chemicals and microorganisms that can cause consumer illness. This chapter addresses the chemical contamination of foods, as opposed to microbiological contamination, which can be found under food-borne illness (http://en.wikipedia.org/wiki/ Food_contaminants). Contaminants are substances that have not been
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intentionally added to food. These substances may be present in food as a result of the various stages of its production, packaging, transport or holding. They also might result from environmental contamination. Since contamination generally has a negative impact on the quality of food and may imply a risk to human health, the EU has taken measures to minimize contaminants in foodstuffs. Community measures have been taken for the following contaminants: mycotoxins (aflatoxins, ochratoxin A, fusarium-toxins, patulin), metals (cadmium, lead, mercury, inorganic tin), dioxins and polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAH), 3-mono-chloropropane-1, 2-diol (3-MCPD) and nitrates (http://ec.europa.eu/food/food/chemicalsafety/contaminants/index_en.htm). During their lifetime animals may be treated with medicines for prevention or cure of diseases. In food producing animals such as cattle, pigs, poultry and fish this may lead to residues of the substances used for the treatment in the food products derived from these animals (e.g. meat, milk, eggs). The residues should however not be harmful to the consumer. To guarantee a high level of consumer protection, Community legislation requires that the toxicity of potential residues is evaluated before the use of a medicinal substance in food producing animals is authorized. If considered necessary, maximum residue limits (MRLs) are established and in some cases the use of the relevant substance is prohibited (http://ec.europa.eu/food/food/chemicalsafety/residues/index_en.htm).
2. TOPICS/CATEGORIES COVERED UNDER EU LEGISLATION 2.1 Food safety The EU integrated approach to food safety aims to assure a high level of food safety, animal health, animal welfare and plant health within the EU through coherent farm-to-table measures and adequate monitoring, while ensuring the effective functioning of the internal market (http://europa.eu.int/comm/food/ intro_en.htm). Recent trends in global food production, processing, distribution and preparation are creating an increasing demand for food safety research in order to ensure a safer global food supply (http://www.who.int/foodsafety/en/). The implementation of this approach involves the development of legislative and other actions in order to (a) assure effective control systems and evaluate compliance with EU standards in the food safety and quality, animal health, animal welfare, animal nutrition and plant health sectors within the EU and in third countries in relation to their exports to the EU, (b) manage international relations with third countries and international organizations concerning food safety, animal health, animal welfare, animal nutrition and plant health and (c) manage relations with the European Food Safety Authority (EFSA) and ensure science-based risk management (http://europa.eu.int/comm/food/intro_en.htm).
2.1.1 HACCP Regulation (EC) No 178/2002 (entry into force 1/1/2005) has three main functions (a) Provides the basis for the assurance of a high level of protection of human
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health and consumers’ interest in relation to food, taking into account the diversity in the supply of food, including traditional products, while ensuring the effective functioning of the internal market. It establishes common principles and responsibilities, the means to provide a strong science base, efficient organizational arrangements and procedures to underpin decision-making in matters of food and feed safety. (b) Lays down the general principles governing food and feed at Community and national level. It established the EFSA. (c) It shall apply to all stages of production, processing and distribution of food and feed. It shall not apply to primary production for private domestic use or to the domestic preparation, handling or storage of food for private domestic consumption. The food law shall aim at the prevention of (a) fraudulent or deceptive practices, (b) the adulteration of food and (c) any other practices which may mislead the consumer. Regulation (EC) No 852/2004 (entry into force 1/1/2006) lays down general rules for food business operators on the hygiene of foodstuffs, taking particular account of the following principles: (a) primary responsibility for food safety rests with the food business operator, (b) it is necessary to ensure food safety throughout the food chain, starting with primary production, (c) it is important, for food that cannot be stored safely at ambient temperatures, particularly frozen food, to maintain the cold chain, (d) general implementation of procedures based on the HACCP principles, together with the application of good hygiene practice, should reinforce food business operators’ responsibility, (e) guides to good practice are a valuable instrument to aid food with food hygiene rules and with the application of the HACCP principles, (f) it is necessary to establish microbiological criteria and temperature control requirements based on a scientific risk assessment and (g) it is necessary to ensure that imported foods are of at least the same hygiene standard as food produced in the Community, or are of an equivalent standard. Another Regulation (EC) No 853/2004 (entry into force 1/6/2006) has no application in relation to (a) primary production for private domestic use, (b) the domestic preparation, handling or storage of food for private domestic consumption, (c) the direct supply, by the producer, of small quantities of primary products to the final consumer or to local retail establishments directly supplying to the final consumer, (d) the direct supply, by the producer, of small quantities of meat from poultry and lagomorphs slaughtered on the farm to the final consumer or to local retail establishments directly supplying such meat to the final consumer as fresh meat and (e) hunters who supply small quantities of wild game or wild game meat directly to the final consumer or to local retail establishments directly supplying the final consumer. According to Regulation (EC) No 854/2004 (entry into force 1/1/2006) audits of good hygiene practices shall verify that food business operators apply procedures continuously and properly concerning at least (a) checks on foodchain information, (b) the design and maintenance of premises and equipment, (c) pre-operational, operational and post-operational hygiene, (d) personal hygiene, (e) training in hygiene and in work procedures, (f) pest control, (g) water quality, (h) temperature control and (i) controls on food entering and leaving the establishment and any accompanying documentation.
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Regulation (EC) No 882/2004 (entry into force 1/1/2006) lays down general rules for the performance of official controls to verify compliance with rules aiming at (a) preventing, eliminating or reducing to acceptable levels risks to humans and animals, either directly or through the environment and (b) guaranteeing fair practices in feed and food trade and protecting consumer interests, including feed and food labeling and other forms of consumer information. A summary of the EU Regulations related to HACCP is given in Table 1.
2.1.2 Pesticide contamination The pesticides have been regulated by three rather old Directives (76/895/EEC, 86/362/EEC, 90/642/EEC), which appeared over a period of 13 years between 1977 and 1990. Directive 76/895/EEC (entry into force 1/1/1977) concerned products intended either for human or, in exceptional cases, animal consumption. For the purposes of this Directive the term ‘‘pesticide residues’’ comprises the residual traces of pesticides, as well as any of the toxic breakdown or metabolized products listed, present in or on the products to which the Directive applies. Member States may not prohibit or impede the putting on the market within their territories of the products referred, on the ground that they contain pesticide residues if the quantity of these residues does not exceed the established MRLs. When a Member State considers that a MRL might endanger the health of humans or of animals other than harmful organisms, that Member State may temporarily reduce that level in its own territory. In that case it shall immediately notify the other Member States and the Commission of the measures taken with a statement of the reasons thereof. The Directive 86/362/EEC (entry into force 7/8/1986) applies to the following crops: wheat, rye, barley, oats, maize, paddy rice, buckwheat, millet, grain sorghum, triticale and other cereals. The MRLs of pesticide residues exceeds 0.1 mg/kg and concerns bromomethane, carbon disulphide, carbon tetrachloride, hydrogen cyanide, cyanides expressed as hydrogen cyanide, hydrogen phosphide, phosphides expressed as hydrogen phosphide. The Directive claims that Member States (a) shall ensure that the products referred do not, from the time they are put into circulation, present a danger to human health as a result of the presence of pesticide residues, (b) may not prohibit or impede the putting into circulation within their territories of the products referred, on the ground that they contain pesticide residues, if the quantity of such residues does not exceed the MRLs, (c) shall prescribe that the products referred, may not contain, from the time they are put into circulation, levels of residues of pesticides greater than those specified and (d) shall take all necessary measures to ensure, at least by check sampling, compliance with the MRLs. There is no application to the products intended for (i) export to third countries, (ii) the manufacture of products other than foodstuffs and (iii) sowing. The Directive 90/642/EEC (entry into force 14/12/1990) also applies to products intended for export to third countries. However, the maximum pesticide residue levels shall not apply in the case of products treated before
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Table 1 Regulations (main points and comments) related to HACCP Title
Main points
Regulation (EC) No 178/2002 (entry into force 1/1/2005) General principles and requirements of food law, establishment of the European Food Safety Authority and report of procedures in matters of food safety
General principles, principles of transparency,
Regulation (EC) No 852/2004 (entry into force 1/1/2006) Hygiene of foodstuffs
Food business operators’ obligations. National and Community guides for good
general obligations of food trade, general requirements of food law. Need for independence, transparency, confidentiality and communication of the EFSA. Rapid alert system, crisis management and emergencies.
hygiene practice. Requirements of imports and exports.
Regulation (EC) No 853/2004 (entry into force 1/6/2006) Specific hygiene rules for food of animal origin
Specific rules on the hygiene of food of animal
origin for food business operators. Not applicable to food containing both products
of plant origin and processed products of animal origin.
Comments
Regulation (EC) No 854/2004 (entry into force 1/1/2006) Specific rules for the organization of official controls on products of animal origin intended for human consumption
Official controls in relation to community
Regulation (EC) No 882/2004 (entry into force 1/1/2006) Official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
Official controls of Member States relatively
establishments, general principles of official controls and actions in the case of noncompliance. Procedures concerning imports or products of animal origin and fishery products.
Source: Adapted from [1] with permission from Blackwell Publishing.
Repeals Directive EU 70/373/EEC 85/591/EEC 89/397/EEC 93/99/EEC 95/53/EC Decisions 93/383/EEC from 1/1/2006 98/728/EC from 1/1/2006 1999/313/EC from 1/1/2006
International Regulations on Food Contaminants and Residues
general obligations, competent authorities, sampling and analysis, crisis management, official controls on the introduction on food and feed of third countries and financing of official controls. Reference laboratories.
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export where it can be satisfactorily proved that (a) the third world country of destination requires that particular treatment in order to prevent the introduction of harmful organisms into its territory or (b) the treatment is necessary in order to protect the products against harmful organisms during the transport to the third world country of destination and storage there. The Directive shall not apply to the products referred, where it can be established by appropriate evidence that they are intended for (a) the manufacture of products other than foodstuffs and animal feed or (b) sowing or planting. The products or the parts of them shall not contain, from the time they are put into circulation, pesticide residue levels higher than those specified. In the case of dried products for which maximum levels are not fixed, the maximum level applicable shall be that laid down in the list, taking into account the residue concentration caused by the drying process. Member States shall ensure, at least by checking samples, compliance with the MRLs. The competent authority or authorities of the Member States shall draw up forward programs laying down the nature and frequency of the inspections to be carried out. All the Directives related to pesticide contamination are given in Table 2.
2.1.3 Radioactive contamination Regulation (EC) No 3954/87 (entry into force 2/1/1988) laid down the procedure for determining the maximum permitted levels of radioactive contamination of foodstuffs and of feedingstuffs that may be placed on the market following a nuclear accident or any other case of radiological emergency which is likely to lead to or has led to significant radioactive contamination of foodstuffs and feedingstuffs. In the event of the Commission receiving official information on accidents or on any other case of radiological emergency, substantiating that the maximum permissible levels are likely to be reached or have been reached, it will immediately adopt, if the circumstances so require, a Regulation rendering applicable those maximum permissible levels. In Regulation (EC) No 2219/89 (entry into force 25/7/1989) the conditions for exporting foodstuffs and feedingstuffs after a nuclear accident or any other radiological situation likely to lead to significant radioactive contamination of foodstuffs and feedstuffs are laid down. Foodstuffs and feedingstuffs in which the level of radioactive contamination exceeds the relevant maximum permitted levels may not be exported. The Member States shall carry out checks to ensure that the maximum permitted levels are observed. Regulation (EC) No 737/90 (entry into force 1/4/1990) applies to milk and dairy products for which the maximum permitted level of radioactivity is 370 Bq/kg The accumulated maximum radioactive level in terms of Cs-134 and Cs-137 shall be 370 Bq/kg for milk and milk products and 600 Bq/kg for all other products concerned. The titles, main points and comments of the EU Regulations about radioactive contamination are summarized in Table 3.
Table 2 Directives (main points and comments) focused on pesticide contamination Comments
Directive 76/895/EEC (entry into force 1/1/ 1977) Setting of maximum residue levels of pesticides in fruit and vegetables
Applicable to products
Amendments — Directive
intended for human and animal nutrition. Not applicable to products exported to third countries. Formal sampling to inspect whether the maximum residue levels are kept.
80/428/EEC (entry into force 11/9/1980), 81/36/EEC
Directive 86/362/EEC (entry into force 7/8/ 1986) Setting of maximum residue levels of pesticides in cereals
Not applicable to
Amendments — Directive
products exported to third world countries, not for edible or drinkable products and for those intended for sowing or planting. Products that contain pesticide residues that exceed the maximum
86/363/EEC (entry into force 7/8/1986), 88/298/EEC
(entry into force 26/2/1981), 82/528/EEC (entry into force 4/8/1982), 88/298/EEC (entry into force 20/5/ 1988), 89/186/EEC (entry into force 10/3/1989), 93/ 58/EEC (entry into force 12/7/1993), 96/32/EC (entry into force 18/6/1996), 97/41/EC (entry into force 1/8/ 1997), 1999/65/EC (entry into force 8/7/1999), 2000/ 24/EC (entry into force 4/5/2000), 2000/57/EC (entry into force 19/10/2000), 2002/66/EC (entry into force 9/8/2002), 2002/71/EC (entry into force 29/8/2002), 2002/79/EC (entry into force 4/11/2002), 2003/60/EC (entry into force 14/7/2003) Regulation (EEC) No 3768/85 (entry into force 1/1/ 1986) and No 807/2003 (entry into force 5/6/2003) Replacement, correction and amendment of articles.
(entry into force 20/5/1988), 93/57/EEC (entry into force 8/7/1993), 94/29/EEC (entry into force 23/7/ 1994), 95/39/EC (entry into force 11/9/1995), 96/33/ EC (entry into force 18/6/1996), 97/41/EC (entry into force 1/8/1997), 97/71/EC (entry into force 18/12/ 1997), 98/82/EC (entry into force 1/11/1998), 1999/ 65/EC (entry into force 8/7/1999), 1999/71/EC (entry into force 1/8/1999), 2000/24/EC (entry into force
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Main points
International Regulations on Food Contaminants and Residues
Title
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Title
Directive 90/642/EEC (entry into force 14/12/ 1990) Setting of maximum residue levels of pesticides in certain products of plant origin including fruit and vegetables
Main points
Comments
levels are prohibited from circulation. Sampling to inspect whether the maximum residue levels are kept.
4/5/2000), 2000/42/EC (entry into force 1/7/2000), 2000/48/EC (entry into force 23/8/2000), 2000/58/EC (entry into force 19/10/2000), 2000/81/EC (entry into force 11/1/2001), 2001/39/EC (entry into force 21/6/ 2001), 2001/48/EC (entry into force 4/7/2001), 2001/ 57/EC (entry into force 21/8/2001), 2002/76/EC (entry into force 27/9/2002), 2002/79/EC (entry into force 4/11/2002), 2002/97/EC (entry into force 7/1/ 2003), 2003/60/EC (entry into force 14/7/2003), 2003/ 62/EC (entry into force 22/6/2003) Regulation (EC) No 807/2003 (entry into force 5/6/ 2003) Filling, amendment and replacement of articles.
Register of list of fruits
Amendments — Directive
and vegetables containing the MRLs and of list containing residues of pesticides and their maximum levels. Commission should be annualy informed for the MRLs.
93/58/EEC (entry into force 12/7/1993), 94/30/EC
(entry into force 23/7/1994), 95/38/EC (entry into force 11/9/1995), 95/61EC (entry into force 18/6/ 1996), 97/41/EC (entry into force 1/8/1997), 97/71/ EC (entry into force 7/1/1998), 98/82/EC (entry into force 1/11/1998), 1999/65/EC (entry into force 8/7/ 1999), 1999/71/EC (entry into force 1/8/1999), 2000/ 24/EC (entry into force 4/5/2000), 2000/42/EC (entry
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Table 2 (Continued )
Products that contain
pesticide residues that exceed the maximum levels are prohibited from circulation.
International Regulations on Food Contaminants and Residues
Source: Adapted from [1] with permission from Blackwell Publishing.
into force 1/7/2000), 2000/48/EC (entry into force 23/ 8/2000), 2000/57/EC (entry into force 19/10/2000), 2000/58/EC (entry into force 19/10/2000), 2000/81/ EC (entry into force 11/1/2001), 2001/35/EC (entry into force 19/5/2001), 2001/39/EC (entry into force 21/6/2001), 2001/48/EC (entry into force 4/7/2001), 2001/57/EC (entry into force 21/8/2001), 2002/5/EC (entry into force 6/2/2002), 2002/23/EC (entry into force 27/3/2003), 2002/42/EC (entry into force 11/6/ 2002), 2002/66/EC (entry into force 9/8/2002), 2002/ 71/EC (entry into force 29/8/2002), 2002/76/EC (entry into force 27/9/2002), 2002/79/EC (entry into force 4/11/2002), 2002/97/EC (entry into force 7/1/ 2003), 2002/100/EC (entry into force 27/1/2003), 2003/60/EC (entry into force 14/7/2003), 2003/62/EC (entry into force 22/6/2003), 2003/69/EC (entry into force 22/7/2003) Regulation (EC) No 806/2003 (entry into force 5/6/ 2003) Amendment of the Directive relatively with the MRLs.
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Table 3
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Regulations (main points and comments) dealing with radioactive contamination
Title
Main points
Comments
Regulation (EC) No 3954/87 Maximum permitted levels Amendment Regulation (EC) (entry into force 2/1/ of radioactive No 2218/89 1988) contamination of Replacement of Determination of maximum foodstuffs and feedstuffs permitted levels of the Annex (entry in case of nuclear accident. radioactive contamination Duration of each into force 25/7/ regulation exceeds up to of foodstuffs and 1989) three months. feedstuffs following a Any foodstuff or feedstuff nuclear accident or any that exceeds the other case of radiological maximum permitted emergency levels is banned from the market disposal. Regulation (EC) No 2219/89 Conditions for exporting foodstuffs and feedstuffs (entry into force 25/7/ after a nuclear accident. 1989) Any foodstuff or feedstuff Special conditions for exporting foodstuffs and that exceeds the feedstuffs following a maximum permitted nuclear accident or any levels is banned for other case of radiological export. emergency Regulation (EC) No 737/90 (entry into force 1/4/ 1990) Conditions governing imports of agricultural products originating in third countries following the accident at the Chernobyl nuclear power station
Application for products
Amendment Regulation (EC) originating from third No 616/2000 countries. Determination of Replacement of maximum accumulated articles (entry radioactivity of Cs134 and into force 24/3/ Cs137. 2000) Control measures in case of non-compliance.
Source: Adapted from [1] with permission from Blackwell Publishing.
2.1.4 Residues of veterinary medicinal products According to Directive 96/22/EC (entry into force 23/5/1996) Member States shall prohibit (a) the placing on the market of stilvenes, stilbene derivatives, their salts and esters and thyrostatic substances for administering to animals of all species and (b) the placing on the market of beta-agonists for administering to animals, the flesh and products of which are intended for human consumption. They shall, also, prohibit (i) the administering to a farm or aquaculture animal of
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substances having a thyrostatic, androgenic or gestagenic action and of betaagonists, (ii) the holding of animals on a farm, the placing on the market or slaughter for human consumption of farm animals or of aquaculture animals that contain the substances referred or in which the presence of such substances has been established, (iii) the placing on the market for human consumption of aquaculture animals to which substances have been administrated and of processed products derived from such animals, (iv) the placing on the market of specific animals and (v) the processing of specific kinds of meat. Member States may authorize (1) the administering to farm animals, for therapeutic purposes, of oestradiol 17a, testosterone and progesterone and derivatives, (2) the administering, for therapeutic purposes, of authorized veterinary medicinal products containing (i) allyl trenbolone, administered orally, or beta-agonists to equidae and pets, (ii) beta-agonists, in the form of an injection to induce tocolysis in cows when calving. The official checks are carried out by the competent national authorities without prior notice. Member States shall also prohibit the importation from third countries (a) farm or aquaculture animals to which products or substances referred have been administered by any means whatsoever or to which substances or products referred have been administered, unless those substances or products were administered in compliance with the provisions and requirements laid down and the withdrawal periods allowed in international recommendations have been observed and (b) meat or products obtained from animals the importation of which is prohibited. In agreement with Directive 96/23/EC (entry into force 23/5/1996) substances having anabolic effect and unauthorized substances are (1) stilbenes, stilbene derivatives, and their salts and esters, (2) antithyroid agents, (3) steroids, (4) resorcylic acid lactones, including zeranol and beta-agonists. Moreover, veterinary drugs and contaminants such as (A) antibacterial substances, including sulfonamides, quinolones, (B) other veterinary drugs (anthelmintics, anticoccidials, carbamates and pyrethroids, sedatives, non-steroidal antiinflammatory drugs, other pharmacologically active substances) and (C) other substances and environmental contaminants (organochlorine compounds, including PCBs, organophosphorus compounds, chemical elements, mycotoxins, dyes). The plan used to carry out the required inspections shall (a) provide for detection of groups of residues or substances according to type of animal, (b) specify in particular the measures for detection of the presence of the substances referred in the animals, in the drinking water of the animals and in all places where the animals are bred or kept and residues of the aforementioned substances in live animals, their excrement and body fluids and in animal tissues and products such as meat, milk, eggs and honey and (c) comply with the sampling rules and levels. Member States may have official random checks conducted (i) during the manufacture of the substances and during their handling, storage, transport, distribution and sale or acquisition, (ii) at any point in the animal feedingstuffs production and distribution chain and (iii) throughout the production chain of animals and raw materials of animal origin covered by this Directive. Where illegal treatment is established, the competent authority must ensure that the livestock is immediately placed under
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official control. Where there is evidence of residues of authorized substances or products of a level exceeding the maximum limit for residues, the competent authority shall carry out an investigation in the farm of origin or departure, as applicable, to determine why the above limit was exceeded. In the event of repeated infringements of MRLs when animals are placed on the market by a farmer or products are placed on the market by a farmer or a processing establishment, intensified checks on the animals and products from the farm and/or establishment in question must be carried out by the competent authorities for a period of at least six months, products or carcases being impounded pending the results of analysis of the samples. Some representative points and comments of the Directives regarding residues of veterinary medical products are given in Table 4.
2.1.5 Contaminants A rather recent Regulation (EC) No 466/2001 (entry into force 5/4/2002) covers several foods both of plant and animal origin (1) fresh spinach, preserved-deep frozen or frozen spinach, fresh lettuce regarding the level of nitrates, (2) roundnuts, nuts, dried fruit, cereals, milk regarding the level of aflatoxins, (3) milk, infant fomulae, meat of bovine animals, sheep, pig and poultry, edible offal of cattle, sheep, pig and poultry, muscle meat of fish, muscle meat of wedge sole, crustaceans, bivalve molluscs, cephalopods, cereals, vegetables, fruit, berries, fats and oils, fruit juices, wines, horsemeat, liver and kidney of cattle, sheep, pig and poultry, rice, soybeans regarding the amount of heavy metals (lead, cadmium, mercury). Member States may, where justified, authorize for a transitional period the placing on the market of fresh lettuces and fresh spinach, grown and intended for consumption in their territory, with nitrate levels higher than those set as maximum levels. Member States shall communicate to the Commission by June 30 of each year, the results of their monitoring and report on the measures taken and the progress made with regard to the application and improvement of codes of good agricultural practice to reduce nitrate levels in lettuce and spinach. This information shall also contain the data on which their codes of good agricultural practice are based. The Regulation (EC) No 466/2001 has been replaced by the Regulation (EC) No 1881/2006. The latter regulation contains the community measures for the following contaminants: mycotoxins (aflatoxins, ochratoxin A, fusarium-toxins and patulin), metals (cadmium, lead, mercury and inorganic tin), dioxins and PCBs, PAHs, 3-MCPD and nitrates. The summary of this Regulation is given in Table 5.
2.1.6 Biological safety Regulation (EC) No 999/2001 (entry into force 1/7/2001) has no application to (a) cosmetic or medicinal products or medical devices, or to their starting materials or intermediate products, (b) products which are not intended for use in human food, animal feed or fertilisers, or to their starting materials or intermediate products, (c) products of animal origin intended for exhibition, teaching, scientific research, special studies or analysis and (d) live animals used in or
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Table 4 Title
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Directives (main points and comments) for residues of veterinary medical products Main points
Comments
The placing on the market Directive 96/22/EC (entry into force 23/5/1996) and the administering to farm animals of substances The prohibition on the use having a thyrostatic action in stockfarming of certain or substances having an substances having a oestrogenic, androgenic or hormonal or thyrostatic gestagenic action and of action and of beta-agonists stilbenes and beta-agonists are prohibited. A certain number of these substances may be used for therapeutic purposes provided their use is controlled.
Repeals Directive EU 81/602/EEC from 1/7/1997 Directive EU 88/146/EEC from 1/7/1997 Directive EU 88/299/EEC from 1/7/1997
The monitoring substances Directive 96/23/EC (entry are divided into two into force 23/5/1996) Measures to monitor certain groups: substances having substances and residues anabolic effect and thereof in live animals and unauthorized substances animal products on the one hand, and veterinary drugs and contaminants on the other. Determination of the official control measures and of those taken in the event of infringement.
Repeals Directive EU 85/358/EEC from 1/7/1997 Directive EU 86/469/EEC from 1/7/1997 Directive EU 89/197/EEC from 1/7/1997 Directive EU 91/664/EEC from 1/7/1997
Source: Adapted from [1] with permission from Blackwell Publishing.
intended for research. Each Member State shall carry out an annual program for monitoring BSE and scrapie. Member States shall inform the Commission of the emergence of a Transmissible Spongiform Encephalopathy (TSE) other than BSE. All official investigations and laboratory examinations shall be recorded. The purpose of Regulation (EC) No 2160/2003 (entry into force 12/12/2003) is to ensure that proper and effective measures are taken to detect and to control Salmonella and other zoonotic agents at all relevant stages of production, processing and distribution, particularly at the level of primary production, including in feed, in order to reduce their prevalence and the risk they pose to public health. This Regulation shall cover (a) the adoption of targets for the reduction of the prevalence of specified zoonoses in animal populations,
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Regulation (main points and comments) related to contaminants
Title
Main points
Comments
Regulation (EC) No 466/2001 (entry into force 5/4/2002) Setting maximum levels for certain contaminants in foodstuffs
Setting of maximum
Amendments Regulation (EC) No 1881/2006 (entry into force 10/1/2007) Replacement of the Annex
levels for certain contaminants in foodstuffs.
Source: Adapted from [1] with permission from Blackwell Publishing.
(b) the approval of specific control programs established by Member States and food and feed business operators, (c) the adoption of specific rules concerning certain control methods applied in the reduction of the prevalence of zoonoses and zoonotic agents and (d) the adoption of rules concerning intra-Community trade and imports from third countries of certain animals and products thereof. Some points of the EU legislation focused on biological safety are stated in Table 6.
2.1.7 Packaging Packaging per se and the food-packaging interactions were considered an issue of crucial importance because several problems, ranging from poisoning to death, occurred over the years. Most of these problems were either due to usage of inappropriate packaging materials or migration (of monomers, oligomers, catalysts, colorants etc.). A great number of Directives (more than ten) were published to fill this gap on packaging issues. Directive 75/106/EEC (entry into force 19/6/1976) relates to prepackages containing the liquid products measured by volume for the purpose of sale in individual quantities of between 5 mL and 10 L inclusive. This Directive shall not apply to prepackages containing the products which are vatted, bottled and labeled in volumes not exceeding 0.25 L and are intended for professional use and which are intended either for consumption on board aircraft, ships and trains or for sale in duty-free shops. The prepackages may be marked with the EEC mark. The actual contents of prepackages may be measured directly by means of weighing instruments or volumetric instruments or, in the case of liquids, indirectly, by weighing the prepacked product and measuring its density. Irrespective of the method used, the error made in measuring the actual contents of a prepackage shall not exceed one-fifth of the tolerable negative error for the nominal quantity in the prepackage. The procedure for measuring the actual contents of a prepackage may be the subject of domestic regulations in each Member State. The checking of prepackages shall be carried out by sampling and shall be in two parts a check covering the actual contents of each prepackage in the sample and another check on the average of the actual contents of the prepackages in the sample. A batch of prepackages shall be considered
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Table 6
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Regulations (main points) with regard to biological safety
Title
Main points
Regulation (EC) No 999/2001 (entry into force 1/7/2001) Rules for the prevention, control and eradication of certain TSEs.
Rules for the prevention, control and
Regulation (EC) No 2160/2003 (entry into force 12/12/2003) The control of Salmonella and other specified food-borne zoonotic agents.
Measures to detect and to control
eradication of TSEs in animals. Application to the production and
placing on the market of live animals and products of animal origin and in certain specific cases to exports thereof. Member States shall carry out an annual program for monitoring BSE and scrapie. Salmonella and other zoonotic agents. Application to production, processing
and distribution. Designation of a competent authority
for each Member State and arrangement of responsibilities. Source: Adapted from [1] with permission from Blackwell Publishing.
acceptable if the results of both these checks satisfy the acceptance criteria. For each of these checks, there are two sampling plans one for non-destructive testing, i.e., testing which does not involve opening the package and the other for destructive testing, i.e., testing which involves opening or destroying the package. Directive 76/211/EEC (entry into force 23/1/1976) relates to prepackages containing products relating to the making-up by volume of certain prepackaged liquids, and intended for sale in constant unit nominal quantities which are equal to values predetermined by the packer, expressed in units of weight or volume, not less than 5 g or 5 mL and not more than 10 kg or 10 L. A prepackage within the meaning of this Directive is the combination of a product and the individual package in which it is prepacked. A product is prepacked when it is placed in a package of whatever nature without the purchaser being present and the quantity of product contained in the package has a predetermined value and cannot be altered without the package either being opened or undergoing a perceptible modification. All prepackages must bear an indication of the weight or volume of the product, known as ‘‘nominal weight’’ or ‘‘nominal volume’’, which they are required to contain. Prepackages containing liquid products shall be marked with their nominal volume and prepackages containing other products shall be marked with their nominal weight, except in the case of trade practice or national regulations which provide otherwise and which are identical in all Member States, or in the case of contrary Community rules. If trade practice
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or national regulations are not the same in all Member States for a category of products or for a type of prepackage, those prepackages must at least show the metrological information corresponding to the trade practice or national regulations prevailing in the country of destination. In Directive 78/142/EEC (entry into force 1/2/1978), the presence of vinyl chloride monomer in, and possible migration from, materials and articles prepared with vinyl chloride polymers or copolymers, hereinafter called ‘‘materials and articles’’, which in their finished state are intended to come into contact with foodstuffs, or which are in contact with foodstuffs and are intended for that purpose is analysed. Materials and articles must not pass on to foodstuffs which are in or have been brought into contact with such materials and articles with any vinyl chloride detectable by the method which complies with the criteria laid down in this Directive. Maximum vinyl chloride monomer level in materials and articles laid down 1 mg/kg in the final product. Criteria applicable to the method of determining the level of vinyl chloride in materials and articles and of determining vinyl chloride released by materials and articles (1) the level of vinyl chloride in materials and articles and the level of vinyl chloride released by materials and articles to foodstuffs are determined by means of gas-phase chromatography using the ‘‘headspace’’ method, (2) for the purposes of determining vinyl chloride released by materials and articles to foodstuffs, the detection limit shall be 0.701 mg/kg and (3) vinyl chloride released by materials and articles to foodstuffs is in principle determined in the foodstuffs. When the determination in certain foodstuffs is shown to be impossible for technical reasons, Member States may permit determination by simulants for these particular foodstuffs. According to Directive 80/232/EEC (entry into force 17/3/1980) the products shall be divided into three groups (a) products sold by weight or by volume, (b) products sold by weight or by volume and put up in the rigid containers and (c) products put up in aerosol form. Food products in which the Directive shall apply are butter, margarine, emulsified or non-animal and vegetable fats (low fat spreads), fresh cheeses except ‘‘petits suisses’’ and other cheeses put up in the same way, table and cooking salt, impalpable sugars, red or brown sugars, candy sugars, cereal products (excluding foods for infants), dried vegetables, dried fruits, ground or unground roasted coffee, chicory and coffee substitutes, frozen products (fruit and vegetables and pre-cooked potatoes for chips, fish fillets and portions, breaded or not breaded, fish fingers). In Directive 82/711/EEC (entry into force 4/11/1982) reference is made to plastic materials and articles, that is to say to materials and articles and parts thereof (A) consisting exclusively of plastics or (B) composed of two or more layers of materials, each consisting exclusively of plastics, which are bound together by adhesives or by any other means, which, in the finished product state, are intended to come into contact or are brought into contact with foodstuffs and are intended for that purpose. For the purposes of this Directive, plastics shall mean the organic macromolecular compounds obtained by polymerization, polycondensation, polyaddition or any other similar process from molecules with a lower molecular weight or by chemical alteration of
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natural macromolecules. Silicones and other similar macromolecular compounds shall also be regarded as plastics. Other substances or matter may be added to such macromolecular compounds. However, the following shall not be regarded as plastics: (i) varnished or unvarnished regenerated cellulose film, (ii) elastomers and natural and synthetic rubber, (iii) paper and paperboard, whether modified or not by the addition of plastics, (iv) surface coatings obtained from paraffin waxes, including synthetic paraffin waxes, and/or micro-crystalline waxes and mixtures of the waxes listed in the first indent with each other and/or with plastics. The tests are to be carried out using all the simulants mentioned below, taking a fresh sample of the plastic material or article for each simulant: distilled water or water of equivalent quality, 3% acetic acid (w/v) in aqueous solution, 15% ethanol (v/v) in aqueous solution, rectified olive oil, if for technical reasons connected with the method of analysis it is necessary to use different simulants, olive oil must be replaced by a mixture of synthetic triglycerides or by sunflower oil. Directive 93/10/EEC (entry into force /1/1994) claimed that it shall apply to regenerated cellulose film which either (a) constitutes a finished product in itself or (b) forms part of a finished product containing other materials, and which is intended to come into contact with foodstuffs or which, by virtue of its purpose, does come into such contact. This Directive does not apply to (a) regenerated cellulose film which, on the side intended to come into contact with foodstuffs or which, by virtue of its purpose does come into such contact, has a coating exceeding 50 mg/dm2, (b) synthetic casings of regenerated cellulose. Printed surfaces of regenerated cellulose film shall not come into contact with the foodstuffs. Member States shall (i) permit, as from 1/1/1994, the trade in and use of regenerated cellulose film which is intended to come into contact with foodstuffs complying with this Directive, (ii) prohibit, as from 1/1/1994, the trade in and use of regenerated cellulose film which is intended to come into contact with foodstuffs not complying with this Directive and (iii) prohibit, as from 1/1/1995, the trade in and use of regenerated cellulose film which is intended to come into contact with foodstuffs. The aim of Directive 94/62/EC (entry into force 31/12/1994) was to harmonize national measures concerning the management of packaging and packaging waste in order, on the one hand, to prevent any impact thereof on the environment of all Member States as well as of third countries or to reduce such impact, thus providing a high level of environmental protection, and, on the other hand, to ensure the functioning of the internal market and to avoid obstacles to trade and distortion and restriction of competition within the Community. To this end this Directive lays down measures aimed, as a first priority, at preventing the production of packaging waste and, as additional fundamental principles, at reusing packaging, at recycling and other forms of recovering packaging waste and, hence, at reducing the final disposal of such waste. This Directive covers all packaging placed on the market in the Community and all packaging waste, whether it is used or released at industrial, commercial, office, shop, service, household or any other level, regardless of the material used. This Directive shall apply without prejudice to existing quality
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requirements for packaging such as those regarding safety, the protection of health and the hygiene of the packed products or to existing transport requirements. For the purposes of this Directive packaging, shall mean all products made of any materials of any nature to be used for the containment, protection, handling, delivery and presentation of goods, from raw materials to processed goods, from the producer to the user or the consumer. Packaging consists only of (a) sales packaging or primary packaging, (b) grouped packaging or secondary packaging and (c) transport packaging or tertiary packaging. Member States shall take the necessary measures to attain the following targets covering the whole of their territory: (a) no later than five years from the date by which this Directive must be implemented in national law, between 50% as a minimum and 65% as a maximum by weight of the packaging waste will be recovered, (b) within this general target, and with the same time limit, between 25% as a minimum and 45% as a maximum by weight of the totality of packaging materials contained in packaging waste will be recycled with a minimum of 15% by weight for each packaging material and (c) no later than 10 years from the date by which this Directive must be implemented in national law, a percentage of packaging waste will be recovered and recycled. The Commission shall promote, in particular, the preparation of European standards relating to (i) criteria and methodologies for life-cycle analysis of packaging, (ii) the methods for measuring and verifying the presence of heavy metals and other dangerous substances in the packaging and their release into the environment from packaging and packaging waste, (iii) criteria for a minimum content of recycled material in packaging for appropriate types of packaging, (iv) criteria for recycling methods, (v) criteria for composting methods and produced compost and (vi) criteria for the marking of packaging. Member States shall ensure that the sum of concentration levels of lead, cadmium, mercury and hexavalent chromium present in packaging or packaging components shall not exceed the following: (A) 600 ppm by weight two years after the date 30/6/1996, (B) 250 ppm by weight three years after the date 30/6/1996 and (C) 100 ppm by weight five years after the date 30/6/1996. Information for users of packaging Member States shall take measures, within two years of the date referred to this Directive, to ensure that users of packaging, including in particular consumers, obtain the necessary information about (i) the return, collection and recovery systems available to them, (ii) their role in contributing to reuse, recovery and recycling of packaging and packaging waste, (iii) the meaning of markings on packaging existing on the market and (iv) the appropriate elements of the management plans for packaging and packaging waste. Directive 97/23/EC (entry into force 29/7/1997) applies to the design, manufacture and conformity assessment of pressure equipment and assemblies with a maximum allowable pressure greater than 0.5 bar. Member States shall not, on grounds of the hazards due to pressure, prohibit, restrict or impede the placing on the market or putting into service under the conditions specified by the manufacturer of pressure equipment or assemblies which comply with this Directive and bear the CE marking indicating that they have undergone conformity assessment. Pressure equipment and assemblies the conformity of
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which has been assessed by a user inspectorate shall not bear the CE marking. The pressure equipment and assemblies referred to may be used only in establishments operated by the group of which the inspectorate is part. The group shall apply a common safety policy as regards the technical specifications for the design, manufacture, inspection, maintenance and use of pressure equipment and assemblies. The user inspectorates shall act exclusively for the group of which they are part. The CE marking shall be affixed in a visible, easily legible and indelible fashion to each item of pressure equipment which is complete or is in a state permitting final assessment. It is not necessary for the CE marking to be affixed to each individual item of pressure equipment making up an assembly. Individual items of pressure equipment already bearing the CE marking when incorporated into the assembly shall continue to bear that marking. Where the pressure equipment or assembly is subject to other Directives covering other aspects which provide for the affixing of the CE marking, the latter shall indicate that the pressure equipment or assembly in question is also presumed to conform to the provisions of this Directive. Pressure equipment must be designed, manufactured and checked, and if applicable equipped and installed, in such a way as to ensure its safety when put into service in accordance with the manufacturer’s instructions, or in reasonably foreseeable conditions. In choosing the most appropriate solutions, the manufacturer must apply the principles set out below in the following order: (i) eliminate or reduce hazards as far as is reasonably practicable, (ii) apply appropriate protection measures against hazards which cannot be eliminated and (iii) where appropriate, inform users of residual hazards and indicate whether it is necessary to take appropriate special measures to reduce the risks at the time of installation and/or use. Where the potential for misuse is known or can be clearly foreseen, the pressure equipment must be designed to prevent danger from such misuse or, if that is not possible, adequate warning be given that the pressure equipment must not be used in that way. The method of operation specified for pressure equipment must be such as to preclude any reasonably foreseeable risk in operation of the equipment. Particular attention must be paid, where appropriate, to (a) closures and openings, (b) dangerous discharge of pressure relief blow-off, (c) devices to prevent physical access while pressure or a vacuum exists, (d) surface temperature taking into consideration the intended use and (e) decomposition of unstable fluids. The manufacturer must ensure the competent execution of the provisions set out at the design stage by applying the appropriate techniques and relevant procedures, especially with a view to the aspects set out below. Preparation of the component parts (e.g., forming and chamfering) must not give rise to defects or cracks or changes in the mechanical characteristics likely to be detrimental to the safety of the pressure equipment. Permanent joints and adjacent zones must be free of any surface or internal defects detrimental to the safety of the equipment. The properties of permanent joints must meet the minimum properties specified for the materials to be joined unless other relevant property values are specifically taken into account in the design calculations. Where there is a risk that the manufacturing process will change the material properties to an extent which would impair the safety of the
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pressure equipment, suitable heat treatment must be applied at the appropriate stage of manufacture. Suitable procedures must be established and maintained for identifying the material making up the components of the equipment which contribute to pressure resistance by suitable means from receipt, through production, up to the final test of the manufactured pressure equipment. Directive 2002/16/EC (entry into force 15/3/2002) is applicable to materials and articles which, in the finished product state, are intended to come into contact or are brought into contact with foodstuffs and are intended for that purpose and which are manufactured with or contain one or more of the following substances: (a) 2,2-bis(4-hydroxyphenyl)propane bis(2,3-epoxypropyl) ether (hereinafter BADGE) and some of its derivatives, (b) bis(hydroxyphenyl)methane bis(2,3-epoxypropyl)ethers (hereinafter BFDGE) and some of their derivatives and (c) other novolac glycidyl ethers (hereinafter NOGE) and some of their derivatives. For the purposes of this Directive, materials and articles are (i) materials and articles made of any type of plastics, (ii) materials and articles covered by surface coatings and (iii) adhesives. This Directive shall not apply to containers or storage tanks having a capacity greater than 10,000 L or to pipelines belonging to or connected with them, covered by special coatings called ‘‘heavy-duty coatings’’. The sum of the migration levels of the following substances: (a) BADGE, (b) BADGE H2O, (c) BADGE HCl, (d) BADGE 2HCl, (e) BADGE H2O HCl shall not exceed the following limits: (i) 1 mg/kg in foodstuffs or in food simulants (analytical tolerance excluded) or (ii) 1 mg/6 dm2 in accordance with this Directive. According to proposal of Regulations COM(2003)0689 and COD(2003)0272, the target of this Regulation is to ensure the effective functioning of the internal market in relation to materials and articles intended to come into contact with foodstuffs, while providing the basis for securing a high level of protection of human health and the interests of consumers. This Regulation shall apply to materials and articles, including active and intelligent food contact materials and articles, which in their finished state (a) are intended to be brought into contact with food or (b) are already brought into contact with food and are intended for that purpose or (c) can reasonably be expected to be brought into contact with foods or to transfer their constituents to food. This Regulation shall not apply to (a) materials and articles which were manufactured and placed on the market before 1/1/1980 and which are supplied as antiques, (b) covering or coating products, such as the products covering cheese rinds, prepared meat products or fruits, which form part of food and may be consumed together with this food and (c) fixed public or private water supply equipment. Active food contact materials and articles means materials and articles that are intended to extend the shelf-life or to maintain or improve the condition of packaged food. They are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food. Intelligent food contact materials and articles means materials and articles which monitor the condition of packaged food or the environment surrounding the food. Materials and articles shall be manufactured in compliance with good
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manufacturing practice so that, under normal or foreseeable conditions of use, they do not transfer their constituents to food in quantities which could (i) endanger human health or (ii) bring about an unacceptable change in the composition of the food or a deterioration in the organoleptic characteristics thereof. For the groups of materials and articles and where appropriate, combinations of those materials and articles, specific measures may be adopted in accordance with the Directive. Those specific measures may include (a) a list of the substances and the use of which is authorized to the exclusion of all others, (b) purity standards for substances, (c) special conditions of use for substances and/or the materials and articles in which they are used, (d) specific limits on the migration of certain constituents or groups of constituents into or onto food, taking due account of other possible sources of exposure to those constituents, (e) an overall limit on the migration of constituents into or onto food, (f) provisions aimed at protecting human health against hazards arising from oral contact with materials and articles, (g) other rules to ensure compliance with this Directive, (h) basic rules for checking compliance with this Directive, (i) rules concerning the collection of samples and the methods of analysis to check compliance with this Directive, (j) additional provisions for ensuring traceability of materials and articles and (k) provisions requiring that the Commission establishes and maintains a publicly available Community Register (‘‘Register’’) of authorized substances, materials or articles. The applicant may, in accordance with the procedure laid down in this Directive, apply for a modification of the existing authorization. The application shall be accompanied by the following: (a) a reference to the original application, (b) a technical dossier containing the new information according to the guidelines, (c) a new complete summary of the technical dossier in a standardised form. The traceability of the materials and articles shall be established at all stages of manufacture, processing and distribution. Business operators shall have in place systems and procedures to allow the identification of the businesses from which and to which the materials or articles and, where appropriate, substances or products used in their manufacture have been supplied. That information shall be made available to the competent authorities on demand. The materials and articles, which are placed on the market in the Community, shall be adequately labeled or identified to facilitate their traceability through relevant documentation or information. List of groups of materials and articles which may be covered by specific measures are active and intelligent materials and articles, adhesives, ceramics, cork, elastomers and rubbers, glass, ion-exchange resins, metals and alloys, paper and board, plastics, printing inks, regenerated cellulose, textiles, varnishes and coatings, waxes and wood. All the EU Directives/Regulations dealing with packaging are given in Table 7.
2.2 Food quality The aim is to provide advice for schemes of best practice and help consumers by promoting best practice amongst assurance schemes. It covers relevant legislation, advice on the setting and delivery of production standards and
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Table 7 Directives (main points and comments) dealing with packaging Main points
Directive 75/106/EEC (entry into force 19/6/1976) Approximation of the laws of the Member States relating to the making-up by volume of certain prepackaged liquids
Directive 76/211/EEC (entry into force 23/1/1976) Approximation of the laws of the Member States relating to the making-up by weight or by volume of certain prepackaged products
Directive 78/142/EEC (entry into force 1/2/1978) Approximation of the laws of the Member States relating to materials and articles which contain vinyl chloride monomer and are intended to come into contact with foodstuffs
Application to packages containing liquid products, measured by volume and intended for sale in unit quantities varying between 5 mL and 10 L. All prepackages must bear an indication of the volume of the liquid which they are required to contain.
Application to prepackages containing products, except liquids, intended for sale in constant unit nominal quantities. All prepackages must bear an indication of the weight or volume of the product.
Materials and articles which are intended to come into contact with foodstuffs can transfer vinyl chloride monomer to these articles in quantities liable to endanger human health. The Directive specifies that such materials and articles must not contain vinyl chloride monomer in a quantity exceeding 1 mg/kg of finished product and must not pass on to foodstuffs more than 0.01 mg/kg of vinyl chloride monomer.
Comments Amendments — Directive EU 78/89/EEC (entry into force 1/1/80) 79/1005/EEC (entry into force 1/1/1981) 85/10/EEC (entry into force 20/12/1985) 88/316/EEC (entry into force 30/6/1988) 89/676/EEC (entry into force 1/7/1990) Replacement and repeal of specific parts of articles and annexes.
Amendment Directive EU 78/891/EEC (entry into force 29/9/1978) Replacements relatively the technical progress in the annexes.
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Title
Directive 80/232/EEC (entry into force 17/3/1980) Approximation of the laws of the Member States relating to the ranges of nominal quantities and nominal capacities permitted for certain prepackaged products
This Directive shall apply to products put up in prepackages. It shall not apply to prepackaged products intended solely for professional use. The products referred to in Directive shall be divided into three groups.
Directive 82/711/EEC (entry into force 4/11/1982) Laying down the basic rules necessary for testing migration of the constituents of plastic materials and articles intended to come into contact with foodstuffs
Directive 93/10/EEC (entry into force 1/1/1994) Relating to materials and articles made of regenerated cellulose film intended to come into contact with foodstuffs
Directive 94/62/EC (entry into force 31/12/1994)
This Directive aims to harmonize national measures concerning the management of packaging and packaging waste in order to prevent any impact thereof on the
Repeal Directive EU 85/339/EEC from 30/6/1996
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This Directive shall apply to regenerated cellulose film. This Directive does not apply to regenerated cellulose film which, on the side intended to come into contact with foodstuffs or which, by virtue of its purpose does come into such contact, has a coating exceeding 50 mg/dm2 and synthetic casings of regenerated cellulose. Printed surfaces of regenerated cellulose film shall not come into contact with the foodstuffs.
Amendments Directive EU 93/8/EEC (entry into force 22/3/93) 97/48/EC (entry into force 1/9/ 97) Lists of simulants which are used in testing migration. Replacement of the annexes.
International Regulations on Food Contaminants and Residues
Relating to testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs. Migration tests may be carried out using simulants. Verification of compliance of migration into foodstuffs shall be carried out under the most extreme conditions of time and temperature foreseeable in actual use.
Repeal Directive EU 83/229/EEC from 1/1/94 Amendments Directive EU 93/111/EEC (entry into force 17/12/1993) Replacement of a part of an article. Directive EU 2004/14/EC (entry into force 24/3/2004) Addition and replacement in articles.
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Table 7 (Continued ) Main points
Packaging and packaging waste
environment, to ensure the functioning of the internal market and to avoid obstacles to trade. This Directive covers all packaging placed on the market in the Community and all packaging waste, whether it is used or released at industrial, commercial, office, shop, service, household or any other level, regardless of the material used. Labeling of packaging. Concentration levels of heavy metals present in packaging.
Comments Amendments Directive EU 97/129/EC (entry into force 12/3/1997) Identification system for packaging materials. Directive EU 97/138/EC (entry into force 12/3/1997) Formats relating to the database system. Directive EU 1999/42/EC (entry into force 3/2/1999) Measures relating to packaging and packaging waste. Directive EU 1999/177/EC (entry into force 28/2/1999) Conditions for a derogation for plastic crates and plastic pallets in relation to the heavy metal concentration levels. Directive EU 1999/823/EC (entry into force 12/12/1999) Confirming the measures notified by the Netherlands on packaging and packaging waste. Directive EU 2001/171/EC (entry into force 1/3/2001) Establishing the conditions for a derogation for glass packaging in relation to the heavy metal concentration levels.
Ioannis S. Arvanitoyannis
Title
Directive EU 2001/524/EC (entry into force 18/7/2001) Publication of references for standards EN 13428:2000, EN 13429:2000, EN 13430:2000, EN 13431:2000 and EN 13432:2000.
Directive 2002/16/EC (entry into force 15/3/2002) Use of certain epoxy derivatives in materials and articles intended to come into contact with foodstuffs
Proposal of Regulation COM(2003)0689 COD(2003)0272 Materials and articles intended to come into contact with food
Materials and articles containing certain substances may transfer significant levels of these substances to foodstuffs (migration), particularly when used as additives, and thus represent a potential risk to human health. It shall apply to plastic materials and articles, materials and articles covered by surface coatings and adhesives. Authorization of the placing on the market of two types of packaging which act ‘‘intelligently’’ in contact with foodstuffs. A positive list of authorized substances may be established. Authorization of the marketing of new plastics by authorizing the use of new substances in their manufacture.
Source: Adapted from [1] with permission from Blackwell Publishing.
Repeal Article 22 of Directive EU 76/ 767/EEC from 29/11/1999.
Repeal Directive EU 2001/61/EC
Repeals Decision EU 80/590/EC Decision EU 89/109/EC
International Regulations on Food Contaminants and Residues
This Directive applies to equipment and assemblies subject to a maximum allowable pressure PS exceeding 0.5 bar. The assessment procedures depend on the risk inherent in the pressure equipment.
Directive 97/23/EC (entry into force 29/7/1997) Approximation of the laws of the Member States concerning pressure equipment
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transparency for consumers. Food safety is the prime condition for food quality and an absolute, non-negotiable must. This is also true of compliance with legally established standards for the environment and animal welfare since they relate to the protection of natural resources and requirements of an ethical nature, in addition to the characteristics of the products (http://europa.eu.int/comm/ agriculture/foodqual/index_en.htm). Food Quality covers food marketing, consumer research, sensory science and nutrition, as well as food research and development and quality assurance. It is aimed at food industry professionals and researchers aiming to match consumer preferences and food quality (http:// www.elsevier.com/wps/find/journaldescription.cws_home/405859/description# description). Legislation in the food safety field started in the 1960s, grew more intense in the 1990s with the advent of the single market. The 1992 and 1999 common agricultural policy reforms emphasised agri-environmental measures and aid for extensification, and also in 1992 there was the introduction of European quality labels. Community legislation cannot and should not take over entirely from that of the Member States and attempt to cover all aspects of quality; rather, it should seek to work in tandem on pursuing a policy to foster quality (http://europa.eu.int/comm/agriculture/foodqual/index_en.htm).
2.2.1 Labeling, general provisions Directive 90/496/EEC (entry into force 1/10/1990) concerns nutrition labeling of foodstuffs to be delivered as such to the ultimate consumer, it shall also apply to foodstuffs intended for supply to restaurants, hospitals, canteens and other similar mass caterers. This Directive shall not apply to natural mineral waters or other waters intended for human consumption and diet integrators/food supplements. Where nutrition labeling is provided, the information to be given shall consist of either group 1 or group 2 in the following order Group 1: (a) energy value, (b) the amounts of protein, carbohydrate and fat. Group 2: (a) energy value, (b) the amounts of protein, carbohydrate, sugars, fat, saturates, fibre and sodium. Directive 2000/13/EC (entry into force 26/5/2000) concerns the labeling of foodstuffs to be delivered as such to the ultimate consumer and certain aspects relating to the presentation and advertising thereof. The following particulars alone shall be compulsory on the labeling of foodstuffs: (1) the name under which the product is sold, (2) the list of ingredients, (3) the quantity of certain ingredients or categories of ingredients, (4) in the case of prepackaged foodstuffs, the net quantity, (5) the date of minimum durability or, in the case of foodstuffs which, from the microbiological point of view, are highly perishable, the ‘‘use by’’ date, (6) any special storage conditions or conditions of use, (7) the name or business name and address of the manufacturer or packager, or of a seller established within the Community, (8) particulars of the place of origin or provenance where failure to give such particulars might mislead the consumer to a material degree as to the true origin or provenance of the foodstuff, (9) instructions for use when it would be impossible to make appropriate use of the foodstuff in the absence of such instructions and (10) with respect to beverages containing more than 1.2% by volume of alcohol, the actual alcoholic
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strength by volume. A summary of the Directives related to labeling is given in Table 8.
2.2.2 Labeling Regulation (EEC) No 1576/89 (entry into force 15/6/1989) lays down the general rules on the definition, description and presentation of spirit drinks. For the purposes of this Regulation spirit drink shall mean an alcoholic liquid intended for human consumption, having particular organoleptic qualities and a minimum alcoholic strength of 15 vol.% and produced either directly by the Table 8
Directives (main points and comments) related to labeling; general provisions
Title
Main points
Comments
Directive 90/496/EEC (entry into force 1/10/1990) Approximation of the laws of the Member States relating to food supplements
Nutrition labeling of
Amendment Directive EU 2003/ 120/EC (entry into force 26/10/1990) Addition of an article.
Directive 2000/13/EC (entry into force 26/ 5/2000) Approximation of the laws of the Member States relating to the labeling, presentation and advertising of foodstuffs
Application to pre-
foodstuffs for the ultimate consumer and for mass caterers. Not applicable to natural mineral waters or any other waters intended for human consumption or to diet integrators/food supplements. Nutrition labeling is not compulsory. The declared energy value and amount of nutrients shall be given in figures using specific units of measurement. packed foodstuffs with final receiver the consumer and mass caterers. No application to products which are exported from the Community. Obligatory recordation of specific elements.
Source: Adapted from [1] with permission from Blackwell Publishing.
Directive EU
79/112/EEC Amendments Directive EU 2001/
101/EC (entry into force 14/12/2001) Addition of an Annex. Directive EU 2003/ 89/EC (entry into force 24/11/2004) Replacement of an article.
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distillation, with or without added flavourings, of natural fermented products, and/or by the maceration of vegetable substances and/or the addition of flavourings, sugars or other sweetening products and/or other agricultural products to ethyl alcohol of agricultural origin and/or to distillate of agricultural origin and/or to spirit as defined in this Regulation or by the mixture of a spirit drink with one or more other spirit drinks, ethyl alcohol of agricultural origin, distillate of agricultural origin or spirit, one or more alcoholic drinks, one or more drinks. Categories of spirit drink and their alcoholic strength are (a) rum (37.5%), (b) whisky/whiskey (40%), (c) grain spirit (35%), (d) wine spirit (37.5%), (e) Brandy or Weinbrand (36%), (f) grape marc spirit or grape marc (37.5%), (g) fruit marc spirit (37.5%), (h) raisin spirit or raisin brandy (37.5%), (i) fruit spirits (37.5%), (j) cider spirit, cider brandy or perry spirit (37.5%), (k) gentian spirit (37.5%), (l) fruit spirit drinks (25%), (m) Juniper-flavoured spirit drinks (25%), (n) caraway-flavoured spirit drinks (30%), (o) aniseed-flavoured spirit drinks (25%), (p) bitter-tasting spirit drinks or bitter, (q) vodka, (r) liqueur, (s) egg liqueur/advocaat/avocat/Advokat and (t) liqueur with egg. Regulation (EEC) No 1601/91 (entry into force 17/6/1991) lays down the general rules on the definition, description and presentation of aromatized wines, aromatized wine-based drinks and aromatized wine-product cocktails. Aromatized wine shall mean a drink (i) obtained from wines with the exception of retsina table wine and possibly with added grape must, grape must in fermentation and/or fresh grape must with fermentation arrested by the addition of alcohol, as defined by Community legislation, (ii) which has been flavoured with the aid of natural flavouring substances and/or natural flavourings preparations or aromatic herbs and/or spices and/or flavouring foodstuffs, (iii) which has generally been sweetened and has possibly been coloured with caramel and (iv) which has a minimum actual alcoholic strength by 14.5 vol.% or more and a maximum actual alcoholic strength by less than 22 vol.% and a minimum total alcoholic strength by 17.5 vol.% or more, however, for those products which bear the description ‘‘dry’’ or ‘‘extra dry’’, the minimum total alcoholic strength shall be set at 16 vol.% and 15 vol.%, respectively. Regulation (EEC) No 2081/92 (entry into force 24/7/1993) lays down rules on the protection of designations of origin and geographical indications of agricultural products intended for human consumption. For the purposes of this Regulation (a) designation of origin means the name of a region, a specific place or, in exceptional cases, a country, used to describe an agricultural product or a foodstuff originating in that region, specific place or country and the quality or characteristics of which are essentially or exclusively due to a particular geographical environment with its inherent natural and human factors, and the production, processing and preparation of which take place in the defined geographical area, (b) geographical indication means the name of a region, a specific place or, in exceptional cases, a country, used to describe an agricultural product or a foodstuff originating in that region, specific place or country and which possesses a specific quality, reputation or other characteristics attributable to that geographical origin and the production and/or processing and/or preparation of which take place in the defined geographical area.
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Furthermore, another Regulation (EEC) No 2200/96 (entry into force 1/1/ 1997) claims that it shall apply to fresh fruits, which are apple, apricot, avocado, grapes, lemon, kiwifruit, tangerine, cherry, melon, water melon, orange, pear, strawberry, nectarine, peach and plum, and vegetables, which are artichoke, bean, carrot, celery, garlic, onion, spinach, chicory, lettuce, pepper, cabbage, cauliflower, vegetable marrow, leek, pea, eggplant, cucumber, tomato and endive. The Regulation (EC) No 2815/98 (entry into force 31/10/2001) concerning marketing standards for olive oil claims that the designation of origin shall relate to a geographical area and may mention only (a) a geographical area whose name has been registered as a protected designation of origin or protected geographical indication and/or (b) for the purposes of this Regulation: a Member State, the European Community, a third country. The designation of origin, where this indicates the European Community or a Member State shall correspond to the geographical area in which the ‘‘extra virgin olive oil’’ or ‘‘virgin olive oil’’ was obtained. However, in the case of blends of ‘‘extra virgin olive oils’’ or ‘‘virgin olive oils’’ in which more than 75% originates in the same Member State or in the Community, the main origin may be designated provided that it is followed by the indication ‘‘selection of (extra) virgin olive oils more than 75% of which was obtained in y (designation of origin)’’. An extra virgin or virgin olive oil shall be deemed to have been obtained in a geographical area for the purposes of this paragraph only if that oil has been extracted from olives in a mill located within that area. Regulation (EC) No 1439/99 (entry into force 14/9/1999) should apply to wine that is sold in the Community. Categories of wine in which the Regulation is set are (a) table wine, (b) table wine with geographical indication, (c) wine produced in specific areas and (d) imported wine. According to Regulation (EC) No 1760/2000 (entry into force 14/8/2000) each Member State shall establish a system for the identification and registration of bovine animals. The system for the identification and registration of bovine animals shall comprise the following elements: (a) ear tags to identify animals individually, (b) computerised databases, (c) animal passports and (d) individual registers kept on each holding. With the exception of transporters, each keeper of animals shall keep an up-to-date register and once the computerized database is fully operational, report to the competent authority all movements to and from the holding and all births and deaths of animals on the holding, along with the dates of these events, within a period fixed by the Member State of between three and seven days of the event occurring. In Regulation (EC) No 1321/2002 (entry into force 1/8/2002), the said producers shall subsequently be inspected regularly. They shall keep current records, for a minimum of six months following dispatch, of the number of birds by type of poultry system showing also the number of birds sold and the names and addresses of the purchasers and quantities and source of feed supply. The definitions given are applicable to the poultry cuts. The sample sizes should be at least as follows: (i) chicken breast: half of the breast, (ii) chicken breast fillet: half of the boned breast without skin and (iii) turkey breast, turkey breast fillet and boned leg meat: portions of about 100 g.
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According to Regulation (EC) No 318/2003 (entry into force 25/2/2003) checks are to be carried out in the packing centres on graded eggs ready for dispatch and not to eggs leaving the packing centres. In order to ensure proper land management and to prevent a build-up of harmful diseases, the open-air runs for laying hens may need to be rotated. Birds should be given even access to the whole paddock area and, where rotation on extensive free range system with at least 10 m2 per hen is practised, each bird should be ensured at all times at least 2.5 m2. The titles, main points and comments of the EU Regulations about labeling are summarized in Table 9.
2.3 Water quality 2.3.1 Directives for water quality The Directive 75/440/EEC claims that surface water shall be assumed to conform to the relevant parameters if samples of this water taken at regular intervals at the same sampling point and used in the abstraction of drinking water, show that it complies with the parametric values for the water quality in question, in the case of 95% of the samples for parameters conforming to those specified and 90% of the samples in all other cases, and if in the case of the 5 or 10% of the samples which do not comply (a) the water does not deviate from the parametric values in question by more than 50%, except for temperature, pH, dissolved oxygen and microbiological parameters, (b) there can be no resultant danger to public health and (c) consecutive water samples taken at statistically suitable intervals do not deviate from the relevant parametric values. The definition of the standard methods of treatment for transforming surface water of categories A1, A2 and A3 into drinking water includes Category A1 (simple physical treatment and disinfection, e.g., rapid filtration and disinfection), Category A2 (normal physical treatment, chemical treatment and disinfection, e.g., pre-chlorination, coagulation, flocculation, decantation, filtration, disinfection) and Category A3 (intensive physical and chemical treatment, extended treatment and disinfection, e.g., chlorination to break-point, coagulation, flocculation, decantation, filtration, adsorption, disinfection). With regard to Directive 79/923/EEC Member States (a) shall within a two year period following the notification of this Directive, designate shellfish waters, (b) may subsequently make additional designations, (c) may revise the designation of certain waters owing in particular to factors unforeseen at the time of designation and (d) shall establish programs in order to reduce pollution and to ensure that designated waters conform to the values set by the Member States, within six years following designation. Where the competent authority records that the quality of designated waters is appreciably higher than that which would result from the application of the values set, the frequency of the sampling may be reduced. According to Directive 98/83/EC, Member States may exempt from the provisions of this Directive the water intended exclusively for those purposes for which the competent authorities are satisfied that the quality of the water has no influence, either directly or indirectly, on the health of the consumers
Table 9 Regulations (main points and comments) for labeling Title
Main points
Regulation (EEC) No 1576/89 (entry into force 15/6/1989) General rules on the definition, description and presentation of spirit drinks
Regulation (EEC) No 1601/91 (entry into force 17/6/1991) Laying down general rules on the definition, description and presentation of aromatized wine, aromatized winebased drinks and aromatized wineproduct cocktails.
Regulation (EEC) No 2081/92 (entry into force 24/7/1993) Protection of geographical indications and designations of origin for agricultural products and foodstuffs
Regulation (EEC) No 2200/96 (entry into force 1/1/1997) Labeling of fresh fruits and vegetables.
The minimum alcoholic strength by volume below which a product may not be marketed in the territory of the EU is laid down. Addition of specific substances to the products.
Three categories of drinks according to their wine content, their alcoholic strength, and whether they contain added alcohol.
No application to wine products or to spirit drinks. Names that have become generic may not be registered. To be eligible to use a protection of designation of origin (PDO) or a protection of geographical indications (PGI) an agricultural product or foodstuff must comply with a specification. Marketing standard for fresh fruits and vegetable. Laying down a list of fresh fruits and vegetable.
Comments Amendments Regulation (EEC) No 3280/92 (entry into force 16/11/1992) Addition to an article. Regulation (EEC) No 3378/94 (entry into force 1/1/1995) Addition of articles.
Amendments Regulation (EEC) No 3279/92 (entry into force 16/11/1992) Regulation (EC) No 3378/94 (entry into force 1/1/1995) Regulation (EC) No 2061/96 (entry into force 3/11/1996) Addition and replacement of articles.
Amendments Regulation (EC) No 535/97 (entry into force 15/4/1997) Regulation (EC) No 1068/97 (entry into force 28/6/1997) Regulation (EC) No 2796/2000 (entry into force 10/1/2001) Completion of articles.
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Table 9 (Continued ) Title
Main points
Regulation (EC) No 2815/98 (entry into force 31/10/2001) Marketing standards for olive oil.
Regulation (EC) No 1439/99 (entry into force 14/9/1999) Marketing standards for wine
Comments
The designation of origin shall relate to a geographical area. The ‘‘extra virgin olive oil’’ and ‘‘virgin olive oil’’ which shall be packaged in an establishment approved for that purpose. Labeling is compulsory and it contains information about the origin of wine. Categories of wine and rules for the marketing standards.
Regulation (EC) No 1760/2000 (entry into force 14/8/2000) Establishment of a system for the identification and registration of bovine animals and regarding the labeling of beef and beef products
Every Member State must set up a cattle identification and registration system. Compulsory labeling of beef and beef products.
Repeal Regulation (EC) No 820/97 Amendment Regulation (EC) No 1825/2000 Addition for traceability, labeling and controls of bovine animals and their products (entry into force 1/9/2000)
Regulation (EC) No 1321/2002 (entry into force 1/8/2002) Marketing standards for poultry meat
Mark of designation of origin for fresh and frozen poultry meat only in the case of imports from countries out of the Community. Inspections for designation of origin in the packaging plants.
Repeal Regulation (EC) No 1906/90
Regulation (EC) No 318/2003 (entry into force 25/2/2003) Certain marketing standards for eggs.
Obligatory sign of designation of origin on eggs packaging originated from a third country. Inspections in the plants of packaging.
Source: Adapted from [1] with permission from Blackwell Publishing.
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concerned and water intended for human consumption from an individual supply providing less than 10 m3 a day as an average or serving fewer than 50 persons, unless the water is supplied as part of a commercial or public activity. All the Directives related to water quality are given in Table 10.
3. TOPICS/CATEGORIES COVERED UNDER USA LEGISLATION 3.1 Food safety Regulating food safety in the United States is complex. This complexity is due largely to the historical division of food safety responsibility amongst different federal agencies and to evolving public attitudes towards the safety of food and concern about the changing nature of food-borne illnesses (http://www.national aglawcenter.org/readindrooms/foodsafety). A food business is defined as being any undertaking regarding food, whether carried out for profit or not, carrying out one or more of the following operations: preparation, processing, manufacturing, packaging, storage, transportation/distribution, handling, offering for sale or supplying a consumer (http://www.eastherts.gov.uk/Buisiness%20Guide% 20to% 20Law%20and%20Practise/Legislation.htm). The Food Safety Act 1990 and regulations specify certain safety standards for the processing and sale of food. It is an offence for anyone to process or sell food, which is harmful to health. The regulations also place an obligation on businesses to ensure that their activities are carried out in a hygienic way. Running a food business means that you have a particular responsibility to protect the health of your customers (http://www.torridge.gov.uk/index.cfm?articleid ¼ 3967).
3.1.1 Food safety legislation In Consumer Product Safety Act (1972) the term ‘‘consumer product’’ means any article or component part thereof, produced or distributed (i) for sale to a consumer for use in or around a permanent or temporary household or residence, a school, in recreation, or otherwise, or (ii) for the personal use, consumption or enjoyment of a consumer in or around a permanent or temporary household or residence, a school, in recreation, or otherwise; but such term does not include (a) any article which is not customarily produced or distributed for sale to, or use or consumption by, or enjoyment of, a consumer, (b) tobacco and tobacco products, (c) motor vehicles or motor vehicle equipment and (d) pesticides. Following Food Quality Protection Act (1996) key issues revolved around: the roles of state, local and tribal governments in pesticide regulation and federal law enforcement; the cost of scientific tests required by the U.S. Environmental Protection Agency (EPA) to support pesticide registration and re-registration, and delays in processing applications for new or amended registrations, especially for minor uses; and long delays in re-registration of older pesticides and the need for fees to support the effort. A lesser issue involved state authority to require training of persons who regularly apply non-restricted pesticides in
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Table 10
Directives (main points and comments) focused on water quality
Title
Main points
Comments
Directive 75/440/ EEC (entry into force16/6/1977) The quality required of surface water intended for the abstraction of drinking water
Demands required for the quality of surface water intended for the abstraction of drinking water. Not applicable to subterranean and subsaline water. Variations in the case of floods or other natural disasters are accepted.
Amendments Directive EU 79/869/EEC Methods of measurement, sampling frequency and parameter analysis. Amendment, repeal and replacement of articles (entry into force 9/10/ 1981) Directive EU 91/692/EEC Standardization of reports concerning the application of specific directives for the environment Articles replacement (entry into force 1/1/ 1993)
Directive 79/923/ EEC (entry into force 5/11/1981) Quality required of shellfish waters
Applicable to coastal and brackish waters in order to support shellfish life. Criteria for minimum demanded quality of shellfish waters. Two year period for Member States to designate shellfish waters. Decline in case of exceptional weather or geographical conditions.
Amendment Directive EU 91/692/EEC Standardization of reports concerning the application of specific directives for the environment (entry into force 1/1/1993)
Directive 98/83/EC (entry into force 25/12/1998) Quality of water intended for human consumption
Application for human health protection from potential contamination. Not applicable to natural, mineral waters and waters that are medicinal products. Demands for limited material migration in plants of drinking water.
Source: Adapted from [1] with permission from Blackwell Publishing.
Repeal of Directive EU 80/778/EEC
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urban and suburban areas. Canceling a pesticide registration, when it is found to cause ‘‘unreasonable adverse effects’’, can be a prolonged process, lasting 4–8 years or more. Another Public Health Security and Bioterrorism Preparedness and Response Act (2002) claims that the Secretary of Agriculture shall by regulation establish and maintain a list of each biological agent and each toxin that the Secretary determines has the potential to pose a severe threat to animal or plant health, or to animal or plant products. In determining whether to include an agent or toxin on the list under this subparagraph, the Secretary shall (1) consider (i) the effect of exposure to the agent or toxin on animal or plant health, and on the production and marketability of animal or plant products, (ii) the pathogenicity of the agent or the toxicity of the toxin and the methods by which the agent or toxin is transferred to animals or plants, (iii) the availability and effectiveness of pharmacotherapies and prophylaxis to treat and prevent any illness caused by the agent or toxin and (iv) any other criteria that the Secretary considers appropriate to protect animal or plant health or animal or plant products and (2) consult with appropriate Federal departments and agencies and with scientific experts representing appropriate professional groups. In Food Safety Act (2002) ‘‘food’’ means food or drink for human consumption, and includes (a) any substance or thing that is manufactured, sold or represented for use as food or drink for human consumption, (b) any substance or thing that is manufactured, sold or represented for use as an additive, ingredient or processing aid in a substance or thing referred to in (a) and (c) any agricultural or aquatic product that is grown, raised, cultivated, harvested or kept for the purpose of producing food or drink for human consumption. An operator is responsible for ensuring that the food in his or her food establishment is safe for human consumption. An inspector who believes on reasonable grounds that food (a) does not meet a prescribed standard for that food or (b) is contaminated or otherwise unfit for human consumption, may (c) seize the food or have it seized and (d) detain it, or have it detained, for examination or inspection that the inspector considers necessary to determine whether that standard has been met or whether the food is contaminated or unfit for human consumption. Some points of the US legislation focused on biological safety are stated in Table 11.
3.2 Technological aspects Factory-made foods have made chemical additives a significant part of our diet. Most people may not be able to pronounce the names of many of these chemicals, but they still want to know what the chemicals do and which ones are safe and which are poorly tested or possibly dangerous. The listing in http://www. cspinet.org/reports/chemcuisine.htm provides the information about most common additives. Over the years, however, improvements have been made in increasing the efficiency and ensuring the safety of all additives. Today food and colour additives are more strictly regulated that at any other time in history. The basis of modern food law is the Federal Food, Drug and Cosmetic Act of 1938,
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US legislation (title, main points) focused on food safety
Title
Main points
Consumer Product Safety Act (1972)
Food Quality Protection Act (1996)
Food Safety Act (2002)
Definitions (consumer product, manufacturer etc.) Consumer product safety standards Commission responsibility — petition for consumer product safety rule Product certification and labeling Financial penalties
Comments Amendments 1976 (Consumer Product Safety Commission Improvement Act) 1978 (Consumer Product Safety Authorization Act) 1981 (Consumer Product Safety Amendments) 1983 (Lead Contamination Control Act) 1988 (Anti-drug Abuse Act) 1990 (Consumer Product Safety Improvement Act) 1994 (Child Safety Protection Act)
It related to pesticide uses The new law will facilitate registrations and reregistrations of pesticides for special (so-called ‘‘minor’’) uses and authorize collection of maintenance fees of support pesticide reregistration.
Definitions (food, food establishment etc.) Licenses for designated food establishments Seizure and destruction of food
Regulation of certain Public Health biological agents and toxins Security and Food safety and security Bioterrorism Preparedness and strategy Notices to States regarding Response Act (2002) imported food Source: Adapted from [2] with permission from Blackwell Publishing.
which gives the Food and Drug Administration (FDA) authority over food and food ingredients and defines requirements for truthful labeling of ingredients (http://www.cfsan.fda.gov/Blrd/foodaddi.html). Another technological aspect is food irradiation. Food irradiation is one means of food preservation that may
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not be familiar to many, but it has been in development since the early decades of the twentieth century. If properly applied, irradiation can be an effective way to treat a variety of problems in our food supply, such as insect infestation of grains, sprouting of potatoes, rapid ripening of fruits and bacterial growth (http://www. fcs.uga.edu/pubs/current/FDNS-E-3.html).
3.2.1 Technological aspects legislation Following Toxic Substances Control Act (TSCA, 1976) the term ‘‘chemical substance’’ means any organic or inorganic substance of a particular molecular identity, including (i) any combination of such substances occurring in whole or in part as a result of a chemical reaction or occurring in nature and (ii) any element or uncombined radical. Such term does not include (a) any mixture, (b) any pesticide when manufactured, processed, or distributed in commerce for use as a pesticide, (c) tobacco or any tobacco product, (d) any source material, special nuclear material, or by-product material, (e) any food, food additive, drug, cosmetic, or device when manufactured, processed, or distributed in commerce for use as a food, food additive, drug, cosmetic, or device. In US Regulatory Requirements for Irradiating Foods (1986), Congress explicitly defined a source of radiation as a food additive. In a report accompanying the legislation, Congress explicitly stated ‘‘Sources of radiation (including radioactive isotopes, particle accelerators, and X-ray machines) intended for use in processing food are included in the term ‘food additive’ as defined in this legislation’’. In early work on food irradiation, sources of sufficiently high energies to induce radioactivity in foods were sometimes used. As research continued, sources whose energies are too low to induce detectable radioactivity were adopted by the international community. Therefore, this issue is of no concern when currently approved sources of radiation are used, but must be addressed if other sources are being considered. Toxicological safety of typical food additives has traditionally been assessed by feeding large amounts of purified substances to laboratory animals and applying a safety factor to the highest dose of a tested substance that causes no toxic effects in any species. Food irradiation (1999) is a technology for controlling spoilage and eliminating food-borne pathogens, such as Salmonella. The result is similar to conventional pasteurization and is often called ‘‘cold pasteurization’’ or ‘‘irradiation pasteurization’’. Like pasteurization, irradiation kills bacteria and other pathogens that could otherwise result in spoilage or food poisoning. The fundamental difference between the two methods is the source of the energy they rely on to destroy the microbes. While conventional pasteurization relies on heat, irradiation relies on the energy of ionizing radiation. The FDA emphasizes that no preservation method is a substitute for safe food handling procedures. The food irradiation process uses three types of ionizing radiation sources (i) cobalt60 gamma sources, (ii) electron beam generators and (iii) X-ray generators. Food Additives Guide (2005) makes clear that food additives are an important component of our food supply. They mean that we can enjoy a wide variety of foods throughout the year. They also have an important role in ensuring that our food lasts longer and is easier to use. There are good reasons for the use of food
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additives. They can be used to improve the quality or stability of a food. For example (a) sorbitol — humectant (420) — may be added to mix dried fruit to maintain the moisture level and softness of the fruit preserve food when this is the most practical way of extending its storage life, (b) sulphur dioxide — preservative (220) — is added to some meat products such as sausage meat to prevent the bugs that cause food poisoning from growing and improve the taste or appearance of a processed food and (c) lecithin — emulsifier (322) — may be added to margarine to help maintain texture. Additives are used in processed foods in relatively small quantities. Many substances used as additives also occur naturally, such as vitamin C or ascorbic acid (300) in fruit and lecithin (322) in eggs or soy beans. Federal Food, Drug and Cosmetic Act (2005) states that the Director of the Center shall (a) conduct postmarket risk assessment of drugs approved under this Act and of biological products licensed under the Public Health Service Act, (b) conduct and improve postmarket surveillance of approved drugs and licensed biological products using postmarket surveillance programs and activities riskbenefit analyses, adverse event reports, the scientific literature, any clinical or observational studies and any other resources that the Director of the Center determines appropriate, (c) determine whether a study is required under this subsection and consult with the sponsors of drugs and biological products to ensure that such studies are completed by the date, and according to the terms, specified by the Director of the Center, (d) contract, or require the sponsor of an application or the holder of an approved application or license to contract, with the holders of domestic and international surveillance databases to conduct epidemiologic and other observational studies, (e) determine, based on postmarket surveillance programs and activities, risk-benefit analyses, adverse event reports, the scientific literature, and any clinical or observational studies and any other resources that the Director of the Center determines appropriate, whether a drug or biological product may present an unreasonable risk to the health of patients or the general public, and take corrective action if such an unreasonable risk may exist, (f) make information about the safety and effectiveness of approved drugs and licensed biological products available to the public and healthcare providers in a timely manner and (g) conduct other activities as the Director of the Center determines appropriate to ensure the safety and effectiveness of all drugs approved under this section and all biological products licensed under the Public Health Service Act. Some representative points and comments of the U.S. legislation about technological aspects are given in Table 12.
3.3 Food quality The goal of the labeling requirement is to increase consumption of domestic commodities and improve the market for U.S. producers. United States Department of Agriculture (USDA) officials appear to have consulted only with opponents of mandatory country of origin labeling before developing cost burden estimates associated with the implementation of the labeling requirements contained in the farm bill (http://enzi.senate.gov/usdaco.htm). The new
Table 12
US legislation (main points, comments) with regard to technological aspects
Title
Main points
Comments
Toxic Substances Control Act (TSCA) (1976)
It establishes the EPA’s toxic substances program. Under TSCA, manufacturers must notify EPA of
Amendment In 1986, TSCA was amended to incorporate the Asbestos Hazard Emergency Response Act to address matters relating to asbestos products in public schools and other buildings.
their intentions to mass-produce a new chemical substance. TSCA also regulates polychlorinated biphenyls, or PCBs. Legal requirements Safety issues (radiological, toxicological,
microbiological, nutritional adequacy) Labeling and packaging of irradiating foods
Food Irradiation (1999)
Food irradiation is the process of exposing food
to ionizing radiation. Food irradiation is a technology for controlling
spoilage and eliminating food-borne pathogens, such as salmonella. FDA approved irradiation for the control of pathogenic microorganisms in red meats. Food Additives Guide (2005)
Use of food additives Food additives are listed according to their
functional or class names Intolerance and food additives
Federal Food, Drug and Cosmetic Act (2005)
Duties of the center for postmarket drug evaluation
Amendments 1997 (Federal Register, 62) 1999 (Federal Register, 64)
International Regulations on Food Contaminants and Residues
US Regulatory Requirements for Irradiating Foods (1986)
and research Publication of progress reports and completed
Source: Adapted from [2] with permission from Blackwell Publishing.
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studies
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US rules require that the FDA conducts inspections to ensure that food manufacturers are complying with practices to reduce or eliminate cross-contact of a food with any major food allergens that are not intentional ingredients of the food (http://www.foodnavigator-usa.com/news/ng.asp?n ¼ 58569-food-labeling-must). Most mandatory labeling legislation around the world has either not yet been fully implemented, or has been implemented in combination with informal moratoria on genetically engineered (GE) foods — the result being that few, if any, GE foods actually carry a label yet. GE food labels are only mandated if the food has any known nutritional difference or food safety risk (including increased toxicity or likelihood of causing allergies; http://www. geo-pie.cornell.edu/issues/intllabeling.html).
3.3.1 Labeling and packaging legislation Fair Packaging and Labeling Act (1967) specifies that packages and their labels should enable consumers to obtain accurate information as to the quantity of the contents and should facilitate value comparisons. Therefore, it is hereby declared to be the policy of the Congress to assist consumers and manufacturers in reaching these goals in the marketing of consumer goods. The separate label statement of net quantity of contents appearing upon or affixed to any package (i) if on a package labeled in terms of weight, shall be expressed in pounds, with any remainder in terms of ounces or common or decimal fractions of the pound; or in the case of liquid measure, in the largest whole unit (quarts, quarts and pints, or pints, as appropriate) with any remainder in terms of fluid ounces or common or decimal fractions of the pint or quart, (ii) if on a random package, may be expressed in terms of pounds and decimal fractions of the pound carried out to not more than three decimal places and is not required to, but may, include a statement in terms of the SI metric system carried out to not more than three decimal places, (iii) if on a package labeled in terms of linear measure, shall be expressed in terms of the largest whole unit (yards, yards and feet, or feet, as appropriate) with any remainder in terms of inches or common or decimal fractions of the foot or yard and (iv) if on a package labeled in terms of measure of area, shall be expressed in terms of the largest whole square unit (square yards, square yards and square feet, or square feet, as appropriate) with any remainder in terms of square inches or common or decimal fractions of the square foot or square yard. The term ‘‘label’’ means any written, printed, or graphic matter affixed to any consumer commodity or affixed to or appearing upon a package containing any consumer commodity. Biotech Food Labeling illustrates three observations made in the theory section of this report. First, to establish successful mandatory labeling requirements, the government must provide or arrange for standards, testing, certification and enforcement. Second, labeling of complex, unclear information will not reduce information and search costs. Third, labeling is not the best policy tool for redressing externalities (even theoretical externalities). Labeling requirements are established by USDA for meat and poultry and by FDA for all other food products. Both agencies require labeling of a biotech food if the food’s composition differs significantly from that of its conventional counterpart. Most
International Regulations on Food Contaminants and Residues
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biotech foods on the market have been found to be essentially equivalent to their conventional counterparts, hence, most biotech foods are unlabeled. As an alternative to segregation, processors could choose to reformulate their products to use ingredients from crops that are exclusively non-biotech, thus minimizing the risk of inadvertently using a biotech variety. For example, corn emulsifiers could be replaced with rice emulsifiers. The cost of any of these options varies greatly depending on the flexibility of the production and marketing systems, the tolerance level for biotech content, the volume of biotech and non-biotech commodities and products processed by the system, and the likelihood of achieving economies of scale. Following Biotech Labeling Guidance (2001) in comments submitted to the FDA, the National Food Processors Association has commended FDA for its draft guidance on voluntary labeling for foods that have or have not been derived through biotechnology. Consumers will benefit only if voluntary biotechnology labeling conforms to standards assuring the information is truthful and not misleading. In this regard, the FDA has provided appropriate and much needed guidance to the food industry through its authority under the misbranding provisions of the Federal Food, Drug and Cosmetics Act. According to Food Labeling (2004) GE food is becoming the rule, rather than the exception, on American grocery-store shelves. By some estimates, two-thirds or more of processed foods now contain ingredients derived from GE corn and soy crops. Unlike the old botanical roulette involved in combining plants to see what traits emerge in their hybrids, genetic engineering is precise. Single traits of one species are spliced into another. The titles and main points of the US legislation about labeling and packaging are summarized in Table 13.
3.4 Water protection and management The Water Act regulates the legal status of water and water estate, the methods and conditions of water management (water use, water protection, regulation of watercourses and other water bodies and protection from adverse effects of water), the method of organizing and performing water management tasks and functions, basic conditions for carrying out of water management activities; powers and duties of Government administration and other Government bodies, local authorities and other legal subjects and other issues of importance to water management (http://en.gmo.hr/index.php/zakonska_regulativa/ hrvatski_zakoni). The Clean Water Act (CWA, 1972) formerly known as the Federal Water Pollution Control Act, intended to restore and maintain the chemical, physical and biological integrity of the Nation’s waters. To accomplish that objective, the act aimed to attain a level of water quality that ‘‘provides for the protection and propagation of fish, shellfish, and wildlife, and provides for recreation in and on the water’’ by 1983 and to eliminate the discharge of pollutants into navigable waters by 1985. The CWA has five main elements (1) a system of minimum national effluent standards for each industry, (2) water quality standards, (3) a discharge permit program that translates these standards into enforceable limits,
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Table 13
US legislation (title, main points, comments) dealing with labeling and packaging
Title
Main points
Comments
Fair Packaging and Labeling Act (1967)
Unfair and deceptive packaging
Amendments
and labeling: scope of prohibition Requirements of labeling, placement, form and contents of Statement of Quantity, Supplemental Statement of Quantity Annual reports to Congress
1992 (The quantity
Biotech Food Labeling (1999)
The biotech labeling example
disclosure on labels of consumer commodities be expressed in both the metric system and the customary inch/pound system of measurement)
illustrates three observations Labeling requirements are
established by USDA for meat and poultry and by FDA for all other food products. Biotech Labeling Guidance (2001)
Labeling of biotechnology food
Labeling Food (2004)
Genetically engineered food is
products Words using in labeling
becoming the rule The new label, reflecting the
definition that organic farmers themselves pushed for will certify that organic food, in addition to being grown without pesticides, contains no genetically engineered ingredients. Source: Adapted from [2].
(4) provisions for special problems such as toxic chemicals and oil spills and (5) a revolving construction loan program (formerly a grant program) for publicly owned treatment works (POTWs). According to Safe Drinking Water Act (SDWA, 1974) the term ‘‘primary drinking water regulation’’ means a regulation which (a) applies to public water systems, (b) specifies contaminants which, in the judgment of the Administrator, may have any adverse effect on the health of persons, (c) specifies for each such contaminant either a maximum contaminant level, if, in the judgment of the Administrator, it is economically and technologically feasible to ascertain the level of such contaminant in water in public water systems or if, in the judgment
International Regulations on Food Contaminants and Residues
Table 14
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US Acts (main points and comments) related to water protection and management
Title
Main points
Clean Water Act (CWA) (1972)
Safe Drinking Water Act (SDWA) (1974)
The CWA was established to restore and maintain the chemical, physical and biological integrity of the nation’s waters. The CWA sets goals to eliminate discharges of pollutants into navigable water, protect fish and wildlife, and prohibit the discharge of toxic pollutants in quantities that could adversely affect the environment.
Comments Amendment The CWA was reauthorized in 1987.
Amendment It seeks to protect sources of the nation’s drinking water and to protect 1996 public health to the maximum extent (Amendments to possible, using proper water treatment the SDWA) techniques. SDWA establishes national primary drinking water standards based upon maximum contaminant levels, and establishes state management programs to enforce the standards.
Source: Adapted from [2].
of the Administrator, it is not economically or technologically feasible to so ascertain the level of such contaminant, each treatment technique known to the Administrator which leads to a reduction in the level of such contaminant sufficient to satisfy the requirements of this section and (d) contains criteria and procedures to assure a supply of drinking water which dependably complies with such maximum contaminant levels, including accepted methods for quality control and testing procedures to insure compliance with such levels and to insure proper operation and maintenance of the system, and requirements as to the minimum quality of water which may be taken into the system and siting for new facilities for public water systems. The main points and comments of CWA and SDWA are given in Table 14.
REFERENCES Scientific sources 1 I.S. Arvanitoyannis, S. Choreftaki and P. Tserkezou, Int. J. Food Sci. Technol., 40 (2005) 1021. 2 I.S. Arvanitoyannis, P. Tserkezou and T. Varzakas, Int. J. Food Sci. Technol., 41 (2006) 130.
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EU LEGISLATION Regulation (EC) No 178/2002 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2002&nu_doc ¼ 178). Regulation (EC) No.852/2004 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2004&nu_doc ¼ 852). Regulation (EC) No 853/2004 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2004&nu_doc ¼ 853). Regulation (EC) No 854/2004 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2004&nu_doc ¼ 854). Regulation (EC) No 882/2004 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2004&nu_doc ¼ 882). EU 76/895/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 78&nu_doc ¼ 895). EU 86/362/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 86&nu_doc ¼ 362). EU 90/642/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 90&nu_doc ¼ 642). Regulation (EC) No 3954/87 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 87&nu_doc ¼ 3954). Regulation (EC) No 2219/89 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 89&nu_doc ¼ 2219). Regulation (EC) No 737/90 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 90&nu_doc ¼ 737). EU 96/22/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 96&nu_doc ¼ 22). EU 96/23/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 96&nu_doc ¼ 23). Regulation (EC) No 466/2001 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2001&nu_doc ¼ 466). Regulation (EC) No 999/2001 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2001&nu_doc ¼ 999). Regulation (EC) No 2160/2003 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2003&nu_doc ¼ 2160). EU 75/106/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 75&nu_doc ¼ 106). EU 76/211/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 76&nu_doc ¼ 211). EU 78/142/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 78&nu_doc ¼ 142). EU 80/232/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 80&nu_doc ¼ 232). EU 82/711/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 82&nu_doc ¼ 711). EU 93/10/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 93&nu_doc ¼ 10). EU 94/62/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 94&nu_doc ¼ 62). EU 97/23/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 97&nu_doc ¼ 23). EU 2002/16/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 2002&nu_doc ¼ 16). Proposal of Regulation COM(2003)0689 COD(2003)0272 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber&1g ¼ en&type_doc ¼ RegulationCOM&an_ doc ¼ 2003&nu_doc ¼ 0689).
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EU 90/496/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 90&nu_doc ¼ 496). EU 2000/13/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 2000&nu_doc ¼ 13). Regulation (EEC) No 1576/89 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 89&nu_doc ¼ 1576). Regulation (EEC) No 1601/91 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 91&nu_doc ¼ 1601). Regulation (EEC) No 2081/92 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 92&nu_doc ¼ 2081). Regulation (EEC) No 2200/96 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 96&nu_doc ¼ 2200). Regulation (EC) No 2815/98 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 98&nu_doc ¼ 2815). Regulation (EC) No 1439/99 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 99&nu_doc ¼ 1439). Regulation (EC) No 1760/2000 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2000&nu_doc ¼ 1760). Regulation (EC) No 1321/2002 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2002&nu_doc ¼ 1321). Regulation (EC) No 318/2003 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus! prod!DocNumber&1g ¼ en&type_doc ¼ Regulation&an_doc ¼ 2003&nu_doc ¼ 318). EU 75/440/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 75&nu_doc ¼ 440). EU 79/923/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 79&nu_doc ¼ 923). EU 98/83/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod! DocNumber&1g ¼ en&type_doc ¼ Directive&an_doc ¼ 98&nu_doc ¼ 83).
e-addresses http://www.ohioline.osu.edu/hyg_fact/5000/5058.html http://www.ncseonline.org/NLE/CRSreports/Pesticides/pest-8.cfm http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname ¼ 109_cong_bills&docid ¼ f:s930:s.txt.pdf http://www.ars.usda.gov/is/timeline/leg.htm?pf ¼ 1S http://www.sice.oas.org/int_prop/nat_leg/Usa/plrt37V.asp http://www.foodstandards.gov.au/mediareleasespublications/factsheets/factsheets1999/ foodadditives.cfm http://www.whitehouse.gov/omb/legislative/sap/107-1/S1731-s.html http://vm.cfsan.fda.gov/Bdms/prodguid.html http://www.epa.gov/radiation/sources/food_irrad.htm http://www.epa.gov/r5water/cwa.htm http://www.epa.gov/history/topics/fqpa/02.htm http://www.wetlands.com/regs/tlpge02a.htm http://www.thecampaign.org/alert-house.php http://www.food-irradiation.com/Pauli1.htm http://pirg.org/ge/GE.asp?id2 ¼ 4778&id3 ¼ ge& http://www.eastherts.gov.uk/Buisiness%20Guide%20to%20Law%20and%20Practise/ Legislation.htm http://www.nationalaglawcenter.org/readindrooms/foodsafety/ http://www.torridge.gov.uk/index.cfm?articleid ¼ 3967 http://www.cspinet.org/reports/chemcuisine.htm
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http://www.cfsan.fda.gov/Blrd/foodaddi.html http://www.fcs.uga.edu/pubs/current/FDNS-E-3.html http://www.mindfully.org/GE/GE4/GE-Legislation-Rep-Kucinich11jun02.htm http://enzi.senate.gov/usdaco.htm http://www.foodnavigator-usa.com/news/ng.asp?n ¼ 58569-food-labeling-must http://www.geo-pie.cornell.edu/issues/intllabeling.html http://www.onderzoekinformatie.nl/en/oi/nod/onderzoek/OND1305164/ http://www.usda.gov/news/pubs/97arp/arp4.htm http://www.i-sis.org.uk/subst.php http://www.mja.com.au/public/issues/172_04_210200/huppleed/leeder.html http://www.the-scientist.com/news/20020206/04 http://www.twnside.org.sg/title2/service169.htm http://www.elsevier.com/wps/find/journaldescription.cws_home/405859/ description#description http://www.food.gov.uk/multimedia/pdfs/foodassureguidance.pdf http://www.fao.org/trade/docs/LDC_foodqual_en.htm http://www.blackwellpublishing.com/journal.asp?ref1471-5732 http://www.mannlib.cornell.edu/collections/policies/food.html http://www.fst.vt.edu/undergraduate.courses.html http://www.washingtonwatchdog.org/documents/usc/index.html http://europa.eu.int/comm/food/intro_en.htm
CHAPT ER
3 Guidelines on Quality Implementation for Analytical Methods Sergio Caroli
Contents
1. Introduction 2. The Role of Quality 2.1 General aspects 2.2 Key aspects of a quality system based on the accreditation criteria 2.3 Key aspects of a quality system based on the Principles of Good Laboratory Practice 3. Conclusions References
77 78 78 79 84 86 87
1. INTRODUCTION The concept that no laboratory can survive in the long term without having a fitfor-purpose quality system in place is nowadays widely accepted by the international scientific community. Experimental information must be, in fact, supported by documented evidence to be valid, credible and comparable. To date, quality systems can be traced back basically to two distinct, yet complementary, approaches, i.e., those adopting the criteria for accreditation, mostly those worked out by the International Standardisation Organisation (ISO), and those based on compliance with the Principles of Good Laboratory Practice (GLP), in particular as developed by the Organisation for Economic Co-operation and Development (OECD) [1–3]. The rationale behind the latter has roots in the need for assessing the integrity of experimental studies, while the former aims at evaluating the competence of a laboratory to perform measurements. In other words, in a GLP system the validity of a completed study is challenged. In so doing, necessarily all the phases of the study are reconstructed and the laboratory performance and Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00003-2
r 2008 Elsevier B.V. All rights reserved.
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facilities are checked. On the other hand, in the case of a quality system based on accreditation, the target is to gain confidence in the ability of the laboratory to generate defendable experimental data. In more detail, a GLP system aims at providing the decision maker with reliable experimental information on new chemical substances so as to allow for a sound assessment of the benefit-to-risk ratio well before chemicals are produced and marketed. The cornerstone of this policy is the implementation of the OECD GLP Principles whenever non-clinical safety studies on new chemicals are undertaken by test facilities. Such Principles can be found in the Decision of the Council concerning the Mutual Acceptance of Data (MAD) in the Assessment of Chemicals [C(81)30(Final)], the Council Decision-Recommendation on Compliance with Principles of GLP [C(89)87(Final)] and the Council. Decision concerning the Adherence of Non-member Countries to the Council Acts related to the MAD in the Assessment of Chemicals [C(97)114(Final)]. Needless to say, the GLP quality system is compulsory resorted to whenever there is, e.g., a registration obligation for the commercialization of a new substance. In turn, an accreditation-oriented quality system is governed by the ISO/IEC Standard 17025 and has to cover the administrative and technical issues specified in the Standard, including internal audits, job descriptions and responsibilities, procedures for equipment/instrument maintenance and calibration, document control, handling of reagents, chemicals and reference materials, sample delivery and storage, validation of test methods, traceability and uncertainty of the test results, training of personnel, client complaints and corrective and preventive actions. Several of these issues are also required for compliance with the OECD GLP Principles either with a different emphasis or with additional requirements. Although the two systems have been designed to meet largely different needs, it is hoped that they can more and more support each other to minimize redundancy and to provide the end user with experimental information as trustworthy as possible.
2. THE ROLE OF QUALITY 2.1 General aspects The food safety policy fits in a wide context that originates at world level and at EU level. At world level the standards for food safety are established in the Codex Alimentarius. The standards for fighting animal diseases are established in the world organization for animal health (OIE). In the white paper on food safety the EU Commission (DG SANCO) outlined a completely new framework for the organization and supervision of food safety. With this new legislation the Commission wants to promote the health of European consumers by establishing food safety provisions that are among the best and most stringent in the world. The white paper comprises an action plan of 84 points. Its objectives can be summarized as follows: (1) (2)
The setting up of a European Food Safety Authority (EFSA). The realization of a coherent European food safety legislation.
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An improved and more coherent organization of the control on the food chain. Permanent dialogue with, and information for the consumer. An international dimension: insist on an implementation of the European food policy at world level. The Food and Veterinary Office (FVO) is responsible at the EU Commission level for supervising the proper application of the EU standards and regulations.
Important decisions are often taken on the basis of experimental data. Hence, it is crucial that such data be comparable, reliable and valid. No laboratory can in fact be run without a fit-for-purpose quality system in place. Quality has been defined by ISO as ‘‘The totality of features and characteristics of a product or service that bear on its ability to satisfy stated or implied needs’’. To date, quality systems are basically inspired either by the GLP Principles or by the accreditation criteria. Laboratory work may be of two different types: (i) the outcome of the investigation are exact figures, to which precision and reproducibility are expected to be attached and (ii) the outcome of the investigation is, in a general sense, complex information which should be credible, reliable and comparable. In the former case, what matters more are the experimental measurements. In this context quality is assessed in terms of precision and reproducibility of the numerical data obtained. The ability of the laboratory to generate such data is thus of primary importance. Quality systems based on accreditation criteria are ideal in this respect. In the latter case, the focus is on the overall study as such. Third parties should be enabled to reconstruct the whole course of the study and to check its integrity so that confidence can be gained in the way the study results have been obtained. Under such circumstances, quality systems based on the GLP Principles apply. Which approach is to be preferred depends only on the scope and goals of the activities performed in the laboratory, although it should not be overlooked that accreditation is basically voluntary, whereas the GLP system is prescribed by law for those Test Facilities (TFs) undertaking non-clinical safety studies. There is still some confusion surrounding the terms of accreditation and certification. As this may well be misleading, consensus has been reached on the following definitions: accreditation is a means used to identify competent testing laboratories, whereas certification is the official approval granted by a given authority.
2.2 Key aspects of a quality system based on the accreditation criteria IUPAC has a long tradition of activities related to quality assurance of analytical measurement results. The formation of the IUPAC/ISO/AOAC Working Party for Harmonization of Quality Assurance (WPHQA) Schemes in 1978 was an important milestone. At that time, efforts were focused on harmonizing requirements related to method validation studies (or laboratory collaborative studies), which had been conducted by a number of organizations around
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the world. Today, after almost 30 years, that working party is the IUPAC Interdivisional WPHQA, which is part of the Analytical Chemistry Division (ACD). As set forth by the IUPAC, ‘‘The international scientific community recognizes that a laboratory must take appropriate measures to ensure that it is capable of providing data of the required quality. Such measures include: (i) internal quality control procedures; (ii) participation in proficiency testing schemes; (iii) validated methods of analysis; (iv) accreditation to an international standard’’ [4]. Accreditation-based quality systems are governed by the international Standard ISO/IEC 17025 [5]. This standard exploits the extensive experience gained in implementing the ISO/IEC Guide 25 and EN 45001 norms and replaces them both. The ISO/IEC Standard sets forth the requirements a laboratory has to meet to be recognized as competent to carry out tests and/or calibrations, including sampling. The pillars of an accreditation system are listed in Table 1. Method validation is central to the accreditation process as reliability and comparability of data are crucial to perform experimental meaningful tests and to achieve credible results which can be profitably used by the client, i.e., the end user. It should be noted that the overall validation process covers all of the pivotal phases of an experimental measurement and not only the mere quantification step, as illustrated in Figure 1. In turn, method validation as such should at least cover the parameters given in Table 2. For the different levels of quality assurance in the accreditation system presented in Table 1, guidelines and requirements are well described in detail by several regulatory bodies and standardization agencies. Relevant guidelines are given in Table 3. Eurachem guides are published on quality in the laboratory in general [5], method validation [6] and proficiency test [7]. A guideline on proficiency test from the joint Eurachem-Eurolab-EA group is also available [8]. At the European level, there is also the CEN, which is working through different technical committees and working groups on standardization of analytical methods for all sectors [9]. At the international level, IUPAC, ISO and AOAC International are distinguished. All three bodies develop validation and standardization frameworks for analytical chemistry. AOAC International introduced the ‘‘AOAC Peer Verified Methods Program’’ [10]. IUPAC, ISO and the AOAC International Table 1
Key aspects of a laboratory compliant with an accreditation system
Service to the client Policy for complaints Control of non-conformities Quality manual Management of records Internal audits Measurement traceability
Motivation of personnel Laboratory setting Validation of methods Equipment Management reviews Test and calibration items Report of results
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Hardware validation
Software validation/qualification
Method validation
System suitability
VALIDATION
Figure 1 Major steps of the validation process.
Table 2
Parameters to be ascertained to validate an analytical method
Applicability Selectivity Calibration and linearity Trueness Accuracy Precision Recovery Range
Limit of detection Limit of quantification Sensitivity Ruggedness Robustness Fitness for purpose Matrix variation Measurement uncertainty
together developed different harmonized guidelines and protocols [11–17], in addition to a number of ISO standards [11,12]. The US FDA, USP and ICH developed guidelines specifically for pharmaceutical and biotechnological methods [18–20]. The international Codex Alimentarius Commission within the United Nations FAO/WHO Food Standards Program has a Codex Committee on Methods of Analysis and Sampling (CCMAS). CCMAS works out criteria for evaluating the acceptability of methods of analysis as well as guidelines on single-laboratory and inter-laboratory validation of methods [21,22]. For single-laboratory validation, CCMAS defends the harmonized IUPAC guidelines [23]. On the international level, also ILAC provides guidelines on proficiency test [5] and on accreditation [24,25] (Table 3). The use of standardized methods of analysis in analytical chemistry is one of the most traditional ways of achieving comparability of measurement results.
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Table 3 Overview of European and international regulatory bodies and their guidelines and standards on different aspects of analytical quality (Adapted from [26]) Body
Guidance on
Reference
Eurachem - A focus for analytical chemistry in Europe Cooperation of International Traceability in Chemistry (CITAC) European Commission for Accreditation (EA) European Committee for Standardization (CE) International Union of Pure and Applied Chemistry (IUPAC) International Standardization Organization (ISO) Association of Official Analytical Chemists (AOAC) US Food and Drug Administration (FDA) US Pharmacopeia (USP) International conference on harmonization (ICH) Food and Agriculture Organization/ World Health Organization (FAO/ WHO) Codex Commission on Methods of Analysis and Sampling (CCMAS) International Laboratory Accreditation Cooperation (ILAC)
Method validation
[5,6,7,8]
Proficiency test Quality assurance Accreditation Standardization
[9]
Method Validation Standardization Internal quality control Proficiency test Accreditation Method Validation
[10,11,12,27]
Method Validation
[21,22]
Proficiency testing Accreditation
[24,25]
[18,19,20]
Source: Adapted with permission from Ref. [10].
Especially in food analysis, agrochemicals, organic analysis and other analytical areas where unstable samples and/or measurands are analyzed, the use of standardized methods is often prescribed by legislation. Two IUPAC internationally harmonized protocols have for many years served as a basis for validation and adoption of standardized analytical methods (procedures). The first is the IUPAC ‘‘Protocol for the Design, Conduct, and Interpretation of Collaborative Studies’’ [26], and the second is the ‘‘Harmonized Protocols for the Adoption of Standardized Analytical Methods and for the Presentation of their Performance Characteristics’’ [28]. These Principles of collaborative studies for method validation are still widely applied by the AOAC International, as well as by the ISO. However, the world is changing rapidly and with the fast development of analytical instrumentation and the availability
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of new analytical techniques and procedures the prescription of methods to be used is sometimes a limiting factor. Responding to the situation, the WPHQA has opened the door for single-laboratory method validation, also known as in-house method validation. The Principles presented in the IUPAC ‘‘Harmonized Guidelines for Single Laboratory Validation of Methods of Analysis’’ [29] and in the proceedings of the Joint AOAC Int./FAO/IAEA/ IUPAC International Workshop on the Principles and Practices of Method Validation, have been accepted as official guidelines by the CODEX Alimentarius Commission. Established internal quality-control practices and regular laboratory participation in proficiency testing constitute another very important pillar of quality assurance in analytical chemistry. Again, the contributions of the WPHQA have been indispensable. Two IUPAC internationally harmonized documents, namely the ‘‘International Harmonized Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories’’ [30] and the ‘‘Harmonized Guidelines for Internal Quality Control in Analytical Chemistry Laboratories’’ [31] still provide the basic rules, which have received wide international acceptance and utilization. Assessment of laboratory performance based on a z-score evaluation introduced in the IUPAC proficiency testing protocol became the most frequently used approach in evaluation of laboratory performance. The protocol has been updated and a revised version titled ‘‘The International Harmonized Protocol for the Proficiency Testing (PT) of Analytical Chemistry Laboratories’’ was published in 2006 [32]. To supplement this so-called classical PT approach, the WPHQA recently initiated a separate project on the Selection and Use of Proficiency Testing Schemes for Limited Number of Participants (Chemical Analytical Laboratories) [32]. In case of a small number of participants, some limitations on statistical applications may appear and this project is aimed at elaborating some additional approaches for evaluation of participants’ results and their reporting. However, neither of the above described external quality assurance schemes replaces the internal laboratory quality control. The ISO/IEC 17025 standard, which serves as a basis for laboratory accreditation, is very general and brief in its Clause 5.9 titled ‘‘Assuring the Quality of Test and Calibration Results’’ [33]. It urges laboratories and accreditation bodies to use separate guidance, specifically prepared for their field of application. One of the most important parameters defining the quality of analytical measurement results is comparability. Comparability of measurement results is based on metrological traceability, which allows results to be compared independently of the time, place, analyst and procedure used. Two facets of this description are of concern. The first is the metrological traceability of chemical measurement results. It is a term often used and cited, but without a firm agreement within the measurement/scientific community regarding associated concepts, their understanding and requirements. Comparability of measurement is the ultimate goal of quality assurance and is a prerequisite for quality assurance implementation.
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The second regards the claim that the traceability chain in chemistry has been broken. This claim is often related to destructive analysis, where the sample and measurand are converted into the physical and chemical form suitable for the selected measurement technique/instrument. Such conversions (digestions, extractions, etc.) may result in the loss of measurand, incomplete conversion into the required chemical/physical form, or even contamination, and are very much dependent on the procedure used.
2.3 Key aspects of a quality system based on the Principles of Good Laboratory Practice In the early 1960s, the US Food and Drug Administration (FDA) became aware that some studies on the safety of new chemicals performed by TFs for regulatory purposes were basically unreliable. Evidence was in fact provided of major adverse effects of such substances which had not been reported at the time when the authorization for production and commerce was granted. In the early 1970s, the US Congress undertook the re-assessment of studies submitted by some TFs to Regulatory Authorities (RAs) and suspected to be fraudulent. Under such conditions thousands and thousands of safety studies on industrial chemicals, pesticides, herbicides, drugs, cosmetics and food and feed additives were
Figure 2 Article published by The Washington Post in 1974 on the falsification of experimental data.
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conducted for years (about 35–40% of all toxicological studies authorized in the USA in that period). As an example, an article published by The Washington Post in 1997 is shown in Figure 2. Senator Edward Kennedy declared at the US Congress of January 20, 1976, that ‘‘y unreliable, undocumented and fraudulent research is the most frightening menace to the health and safety of people. That research be wrong because of technical problems or because of the lack of competence or even due to criminal negligence is less important than the very fact that it is wrong y’’. The GLP Principles were conceived to harmonize the conduct of non-clinical safety studies and to minimize the risk of fraud. Since the early years, this matter became a priority for the OECD which set up the GLP Principles in order to promote and manage the mutual acceptance of non-clinical safety studies in the Table 4
The OECD series on the GLP Principles and compliance monitoring OECD Principles of Good Laboratory Practice
No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 No. 11 No. 12 No. 13 No. 14 No. 15
Principles of Good Laboratory Practice and Compliance Monitoring (as revised in 1995) Revised Guides for Compliance Monitoring Procedures for Good Laboratory Practice (1995) Revised Guidance for the Conduct of Laboratory Inspections and Study Audits (1995) Quality Assurance and GLP (as revised in 1999) Compliance of Laboratory Suppliers with GLP Principles (as revised in 1999) The Application of the GLP Principles to Field Studies (as revised in 1999) The Application of the GLP Principles to Short Term Studies (as revised in 1999) The Role and Responsibilities of the Study Director in GLP Studies (as revised in 1999) Guidance for the Preparation of GLP Inspection Reports (1995) The Application of the Principles of GLP to Computerized Systems (1995) The Role and Responsibilities of the Sponsor in the Application of the Principles of GLP (1999) Requesting and Carrying Out Inspections and Study Audits in Another Country (2000) The Application of the OECD Principles of GLP to the Organization and Management of Multi-Site Studies (2002) The Application of the OECD GLP Principles to in vitro Studies (2004) Establishment and Control of Archives That Operate in Compliance with the Principles of Good Laboratory Practice (2007)
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Key aspects of a Test Facility compliant with a GLP system
Director of the Test Facility Study Director Quality Assurance Unit Operative Procedures Archivist Sponsor Test and reference items
Study plan Final report Standard Test Site (if applicable) Principal Investigator (if applicable)
Member Countries. According to OECD, the Principles of GLP are a quality system concerned with the organizational process and the conditions under which safety studies are planned, conducted, controlled, recorded, reported and archived. In practice, they form a body of reciprocally dependent documented items that make the falsification of a study more time-consuming and expensive than its actual correct performance. GLP embodies a set of Principles that provides a framework within which laboratory studies can be reliably carried out. These studies are undertaken to generate data by which the hazards and risks to users, consumers and third parties, including the environment, can be assessed for pharmaceuticals, agrochemicals, cosmetics, food and feed additives and contaminants, novel foods and biocides. GLP helps assure regulatory authorities that the data submitted are a true reflection of the results obtained during the study and can therefore be relied upon when making risk/safety assessments. As a part of the permanent activities of its Environment, Health and Safety Programme, the OECD also prepares and publishes Test Guidelines for Chemical Substances to be used when performing GLP studies and thus enhance their reliability. The Series on the GLP Principles and compliance monitoring consists at present of 15 monographs, as detailed in Table 4, whereas Table 5 details the pillars of a GLP system [34,35]. These guides form the core of the legal provisions of the European Union in the field of GLP [36,37].
3. CONCLUSIONS The two quality systems have been conceived to meet quite different needs. In other words, the accreditation criteria are designed to manage activities in a laboratory where routine quantitative measurements (such as analytical determinations) are carried out, whereas the GLP Principles are intended to guarantee the integrity of data generated in non-clinical safety studies. Their respective fundamental characteristics are summarized in Table 6. From this standpoint, it is worth mentioning that, e.g., the GLP system prescribes that the Director of the TF, the person responsible of the Quality Assurance Unit, the Study Director and the Archivist be all independent of each other to fully
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Key elements of the accreditation and GLP system
Accreditation quality system
GLP quality system
Overlapping aspects
Management of complaints Uncertainty of measurements Proficiency testing Preventive actions Service to the client Sampling
Master schedule Study director Archivist Quality assurance unit Study plan Test article Reports
Management Motivation Training Reference materials Equipment and maintenance Method validation Chain of custody Quality control Procedures Corrective action Audits Sample reception
guarantee the fair conduct of the study, while in the case of the accreditation system the first two functions can coincide and the third one does not exist. On the other hand, in the accreditation system, it is imperative to have a quality manual, which in turn is not formally requested in the GLP system, although in the latter the Standard Operating Procedures play basically the same role. Moreover, a study plan, compulsory in the GLP system, is not needed in the accreditation one, not to speak of the fact that management of complaints and participation in proficiency testing is compulsory in the latter, but not necessary in the former. As regards validation of methods, the GLP system requires that validated methods are in place, but does not impose that such methods are set up according to the GLP Principles, any other fit-for-purpose quality system being acceptable to this end. All this provides clear evidence of the profound diversity in the approaches and goals of the two systems, although some common aspects are present. In this regard, in recent years, the OECD has established a dialogue group to verify where the two systems can actually interact, thus minimizing useless duplication of efforts. In conclusion, the selection of the quality system to be adopted should be carefully made on the basis of the prevailing activities carried out in the laboratory. Quality is inescapable, but it has a cost: a wrong decision can only lead to failure.
REFERENCES 1 Ph. Quevauviller, E.A. Maier and B. Griepink (Eds.), Quality Assurance for Environmental Analysis, Elsevier, Amsterdam, The Netherlands, 1995, xx–649. 2 J.P. Seiler, Good Laboratory Practice. The Why and the How, Springer, Berlin, Germany, 2001, x–395. 3 Ph. Quevauviller (Ed.), Quality Assurance in Environmental Monitoring. Sampling and Sample Pretreatment, VCH Weinheim, New York, NY, USA, 1995, xv–306.
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4 IUPAC Technical Report, Pure Appl. Chem., 74(5) (2002) 835–855. 5 CITAC/Eurachem Guide: Guide to Quality in Analytical Chemistry – An Aid to Accreditation, 2002. Available at http://www.Eurachem.bam.de 6 Eurachem Guide: The fitness for purpose of analytical methods. A laboratory guide to method validation and related topics, LGC, Teddington, Middlesex, UK, 1998. Available at http://www. Eurachem.bam.de 7 Eurachem Guide: Selection, use and interpretation of PT schemes by laboratories, 2000. Available at http://www.Eurachem.bam.de 8 Eurachem/Eurolab/EA Guide EA-03/04: Use of proficiency testing as a tool for accreditation in testing, August 2001 – rev. 01, 18pp. 9 CEN, European Committee for Normalisation, 2004. Available at http://www.cenorm.be/cenorm/ index.htm 10 AOAC International, Method Validation Programs (OMA/PVM Department), including Appendix D: Guidelines for collaborative study procedures to validate characteristics of a method of analysis, 2000. Available at http://www.aoac.org/vmeth/devmethno.htm 11 M. Thompson, S. Ellison and R. Wood, Pure Appl. Chem., 74 (2002) 835. 12 M. Thompson and R. Wood, Pure Appl. Chem., 67 (1995) 649. 13 W. Horwitz, Pure Appl. Chem., 67 (1995) 331. 14 M. Thompson and R. Wood, Pure Appl. Chem., 65 (1993) 2123. 15 L.A. Currie, Pure Appl. Chem., 67 (1995) 1699. 16 L.A. Currie and G. Svehla, Pure Appl. Chem., 66 (1994) 595. 17 W.D. Pocklington, Pure Appl. Chem., 62 (1990) 149. 18 ICH-Q2A, Guideline for Industry: Text on Validation of Analytical Procedures, 1995. Available at http://www.fda.gov/cder/guidance/index.htm 19 ICH-Q2B, Guidance for Industry: Validation of Analytical Procedures: Methodology, 1996. Available at http://www.fda.gov/cder/guidance/index.htm 20 FDA/CDER/CVM, Guidance for Industry – Bioanalytical Method Validation, 2001, 22pp. Available at http://www.fda.gov/cder/guidance/index.htm 21 CX/MAS 01/4, Codex Alimentarius Commission, Codex Committee on Methods of Analysis and Sampling, Criteria for evaluating acceptable methods of analysis for Codex purposes, Agenda Item 4a of the 23rd Session, Budapest, Hungary, 26 February–2 March 2001. 22 CX/MAS 02/12, Codex Alimentarius Commission, Codex Committee on Methods of Analysis and Sampling (FAO/WHO), Validation of methods through the use of results from proficiency testing schemes, Agenda Item 8c on the 24th Session, Budapest, Hungary, 18–22 November 2002. 23 W. Horowitz, Pure Appl. Chem., 60 (1988) 855. 24 ILAC-G13, Guidelines for the requirements for the competence of providers of proficiency testing schemes, ILAC Technical Accreditation Issues Committee, 2000, 23pp. Available at http://www. ilac.org/ 25 ILAC-G18, The scope of accreditation and consideration of methods and criteria for the assessment of the scope in testing, ILAC Technical Accreditation Issues Committee, 2002. Available at http:// www.ilac.org/www 26 I. Tavernier, M. De Loose and E. van Bockstaele, Trends Anal. Chem., 23 (2004) 535. 27 ISO/IEC 17025, General Requirements for the Competence of Testing and Calibration Laboratories. 28 M. Thompson, S.R.L. Ellison and R. Wood, Pure Appl. Chem., 74 (2002) 838. 29 A. Fajgelj and A. Ambrus (Eds.), Principles and Practices of Method Validation. The Royal Society of Chemistry, Special Publication No. 256, Cambridge, 2000, ISBN 0-85404-783-2. 30 M. Thompson and R. Wood, Pure Appl. Chem., 67 (1995) 649. 31 M. Thompson, S.R.L. Ellison and R. Wood, Pure Appl. Chem., 78 (2006) 145. 32 www.iupac.org/projects/2005/2005-019-2-500.html 33 ISO/IEC 17025 International Standard: General Requirements for the Competence of Testing and Calibration Laboratories, International Organization for Standardization, Geneva, 2005, ICS 03. 120.20.
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34 OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98)17. 35 S. Caroli (Ed.), The New Principles of Good Laboratory Practice: Priorities, Problems, Perspectives, Ann. Ist. Super. Sanita`, 2002, Vol. 38, 110pp. 36 Directive 2004/9/EC of the European Parliament and Council, 11 February 2004, Off. J. EU, L50 (2004) 28–43. 37 Directive 2004/10/EC of the European Parliament and Council, 11 February 2004, Off. J. EU, L50 (2004) 44–59.
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CHAPT ER
4 Immunochemical and Receptor Technologies: The Role of Immunoassay, Immunoaffinity Chromatography, Immunosensors and Molecularly Imprinted Polymeric Sensors ´ Marinel la Farre´, Elena Martı´nez and Damia` Barcelo
Contents
1. 2. 3. 4.
Introduction Definitions and Concepts Immunoassay Formats Immunoassay Development 4.1 Hapten synthesis 4.2 Antibody production 4.3 Assay development and assay optimization 4.4 Validation 5. Immunochemical Technologies 5.1 Radioimmunoassays (RIAs) 5.2 Enzyme-based immunoassays (EIAs) 5.3 Fluorescence polarization immunoassay (FPIA) 5.4 Chemiluminescent magnetic immunoassay (CMIA) 5.5 Flow-injection immunoassay (FIIA) 5.6 Immunoaffinity chromatography 5.7 Immunosensors 6. Biomimics: Molecularly Imprinted Polymers (MIPs) 6.1 MIPs in preparation/preconcentration and separative applications 6.2 Development and application of MIP-based sensors 7. Commercial Instrumentation and Future Perspectives References
Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00004-4
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1. INTRODUCTION The food industry faces an unprecedented level of scrutiny. Consumers are not only concerned with the safety and quality of food products but also with the way in which they are produced. At the same time, the food industry has developed new ways of assuring appropriate standards for its products and their methods of production, developing systems such as Total Quality Management (TQM) and Hazard Analysis and Critical Control Point (HACCP) to identify and manage key steps in production. On the other hand, the adulteration of food has progressed from being a simple means of fraud to a highly sophisticated and lucrative business. The problem is further compounded by the lack of clear international definitions for enforcement purposes. Adulteration of food has ramifications within society and cannot be ignored since interference with foodstuffs may potentially lead to the production of food that is harmful to health. For all these reasons food safety authorities force a major control. Nowadays quality control analysis is an essential tool in food industry, and due to these reasons a significant number of rapid analytical techniques have arisen during the past decade. One of the most successful groups of these rapid techniques are immunoassays. Immunoassays are based on the binding properties of antibodies (Abs) with antigens (Ags). An antibody is an immunoglobulin (Figure 1) used by the immune system to identify and neutralize foreign objects. An antibody contains two sites called paratopes that bind antigens. These structures can be thought of as similar to locks and are specific for just one particular part of the antigen called an epitope, which can be thought of as similar to a key.
Antigen binding site
Antigen binding site
Variable Constant
Light chain
Heavy chain
Figure 1 Antibody scheme.
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The Ab–Ag interaction is reversible, as determined by the law of mass action, and is based on electrostatic forces, hydrogen bonding, hydrophobic and Van der Waals forces. The binding properties of an antibody to an antigen have been used for the development of a wide variety of analytical techniques applicable in clinical chemistry, endocrinology, food analysis and environmental control. This chapter summarizes the most representative immunochemical technologies applied to food analysis.
2. DEFINITIONS AND CONCEPTS Immunoassays are applied for the measurement of both single and multiple analytes [1]. Chemical pesticides, polychlorinated biphenyls (PCBs) and antibiotics are typical examples of contaminants, which can be rapidly and efficiently determined by antibody-based methods. Antibodies enable not only a rapid detection of analyte in water, body fluids, soil or food extracts, but they can also be exploited in sample preparation prior to analyte detection. Because antibody affinity and specificity determine primarily the analytical capability of the immunochemical method, the properties of the antibodies represent an important innovative factor in developing an analytical system. Highly sensitive detection of toxic analytes can be performed by enzyme immunoassays, immunosensors and related techniques, whereas immunoaffinity chromatography and flow-injection immunoassay (FIIA) systems enable the concentration and clean up of the analytes in question. The manner of chemical binding of the hapten to a protein determines the character of the antibody specificity. Various types of hapten derivatives conjugated to proteins have been used for antibody development. Different types of Abs are involved in immunoassays: polyclonal, monoclonal and recombinant. Polyclonal Abs are obtained from the serum of animals immunized with a particular Ag. The Ab pool obtained from serum is the result of many B-cell clones, each secreting one specific Ab. Antiserum refers to a pool of serum containing all of the Ab fraction plus other serum proteins. Whereas, monoclonal antibodies are produced following the fusion of myeloma cells with Ab-secreting B-cells (Figure 2). The resultant continuous cell line (hybridoma) produces large quantities of the homogeneous, well-defined, single-epitope Ab. The availability of large quantities of continuously produced Ab allows greater standardization and quality control of the Ab reagent. Therefore, monoclonal antibodies are more precisely characterized, legally protected and have greater acceptance by regulatory agencies. Preparation of recombinant antibody fragments with novel binding properties was a primary goal of gene technologies. Their major asset lies in the possibility to focus mutagenesis on that part of gene that determines the structure and affinity of antibody binding site. Large phage libraries expressing antibody fragments on the surface of individual phage particles were used for preparation of recombinant antibodies. The systems enable separation of individual phage
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Immunization Tissue culture
Antibody-forming
Tumor cell
Fusion
Hybridoma Hybridoma screening for antibody Antibody-producing
hybridoma cloned
Monoclonal antibodies isolated for cultivation
Figure 2 Monoclonal antibodies production scheme.
particles and subsequent selection of phage antibodies from a large number of expressed phage particles. Immunoassays offer a number of advantages in food contaminants analysis over the conventional methods, such as liquid- or gas chromatography-based methods, because immunoassays can provide fast, simple and cost effective detection, with sensitivity in most cases comparable or better to conventional techniques. In addition, the possible automation and the versatility in the applications of immunochemical techniques have also made these techniques more popular in recent years. Main advantages of these methods are: Reduced time of analysis Low detection limits High throughput of samples
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Cost effectiveness for large numbers of samples Adaptability to field use Small volumes of sample and solvents are required. Another relevant advantage is the cost effectivity of immunochemical approaches. In general, the major costs involved in food contaminants analysis are related to instruments (including maintenance), skilled personnel, solvents, reagents and time (for both sample pre-treatment and analysis). On the contrary, the instrumentation required by immunochemical approaches is economical in comparison to classical instrumental method, and involved manipulation are easy and rapid, requiring none or minimal quantities of solvent as well as little amount of sample. All these factors reverberate in more than 10 times lower cost of analysis using immunoassays than using classical instrumental analysis. On the other hand, a series of limitations should be also pointed out.
Immunoreagent preparation Lack of implementation Lack of immunoreagent stability Cross-reactivity Matrix effects.
The lengthy development time that can be required for immunoreagent preparation and the lack or limited response in front of some contaminants is sometimes an important limitation of immunochemical techniques, e.g., almost no immunological response can be obtained in front of per-fluorinated compounds and no antibodies against these contaminants can be obtained. The lack of stability (especially thermal, and in front of extreme pH ranges) is a limitation for the use of immunochemical analysis in some cases. Cross-reactivity is the lack of specificity of an immunoassay in front of an analyte when the immunoassay can react with other structurally related compounds, this is a characteristic that directly depends on the antibody. However, when the object of the analysis is screening a group of related substances, this fact can be an advantage.
3. IMMUNOASSAY FORMATS Immunoassays may be carried out according to different formats. Figure 3 shows the most widely used. Competitive assays are the most common and can be performed in different ways, such as the analyte and the tracer competing for a limited number of binding sites (direct), or the analyte and the immobilized ligand (antigen) competing for a limited number of binding sites (indirect). Non-competitive immunoassays are also performed in sandwich-type immunoassays, in which the analyte is recognized by two different antibodies.
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Figure 3 Enzyme-linked immunosorbent assay formats.
4. IMMUNOASSAY DEVELOPMENT The main steps for immunoassay development are: 1. 2. 3. 4.
Hapten synthesis Antibody production Assay optimization and characterization Validation.
4.1 Hapten synthesis Small molecules, such as most food contaminants, with molecular weights lower than 5,000–10,000 do not elicit immune responses. In those cases, the molecules are named haptens because it is necessary to attach them to a large carrier protein in order to stimulate the immune response. Sometimes, the hapten is used as detector, then it is attached to an enzyme. In both cases, as immunogen, or as detector, the attachment to the protein is through a covalent bond, and a linker or spacer arm is often used to enable greater antibody recognition. Antibody specificity is directed to sites on the hapten that are distal to the point of linker attachment [2,3]. A good displacement of hapten by the spacer arm generally produce antibodies with high titer, but sometimes fails to provide the required sensitivity in an immunoassay. A wide variety of proteins are available for the synthesis of immunogens or antigens including bovine serum albumin (BSA), and human serum albumin, ovalbumin, conalbumin, thyroglobulin, keyhole limpet hemocyanin (KLH) or horseshoe crab hemocyanin, and the synthetic polypeptides poly-l-lysine
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and polyglutamic acid. Among them, KLH is often the first choice as an immunogen carrier protein because it is large (approximately 106 kDa), highly immunogenic and easy to conjugate because their high abundance of functional available groups. Thyroglobulin has been increasingly used as an immunogenic carrier protein owing to its high water solubility. Another frequently used protein in immunoassay is BSA, specially as a coating antigen carrier. Advantages of BSA include its wide availability in pure form, low cost, stability, relatively resistant to denaturation and is suitability for some conjugation procedures that involve organic solvents. BSA has a molecular weight of 64,000 Da. A spacer arm is preferable with an alkyl chain of three to six carbons. The use of bulky functionalities, such as aromatic rings, conjugated double bonds in spacer arms should be avoided to minimize the recognition of this region by the antibodies [3]. Propionic, succinic and caproic acid are the most commonly used spacer arms for contaminant immunoassay development. The selection of conjugation method is dependent on the functional group on the hapten (e.g., carboxylic acid, amine, aldehyde). A hapten with a carboxylic acid group can conjugate with a primary amino group of a protein using the carbodiimide, activated N-hydroxysuccinimide (NHS) ester or mixed anhydride methods. Haptens with free amino groups can be coupled to proteins using glutaraldehyde condensation or diazotization. Haptens that have been designed to contain spacers may be linked directly to the protein with methods such as the mixed anhydride, whereas haptens lacking a spacer should be coupled using methods that insert a linker between the hapten and the protein such as with glutaraldehyde. Typical procedures are:
Anhydride mixed method Carbodiimide method NHS method Carboxylic methods Hydroxyl groups.
Methods for linking hapten carboxyl groups to amine groups of antigenic proteins include activation by carbodiimides, isobutyl chloroformate or carbonyldiimidazole. In the widely used carbodiimide method, the carbodiimide activates the carboxylic acid to speed up its reaction with the amine. Acidic conditions catalyze the formation of the active O-acylurea intermediate while the protein is more reactive at higher pH, when the lysine amino groups are unprotonated. Therefore, as a compromise, a pH near 6 is used. The choice of the carbodiimide is dependent on the reaction conditions. For example, dicyclohexylcarbodiimide (DCC) is used in nonaqueous media with nonpolar, water insoluble haptens where the carrier protein, in aqueous solution, is added to the activated hapten in a two-step reaction. For more water-soluble haptens, water-soluble derivatives of DCC such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or 1-cyclohexyl-3-(2-morpholino ethyl) carbodiimide
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metho-p-toluenesulfonate (CMC or Morpho CDI) are used in one-step reactions. However, the 1-ethyl-3-(3-dimethylaminopropyl)-3-ethylcarbodiimide react directly with the protein, and some antibodies are generated against the resulting highly immunogenic protein–urea complex. Formation of these antibodies is not a drawback as long as a different coupling chemistry is used to prepare the plate coating antigens. Activated NHS esters of carboxylic acids are prepared by reacting the acid with NHS in the presence of DCC. NHS esters are stable when kept under anhydrous and slightly acidic conditions, and they react rapidly with amino groups to form an amide in high yield. Like the carbodiimide method, the mixed anhydride method [4] results in an amide complex. The acid-containing hapten is dissolved in a dry, inert, dipolar, aprotic solvent such as p-dioxane, and isobutyl chloroformate is added with an amine catalyst. The activated mixed anhydride is chemically stable and can be isolated and characterized. The aqueous protein solution is added to the activated acid and the pH is maintained at around 8.5. A low temperature (around 101C) is necessary during the reaction to minimize side reactions. Amine groups in haptens, carrier proteins or both can be modified for conjugation through homo- or heterobifunctional crosslinkers such as succinic anhydride, succinyl chloride, or glutaraldehyde. Glutaraldehyde condensation has been used widely to produce protein–protein and hapten–protein conjugates. The glutaraldehyde reagent should not have undergone polymerization. Aromatic amine-containing haptens are converted to diazonium salts with ice-cold nitrous acid. Diazonium salts can then react with a protein in alkali through electrophilic attack of the diazonium salt at histidine, tyrosine and (or) tryptophan residues of the carrier protein. Other reactions can also be used to couple haptens to proteins, e.g., the periodate oxidation is suitable for compounds possessing vicinal hydroxyl groups such as some sugars. The conjugates characterization is based on the determination of the haptens density, which is important for both immunization and assay performance. The degree of conjugation can be determined by established methods, such as characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or by the use of a radio labeled hapten. If the hapten has a similar lmax to the protein, the extent of incorporation can still be estimated when the concentration of the protein, and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to the amount of hapten conjugated [5]. Hapten density can also be determined indirectly by measuring the difference in free amino groups between conjugated and unconjugated protein using trinitrobenzenesulfonic acid [6]. These methods can be used as a rough estimation because the process of conjugation usually alters the apparent number of amine or sulfhydryl groups on the protein. On the other hand, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been applied to estimate the number of
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covalently bound haptens [7]. Although these methods offer an accurate approach, large proteins cannot be analyzed. Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis. The optimum hapten ratio may depend on the study objectives, the nature of the antigen, immunization protocol, etc. A general rule of thumb is to target high hapten ratios for immunogens and low hapten ratios for coating antigens or enzyme tracers. For immunogens, a high hapten ratio implies greater exposure of the immune system to the hapten; for coating antigens or enzyme tracers, a lower hapten density implies fewer haptens to compete with the analyte in the assay. Optimum hapten density is often determined empirically with checkerboard titration procedures. Such procedures are very rapid and are normally adequate to optimize enzyme-linked immunosorbent assays (ELISAs) without knowing the exact hapten density. However, for the development of new immunosensor devices the determination of exact hapten densities may become increasingly important.
4.2 Antibody production Any vertebrate can be used as a source of antibodies. However, the most used animals for antibody production are rabbits, goats and sheep for polyclonal antibodies, and mice for monoclonal antibodies. For example rabbits are easy to care for and produce a moderate amount of serum often with high antibody titers. Goats or sheep also produce high-quality antiserum in larger amounts. Monoclonal antibodies are obtained from a mouse cell line. These antibodies are produced by fusing single antibody-forming cells to tumor cells grown in culture. The resulting cell is called a hybridoma. Each hybridoma produces relatively large quantities of identical antibody molecules. By allowing the hybridoma to multiply in culture, it is possible to produce a population of cells, each of which produces identical antibody molecules. Although it is attractive to have a permanent supply of antibody with constant specificity and affinity, these cell lines may contain an unstable chromosome complement and their immortality depends upon proper storage and maintenance. The advantages, disadvantages and production of monoclonal antibodies have been discussed in several book chapters and reviews [8,9]. Antibody engineering and production of recombinant antibodies is a very promising field both for research and application [10,11]. Basic synthetic ways for preparation of the hapten derivatives (hapten design) were explored primarily in steroids more than 30 years ago [12]. Hapten immunochemistry thus represents a consistent area for the development and preparation of conventional antibodies. As new impulses for progress are largely depleted, further experimental strategies have been sought outside the classical immunochemistry area. In the past decade, molecular biology has generated fundamental changes in antibody production.
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The discovery of polymerase chain reaction (PCR) simplified the cloning of monoclonal antibody genes from mouse monoclonal cell lines. These functional recombinant antibody fragments could be expressed in bacteria for use [13]. To take advantage of recombinant technology, efficient, large-scale screening techniques must be used. Immunization procedures and schedules vary depending on the laboratory [14]. Usually an initial series of injections is followed by booster injections some weeks later. Animals are generally bled 7–14 days after each booster injection to determine the characteristics of the serum. Serum is collected or pooled following numerous booster injections and (or) the animal may be exsanguinated. For long-term storage, antibodies are best stored frozen either in solution or as a lyophilized powder. Antibodies can be kept in solution containing 0.1% sodium azide (to prevent growth of microorganisms) in a refrigerator for up to a year. Solutions can also go through freeze–thaw cycles several times without too much loss of activity. Although antibodies are relatively hardy proteins, the concentration should be kept above 1 mg/mL during storage, solutions should be frozen quickly in liquid nitrogen before placing in a standard freezer and for long-term storage antibodies are lyophilized and stored in containers sealed under dry nitrogen.
4.3 Assay development and assay optimization The different steps and procedures to develop and optimize one of these assays are linked to the type of immunoassay, such as immunoreagent immobilization in case of immunosensor development, these steps will be reviewed in later sections focused on the different techniques. However, in immunoassay development, there are always two general steps that can be summarized as Determination of optimal immunoreagent concentrations and Determination of optimum general assay conditions of pH, solvents, ionic strength and cross-reactivity. The first stage is the determination of the optimum coating antigen/hapten– enzyme conjugate and anti-analyte antiserum concentrations. This stage involves, in most cases, the development of an ELISA, using a checkerboard titration. In the 96-well plate, the coating antigen concentration is varied by row and the antibody concentration is varied by column so that each well has a different combination of antigen and antibody concentrations. The concentrations that yield a reasonable signal and at which the system is not saturated can be estimated by plotting the resulting absorbance values versus reagent concentration. Using the optimum reagent concentrations, the assay is tested for inhibition by the target analyte. If a useable IC50 is obtained, then further optimization is conducted. This second stage of optimization includes determining the optimum assay temperature and incubation times and the effect of potential interferences (e.g., solvent, salt, pH and cross-reactivity).
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The percentage of cross-reaction produced by the structurally close-related compounds is of high importance because they may cross-react with the antibody, yielding inaccurate results.
4.4 Validation Initial validation involves an evaluation of the sensitivity and specificity of the immunoassay, whereas later validation includes comparison with a reference method (in general based on chromatography) with a known accuracy using an identical sample set. When comparing two methods, it is important to be aware of the strengths and weaknesses of each one. Validation should also include testing the effects of sample matrices. Matrix effects are determined by running calibration curves in various dilutions of matrix and comparing the results with those obtained for corresponding calibrations curve run in buffer. Overlapping curves indicate no effect of matrix. Parallel curves are an indication that matrix interference is binding the antibody in the same manner as the analyte. Nonparallel curves are indicative of nonspecific matrix interferences. A second test for matrix effects is to analyze a sample before and after a known amount of analyte has been added (test of additivity). If the values for the ‘before’ and ‘after’ samples are not additive a matrix effect is presumed. If matrix effects are present, then adjustment of the immunoassay method, such as running the calibration curve in the matrix or further sample preparation, is necessary. The final steps in validation involve testing a limited number of samples containing incurred residues to determine if the method provides reliable data.
5. IMMUNOCHEMICAL TECHNOLOGIES Table 1 summarizes the most representative immunochemical technologies applied to food analysis.
Table 1
General scheme of immunochemical techniques related to food control analysis
Basic immunoassays – Fluorescent immunoassays – Chemiluminescence immunoassays (CIA) – Radioimmunoassays (RIA) – Enzyme-lynked immunosorbent assays (ELISAs) – Fluorescence polarization immunoassay (FPIA) Immunoaffinity chromatography Flow injection immunoassays Immunosensors
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5.1 Radioimmunoassays (RIAs) The development of practical immunoassays began in the 1960s with the application of radioimmunoassays (RIAs). RIAs utilize radioactive isotopes as a label, and the amount of radioactivity measured is indicative of the amount of analyte present. RIAs are still used today, particularly for detection of very low quantities of analytes. However, due to the inherent complications of handling and disposing of radioactive materials, RIAs are less often used in the laboratory than other types of immunoassays, such as enzyme-based immunoassays (EIAs).
5.2 Enzyme-based immunoassays (EIAs) Whereas RIAs use radioactivity to measure the concentration of analyte, EIAs typically use a change in color, emission of light, or other signal. Specific equipment is required to quantify the amount of enzyme present by measuring the signal change that occurs. EIAs offer numerous advantages over other immunotechniques because their signal is amplified by forming a great amount of product molecules, and they are widely used for food purposes, especially those based on heterogeneous conditions, such as ELISAs. The main enzymes used are horseradish peroxidase (HRP), alkaline phosphatase and b-galactosidase. Due to the selectivity and sensitivity, as well the simplicity of performing ELISAs, a wide number of assays for food contaminants have been reported in the literature, including low-weight analytes, such as phenols [15] and pesticides [16]. Table 2 summarizes some examples of ELISAs for food contaminants analysis. After the development of a new immunoassay, an important stage is the optimization for its application to real samples. These studies are based in setting the ideal conditions for real work, the evaluation of matrix effects and the establishment of the stages of sample pretreatment that could be necessary. It is also necessary to establish the selectivity of the assay for real samples under real working conditions, and the percentage of recovery. Finally, the validation can be carried out comparing the results of the EIA analysis of natural samples with those obtained by well-established methods, such as chromatographic approaches. Following the previous described scheme, a high number of works have been published showing the optimization and validation of new immunoassays for food analysis applications. Garce´s-Garcı´a et al. [17] developed and evaluated four plate immunoassays for routine determination of pesticide residues (diazinon, fenthion, malathion and chlorpyrifos) in extra virgin olive oil. A good correlation was found between the proposed immunoassays with the reference chromatographic (GC–MS) method. Nunes et al. [18] described a direct application of an ELISA for carbaryl determination in fruit and vegetables, with a limit of detection from 0.13 to 6.05 mg/L. A good correlation was found in comparison with a liquid chromatography postcolumn reaction fluorescence detection method. Shen and Jian [19] performed an study on screening, determination and confirmation of chloramphenicol (CAP) in 10 kinds of matrices, including seafood, meat and honey by
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Summary of ELISAs applied to food analysis
Class
Name
Matrix
Reference
Herbicide
Bensulfuron-methyl Chlorpropham Chlorsulfuron Cyclohexanediones Dichlobenil Dichlorprop methyl ester Fluometuron Hexazinone Isoproturon Metsulfuron-methyl Propanil Triazines Trifluralin
Water Food
[20] [21] [22] [23,24] [25] [26] [27] [28] [29] [30] [31] [32] [33]
Acetamiprid Allethrin Azidirachtin Azinphos methyl Azinphos methyl Carbofuran Chlorpyrifos DDT Esfenvalerate Fenitrothion Flucythrinate Imidacloprid Organophosphates Oxamyl Permethryn Pymetrozine Pyretroids Spinosad
Food, water
Insecticide
Fungicide
Antibiotics
Soil Water Water Water Water Food Food
Food, water, sediments
[34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46,47] [48] [49] [50] [51] [52]
Benalaxyl Captan Chlorothalonil Imazalil Myclobutanil Procymidone Tebuconazole Thiabendazole Thiram
Food, Food, Food, Food Food, Food Food Food Food
[53] [54] [55,56] [57] [58] [59] [60] [61] [62]
Gentamicin Kanamycin
Milk Milk
Food Water Water Food Water Food, soil Water Food, water Food, water Food Water Plants
water water water water
[63] [64]
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Table 2 (Continued ) Class
Name
Matrix
Reference
Neomycin Oxytetracycline Streptomycin Tetracycline Tylosin
Milk Pork meat Milk Water Water
[64] [65] [66] [67] [68]
Microorganisms
Escherichia coli Toxoplasma gondii Listeria monocytogenes
Food Pork Milk, ice-creams
[69] [70] [71]
Mycotoxins
Fumonisin Citrin
Food Grains
[72] [73]
ELISA, high-performance liquid chromatography with UV detector (HPLC–UV) and gas chromatography in combination with electronic capture detector (GC– ECD) and MS detector using electronic ionization and negative ion chemical ionization modes with selected-ion monitoring acquisition (GC–MS–EI–SIM and GC-MS–NCI–SIM). A number of immunoassays have been developed for detection of antibiotics and coccidiostats in milk. Watanabe et al. [74] developed a monoclonal ELISA for the detection of monesin in chicken plasma and cattle milk, with a limit of detection of 1 ng/mL and relative standard deviations between 2.1–6.3% intraassay and 5.9–12.9% inter-assay. At present different commercial kits are available in the market. Matthews and Haverly [51] evaluated three commercial kits in tube format for the determination of methyl-chlorpyrifos, methyl-pirimifos and fenitrothion in samples of grain. They realized linearity assays, and reproducibility, matrix effects studies and they determined the cross reactivity of related compounds, and they compared the performance of these commercial immunoassays with those from GC. In this work, the ELISA for Methyl-Chlorpyrifos was the most sensitive. Some of these immunoassays have been developed for food quality evaluation, for example, a Performance Tested MethodSM multiple laboratory validations for the detection of peanut protein in four different food matrixes were conducted under the auspices of the AOAC Research Institute [75]. In this blind study, three commercially available ELISA test kits were validated: Neogen Veratoxs for Peanut, R-Biopharm RIDASCREENs FAST Peanut and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream and milk chocolate spiked at 0 and 5 ppm peanut. All three commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 mg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be o90%. The probability that a test sample contains an allergen given a prevalence rate of
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5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When two test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least two of the validated kits. During recent years a high number of assays have been developed using the dipstick format [26,27,76,77]. Cho et al. [78] developed a direct competitive ELISA for fenthion in microtiter plate and dipstick format. The microtiter plate ELISA showed an IC50 value of 1.2 mg/L with a detection limit of 0.1 mg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using a support membrane allowed the quick visual detection of fenthion in concentrations W10 mg/L. The IC50 value of the dipstick format using reflectance detection was 15 mg/L with a detection limit of 0.5 mg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87–116%. Sharma et al. [79] have evaluated the suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in several foods. The two lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), were based on the immuno-detection of BoNT types A, B and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/mL of BoNT types A and B and 20 ng/mL of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. The assays successfully detected BoNT types A, B and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples. Sometimes these assays present the advantage of avoiding any sample pretreatment; however, in some cases sophisticated extraction procedures can be also necessary in order to extract residues from complex matrices. This is the case of the extraction of pesticide residues in food samples presented by Lopez-Avila et al. [80] who performed a fluid solid extraction and ELISA procedure. General recoveries were around 70% except for carbaryl with a recovery percentage of 49.4%. Urruty et al. [81] applied a method based on solid-phase microextraction (SPME) followed by and immunoassay based on magnetic particles for the determination of procymidone residues in wine. The linear range of this assay was from 2 to 100 mg/L in fortified samples. The recoveries by ELISA and using the official method by GC were 109 and 103%, respectively.
5.3 Fluorescence polarization immunoassay (FPIA) Fluorescence polarization immunoassay (FPIA) is a type of homogeneous competitive fluorescence immunoassay. With competitive binding, antigens from the specimen and antigen-fluorescein (AgF) labeled reagent competes for binding sites on the antibody. As an homogeneous immunoassay, the reaction is carried
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out in a single reaction solution, and the bound Ab–AgF complex does not require a wash step to separate it from ‘free’ labeled AgF. This immunoasay is utilized to provide accurate and sensitive measurement of small toxicology analytes such as therapeutic drugs, and some hormones. FPIA utilizes three key concepts to measure specific analytes in a homogeneous format Fluorescence: Fluorescein is a fluorescent label. It absorbs light energy at 490 nm and releases this energy at a higher wavelength (520 nm) as fluorescent light. Rotation of molecules in solution: Larger molecules rotate more slowly in solution than do smaller molecules. This principle can be used to distinguish between the smaller antigen-fluorescein molecule, AgF, which rotates rapidly, and the larger Ab–AgF complexes, which rotate slowly in solution. Polarized light: Fluorescence polarization technology distinguishes AgF label from antibody bound-antigen-fluorescein (Ab–AgF) by their different fluorescence polarization properties when exposed to polarized light. Polarized light describes light waves that are only present in a single plane of space. When polarized light is absorbed by the smaller AgF molecule the AgF has the ability to rotate its position in solution rapidly before the light is emitted as fluorescence. The emitted light will be released in a different plane of space in which it was absorbed and is therefore called unpolarized light. With the larger sized Ab–AgF complex, the same absorbed polarized light is released as polarized fluorescence because the much larger Ab–AgF complex does not rotate as rapidly in solution. The light is released in the same plane of space as the absorbed light energy, and the detector can measure it. A FPIA method based on a monoclonal antibody for the detection of parathionmethyl was developed and optimized by Kolosova et al. [82], with a linear range from 25 to 10,000 mg/L. The detection limit was 15 mg/L. Recovery in vegetable, fruit and soil samples averaged between 85 and 110%. The method developed showed a high specificity and reproducibility (coefficient of variation ranged from 1.5 to 9.1% for inter-assay and from 1.8 to 14.1% for intra-assay). The same authors developed another FPIA also based on monoclonal antibodies for the determination of ochratoxin A (OTA) [83]. This assay was highly specific and the cross-reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T-2 toxin) was negligible (o0.1%). However, when the assay was used in naturally contaminated barley samples in comparison with an indirect competitive ELISA some disagreement was observed between the results.
5.4 Chemiluminescent magnetic immunoassay (CMIA) Chemiluminescent compounds can also be used to label analytes. A chemiluminescent label produces light when combined with a trigger reagent. A chemiluminescent magnetic immunoassay (CMIA) for atrazine analysis was developed and evaluated by Fischer-Durand et al. [84], and this study demonstrates the feasibility of pesticide determination using CMIA, although the sensitivity of current CMIA format does not reach the required levels.
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5.5 Flow-injection immunoassay (FIIA) FIIA, is based on the introduction of the sample into a carrier stream, which enters the reaction chamber where the immunoreaction takes place. In general in FIIA, the antibodies are immobilized to form an affinity column and analyte is pumped over the column. The loading of the antibodies with analyte is followed by pumping over the column enzyme tracers that compete with the analyte for the limited binding sites of the antibodies. Generally, the indirect format produces a result inversely proportional to the analyte concentration. FIIA can be used with electrochemical, spectrophotometric, fluorimetric and chemiluminescence detection methods. Conventional UV visible spectrophotometry is also suitable for the FIIA detection of bioligand interactions. FIIA has been used for the detection of diuron and atrazine in water. This method was developed as a cost-effective screen for determining compliance with the European drinking water directive. FIIA has been successfully used for the detection of pesticides (e.g., triazines [85,86]). At present, FIIA is integrated into different immunosensors.
5.6 Immunoaffinity chromatography This technique uses the binding properties between Ab–Ag for the selective extraction of an analyte from complex food and environmental matrices. The technique is especially useful for polar organic analytes. Immunoaffinity sorbents are used for pre-concentration of closely related compounds, that are later eluted, separated and analyzed; an example of application is the detection of isoproturon in water samples, allowing determination of 0.1 mg/L [87], or of bisphenol A [88]. The main limitations of these procedures are the effects of the matrix on immunoreagents, the small volume admitted by the immunosorbents and the desorbtion step that is sometimes difficult. Recent applications in food analysis have been focused on aflatoxins and ocratoxine. Prado et al. [89] performed the analysis of OTA in beers by immunoaffinity column and HPLC using a fluorescence detector. Recoveries of OTA from beer samples spiked at levels from 8.0 to 800 pg/mL ranged from 81.2 to 95.0%, with coefficient of variation between 0.1 and 11.0%. Detection limit and quantification limit were 2.0 and 8.0 pg/mL, respectively.
5.7 Immunosensors Immunosensors are a specific case of biosensors, and they constitute the most advanced technologies based on the binding properties of antibodies. In this context, nanotechnology, miniaturization, multi-sensor array development and, especially, biotechnology arise as fast-growing areas that will have a marked influence on the development of new immuno-sensing strategies in the near future. A biosensor is defined by IUPAC as a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical
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information using a biological recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element. In the special case of immunosensors, the biochemical receptor is an antibody. This type of device combines the principles of solid-phase immunoassay with physicochemical transduction elements (electrochemical, optical, piezoelectric, evanescent wave and surface plasmon resonance (SPR)). In this section most relevant transduction principles coupled to immuno-affinity properties used in food analysis applications will be summarized.
5.7.1 Electrochemical transduction Small molecular weight organic residues in food (such as pesticides or their metabolites) have few distinguishing optical or electrochemical characteristics, the detection of stoichiometric binding of these compounds to antibodies is typically accomplished using competitive binding assay formats. The main limitation of these techniques is the electrochemical detection of the immunoreaction because it is necessary to use enzymes that will generate electrochemical active compounds. Electrochemical immunosensors have been widely used for food analysis in amperometric, potentiometric and conductimetric configurations. Some examples of new developments are the disposable screen-printed electrodes for the detection of polycyclic aromatic hydrocarbons (PAHs) [90] and the use of recombinant single-chain antibody fragments [91] for atrazine determinations. Alarcon et al. [92] described a direct, competitive electrochemical ELISA development for the quantitative determination of OTA in wine using polyclonal antibodies. The assay is carried out on carbon-based screen-printed electrodes. Recently, a novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads has been presented by Zacco et al. [93]. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads — such as those based on the use of Protein A or carboxylate modified magnetic beads, the best strategy was the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP for the enzymatic labeling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite–epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator.
5.7.2 Acoustic transduction These transducers have also been applied in immunosensors for food or water analysis [94]. The resonant frequency of an oscillating piezoelectric crystal can be affected by a change in mass at the crystal surface. Piezoelectric immunosensors are able to measure a small change in mass. Novelty contribution is the use of
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magneto elastic transduction, an example is a mass-sensitive magneto elastic immunosensor for detection of Escherichia coli [95]. However, most of the past publications have been based on immunosensors using quartz crystal microbalance (QCM) for the detection of trace amounts of chemical compounds, such as dioxins [96]. Other examples of application are focused on the determination of pathogens such as the work presented by Liu et al. [97], in this work the detection of E. coli O157:H7 was accomplished using a QCM immunosensor with nanoparticle amplification. The quartz crystal is a highly precise and stable oscillator. It has been used widely in electric circuits as a frequency standard clock in computers, communication systems and frequency measurement systems. The quartz crystal is the crucial component of the QCM because it registers and reports the mass deposited on its electrodes quantitatively: the mass changes its oscillation frequency. It is also used for immunosensor techniques, for film thickness metres and for chemical sensors. A variety of surface-based detection principles are employed for immunosensors based on QCM. Preparation and characterization of such layers for useful deposition of detector molecules require sophisticated surface science input. Furthermore, commercialization for use in the food analysis, medical diagnostics and environmental monitoring fields necessitates stability and reliability of the bio-interface. Therefore, antibody immobilization method and its stabilization are very important to prepare the bio-functional interface of QCM immunosensors [98,99]. These methods are classified into three main categories Immobilization of the antibody on the crystal precoated with a suitable material Immobilization via entrapment in polymer membranes Immobilization via glutaraldehyde cross-linking. The prerequisites for the active surface of a QCM immunosensor include that it be chemically active of or toward the immobilized antibody or antigen. Furthermore, the coatings achieved using the immobilization method must be as uniform and thin as possible. These features are particularly important in QCM immunosensors because high sensitivity can only be achieved using active, thin and rigid layers. Additionally, leakage of the immobilized materials must not occur to any extent during the immunosensor use. Although many immobilization methods have been tried with QCM immunosensors, no one ideal method exists that gives high immobilization yield and good stability. Furthermore, the complexity and diversity of antibody or antigen that is useful for different purposes render it difficult to devise one optimum method. Therefore, it is necessary to find a suitable immobilization method for an antibody or antigen for a particular application. The development of QCM immunosensors for food analysis is in an initial phase of development, but it is one of the most promising fields. Up to now most of the applications have been centred in the analysis of pathogenic microorganisms [100].
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5.7.3 Optical transduction Many optical devices have been developed. On the basis of solid-phase fluoroimmunoassays combined with an optical transducer chip chemically modified with an analyte derivative for the measurement of different pesticides in water control analysis [101,102], even for multidetection measuring pollutants at low nanogram per litre levels in single few-minutes analysis [103]. Different optical immunosensors have been constructed for the control of bacterial growth [104]. A variety of devices have been developed for bacterial detection, using a range of transduction elements. Seo et al. [105], described an immunosensor for Salmonella typhimurium detection in chicken carcass wash fluid, using a flowthrough cell that comprised two channels, one with immobilized anti-Salmonella antibodies through a silane-derivatized surface, and a reference channel with immobilized human IgG. An integrated optic interferometer allowed the specific measurement of Salmonella by comparing the phase shift generated by the refractive index variation with the reference channel. Although the assay time was 10 min, 12-h pre-incubation time is needed in order to allow a cultural growth level detectable by the sensor. DeMarco et al. [106] developed a portable polystyrene fibre optic device for E. coli O157:H7 and staphylococcal enterotoxin B analysis in 20 min. This rapid and sensitive device is now commercially available from Research International. The basic principle of optical waveguide lightmode spectroscopy (OWLS) technique is that the light polarized of He–Ne laser is coupled by diffraction grating into the wave-guide layer, if the incoupling condition is fulfilled. This incoupling resonance phenomenon occurs at the precise angle of incidence. This angle depends on the refractive index of the medium covering the surface of the wave-guide. In the wave-guide layer, the light is guided by total internal reflection to its ends where it is detected by photodiodes. By varying the angle of incidence of the light the mode spectrum can be obtained and the effective refractive index can be calculated for both the electric and the magnetic mode. OWLS is a label-free technique for investigating surface adsorptions, binding and adhesions processes as occurs in an immuno-reaction. This technique has been applied to food analysis such as the detection of Aflatoxin and Ochratoxin in wine using both competitive and indirect immunoassays. The sensitive detection range of the competitive detection method was between 0.5 and 10 ng/mL in both cases [107]. Several approaches are based on the principle of total internal reflection fluorescence (TIRF). This principle is based on incident plane waves that generate an evanescent wave, which excites molecules near a sensor surface, with a local distribution proportional to the evanescent electric field intensity. After a characteristic excited-state lifetime, these molecules emit fluorescent radiation with a local distribution in the surface similar to the excitation distribution. The light of the fluorescent evanescent wave is coupled back as a plane wave in the same way as the primary process, when a plane wave generates an evanescent wave. A recent work reports a sandwich immunoassay coupled to TIRF for the determination of Campylobacter and Shigella bacteria with limits of detection of
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4.9 104 cfu/mL for Shigella dysenteriae, and lower than 9.7 102 cfu/mL for Campylobacter jejuni [108]. Recently, a TIRF-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal antiprogesterone antibody as biological recognition element [109,110]. SPR is an optical electronic technique in which an evanescent electromagnetic field generated at the surface of a metal conductor is excited by light of a certain wavelength at a certain angle. Interest in the development of SPR-based immunosensors for detection and monitoring of low-molecular-weight analytes in food and environmental fields has been rapidly increasing over the past 10 years. By combining the advantages of the specific antigen–antibody immunoreaction and the high sensitivity and reliability of SPR signal transduction, SPR immunoassays offer exceptional sensitivity, specificity, speed and multianalyte detection in complex analytical matrices, such as food. Advances in the technology of antibody production and the signal transduction provide a promising scope for SPR immunosensors to lead in the next generation biosensors. SPR is associated with the evanescent electromagnetic field generated on the surface of a thin metal film when excited by an incident beam of light of appropriate wavelength at a particular angle, and it is explained as a charge density oscillation occurring at the interface between two media of oppositely charged dielectric constants. As the evanescent field generated under total internal reflection conditions is strongest at the interface and diminishes exponentially with increasing the distance of penetration from the interface, SPR promotes the detection of only surface-confined molecular interactions occurring on the transducer surface. There are two kinds of configuration used for excitation of surface plasmons: Kretchmann [111] and Otto [112]. Most of the SPR instruments use Kretchmann configuration working at attenuated total reflectance for excitation of surface plasmons. In general, an SPR immunosensor consisted of several important components: a light source, a detector, a transduction surface (usually gold-film), a prism, the bio-molecule (antibody or antigen) and a flow system. Figure 4 shows a simple scheme of the principle and operation of SPR immunoassay technique. The transduction surface is usually a thin gold-film (50–100 nm) on a glass slide optically coupled to a glass prism through a refractive index matching oil. In addition to gold, various metals can be used including silver, copper and aluminium. However, gold is highly preferred due to its chemical stability and free electron behaviour. Plane polarized light is directed through a glass prism to the gold/solution dielectric interface over a wide range of incident angles and the intensity of the resulting reflected light is measured against the incident light angle with a detector. At certain incident light wavelength and angles, a minimum in the reflectivity is observed at which the light waves are coupled to the oscillation of surface plasmons at the gold/solution interface. The angle at which the minimum reflectivity occurs is denoted as an SPR angle. This critical angle is very sensitive to the dielectric properties of
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Flow Channel
Optical detection unit Reflected light I (initial)
Polarized light
Reflected light II (after binding)
Prism
Sensor chip with gold film
Intensity
Signal resonance II I
II I Angle
Time
Figure 4 SPR scheme based on total internal reflection (Kretschmann configuration).
the medium adjacent to the transducer surface apart from its dependence on the wavelength and polarization state of the incident light. In particular, the resonance condition is extremely sensitive to the refractive index of the sample in contact (around 200 nm) with the metal surface, because the optical electric fields are localized within 250 nm from the gold surface. The resonance conditions are influenced by the bio-molecules immobilized on the gold layer. Thus, adsorption of bio-molecules (antigen or antibody) on the metallic film, any followed conformational changes of the adsorbed bio-molecules (or subsequent modification) and molecular interactions with relevant substances can be accurately detected. When a surface immobilized antibody binds with an analyte, the change in the interfacial refractive index can be detected as a shift in the resonance angle. These changes are monitored over time and converted into a sensorgram, from which the kinetics and affinity constants of the interaction can be determined. The resonance angle shift can provide information on the amount of bound analyte, the affinity of analyte for the antibody and the association (or dissociation) kinetics between the antibody and analyte. Microfluidic systems are important in bringing antibody or antigen solutions over the gold surface. Since the SPR signal is highly sensitive to the structure, configuration and reactivity of the surface-immobilized species, a slight change in any of these properties will be transformed into an SPR signal in real-time. Thus, the use of a uniform, well-regulated microfluidic component is necessary
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in studying the bio-molecular association/dissociation reactions with precise kinetic information and/or configuration changes. SPR offers the following advantages compared to other transduction techniques for application as a high throughput tool in food analysis: SPR does not require labeling of the reagents. SPR analyzers are capable of producing continuous real time responses to biomolecular interactions occurring at the interface. This will lead to rapid evaluation of the analytical systems. Sensor surface can be regenerated for repeated multiple use. The SPR instrumentation is feasible for miniaturization and multispot detecting, which is highly useful in the fabrication of portable sensors. By combining the advantages of the specificity of the biorecognition reactions, the SPR immunosensor could be developed for detecting any analyte with error-free measurements. In addition to these advantages, the main limitations should also be mentioned: Specificity: A high cross-reactivity with interfering substances can be produced, if density and the compact degree of the monolayer immobilizing the sensing element is not the optimal one. The percentage of cross-reactivity of this type of immunoassay is also directly linked to the specificity of immunoreagents involved. Interferences: The SPR sensors are sensitive to any effect that produces a variation in the refractive index, fluctuations of temperature, or different composition of the samples. In the detection of various small-molecular organic compounds related to food quality, SPR immunosensors based on the indirect competitive immunoreaction principle were predominantly applied. Some examples of immunosensors reported for food analysis are summarized in Table 3. Different applications have been published for aflatoxins control [113,114], and pesticides residues in water and food [115,116]. Some applications of SPR immunosensors have been developed for the food quality control, such as the casein content in milk products [117], for food adulteration control [118], for the detection of genetically modified organisms (GMO) [119] and for the detection of pathogens in food and water [120–123], for example, a prototype SPR analyzer developed by Homola et al. [124] showed good stability and high sensitivity in the determination of the microbe, Staphylococcal enterotoxin B. Some examples of immunosensors reported for food analysis are summarized in Table 3.
6. BIOMIMICS: MOLECULARLY IMPRINTED POLYMERS (MIPS) Due to the frequently poor stability (thermal, pH) and short life times of biological components, synthetic molecules with high affinity properties, similar
114
Table 3 Examples of immunosensors for food contaminants analysis Analyte
Type and basis
Isoproturon Simazine
Indirect assay and TIR-fluorescence detection Competitive immunoreaction and potentiometric detection
Triazines Triazines
Inhibition assay and SPR detection Inhibition assay and surface refractive index change detection Flow-through amperometric immunosensor based on peroxidase chip and enzyme channeling system Competitive immunoassays and fluorescence detection
Triazines
Pesticides Mycotoxins Aflatoxins Aflatoxins Deoxynivalenol Fumonisin B1 Ochratoxin A
Direct immunoassay and fluorometric detection Indirect assay and SPR Indirect assay and SPR Direct assay and SPR detection Inhibition assay and SPR detection
Sensitivity
Reference
Water Water Water Water Water Aqueous solution water Water Meat extract, milk, tomatoes, cucumbers, potatoes Water Water
0.03 mg/L 1–10 mg/L 0.06–0.2 mg/L 20 ng/L 1.38 mg/L 0.01 ng/mL
[125] [126] [127] [63] [62] [128]
0.01–0.14 mg/L 3 ng/mL
[129] [34]
0.16 mg/L 15 mg/mL
[130] [131]
1 ng/L
[132]
Aqueous solution
Aqueous solution
Aqueous solution Food Grain Aqueous solution Liquid food samples
[133]
0.01 mg/L 2.5 mg/L 15 mg/mL 5 ng/mL
[134] [60,61] [129] [135] [136]
Marinel la Farre´ et al.
Pesticides and herbicides Atrazine Inhibition assay and electrochemical detection Atrazine Inhibition assay and optical detection Atrazine Indirect assay and TIR-fluorescence detection Atrazine Indirect assay and SPR Carbaril Indirect assay and SPR Chlorsulfuron Competitive assay and amperometric detection
Matrix
Food borne microorganisms and bacterial toxins Staphylococcal Sandwich assay format
Tomatoes, sweet corn, green beans, mushrooms and tuna
Biosensor arrays
Enterotoxin B Botulinum toxoid A
Indirect inhibition assay and SPR detection Sandwich assay format and Biosensor arrays
Food Tomatoes, sweet corn, green beans, mushrooms and tuna
Domoic acid Domoic acid Cyanobacter hepatotoxins microcystins E. coli enterotoxin E. coli enterotoxin E. coli O157:H7
Indirect inhibition assay and SPR Indirect inhibition assay and SPR Recombinant antibodies
Shellfish Shellfish Food and drinking water
QCM SPR Anti E. coli covalently immobilized to Biodyne membrane and amperometric detection
Food Food Aqueous suspension
[137]
Whole tomatoes 0.1 ng/ml Mushrooms 0.5 ng/ml Sweet corn 0.5 ng/ml Green beans 0.5 ng/ml Tuna 0.5 ng/ml Tomato juice (buffered) 20 Whole tomatoes 50 Mushrooms 100 Sweet corn 50 Sweet corn juice 50 Green beans 250 Green bean juice 100 Tuna 500 0.5–150 ng/L 2–3.3 mg/L
35 pM 70 pM 100 cells/mL
[71] [138]
[139] [140] [141]
[142] [143] [144]
Immunochemical and Receptor Technologies
Enterotoxin B
Tomato juice (buffered) 0.1 ng/ml
115
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Table 3 (Continued ) Type and basis
Matrix
Sensitivity
Reference
E. coli O157:H7
Anti E. coli immobilized in a fibre optic and fluorescence detection SPR sensor chip-MALDI-TOF detection Array biosensor
Beef
3-30 cfu/mL
[145]
Milk, mushrooms Food
1 ng/ml
[146] [147]
Chicken carcass wash fluid
1.104 cfu/mL
[148]
Aqueous suspension
1.106 cfu/mL
[149]
Food
10 ng/g
[64]
Milk
0.5 ng/mL
[150]
Enterotoxin B Pathogens (Campylobacter jejuni) and mycotoxins Salmonella typhimurium S. typhimurium
Anti-Salmonella antibody covalently bound to sensor surface refractive index change detection Anti-Salmonella antibody adsorbed to gold electrode QCR detection Sandwich assay and evanescent wave detection
Staphylococcal enterotoxin A Staphylococcal Sandwich assay and SPR enterotoxin B Staphylococcal Automated fibre optic enterotoxin B Clostridium Paramagnetic bead-based botulinum toxins A, electrochemiluminescence B, E and F C. jejuni Array Biosensor; Sandwich immunoassay
Food
[151]
Food
50 pg/ml for serotype A [152] to 50–100 pg/ml for serotypes B, E and F Yogurt 1880 [153] Milk 469 Ground turkey sausage 469 Ground turkey ham 3750
Food
Marinel la Farre´ et al.
Analyte
Antibiotics Chloramphenicol Chloramphenicol Ciprofloxacine Enterofloxacine Penicillin and its derivatives Sulphadiazine
Inhibition assay based on indirect immobilization of sulphamethazine to a sensor chip and SPR detection Inhibition assay and SPR detection Inhibition assay and SPR detection Inhibition assay and SPR detection Inhibition assay based on indirect immobilization of Sulphamethazine to a sensor chip and SPR detection
Milk
1 ng/mL 5 106 M
[154] [155]
Milk Milk Milk
1.5 mg/Kg 1.5 mg/Kg 2 mg/Kg
[154] [154] [156]
Pig bile
0.02 mg/mL
[157]
Milk Milk Milk Milk
1 mg/L 1.7 mg/Kg 1 mg/Kg 0.5 mg/Kg
[158] [159] [160] [161]
Immunochemical and Receptor Technologies
Sulphamethazine Sulphamethazine Sulphamethazine Sulphamethazine
Inhibition assay and SPR detection Label-free detection, quartz crystal microbalance measurement Inhibition assay and SPR detection Inhibition assay and SPR detection SPR, direct inhibition assay
117
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to biological ones, are being introduced. One of the most promising groups of biomimetic materials are molecularly imprinted polymers (MIPs). MIPs are a class of highly cross-linked polymer-based molecular recognition elements engineered to bind a single target compound or a class of structurally related compounds with high selectivity. By engineering both the binding site and the polymer backbone a wide range of optimized separation phases can be produced. Selectivity is introduced during MIP synthesis in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities or imprints that are sterically and chemically complementary to the desired target analyte(s). Non-covalent imprinting, in particular, has a great range of applications because of the theoretical lack of restrictions on size, shape or chemical character of the imprinted molecule. The possibility of tailor-made, highly selective receptors at low cost, with good mechanical, thermal and chemical properties makes these polymers appear ideal chemoreceptors. There are great hopes for development of a new generation of chemical sensors using these novel synthetic materials as recognition elements [128,162]. The selected ligand or print molecule is first allowed to establish bond formations with polymerizable functionalities, and the resulting complexes or adducts are subsequently copolymerized with cross-linkers into a rigid polymer. Following the extraction of the print molecule, specific recognition sites are left in the polymer, where the spatial arrangement of the complementary functional entities of the polymer network together with the shape image corresponding to the imprinted molecule. Molecular imprinted polymers can be prepared by self-assembly, where the prearrangement between the ligand and the functional monomers is formed by non-covalent bond, or metal coordination interactions; or by a preorganized approach, where the aggregates in solution prior to polymerization are maintained by reversible covalent bonds. By use of a high percentage of crosslinker, polymers of substantial rigidity and complete insolubility are obtained. MIPs allow for selective extraction of low levels of target compounds in the presence of a mixture of potentially interfering matrix components. Compared to biological receptors, MIP polymer recognition systems have the advantage of superior chemical and mechanical stability being compatible with most solvents, pressures and pH conditions. The materials can be engineered for an almost unlimited variety of small molecules, such as drugs, natural products, pharmaceuticals, peptides and other types of molecules. The main characteristics of these materials is their resistant, mechanical stress, high temperatures and pressures, resistant against treatment with acid, base, or metal ions, stability in a wide range of solvents, and can be used repeatedly.
6.1 MIPs in preparation/preconcentration and separative applications MIPs are recognition elements that have been successfully applied in analytical chemistry as an alternative to immunoaffinity systems. They have been applied in affinity chromatography, binding assays and sensor developments.
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The past decade is characterized by the transition of molecular imprinting techniques from a predominantly theoretical research to practical analytical applications. The first application of MIPs was as stationary phases in affinity chromatography, in particular for the enantio separation of racemic mixtures of chiral compounds, and much of the early work on MIPs was devoted to this aspect. Capillary electrochromatography (CEC) is a hybrid separation method that couples the high separation efficiency of capillary zone electrophoresis (CZE) with liquid chromatography, and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed. Since there is no back pressure it is possible to use small diameter packing and thereby achieve very high efficiencies. CEC might be one of the more promising chromatographic techniques to be used in combination with MIPs, in particular for chiral separations. MIP-based CEC profits from the inherent separation power of this method; compared with MIP-based HPLC, appreciable more resolution can be achieved. One of the most studied areas of application was preparation/preconcentration techniques. Sellergren in 1994 [163] published one of the first relevant works on MIP-based solid-phase extraction (SPE), since then a new window was open, and intensive labor of development have been carried out on molecularly imprinted solid-phase extraction (MISPE). The potential advantages of this technique compared to conventional SPE sorbents are the specificity, batch reproducibility, can be regenerated and cost-effective synthesis. Due to these reasons, during previous years the potential of MIPs application for SPE materials has been thoroughly investigated culminating in the commercial availability of MIP-based SPE cartridges by companies, such as ELIPSA (Germany) [164] and MIP Technologies (Sweden) [165]. However, some limitations of these new SPE materials have also been reported [166] such as diminution of recovery percentage after repetitive regenerations. For food analysis some relevant contributions have been done using MISPE in different areas, such as the analysis of flavones in wine [167,168], or in food toxicant analysis. Main applications in food toxicants analysis have been carried out in three specific areas: Residues of pharmaceutical compounds (antibiotics, b-agonists) Microbial pathogens and mycotoxins Residues of pesticides. The class selective MIP SPE-b-agonists has shown to be a valuable tool in screening and quantitative determination of b-agonists in muscle tissue from several kinds of animals [169]. The extraction of mycotoxins demonstrates the versatility of MIPs even for more complex natural compounds such as deoxynivalenol and zearelenone [148], or OTA [138]. A high sensitivity (0.05–0.2 mg/L) method was developed for the analysis of chlorotriazine in environmental samples by Ferrer and Barcelo´ [170].
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Applications of MIPs for SPE and packing column materials for food analysis
Analyte
Analysis format
Functional monomer
Reference
(+)-Catechin 4-Nitrophenol Chloramfenicol Chloro triazines Clembuterol
MIP-based SPE MIP-based SPE MIP-based SPE MIP-based SPE MIP-packed HPLC column MIP-based SPE Polymeric microcapsules MIP-based SPE MIP-based SPE MIP-based SPE MIP-based SPE MIP-packed HPLC column MIP-based SPE MIP-based SPE MIP-based SPE On-line MIP-based SPE RAM-MIP-SPE MIP-based SPE
EDMA 4-VP NS
[171] [172] [173] [174] [175]
Flavonoids Listeria monocytogenes Micotoxins Microsystin-LR Ochratoxin A Ochratoxin A Penicillin V, Penicillin G, Oxacillin Propazine Simazine b-blockers Sulfamethazine Triazines Triazines
MAA
4-VP+EDMA [176] Poly(allilamine) [177]
PAM EDMA MAA+4-VP
MAA MAA+EDMA
[154] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187]
Note: 4-VP, 4-vinylpyridine; EDMA, ethylene glycol dimethacrylate; MAA, methacrylic acid; PAM, N-phenylacrylamide; DEAEM, diethylamino ethyl methacrylate.
The synthesis, evaluation and comparison of different MIPs based on the use of preformed silica beads, to be used as selective HPLC stationary phases able to recognize and separate several phenylurea herbicides at low concentration levels directly from vegetable sample extracts without any previous clean-up step, have been reported by Tamayo and Martin-Esteban [143]. A clembuterol-selective MISPE cartridge has been released by MIP Technologies, for detection of trace concentrations of this illegal growth promoter in meat products. Recently, membranes become increasingly attractive materials for efficient affinity separations [188]. Some examples of reported applications of MIPs for SPE and packing column materials are shown in Table 4.
6.2 Development and application of MIP-based sensors During the past decade the number of application of MIP-based sensors has increased drastically.
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The high selectivity and affinity properties of MIP for target analytes make them ideal recognition elements in sensors development [63]. Capacitive [189], conductimetric [190], field effect [191], amperometric [192] and voltammetric [193] electrochemical transduction systems have been used. On the basis of conductimetric transduction by Piletsky et al. [190] sensors for herbicide analysis have been developed with a linear range of 0.01–0.50 mg/L for atrazine, without interference of simazine. Chloroaromatic acids were determined by Lahav et al. [191] using a TiO2 sol-gel system, and using a voltammetric transduction Pizzariello et al. [193] developed a sensor for clembuterol analysis. MIP-based sensors coupled to piezoelectric transducers are one of the most promising areas. Different devices have been developed for their use in food industry, such as a supported-piezoelectric detection based on MIP for the quantification of caffeine content in coffee and tea samples [194], the detection of sorbitol [195], antibiotics in milk [174], and the detection of toxicant compound such as PAHs [196]. Another optical transducers reported is a chemiluminescent sensor for clembuterol determination [197]. Applications of MIP-based sensors for contaminant analysis are summarized in Table 5. In spite of the development in this field, MIPs still have a series of limitations: Unless the print-material is inexpensive, their preparation is very costly There is a lack of robustness and sensitivity, due to inefficient removal of the print molecules during MIP preparation Several times there is a lack of reproducibility General MIP preparation procedures are adequate at the laboratory level, and now these preparation procedures need to be developed for scaling up to commercial production. Table 5
Examples of MIP-based sensors for food toxicant analysis
Analyte
Functional monomer
Transduction
Reference
o-Xilene 2,4-Dichlorophenoxyacetic-acid
NS 4-VP
[198] [199]
Atrazine
MAA+EDMA
QCM Electrochemical detection using screen printed electrodes Amperometric detection Conductimetric sensor SPR-QCM
[202]
SPR (SPREETA) Voltammetric BAW
[203] [204] [140]
Trichloroacetic acid 4-VP+EDMA and haloacetic acids Domoic acid 2-(Diethylamino) ethyl methacrylate Ochratoxin A Polypyrrole film Sulphamethazine MAA Caffeine MAA+EDMA Note: PQC, piezoelectric quartz.
[200] [201]
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However, new polymeric strategies have been proposed in order to overcome these limitations. For example, Jodlbauer et al. [138] reported the development of an OTA-selective MIP material, specifically designed to recognize and bind OTA under polar protic conditions, using as a functional monomer Q-MMA, auxiliary monomer tBu-MAA and the OTA-mimicking template. Recently, Maier et al. [180] reported the application of this material for the investigation of OTA in red wines using a two-dimensional SPE clean-up protocol on C18-silica and a target-selective MIP. The combined protocol afforded extracts suitable for sensitive OTA quantification by HPLC-fluorescence detection. In this study, problems inherent to MIP-based SPE have been addressed including the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavourable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding and slow analyte binding kinetics.
7. COMMERCIAL INSTRUMENTATION AND FUTURE PERSPECTIVES The majority of reported biosensor research has been directed towards the development of devices for clinical markets; however, driven by the need for better methods for food surveillance, research into this technology is also expanding to encompass food applications. Table 6 shows some examples of commercial biosensor instrumentation available for toxic compounds and pathogens analysis in food. A number of instruments for food analysis are already commercially available. However, the commercial success of biosensors is limited to a small number of applications, where the market size justified more research, validation and development investment. These commercial devices are focused on few applications, such as the determination of saccharides (YSI), or the detection of bacterial toxins or pathogens (Research International). The future commercial status and general acceptance of this technology will depend on the performance characteristics, sample throughput, associated costs, validation and acceptance by regulatory authorities. A variety of laboratory prototype biosensors have been reported which measure a fairly broad spectrum of food contaminants and residues. Immunoassays traditionally have been used as a single-analyte method, which has often been considered a limitation of the technology. However, several approaches are possible to overcome this limitation. An approach is to use a compact disk (CD)-based microarray system [139]. A microdot system was developed that utilized inkjet technology to print microdots on a CD. The CD was the solid phase for the immunoassay, and laser optics were used to detect the near-infrared fluorescent label. The advantage of the CD system is the ability both to conduct assays and to record and/or read data from the same CD. Since the surface of a single CD can hold thousands of dots, thousands of analyses can be made on a single sample simultaneously. Such high-density analyses could lead to environmental tasters where arrays of immunosensors are placed on Chips [205] or high-density plates. Because the CD
Table 6
Commercial devices
Name
Analyte
Description
Company
Analyte 2000t
Staphylococcal enterotoxin B, Escherichia coli O157:H7, spores
Research International, 18706 142bd Ave, N.E., Woodinville, WA, 98072, USA (www.resrchintl.com)
ANDREA
Direct measurement of molecular locking and DNA-hybridization
Autolab SPR SPRIT
Generic sensor for studding binding interactions with low molecular weight molecules Hormones, vitamins, mycotoxins, antibiotics, low weight molecules
4-Channel, single wavelength fluorometer optimized for performing evanescent-wave fluoroimmunoassays The measurement of antibody– antigen reactions is done by a new technique on the basis of ellipsometry. Ellipsometry is an optical method, which simply measures the state of polarization of light that is reflected from a substrate. SPR affinity based biosensor
SPR affinity based biosensor
BIAcore AB, Rapsgatan 7, Uppsala, Sweden (www.biacore.com)
Generic sensor for studding binding interactions with low molecular weight molecules
SPR affinity based biosensor
IBIS
Generic sensor for studding binding interactions with low molecular weight molecules
SPR affinity based biosensor
MORITEX, SPR-670M
Generic sensor for studding binding interactions with low molecular weight molecules Staphylococcal enterotoxin B, Bacillus anthracis, Yersinia pesti, micotoxins, spores Generic sensor for studding binding interactions with low molecular weight molecules
SPR affinity based biosensor
QUANTECH LTD, 815 Northwest Parkway, Suite 100, Eagan, MN, 55121, USA (http://www.quantechltd.com/) Windsor Scientific Limited, 264 Argyll Avenue, Slough Trading Estate, Slough, Berkshire SL1 4HE, United Kingdom. (www.windsor-ltd.co.uk) Moritex Inc., Japan (www.moritex.co.jp)
RAPTORt
Miniaturized SPR affinity based biosensor
Research International, 18706 142bd Ave, N.E., Woodinville, WA, 98072, USA (www.resrchintl.com) Texas Instruments Inc., 12500 TI Boulevard, Dallas, TX, 75243-4136, USA. (www.ti.com)
123
SPREETAt
Portable fluoroimmuno biosensor
Windsor scientific (http://www.ecochemie.nl/)
Immunochemical and Receptor Technologies
BIAcore (1000, 2000, 3000, Q, A100, Flexchip) FasTraQ is a biosensor
DRE — Dr. Riss Ellipsometerbau GmbH, Feldstr. 14 D-23909, Ratzeburg, Germany (www.dre.de)
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format has the potential for high-density analyses, there will be the opportunity for easily generating multiple replicates of the same sample, including more calibration standards, thus improving data quality. The development of class-selective antibodies is another approach to multianalyte analysis. The analyst may design haptens that will generate antibodies that recognize an epitope common to several compounds. Examples of classselective immunoassays that have been developed are mercapturates [206], glucuronides [207], pyrethroids [208], organophosphate insecticides [209] and benzoylphenylurea insecticides [210]. Rather than have one antibody that can detect a class, a third approach is to analyse a sample using multiple immunoassays, each with a known crossreactivity spectrum, and determine the concentration of the analytes and confidence limits mathematically [211]. A drawback to using class-selective assays or assays with known cross-reactivity is that for a given antibody, the sensitivity for each analyte vary, and the sensitivity for some analytes may not be sufficient, hence selection of well-characterized antibodies will be a critical step. Some of the other important keys in the future of immunoassays and immunosensors development are to allow more stability of biological components, more robustness assays, more repeatability between different batches of production when disposable elements are involved, and the integration of new technologies coupled to biosensors, such as the PCR. On the other hand, there are at least two other developments that are expected to have significant impact, the laboratory on a chip (LOC) [212] and nanotechnology [213]. The concept of LOC entails miniaturization of all the essential components of analytical instrumentation (e.g., sample preparation, components, reaction with appropriate reagents and detection) by micro fabrication on a chip. Some of the components in LOC technology have already been released on the market (GeneChips from Affimatrix). Nanotechnology refers to the exploitation of processes to generate and utilize structures, components and devices with a size range from 0.1 nm (atomic and molecular scale) to 100 nm or larger in some cases, by control at atomic, molecular and macromolecular levels. It has been suggested that nanoscale sensors and ultra miniaturized sensors could lead to the next generation of biotechnology-based industries. There are several companies manufacturing SPR instruments for studying bio-molecular interactions [214,215]. Each company produces different SPR systems equipped with a variety of options usable for specific applications. Some of these companies are Biacore, SENSIA, Windsor scientific, Quantech, Texas, NTT and Moritex (formerly, Nippon Laser and Electronics). SPR instruments from Biacore have been widely used by the sensor researchers around the world. Finally, among the variety of biomimetic recognition schemes utilizing supramolecular approaches MIPs have proven their potential as synthetic receptors in numerous applications, and their advantages compared to biochemical recognition systems include thermal stability, storage endurance and lower costs. In particular, the areas of, food and beverage analysis require analytical tools capable of discriminating chemicals with high molecular specificity. Furthermore, food and process safety control issues favour the application of on-line in situ
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analytical methods with high molecular selectivity. While biorecognition schemes frequently suffer from degrading bioactivity and long-term stability when applied in real-world sample environments, MIPs serving as synthetic antibodies have successfully been applied as stationary phase separation matrix, and biomimetic recognition layer in chemical sensor systems. Current research demonstrates the progression of MIP chemistry and the potential of these materials to solving a wide variety of food analytical problems. The combination of MIPs with a range of transducers to produce on line and real time sensors is expected, and their potential has been demonstrated, but limitations described previously should be addressed. The majority of applications of MIPs are directed to low molecular weight, organic soluble analytes of interest in the pharmaceutical industry (e.g., chiral drug separation), but new applications have been developed in environment and food industry. Several new approaches to MIP production (e.g., surface imprinting) are expected to allow MIPs to be prepared in aqueous media. Other new approaches in development are Magnetic MIPs and Cathalytic MIPs (also referred to as enzyme mimics or plastizyme).
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CHAPT ER
5 Advanced Sample Preparation Techniques for the Analysis of Food Contaminants and Residues Pat Sandra, Frank David and Gerd Vanhoenacker
Contents
1. Introduction 2. Sample Preparation Methods for VOCs 2.1 Static headspace (SHS) 2.2 Dynamic headspace (DHS) 2.3 Solid phase microextraction (SPME) 2.4 Headspace sorptive extraction (HSSE) 2.5 Selected applications 3. Sample Preparation Methods for SVOCs and NVOCs 3.1 Solid samples 3.2 Liquid samples 4. Fractionation and Clean-Up 4.1 Chemical methods 4.2 Chromatographic methods 4.3 Gel permeation chromatography 4.4 On-line techniques 4.5 Selected applications 5. Evaporation 6. Derivatization 7. Conclusion References
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1. INTRODUCTION In the analysis of contaminants and residues in food samples, enrichment is of vital importance because samples are too dilute (e.g., beverages) or too complex (e.g., meat) for direct analysis and need to undergo a chain of specific treatments to make them compatible with the analytical techniques. While the dictum ‘‘the Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00005-6
r 2008 Elsevier B.V. All rights reserved.
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best sample preparation is no sample preparation’’ is also true for food analysis, for the determination of traces of contaminants and residues, direct analysis is only applicable in some exceptional cases, e.g., the direct analysis of polycyclic aromatic hydrocarbons (PAHs) in drinking water by liquid chromatography (LC) with fluorescence detection and the determination of haloforms in drinking water with capillary gas chromatography with electron capture detection (CGCECD). Extraction, fractionation/clean-up, concentration and/or derivatization steps preceding the analysis, mostly by chromatographic techniques, are mandatory. A typical flow diagram for the determination of contaminants and residues in foodstuffs is shown in Figure 1. Sampling and sample preparation remain among the more time-consuming, error-prone and contamination-prone aspects of the flow diagram. Obtaining a representative sample and proper storage of the sample are important parts of any analysis. Both are often overlooked by analytical chemists, who regard them as self-evident secondary problems, with the chromatographic analysis being of primary interest. However, errors or faults in the sampling protocol cannot be corrected at any point in the analysis. For the analytical data to be meaningful, a plan for acquiring samples and storing samples should be implemented and, if possible, validated by statistical techniques. A discussion on sampling is beyond the scope of this chapter. On the other hand, problems of storage mostly occur in the analytical laboratory and are the responsibility of the analyst. Processes that occur in the sample between the time of sample collection and analysis such as adsorption on the container walls, vaporization loss, photoreactions, and microbial action, can invalidate the data. Samples should be properly stored in the dark in brown glass vials or containers and maintained at 41C or lower. Especially for liquid samples adsorption on the glass wall should be controlled and, if needed, suppressed by adding a polar modifier like methanol, e.g., in beverages with no or low alcohol content.
Sampling
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Figure 1 Flow diagram for the analysis of contaminants and residues.
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In recent years, great efforts have been made to develop sample preparation techniques that guarantee high recovery and reproducibility and that moreover are faster, cheaper, greener and easier to automate than older methods. A recent trend is also the development of multi-residue methods (MRMs) for a great variety of matrices such as the QuEChERS method [1,2] and sorptive extraction [3,4]. In this chapter the currently used sample preparation methods for chromatographic analysis of contaminants and residues in foodstuff are reviewed and the new methods are discussed in depth and illustrated with examples from the laboratories of the authors. The subdivision of the chapter is, in first instance, based on the target compounds to be determined which can be divided into three main classes: (1) volatile organic compounds (VOCs) that can be analysed via headspace techniques eventually after derivatization, (2) semivolatile compounds (SVOCs) that are GC amenable (thermally stable) like polychlorobiphenyls (PCBs), PAHs, most of the pesticides, etc. that require an extraction step and finally (3) non-volatile or thermally labile compounds (NVOCs) the analysis of which should be performed by LC after extraction. Also the different matrixes from which the target solutes are enriched should be taken into account and differentiated. In general, matrixes can be divided based on the absence or presence of fatty material. Non-fatty foodstuffs include non-alcoholic beverages, alcoholic beverages (ethanol content from 3% to 50% ethanol), fruits and vegetables and herbs, while the fatty food includes milk, vegetable oils and diary products. It has to be noted that in recent years the sample preparation strategies have changed drastically. On the one hand, analysts are confronted with the continuous decrease of legislative limits for food contaminants and residues accompanied with higher requirements for more precise and accurate results and, on the other hand, the performance of analytical tools in terms of qualitative and quantitative analysis, has increased tremendously. Mass spectrometers are a must in trace analysis and because of their ‘‘recognition’’ features they have partly taken over the ‘‘selectivity’’ of classical sample preparation methods. Notwithstanding this, quantification is still challenging and both in target and multi-residue methods, isotope dilution and the method of standard addition provide the most accurate results by compensating for matrix effects (sample characteristic) and ion suppression in mass spectrometry (MS) (sample preparation characteristic). The cleaner the extract, the better are the quantitative data, stressing the importance of sample preparation. Several sample preparation techniques will only be covered briefly in this chapter. For more details, refer to Refs. [5,6] and two recent special issues of J. Chromatogr. A [7,8] including a review on Sample Preparation Techniques for the Determination of Trace Residues and Contaminants in Foods by Ridgway et al. [9].
2. SAMPLE PREPARATION METHODS FOR VOCS VOCs in liquid and solid food samples are best analyzed by headspace techniques. Injecting only the vapour phase gives clean chromatograms and the
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analytical system is not contaminated by non-volatile compounds generally present in liquid extracts. Different approaches are available for headspace sampling and the selection is most often dictated by the sensitivity required. One of the main problems in headspace analysis is quantification as the concentration of the analytes in the vapour phase is matrix dependent. The methods of standard addition and, if possible, of isotope dilution guarantee the most precise and accurate results. Headspace sampling is intensively used for the analysis of off-flavors in foods and beverages but can also be applied to investigate residues from packaging materials [10], benzene in bottled sparkling water, chloropropanols in soy-derived products [11], furan in food products [12], etc.
2.1 Static headspace (SHS) In SHS, an aliquot of a solid or liquid sample is placed in a vial and the sealed vial is heated at a given temperature for a defined amount of time. Heating is required to increase the concentration of the target compounds in the vapour phase. A portion of the headspace is then injected into a GC instrument via a loop or a gastight syringe. Precise replication of the headspace sampling conditions is mandatory to obtain reproducible results.
2.2 Dynamic headspace (DHS) The difference between SHS and DHS analysis mainly lies in the sensitivity that can be reached. In the static mode, part of ‘‘one equilibrium’’ is injected whereas in the dynamic mode the equilibrium concentration is continuously removed and trapped resulting in exhaustive extraction and thus high sensitivity for the analysis of volatiles. For liquid samples, DHS sampling is mostly carried out by purge-and-trap analysis. An inert gas is bubbled through the liquid sample and the purgeable organics are moved from the aqueous to the vapor phase. The volatile compounds are then trapped (solid phase extraction, SPE) on an adsorbent such as Tenax or active charcoal. The trap containing the adsorbent is built in a desorption chamber equipped with a powerful heating mechanism which when activated, permits desorption (gas phase extraction) of the trapped compounds. This technique has the distinct merit of providing a clean sample, free from its often complex matrix. A purge-and-trap device can easily be mounted on a GC equipped with a flame ionization detector (FID), flame photometric detector (FPD) or MS. This technique is most appropriate for mg/L (ppb) level analysis of low molecular weight, slightly water-soluble volatile organics with a boiling point below 2001C. A variation of purge-and-trap is closed-loop stripping (CLS) analysis [13] which is a combination of gas phase extraction with SPE in a closed system. CLS allows ng/L (ppt) analysis of contaminants in drinking water. A recently introduced DHS sampling method is in-tube extraction (ITEX; CTC Analytics, Zwingen, Switzerland) [14]. The enrichment of volatiles can be fully automated as the ITEX device can be mounted on a CTC Combipal sampler. The principle of ITEX is illustrated in Figure 2.
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Figure 2 Principle and operation of in-tube extraction (ITEX).
The sample is heated and agitated in a sealed vial until equilibrium is achieved. A special syringe equipped with a trap containing an adsorbent (Tenax, Carbosieve, etc.) is pierced through the vial septum and the syringe pumps the headspace through the adsorbent, e.g., 10 times 1 mL. The trap is then flashheated and desorbed in the GC injector. Afterwards, the hot ITEX trap is cleaned with inert flush gas before the next sampling. The comparison SHS and ITEX will be presented in Section 2.5.1 for the determination of diacetyl, responsible for the cardboard flavour, in beer.
2.3 Solid phase microextraction (SPME) SPME is intensively applied in headspace analysis. A number of books are reviewing the fundamental aspects and the applications of SPME [3,15]. On polymers such as polydimethylsiloxane (PDMS) and polyacrylate a partitioning mechanism applies while on divinylbenzenestyrene copolymers or mixtures of Carboxen (CAR) and PDMS, adsorption controls the enrichment. In headspace SPME, two mechanisms apply, firstly the partitioning of analytes between the sample matrix and the headspace, and secondly the partitioning between the headspace and the fibre coating. SPME is thus not an exhaustive extraction technique but an equilibrium technique. Maximum sensitivity is obtained at equilibrium, although commonly non-equilibrium conditions are used. This requires optimization of temperature, time and agitation for each application.
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Gandini and Riguzzi applied HS-SPME for the analysis of methyl isothiocyanate, an illegal antifermentative agent, in wine [16]. Page and Lacroix applied HS-SPME to the analysis of fruit juices, soft and fruit drinks, and milk for the determination of volatiles ranging from vinyl chloride to hexachlorobenzene [17]. Recent applications include the determination of furan in baby food [18] and formaldehyde in fish [19].
2.4 Headspace sorptive extraction (HSSE) A new approach for sorptive enrichment of analytes from the headspace of aqueous or solid samples, referred to as headspace sorptive extraction has recently been developed [20,21]. The technique implies the sorption of volatile compounds into a large amount of PDMS (ca. 50 mg) placed on a glass rod support. The PDMS-coated glass bar is then thermally desorbed on-line with capillary GC-MS. Using a large amount of sorptive phase, highly volatile as well as SVOCs can be efficiently enriched and compared to SPME a significant increase in sensitivity is achieved. Bicchi et al. [22] compared the performance of HSSE, through the determination of the recoveries and relative abundances of 16 components in coffee headspace, with SHS and HS-SPME applying the fibres PDMS 100 mm, Carbowax/divinylbenzene 65 mm (CW/DVB), Carboxen/PDMS 75 mm (CAR/ PDMS), polyacrylate 85 mm, PDMS/divinylbenzene 65 mm (PDMS/DVB) and Carboxen/divinylbenzene/PDMS 30 mm (CAR/PDMS/DVB). In all cases, HSSE gave higher recoveries, which is entirely due to the high amount of extracting phase applied.
2.5 Selected applications 2.5.1 In-tube headspace extraction for the determination of the diacetyl offflavor in beer In the quality control of beer, several VOCs are monitored. These compounds include C3–C5 alcohols, C2–C5 esters, dimethyl sulfide and 1,2-diketones (diacetyl, 2,3-pentanedione). These compounds are present at different concentration levels ranging from tens of ppm (alcohols) to ppb level (diacetyl). Beer samples are normally analyzed by SHS in combination with GC. In order to cover all solutes and concentration levels, often several runs are needed per sample. Alternatively, the analysis is performed using effluent splitting to three detectors: FID for alcohols and esters, selective sulfur detection (FPD, PFPD) for dimethyl sulfide (DMS) and ECD for diketones. The three detectors allow sufficient sensitivity and selectivity, but this set-up is rather complicated and problems with splitters and thus robustness are encountered. With mass spectroscopic detection, all solutes can be detected using the simultaneous scan and selected ion-monitoring (SIM) acquisition of state-of-the-art MS systems. However, for some solutes, the sensitivity of mass spectroscopic detection is at the limit, especially in combination with SHS.
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The total ion chromatograms obtained for a beer sample using classical SHS sampling and ITEX sampling are compared in Figure 3A and 3B, respectively. Both chromatograms represent the datafiles obtained in scan acquisition mode. Ethanol, the most abundant peak, elutes at 6 min. The peak at 4 min corresponds to the air peak (MS scan from m/e 29). It is clear that more peaks are detected using the ITEX sampling. The following solutes could be identified using the mass spectra: 1-propanol (peak 1), ethyl acetate (peak 2), 2-methyl-1-propanol Abundance 800000
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Figure 3 Total ion chromatograms for beer by SHS (A) and ITEX (B) sampling, and SIM chromatograms at m/z 86 for diacetyl by SHS (C) and HSSE (D). Belgian lager beer (10 mL) was placed in a 20-mL headspace vial and the samples were analysed as such. The SHS conditions were as follows: sample conditioning at 801C for 15 min, 2.5 mL headspace; the ITEX conditions were sample conditioning at 801C for 15 min, 10 extraction strokes of 1 mL at 50 mL/s and desorption at 2501C with 1 mL headspace at 50 mL/s. The analyses were performed on an Agilent 6890 GC — 5975 MSD combination equipped with a 20-m L 0.18-mm ID 1 mm df DB-VRX column. The carrier gas was helium at 200 kPa constant pressure. Injection was split at 1/25. The oven was programmed from 401C, 5 min, at 101C/min to 2501C, 10 min. The MSD transfer line was set at 2501C. The MS was operated in the scan/SIM mode with 50 ms dwell times.
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(peak 3), ethyl propanoate (peak 4), 3-methyl-1-butanol (peak 5), 2-methyl-1butanol (peak 6), 2-methyl propyl acetate (peak 7), ethyl butyrate (peak 8), 3-methyl butyl acetate (peak 9) and 2-methyl butyl acetate (peak 10). Also DMS could be detected at 7.7 min (Figure 3B). Using an extracted ion chromatogram (EIC), the peak can be quantified without problem in the beer sample. The signal-to-noise, measured on ion m/z 62 was 70. The concentration of DMS in this sample was 8 ppb. In the chromatogram obtained by SHS, DMS was difficult to detect (Figure 3A). Only in an extracted ion trace, a small peak with signal-to-noise of 5 could be detected, but no library search confirmation was obtained. The sensitivity was thus increased by a factor of more than 10 for this compound using ITEX sampling. The EICs for ion m/z 86, typical for diacetyl, obtained by GC-MS in SIM mode are compared in Figure 3C and 3D. At 13.1 min, diacetyl can be detected in the chromatogram obtained by SHS only as a trace (S/N ¼ 8 in Figure 3C). Using ITEX, the peak can be detected more easily (Figure 3D) and confirmation of the identity through the relative ratios of target and qualifier ions is possible. The S/N value obtained by ITEX was 44 or 6 times higher than with SHS. The concentration of diacetyl was in the order of 10 ppb.
2.5.2 Comparison of SHS and HSSE in the analysis of fragrances The selection of an HS technique strongly depends on the sensitivity required which is linked to the instrumentation, and especially to the detectors available in the laboratory. In our experience [22], of the recently introduced HS techniques, ITEX, SPME and HSSE, the latter is the most sensitive technique and enrichment factors compared to SHS are increased drastically. This is illustrated with the analysis of some fragrances in a lotion. In Figure 4A, the total ion chromatogram using classical SHS sampling is shown. The internal standards, added at the same concentration level, are detected at 10.3 and 23.1 min, respectively. The response for the first internal standard is higher in comparison to the second internal standard, corresponding to their relative volatility. In this chromatogram, linalool (peak 1) and hexyl cinnamaldehyde (peak 6) are easily detected. Other solutes are only detected as traces and confirmation of their presence by mass spectral comparison with a library spectrum is difficult. The chromatogram obtained by HSSE sampling on PDMS is shown in Figure 4B. Excellent enrichment is obtained considering that the abundances in both chromatograms differ by a factor 10. The structures of all solutes could be elucidated through the MS spectra and some important compounds are citronellol (peak 2), alpha isomethyl ionone (peak 3), lilial (peak 4) and amyl cinnamaldehyde (peak 5).
3. SAMPLE PREPARATION METHODS FOR SVOCS AND NVOCS Food matrixes encompass a broad range of physical types and the selection of a sample preparation method is, in first instance, dictated by the sample structure, e.g., powders, vegetables, fruits, meat and fatty or non-fatty beverages (e.g., milk and yoghurt versus soft drinks and wines). With the exception of food samples directly soluble in an organic solvent, e.g., vegetable oils, all samples analysed for
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Figure 4 Headspace sampling of fragrances by SHS (A) and HSSE (B). A sample of 100 mg was placed in a 20-mL headspace vial. Two internal standards (1,4-dibromobenzene and 4,4u-dibromobiphenyl) were added at the 10 ppm level. The SHS conditions were sample conditioning at 801C for 15 min, HS needle at 901C and injection of 1 mL at 350 mL/s with a 1/10 split ratio. For HSSE, the sample was conditioned at 801C for 15 min with a 10 mm 0.5 mm df PDMS Twister (Gerstel, Mulheim a/D Ruhr, Germany). The Twister was desorbed at 2501C during 10 min in the splitless mode and the released solutes were cryo-focussed at 1001C. The PTV injector was heated from 1001C at 6001C/min to 2501C and the split ratio was 1/10. The analyses were performed on an Agilent 6890 GC — 5975 MSD combination equipped with a 30-m L 0.25-mm ID 0.25 mm df HP-5MS (Agilent Technologies). The carrier gas was helium at 168 kPa constant pressure. Injection was at 2501C with a 1/10 split. The oven was programmed from 501C, 1 min, at 81C/min to 2701C. The MSD transfer line was at 2501C. The MS was operated in the scan mode between 40 and 350 amu.
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NVOCs or SVOCs require a solvent extraction step to enrich/isolate the target solutes from the matrix. Extraction is commonly followed by a clean-up or fractionation step to selectively remove interferences that disturb the chromatographic analysis. However in recent years, the selective nature of mass spectrometers and especially of MS-MS techniques, is more and more exploited to eliminate or reduce these steps. However, care should be taken because these interferences can have a pronounced effect on the MS ionization efficiencies and isotope dilution should be applied. Because both solid and liquid samples require enrichment in an organic solvent, it seems logical to discuss first the sample preparation techniques for solid and semi-solid samples and later those for liquid samples.
3.1 Solid samples Before extraction, solid samples require some extra steps such as grinding, sieving and drying. Extraction is then performed as such or after mixing with a solid matrix (see matrix solid phase dispersion).
3.1.1 Shake flask extraction, Soxhlet and ultrasonic extraction The extraction of solid samples is most commonly done using traditional liquid– solid extraction methods such as shake flask extraction, classical Soxhlet extraction (introduced in 1870!) and its more modern forms Soxtec and Soxtherm. Soxhlet extraction is still considered as a rugged method because it has very few variables that can adversely affect extraction recovery. The modern forms allow equivalent extraction efficiency in about 2 h. It is not the aim to detail all these methods but rather to concentrate on ultrasonic extraction (UE). UE uses mechanical energy in the form of shearing action that is produced by a low-frequency sound wave. The sample is immersed in an ultrasonic bath with a solvent and subjected to ultrasonic radiation for 15–60 min. UE often provides very good results for solid food samples. Advantages over other techniques are simplicity, speed, productivity and low cost. The selection of the solvent is of utmost importance and heating can be applied if needed although self-heating is already generated by the sonication process. Its application will be illustrated and its performance compared to microwave assisted solvent extraction (MASE) and accelerated solvent extraction (ASE) for the analysis of PCBs in food samples (Section 3.2.6).
3.1.2 Supercritical fluid extraction (SFE) The excellent properties offered by a supercritical fluid namely tuneable solvating power (selectivity), high diffusivity and low viscosity have resulted in analytical usage for many years. Despite early promise, the utility of supercritical fluids for extraction was dormant for many years until the mid1980s when analytical-scale SFE instrumentation became commercially available. SFE was received with high expectations by the analytical community, but in recent years enthusiasm has declined as the disadvantages have become clearer. The most important disadvantage is definitely the complexity of the extraction
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procedure which is strongly matrix dependent and needs careful optimization. A procedure developed for one particular matrix does not automatically work for another matrix. Chuang et al. [23] applied SFE for the analysis of pesticides in baby food but they could not obtain quantitative recoveries for all monitored pesticides. SFE is therefore not really accepted for routine work and remains a research tool. The determination of PAHs in vegetable oils by SFE was recently described by Lage and Cortizio [24]. In Section 3.2.5, the determination of pesticides by supported liquid-SFE will be presented.
3.1.3 Pressurized liquid and accelerated solvent extraction (PLE, ASE) PLE encloses a number of extraction techniques that use solvents at elevated temperatures and pressures. Best known is ASE that originates from SFE and uses organic solvents at high temperature and high pressure to leach out the organics from solid matrixes. Compared to extractions at or near room temperature and at atmospheric pressure, ASE delivers enhanced performance by the increased solubility, the improved mass transfer and the disruption of surface adsorption by the conditions applied. Dionex Corp. introduced in 1995 a fully automated sequential extraction system that was very soon after its introduction recommended by the US Environmental Protection Agency (EPA) for the extraction of solid waste [25]. Typical conditions for an ASE extraction are: temperature 1001C, pressure 2,000 psi, extraction time 5 min equilibration and 5 min static extraction with a solvent composed of dichloromethane and acetone in ratio 1:1. The speed of the extraction process is greatly increased compared to conventional liquid– solid methods and virtually all organics can be extracted. Disadvantages of ASE are the lack of selectivity which means that further clean-up is needed and that the sample is too dilute for direct analysis and further concentration is required. Recent developments to circumvent these shortcomings are the combination of ASE with SPE and the application of large volume injection. The application of PLE for food (and biological) sample analysis has been reviewed by Carabias-Martinez et al. [26]. Recent applications include the determination of ochratoxin in bread [27] and bisphenol A diglycidyl ether in canned food [28]. Another variant of PLE is extraction at high temperatures and pressures with water. Super-heated water extraction (SHWE) including many applications was recently reviewed by Smith [29]. Although SHWE is by far the most ‘‘green’’ extraction technique, main disadvantages of SHWE over ASE are that the solutes are obtained in dilute aqueous medium and further extraction with an organic solvent is required. A large number of matrix compounds are extracted as well, so that further clean-up is needed and last but not least that the thermal stability of the target solutes under SHWE conditions should be carefully evaluated.
3.1.4 Microwave assisted solvent extraction (MASE) MASE utilises electromagnetic radiation to desorb organics from their solid matrices. MASE typically operates at 2.45 GHz. The use of a microwave oven for sample preparation originates from inorganic or elemental analysis. In this case the electromagnetic radiation is used to destroy inorganic and organic matter using a
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combination of strong acids and peroxides. The first application of microwaves for the extraction of organics from solid material appeared in 1986 [30]. In recent years, different systems have become commercially available and they are based on extraction in a closed high pressure vessel with microwave absorbing solvents, extraction with a non-microwave absorbing solvent in an open vessel and/or extraction with a non-microwave absorbing solvent in a closed vessel applying a Weflon stir bar that heats the solvent. The performance of MASE has been compared to other recently introduced techniques like ASE and SFE and similar recoveries were obtained for soil and sediment samples [31]. For food analysis, the same disadvantages mentioned for ASE apply namely lack of selectivity and the dilution effect. Moreover, care should be taken with solutes that are thermolabile or can rearrange under the influence of electromagnetic radiation. The performance of MASE has also been evaluated for the Belgian dioxin crisis (Section 3.1.6).
3.1.5 Matrix solid phase dispersion (MSPD) MSPD first reported by Barker et al. [32] is a powerful technique for disrupting and extraction of solid, semi-solid and viscous samples. A small amount of sample is homogenized with bulk-bonded silica sorbent (typical ratio 1:4) with a glass pestle in a glass mortar. The shearing forces disrupt the structures of the matrix and disperse the sample over the surface of the sorbent. The homogeneous blend of sorbent and sample is then packed in a syringe-type cartridge (like an SPE cartridge) followed by elution of targets and interfering compounds (or vice versa) by consecutive washings with appropriate solvents. For non-fatty matrices, a simultaneous clean-up often occurs during the MSPD process which allows direct analysis of the extracts [33] while further clean-up is required for MSPD extracts of fatty matrices. An elegant way to remove fat traces was described by Ferrer et al. [34]. Olives and the acetonitrile extract of olive oil were homogenized with aminopropyl silica and the obtained powder was loaded on a Florisil SPE cartridge. Elution with acetonitrile gave a nearly fat free extract. This method is compared to the conventional procedure of size exclusion clean-up. The practical and theoretical aspects of MSPD are described in Ref. [35]. A recent review by Barker [36] contains numerous applications illustrating that the method has found wide application in the analysis of contaminants and pesticide residues from animal tissues, fruits, vegetables and other matrices.
3.1.6 Selected applications 3.1.6.1 The determination of PCBs in foodstuffs. In the European Community, 1999 will be remembered as the year of the Belgian dioxin crisis. The dioxins (mainly polychlorinated dibenzofurans or PCDFs) originated from the presence of used transformer oil in animal feed and many thousands of food samples had to be analysed for PCBs. During the crisis, the sample preparation technique applied at the Research Institute for Chromatography, Kortrijk, Belgium, was continuously updated and a high-throughput method based on UE was developed. The implementation of the method had to be accompanied with validation studies and the analysis of certified samples.
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The analytical scheme of the official method Beltest I 014 for the analysis of PCBs in food consists of different steps: sample drying, extraction, clean-up and CGC analysis. Sample drying can be performed by freeze-drying or chemically by the addition of sodium sulphate. In the second step, the lipophilic contaminants are extracted from the matrix using an apolar solvent. The extract contains the lipids, PCBs, PCDDs, PCDFs and other apolar solutes such as organochloro-pesticides (OCPs), PAHs and mineral oil. Different extraction techniques may be applied namely Soxhlet extraction (or the automated versions Soxtec or Soxtherm), UE, ASE, MASE and SFE. All these techniques perform equally well for the extraction of fat and PCBs as will be illustrated further. It is obvious that especially in a crisis situation the selection should be based on sample throughput and cost. Next the PCBs are fractionated from the (co-extracted) fat matrix. For this fractionation, column chromatography on acidic silica gel and aluminium oxide is advised although other techniques such as gel permeation chromatography (GPC) and SPE may be applied if validated. Both sample extraction and clean-up require a concentration step. Finally, the cleaned extract is analysed by CGC-ECD and positive samples are confirmed by capillary gas chromatography with mass selective detection (CGC-MS). UE and clean-up by a dispersive solid phase extraction (DSPE) technique nowadays also applied in the QuEChERS method, was performed as follows. Samples were homogenised using a blender. From fat samples (chicken or pork fat) 1 g sample was weighed in a 20-mL headspace vial. From eggs, 3 g egg yolk was taken. For animal feed samples or other meat products, a sample size corresponding to 200–500 mg fat was taken. To the sample, 2 g anhydrous sodium sulphate and 10 mL petroleum ether were added. Tetrachloronaphthalene or octachloronaphthalene may be added as internal standard to the sample at this stage although external standardization can also be applied. The headspace vial is closed and placed in an ultrasonic bath at 301C for 30 min. In this step, the fat and PCBs are transferred from the matrix in the petroleum ether phase. The sodium sulphate adsorbs the water present in the sample. After extraction, the sample is allowed to settle. An aliquot (typically 5 mL) is transferred to a test tube and another aliquot (2 mL) is used to determine gravimetrically the fat content of the extract. To the test tube, 2 g of acidic silica gel (44% sulphuric acid) is added and the tube is placed in an ultrasonic bath for 30 min. This technique is nowadays called DSPE. In column chromatography and in SPE, the analytes are eluted through a bed and the fat is retained. In DSPE, the fat matrix is allowed to bind on the adsorbent that is mixed with the sample, while the solutes of interest stay in solution. For the fractionation of fat from PCBs, this method works very efficiently. After settlement of the adsorbent or centrifugation, an aliquot of the clear solution is transferred to an autosampler vial. The final volume is not important, since the concentrations are calculated to the initial 10 mL solvent and the sample or fat weight. The whole sample preparation takes approximately one hour and several samples can be prepared in parallel. One technician can handle more than 50 samples per day.
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The extracts are analysed by CGC-ECD and/or CGC-MS. For MS detection, SIM mode is performed on two ions per congener group. The limit of detection (S/NW3) of the micro-ECD and the MS were 0.2 and 0.5 pg, respectively. Figure 5 shows the RTL-CGC-ECD profiles of Aroclor 1260, egg, pork fat and mink fat recorded with a time interval of nearly one month. PCB congeners 153, 138 and 180 are the pre-dominant congeners. The chromatogram of the mink fat extract is interesting. The minks were fed with contaminated eggs and although the same PCB profile (mainly Arochlor 1260) is present, some differences are noted in the relative abundances of the PCB congeners. This can be explained by a different metabolism between the animal species. In the mink sample, the PCB concentration measured as the sum of the seven congeners was as high as 25 ppm (25 mg/kg fat). This concentration was fatal for most minks. Some 4,000 samples, including animal feed, eggs, chicken fat, pork fat, pork meat, meat products (ham, sausages), etc. were analysed using this methodology. From a practical point of view, the splitless liner was replaced after 100 analyses and the column after 1,000 injections. On the column selected and with the
Hz 2000
PCB 153
PCB 138
PCB 180
A
1000 0 6000 4000 2000 0
B
6000 4000 2000 0
C
15000 10000 5000 0
D
20
22
24
26 Time (min)
28
30
32
Figure 5 Retention time locked CGC-ECD analyses of Aroclor 1260 (A), egg extract (B), pork fat extract (C), mink fat extract (D). Analyses were performed on a HP 6890 GC (Agilent) equipped with split/splitless inlet and micro-ECD detection. Separations were carried out on a 30-m L 0.25-mm ID 0.25 mm df HP-5MS column (Agilent). Injection of 1 mL was done in the splitless mode at 2501C. The carrier gas was hydrogen at an initial head pressure of 71 kPa hydrogen. The pressure was adjusted via the RTL software to obtain a retention time of 26.999 min for p,pu-DDT. The oven was programmed from 701C (2 min) to 1501C at 251C/min, to 2001C at 31C/min and to 3001C (2 min) at 81C/min. Nitrogen at 40 mL/min was used as detector make-up gas. The detector was set at 3201C. Reprinted from Ref [52] with permission from Elsevier.
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chromatographic conditions applied the PCB congeners 28 and 31 are not separated but this was not critical as both congeners were not relevant for this PCB pollution. The performance of UE was compared with ASE and MASE. The extraction efficiency of the three methods was evaluated with three egg samples contaminated at different levels (low, medium and high). For ASE, a Dionex ASE 200 system (Dionex Corp., Sunnyvale, CA, USA) was used. A 1-g sample was extracted at 1001C and 1,500 psi using petroleum ether as solvent. The extraction time was 5 min oven heat-up time, 5 min static extraction and 3 cycles with 60% of the extraction cell volume (22 mL). The extract was then concentrated to 10 mL. For MASE, an ETHOS SEL system (Milestone, Analis, Gent, Belgium) was applied. A 1-g sample was extracted after 20 min at 951C. The extraction solvent was n-hexane (10 mL in extraction thimble, 10 mL outside thimble) using a Weflon stir bar to absorb the microwave energy in combination with the nonmicrowave absorbing solvent. After extraction, the extract was filtered and concentrated to 10 mL to obtain the same final concentration factor as the UE and ASE. Clean-up and analysis was done in the same way for the three extracts of the three samples. The results based on duplicate analysis are summarized in Table 1. For the sample with the lowest concentration (egg 1) the relative standard deviation (RSD) on the PCB sums obtained by the three techniques is 12%; for the two other samples the RSDs are less than 6%. For the individual values, some small differences are noted but in general these differences are within 10% of the average values. These results clearly demonstrate that there is no statistically significant difference between the three techniques and that equally good results are obtained. The ultrasonic method exhibits by far the highest throughput and is extremely cheap compared to ASE and MASE. The repeatability of the method (n ¼ 6) was evaluated by the analysis of a contaminated egg and fat sample (Table 2). These samples were distributed in a round robin test for Belgian laboratories during the crisis. The RSDs are all below 10% for the congeners PCB 118, 153, 138 and 180 and below 5% for the sum of the congeners. The linearity and method sensitivity were determined by spiking a blank pork fat sample at 6 levels with the individual PCB congeners. The spike levels were 5, 10, 25, 50, 100 and 200 ppb (ng/g fat) per congener. The recovery was determined versus an external standard and the linearity was measured by plotting the Table 1
Comparison of UE, ASE and MASE for PCB enrichment
PCB
118 153 138 180 Sum
Egg 1 (ppb)
Egg 2 (ppb)
Egg 3 (ppb)
UE
MASE
ASE
UE
MASE
ASE
UE
MASE
ASE
170 263 240 111 783
68 309 320 166 863
147 210 234 93 682
556 1,142 1,019 696 3,412
410 1,051 1,120 552 3,133
550 1,184 1,105 686 3,525
945 1,811 2,031 1,015 5,803
1,023 2,032 2,323 1,166 6,544
922 2,042 2,128 1,259 6,349
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Repeatability for a contaminated egg and fat sample (1999 Belgian dioxin crisis)
PCB
118 153 138 180 Sum
Egg sample
Fat sample
Concentration (ppb)
RSD (%)
Concentration (ppb)
RSD (%)
141 333 342 165 982
7 2 4 5 2
20 274 318 165 776
4 6 3 4 4
Table 3
Figures of merit for the fast PCB method
CB
Mean % recovery
Linearity
S/N at 5 ppb
28 52 101 118 153 138 180
110 88 91 105 101 104 105
0.9999 0.9903 0.9991 0.9996 0.9994 0.9991 0.9998
9 8 12 12 13 11 18
absolute peak areas versus the spiked concentration. The signal-to-noise ratio was also measured at the lowest spiked concentration. The results are summarised in Table 3. All recoveries are within 88–110% for the individual congeners. The linearity is better than 0.995 except for PCB 52 for which an interfering peak was observed in the CGC-ECD trace. The signal-to-noise ratio was better than eight for all congeners at the 5 ppb level. Lastly, the reproducibility and accuracy of the ultrasonic method was evaluated with the determination of the PCB content in two certified reference materials of the European Community namely the cod liver oil sample CRM 349 and the mackerel oil sample CRM 350 (IRMM, Geel, Belgium). The analyses of these reference materials were performed by four laboratories to which the method was transferred and having the same CGC-ECD and CGC-MS instrumentation. The results are summarised in Table 4 for cod liver oil and in Table 5 for mackerel oil. Most values are within 88% and 110% of the certified samples. The values outside these ranges are noted in italic. For all laboratories and for both techniques, the sum values were always between these limits. The relatively long analysis times (see Figure 7) prompted us to evaluate fast high resolution capillary GC for PCB analysis [37]. This reduced the chromatographic analysis time to less than 8 min. Later, extracts were directly introduced in a mass spectrometer operated in the negative chemical ionization mode. This reduced the analysis time to less than 2 min [38].
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Table 4 PCB
28 52 101 118 153 180 Sum
Table 5 PCB
28 52 101 118 153 180 Sum
Accuracy and reproducibility test for the cod liver oil sample Certified Concentration (ppb)
68 149 370 454 938 280 2,259
Lab 1
Lab 2
Lab 3
Lab 4
ECD
MS
ECD
MS
ECD
MS
ECD
MS
61 126 333 390 975 252 2,137
65 165 394 467 989 295 2,375
73 141 385 421 886 283 2,189
64 164 404 448 1,010 312 2,402
104 144 296 479 790 270 2,083
68 199 437 508 1,030 326 2,568
75 159 356 397 810 288 2,085
70 148 373 458 1,016 273 2,338
Accuracy and reproducibility test for the mackerel oil sample Certified Concentration (ppb)
22.5 62 164 142 317 73 778.5
Lab 1
Lab 2
Lab 3
Lab 4
ECD
MS
ECD
MS
ECD
MS
ECD
MS
25 75 152 163 337 73 825
24 72 181 152 345 79 853
21 56 175 117 319 75 763
21 56 175 117 319 75 739
21 65 152 138 287 76 739
19 71 143 125 350 83 791
19 54 150 131 290 70 714
16 63 172 134 316 60 761
3.1.6.2 The determination of nitrofuran metabolites in scrimp and poultry. Nitrofuran antibiotics have been widely used as food additives for treatment of Gram positive and Gram negative bacteria in poultry and fish. The parent compounds and metabolites are suspect carcinogens and they have been banned around the world. The Rapid Alert System for Food and Feed Annual report 2005 [39] shows that these compounds and metabolites continue to be detected in food samples thus remaining today a major concern in food safety. The nitrofuran antibacterial drugs furazolidone, furaltadone, nitrofurazone and nitrofurantoin have been found to metabolize rapidly and the metabolites bind to muscle tissue. The determination of metabolites and not the parent compounds is required in samples of animal origin. The structures of the parent compounds, the metabolites and the derivatives are shown in Figure 6. The European Union set a minimum required performance level (MRPL) at 1 mg/kg for each metabolite [40]. Such levels can easily be determined using stateof-the-art MS systems (ion trap, triple quad and high resolution TOF) if an appropriate sample preparation technique is applied. Sample preparation should include a homogenization step, the release of the metabolites from the proteins, transformation into derivatives to facilitate chromatographic elution and UV
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N
O 2N
N
O
O
O
O
N
H2N
N
O
O
H2N
NH
NO2
SEM
N
NH
N
2
NH
O
NO2 N N
O
Nitrofurantoin
2
NBA-SEM O
H2N
O
O N
NH O
O N
N NH
NH2 O
Nitrofluranzone
O
O
NBA-AMOZ
NH
NH2
O
N O
AMOZ
O
O2N
N N
O
O Furaltadone O 2N
NO2
N
N
NH
O
AHD
NBA-AHD NO2
O
N
N O
Furazolidone
O
H2N
N
O
N N
O AOZ
O
O NBA-AOZ
Figure 6 Structures of the nitrofuran antibiotics, metabolites and formed derivatives.
(the metabolites are not UV absorbing) or MS detection and transfer to an organic phase for analysis. Fortunately, the labelled standards NBA-d5AMOZ (used to quantify NBA-AMOZ) and NBA-d4AOZ (used to quantify the other metabolites) are commercially available simplifying drastically their determination. A typical sample preparation protocol is as follows. One gram of homogenized tissue (e.g., poultry and shrimps) is mixed with 5 mL of a 0.2-M hydrochloric acid and 50 mL of a 2-nitrobenzaldehyde (2-NBA) solution (100 mM in methanol) and incubated 16 h at 371C. The mild acidic conditions release the metabolites via the imine bond from the tissue and the formed amino groups react with 2-NBA to form an aromatic imine bond. The acidic medium is neutralized with 500 mL of a 0.3-M Na3PO4 solution in water and the pH is adjusted to 7 with a 2-M NaOH solution. The sample is twice extracted and centrifuged with 4 mL ethyl acetate and the combined extracts are evaporated to near dryness and reconstituted in 500 mL of initial LC mobile phase. Reversed phase LC-MS (ITD, QqQ or HR-TOF) with electrospray ionization in the positive mode is applied. A typical LC-QqQ analysis is shown in Figure 7. A fish sample (genus Tilapia) was spiked with 0.5 ng/g of the four metabolites and extracted as described [41].
3.2 Liquid samples Extraction of liquid samples (aqueous food samples or extracts obtained from the extraction of solids — see Section 2) is based on partitioning into an immiscible extracting phase which can be a liquid like in liquid–liquid extraction (LLE) or a solid like in SPE.
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Abundance
NBAAMOZ
NBAAOZ
1000 NBASEM
0
5
10 Time (min)
NBAAHD
15
Figure 7 LC-QqQ analysis of nitrofurans of a fish extract spiked at the 0.5-ng/g (ppb) level. The sample was analyzed on a reversed phase column with a mobile phase gradient from 20% acetonitrile/80% formic in water to 99% acetonitrile in 10 min with electrospray ionization in the positive mode. Reprinted from Ref. [41] with permission from Agilent Technologies.
3.2.1 Liquid–Liquid extraction The most commonly used enrichment method for aqueous samples is LLE. LLE may be carried out manually by shaking the water sample with an organic solvent in a separation funnel or automatically, using a continuous liquid–liquid extractor. Depending on the extraction conditions used, extracts can contain intermediate to low polarity components (universal extraction for neutral solutes) or acid and base compounds (selective extraction) by adjusting the pH. LLE is very time-consuming and often uses toxic solvents. The volume of the extract is usually too large for direct injection and in order to obtain sufficient sensitivity, an additional evaporation–concentration step is necessary. Particular care needs to be taken in both the solvent extraction and concentration procedures to avoid contamination of the sample. Moreover, solvent impurities will be concentrated as well often masking the target solutes. For a number of applications automated micro LLE in the vial of an injection autosampler can offer a simple and robust alternative. By using highly sensitive and selective detectors, eventually in combination with large volume injection, sub-ppb sensitivities can be reached. Micro LLE has been used for the analysis of trihalomethanes in drinking water [42].
3.2.2 Supported liquid–liquid extraction SLLE uses a diatomaceous earth material placed in a cartridge to adsorb the aqueous phase by hydrophilic interactions. An efficient LLE occurs when a water immiscible solvent is applied. The high surface area at the interface between the organic and aqueous phase increases efficiency while eliminating the possibility
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of emulsion formation (e.g., in extracting milk). The analytes are then eluted as the solvent passes through the adsorbent. Selective extraction for acids and bases can be obtained by adjusting the pH of the aqueous solution. Two examples of SLLE, one applying a liquid and one a supercritical fluid are described. SLLE was experienced to be an excellent method for the determination of chloramphenicol (CAP) in different matrices. No additional clean-up was required. Moreover emulsion formation that was observed for several matrices was completely suppressed. The procedures for honey and shrimps are detailed. A 5-g sample of honey was diluted to 20 mL with water and an appropriate amount of the internal standard d5-chloramphenicol (CAP-d5) was added. The solution was loaded on an ISOLUTE HM-N cartridge from (IST Hengoed, UK, Part-nr. 800-1300-FM) and allowed to stand for 5 min. Elution was performed with 50 mL ethyl acetate. The eluate was collected and the solvent was evaporated under a nitrogen stream at 401C. The residue was redissolved in 1 mL water/methanol (9:1, v/v) and placed in an ultrasonic bath for 1 min. The solution was filtered by a syringe filter before injection. For shrimps, a portion of at least 10 g of frozen shrimps was defrosted and mixed. To 10 g of the mixed shrimps, 30 mL of water and the internal standard was added. After centrifugation for 10 min at 2,000 rpm, a 20-mL portion of the supernatant was loaded on the ISOLUTE HM-N cartridge and allowed to stand for 5 min. After this, the same elution and concentration procedures as described for honey were applied. Both a single quad and an ion trap MS have been used for CAP determinations and a comparison is given in Ref. [43]. Figure 8A shows the analysis of a standard mixture containing 2.5 ppb CAP and 5 ppb CAP-d5. By applying a fragmentor voltage of 160 V, a fragment ion corresponding to [M-HCOCl] is detected at m/z 257 and 262 for CAP and CAP-d5, respectively which are monitored for confirmation purposes. The extraction procedure was evaluated for repeatability and linearity. For repeatability, blank honey was spiked with 1 ppb CAP and 1 ppb CAP-d5. The extraction procedure was carried out six times and the recovery was calculated. The recovery for CAP varied from 85.3% to 94.9% and the mean recovery was 90.6%. The RSD on the recovery taking the internal standard (IS) into account was 3.4%. A calibration curve was constructed with blank honey samples spiked with 0.1, 0.2, 0.5, 1.0 and 2.0 ppb CAP. The samples contained 1 ppb of the IS. The correlation coefficient was 0.9997. The limit of detection (LOD) for the quadrupole was found to be 0.5 ppb corresponding to 50 pg on-column. Taking the sample preparation into account, CAP can be detected in honey samples containing 0.1 ppb. Figure 8B shows the analysis of a CAP polluted honey sample at a concentration of 0.6 ppb. The matrix of honey is rather complex and lower LODs can be obtained by MS-MS techniques (ITD in Ref. [43] and QQQ in Ref. [44]). The same method was applied to the analysis of shrimps. The sample matrix showed less interference compared to honey samples. An example of an analysis of shrimps is shown in Figure 8C. Due to the reduced matrix effect, the LOD with the quadrupole instrument was lowered to ca. 0.05 ppb in the sample. A concentration of 0.35 ppb was recovered in the shrimp sample. Extraction recovery was 85%.
Abundance 7000 321 m/z 4000
CAP (2.5 ppb)
A
323 m/z 257 m/z
1000 14000
326 m/z CAP-d5 (5 ppb)
8000
328 m/z
2000
262 m/z 4
14000
5
Time (min)
EIC 257, 321, 323 m/z (CAP)
Honey B
2000
15000
EIC 262, 326, 328 m/z (CAP-d5)
0 4
5
6
7
Time (min) 14000
EIC 257, 321, 323 m/z (CAP)
Shrimp C
2000 15000
EIC 262, 326, 328 m/z (CAP-d5)
0 4
5 Time (min)
6
7
Figure 8 LC-MS analysis of 2.5 ppb CAP and 5 ppb internal standard (A), of CAP in a honey (B) and a shrimp (C) sample. The column was an Eclipse XDB C18, 150-mm L 4.6-mm ID, 5 mm dp (Agilent) operated at a flow of 0.9 mL/min with the following gradient between 10 mM ammonium acetate in water (solvent A) and methanol/acetonitrile 1/9 (solvent B): 0–1 min 30% B, 1–8 min 30–70% B, 8–8.5 min 70–100% B, 8.5–12 min 100% B and post time 4 min at 30% B. Column temperature was 301C and the injection volume 100 mL. MS settings were: negative ESI, drying gas temperature 3401C and flow-rate 11 L/min, nebulizer pressure 50 psig, Vcap 3,500 V, MSD acquisition on between 3 and 7.5 min, fragmentor 160 V, SIM ions 257, 321, 323 m/z for CAP and ions 262, 326, 328 m/z for CAP-d5.
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Table 6 Quantification data for pesticides spiked in orange juice and determined by SFE-CGC-MS Pesticide
Measured concentration (ppb)
RSD (%)
Spiked concentration (ppb)
Recovery (%)
Thionazin Methyl parathion Fenthion Methidation Pyrazophos Phosalone Vinclozolin Procymidone
17 107
3.5 2.9
20 101
85 103
92 44 28 429 273 130
4.1 3.6 4.8 3.3 3.7 4.1
103 51 30 505 303 130
89 86 93 85 90 100
Liquids can also be enriched by SFE after mixing them with a solid support such as Chromosorb W (60–80 mesh). The method was applied to the determination of pesticide residues in orange juices. A 2-g amount of Chromosorb W was placed in a 7-mL SFE thimble and 2 mL of orange juice was added on top of the adsorbent followed by another 0.5 g Chromosorb W. SFE was performed at a flow rate of 1 mL/min pure CO2 with a density of 0.75 g/mL and at a temperature of 501C for 5 min of static and 30 min of dynamic operation. The extracted solutes were collected on a trap packed with octadecylsilica and the trap was then rinsed with 1 mL chloroform. The extract was analysed by capillary GC-MS operated in the scan mode. The selectivity of SFE was fully exploited, i.e., the pesticides are soluble in neat CO2 at the selected density while unwanted polar and high molecular mass solutes like triglycerides were retained on the Chromosorb W or octadecyl-modified silica support (ODS) adsorbents. Some data are summarized in Table 6 giving the measured concentration (mean value of 3 experiments), the RSD%, the amount spiked in the sample and the recovery. Recoveries ranged between 85% and 103% with precisions lower than 5% for the total analytical procedure. Working in the SIM mode, determinations at the low ppb level are possible. The total analysis time was 1 h and both the sample preparation and analysis were fully automated [45].
3.2.3 Solid phase extraction This extraction procedure is more and more applied, being much faster, cheaper, and more versatile than most classical techniques. Moreover, SPE procedures are easily automated using robotics, or can be coupled on-line with chromatographic techniques. The principle of retention (enrichment) is analogous to LC and is suitable for low, intermediate and high polarity solutes. Commonly, SPE media are based on normal phase, reversed phase or ion exchange chromatography. Giving an overview of the different SPE sorbents and their applications is not within the scope of this chapter. Hundreds of SPE cartridges are commercially available and the catalogues of the manufacturers describe their features and
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applications. The principles and practice of SPE have been described in books by Thurman and Mills [46], Fritz [47] and Simpson [48]. Present designs of SPE devices are the syringe-type cartridge, the Empore disks and the Empore disk cartridges. The syringe cartridge is the most common arrangement and cartridges ranging in mass of sorbent from 50 mg to 10 g are commercially available. Solvent flow through is typically done using a commercially available vacuum manifold. Permeability is no problem because sorbents are mostly based on irregular particles with a particle diameter between 30 and 60 mm. In the Empore disk format, 5–10 mm particles are intertwined with fine threads of Teflon giving a disk with a thickness of ca. 0.5 mm and a diameter in the range 47–70 mm. Manifolds are also commercially available for multiple sample extraction using Empore disks. The SPE disk allows rapid flow rates of sample and of solvents. One litre of water can be passed through an Empore disk in ca. 10 min whereas with the syringe cartridge the same volume of water can take more than 1 h. Both approaches need a relatively large volume of desorbing liquid and further concentration is required. In the disk cartridge format, a disk of 10 mm diameter is placed in a cartridge between two polyethylene frits. Only small volumes of solvent are needed for solid phase stripping eliminating the need for an additional evaporation step hereby considerably reducing the risk of losses and contamination. The principle of SPE is illustrated with the determination of triazines in drinking water applying large volume injection capillary GC-MS. For the enrichment of triazines from water a 10-mm/6 mL ODS Empore disk cartridge was chosen. This means that enrichment is based on the reversed phase mechanism. For capillary GC, the operation can be divided in six steps namely wetting and stripping the sorbent, conditioning the sorbent, loading the sample, rinsing the sorbent to elute extraneous material, drying of the sorbent and finally elution of the analytes of interest. The disk was first rinsed with 0.5 mL of methanol (wetting and stripping), followed by 1 mL distilled water (conditioning). Twenty-five milliliter water spiked at the 1 ppb level with eleven triazines was passed through the cartridge at a flow of 50 mL/min (loading). Five milliliter distilled water was then passed through the cartridge to elute polar extraneous solutes (rinsing). After drying under vacuum for 10 min (drying), desorption was performed directly in a 2-mL autosampler vial with 0.5 mL ethyl acetate. Forty microliter of the extract was directly injected, this means without further concentration, into the capillary GC system equipped with a programmed temperature vaporizing (PTV) injector operated in the solvent vent mode. The detection limits set by the European Community (0.1 ppb) could easily be reached with scan MS while ion-monitoring allowed determinations down to the ppt or ng/L level. Several approaches allow the tuning of the selectivity of the SPE process. pH control is important to suppress ionization as illustrated for the analysis of veterinary drug residues in shrimps by Li et al. [49]. For ionic solutes the ion exchange mechanism can be utilized. Chlormequat (chlorocholine chloride) belongs to the family of quaternary ammonium salt pesticides. Chlormequat was extensively used for the control and management of terrestrial and aquatic
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vegetation since the mid-1950s but its use has been restricted because of its toxicity to man. Nevertheless, in the late 1990s concentrations as high as 7 ppm were measured in pears. Because of the zero tolerance for pesticides in baby food, often containing pears, a robust and reliable analytical method down to the ppb level is needed. In the procedure presently used in our laboratory, chopped and homogenized pears are mixed with methanol containing the internal standard d4-chlormequat. The mixture is filtered and an aliquot is extracted by SPE on a strong cationic exchanger (BondElut SCX, Varian, CA, USA). After rinsing with a mixture of MeOH-H2O (1:1), chlormequat and the internal standard are eluted with MeOH-H2O (0.2 M NH4OAC) (4:6). Analysis is performed by LC/MS-MS on a cyanopropyl column with electrospray MS in the positive mode. Monitored ions are m/z 122 and daughters m/z 58:60. Other selective SPE methods include the use of restricted access media (RAM), immunosorbents and molecularly imprinted polymers (MIP). These sorbents have mainly been applied for biological and environmental samples and food applications to date are rather limited. Pharmaceuticals in milk were determined by Souverain et al. [50] using RAM SPE and SPE with an immunosorbent was applied by Pichon et al. [51] for the determination of phenylurea pesticides in fruit juices. DSPE in which the solid material added to the liquid sample is more and more applied. The main reason is the high-throughput possibilities DSPE offers. As outlined in Section 3.1.6, DSPE was intensively used in 1999 in our laboratory for the Belgian dioxin crisis [52,53]. Similar procedures are also used in the QuEChERS multi-residue method [1]. In recent years, regulatory agencies emphasize more and more the need for development and use of analytical methods able to determine in food products as many residues as possible out of the whole pattern of insecticides, fungicides and other compounds applied in agricultural practice. At present, single residue methods, i.e., the determination of one pesticide, e.g., chlormequat, or selective residue methods (MRMs), i.e., the determination of a relatively small number of chemically related compounds, e.g., N-methylcarbamate insecticides, are intensively applied for pesticide residue determinations in large number of samples whose pesticide treatment history is known. The use of single residue methods and sMRMs will definitely continue but the development and utilization of MRMs, i.e., the determination of as many pesticides as possible with preferably a single sample preparation method and one GC and one LC method both hyphenated to MS, recognizes a momentum. In the past, a variety of capillary GC-MS-based MRMs have been developed. For example, working group 4 of the Technical Committee (TC 275) of the European Committee for Standardization (CEN) provides information on five MRMs for non-fatty foods (EN 1528:1996) [54]. All methods require extraction with organic solvents such as acetone [55–57], acetonitrile [58] and ethyl acetate [59] and with the exception of Ref. [59] they all require partitioning in a solvent mixture and further clean-up by column chromatography or GPC is advised. The multi-residue capillary GC-MS method that was applied in our laboratory until the application of stir bar sorptive extraction (SBSE – see Section 3.2.5) was a
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method used by the laboratories of the Dutch Inspectorate for Health Protection [59]. This method is similar to the Luke method [55] but the extraction procedures have been miniaturized to reduce solvent consumption. Other MRMs for the GC determination of residues in fruit and vegetables include the procedures described by Stan et al. [60,61] for 385 pesticides using extraction of the samples with acetone followed by salting-out and a multi-bed clean-up and the procedure described by Fillion et al. [62] for 251 pesticide and degradation products. The sample preparation of the later method comprises extraction with acetonitrile, a salting-out step, clean-up by SPE on octadecyl and on carbon/aminopropyl silica and a concentration step. An MRM for animal tissues was recently described by Pang et al. [63] using gel permeation chromatographic clean-up followed by GC-MS and LC-tandem MS. The procedure on clean-up will be described in Section 4.4. Today, the QuEChERS method is by far the best developed MRM for a wide variety of pesticides in several common non-fatty and, since recently, fatty food matrices. The QuEChERS process, as initially developed for fruit and vegetables, is outlined in the flow diagram in Figure 9 [1]. DSPE is a key aspect of the method and the type of adsorbents, as well as the pH and polarity of the solvent, can be adjusted for differing matrices and difficult analytes.
Homogenization in Dry Ice 10 g Sample
10 mL Acetonitrile
Add 4 g MgSO4 + 1 g NaCl
Add IS solution
Dispersive SPE PSAm + MgSO4
(Add 0.1% HAc and Analyte Protectants)
CGC-MS or LC-MS
Figure 9 Flow diagram of the QuEChERS procedure.
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In step 1, a 10-g sample obtained by homogenization in a powerful chopping device using dry ice is vigorously shaken with 10 mL acetonitrile for 1 min. Salts are added in step 2 to induce the phase separation. MgSO4 reduces the water phase while NaCl controls the ionic strength of the extracting solvent. After shaking for 1 min, an internal standard solution is added (step 3). Step 4 is DSPE and typically 50 mg adsorbent is added to a 1-mL aliquot of the acetonitrile phase. The sample is centrifuged, and acetic acid and GC protectors (sorbitol, gulonolactone and ethyl glycerol) are added before injection. For LC analysis, GC protectors are not needed. In recent years, several modifications of the QuEChERS method have been made for specific applications and matrices. Different extraction solvents can be applied and DSPE, commonly performed with PSAm (primary secondary amine) which mainly removes sugars and fatty acids, can be complemented with graphitized carbon black (remove pigments and sterols) and/or octadecylsilica (remove lipids). An important note is that ‘‘QuEChERS-kits’’ have been assembled commercially by Sigma Aldrich/Supelco (Bellefonte, Pennsylvania), Restek (Bellefonte, Pennsylvania) and United Chemical Technologies (Bristol, Pennsylvania). The QuEChERS technique has proven successful for the determination of pesticides in a variety of fruit and vegetables, e.g., [64–66] and also recently in fatty matrices such as olive oil [67], and milk and eggs [68]. The technique was also used to analyse acrylamide in various food matrices [69] and ivermectin in salmon and antioxidants in pet food [70]. In the European Community (EU), QuEChERS round robin tests have been conducted with over 100 laboratories participating. These tests showed that the method can be transferred among laboratories with success [71]. In the US, the QuEChERS method has been adopted First Action as AOAC International Official Method 2007.01. The QuEChERS method is an excellent method to determine pesticides at the maximum residue limits (MRLs) set by regulatory authorities for human consumption. However, for very low MRLs such as set by the European baby food directive (10 ng/g), the method is inadequate. In a recent article, Lehotay et al. [72] described the analysis of a mixture of vegetables and fruit spiked with 36 organophosphorus and organochlorine pesticides at the 10-ng/g level. With the original QuEChERS method only 12 of the 36 pesticides could be determined at the 5-ng/g level. A new technique called Solvent in silicone tube extraction (SISTEx) was introduced. This is a combination of sorptive extraction and solvent collection and similar in its operation as membrane-assisted extraction. The number of elucidated pesticides increased to 24 with a 44-fold improvement in detection threshold.
3.2.4 Solid phase microextraction The literature on application of SPME in food analysis is overwhelming. The reader is referred to the many application notes of Supelco, the producer of the SPME devices. A review by Wardencki et al. [73] describes several applications of SPME in food analysis. However, most applications use headspace SPME (e.g., the off-flavours geosmin and methylisoborneol in drinking water, the cork
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flavour caused by trichloroanisol in wine, off-flavours in rancid oils and fats, caffeine in decaffeinated coffee and the sunstruck flavour in beer) and direct immersion SPME of water samples. The application of the latter technique to complex liquid food samples can be difficult because of protein, sugar, etc., adsorption limiting the lifetime of the fibres. This could be solved by incorporating the fibre inside a hollow cellulose membrane but this technique requires much longer extraction times [74]. SPME liquid desorption in combination with LC is another alternative, also applicable for the analysis of thermolabile pesticides. SPME-LC for the analysis of pesticides has been reviewed by Aulakh et al. [75]. SPME-LC was applied by Blasco et al. [76] to determine post-harvest fungicides in fruits.
3.2.5 Stir bar sorptive extraction In SBSE a glass-lined magnetic bar is covered with a thick layer of PDMS [77,78]. Through magnetic stirring of the bar in the sample solution the components are enriched in the PDMS phase. The typical manipulations in SBSE are (1) the stir bar is introduced in the liquid and stirred for a given time at a selected stirring speed and temperature, (2) removed with tweezers, (3) rinsed with distilled water to remove matrix compounds such as sugars, proteins, etc., (4) dried on lint-free tissue and finally (5) introduced in desorber tubes for thermal extraction (for capillary GC analysis) or in a mini vial for liquid desorption (for LC analysis). Advantages of this technique are ease-of-use, high sensitivity, high accuracy of analysis and reduced risks of contamination compared with other sample preparation techniques. Like all sorptive extraction methods SBSE is an equilibrium technique. Enrichment factors at equilibrium can be predicted from the log P values [79,80]. In practice, it is not necessary to work at this point and extractions are commonly performed for a much shorter defined period of time. Stir bars have been commercialized under the name TWISTER and are, together with the analytical tools, available from Gerstel (Mu¨lheim a/d Ruhr, Germany). SBSE has been applied for quality control, and trace (ppb) and ultra-trace (ppt) analysis of water samples, beverages, diary products, biological fluids, etc. A recent review by F. David and P. Sandra describes the applications of SBSE for environmental analysis, food analysis, and biomedical and life science applications [81]. The performance of SBSE in combination with liquid extraction and LC-MS analysis has been compared to SPME for the determination of organophosporus insecticides in honey [82] and to SPE for fungicide residues in grapes [83]. Two typical examples are discussed, namely elucidation of endocrine disrupting chemicals (EDCs) in cheap red wines and the analysis of benzoic acid in yoghurt. In a cheap red wine sample some EDCs were detected by SBSE-CGC-MS. The question was from where did these EDCs originate? The red wine sample was closed by a plastic stopper and 10 mg of the stopper was directly analysed by thermal desorption-CGC-MS analysis (Figure 10). The main peak is diisobutylphthalate (DiBP) (peak 5). The other compounds are the nonylphenols (peak group 4), benzoic acid (peak 2) and the typical wine components ethyl
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Abundance 2 1 5
2x107 3 1x10
4
7
10
15 Time (min)
20
Figure 10 TD-CGC-MS analysis of 50 mg of a plastic stopper. A 10-mg sample of the stopper was thermally desorbed at 2501C and the released volatiles were analysed by capillary GC-MS on a 30-m L 0.25-mm ID 0.25 mm df HP5-MS. The MS was operated in the scan mode and ion m/z 149 was selected.
Abundance BA 3000000
2000000
1000000
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16
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Time (min)
Figure 11 SBSE analysis of a yoghurt sample on the presence of benzoic acid. Extraction time was 60 min at 1,400 rpm and analysis was performed by capillary GC-MS on a 30-m L 0.25-mm ID 0.25 mm df HP-FFAP (Agilent) column.
octanoate (peak 1) and ethyl decanoate (peak 3) that were adsorbed on the stopper. A yoghurt sample, claimed to be free of preservatives, was diluted 1:3 with distilled water and acidified to pH 2 with 1N hydrochloric acid. SBSE extraction was performed followed by CGC-MS analysis (Figure 11). The concentration of benzoic acid in the yoghurt sample was 28 ppm. Because of a log K(o/w) for benzoic acid of 1.87 maximum recovery is 40% at equilibrium. For quantification, the standard addition method was applied. The limit of quantification (LOQ)
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(at signal-to-noise level S/N10) was 8 ppb in the scan mode and 0.5 ppb in the ion-monitoring mode.
3.2.6 Selected applications 3.2.6.1 Multi-residue screening of pesticides in vegetables, fruit and baby food applying SBSE. An MRM has been described based on SBSE followed by capillary GC-MS using retention-locked conditions [4]. The method provides detection from the mg/kg (ppm) to the sub mg/kg (ppb) level hereby complying with the MRL set by regulatory organizations for pesticides in different matrices. A schematic overview of the original method is presented in Figure 12. Approximately 15 g of a vegetable, fruit or baby food sample is accurately weighed in a 100-mL flask and 30 mL of methanol is added. The mixture is homogenized using an Ultra Turrax mixer for 5 min and the flask is placed in an ultrasonic bath for 15 min. A fraction (approximately 10 mL) of the blend is placed in a closed 20-mL vial and centrifuged for 5 min at 5,000 rpm. One milliliter of the supernatant methanol phase is put in a 20-mL headspace vial and 10 mL of water is added. A stir bar 10 mm long coated with a 0.5 mm PDMS layer is added and the mixture is stirred for 60 min at 1,000 rpm. After sampling, the stir bar is taken out with tweezers, dipped in distilled water, placed on lint-free tissue to remove residual droplets and finally put in a liner of a thermal desorption system. For quantification, 5 mL of appropriate pesticide standard solutions in methanol are added to the sample before Ultra Turrax mixing and ultrasonic treatment. Analysis is performed by retention time locked (RTL) GC-MS using automatic RTL screener software in combination with the Agilent RTL pesticide library. Modifications can be done according to specific needs in different steps of the procedure. For example, sample extraction can be done with acetonitrile or after pH adjustment for acidic or basic pesticides; new developments in GC and MS 15 g Sample + 30 mL MeOH
Ultraturrax + Ultrason (15 min)
1 mL Extract + 10 mL Water
SBSE (60 min)
TD-RTL-CGC-MS
Figure 12 Flow diagram of the multiresidue SBSE method for pesticides in fruit and vegetables.
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software, i.e., the deconvolution reporting software (DRS) can be implemented in the procedure, etc. Figure 13A shows the recorded total ion chromatogram of the SBSE-TDcapillary GC-MS analysis of a lettuce sample. The main peaks (1–3) correspond to Abundance
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Abundance -4
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1. Vinclozolin (175 ppb) 2. Toclofos-methyl (17 ppb) 3. Procymidone (249 ppb)
2 Reproducibility (n=6) Vinclozolin: RSD = 5.4 % Toclofos-methyl: RSD = 8.8 % Procymidone: RSD = 4.6 %
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Figure 13 Total ion chromatogram of the SBSE extract of a lettuce sample (A) and EIC (B) using the RTL and MS libraries. Splitless thermal desorption was performed by heating the tube from 401C to 2801C (5 min) at a rate of 601C/min. The analytes were cryo-focused in a PTV at –1501C with liquid nitrogen prior to injection. An empty baffled liner was used in the PTV injector. For splitless injection (2 min) the PTV was ramped from –1501C to 2801C (2 min) at a rate of 6001C/min. Capillary GC analysis was done on a 30-m L 250-mm I.D., 0.25 mm df HP-5MS column (Agilent Technologies). The oven was programmed from 701C (2 min) at 251C/min to 1501C, at 31C/min to 2001C and finally at 81C/min to 3001C. This is the temperature program required for the RTL screener option (Agilent Technologies). Helium was used as carrier gas. The head pressure was calculated, using the RTL software, so that p,pu-DDT was eluting at a constant retention time of 26.98 min. An Agilent 5973 MSD was used in the scan mode (m/z 40–500). Reprinted from Ref. [4] with permission from Elsevier.
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C16–C18 fatty acids. Detection and identification of trace levels of pesticides in this complex profile can be very time-consuming and laborious. The presence of pesticides is elucidated automatically via the RTL screener software in combination with the RTL-MS library for pesticides and endocrine disruptors selecting four qualifier ions for positive identification (presently with the DRS software the full spectrum is taken into account). Toclofos-methyl, vinclozolin and procymidone were detected by the RTL in the lettuce sample. The EIC at m/z 212, 265 and 283 for vinclozolin, tolclofos-methyl and procymidone, respectively, is shown in Figure 13B. These pesticides have thus positively been identified in the lettuce sample and only now can MRM quantification be performed. There are different ways to quantify accurately positive findings. Conventional methods in food analysis are single calibration with a standard the concentration of which is close to the estimated concentration and prepared in a blank matrix to compensate for matrix effects, internal standard addition of D- or C13-labelled pesticides and standard addition at five or six concentration levels. The first method requires a blank sample to compensate for matrix effects but as strange as this may look such samples are not readily available. The second approach cannot be applied in an MRM because labelled standards of only a few pesticides are commercially available. The last method is by far the easiest to use in a routine environment. However, this method is time-consuming and thus costly. Precise quantification in fact is only needed when the detected quantity is expected to exceed the maximum allowable level. MRL in foodstuffs, with the exception of baby food, are relatively high and with semi-quantitative methods elucidation of negative, i.e., far below the MRL, and positive samples, i.e., concentration around the MRL values, can easily be made. Only accurate quantification is needed for positive samples. For the lettuce sample, quantification was made by standard addition at six levels and the correlation coefficients were all higher than 0.99. Vinclozolin, tolclofos-methyl and procymidone were quantified at 175, 17 and 249 mg/kg, respectively. These are mean-values of six complete analyses (n ¼ 6) and the RSDs were 5.4%, 8.8% and 4.6%, respectively. The residue levels are far below the MRL of the European Community, i.e., 5 mg/kg for vinclozolin and procymidone and, 0.5 mg/kg for tolclofos-methyl in lettuce and in fact accurate quantitation was not required because the lettuce sample could be considered negative taking the abundances of the MS-trace into account. MRLs in baby food become more and more stringent and ultra-trace level analysis (mg/kg and sub mg/kg) is required. Baby food is a more complex matrix because, besides vegetables or fruits, small quantities of meat and thus fat are also present. This affects the efficiency of the pre-extraction in methanol and the SBSE recovery. Standard addition calibration is the only valid alternative for baby food. As illustration, the analysis of a baby food containing vegetables, complete rice and chicken using SBSE-TD-GC-MS is presented. Preliminary screening was performed with the MSD in the scan mode, hereby elucidating the presence of small traces of piperonyl butoxide. For accurate quantitation, the MSD was used in the SIM mode at m/z 176. Six sub-samples of the baby food
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Abundance 120
Non-spiked 1
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19 Time (min)
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Figure 14 SBSE extract of baby food contaminated with piperonyl butoxide (for conditions, see Figure 13). Reprinted from Ref. [4] with permission from Elsevier.
sample were spiked with a piperonyl butoxide standard in methanol at concentrations of 0, 2, 5, 10, 20 and 50 mg/kg. Figure 14 shows the selected ion chromatogram at m/z 176 of the unspiked sample (A) and of the sample spiked at 2 mg/kg piperonyl butoxide (B). The correlation coefficient of the standard addition curve was R2W0.99 and the concentration in the sample was calculated at 1.1 mg/kg.
3.2.6.2 Determination of iprodione in white wines using SBSE-liquid desorptionLC/MS. The pesticide iprodione partially decomposes to 3,5-dichlorophenyl hydantoin during GC analysis. LC-MS is therefore the method of choice [84]. For its determination in wine samples, SBSE was combined with liquid desorption followed by LC-atmospheric pressure chemical ionization mass spectroscopy. Undiluted wine (10 mL) was poured into a headspace vial of 20 mL and a twister containing 25 mg PDMS was stirred in the sample for 40 min at a speed of 1,400 rpm. The stir bar was extracted with 1 mL acetonitrile in an ultrasonic bath for 15 min. Five microliter of the extract was injected for LC-MS analysis. Figure 15 shows the analysis for an Italian sparkling wine. The calculated concentration of iprodione via standard addition was 98 mg/L (RSD 4.6% triplicate analysis).
4. FRACTIONATION AND CLEAN-UP Solvent distribution and LC (or its derivative SPE) are commonly applied to further fractionate or clean-up a solvent extract.
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Abundance Iprodione (98 ppb) 200000
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0
0.5
1
1.5 Time (min)
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Figure 15 LC–MS analysis of the liquid extract of SBSE of a wine sample. LC–APCI–MS analyses were carried out on a benchtop HP1100 Series LC–MSD instrument (Agilent Technologies, Waldbronn). A Phenomenex Luna C 18 column, 250-m L 4.6-mm I.D., 5 mm particle size was used. The mobile phase consisted of water (solvent A) and 10% tetrahydrofuran in methanol (solvent B). A gradient from 70% B at 0 min to 80% B at 20 min was applied. The flow-rate was 1 mL/min and the analyses were performed at 221C. APCI was carried out in the negative mode at a mass range between m/z 200–350. The fragmentor voltage was set to 70 V. The nitrogen drying gas was at 3501C with a flow-rate of 5 L/min. The nebulizer pressure was 60 psig. The capillary voltage was 4,000 V and the corona current was 25 mA. Analyses in the SIM mode for iprodione were carried out at m/z 242.9, 245.0 and 246.8.
4.1 Chemical methods In solvent distribution the principle of ‘‘like likes like’’ is applied and the partitioning is based on polarity or selective interactions. As an example, the separation of apolar/medium polar and polar compounds can be performed by distribution in a hexane–methanol/water (95:5) mixture. Acidic compounds such as phenols can be extracted with 6% NaHCO3 while alkaline compounds can be recovered with 5% HCl. Alcoholic contaminants can be fractionated via an isocyanate reaction while solutes with an aldehyde or ketone function can be fractionated with a Schiff base like phenylhydrazine. The chemical methods are more and more replaced by chromatographic methods.
4.2 Chromatographic methods The different modes of LC are used to further fractionate or clean-up samples after solvent extraction. The separation mechanism can be based on polarity (normal phase LC), on hydrophobicity (reversed phase LC), on the ionic character of the solutes (ion exchange LC), on their shape (graphitized carbon elution) or on their size (size exclusion or GPC). Low pressure LC, better known as column chromatography, high pressure LC or SPE are all used depending on the aim of the clean-up and the complexity of the sample. Column chromatography is somewhat old-fashioned and large amounts of (toxic) solvents have to be used to elute the solutes of interest. Moreover, concentration is very time-consuming and
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Fat
GPC on Biobeads SX-3 (to remove fat)
Alumina (to remove apolar compounds)
Graphitized Carbon (to remove the PCBs)
Analysis CGC-High Resoltion MS
Figure 16 Flow diagram for the fractionation and clean-up of dioxins and furans from fat samples.
prone to artifacts. HPLC is the method of choice when the fractionation is critical and high resolution is needed in this step. SPE is the best method for highthroughput analysis using minimum amounts of solvents. Frequently employed adsorbents are silica gel, alumina, Florisil and graphitized carbon. A scheme for the clean-up of dioxins (PCDDs) and furans (PCDFs) from fats is shown in Figure 16. Presently, automated sample preparation systems are available for the enrichment of PCDDs/PCDFs and PCBs in a single step. Automated and on-line systems will be presented in Section 4.4. A technique with excellent features for the fractionation of contaminants and pesticide residues from fatty food samples is size exclusion chromatography (SEC) or GPC.
4.3 Gel permeation chromatography For fatty food matrices, the main problem is the co-extraction of fats. The amount of fat extracted strongly depends on the nature of the target solutes and the solvent to be selected to efficiently extract them. For example, the determination of PCBs in meat requires extraction with an apolar solvent like cyclohexane that will solubilize the fat completely while extraction of phenylurea pesticides can be performed with acetonitrile, only partly solubilizing the fat. When the fat content is relatively low, but still disturbing chromatographic analysis, clean-up by SPE on octadecylsilica can be applied. Lipid traces are strongly retained on ODS and do not elute even with pure methanol. For high lipid concentrations and/or when the polarity of the target solutes is the same as of the lipids (phthalates in edible oils), GPC is an excellent technique. In GPC, compounds are separated according to the molecular volume (E molecular mass). Larger molecules cannot
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enter the pores of the GPC packing material (polystyrenedivinylbenzene) and elute before smaller molecules that can enter the small pores. Already in the 1980s, low pressure GPC was used for the fractionation of pesticides from lipid matrices [85,86]. Classical GPC separations are performed on 40-cm L 25-mm ID low pressure columns packed with Biobeads SX-3 and operated at 5 mL/min. A similar set-up was recently used by Pang et al. [63] in a validation study on 660 pesticide residues in animal tissues. In this method, 10 g animal samples were mixed with 20 g sodium sulphate and extracted with 35 mL cyclohexane/ethyl acetate (1:1) twice by blender homogenization, centrifugation and filtration. After evaporation, the extract was injected on a S-X3 GPC column (40-cm L 25-mm ID) with cyclohexane/ethyl acetate (1:1) as mobile phase at 5 mL/min. The 22–40 min fraction was collected for subsequent analysis by GC-MS and LC-tandem MS. GPC can be miniaturized and solvent consumption largely reduced by using HPLC equipment and high-pressure GPC columns. Automated GPC clean-up can be performed using a system consisting of an isocratic HPLC pump, an autosampler allowing the injection of 500 mL fat extract, a temperature controlled column oven, a variable UV or RI detector and a fraction collector. In our laboratory, GPC is performed on small bore columns (e.g., 300-mm L 7.5-mm ID) packed with PL-Gel with 5 mm particles having 5 nm pore size and operated at 1-mL/min dichloromethane. The 5-nm pore size is important since it gives the highest resolution and the largest elution window for compounds in the 100–1,000 Da mass range. The resolution power is further increased by using two columns in series (60-cm L). Solvent consumption is approximately 5–10 times lower in comparison to the classical GPC method. The method is illustrated with the analysis of palm oil spiked at the 10-mg/kg level with some PAHs. A 50-mg sample of the palm oil was diluted with 1 mL dichloromethane and 500 mL was injected on a 60-cm L 7.5-mm ID PL-Gel column. Figure 17A shows the GPC chromatogram of the spiked palm oil superimposed by the EPA GPC evaluation mixture consisting of corn oil (peak 1), diethylhexylphthalate (DEHP) (peak 2), methoxychlor (peak 3), perylene (peak 4) and sulphur (peak 5). The fraction eluting between 18 and 21 min (3 mL) was evaporated with 100 mL isooctane as keeper and 1 mL was analysed by GC-MS on a 30-m L 0.25-mm ID 0.25 mm df HP-5MS. Figure 17B shows the chromatograms obtained by single ionmonitoring for some high molecular mass PAHs and ppb level determinations are possible.
4.4 On-line techniques The urgency to increase the capacity of analytical laboratories and to reduce manual labour has resulted in the construction of sample preparation stations for high-throughput analysis. Automated sample preparation is often accompanied with more reliable and rugged data compared to manual sample preparation methods. Well known is the ‘‘dioxin street’’ in which ASE, a ‘‘Power-Prep’’ workstation (Fluid Management Systems, Waltham, MA, USA), a solvent evaporation unit and a GC-HRMS are implemented. In the automated prep
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Figure 17 GPC chromatogram of the EPA standard (dashed line) and a palm oil sample spiked with PAHs (A) and GC-MS analysis of the collected fraction (B). A sample of 1 mL was analysed on a 30-m L 0.25-mm ID 0.25 mm df HP-5MS using single ion-monitoring for some high molecular mass PAHs.
station system, a multi-step SPE procedure based on well-established sets of adsorbents such as acidic, basic and neutral silica, basic alumina, Florisil and graphitised carbon black are combined depending on the target analytes. Nowadays, laboratories performing dioxin analysis of fatty food samples must also produce PCB data and the use of a single sample preparation step for PCDFs/PCDDs and PCBs has been described [87–89].
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Fully automated on-line LC-GC systems for fractionation and clean-up have been described [90]. The systems can be based on large volume injection via a cool on-column injector or a PTV injector. The first system allows the enrichment of both volatiles and semi-volatiles, while the second is restricted to the analysis of semi-volatiles. De Paoli et al. [91] constructed an on-line GPC-normal phase LC-GC-FPD system for the determination of organophosphorus pesticides in fruit. Hyotylainen et al. [92] described an on-line microporous membrane LLEGC-MS system for the determination of pesticides in red wines. Jongenotter and Janssen [93] applied on-line GPC-GC-FPD for the determination of organophosphorus pesticides in edible oil. A similar system using MS as detection was used by Liu et al. [94] for measuring multi-residual pesticides in agricultural products. Pesticides were extracted from homogenised food samples with acetonitrile and decontaminated via DSPE with PSAm as sorbent. The on-line LC-PTV-GC-NPD system used in our laboratory for the determination of pesticides in essential oils [95] is presented in Section 4.5.
4.5 Selected applications 4.5.1 Analysis of pesticides in orange oil by on-line LC-GC The principle of LC-PTV-GC-NPD is illustrated with the determination of ethion and chlorpyriphos in orange oil used to prepare soft drinks. The essential oil is fractionated by normal phase LC, resulting in a separation according to polarity and the fraction containing the pesticides and leaving the LC detector is directed via a capillary tube with well-defined dimensions, in a T-shaped flow cell. The cell is equipped with a septumless sampling head through which a syringe needle can be introduced. The sampler is completely computer controlled. To transfer a selected LC fraction, the transfer start and stop times, measured on the LC chromatogram, are introduced in the controller software. The time delay between the LC detector and the flow cell is automatically calculated from the connecting tubing dimensions and the LC flow rate. At the time the LC fraction of interest passes through the flow cell, the syringe needle penetrates the septumless head and samples the LC fraction at a speed equal to the LC flow rate. Volumes up to 2 mL can be collected in the syringe. After collection, the needle is withdrawn from the flow cell and a large volume injection is made using the PTV in the solvent vent mode. Depending on the fraction volume and solvent type, the sample introduction parameters (inlet temperature, vent flow, vent time, injection speed, etc.) are calculated by the PTV calculator program. The LC profile for the orange essential is shown in Figure 18A. The apolar mono- and sesquiterpenes elute first, followed by the terpenoids and after 16 min the flavanoids also elute. These last compounds especially give unwanted interferences in direct GC analysis because they have similar molecular weights as the pesticides. Using the same conditions, ethion and chlorpyriphos standards eluted at 4.6–4.7 min. The fraction eluting from 4.4 to 4.9 min (volume ¼ 0.5 mL) was automatically transferred to the GC inlet. The LC-GC interface was programmed to take the sample at a 1,000 mL/min sampling speed (the same as the LC flow rate). The complete fraction of 500 mL was injected in the PTV inlet
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Figure 18 On-line NPLC-CGC-NPD for the characterization of chlorpyriphos and ethion in orange oil, (A) NPLC analysis, (B) capillary GC-NPD analysis. HPLC parameters used: column 250-mm L 4.6-mm ID 10 mm dp Lichrospher 100 DIOL, injection volume 20 mL, mobile phase in a gradient from 100% hexane for 10 min, to 40% isopropanol at 20 min (2 min hold) at a flow rate of 1 mL/min and UV detection at 220 nm. Capillary GC-NPD analysis was performed on a 30-m L 0.25-mm ID 0.25 mm df HP-5MS column and the oven was programmed from 701C (2 min) to 1501C at 251C/min and then to 2801C at 81C/min (10 min). The detector was set at 3201C with 3 mL/min hydrogen, 80 mL/min air and 30 mL/min helium make-up flow.
at 250 mL/min. This injection speed corresponds to the injection speed calculated by the PTV software program. Figure 18B shows the resulting GC-NPD chromatogram and both ethion and chlorpyriphos are detected. The chromatograms are very clean and no interferences are present. This demonstrates the excellent selectivity of the LC-GC combination. The concentrations of ethion and chlorpyriphos were calculated by external standard analysis and are 1.9 ppm for chlorpyriphos and 0.8 ppm for ethion.
4.5.2 Determination of phthalates in milk and edible oils by off-line GPC-GC [96] Approximately 5 g of milk samples or milk powders dissolved in water (1g/10 mL) are extracted on a shaking machine for 30 min with 20 mL of a 1:1 cyclohexane/acetone mixture in a 40-mL vial. After completion, the vials are
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centrifuged and the supernatant is transferred to a pre-weighed vial. The solvent is evaporated under nitrogen and the fat content is measured. The residue is dissolved in dichloromethane and the internal standard (d4-DEHP) is added. The amounts of solvent and internal standard are adjusted to give approximately 50 mg fat and 100 ng internal standard per mL dichloromethane. The solution is then homogenised in a vortex agitator. For the determination of phthalates in edible oils and fat, the oil or fat sample can be diluted directly in dichloromethane to a concentration of 50 mg fat per mL. For GPC separation of the dichloromethane solution, 500 mL is injected onto a column combination consisting of 5-cm L 7.5-mm ID PL-Gel pre-column and two 30 cm 7.5-mm ID 5 mm PL-Gel 5 nm columns. The mobile phase is dichloromethane at 1 mL/min flow rate. UV detection at 220 nm is used to monitor the effluent. Phthalates typically elute in a window between 20 and 23 min (3 mL fraction). This fraction is automatically collected and dichloromethane is evaporated under a nitrogen stream and the residue is redissolved in 100 mL isooctane. A 1-mL sample of the extract was analysed by capillary GC-MS. The limit of quantification for milk samples with 3% fat content is 1 ng/g for DiBP and dibutylphthalate (DBP) and 3 ng/g for DEHP. The recovery of the GPC clean-up method was tested by spiking an olive oil sample at a 500 ppb level with dimethylphthalate (DMP), diethylphthalate (DEP), DiBP, DBP, butylbenzylphthalate (BBzP) and DEHP, and at a 5-ppm level with diisononylphthalate (DiNP) and diisodecylphthalate (DiDP). The olive oil was tested before and no phthalates were detected at concentrations above 50 ppb (500 ppb for DiNP and DiDP). The results from the non-spiked oil were therefore considered as the ‘‘procedure blanks’’. d4-DEHP (internal standard) was added to the dichloromethane solution. The sample was analysed in triplicate. The linearity was tested by spiking an olive oil sample at 8 levels (100, 250, 500, 1,000, 2,500, 5,000 and 7,500 ng/g fat) ( 10 for mixed isomers). The mean recovery, standard deviation (RSD%) and linearity (correlation coefficient r2) are listed in Table 7. In general, good recoveries (W80%) are obtained, except for butylbenzylphthalate where the recovery is lower. This is due to a slightly different behaviour of this compound in sample clean-up (GPC). The correlation coefficients are better than 0.99 for all phthalates, except for DiNP (0.98). This correlation shows that the method can be used in the concentration range from 100 to 7,500 ppb for the single isomer phthalates and from 1 to 75 ppm for the mixed isomer phthalates. The major problem in phthalate analysis is the contamination problem, resulting in false positive results or over-estimated concentrations. The risk of contamination is present in the whole analytical scheme, including sampling, Table 7
Validation data for the determination of phthalates in olive oil
Recovery (%) RSD (%) Linearity (r2)
DMP
DEP
DiBP
DBP
BBzP
DEHP
DiNP
DiDP
103 15.7 0.996
134 15.7 0.995
104 9.5 0.999
138 11.8 0.996
69 16.0 0.995
127 12.1 0.998
85 11.6 0.984
81 10.6 0.990
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sample preparation and chromatographic analysis. Due to the fact that phthalates are widely used, they are present in air, in water and organic solvents, in plastics and adsorbed on glass or other materials. Running continuous blanks is the only solution to this problem.
5. EVAPORATION Liquid extraction or desorption techniques often needs solvent evaporation to concentrate the target solutes. Solvent evaporation can only be applied for concentrating semi-volatiles and even then care must be taken or solutes of interest will be lost. Moreover contamination can occur and impurities in the extracting solvent are enriched hence the need for ultra-pure solvents. Most common approaches for solvent evaporation are gas blow-down, rotary evaporation, Kuderna-Danish evaporative concentration, automated evaporative concentration [97], and TurboVap concentration (Zymark Corporation, Hopkinton, MA, USA). The loss of solutes can partly be overcome by adding a keeper, i.e., a small amount of an organic solvent of the same polarity as the extracting solvent but with a higher boiling point, e.g., isooctane in n-hexane or cyclohexane.
6. DERIVATIZATION For single residue and selective residue determinations, derivatization can be useful to improve the chromatographic analysis as well as the extraction yield and to decrease detection. A typical example is the analysis of amitrol [98]. Derivatization with hexylchloroformate introduces hydrophobicity in amitrol and the derivative can be extracted by SPE on octadecyl silica, analysed by reversed phase LC and, moreover, the derivative shows better ionization in MS compared to the original molecule. Another example of derivatization in LC was presented in Section 3.1.6.2 for the analysis of nitrofurans. Derivatization in GC is most often applied to ‘‘cap’’ the polar groups by substituting the active proton so that volatility increases and irreversible or reversible adsorption into the column is avoided. However, derivatization is more and more applied in sorptive extraction techniques to increase the recoveries. For example, chlorophenols are derivatized into the acyl compounds by adding a base like potassium carbonate to the solution followed by acetic acid anhydride. The acyl derivatives possess higher affinities for the extraction phase in headspace and direct emerging SPME and SBSE. Figure 19 shows the analysis of a water sample spiked at the 10-ng/L level (10 ppt) with monochlorophenols (ion m/z 128), dichlorophenols (ion m/z 164), trichlorophenols (ion m/z 196), tetrachlorophenols (ion m/z 232) and pentachlorophenol (ion m/z 266). Extraction was performed by emerging SBSE using a 10 mm 0.5 mm df PDMS stir bar on a 10-mL sample after addition of 0.5 g K2CO3 and 0.5 mL acetic acid anhydride. Extraction conditions were 45 min stirring speed at 1,000 rpm and room temperature. The derivatives were
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Abundance
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TCP 3000 TeCP 2000
1000
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16 18 Time (min)
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Figure 19 SBSE analysis of chlorophenols in drinking water after in-situ acylation. The derivatives were thermally desorbed in the splitless mode and analyzed on a 30-m L 0.25mm ID, 0.25 mm df HP5-MS column using MS detection in the ion-monitoring mode (for other conditions, see Figure 13). Reprinted from Ref [81] with permission from Elsevier.
thermally desorbed in the splitless mode and analyzed on a 30-m L 0.25-mm ID, 0.25 mm df HP5-MS column using MS detection in the ion-monitoring mode. SBSE was also applied for the determination of pyrimethanil, chlorothalonil, vinclozolin and chlorfenvinphos in honey samples [81]. Sample preparation was only dilution of 1 g honey with water. Campillo et al. applied a similar derivatization method for the headspace SPME analysis of chlorophenols in honey samples [99]. Numerous derivatization reactions can be applied and for more information we refer to the book by Blau and Hacket [100]. It is important to note that nowadays, derivatization can be fully automated and often placed on-line with the chromatographic analysis.
7. CONCLUSION Notwithstanding the tremendous power of state-of-the-art analytical instrumentation, and especially the recent progress made in MS, sample preparation is still a key step to ensure the validity of the measurements. Increasing demand for high-throughput (faster and simpler) determinations of contaminants and residues has seen the introduction of many new sample preparation techniques. Much has been published on sample preparation in recent literature and a considerable database of applications has evolved. The methods used in routine laboratories are often the ones dictated by regulatory organizations and implementation of new developments can be a slow process because of validation and robustness issues. In the era of green
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chemistry, an argument to speed up the application of recent methods is the drastically reduced solvent consumption making them environmentally friendly. An extremely important aspect is quantification. The degree of accuracy and precision will always remain a function of the number of targets to be determined. Single residue methods and sMRMs provide more accurate results than MRMs. For single residue methods and sMRMs, often labelled standards are available that can be added to the sample before or during the sample preparation steps. Examples are the determination of chlormequat in fruit and vegetables, of CAP in honey, of dioxins in milk, of nitrofurans in fish. In MRMs compromises have to be made but this does not automatically mean that the obtained data are less important. In the first instance, a semi-quantitative idea is obtained allowing one to decide whether or not precise quantification is needed. When the concentration is far below the MRL value further work in this respect is a waste of time. The main advantage of MRM is that a broad range of contaminants and residues is screened and when positive elucidation, both in terms of nature and close to the MRL level, is obtained accurate quantification can be made by standard addition.
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CHAPT ER
6 Recent Developments in Chromatographic Techniques Katerina Mastovska
Contents
1. Introduction 2. Fast Chromatographic Separation 2.1 Fast gas chromatography 2.2 Fast liquid chromatography 3. Two-Dimensional Chromatographic Separation 3.1 Comprehensive two-dimensional gas chromatography 4. Overcoming Adverse Matrix Effects 4.1 Approaches to overcome matrix effects in gas chromatography 4.2 Approaches to overcome matrix effects in liquid chromatography-mass spectrometry 5. Future Trends References
175 176 178 184 188 189 191 192 194 196 197
1. INTRODUCTION Chromatographic techniques play a significant role in the determination of analytes in complex matrices, separating individual sample components before their detection [1]. In the analysis of contaminants and chemical residues in food, gas chromatography (GC) and liquid chromatography (LC) are two main chromatographic methods employed in practice. Other chromatographic techniques, such as thin-layer chromatography (TLC) [2], supercritical fluid chromatography (SFC) [3], or capillary electrochromatography (CEC) [4], may be suited for some special applications but did not find a wider use in routine analysis of food contaminants and residues. GC is a suitable technique for thermally stable compounds that can be readily volatilized. This includes mainly halogenated contaminants, such as polychlorinated dibenzo-p-dioxins — PCDDs, polychlorinated dibenzofurans — PCDFs,
Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00006-8
r 2008 Elsevier B.V. All rights reserved.
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polychlorobiphenyls — PCBs, polybrominated diphenylethers (PBDEs), semivolatile pesticides, polycyclic aromatic hydrocarbons (PAHs), or phthalates. A group of LC-amenable contaminants and residues comprises polar, thermally labile and/or larger molecules that cannot be easily volatilized, such as the majority of veterinary drug residues, mycotoxins and other toxins, polar pesticides or large PAHs. As compared to LC, GC generally provides more separation efficiency. Also, GC has been traditionally combined with more selective detectors for contaminant and residue analysis. In particular, the well-established conjunction of GC with mass spectrometric (MS) detection and availability of relatively affordable benchtop GC-MS instruments made GC a number one choice for multicomponent contaminant and residue analysis, even though the polar analytes had to be derivatized before the GC step. In recent years, the situation has dramatically changed thanks to the advances in LC-MS, which brought this powerful technique to routine laboratories and opened the door to direct, selective and sensitive analysis of polar compounds. For instance, LC-MS is gradually replacing immunochemical and microbial methods in the analysis of veterinary drugs [5]. Also, LC-MS is competing with GC-MS for the status of a reference technique in the multiclass, multiresidue analysis of pesticides [6,7]. In fact, many pesticide residues are not directly amenable for GC and their permanently increasing number reflects a trend in pesticide product development, i.e., transition from the use of persistent and less polar compounds to more readily degradable, more (sometimes very) polar, low volatile and/or thermolabile pesticides. Determination of these ‘‘modern’’ pesticides and their metabolites was rather difficult in the past [8]. GC- and LC-MS(-MS) provides several advantages over the use of other GC or LC detectors, which mainly include simultaneous quantitation and structural identification of detected analytes, detection of a wide range of compounds independent of their elemental composition or structure, and possibility to spectrometrically resolve co-eluting peaks. All these features can substantially contribute to the increased sample throughput in the analysis of multiple chemical residues or contaminants, with the latter trait being extremely important in fast chromatographic separations. Thus, the increasing availability, affordability, and performance of GC-MS and LC-MS instruments have led to a growing interest in fast GC and LC separations in recent years. Also, fast separation and detection play an important role in two-dimensional (2D) separations, particularly in comprehensive 2D chromatography. This chapter discusses recent developments in fast and 2D chromatographic techniques and their application in the GC and LC analysis of contaminants and chemical residues in foods. Furthermore, it also reviews current and some novel approaches to overcome matrix effects that may adversely affect quantitative GC and LC-MS analysis of trace components in food matrices.
2. FAST CHROMATOGRAPHIC SEPARATION Faster chromatographic separation reduces the analysis time, which can translate in a significantly increased sample throughput, providing that the entire
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laboratory operation, including sample preparation and data analysis, is streamlined to keep up with the fast GC or LC determinative step. The GC or LC separation time, given as the retention time tR of the last analyte, can be described by L (1) tR ¼ t0 ðk þ 1Þ ¼ ðk þ 1Þ u¯ where t0 is the time required for the elution of an unretained component, k the solute retention (or capacity) factor, L the column length, and u¯ the average linear velocity of the mobile phase. Therefore, the separation time is decreased by reducing L, decreasing k, and/or increasing u¯. In practice, an increase in separation speed is not for free and some sacrifice must be made in chromatographic resolution (Rs) and/or sample capacity (Qs) in most cases. An existing method can firstly be speeded by minimizing Rs (of important peaks) to a value that is just sufficient for our application, employing for instance shorter columns, higher than optimum mobile phase flow rates, faster temperature programming in GC or steeper mobile phase gradients in LC. A reduction of L decreases the number of theoretical plates N (a measure of separation efficiency) in a directly proportional relationship (L ¼ H N, where H is the plate height) but the impact on Rs is less severe because it is proportional to OL. Thus, practically all fast GC and LC methods utilize shorter columns in combination with other approaches. In GC-MS and LC-MS, Rs is not necessarily the limiting factor in speed because MS has the ability to distinguish between analytes that have some differences in their MS spectra. Thus, except for certain applications, such as chiral separations or congener-specific analysis of dioxins and PCBs, MS can resolve co-eluting peaks spectrometrically. As indicated in Figure 1, MS adds another adjustable degree of control in selectivity (and sensitivity), which allows compensation for a potentially worse GC or LC performance in faster separations [9]. If the gain in speed at the minimum acceptable Rs is not sufficient, the analysis time can be
compensation
Selectivity
Selectivity
(Resolution)
GC or LC
Speed MS
Sensitivity
Sensitivity (Sample Capacity)
compensation
Figure 1 The combination of GC or LC with MS for optimization of speed. The sensitivity and selectivity of each approach can be used to compensate for losses in the other to provide a faster analysis of potentially the same quality. Modified with permission from Ref. [9].
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further reduced by shifting the optimum mobile phase velocity (u¯opt) to higher values. Fast GC and LC approaches that can maintain Rs while speeding the analysis are discussed below in greater detail, including the major instrumentation requirements for their practical use. General requirements for instrumentation in fast GC and LC involve the ability to provide and handle the speed. As the speed increases, the peaks become narrower, which poses greater demands on the used instrumentation. Extracolumn contributions to band broadening (in the injector, connections, or detector) may become of critical importance in limiting the potentially available column performance. Also, the detector must be able to record the narrower peaks with an acceptable precision, providing reproducible quantitation and identification characteristics.
2.1 Fast gas chromatography Table 1 lists possible approaches to fast GC derived from the GC theory [9–14] and includes practical considerations associated with their application [10], indicating mainly specific instrument requirements and sacrifices made in Rs and/or Qs. In terms of the speed, modern, commercially available GC instruments offer sample introduction, pressure control, temperature programming, and data acquisition rates sufficient for fast GC with typical separation times of 1–10 min and peak widths (full-width at half-maximum, FWHM) of 0.2–1 s [15]. For very fast and ultra-fast GC (separation times in the range of less than 1 or 0.1 min, respectively), specially designed or exceptional instrumentation is mostly needed (mainly for sample introduction and pressure regulation), thus the application of these approaches in practice is currently very limited [10]. As mentioned earlier, the easiest way to speed an existing method involves minimizing Rs to the extent that is still acceptable for a given purpose. In GC, this can be achieved by employing for instance higher than optimum carrier gas velocities, faster temperature programming rates and/or shorter capillary columns. These simple approaches are often combined with other fast GC techniques to provide an additional gain in speed. Fast temperature programming can be accomplished with conventional GC ovens [16,17], by resistive heating [17–23], or less intensively studied microwave GC ovens [24,25]. Modern oven-based GC instruments provide maximum temperature programming rates of 1–21C/s, but these maximum declared rates for a given instrument are reached only at relatively low temperatures. As the temperature increases, heat losses from the oven to the surrounding air also increase, which may lead to larger time lags between the actual and set column temperatures and less reproducible heating at faster temperature programming rates, resulting in longer analysis times vs. the expected ones and decreased precision of tR measurements [17,26]. In resistive heating, electrical current is employed to heat a conductive material (a metal) that encases the analytical column, thus the thermal mass of the heater is minimized and the heat-up and cool-down rates can be very fast and precise. Commercial systems are available, in which a fused silica capillary
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Table 1 Possible approaches to faster GC separation and practical considerations associated with their application (see the chapter text for the symbol definitions) Possible approaches to faster GC separation
Practical considerations
(A) kL, reduced column length
Decreased resolution (Rs p OL)
(B) kk, decreased retention factor
Decreased resolution (Rs p k/(k+1); less significant for k W 3) For isothermal analysis only (restricted to analytes with a narrow boiling-point range) Special instrumentation required for temperature rates W 1–21C/s Higher elution temperatures (potential greater thermal breakdown of susceptible analytes) Limited by stationary phase availability (difficult for a wide range of analytes) Special instrumentation required for a coupled-column operation Reduced sample capacity (Qs p df) and ruggedness Increased sample capacity (Qs p d3c ) and ruggedness
(1) Higher isothermal temperature
(2) Faster temperature programming
(3) Altered stationary phase (of a single column or coupled columns) to improve selectivity (4) Thinner film of the stationary phase (5) Larger diameter capillary column (for fixed column length) (C) mu¯, increased carrier gas velocity (1) u¯ W u¯opt, higher than optimum carrier gas velocity (2)mu¯opt, increased optimum carrier gas velocity (a) Hydrogen as a carrier gas
(b) Vacuum outlet operation (low-pressure GC)
(c) Smaller diameter capillary column (for fixed resolution)
Decreased resolution (H W Hmin, Rs p 1/OH) Instrumentation limited (GC pressure limit, MS pumping capacity and sensitivity) Constant resolution (H ¼ Hmin, Rs ¼ constant) Safety concerns Potential inertness problems Reduced GC inlet pressure requirements Source of vacuum required for operation (inherent to MS) Restriction capillary or special injection technique required for sample introduction into the column kept at subambient pressures Reduced sample capacity (Qs p d3c ) and ruggedness Increased GC inlet pressure requirements
Source: Reprinted with permission from the publisher Elsevier from Ref. [10].
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column is coiled together with a heating wire or inserted into a resistively heated metal tube, achieving temperature programming rates up to 201C/s [17–23]. A practical drawback of these systems is the difficulty in accessing the column to perform routine maintenance. Besides much faster temperature programming rates, the resistive heating technique provides very good tR repeatability and rapid cooling down, which results in higher sample throughput, even if the same temperature programming rate is applied in an oven-based GC as demonstrated in rapid pesticide multiresidue analysis in various food crops [17–19]. Another possible approach to faster GC is to optimize selectivity (ability to distinguish between compounds) with respect to the utilization of time. In GC, the use of one type of stationary phase over another may increase the separation speed only to a small degree. However, a sequential combination of two different GC columns (2D GC) may provide improved or equivalent separation selectivity in a shorter amount of time. Among 2D GC approaches, pressure-tunable (flow modulation) GC-GC developed by Sacks et al. [27–29] is predominantly aimed at analysis time reduction. In this technique (commercially available as stop-flow GC [30]) the carrier gas pressure is programmed at the junction of two, serially coupled columns with different stationary phase chemistries. An increase in the junction point pressure reduces the flow in the first column and accelerates the flow in the second column, resulting in increased residence time in the first column and decreased residence time in the second column. This increases the influence of the stationary phase chemistry of the first column and decreases the influence of the second column (a reduction in the junction point pressure has the opposite effect). Thus, pressure-tunable GC-GC can alter retention patterns by programming the column ensemble selectivity, which can be used to improve the quality of the separation with respect to the utilization of time. A drawback of this approach involves a tedious method development including optimization of pressure programming and selection of suitable columns, especially if complex compound mixtures have to be separated. The GC analysis can be speeded without sacrificing Rs by increasing the diffusivity of the solute in the mobile phase (using hydrogen as a carrier gas or operating the column at lower pressures) or using narrower columns. In addition to the increased u¯opt, a reduction of column inner diameter (dc) of a thin-film capillary column also results in a proportionally decreased H, thus a higher efficiency per L. Therefore to maintain Rs while increasing the analysis speed, the use of a narrower column must be accompanied by a proper reduction in L (L to dc ratio should be kept constant [12]). A significant disadvantage of this approach involves substantial sacrifice in Qs, which is proportional to d3c. Moreover, a thinner film of the stationary phase (df) is often used in combination with narrower columns (to keep the phase ratio constant), resulting in a further decrease in Qs (directly proportional to df). With respect to dc, capillary columns can be divided into several groups [9]: (i) megabores (dcX0.5 mm; commercially available 0.53 mm); (ii) wide bores (0.3pdco0.5 mm; 0.32 and 0.45 mm); (iii) narrow bores (0.2pdco0.3 mm; 0.20, 0.25, and 0.28 mm); (iv) microbores (0.1pdco0.2 mm; 0.10, 0.15, and 0.18 mm); and (v) sub-microbores (dco0.1 mm; various dc commercially available). Table 2
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Table 2 Relative speed, Qs and column head pressure (p) calculated for different dc at the same separation efficiency (L/dc ¼ 100,000) and phase ratio (dc/df ¼ 1,000) using He carrier gas at speed-optimized flow rate (SOF) and 1001C column temperature (see the chapter text for the symbol definitions) dc (mm)
L (m)
SOF (mL/min)
t0 (min)
Relative speed
Relative Qs
p (psi)
0.53 0.32 0.25 0.20 0.10 0.05
53 32 25 20 10 5
4.24 2.56 2.0 1.6 0.8 0.4
2.72 1.25 0.89 0.67 0.30 0.15
1 2.18 3.05 4.06 8.97 8.5
100 22 10.5 5.4 0.67 0.084
6.7 14.9 21.3 28.9 68.7 150.2
Source: Modified with permission from Ref. [12].
compares relative speeds that can be achieved for columns of different dc at the same separation efficiency and phase ratio. This table also demonstrates the sacrifice in Qs (hence sensitivity) that has to be made for an increase in speed using microbore or sub-microbore columns. For instance, as compared to a standard 0.25 mm i.d. column, 3- and 6-fold gains in speed are obtained at the cost of 16- and 125-fold lower Qs in the cases of 0.10 and 0.05 mm i.d. columns, respectively. In this respect, short megabore columns may offer a viable alternative to microbore columns in fast analysis of trace components, especially when combined with MS to compensate for decreased separation efficiency.
2.1.1 Fast gas chromatography with short microbore columns The use of short microbore columns represents an attractive approach to fast GC but several practical aspects have to be considered, especially in routine analysis of trace components in complex matrices, such as in food samples. Important concerns associated with application of columns with a smaller dc include pressure requirements necessary for their operation, injection difficulties, analyte detectability, and method ruggedness [12,31–33]. Table 2 shows increasing pressure requirements necessary for operation of columns of a smaller dc. Most common GC instruments allow maximum column head pressure of 100 psi (690 kPa), some provide 150 psi. Thus, the calculated head pressure value for a 0.05-mm i.d., 5 m long capillary column (at 1001C, He as a carrier gas) is at the limit of the commercially available GC instruments at this time. The use of H2 instead of He can lower the pressure requirements. For instance, if H2 was used instead of He at the same flow, the column head pressure for a 0.05-mm i.d. column in Table 2 would drop from 150 to 102 psi. However, the operation of the sub-microbore columns is restricted to low flow rates and temperatures even if H2 is employed as a carrier gas. Thus currently, the use of microbore columns (dcX0.1) seems to represent a practical limit for common GC instrumentation.
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Injection is a crucial step in fast GC because the introduced sample vapor plug has to be sufficiently narrow to minimize the input band width. Split injection is generally favored over non-splitting injection techniques because of its speed, which is however achieved at the expense of reduced sensitivity, especially in the case of very high split ratios. In splitless injection, only the column flow is utilized for sample transfer from the injector liner to the column, thus effective refocusing of the initial band is required. In columns with a smaller dc, refocusing becomes more difficult because the length of the flooded zone increases with decreasing dc. Therefore, cryofocusing or coupling with a short retention gap of a larger dc (also suitable for on-column injection in fast GC) are often necessary to prevent severe discriminations and peak distortions in this case [33]. A programmed temperature vaporizing (PTV) injection in solvent vent mode can eliminate the problems caused by column flooding, however other practical limitations associated with the use of microbore columns remain. In addition to instrumental requirements, analyte detectability and method ruggedness are two important concerns in real-world analysis using columns with a smaller dc. Microbore columns produce sharper, thus taller peaks, but this effect usually does not fully compensate for the lower sample amount injected. Moreover, the peak-sharpening effect does not improve detectability (signal to noise ratio, S/N) in applications, in which matrix interferences are the limiting source of noise. Also, the repeated injections of matrix extracts deteriorate the performance of microbore columns faster as compared to the columns with a larger dc, resulting in a more frequent need for column maintenance [26]. In many respects, the use of 0.15 mm i.d. columns seems to be a reasonable compromise between speed and other issues in real-world analysis of sample extracts, such as in the analysis of pesticide residues in food samples [31]. Various applications of microbore columns in the analysis of food contaminants and chemical residues (such as pesticides, PCBs, or PAHs) have been reported in the literature [9,14], often combining microbore columns with a high-speed time-offlight (TOF) MS instrument that provides fast data acquisition rates (up to 500 spectra/s is currently available with a commercial instrumentation) for adequate recording of narrow peaks. A unique application of short, (sub-)microbore columns involves their use in the comprehensive GC GC technique, where they serve as second dimension columns for very fast separations of modulated peaks eluting from the first dimension (see the discussion on GC GC later in this chapter).
2.1.2 Fast gas chromatography with short megabore columns As discussed earlier, short megabore columns provide several benefits as compared to microbore columns, including higher Qs, improved ruggedness and less instrumentation problems associated with their use [26,34]. However, they have to be combined with a highly selective detection technique, such as MS, to compensate for decreased separation efficiency in fast GC applications. When connected to MS as a source of vacuum, short megabore columns can be operated at lower pressures along the entire column length. Lower column pressures lead to higher diffusivity of the solute in the gas phase, which shifts
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u¯opt to a higher value, resulting in faster GC separation as compared to the use of the same column operated at atmospheric outlet pressure conditions [35]. A practical problem associated with the direct connection of a short megabore column to MS involves extension of sub-ambient conditions to the injector unless precautions are made. Special injection techniques for sub-atmospheric pressure conditions have been tested with varying degrees of success [36,37], but the simplest way to solve this injection problem is to employ a short, narrow restriction capillary connected to the front of the wider analytical column [37,38]. In this manner, the analytical column is kept under low-pressure conditions, but the inlet remains at usual GC inlet pressures, thus the same injection methods can be used as in conventional GC. An additional benefit is that the restriction column also serves as a retention gap (or guard column) in the analysis of relatively dirty samples [39]. As for the analysis of food contaminants and residues, low-pressure (LP) GC-MS has been successfully applied mainly in the analysis of pesticide residues in various produce matrices using single quadrupole [26,39], TOF [40], triple quadrupole [41], or ion trap [42–44] MS instruments. As mentioned earlier, the use of megabore columns in LP-GC-MS provides increased Qs and improved ruggedness. However, the sensitivity of conventional MS instruments at higher flow rates in LP-GC-MS may be a limiting factor in the attainable speed in practice [39]. Conventional MS instruments work optimally at 1–2 mL/min effluent flows and, even if the MS pumping capacity is not exceeded, sensitivity can be significantly reduced at higher flow rates. This is not true for the supersonic molecular beam (SMB) GC-MS technique (developed by Amirav et al. [45–49]), which is well suited for the use of high flows because SMB-MS requires high gas flow rates for its operation (up to 240 mL/min is possible with the prototype instrument). The high column flows in SMB-MS enable very fast analyses, especially when combined with a short megabore column. They also facilitate very fast sample introduction using splitless injection without a need for cryofocusing (as compared to a microbore column operated at low flow rates), thus eliminating cool-down times. The use of a megabore column results in wider peaks that can be recorded using a conventional quadrupole MS even for ultrafast run times. In addition to an increase in speed, the fast flow rates and other unique features of GC-SMB-MS also enable analysis of thermally labile and lowvolatility analytes, such as carbamate pesticides [46] or large PAHs [48], thereby extending the scope of GC-MS to compounds currently done only by LC. In GC-SMB-MS, organic molecules pass through a small opening (about 0.1 mm i.d.) placed between the GC outlet and the MS, form an SMB by coexpansion with a carrier gas into vacuum and are vibrationally supercooled in the process. As a result, an enhanced molecular ion occurs practically for all molecules, increasing the MS detection selectivity in electron ionization (EI). SMBMS still provides typical EI fragmentation patterns, thus enabling conventional MS library searching, but with a higher confidence in correct compound identification due to the presence of a prominent molecular ion [49]. The potential and beneficial features of GC-SMB-MS have been demonstrated in numerous applications; however, the lack of commercial availability is
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currently a severe limitation in the applicability of this fast GC-MS approach in practice.
2.2 Fast liquid chromatography Basic approaches to fast LC are very similar to fast GC approaches presented in Table 1. Of course, differences exist due to a simple fact: LC employs liquid not gaseous mobile phases. Apart from different physico-chemical properties of the mobile phase, LC is much more versatile technique than GC because both stationary and mobile phase affect the separation. A myriad of stationary and mobile phase combinations can be used, enabling selectivity optimization for enhanced separation and/or increased speed. Similarly to GC, an existing method can be speeded up by employing higher than optimum mobile phase flows and/or shorter columns. Faster temperature programming in GC can be compared to faster (steeper) mobile phase gradients in LC, resulting in faster analyte elution as the mobile phase strength increases more rapidly. Also, fast isocratic runs can be used for analytes of relatively narrow polarity range. The benefit of isocratic elution is the elimination of column reequilibration to the initial mobile phase conditions, which greatly improves the throughput. A fast isocratic run in LC is an analogy to a fast isothermal analysis in GC, which also provides high-throughput due to the elimination of cool-down times, but is restricted to analytes with a narrow boiling point range. High sample throughput represents an ultimate goal in many applications. Pharmaceutical industry currently drives the development and utilization of highspeed, high-throughput LC and LC-MS approaches, especially in the area of drug discovery and development [50]. Even if very short columns and ballistic gradients are used, a lot of time can be wasted during column cleaning and reequilibration, injection of the next sample, etc. Thus to fully utilize the detector time, several columns can be operated in parallel, with only the chromatographic window of interest from each LC separation being transferred into the detector (usually MS) [51]. The simplest approach involves the use of an alternating twocolumn (dual-column switching) system that separates analytes on one column using a gradient provided by a binary pump, whereas the second column is being re-equilibrated for the next run with a simple isocratic pump [52]. More complex techniques, such as staggered injections [53] or multiplexing [54], require more sophisticated instrumentation to minimize the detector downtime, resulting in significantly increased sample throughput mainly in single-analyte applications. As in GC, fast LC approaches that do not necessarily sacrifice separation efficiency for speed are attractive in practice, especially in multicomponent analysis. The use of LC-MS(-MS) may allow compromises in Rs, but a certain degree of separation is beneficial for creating different analyte windows (e.g., for single-run analysis of compounds ionized in positive and negative modes) and/or for separation from matrix components that can affect analyte ionization (as discussed later in this chapter in the section dealing with matrix effects). As discussed earlier, the LC analysis can be speeded up without sacrificing Rs if the optimum mobile phase velocity (u¯opt) is shifted to higher values by improving
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mass transfer. In practice, this can be accomplished by employing columns with small particle sizes (dpo 2 mm), monolithic columns, and/or mobile phases at elevated temperatures.
2.2.1 Fast liquid chromatography using small particle sizes According to the chromatographic theory, as dp decreases, the separation efficiency increases because N is inversely proportional to dp, and so is u¯opt. Thus, columns with smaller dp can provide an increase in speed due to higher u¯opt. Although this theory has been well-known for decades, the practical application of this approach has been difficult for several reasons. The operation of small dp columns at high flow rates creates high backpressures (often exceeding 10,000 psi), requiring special instrumentation that enables injection of samples and reproducible mobile phase pumping at these pressures. Moreover, the entire LC system must have very low extra-column volumes to preserve the separation efficiency. Another challenge involves the design and development of sub-2 mm particles and their packing into reproducible and rugged columns [55]. Also, the samples must be well filtered to prevent clogging of the frits holding the particles in the column. Recently, two manufacturers introduced columns with smaller particles (1.7 and 1.8 mm) together with compatible LC systems, providing adequate pressure limits, injection systems, low extra-column volumes, as well as fast MS detection for narrow peaks. One manufacturer termed the technology as ultra performance liquid chromatography (UPLC) [56], whereas the other describes the columns as suitable for rapid resolution LC. The UPLC term and technology is spreading throughout the scientific community, which can be demonstrated by a number of studies evaluating this approach in various applications, including mainly those in the pharmaceutical and bioanalytical fields, but also in multiresidue analysis of pesticides [57,58], veterinary drugs [59] or heterocyclic amines [60] in various food matrices. Figure 2 compares high-performance liquid chromatography (HPLC)- and UPLC-MS/MS multiresidue analyses of 17 selected pesticides in a baby-food sample. Very recently, ‘‘fused-core’’ technology has been introduced, which produces 2.7 mm particles with a 0.5-mm porous shell fused to a solid core [61]. The porous shell decreases the diffusional mass transfer path as compared to totally porous particles. Thus, similar resolving power might be achieved as with sub2 mm totally porous particles at comparable or even higher speeds without high backpressures. Another, more explored alternative is the use of monolithic columns.
2.2.2 Fast liquid chromatography using monolithic columns Monolithic columns consist of one piece of an organic polymer or silica with flowthrough pores [62,63]. In this continuous phase of porous material, the size of flow-through channels and the depth of the pores that are accessible only by diffusional mass transfer may be optimized independently, enabling simultaneous maximization of column permeability and mass transfer. For example, recently
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Figure 2 Comparison of (i) HPLC-MS/MS and (ii) UPLC-MS/MS analysis of 17 pesticides in a potato-based baby food matrix. Insets show expanded peaks for terbufos sulfone (0.01 mg/kg), demonstrating the peak width differences. Reprinted with permission from Ref. [57].
reported silica monoliths with permeability similar to that of a column packed with 5 mm particles were able to attain separating power of columns packed with 2–2.5 mm particles [64]. As a result, monolithic columns can offer highly efficient separations with low resistance to hydraulic flow, thus operation at high flow rates without high backpressures. Figure 3 compares monolithic columns to
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Figure 3 Comparison of (a) highly permeable and less-efficient packed columns with large particles, (b) less-permeable and highly efficient packed columns with small particles, and (c) highly permeable and highly efficient monolithic columns. Reprinted with permission from Ref. [63].
columns packed with large and small particles, demonstrating the differences in column permeability and efficiency. Monolithic columns have been used mainly for fast separations of large molecules, such as proteins or peptides, which significantly benefit from improved mass transfer because their diffusion coefficients are several orders of magnitude smaller than the diffusion coefficient of smaller molecules [63]. Although monolithic columns are commercially available, the current selection of stationary phases is rather limited. However, this situation may change in the future due to the increasing trend towards faster LC separations.
2.2.3 Fast high-temperature liquid chromatography The application of elevated temperatures in high-temperature LC (HTLC) decreases viscosity of the mobile phase and increases analyte diffusivity, which leads to lower backpressures and increased mass transfer, respectively [65]. As a result, LC columns can be operated at higher flow rates, reducing the analysis time without making significant sacrifices in the separation efficiency. Moreover, elevated temperatures can be employed in the combination with small particle size columns, thus providing an additional gain in speed or separation efficiency, while keeping the backpressure within reasonable limits. Also, temperature programming of the column can change separation selectivity, which may improve analyte separation or serve as an alternative to solvent gradient. Another attractive aspect involves the use of superheated water (at 100–200 1C) as a potential replacement for medium-polarity mobile phases (such as acetonitrile–water mixtures) in reversed-phase LC [66,67]. This would reduce solvent and waste costs and simplify system operation. Under high-temperature conditions, water behaves as a moderate polarity solvent because its dielectric constant decreases from about 80 to about 35 over the range 25–2001C [68], while it retains an appreciable density (W 0.85 g/mL), cohesive energy, and hydrogen bonding potential [69]. Until recently, the application of HTLC was rather difficult in practice due to instrument and column limitations. Instrumentation for HTLC is now available, which allows operation at temperatures up to 2001C with mobile phase
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preheating to eliminate thermal mismatch [70]. The thermal mismatch occurs when the cool mobile phase enters the heated column and warms up faster along the walls, leading to band broadening due to the faster flow of the mobile phase along the column wall than in the centre [71]. Another practical concern involves the stability of the stationary phase at the elevated temperatures. Conventional, reversed-phase LC columns with silicabased stationary phases are stable at temperatures up to 901C (50–601C limit is more common). Stationary phases with higher temperature stability are based mainly on zirconium oxide, although graphitized carbon or rigid polystyrene– divinylbenzene phases can also tolerate elevated temperatures [70]. With the recent advancements in column technology and instrumentation, HTLC may soon become a practical tool, especially in the combination with narrower columns and/or columns with small particle sizes and in applications that can tolerate higher temperature with respect to the analyte stability.
3. TWO-DIMENSIONAL CHROMATOGRAPHIC SEPARATION 2D GC or LC separations involve the use of two coupled columns of different selectivity (or capacity) to provide improved separation for sections (heartcuts) of one-dimensional (1D) chromatogram or for the whole first dimension separation (in comprehensive 2D chromatography) [1]. In standard heart-cutting techniques, only a single or a few small fractions of the first dimension effluent are transferred to the second column for further separation to enhance resolution of compounds eluting in specific region(s). In GC, heart-cutting is employed (using somewhat complex instrumentation) mainly for detailed characterization of petroleum products or flavors and fragrances [72]. It can be also utilized in congener-specific analysis of PCBs [73]. In standard 2D LC, the two columns are usually coupled using a low-dead volume switching valve (or a series of valves in advanced systems) that manages the mobile flow direction, including transfer of sample components between the columns. The basic requirement involves compatibility of the mobile phase flow and strength with the transfer to the second column. Thus, the mobile phase eluting from the first column should have a lower flow rate, should be weak in respect to the secondary column separation mechanism, and should be miscible with the mobile phase for the second column. As a result, only a limited number of stationary phases of different selectivity are compatible. Examples of compatible columns include ion-exchange with reversed-phase or size-exclusion with normal, reversed-phase, and/or ion-exchange. In a sense, clean-up procedures, such as solid-phase extraction (SPE), size-exclusion or turbulent flow chromatography, coupled on-line to LC can also be considered 2D separations, introducing a fraction containing analytes to the analytical column and diverting the removed matrix components to the waste. Using special interfaces enabling large volume injections of LC effluents, LC can be coupled to GC [74]. In LC-GC, the role of LC is to perform selective cleanup, concentration and/or fractionation of the sample, whereas GC serves as a
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determinative step. Recently, the applicability of comprehensive LC GC has been evaluated, showing a great potential of this technique for characterization of certain complex samples, such as edible oils and fats [75]. As opposed to the heart-cutting techniques, comprehensive 2D chromatography requires each component or fraction eluting from the first column to be subjected to a second separation system using different, ideally independent separation mechanism (orthogonal selectivity). As a result, the peak capacity of comprehensive 2D chromatography is significantly increased and can be estimated by multiplying the peak capacity of the first and second dimension separations [76]. The introduction and commercialization of comprehensive 2D GC (GC GC) are probably the most exciting recent developments in GC. As for comprehensive 2D LC (LC LC), this technique is still more or less at the stage of exploration, but its potentials have been already demonstrated in a number of complex separations, including mainly those in the area of proteomics and metabolomics [77,78].
3.1 Comprehensive two-dimensional gas chromatography Comprehensive 2D GC, GC GC, employs two columns of different selectivity coupled by a special interface or modulator that can sample or collect the effluent from the first column and introduce it periodically (about every 3–6 s) to the second column [79–85]. In terms of column dimensions, the first column is usually a conventional GC capillary column (typically 15–30 m 0.25 mm i.d.), whereas short (sub-)microbore columns are used as the second dimension columns (0.5–2 m 0.1 mm i.d. or even 0.05 mm i.d.) to provide very fast separations (in a few seconds) with minimum broadening of the very narrow bands generated by the modulator. To create an orthogonal-selectivity system in GC, the first column should have a non-polar stationary phase (typically 100% dimethylpolysiloxane or 5% phenyl/ 95% dimethylpolysiloxane), which provides separation based solely on volatility (boiling point) of the analytes. As a result, each small fraction eluting from the first column will contain analytes of very similar volatilities. The runs in the second dimension are very fast, thus performed almost isothermally. Therefore, there is practically no contribution of boiling-point-based separation in the second column, with the analyte retention being based on specific interactions with the stationary phase, such as hydrogen bonding, p–p interaction, steric effects, etc. Typical stationary phases for the second dimension include W35% phenyl/o65% dimethylpolysiloxane or polyethyleneglycol (Carbowax). It should be noted that, in certain specific applications, non-orthogonal systems (such as polar nonpolar or polar polar columns) may provide some benefits [86], but the orthogonal column combination described above (non-polar polar columns) is generally preferred in practice, especially for group-type analysis or classification purposes. Various GC GC modulators have been developed, mainly based on cryogenic focusing. A current, prevalently used commercial system employs a nonmoving quad-jet dual stage thermal modulator [87] consisting of two sets of dual
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(A)
Stage 1 cold
Stage 2 cold
(B) Stage 2
I
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tI hot
cold tI
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III tIII cold
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Figure 4 Illustration of (A) the GC GC modulation process using a non-moving quad-jet dual stage thermal modulator located at the junction of the first and second column and (B) the modulation period, including hot and cold pulse durations. A single modulation period consists of four distinct steps: (I) a fraction of solutes eluting from the first column is trapped using a cold pulse produced by the first trapping zone (stage 1) of the modulator; (II) a hot pulse then releases the trapped solutes to the second trapping zone (stage 2) that is cold at that moment; (III) stage 2 continues to retain the solutes while stage 1 cools again; and (IV) stage 2 releases the solutes with a hot pulse to the rest of the secondary column. The duration of each step are symbolized by tI–tIV. Reprinted with permission from Ref. [96].
(‘‘cold’’ and ‘‘hot’’) jets that create two trapping zones (stages) at the beginning of the second column. Cold and hot jets supply in pulses N2 (cooled by liquid N2) and heated air, respectively. Figure 4 shows four distinct steps that take place during a single modulation period using the quad-jet dual-stage thermal modulator. The modulation period must be short enough to preserve the 1D separation. In this respect, it is recommended to take at least three samples (slices) across a peak eluting from the first column [88]. The peaks eluting from the second column are very narrow (typical basewidths of 100–200 ms), thus require the use of fast detectors with small internal volumes, such as flame ionization detectors (50–200 Hz data acquisition frequency), micro electron capture detector (50 Hz), high-speed TOF MS (up to 500 spectra/s) or quadrupole MS with a limited scan range [89]. High-speed TOF MS is probably the most popular detector, providing full MS spectra information and enough data points for adequate characterization of the narrow secondary peaks. Figure 5 illustrates the process involved in generation and visualization of a GC GC chromatogram. The outcome of a GC GC run is a series of fast secondary chromatograms, which are usually stacked side-by-side to form a 2D plot (array) with the first dimension representing the retention time on the first column and the second dimension, the retention time on the second column. After this transformation, it is possible to visualize the chromatograms using contour
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Figure 5 Generation and visualization of a comprehensive GC GC chromatogram. Reprinted with permission from Ref. [83].
plots, where the peaks are displayed as spots on a plane using colors, shading, and/or contours to indicate the signal intensity [83]. The peak intensity can be used as the third dimension in 3D plots, which generally have less practical value but provide attractive figures in data presentations. Numerous applications of GC GC (mainly GC GC-TOF MS) have been reported in the literature. The applications pertinent to the analysis of contaminants and chemical residues in food include mainly the analysis of pesticide residues [90,91], PCBs [92–94], PCDDs and PCDFs [94–96], or PBDEs [97]. In addition to the improved selectivity (increased analyte–analyte and analyte–matrix separation), GC GC also provides improved sensitivity due to the focusing effect in the modulator. As a result, analyte detectability can be improved significantly.
4. OVERCOMING ADVERSE MATRIX EFFECTS In general, matrix effects are all adverse phenomena caused by unavoidable presence of co-extracted matrix components, leading to inaccurate quantitation,
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decreased method ruggedness, low analyte detectability and/or even reporting of false positive or negative results [98]. The most obvious matrix effects are coelutions of matrix components with analytes that affect analyte detection. Those effects can be overcome by improved selectivity of detection (e.g., using MS or MS-MS), separation (e.g., using 2D chromatography), and/or sample preparation. There are, however, matrix effects that are more difficult to manage because the culprit cannot be easily eliminated. These effects include mainly matrix-induced response enhancement in GC and signal suppression or enhancement in LC-MS with atmospheric pressure ionization (API) techniques.
4.1 Approaches to overcome matrix effects in gas chromatography Matrix-induced response enhancement, first described by Erney et al. [99], is a matrix effect well-known mainly in the GC analysis of pesticide residues in food, negatively impacting quantitation accuracy of certain susceptible analytes [98]. When a real sample is injected, the matrix components tend to block active sites (mainly free silanol groups) in the GC inlet and column, thus reducing losses of susceptible analytes caused by adsorption or degradation on these active sites. This phenomenon results in higher analyte signals in matrix-containing vs. matrixfree solutions, thus precluding the convenient use of calibration standards in solvent only, which would lead to overestimations of the calculated concentrations in the analysed samples. In theory, elimination of active sites or matrix components would overcome the matrix-induced enhancement effect; however, absolute and permanent GC system deactivation or thorough sample clean-up are virtually impossible in practice [100]. Careful optimization of injection and separation parameters (such as the injection technique, temperature, and volume; liner size and design; solvent expansion volume; column flow rate; and/or column dimensions) can lower the number of active sites (due to a decreased surface area) or shorten the analyte interactions with them. This results in a reduction but rarely complete elimination of the effect. For instance, application of a pressure pulse or temperature programming during the injection (to reduce residence time or thermal degradation in the injection port) may serve as examples of this effort [101–104]. Since an effective elimination of the sources of the matrix-induced response enhancement is not likely in practice, the analysts often try (or are required) to compensate for the effect using alternative calibration methods. The current compensation approaches include the use of matrix-matched standards, standard addition method, and isotopically labelled internal standards (not feasible in multianalyte analysis due to their unavailability and/or prohibitive price). All of these techniques require extra labour and costs; moreover, they may still lead to quantitation inaccuracies because the extent of the effect depends on analyte concentration and matrix composition [105] (problems in the case of standard addition and matrix-matching, respectively). Matrix-matched standardization is thus far the most widely used approach in spite of its imperfections including a rather time-consuming and laborious
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preparation of matrix-matched standards and need for an appropriate blank material (ideally the same as the analysed samples). The matrix-matching procedure becomes especially onerous when different commodity types are to be analysed in one batch of samples, which is often the case in routine pesticide residue analysis. Recently, a concept of ‘‘analyte protectants’’ has been introduced in the GC analysis of pesticide residues [106,107], but can be applied to other analytes as well. Analyte protectants are compounds that strongly interact with active sites in the GC system, thus decreasing degradation and/or adsorption of co-injected analytes. The concept idea is to add suitable analyte protectants to sample extracts as well as matrix-free (solvent) standards to induce an even response enhancement in both instances, resulting in effective equalization of the matrixinduced response enhancement effect. A mixture of 3-ethoxy-1,2-propanediol, gulonic acid g-lactone, and sorbitol was found to be the most effective for the volatility range of GC-amenable pesticides [107] as demonstrated in Figure 6, which compares peak shapes and intensities of three selected pesticides obtained in solvent standards and matrix extracts with and without the addition of the above mixture of analyte protectants. In addition to the compensation for matrix-induced response enhancement, the application of analyte protectants can also significantly reduce another matrix effect called matrix-induced response diminishment [26,108]. This effect is caused by gradual accumulation of non-volatile matrix components in the GC system, resulting in formation of new active sites and gradual decrease in analyte responses. The use of analyte protectants provides GC system deactivation in each injection, resulting in improved ruggedness and a less frequent need for the GC system maintenance [107]. Another way how to overcome gradual build-up of non-volatile matrix components is to remove them from the system after each analysis. This can be accomplished using a special injection technique called direct sample introduction (DSI) [109]. In DSI, a liquid (or solid) sample is placed in a disposable microvial. After this step, the microvial is introduced into the injection port using a manual probe or more recently using an autosampler. In the automated version, called DMI (difficult matrix injection) or recently introduced Linex (automated liner exchanger), the liquid sample (up to about 20–30 mL) is injected into the microvial placed in a liner, which is then inserted into the inlet [110–114]. After solvent evaporation, the inlet is rapidly heated and analytes transferred to the column for a GC separation. When the GC run is completed, the liner with the microvial is removed from the system together with non-volatile matrix components, which remain in the disposable microvial. In comparison with other injection techniques, DMI incorporates greater glass surface area in the inlet, which needs to be deactivated. Also, the activity of the inlet may vary greatly throughout the GC sequence because a new (different) liner and microvial are introduced into the system each time. In this respect, the use of analyte protectants was demonstrated to offer more convenient and effective solution than standard silanization of glass surfaces, thus another benefit in addition to the compensation for matrix-induced response enhancement [113].
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A) without analyte protectants
B) with analyte protectants
1.12x
a) Lindane Cl Cl
Injection in: matrix solvent
Cl
Cl
Cl Cl
b) Phosalone S
Cl N O
S
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O
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Figure 6 Comparison of peak shapes and intensities of three selected pesticides (with different susceptibility to the matrix-induced enhancement effect) obtained by injection in matrix (fruit extract) and solvent (acetonitrile) solutions (A) without and (B) with the addition of analyte protectants (ethylglycerol, gulonolactone, and sorbitol). The numbers demonstrate signal (peak height) enhancement factors (signal in matrix vs. solvent) obtained without the use of analyte protectants and improvement in o-phenylphenol signal intensity in matrix with the use of analyte protectants. Reprinted with permission from Ref. [107].
4.2 Approaches to overcome matrix effects in liquid chromatographymass spectrometry In LC-API-MS, the co-eluting matrix components can suppress or enhance analyte ionization, leading to lower or higher signals in the presence of matrix [98,115,116]. The exact mechanism of matrix suppression/enhancement is not known, but the matrix components are assumed to interfere in the spraying, ionization, and evaporation processes, including droplet formation, competition for the droplet surface and proton transfer, or ion-pair formation [117–119].
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The signal suppression effect is observed mainly in electrospray ionization (ESI), whereas the enhancement effect is more likely to occur when atmospheric pressure chemical ionization (APCI) is employed [120]. For analytes that provide acceptable signals in different ionization modes, a change from ESI to APCI or from positive to negative-ion mode (and vice versa) may provide a feasible approach to elimination or reduction of the matrix effects [121]. Also, the optimization of mobile phase composition may be helpful in certain applications [122]. In general, the matrix effects in LC-API-MS can be eliminated if the co-eluting matrix components are removed from the sample extract or separated from analytes in the chromatographic run. In practice, improved sample preparation (more selective extraction and/or clean up) or LC separation can be a solution to the suppression/enhancement effect in the analysis of a smaller number of analytes, but the elimination is more difficult in multicomponent analysis, such as in the analysis of pesticide residues. Another possibility is to reduce the concentration of co-eluting matrix components by diluting the sample and/or injecting a smaller sample volume [122–124], which reduces the matrix effects, but also decreases analyte detectability. Similarly, reduced suppression was observed in nano-ESI [125] or with post-column splitting to reduce the LC flow rate in ESI [126]. As in GC, compensation approaches, such as the use of matrix-matched standards, standard addition method, or isotopically labelled internal standards,
Figure 7 An example of echo and sample peaks obtained for the same analyte (a pesticide, propoxur) using the echo peak technique, involving two subsequent injections of a sample extract and solvent standard with the same propoxur concentration. Reprinted with permission from Ref. [120].
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are often employed in practice with similar practical drawbacks and limitations as described earlier. As mentioned earlier, isotopically labelled internal standards are very effective, but their application is limited due to their unavailability or high price. An interesting alternative to the labelled internal standards is so-called echo peak technique [127], which has been evaluated in the analysis of pesticide residues [120,128] and antibiotics [129]. The echo peak technique involves injections of the sample and standard solution within a short time period using a relatively simple instrumentation [120,127–129]. As a result, the analyte peak from the sample elutes in a very close proximity to the analyte peak from the standard solution, which forms an echo peak (Figure 7). It is expected that these two closely eluting peaks are affected by the co-eluting matrix components (usually larger peaks) to the same extent. This approach can be quite effective, but 100% compensation may not be achieved for all analytes/matrices. Still, it represents an attractive method in routine pesticide or veterinary drug screening, because it offers some additional benefits, such as direct comparison of analyte concentrations in the sample with critical standard concentrations (e.g., with lowest calibration levels or maximum residue limits) and a quality control measure present in each LC run to check for retention time shifts, sensitivity or other LC-MS instrument problems.
5. FUTURE TRENDS Current trends towards high-speed, high-throughput analysis are likely to continue spearheading the development in both GC and LC, especially in the combination with MS detection. Very fast separations also play a pivotal role in comprehensive 2D chromatography. GC GC has already made the transition from research to routine laboratories. And, it is only a matter of time when LC LC and LC GC become techniques of choice in applications that can benefit from their separation power. Current, commercially available instrumentation reflects the trend towards increased speed, providing faster data acquisition rates in MS, low dead volumes, new column technologies, rapid and precise temperature and pressure programming in GC or fast mobile phase gradients and column heating in LC. This trend will continue for both hardware and software components. In particular, fast and reliable automated data processing is highly desirable in routine practice because it is currently one of the limiting factors in the implementation of fast and 2D chromatography. Furthermore, routine laboratories will start fully benefiting from fast chromatography if the sample preparation becomes also faster. In this respect, techniques and instrumentation that offer more rugged, sensitive, and selective analysis have a better chance of being implemented because they can tolerate dirtier, minimally processed samples. The obvious trend towards minimum sample preparation leads to higher chances of more pronounced matrix effects that need to be managed properly. In this respect, techniques, such as echo peak in LC-MS or analyte protectants with DSI/DMI in GC-MS, may provide convenient and quite
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effective alternatives to the current approaches, such as matrix-matched standardization.
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CHAPT ER
7 New Approaches in Mass Spectrometry Yolanda Pico´
Contents
1. Introduction 201 2. High Resolution and Accurate Mass Analysers 204 2.1 Time-of-flight mass spectrometry 205 3. Tandem Mass Spectrometry 211 3.1 Triple quadrupole mass analysers 211 3.2 Ion-trap mass spectrometers 212 3.3 Linear ion trap 213 4. High Resolution Mass Spectrometers in Tandem 219 4.1 Quadrupole time-of-flight mass spectrometer 219 4.2 Fourier transform ion cyclotron resonance and orbitrap mass analysers 222 5. Conclusions and Future Trends 227 References 228
1. INTRODUCTION Nowadays, mass spectrometry (MS) is the most used technique to determine food contaminants [1–3]. Normally, this technique is combined with a separation one, such as gas chromatography (GC) [2,4,5], liquid chromatography (LC) [6–8] or capillary electrophoresis (CE) [9,10], because of the high number of organic pollutants and their low concentrations in food samples. Among the different MS techniques and their combinations with chromatographic separation, GC-MS is the routine, preferred analytical method for determining food contaminants and residues since the end of the 1970s. A large number of publications have resulted from research on food applications of GC-MS [4]. This technique is frequently used to study the behaviour of contaminants and residues as well as to monitor their presence in food. GC-MS is currently a mature Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00007-X
r 2008 Elsevier B.V. All rights reserved.
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technique applied in the analysis of a significant number of pollutants [2,4,11,12]. The compounds most commonly analysed include alkanes, polycyclic aromatic hydrocarbons (PAHs), pesticides, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), as well as other endocrinedisrupting chemicals such as phthalates and short ethoxy alkylphenol etoxylates [5,13–18]. GC-MS is also the technique of choice for the analysis of emerging pollutants, such as polybrominated diphenyl ethers (PBDEs) or polychlorinated alkanes [17]. However, the development of LC-MS in the past 20 years has made the detection of many harmful compounds possible. The primary advantage LC-MS has over GC-MS is that it is capable of analysing a much wider range of components. Compounds that are thermally labile, exhibit high polarity or have a high molecular mass may all be analysed using LC-MS, even proteins may be routinely analysed. Several recently published comprehensive papers summarized the current state-of-the-art in LC-MS analysis of specific classes of contaminants (e.g., veterinary drug residues, banned growth promoters, perfluorinated compounds, and pesticides and their degradation/transformation products and food packaging migrating materials) [6,19–27]. These two approaches can be considered the established ones. There are more emerging techniques, such as CE-MS or ambient MS, which are very promising, helping to improve MS features for the analysis of food contaminants, even though they are not well ascertained yet in the field of food contaminants and residues analysis [28–30]. The introduction of novel methods and expanding applications to diverse areas highlight truly impressive progress in MS. Table 1 summarizes the current state-of-the-art in MS for food contaminants and residue analysis and the expected improvements. These developments are illustrated here by two seemingly different areas of research: innovative ionization methods and mass analysers. Some emerging technologies in the field of the ionization sources have shown great potentials to further enhance the capabilities of high throughput analysis by MS, including CE-MS and ambient MS [31,32]. The recent development on CE-MS has been produced in the field of micro- and nano-electrospray ionization (ESI) ion sources, which have radically improved the CE/ESI-MS sensitivity level. In such devices the need of make-up flow is eliminated because the sample is sprayed into the MS either directly from the tip of the separation capillary or from a tapered micro- or nanoemitter butted to the CE column. In both cases, the achieved flow rates are situated in the range of nanolitre per minute . The effects of the use of this kind of micro/nanosprayers, which include higher analyte concentration, reduced spraying potential and closer positioning of the sprayer to the orifice of the mass spectrometer, render a substantially improved mass transfer into MS, better ionization and desolvation of the generated droplets and therefore, superior sensitivity [33]. Because of the rather low sensitivity provided by the sheath flow CE-MS, for applications where limited amount of material is available, sheathless on-line CE/ESI-MS is a reliable alternative, even if the tedious optimization procedures make it often a non-user-friendly approach. The lack of commercial
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Table 1 Summary of the improvements of mass spectrometry in food contaminants and residues GC-MS
LC-MS
CE-MS
Current state-of-the-art in mass spectrometry Well-established Well-established The dominant ionization ionization interfacing sources: EI sources: EI system is the and CI and CI sheath interface
Variety of mass Variety of mass Variety of mass analysers MS, analysers MS, analysers MS, MS2 (quadrupole, MS2 (quadrupole, MS2 (sector, ion trap, sector) ion trap, triple triple quadrupole, quadrupole) ion trap, time-of-flight)
Routine technique Routine technique No real applications
MS
Method for the analysis of solids are still under development (DART) MALDI is the well-developed techniques always combined with time-of-flight and not appropriate for small molecules Not appropriate for food contaminants and residues which are small molecules
Expected improvements in mass spectrometry New mass New mass New interfacing New interfacing analysers (triple analysers systems system (sheathless quadrupole, (time-of-flight, (desorption interface, liquid time-of-flight, linear traps, electrospray junction interfaces, etc.) quadrupole ionization) etc.) time-of-flight, orbitrap, etc.) Already a reality Already a reality The available First applications approaches of commercial require devices improvements Note: EI, electron ionization; CI, chemical ionization; DART, direct analysis in real time; MALDI, matrix assisted laser desorption.
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sheathless interfaces or completely standardized procedures for smooth and fast in-house production, along with the deficit in methodological protocols for running different analytes, stimulated continuously the creativity of the CE-MS scientific community. So far, a remarkable number of sheathless setups were conceived and their potential for various applications tested [34]. These combinations are still little applicable to food contaminants and residue analysis because the sensitivity is not optimum yet and will not be covered in this chapter [9,10]. Recently introduced ambient MS represents a new family of ionization techniques that create ions under ambient conditions without vacuum constraints, including desorption electrospray ionization (DESI) [31] and direct analysis in real time (DART) [35], which have already been detailed in Chapter 1. In DESI experiments, a spray of charged solvent droplets hits the surface of the sample and ionizes the sample molecules (small molecules and large biomolecules) [35]. In DART experiments, a plasma of excited-state atoms and ions from nitrogen/ helium desorb/ionize small molecules from the surface of the sample. These techniques can be used to rapidly obtain mass spectra of compounds without any sample preparation [35]. Some encouraging results on analysis of food with ambient MS have recently been reported in the literature [36]. These techniques are very promising and further developments in this area will establish the use of ambient MS for high-throughput analysis, accurate mass measurement and other aspects of structural characterization of food contaminants and residues. This chapter is, thus, aimed at discussing the progress of MS analysers in food contaminants and residue analysis and reporting the recent applications for the trace analysis of selected contaminants. Two emerging trends in the mass analysers design are valuable to get better quality and quantity of structural information: High-resolution (HR) mass spectrometers, which calculate elemental composition and nominal mass, because they can determine exact mass of analytes. They are a tool to identify unknown substances in the samples. These spectrometers are represented by time-of-flight (TOF). Tandem mass spectrometry (MS/MS), which allow the mass spectrometer to analyse an ion and obtain its mass spectrometer again. The mass spectrometers capable of carrying out MS/MS are QqQ, ion trap, quadrupole time-of-flight (QqTOF) and linear ion trap (LIT).
2. HIGH RESOLUTION AND ACCURATE MASS ANALYSERS High resolution and accurate mass mainly include four types of instrumentation — double-focusing magnetic-sector (resolving power of 10,000–40,000 full width at half maximum (fwhm) with 2–5 ppm accuracy), TOF (resolving power of 10,000–20,000 fwhm and an accuracy of 2–5 ppm at m/z 1,000), the LITOrbitrap (resolving power of 60,000–100,000 fwhm) and Fourier transform ion cyclotron resonance (FTICR) MS systems (W100,000 fwhm and 0.5–2 ppm). Although all four instruments work readily with GC or LC systems, the magnetic-sector instruments were developed initially to operate with GC-MS.
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High resolution and accurate GC-MS with a magnetic sector instrument is already a well-developed technique to determine some organic contaminants such as PCBs, PBBs and PCDD/Fs, as described in detail in Chapters 14 and 15 of this book and hence is not discussed in this Chapter. The more recent highaccuracy TOF systems have been applied indistinctly to LC-MS or GC-MS. The QqTOF, FTICR-MS and LIT-Orbitrap systems are currently considered research tools in LC-MS and are also susceptible to carry out MS/MS. These instruments and their applications are described in next section [37].
2.1 Time-of-flight mass spectrometry The current trend in the trace analysis of food contaminants and residues is to use TOF-MS systems because they are easier and less expensive to operate compared with the other high resolution and high accuracy mass spectrometers [5,37–41]. Recent advances have reduced instrument costs and have made analysis less complicated, more accurate and more quantitative [15]. At present, several commercial benchtop instruments are available with high resolution and accurate mass that are suitable for food analysis coupled either with GC or LC [5,41]. TOF-MS offers the advantage of a comprehensive mass analysis in a broad dynamic range, because TOF-MS is a non-scanning technique, all ions included in the mass range are virtually recorded at the same time and are represented at the same point on the chromatographic peak profile. Constant ion ratio across the GC or LC peak is thus ensured. High-quality mass spectra are produced and deconvoluted if more than one compound is present at the time mass spectra are recorded, as is the case when two compounds chromatographically co-elute. Other TOF-MS advantages are high sensitivity, speed and ease of use, simple experimental design (no tuning) and that it can achieve resolving powers (m/Dm) of Z3,500, and exact parts per million mass measurements. Those spectrometers are the best available instruments for routine HRMS, not as accurate as a sector, but much easier to handle [5,37–41]. TOF-MS analysers provide high specificity due to both high mass accuracy and mass resolution. This technique can generate high specificity without limiting the number of simultaneously observed target compounds. Both the accurate mass of each scan and the higher mass resolving power provide a greater degree of reduction in chemical noise, thereby enhancing selectivity. The advantage of a TOF-MS analyser for screening is its ability to examine a data file for a theoretically unlimited number of food contaminants [41]. TOF-MS combined with LC is used in food contaminants and residue analysis because, in contrast to conventional triple quadrupole (QqQ) instruments working in selected reaction monitoring (SRM), it offers several benefits such as: A large number of targets can be screened at the same time without loss of sensitivity [42–44]. Unknown peaks can be identified based on accurate mass and isotopic profile evaluation [45–47]. Data can be reprocessed a posteriori for additional compounds, which had not yet been investigated [41].
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The LC-TOF-MS approach enables screening for several hundreds of compounds with high sensitivity within one run. The selectivity is based on the accurate mass measurements with mass traces defined within 0.005 Da over a dynamic range of about three orders of magnitude [48–50]. It is also important to remark the feasibility of LC-TOF-MS/MS and elemental database searching as spectral library [51]. Figure 1 illustrates an example of the LC-TOF-MS application to identify an unknown chlorinated compound. In a grapefruit extract and a pear extract, a doubly chlorinated species was detected with m/z 311 at a retention of 18.2 min. Figure 1(a) shows the total ion chromatogram (TIC) of the grapefruit extract (accurate mass spectrum as insert) and Figure 1(b) shows the accurate mass spectrum of this doubly chlorinated species in the pear extract. The accurate m/z of the main peak was 311.0712 with an A+2 isotope signal at m/z 313.0681, which has a relative intensity consistent with the presence of two chlorine atoms. Although all available databases (e.g., The Merck Index, ChemIndex and e-catalogues) were used, no match with the candidate elemental compositions was obtained. The authors, based on previous experience, indicate that the most likely elemental composition was C15H17N2OCl2. This elemental composition was rather similar to that of imazalil (a common post-harvest fungicide); there was only a difference of a methylene group (CH2) that reasonably attributes to a methyl group substitution. The combination of GC with TOF-MS technique resulted in the introduction of two types of spectrometers differing in their basic characteristics: Instruments using HR analysers characteristic with only moderate acquisition speed. Unit-resolution instruments that feature a high acquisition speed (HSTOF). The potential application of these approaches is obviously complementary. Recently GC-TOF-MS, employing both detectors, has been demonstrated as a powerful and highly effective analytical tool in various fields, and the analysis of food contaminants is one of them [5]. Due to the availability of spectral information even at very low levels for a particular compound (high mass analyser efficiency), this technique can be used not only for quantification/confirmation of target analytes but also for identification of non-target sample components even in very complex mixtures [12,13,16,17,25,52–54]. The moderate acquisition rates of the HRTOF instruments predetermine their use as the detector for conventional and fast GC; the HSTOF instruments are suitable for detection of very narrow chromatographic peaks generated by very fast and ultra-fast GC or GC GC. HSTOF-MS instruments provide only a unit mass resolution, whereas HRTOF analysers offer resolution as high as 7,000 fwhm and even more. The advantage of high resolution is the possibility of partially or completely resolving matrix components yielding ions with the same nominal mass as that of the target analyte, hence, significantly reducing background interferences and, consequently, improving the analyte identification. The utility of high speed is the possibility to couple a selective detector with GC GC improving the capabilities of the technique.
207
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tive
Figure 1 Identification of an unknown chlorinated specie: (a) total ion chromatogram and (b) accurate mass spectrum (m/z 311); a case study of a methyl derivative of imazalil. Reproduced from Ref. [47] with permission from Wiley.
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TOF-MS combined with conventional GC is applied in food contaminants and residue analysis. The HRTOF-MS instruments achieve mass accuracy as low as 5 ppm. Under these conditions, the determination of elemental composition is possible; also the specificity for the identification of unknowns is enhanced. This detector has been widely applied to the determination of pesticide residues, acrylamine and environmental contaminants, such as PCBs and PBDEs [12,13,16,17,25,52–54]. The high mass resolution and monitoring of the exact mass of target analytes, can reduce significantly the chemical noise originating from various sources (e.g., matrix coextractants, column filled) resulting in an improved limit of quantification (LOQs) [2,12]. Figure 2 shows negative ion chemical ionization (NICI) chromatograms obtained by HRTOF-MS examination of purified fish (perch) extract and standard of PBDEs [17]. This study demonstrated that operating the instrument in the NICI mode provides not only more sensitive detection (20–100fold) compared with electron ionization (EI) but also results in increased selectivity. Although the low m/z bromine ions used for identification/ quantification of target analytes may not seem specific enough (compared with high molecular ions produced by the EI), their selectivity is relatively high since only a limited number of GC-amenable compounds, potentially present in food samples, are prone to yield ions capable of efficient electron capture in NICI [17]. Moreover, thanks to the availability of full spectral information, other analytes (particularly, the case of major PCB congeners) occurring in this sample could be determined without any instrumental modification, simply by retrieval of stored data. To obtain comparable LOQs by quadrupole operated in selected ion monitoring (SIM) mode (ions m/z 79 and 81 monitored only), as those achieved by HRTOF-MS, a 10 times higher sample equivalent had to be introduced into the system. In other words, HRTOF-MS was more sensitive by one order of magnitude in the analysis of PBDEs. However, in this field, GC GC coupled with HSTOF-MS has been proved in the past few years to be a powerful technique for achieving unsurpassed separation and has been therefore applied to any challenging separation needs for unravelling complex mixtures, for which, conventional GC showed its limitation. In particular, the determination of PCBs, PBBs and PCDD/Fs is taking great advantage in using this technique. In this context, TOF-MS offers several important features over conventional MS detectors to be combined with GC GC: Full mass spectral information is available across the chromatogram, with detectability fully comparable with conventional MS detectors. The absence of spectral skew and constant ion ratios across the peak allows application of the software algorithm of peak finding and spectral deconvolution, enabling the applications in non-target analysis. Being the fastest MS detector, TOF can be used in GC GC without any compromise.
6.47
100
CB 180
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209
m/z 393.803 Mass window: 0.02 Da (A)
% 6.75 6.03
6.71
6.42 6.07
7.03
7.23
0 5.73
6.47
m/z 359.842 Mass window: 0.02 Da
CB 180
CB 153
Rel. abundance
100
5.96
(B)
6.41
%
6.22 5.98 5.68
6.72 5.82
6.03
6.75
BDE 49
% 5.89 6.06 6.12
6.38 6.32
6.69
(C)
BDE 99
BDE 28 5.63
m/z 80.916 7.26 Mass window: 0.02 Da BDE 100
6.56
100
BDE 47
0
7.48 6.90
7.62
7.13
0 5.60
5.80
6.00
6.20
6.40
6.60
6.80
7.00
7.20
7.40
Time (min)
Figure 2 GC-TOF-MS chromatograms of selected PCBs and PBDEs in diluted fish extract (0.32 mg of original matrix equivalent injected) in NICI mode. Ions extracted by a mass window of 0.02 Da. (A) Chromatogram of ion m/z 393.803 corresponding to heptachlorobiphenyls, (B) Chromatogram of ion m/z 359.842 corresponding to hexachlorobiphenyls and (C) Chromatogram of ion m/z 80.916 corresponding to PBDEs. Reproduced from Ref. [17] with permission from Wiley.
As an interesting example, Focant et al. [55] developed and tested a comprehensive gas chromatography-isotope dilution-time-of-flight mass spectrometry (GC GC-ID-TOF-MS) method for the measurement of selected PCDD/Fs and PCBs in foodstuffs (fish, pork and milk samples). The set of compounds consisted of seven 2,3,7,8-substituted PCDDs, ten 2,3,7,8-substituted PCDFs, four non-ortho-PCBs, eight mono-ortho-PCBs and six indicator PCBs (Aroclor 1260), from a total of 35 compounds. GC GC-ID-TOF-MS results were compared to the confirmatory reference gas chromatography isotope dilution highresolution mass spectrometry (GC-ID-HRMS) method, to the alternative screening gas chromatography isotope dilution quadrupole ion storage tandem mass spectrometry (GC-ID-QIST-MS/MS) method, and to the DR-CALUXs
210
Yolanda Pico´
bioassay screening method. This comparison indicates the good performance of GC GC-ID-TOF-MS. Hoh et al. [56] presented a remarkable study focused on an extensive optimization of GC GC-TOF-MS conditions for analysis of PCDD/Fs by evaluating primary GC oven temperature programs, carrier gas flow rate, GC GC conditions and MS conditions to maximize sensitivity and selectivity in the detection and separation of the 17 PCDD/Fs, in the presence of potentially interfering PCBs. Figure 3 shows how to preserve the separation, HxCDF II eluted immediately after HxCDF I in the 1D arrangement, and HxCDD II eluted just after HxCDD I. These two congener pairs (HxCDF I and II, HxCDD I and II) have identical mass spectra, so they must be chromatographically separated for the congener-specific analysis. The 1D separations were preserved in r4 s modulation periods, as shown in Figure 3A and B, but they co-eluted at the 5 s modulation period, as shown in Figure 3C. The separation gained from the 2D column can be seen in the 2D-axis (on the lower left) in Figure 3D. The resolution (RS) was not much different among the modulation periods, and the separation of the pair was achieved in all the modulation periods, even in the longest modulation period of 4 s (see Figure 3D).
Figure 3 GC GC contour maps of HxCDD I and HxCDD II (m/z 390) obtained from different modulation periods: (A) 1 s; (B) 4 s; (C) 5 s and (D) GC GC surface map for 6.7 pg injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (m/z 322) and 58 pg of CB126 (m/z 254) with 4 s modulation period. Reproduced from Ref. [56] with permission.
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3. TANDEM MASS SPECTROMETRY It has been widely recognized that MS/MS (or MS2) offers very good sensitivity and selectivity in trace analysis of food contaminants. These mass analysers are already used routinely combined with LC in many food safety fields, where LC-MS2 techniques, such as QqQs and ion traps (ITDs), are in common use. More recent approaches in LC-MS2 are LITs, new-generation QqQs and hybrid instruments, quadrupole time-of-flight (QqTOF) and Q-linear traps (Qq-LITs), which are gaining widespread acceptance in several application areas. These instruments offer advantages such as high scanning speeds, accurate mass measurement (QqTOF) and increased sensitivity (LITs and new-generation QqQs).
3.1 Triple quadrupole mass analysers QqQ mass analysers have become the most widely used analytical tool in food analysis. A number of analytical methods, using LC-MS (QqQ) have been developed for the analysis of different classes of priority and emerging contaminants in food samples. LC-MS2 (QqQ) has mostly been applied for the determination of target analytes, using the SRM and achieving LODs of typically nanogram per kilogram (ng/kg) (e.g., in the analysis of growth promoters, the use of QqQ in LC-MS2 has substantially increased the selectivity and the sensitivity of the determination, therefore allowing detection of low, but nevertheless toxicologically relevant, concentrations in the sub-ng/kg range). Their application to food contaminants analysis has allowed the determination of a great number of compounds, especially polar ones that were previously difficult or even impossible to analyse. LC-QqQ-MS/MS is an already well-established technique that presents many applications in food safety. The reader is referred to the chapters on particular contaminants (pesticides, veterinary drugs residues, myco and phycotoxins, growth promoters or food contact materials), where a number of interesting applications are described. Hence, it is not discussed in this chapter. Recent combination of QqQ with GC is still quite a new approach within the field of MS. These instruments have been successfully applied mainly to the analysis of pesticides and PCBs [57]. Unquestionably, MS/MS gives a much higher degree of certainty in analyte identification than any single-stage MS technique, because isobaric interferences are avoided and multiple-component spectra can be resolved. Thanks to this, the confirmation of target analytes can be achieved with a higher level of confidence. Among the different mass analysers that can perform MS/MS, QqQ mass spectrometers have recently been proposed for the determination of pesticide residues in crops [58–60]. A remarkable advantage of the QqQs, in comparison with previously used ion traps, is the possibility of operating in SRM, which is a faster scan mode than product ion scan available on the ion traps. Modern quadrupole instruments are capable of sufficiently fast scan rates for fast-GC-MS. Although considered as one of the most powerful techniques, there are not many references in the literature to the use of gas chromatography/QqQ-MS/MS in the analysis of pesticide residues. Garrido Frenich et al. [58] studied the potential of
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GC with QqQ MS/MS for the multi-residue analysis of pesticides in vegetables, validated the method on cucumbers, then expanded the scope of the method, and validated on strawberries [59]. Okihashi et al. [60] dealt with the simultaneous analysis of 260 pesticide residues in agricultural products. Two MS/MS transitions were selected for each analyte using the intensity ratio obtained from them as a confirmatory parameter. The sensitivity of this method was lower than with most of the selective GC detectors, such as flame photometric or single MS. The selectivity of QqQ gives a very clean chromatogram, making compound identification and confirmation easy. Other examples are the determination of xenoestrogens (pesticides, PCBs and polybrominated diphenyl ethers) in human breast tissues [61], organochlorine and organophosphorous pesticides in animal liver [62] and meat samples [63]. Also, low-pressure gas chromatography/triple quadrupole tandem mass spectrometry (LP-GC-MS/MS) conditions were developed for 78 pesticides but validation data were generated for only 12 pesticides in tomato [64]. In the field of PCBs, GC GC-QqQ-MS/MS is presented by Bucheli and Brandli [15] as a robust, selective and sensitive method for the accurate quantification of enantiomeric fractions of atropisomeric PCBs in several foods. GC GC-MS/MS proved to be superior to coupling technique with less dimensions to quantify atropisomeric PCBs, almost irrespective of their origin and cleanup quality. However, modern QqQ are not fast enough and can only be combined with GC GC in very specific situations.
3.2 Ion-trap mass spectrometers The conventional 3D ion-trap (Paul trap) mass spectrometer has only three electrodes, one ring electrode between two hyperbolic endcap electrodes, which form a three-dimensional trap. The oscillating potential difference established between the ring endcap electrodes forms a substantial quadrupolar field. The ions can be stored for milliseconds or longer in an ITD. Raising the DC potential will isolate ions of a particular m/z by ejecting all of the other ions from the trap. A subsequent increase of the rf potential will eject the stored ions from the trap and send them to the detector. ITD can simultaneously store positive and negative ions for extended periods of time. The principal advantage of ion trap is its ability to perform multiple stages of MS, which increase the amount of information obtainable from a molecule. This characteristic facilitates the identification of unknown compounds making ITD an ideal tool to study fragmentation processes and pathways, increasing, at the same time, sensitivity and selectivity. The fragmentation of isolated ions in an ion trap depends on the frequency of the resonance excitation voltage, the magnitude rf and the duration and trapping rf voltage during resonance excitation. Those features make ITD an attractive option to perform different studies. In fact, the ITD is already a well-established MS technique that can be combined with both LC and GC [65–74]. There are a number of publications using this detector for almost all the possible food contaminants and residues. However, it should be mentioned in this Chapter because it is a very recognized instrument and their ability as confirmation technique makes it almost indispensable in several types of applications.
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The drawbacks of ITD are its inability to trap product ions below m/z 50 and the fragment ions with masses in the lower third of the mass range will not be detected, this drawback is relevant for acrylamide. The reported differences between the product ion mass spectra obtained by this instrument and those obtained by the most conventional and routine QqQ. When fragmentation on an ITD and the QqQ is compared, two factors are to be considered: (i) the amount of energy that can be deposited in an ion — the relative amount of energy that can be deposited within a molecule is greater for a QqQ (uses Ar as collision gas) than for an ITD (uses He), and (ii) the time that the ion is stored on the ITD or the time that the ion spends in the collision cell for the QqQ .The formation of rearrangement product ions versus cleavage product ions is favoured in the ITD. Another drawback is that the ion trap requires clean extracts and more than periodical cleaning of the system. An interesting and recent example of the applicability of gas chromatography coupled with ion trap MS/MS (CG-MS-MS) for the analysis of PCDD/Fs and dioxin-like PCBs in samples containing low levels of these compounds illustrates very well this problem [75]. Vegetable oils were selected for this study because European Union (EU) legislation has established a very low value of maximum concentration levels for them: 0.75 pg WHO-TEQ/g for dioxins and furans and 1.50 pg WHO-TEQ/g for the sum of PCDD/Fs and dioxine-like PCBs (dl-PCBs). The importance of the clean-up when using ion-trap instruments is directly related to the normal operation of this analyser that introduces into the trap a number of ions of m/z range regardless of whether they are from the target compounds or the interfering ions. The decrease of the number of precursor ions produces a reduction of the product ions and, as a consequence, of the signal-to-noise ratio. Therefore, an inadequate clean-up decisively affects the quality of the final results and false negatives may be obtained. The presence of lipid components in the oil extracts due to an insufficient clean-up drastically affected the sensitivity of the ion trap. This problem was only observed in the analysis of the mono-ortho PCB fraction because the polarity of the solvents used (n-hexane:dichloromethane 98:2 (v/v) and dichloromethane:Hexane 50:50 (v:v)) for eluting these compounds from the alumina and carbon adsorbent column favoured the presence of small amounts of lipids. As a result, the chromatograms obtained were characterized by the presence of deformed peaks and elevated signal-to-noise ratios, as can be seen in Figure 4a where the chromatogram of an oil sample is given. In contrast, in Figure 4b nice chromatograms with well-defined peaks were obtained using an additional clean-up step, using a 2-g acid silica (44% sulfuric acid) column and eluting the target compounds with 5 mL of n-hexane. So, GC-MS/MS can be considered a suitable technique for the analysis of PCDD/Fs and dl-PCBs in vegetable oils, but an exhaustive clean-up process of the extracts is required.
3.3 Linear ion trap The LIT mass spectrometer, a recent addition to the MS family, has increased ion capacity, improved ion trapping efficiency and faster cycle times compared
214 Yolanda Pico´
Figure 4 GC-MS/MS chromatograms of mono-ortho PCBs fraction for a vegetable oil sample with: (a) problems in the clean-up process and (b) suitable clean-up process. Reproduced from Ref. [75] with permission.
New Approaches in Mass Spectrometry
215
to the previous one. LIT use a set of quadrupole rods to confine ions radially and a static electrical potential on end electrodes to confine the ions axially. The linear form of the trap can be used as a selective mass filter or as an actual trap by creating a potential well for the ions along the axis of the electrodes. Advantages of the linear trap design are increased ion storage capacity, faster scan times, and simplicity of construction. Recently, LITs have been combined with quadrupole, TOF and FTICR-MS. LITs can be used either as ionaccumulation devices or as commercially available, stand-alone mass spectrometers with MSn capabilities. The combination of QqQ MS with LIT technology in the form of an instrument of configuration Qq-LIT (like a QqQ but the third quadrupole is substituted by the LIT), using axial ejection, is particularly interesting because this instrument retains the classical QqQ scan functions such as SRM, product ion (PI), neutral loss (NL) and precursor ion (PC), while also providing access to sensitive ion-trap experiments. For small molecules, quantitative and qualitative analysis can be performed using the same instrument. Several studies such as determination of Ochratoxin A in wine and beer [76], avermectin residues in food [77] and fluoroquinolones in animal tissues [78], have already been developed using linear ion-trap MS. All these studies showed the good performance characteristics of this system that is replacing the popular QqQ. In this way, Garcia-Reyes et al. [42] presented a new approach based on LC-TOF-MS and LC-MS/MS analysis for the comprehensive screening of target pesticides. The proposed approach consists of three steps. First, samples are analysed by LC-TOF-MS and the proposed method provides the screening and preliminary identification of the species by retention time and m/z mass window, followed by subsequent identification (if positive results) by LC-TOF-MS accurate mass analysis (second step). Final confirmation and reliable quantitation is accomplished in the final step by LC-MS/MS analysis with two SRM transitions. This scenario, proposed for target pesticides, is complemented with the ability of LC-TOF-MS for the analyses of both non-target and unknown pesticides and the possibility of recording a data file as a history of each individual sample. The identification criteria, quantitation capabilities, and effectiveness of the proposed comprehensive screening method have been evaluated with various spiked food extracts and also in real samples obtaining satisfactory results. The potential of the proposed approach was tested with a large number of pesticides (i.e., 100). The optimization of MS parameters (declustering potential (DP), entrance potential (EP), collision cell entrance potential (CEP) for precursor ions, collision energy and collision cell exit potential (CXP) for product ions) was performed by flow injection analysis (FIA) for each compound. Table 2 shows the values of the parameters optimized and the SRM transitions used for the confirmation and quantification of the selected pesticides by LC-LIT-MS. For confirmation of the studied pesticides, two SRM transitions were used, and for quantification, the most intense SRM transition was selected.
Pesticide
MS parameters
tR (min)
SRM1
SRM2
Dwell time (ms)
DP
EP
CEP
Collision energy CXP (SRM1, (SRM1, SRM2) SRM2)
6.2 9.7 8.8 8.8 9.7 10.2 8.8 11.5 15.0 11.5 11.2 13.8 9.8 15.3 12.5 16.3 16.01 18.1 17.3 18.4 17.6 18.2 16.8
167.0/85.1 223.1/106.2 192.4/160.2 202.0/131.2 220.1/72.2 223.2/148.1 271.2/99.1 163.3/106.1 222.0/92.0 292.2/211.2 242.2/185.2 203.1/175.3 303.1/217.3 256.3/209.2 316.2/159.1 230.1/199.2 223.2/126.2 199.1/128.2 266.2/234.1 213.2/75.0 258.2/122.1 253.2/126.1 297.1/159.1
167.0/108.1 223.1/63.0 192.4/132.1 202.0/175.2 220.1/90.1 223.2/86.0 271.2/225.1 163.3/88.1 222.0/77.0 292.2/132.1 242.2/170.2 203.1/104.1 303.1/261.2 256.3/175.1 316.2/191.1 230.1/125.1 223.2/56.1 199.1/111.0 266.2/191.2 213.2/156.0 258.2/201.1 253.2/99.0 297.1/109.2
5 8 5 8 8 5 5 5 5 5 5 5 5 5 5 5 5 8 5 5 10 5 5
37 35 52 59 37 45 51 20 60 41 42 42 66 56 63 38 50 32 64 77 44 52 20
5 6 5 5 6 4 4 5 3 3 5 4 7 5 4 2.6 5 4 6 6 5 5 5
14.90 16.47 15.61 15.88 16.39 16.48 17.82 14.80 16.44 18.41 17.01 15.91 18.71 17.40 19.08 16.67 16.48 15.80 17.68 16.20 17.46 17.32 18.55
(24, 28) (10, 16) (24, 42) (35, 42) (11, 21) (12, 20) (14, 20) (14, 14) (47, 38) (18, 24) (16, 32) (27, 23) (38, 22) (24, 26) (41, 26) (27, 14) (27.5, 31) (12, 23) (26, 46) (18, 12) (24, 11) (27.5, 56) (28, 26)
(2, 2) (1, 1) (3, 2) (2, 2) (1, 1) (3, 2) (3, 1) (1.8, 1.8) (1, 1) (3, 2) (3, 3) (1, 2) (3, 4) (2.5, 2) (2, 2) (2.2, 2.9) (2, 2) (3, 2) (2, 4) (1, 3) (2, 3) (2, 2) (2, 2.5)
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Cyromazine Butoxycarboxin Carbendazim Thiabendazole Oxamyl Aldicarbsulfone Nitempyram Methomyl Chloridazon Thiametoxam Methiocarbsulfoxide Metamitron Cambendazole Imidacloprid Oxfendazole Dimethoate Acetamiprid Cymoxanil Albendazole Butocarboxin Methiocarbsulfone Thiacloprid Imazalil
Identification
216
Table 2 LC-MS/MS method: SRM transitions and values of MS parameters optimized for SRM operation mode using a hybrid triple quadrupole linear ion trap system (QTRAP)
296.2/264.3 213.2/89.1 279.2/219.2 202.1/132.2 255.0/237.2 199.2/72.2 235.2/153.3 200.3/67.0 298.0/144.3 218.3/174.3 242.3/158.2 300.0/268.2 222.3/165.3 213.2/72.0 224.2/109.1 732.7/142.2 233.2/72.1 216.1/174.1 415.1/159.1 280.2/220.2 207.2/72.1 287.1/123.2 233.1/72.1 215.1/148.3 226.2/107.1 746.5/142.2 259.0/170.1 388.2/301.2 408.0/182.3
296.2/105.3 213.2/116.2 279.2/102.1 202.1/124.2 255.0/209.0 199.2/126.1 235.2/136.3 200.3/77.1 298.0/100.0 218.3/160.3 242.3/200.2 300.0/159.3 222.3/123.2 213.2/140.1 224.2/167.3 732.7/189.3 233.2/160.2 216.1/104.1 415.1/227.2 280.2/192.3 207.2/165.2 287.1/72.1 233.1/160.2 215.1/126.1 226.2/164.2 746.5/189.2 259.0/148.2 388.2/165.2 408.0/226.8
5 5 8 5 10 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 8 5 5 5
52 30 54 50 40 26 33 45 43 69 18 55 40 54 31 20 41 47 40 50 56 48 41 40 28 20 39 65 32
8 5 4 5 3 5 5 5 8 7 5 4 5 5 5 5 5 4 6 4 7 5 5 5 5 5 5 5 5
18.52 16.20 18.04 15.89 17.37 15.80 16.81 15.84 18.57 16.34 17.01 18.63 16.45 16.20 16.48 30.74 16.76 16.28 21.85 18.04 16.03 18.27 16.76 16.25 16.56 31.13 17.48 21.01 21.65
(26, (22, (13, (23, (13, (32, (25, (55, (29, (40, (22, (25, (16, (39, (10, (40, (34, (20, (40, (21, (33, (35, (34, (18, (21, (46, (26, (28, (30,
45) 15.8) 12) 25) 24) 34) 43) 58) 43) 44) 33) 46) 20) 32) 26) 42) 35) 40) 26) 22) 18) 23) 35) 22) 8) 40) 21) 40) 30)
(3, 2) (2, 2) (2, 2) (1.7, 1.79) (4, 2) (2, 2) (2, 2) (1, 1) (2, 1) (2, 2) (3, 3) (2, 2) (2, 2) (1, 2) (1, 4) (2, 2) (2, 2) (2.5, 2) (2, 2) (2, 3) (2, 7) (1, 2) (2, 2) (2, 2) (1.8, 2) (2, 2) (3, 3) (2.7, 2.3) (3, 4)
217
17.9 19.2 19.2 19.9 33.3 19.6 19.8 21.9 18.8 21.5 21.29 20.5 21.6 21.6 21.7 19.5 22.06 22.3 20.0 22.3 22.3 22.0 22.5 22.9 22.8 20.2 23.5 23.5 23.2
New Approaches in Mass Spectrometry
Mebendazole Aldicarb Oxadixyl Simazine Fluroxypyr Monuron Lenacil Pyrimethanil Spiroxamine Ethoxyquin Prometryn Fenbendazole Carbofuran Chlorotoluron Bendiocarb SpinosynA Fluometuron Atrazine Miconazole Metalaxyl Isoproturon Difenoxuron Diuron Monolinuron Ethiofencarb SpinosynD Metobromuron Dimethomorph Flazasulfuron
218
Pesticide
Prochloraz Propazine Cyproconazole Methiocarb Terbuthylazine Chloroxuron Bromuconazole Linuron Methidathion Fenamiphos Chlorbromuron Azoxystrobin Promecarb Tebuconazole Diflubenzuron Iprodione Triflumizol Malathion
Identification
MS parameters
tR (min)
SRM1
SRM2
Dwell time (ms)
DP
EP
CEP
Collision energy CXP (SRM1, (SRM1, SRM2) SRM2)
24.0 24.7 24.7 24.9 25.2 24.7 25.1 25.4 25.3 25.1 25.9 25.1 25.68 26.1 26.3 29.0 27.8 27.1
376.1/308.1 230.2/188.2 292.2/70.1 226.2/121.2 230.2/174.3 291.0/72.1 376.0/159.2 249.1/160.0 302.9/145.2 304.2/217.2 293.1/204.1 404.0/372.0 208.1/109.2 308.2/70.0 311.1/141.0 330.0/245.0 346.2/278.2 331.0/127.2
376.1/266.1 230.2/146.2 292.2/125.1 226.2/169.2 230.2/146.1 291.0/218.1 376.0/70.0 249.1/182.2 302.9/84.9 304.2/234.2 293.1/182.2 404.0/344.0 208.1/151.3 308.2/125.2 311.1/158.2 330.0/101.0 346.2/73.1 331.0/285.2
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
40 48 63 20 48 62 47 50 20 63 48 40 32 48 40 50 30 43
5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5
20.76 16.67 18.41 16.56 16.67 18.38 20.76 17.20 18.71 18.74 18.43 21.54 16.05 18.86 18.94 19.47 19.92 19.50
(16, (31, (49, (24, (23, (40, (28, (22, (13, (26, (24, (20, (20, (40, (40, (15, (12, (16,
Source: Reproduced from Ref. [42] with permission
21) 21) 43) 13) 31) 34) 44) 19) 22) 20) 20) 33) 14) 49) 20) 32) 22) 12)
(3.2, 3) (2, 3) (1, 2) (2, 2) (3, 2) (3, 2) (2, 1) (3, 3) (1.3, 1.8) (2, 3) (3, 3) (3, 3) (3, 3) (1, 2) (1.5, 1.8) (4, 2) (3, 2) (2, 3)
Yolanda Pico´
Table 2 (Continued )
New Approaches in Mass Spectrometry
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4. HIGH RESOLUTION MASS SPECTROMETERS IN TANDEM 4.1 Quadrupole time-of-flight mass spectrometer The quadrupole time-of-flight (QqTOF) mass spectrometer has been developed and introduced with the analysis of biopolymers, especially proteins, in mind. Nevertheless, it is also a useful instrument for the determination of small molecules. The QqTOF mass spectrometer consists of an MS1 and collision region adapted from a QqQ instrument and a reflectron-type orthogonal acceleration TOF analyser for MS2. It can basically be seen as a QqQ system with the last quadrupole replaced by a TOF analyser. In recent years, QqTOF instruments for environmental analysis have been accepted more significantly and the number of screening, quantitative and confirmatory methods reported in the literature is steadily increasing. Recent publications include application of LC-QqTOF-MS for: Screening, confirmation and quantitative analysis of target environmental contaminants (e.g., Sudan azo-dyes [25], pesticides and metabolites [79–81]). Screening and elucidation of unknown contaminants present in food [43,82]. Characterization of degradation products of selected contaminants [79,83]. The application of a hybrid QqTOF-MS technique to food contaminants analysis attains an unequivocal confirmation of the contaminants detected. The elimination of false positives and avoiding interpretation ambiguities are possible because of its unique characteristic of generating full-scan product-ion spectra with exact masses. The main fields of application are identification of unknowns and elucidation of structures proposed for transformation products, where the amount of information obtained allows secure identification of the identity of compounds. The advantages of this mass analyser are High resolution (5,000 fwhm), high sensitivity (fmol-amol), impressive mass accuracy, simplicity of calibration and high speed in MS/MS analysis with parallel scanning of MS1 and MS2. QqTOF delivers simple exact mass measurement of precursor and product ions with maximum sensitivity to yield the highest confidence in structural elucidation and databank search results. Compared to the QqQ instruments, QqTOF-MS/MS has the unique capability of determining accurate mass on the fragment ions generated in the collision cell, this ability is particularly important in the structural elucidation of unknowns. For example, the analysis of a pear sample, conventionally treated and taken from a supermarket, yielded the ultra high-performance liquid chromatography UPLC-QqTOF-MS chromatograms depicted in Figure 5, which illustrates the TIC, the extracted ion chromatograms (XICs) of the peaks that could be of interest, the mass spectra of these peaks and the tentative structure. Full-scan data acquisition ranging from m/z 100–500 pointed forward the presence of several small peaks at
220
Yolanda Pico´
Figure 5 (A) Total ion chromatograp (TIC) obtained from the UPLC-TOF-MS analysis of the studied pear extract, (B) extracted ion chromatogram (XIC) of m/z 149, (C) XIC of m/z 202, (D) XIC of m/z 192, (E) XIC of m/z 218 and (F) XIC of m/z 297. The mass spectrum corresponding to each peak is shown as an insert with the most probable structure. Reproduced from Ref. [84] with permission.
221
New Approaches in Mass Spectrometry
the beginning of the chromatogram and a series of main peaks between 3.5 and 6.4 min (Figure 5A). The mass spectra of all the signals visible in the chromatogram were analysed. Tentative identification has been carried out searching those chemically coherent elemental compositions in the Google website or in the ‘‘Merck Index Data Base’’. The peaks that have not been labelled in Figure 5A were identified as different phthalate esters (data not shown), which are extensively used as plasticizers in a variety of materials. On the others, three main peaks (number 2, 6 and 7) present the common mass spectra shown as an insert in Figure 5B, the ions at m/z 149.0209 and 177.0522 correspond with the protonated molecules of N,Nu-dimethylthioxamide and N,Nu-tetramethyldithioxamide. Both chemicals are used as rubber and latex additives and as components of the polymers used in electronic devices like polymer light-emitting diodes. This identification together with the fact that the three peaks eluted at similar time intervals indicated that they come from a plastic contamination. The hypothesis was confirmed later because methanol chromatograms started to show the same mass spectrum after a few injections of the same vial. The main drawbacks of the QqTOF according to these studies are the low dynamic range (response was never found linear for more than two orders of magnitude) and high detection limits compared with those obtained by the QqQ. Table 3 lists the key parameter: LOQs, for a group of selected pesticides which were the same for all the tested matrices [79]. These limits were calculated according to the guidelines of the European Commission for residue analytical methods for post-registration control and monitoring as the lowest analyte concentration that provides acceptable recoveries (W70%) and precision (o20%). QqQ working in SRM reaches, at least, 10-fold higher sensitivity for the majority of the compounds compared to the ITD and QqTOF. It is best suited to attain Table 3
LOQs obtained in fruit after PLE by the three instruments
Compound
Acrinathrin Bupirimate Buprofezin l-cyhalothrin Cyproconazole Fluvalinate Hexaflumuron Kresosim methyl Propanil Pyrifenox Pyriproxyfen Tebufenpyrad
LOQs (mg/kg) QqQ
ITD
QqToF
0.01 0.0001 0.0001 0.01 0.002 0.001 0.01 0.0005 0.001 0.0005 0.002 0.0005
0.01 0.005 0.01 0.4 0.01 0.2 0.02 0.01 0.02 0.01 0.02 0.01
0.02 0.01 0.05 0.3 0.05 0.4 0.04 0.05 0.07 0.03 0.05 0.05
Source: Reproduced from Ref. [79] with permission
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LOQs well-below the strict MRLs regulated for these pesticides, which range from 0.01–5 mg/kg in the different selected fruits. However, it should be noted that for ITD, LOQs are higher than MRLs only for l-cyhalothrin in all matrices, apart from stawberry, and for fluvalinate in strawberry and cherry. For QqTOF, LOQs are higher than MRLs only for four pesticides, which are buprofezin in all matrices except orange, l-cyhalothrin in all matrices except strawberry, cyproconazole in orange, strawberry and cherry, and fluvalinate in strawberry and cherry. The main advantage attributed to this instrument is the unambiguous identification obtained by accurate MS/MS, as illustrated in Figure 6 for carbendazim and ethoxyquin.
4.2 Fourier transform ion cyclotron resonance and orbitrap mass analysers An orbitrap mass analyser is the most recent addition to the set of tools that can be applied to identification, characterization and quantitation of compounds in complex systems. With its ability to deliver low-ppm mass accuracy and extremely high resolution, all within a time scale compatible with nano-LC separations, the orbitrap has become an instrument of choice for many proteomic applications since its commercial introduction in 2005 [85,86]. However, its application to determine food contaminants residues is still rare [24]. Orbitrap is an unconventional ion trap because there is neither radio frequency (RF) nor a magnet to hold ions inside. Instead, moving ions are trapped in an electrostatic field [87,88]. The electrostatic attraction towards the central electrode is compensated by a centrifugal force that arises from the initial tangential velocity of ions: very much like a satellite on orbit. The electrostatic field that the ions experience inside the orbitrap forces them to move in complex spiral patterns. The axial component of these oscillations is independent of initial energy, angles and positions, and can be detected as an image current on the two halves of an electrode encapsulating the orbitrap. A Fourier transform is employed to obtain oscillation frequencies for ions with different masses, resulting in an accurate reading of their m/z. Such measurements achieve very high resolution matching that of FTICR instruments, and surpassing, by an order of magnitude, the resolution presently obtainable with TOF-MS. The orbitrap functions very efficiently as a high-resolving accurate mass detector. Although it may be possible to fragment ions in the orbitrap, it is more practical and faster to manipulate them in another mass analyser to which the orbitrap can be linked as a detector. Several ideas have been tried out to create a powerful hybrid system. Ultimately, it was determined that a LIT is an ideal partner for the orbitrap — it is exquisitely sensitive, very fast and capable of multiple levels of fragmentation commonly referred to as MSn. Often it is advantageous not to stop at MS2. The LTQ-Orbitraps is the only commercially available system featuring the orbitrap mass analyser. It consists of three main parts (Figure 7). The first section after the source is a LIT capable of detecting MS and MSn spectra at very high
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Figure 6 Accurate product ion mass spectrum and proposed fragmentation of (A) carbendazim and (B) ethoxyquin. Proposed fragmentations are shown as an insert. Reproduced from Ref. [84] with permission.
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API ion source
Linear ion Trap
C-Trap
Differential pumping
Orbitrap
Differential pumping
224
Figure 7 Schematic diagram of the LTQ-Orbitrap mass spectrometer.
sensitivity but relatively low resolution and mass accuracy. Ions accumulated in the LIT can be transferred into an RF-only quandrupole called C-trap due to its letter ‘C’ shape. The C-trap accumulates and stores the ions. This additional storage improves the analytical capabilities of the instrument. Also, higher energy collision fragmentations can be done in the C-trap, which is of considerable interest. The last part of the system is the orbitrap itself. It is filled by a pulse of ions accumulated in the C-trap. So in fact, this hybrid contains two complete mass analysers capable of detecting ions and recording spectra: the LIT and the orbitrap. Depending on the requirements for the analysis, the two analysers can be used independently or in concert. Although its application to determine food contaminants and residues is still rare, its wider acceptability is expected. LIT and LTQ-Orbitrap mass analysers were compared for determination of stanozolol and its analogues or metabolites 17-epistanozolol, 3-OH-stanozolol, 4-OH-stanozolol, 17-epi-16-OH-stanozolol, 16-OH-stanozolol, and the synthetic analogues 4-dehydrostanozolol, 17-ketostanozolol and methyl-3-OH-stanozolol [89]. Their structural elucidation by MS/MS provides information that can be utilized to identify further unknown metabolites or artificially modified analogues of stanozolol, such as the recent discoveries of synthetic growth promoters on the basis of the so-called designer steroids. Nielen et al. [90] compared LC coupled with orbitrap with different accurate mass alternatives such as TOF-MS or FTICR. In this study, mass resolution and accuracy are discussed for LC-MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designed b-agonist ‘‘Clenbuterol-R’’ as model substances. LC/QqTOF-MS/MS of stanozolol provides a full accurate mass spectrum of product ions. The ion at m/z 81.0494 confirms the C4H5N2 elemental composition of the pyrazole ring substructure (mass error +4.7 mDa or +58 ppm). However, the higher mass product ions obtained do not confirm stanozolol. The origin of strange accurate masses in the QqTOF-MS/MS analysis of stanozolol soon became apparent: as can be seen in Figure 8b, most of the product ions of stanozolol are
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Figure 8 Characteristic details of (a) the QTOF-MS/MS and (b) LTQMS2/FTICR product ion mass spectra of stanozolol representing one example of the doublet product ions. Reproduced from Ref. [90] with permission.
actually doublets of distinct product ions having only minor exact mass differences. In FTICR and in FT Orbitrap MS these doublets are easily resolved but they will overlap at least partially at 5,000 fwhm mass resolution for m/z 161, as shown by the QqTOF-MS data in Figure 8a. As a result, an artificial single product ion is obtained showing an accurate but wrong average mass value upon mass measurement. This will be a typical result for bench-top (Q)TOF-MS
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instruments; only high-end QqTOF-MS systems providing more than 13,000 resolution (fwhm) at m/z 160 would have resolved these doublets as well. The width of the signal at m/z 161 in Figure 2a might have been a trigger for being an artificial composite from a doublet. It should be noted, however, that such information is not always available since current TOF-MS instruments often show automatic centroiding and mass measurement routines in which detailed information about the peak shape is lost. As an example, a raw isolate of Clenbuterol-R was injected into an LTQMSn/FT Orbitrap MS system operated at a mass resolution of 100,000–200,000 fwhm and the accurate mass product ions thus obtained were compared with the QqTOF-MS/MS data at a mass resolution of 5,000 fwhm. Data shown in Table 4 pointed out that the QqTOF-MS/MS product ions are confirmed by the high resolution and higher mass accuracy data from the LTQMSn/FT Orbitrap MS system. Contrary to stanozolol, no doublet product ions showed up at higher mass resolution. A remarkable difference is the presence of the low intensity m/z Table 4 Comparison of accurate mass MSn and MS/MS data of the designed b-agonist Clenbuterol-R Product Elemental ion (m/z) composition
LTQMSn/FT LTQMSn/FT LC/QTOFMS/ Orbitrap MS Orbitrap MS MS measured measured mass error (ppm)
LC/ QTOFMS/MS mass error (ppm)
471 415 397 385 384 378 350 348 322 304 293 203 195 190 168 167 151 132 94 86
471.1721 415.1092 397.0976 385.0739 384.0665 378.1132 350.1049 n.d. 322.0503 304.0402 293.0241 203.0131 195.0910 189.9814 168.0441 167.0362 151.0175 132.0675 n.d. n.d.
+0.4 +1.0 n.d. +5.1 +6.8 +6.5 2.5 +6.8 +3.0 +3.3 +4.1 +2.8 +6.8 +6.9 +26.3 +12.8 6.1 +3.0 5.6 +12.5
C25H29N4OCl2 C21H21N4OCl2 C21H19N4Cl2 C20H17N3OCl2 C20H16N3OCl2 C19H22N3OCl2 C20H17N3OCl C20H15N3OCl C15H14N3OCl2 C15H12N3Cl2 C14H11N2OCl2 C8H9N2Cl2 C13H11N2 C7H6NOCl2 C8H9N2Cl C8H8N2Cl C8H6NCl C8H8N2 C6H8N C5H12N
Source: Reproduced from Ref. [90] with permission. Note: n.d.: not detected.
+1.7 +1.2 1.3 1.1 +0.0 0.6 1.6 n.d. 1.7 0.3 0.7 3.1 3.5 3.7 1.7 5.1 5.5 5.3 n.d. n.d.
471.1715 415.1091 n.d. 385.0763 384.0691 378.1159 350.1046 348.0922 322.0518 304.0413 293.0255 203.0143 195.0930 189.9834 168.0493 167.0392 151.0174 132.0686 94.0646 86.0975
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397.0976 product ion in the LTQMS2/FT Orbitrap MS spectrum of m/z 471, becoming slightly more abundant in the LTQMS3/FT Orbitrap MS spectrum of m/z 471-415. The apparent loss of water from m/z 415 can be explained by rearrangement only since Clenbuterol-R does not have a hydroxyl but an ether group. The second discrepancy between the QqTOF-MS/MS and the LTQMSn/FT Orbitrap MS results is the product ion at m/z 348. This ion was suggested to be originated from m/z 384 by the neutral loss of HCl. However, contrary to the radical cation at m/z 385.0739, the product ion m/z 384 was hardly formed in LTQMS3/FT Orbitrap MS. The most abundant MS3 product ions are m/z 322.0508, 304.0402, 203.0131 and 195.0910. Note that LTQMS4/FT Orbitrap MS experiments of m/z 471-415-322 confirmed that m/z 304.0402 is not formed by the loss of water from m/z 322.0508 but obviously directly from m/z 415 following rearrangement.
5. CONCLUSIONS AND FUTURE TRENDS MS, combined with chromatography or not, is really needed in a routine food laboratory carrying out monitoring of contaminants and residues. Both GC-MS and LC-MS are indispensable tools to determine food contaminants and residues. Nowadays, LC-MS has become a priority without substituting GC-MS. They are not competitive but complementary techniques. A laboratory for food contaminants and residue analysis requires, at least, both types of instruments because each one has its own field of work. However, advances in MS are not so fundamental for GC-MS because the classical quadrupole with electron impact is still an appropriate technique for many determinations. Advances in GC-MS technology have led to dramatic improvements in sample throughput and sensitivity analytical performance. GC-MS produces a very consistent ionization that makes possible very large electronic libraries of verified standards for sample matching and confirmation of identity. The use of bench-top instruments with QqQ or ITD will certainly continue and increase in future years because of their relative ease of operation, selectivity, unequivocal identification capability and detection limits down to parts per trillion (ppt). GC-MS instruments used for food contaminants and residue analysis range from simple EI quadrupoles to GC GC-TOF-MS instruments that can decrease the detection limits, which will probably become routine in the near future. The new generation of fast-scanning TOF-MSs capable of working at high scan rates (500 scan/s) offers the ideal detection technique to couple with GC GC. This coupling will provide a powerful technique for the qualitative and the quantitative analysis of complex food samples, although software programs for automated handling and processing of the tremendous quantity of data generated by these systems will be required. True computer-assisted chemical analysis by GC GC, with mass spectral detection, appears plausible in the near future. Nowadays, there is a clear trend to increase the number of liquid chromatography applications in food contaminants and residue analysis, both in specific/individual and in multi-residue methods, especially after the
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introduction into the market of robust, easily operated LC-MS instruments that provide a new way of analysing contaminants more sensitively and efficiently. This trend is closely bound to the evolution of modern LC-MS instruments and the improvements in the quality of their performance. MS research is focused mainly on expanding the implementation of mass analysers instead of on ionization sources, as it was 10 years ago. The sensitivity and specificity of pesticide analysis in food matrices have advanced by combining different designs of analysers in order to enlarge versatility and to increase application of MS2 (QqQ, ITD, LIT, QqTOF). In this situation, there is still a question over which instrument should be selected. The answer seems a bit complex, but exact mass seems required more and more in food laboratories. Over the past few years, there has been substantial progress in technologies employing the orthogonal acceleration TOF-MS for improved performance. TOF analysers represent a complementary approach to the routine QqQ for target as well as non-target analysis of a wide range of (semi)volatile organic compounds present in food. TOF and Qq-TOF instruments provide sufficiently accurate mass to carry out unequivocal confirmation of target compounds and investigate unknown contaminants. In this respect, and to clarify it, it is rather easy with a TOF to look for a comprehensive list of metabolites of a known pesticide in a complex sample but it is certainly much more difficult to investigate a real unknown compound. TOF instrumentation still needs to increase the availability of searchable databases of elements. The use of other types of high resolution and accurate mass instruments (e.g., Orbitrap and FT-ICR) is still rare because of their high cost that makes them unaffordable to most food laboratories. In that way, several works proposed an analytical strategy for the monitoring of food contaminants and residues consisted of a comprehensive approach to tackle this analytical problem. Target analysis of contaminants by LC-MS/MS provided accurate quantitative results for a group of selected compounds. Besides, LC-TOF-MS offers unique capabilities for identifying both non-target and unknown compounds, providing a dynamic methodology able to increase the scope of the monitoring program in a fast and simple way, with the inclusion of new compounds [42]. That means, at least, two LC-MS systems will be required for a comprehensive analysis.
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CHAPT ER
8 Capillary-Based Separation Techniques Peter Viberg
Contents
1. 2. 3. 4.
Introduction Miniaturization and the Capillary Format Capillary Liquid Chromatography Capillary Electroseparations 4.1 Instrumentation 4.2 Electroosmotic flow and Joule heating 4.3 Band broadening 4.4 Capillary electrophoresis 4.5 Capillary chromatographic electroseparation techniques 5. Detection Techniques in Capillary-Based Separations 6. Automation and On-Line/In-Capillary Coupled Solid-Phase Extraction in Capillary Electrophoresis 7. Applications in Determination of Food Contaminants Using Capillary Electrophoresis References
231 232 233 235 235 236 239 239 241 250 251 252 253
1. INTRODUCTION The rapid advances in analytical chemistry place great demands on the development of new efficient separation techniques. Frequently, numerous samples, which are provided in very small volumes, need to be analysed rapidly, and the samples often consist of analytes at low concentration in complex mixtures of matrix constituents. One way to tackle these needs is to use highly efficient miniaturized separation techniques, and such techniques have been developed during past decades. This chapter will focus on describing different capillary-based separation techniques. Capillary-based separation techniques comprise several different modes of separation that have gained in popularity due to different miniaturization Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00008-1
r 2008 Elsevier B.V. All rights reserved.
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advantages. The capillary-based separation techniques are associated with low solvent and sample consumption, short analysis time, and sometimes high detection sensitivity. Furthermore, separations obtained using capillary-based separation techniques often show excellent separation efficiencies. These characteristics make them suitable for analysis of small sample amounts, small sample volumes, trace analysis, and high sample throughput analysis. Several capillary-based separation techniques will be described in this chapter, including capillary liquid chromatography (CLC), capillary electrophoresis (CE), capillary electrochromatography (CEC), and capillary electrokinetic chromatography (EKC). Theoretical aspects and separation mechanisms will be discussed together with instrumentation descriptions and different modes of sample treatment. Automation and on-line coupling will also be discussed and practical tips will be included in the text. The different capillary-based techniques will be compared with each other as well as with other traditional separation techniques. The performance of the capillary-based techniques is very beneficial in the field of determination of food contaminants, and different applications within this field will be mentioned.
2. MINIATURIZATION AND THE CAPILLARY FORMAT The trend in analytical chemistry over several decades has been to decrease the dimensions of the analytical equipment. For example, the development of miniaturized liquid chromatography (LC) techniques has evolved from traditional analytical-sized systems, over micro-bore LC, to capillary-based systems (Table 1). It is evident that as the column diameters decrease from analyticalsized columns to capillary columns, the surface-to-volume ratio is dramatically increasing. As a consequence, a potential risk with miniaturized columns is that the surface of the column has undesired effects on the separation. For example, analytes may adsorb to or interact with the capillary wall resulting in spreading and tailing of the analyte zone with resulting less-efficient separations. On the other hand, in electroseparation systems, the large surface-to-volume ratio associated with capillaries is beneficial as it allows for efficient transfer of Joule heat [1] from the capillary (Joule heat appears from the electrical resistance of the Table 1
a
Different column dimensions used for LC
LC type
Internal diameter (mm)
Column volumea (mL)
Surface-to-volume ratio (mm1)
Analytical Microbore Capillary (larger bore) Capillary (smaller bore)
4,600 1,000 100 10
16,600 790 7.9 0.079
0.87 4 40 400
Calculated per metre column.
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background electrolyte when a high voltage is applied over the capillary). Furthermore, the large surface-to-volume ratios in combination with the small column dimensions make open-tubular columns practical. Open-tubular columns have some benefits that will be discussed later. The very low volumes of the separation capillaries require that the dead volumes in the system are kept as small as possible. Connecting tubes must be kept as short as possible and have small internal diameters, or they may result in severe band broadening. Great care must also be taken towards using tubeconnectors that introduce as small dead volumes as possible. ‘‘Zero-dead volume’’ connectors are commercially available, but great care must be taken when cutting the capillaries or tubes to get smooth and flat ends, or else significant dead volumes will be introduced. A 100-mm transfer tube (7.85 mL per meter tube) may add little to the dead volume for an HPLC column with an internal diameter of 4.6 mm, but with a capillary column with a 50-mm internal diameter the dead volume from the tube would be devastating and the analytes risk to spend as long time in the tube as in the columns, with resulting band broadening caused by longitudinal diffusion of the analytes. A great benefit with capillary columns is that they, as a result of their flexibility, often can be connected directly to the valves and detectors without any additional tubes connecting them (providing that the column is not too short). With UV-VIS absorption detection, on-column detection is preferred as it does not add to the dead volume. The small volumes of the capillary columns (Table 1) also results in a low consumption of mobile phases and buffers, which can considerably reduce the costs for the analysis and make it more environmentally friendly. For example, a capillary column with a 100-mm internal diameter consumes considerably less than 1% of the solvent consumed by an analytical-sized column with a diameter of 4.6 mm. When capillary-based separation techniques are discussed, the term capillary usually refers to a capillary made of fused silica coated with polyimide, in which the analytes are separated. Such fused silica capillaries are strong, long-lasting, and robust over wide ranges of buffers and solvents. The polyimide coating protects the capillary from breaking and makes it easy to handle. The capillaries are relatively cheap and readily available in several dimensions. The most widely used separation capillaries have internal diameters ranging from 10 to 200 mm, but several other dimensions exist. In addition to this, capillaries with crosssections other than circular are commercially available, for example, squared capillaries. Fused silica is an ideal material for electroseparations as it provides sufficient electrical insulation, and because it is transparent to UV-VIS light, which enables on-column detection.
3. CAPILLARY LIQUID CHROMATOGRAPHY CLC is a miniaturized version of traditional HPLC, and a CLC system is, therefore, similar to an ordinary HPLC system (Figure 1). The preparation of packed capillary columns with high separation efficiencies have gradually
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INJECTOR SPLITTER
COLUMN
PUMP DETECTOR Mobile phase reservoirs
Waste
Figure 1 Schematic diagram of an example of a typical instrumentation used for LC separations. The splitter can be either included in the pump or it can be externally fitted. In the case of isocratic LC, the waste flow from the splitter may be recirculated back to the mobile phase reservoir and thus reused. The detector may be of on-column or post column type. If a mass spectrometer is used as detector, there will not be a flow from the detector to waste. Injection of sample may be performed either from an external syringe, or from an internal injection device.
developed from columns with internal diameters of several hundreds of micrometer [2,3] down to diameters well below 100 mm [4–6]. The separation in LC is based on partitioning of the analytes between a stationary phase and a mobile phase. Reversed phase chromatography is the most common, but also normal phase and ion-exchange chromatography are frequently performed. The stationary phase is often immobilized on 3–10 mm silica-based particles, but monolithic phases are also commonly used. The very small volumes of the capillary columns (Table 1), require much lower volumetric flow rates of the mobile phase, than analytical-sized equipments do. Typical volumetric flow rates for capillary columns are found in the range from a few mL min1 down to nL min1. Such low flow rates require either special pumps, or that a larger flow is split before entering the separation column. In isocratic LC, splitting may be performed using a fixed splitter. However with gradient LC, due to the changes in the viscosity of the mobile phase over the gradient, a dynamic splitter should be used to maintain a constant volumetric flow rate. Commercial pumps with flow-meters and automatically adjusted splitters are available for such purposes. The small volumes of the capillary columns (and the corresponding small volume of the stationary phase) require small injection volumes of much less than 1 mL to avoid stationary phase over-loading and to avoid peak broadening. Commercial loop injectors are available for sample injections down to tens of nanoliter, but for injection of smaller volumes, timed injections are required. However, larger injection volumes can be tolerated if the analytes are dissolved in a solvent with very low elution strength, in which case the analytes will be stacked on the top of the
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column and the volume will be of less importance. However, there is a risk of over-loading the stationary phase during stacking. A lot of effort has been put into the development of both improved solid phase particles, and of new solid phases for LC. Different types of columns and stationary phases are discussed below under ‘‘CEC with immobilised stationary phases’’.
4. CAPILLARY ELECTROSEPARATIONS Capillary electroseparation techniques include both chromatographic and electrophoretic separations, as well as combinations of the two modes [7,8]. Therefore, analytes can be separated by capillary electroseparation techniques on the basis of either their charge-to-size ratios, or their chromatographic properties.
4.1 Instrumentation The instrumentation normally used for capillary electroseparations is illustrated in Figure 2. The instrumentation includes a capillary (that may contain a stationary phase) in which the separations take place, and a high voltage supply that is used to produce an electroosmotic flow (EOF) through the capillary, and to electrophoretically separate charged analytes. A detector and a means of injecting High Voltage −
+
Capillary
Electrode EOF
Po
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Pi
Inlet vial
Figure 2 Schematic diagram of a typical instrumentation used for capillary electroseparations. The ends of an electrolyte-filled capillary are submerged in electrolytecontaining vials and a high voltage potential is applied over the vials via electrodes. One or both vials can be pressurised (P0 and Pi) to facilitate injections or modifications to the flow velocity during the separation. On-column absorbance detection usually takes place on the outlet region of the capillary. In case of on-line ESI-MS detection, the outlet end of the capillary terminates at the interface of the mass spectrometer instead of in the outlet vial.
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the sample are other important parts of the system. Small and precise volumes can be injected, for example, by applying pressure to the inlet vial for welldefined times (Equation (1) can be used to calculate the injection volume for open-tubular capillaries applied in, for example, CE), or by electrokinetic pumping (where the EOF is used to pump sample into the capillary). During separation, the inlet end of the capillary is submerged in an electrolyte-containing vial, while the outlet end of the capillary terminates in either an electrolytecontaining vial or at the interface to a mass spectrometer. Vinj ¼
DPpd4 t 128ZL
(1)
where Vinj is the injection volume, DP the pressure difference over the capillary, d the internal diameter of the capillary, t the injection time, Z the viscosity of the electrolyte, and L the length of the capillary.
4.2 Electroosmotic flow and Joule heating During capillary electroseparations, a high voltage potential is applied over both ends of the separation capillary. This potential results in electrophoretic transport of electrolyte through the capillary, an EOF [1], as illustrated in Figure 3. In an High Voltage
Electric Double layer
Figure 3 Schematic diagram of an electrolyte-filled fused silica capillary with a high voltage applied over the ends. An electric double layer of cations is formed close to the negatively charged capillary wall (inset), and as a result, a flow (EOF) is created through the capillary. The obtained flat flow velocity profile is illustrated by the arrows. For clarity, anions in the electrolyte are not drawn.
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electrolyte-filled capillary, the negative silanol groups of the fused silica (present above approximately pH 2) will attract cations from the electrolyte resulting in a decreasing concentration gradient of cations from the capillary wall towards the centre of the capillary. Due to ion-pairing between the cations from the electrolyte and the negative charges on the capillary wall, the cations closest to the capillary wall will form a fixed layer known as the Stern layer. The ions just outside of the Stern layer ions will form a diffuse layer, and together these two layers form the electric double layer. In the electric field created in the capillary by the high voltage applied over the capillary, cations in the diffuse layer will move towards the cathode, dragging the bulk liquid along, which results in a flow with a characteristic flat flow velocity profile. The flat flow velocity profile is believed to exist as long as the distance between the charged surfaces (or the diameter of an open-tubular separation capillary) is at least 10 times larger than the width of the electrical double layer [1,7]. This is the case in most practical open-tubular capillary electroseparation capillaries, but may not be the case in the passages between the stationary phase particles in a packed particle column. A flat flow velocity profile is beneficial for separation purposes as it does not add to band broadening [1]. Figure 4 illustrates the difference between the velocity profiles of the EOF and a hydrodynamically pumped flow. A hydrodynamically pumped flow has the highest velocity in the centre of the channel and the flow decreases towards the walls of the channel. Therefore, analytes present at different distances from the channel wall will move at different rates resulting in the spreading of the sample zone. The magnitude of the EOF can be altered by changing the viscosity and permittivity of the electrolyte, being for example, a function of the amount of organic modifiers and the ionic strength [9,10]. Furthermore, the magnitude of the EOF shows a strong dependence with the pH in the electrolyte, as the number of charges of the silanol groups on the capillary wall will change with pH. At a pH below 2, the capillary wall will not be negatively charged and consequently there will be no EOF. The charges on the capillary wall can also be affected by using different coatings adsorbed or bound to the capillary wall [11–15]. The coatings can be, for example, polymers or surfactants, and the aim is often to yield a capillary that produces an EOF that is independent of the pH. By using cationic polymers the capillary wall can be coated with positive charges instead of negative charges and the direction of the EOF can be reversed [16]. The volumetric flow velocity of the electrolyte through the capillary can also be changed by applying pressure to either the inlet or the outlet end of the capillary [17]. Modifications to the volumetric flow velocity can be made, for example, in order to obtain a high enough flow rate to support electrospray ionization (ESI) mass spectrometric (MS) detection (ESI requires a minimum flow rate to result in a stable and quantitative MS detection). To obtain a stable and reproducible EOF, the capillary should be washed with sodium hydroxide solution prior to use, in order to free the negative charges on the capillary wall, followed by careful washing with water and finally running electrolyte.
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Hydrodynamic flow velocity profile (pressure driven)
Flow velocity profile (electrokinetically driven)
Figure 4 Top: Flow velocity profile for a hydrodynamically driven flow that is driven by a pressure gradient over the capillary. Lower: Flow velocity profile for EOF, which is driven by a voltage difference over the capillary.
The electrical current resulting from the high voltage applied over the capillary, in combination with the electrical resistance of the electrolyte, produces heat during an electroseparation. This Joule heating [1,10] is disadvantageous as it results in a heat gradient from the centre of the capillary to its walls, with a corresponding change in viscosity, which disturbs the flat flow velocity profile. Thus, if the Joule heating is not kept low it may lead to band broadening and loss in separation efficiency. If the Joule heating is too strong it may even result in bubble formation or even boiling in the capillary which will destroy the separation and likely result in a current break-down. However, the high surface-to-volume ratio of the capillary allows for efficient heat-transfer from the capillary which enables favourably high voltages to be used, compared to, for example, traditional gel-electrophoresis. The Joule heat can be further controlled by decreasing the current through the capillary by, for example, using
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low ionic strengths of the background electrolyte. Normally, acceptable buffering capacities are obtained with electrolytes with ionic strengths around or below 50 mmol L1.
4.3 Band broadening CE has gained a high interest partly because of its capacity for very high efficiency separations in short times. The small diameter capillaries allow for high field strength to be used, with resulting rapid separations, without being disturbed by Joule heating. Furthermore, the open-tubular column employed eliminates the multiple path term in the van Deemter equation, and the lack of stationary phase eliminates mass transfer associated band broadening. Because of this and the flat flow velocity profile in CE, the main reason for band broadening in CE is longitudinal diffusion of the analytes, which is why CE often yields extra high separation efficiencies with macromolecules that have very slow diffusion rates (minimising band broadening from longitudinal diffusion in the capillary). Also CEC and EKC benefit from flat flow velocity profiles, but the use of stationary phases in CEC and in CLC, and pseudostationary phases in EKC, will add to the band broadening. Band broadening is partly the result of limitations in mass transfer with these chromatographic techniques. The limitations in mass transfer results in some analytes being swept away by the mobile phase (or the background electrolyte) before they have time to equilibrate with the stationary (or pseudostationary) phase, while other analytes still remain in the stationary (or pseudostationary) phase. By reducing the particle diameters in packed columns the distances between the particles will be reduced, which results in improved mass transfer. However, particularly with CLC, the pressure drops with small particles add practical lower limits around 3 mm particles or slightly below. With pseudostationary phases in CEC and EKC systems however, no pressure drops occur and very efficient separations can be obtained with, for example, nanoparticles or micelles that have very small interparticle distances in the background electrolyte. Several other effects also affect the band broadening in capillary separations, for example, the monodispersity of the stationary phase particles. Also Eddy diffusion and the porosity of stationary or pseudostationary phases will affect band broadening.
4.4 Capillary electrophoresis Electrophoresis was first described as a separation technique by Tiselius in the first part of the twentieth century [18–20]. The first step towards miniaturization of electrophoretic separation was taken by Hjerte´n in the 1960s, when he described electrophoresis in narrow bore tubes [21]. CE in its modern form occurred in the early 1980s [22–24]. CE, as other electrophoretic separation techniques, is based on different mobilities of analytes with differences in their charge-to-size ratios, in an electrical field. Therefore only charged analytes, and
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not neutrals, can be separated in CE. An ion in an electrical field has a mobility expressed by Equation (2). q (2) mep ¼ 6pZR where mep is the electrophoretic mobility of the ion, q the charge of the ion, Z the viscosity of the electrolyte, and R the ion radius. Thus, according to Equation (2), the larger the size of the ion the smaller the electrophoretic mobility, and the higher the charge, the larger the electrophoretic mobility. The reasons for this being that a larger analyte ion has a greater friction in the background electrolyte than a smaller and that a higher charge gives the ion a stronger force in the electric field. The relation between electrophoretic velocity, electrophoretic mobility, and electric field strength is given by Equation (3). uep ¼ mep E
(3)
where uep is the electrophoretic velocity of the ion and E the electric field strength. During a CE separation, a charged analyte will have a velocity and a mobility through the capillary that is affected both by the electrophoretic mobility of the ions and the EOF. The apparent mobility of an ion is the sum of the electrophoretic mobility of that ion and the electroosmotic mobility, which can be obtained from experimental data by calculating the velocity for the analyte and that of an uncharged species, respectively and converting these velocities into mobilities. A drawback with CE is its limitations in the analysis of diluted samples. The lack of a stationary phase in CE does not allow for sample stacking, as is commonly employed in chromatographic techniques. Instead, several sample concentration techniques have been developed for CE: Field amplified stacking (FASS) [25–27] is used as an easy and straightforward means to concentrate the analytes in the sample plug in the capillary. FASS relies on a much lower ionic strength in the sample compared to the background electrolyte surrounding the sample in the capillary. A lower ionic strength means a lower electrical conductivity and therefore a higher electrical resistance, resulting in higher field strength in the sample plug compared to in the surrounding background electrolyte. According to Equation (3), an increase in field strength will result in a higher velocity for charged species. In practise, this means that analyte ions will move fast through the sample plug until they reach the interface with the background electrolyte where they will be subjected to a lower field strength resulting in a lower velocity and consequently the analytes will be concentrated, or stacked, in a thinner band. It is therefore often recommended for CE that samples are prepared with a low conductivity, or that samples with high salt concentrations are desalted prior to analysis. In isoelectric focusing (IEF) [28], the separation is based on different isoelectric points (pI) of the analytes. A pH gradient is maintained in the capillary and the analytes will, when a high voltage is applied over the capillary, move in the gradient until they reach the point where their pI equals the pH. There they will be electrically neutral and their movement will be terminated
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and the analytes will be concentrated. Other efficient concentrating techniques applicable to the capillary format include isotachophoresis [29,30] and sweeping [31]. CE can also be combined with different types of on-line or in-capillary solid phase extractions (SPE) techniques. In addition to these sample concentration techniques different improvements can be made to the detection of diluted samples (see section Detection techniques in capillary-based separations).
4.5 Capillary chromatographic electroseparation techniques In capillary chromatographic electroseparation techniques, the separation is based on partitioning of analytes between two phases that move at differential velocities [1,8]. In that sense, capillary chromatographic electroseparation techniques are similar to other types of chromatographic techniques. However, in contrast to, for example, LC, the flow through the column is generated by electroosmosis [7]. Often one of the phases is a solid stationary phase and the other a liquid mobile phase. The most straightforward example of capillary chromatographic electroseparation techniques being the use of a capillary column packed with stationary phase particles through which a mobile phase is pumped by EOF [23,32], in which case the technique generally is referred to as CEC. Other examples include the use of a moving stationary phase, or a pseudostationary phase. Such techniques are generally referred to as EKC techniques. Because of the stationary phase, neutral analytes can be separated in capillary chromatographic electroseparation techniques, but also charged analytes, which can be separated by combined forces of chromatographic and electrophoretic separation mechanisms. Capillary electrochromatographic separation techniques combine the excellent separation resolution of HPLC with the high-efficiency separations obtained with CE, which make these techniques very advantageous.
4.5.1 CEC with immobilized stationary phases The first successful development of CEC was performed in the 1980s, using 170 mm i.d. capillary columns packed with 10 mm particles [23]. Since then, improved columns have been developed, with smaller internal diameters and smaller stationary phase particles. The capillary columns employed for CEC are often the same as can be used with CLC systems. Because electroosmosis is used to pump the mobile phase in CEC, instead of a pressure difference as in LC, the pressure drop over the column is not as important in CEC as it is in LC, and very small stationary phase particles can be used in the CEC columns. During the past 10 years, several monolithic stationary phases have been developed. A monolith (Figure 5), also known as a continuous bed, has a spongelike structure and it can be made from several different materials, including plastic polymers [33–36] and silica [37]. The monolithic columns often result in lower back pressures compared to packed stationary phases, resulting in more rapid conditioning of the column.
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Figure 5 Scanning electron micrographs of a monolithic packing in a 75-mm inner diameter fused silica capillary. The micrograph is taken at the end of the cut capillary. The inner wall was treated, prior to polymerization of the monolith, with g-(trimethoxysilyl)propyl methacrylate containing the inhibitor DPPH. Reproduced with permission from Ref. [97]. Copyright (1999) Elsevier.
Open-tubular stationary phases for CEC have been prepared by in-situ polymerization of monolithic plastic polymers [38] (Figure 6), and by adsorption of gold nanoparticles to the capillary wall [39]. Open-tubular columns have the benefit of a flat flow velocity profile over its whole crosssection but suffer from lower sample capacities than packed or monolithic columns do.
4.5.2 CEC with pseudostationary phases — electrokinetic chromatography In the mid-1980s, the first attempt of EKC using micelles as a pseudostationary phase in micellar EKC (MEKC) was presented [40,41]. Excellent separations of neutral analytes were obtained, as illustrated by Figure 7. The separation in MEKC is based on surfactants that form micelles acting as a pseudostationary phase in the background electrolyte. Several other pseudostationary phases have also been employed, including cyclodextrins [42,43], proteins [44], liposomes [45], microemulsions [46–48], polymeric micelles [49], and polymeric microparticles and nanoparticles [50–52]. In order to function efficiently, the pseudostationary phases must form stable solutions or suspensions and they must have properties that allow them to interact with the analytes. The interactions can be based on, for example, reversed phase mechanisms, as in Ref. [41] (Figure 7). Figure 8 illustrates that excellent chromatographic resolution can be obtained with microemulsion EKC (MEEKC) [46]. In MEEKC, an organic solvent that is
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Figure 6 Scanning electron micrographs of three molecularly imprinted monolithic polymer coatings on the wall of a 50-mm open-tubular capillary column used for CEC. The three coatings were synthesized using different solvents, or porogens, but with otherwise identical prepolymerization mixtures and conditions: A, toluene; B, dichloromethane; C, acetonitrile. The white bars indicate a 5-mm distance. Reprinted with permission from [38]. Copyright (2002) American Chemical Society.
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4 6 5 2
14 12 1315
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Figure 7 MEKC separations using SDS-micelles as pseudostationary phase. (1) water, (2) acetylacetone, (3) phenol, (4) o-cresol, (5) m-cresol, (6) p-cresol, (7) o-chlorophenol, (8) m-chlorophenol, (9) p-chlorophenol, (10) 2,6-xylenol, (11) 2,3-xylenol, (12) 2,5-xylenol, (13) 3,4-xylenol, (14) 3,5-xylenol, (15) 2,4-xylenol, (16) p-ethylphenol. Please see Ref. [41] for further details. Reprinted with permission from [41]. Copyright (1983) American Chemical Society.
incorporated in micelles is added to the background electrolyte. Other binding or interaction mechanisms can also be used with EKC. The use of chiral cyclodextrins is an efficient means of obtaining enantiomeric separations through host–guest complexations, because of differences in interaction properties between the L and D form of analytes [43]. Figure 9 illustrates separations of optical isomers obtained through the use of cyclodextrins as pseudostationary phase in EKC. Molecularly imprinted polymeric microparticles suspended in background electrolyte have also been used in enantiomer separations [50], as illustrated by Figure 10. Molecular imprints are cavities created in polymers that have a predetermined selectivity towards special analytes that is based on, for example, size, charge, or hydrogen bonds [33,53]. The use of polymeric nanoparticles as pseudostationary phases is of growing interest [54]. A nanoparticle-based CEC technique combined with ESI-MS detection, termed ‘‘continuous full filling (CFF)’’ was reported in 2002 [51]. This CFF technique involves the use of stable suspensions of nanoparticles (approximately 50–400 nm in diameter) in a background electrolyte. In CFF experiments, the sample solution is injected into a capillary that is filled with a nanoparticle suspension, and the separation is performed with nanoparticle suspension as running electrolyte, resulting in a continuous flow of nanoparticles through the capillary throughout the analysis. Therefore, an orthogonal ESI source was used to separate the analytes from the nanoparticles and thereby to
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mAU A 40
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Figure 8 Comparison of microemulsion EKC and MEKC. Capillary: 50 mm inner diameter, 48.5 cm long (40 cm to detector). Voltage, 20 kV; detection, 215 nm. (A) Microemulsion EKC: 0.8% 5-chloropentane, 3.3% SDS, 6.6% n-butanol, and 89.3% 10 mM sodium tetraborate pH 9.2. (B) MEKC: 100 mM SDS in 10 mM sodium tetraborate pH 9.2. The peaks are: 1, caffeine; 2, terbutaline; 3, tropic acid; 4, cinnamic acid; 5, pindolol; 6, hydrocortisone; and 7, prednisolon. Reproduced with permission from Ref. [47]. Copyright (2003) Elsevier.
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7 8
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migration time (min)
Figure 9 Electropherograms obtained from EKC separations of optical isomers with cyclodextrins as pseudostationary phase. Left: norephedrine and ephedrine. Right: norepinephrine, epinephrine, and isoproterenol. 1, ()norephedrine; 2, (+)norephedrine; 3, ()ephedrine; 4, (+)ephedrine; 5, ()norepinephrine; 6, (+)norepinephrine; 7, (–)epinephrine; 8, (+)epinephrine; 9, ()isoproterenol; and 10, (+)isoproterenol. Further information can be found in Ref. [42]. Reproduced with permission from Ref. [42]. Copyright (1989) Elsevier.
Figure 10 Electrochromatogram of an enantiomer separation of propranolol, obtained by CEC using molecularly imprinted microparticles as pseudostationary phase. The microparticles were suspended in the electrolyte to 5 mg mL1 and were introduced by applying 50 mbar for 4 s, which corresponds to 11.8 cm of the capillary length. The capillary was of 100 mm i.d., 35 cm total length, and 26.5 cm effective length. Sample (25 mmol L1) was injected electrokinetically at 5 kV for 4 s. The separation was performed by applying 15 kV (429 V cm1) at 5 bar overpressure. Obtained from Ref. [50] — Reproduced by permission of The Royal Society of Chemistry.
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hinder the nanoparticles from entering and contaminating the mass spectrometer (Figure 11). Thus, MS detection is enjoyed with its high sensitivity, and other benefits, without the detection being disturbed by the nanoparticles. While the first report on CFF-CEC [51] demonstrated the proof-of-principle for the technique with ion exchange chromatographic separations, later studies focused on reversed phase separations [52,55,56]. Reversed phase separations were made possible through the use of charged nanoparticles having a hydrophobic core and a hydrophilic surface, thus enabling stable nanoparticle suspensions to be made in background electrolytes with low concentrations of organic modifiers. Separations of neutral phthalate esters were performed that resulted in very high separation efficiencies with up to over one million theoretical plates per meter column [57]. The high separation efficiencies are partly believed to be the result of high concentrations of small nanoparticles in the suspensions with resulting small distances between the nanoparticles and therefore efficient mass transfer. The interparticle distances in the study were approximately 0.7 mm [58].
Nanoparticle Analyte ions Electrospray Plume
Entrance to mass spectrometer
Flow direction in capillary
End of capillary
Flow of nebulizer gas
Flow of sheath liquid
Figure 11 Schematic illustration of the orthogonal electrospray interface during continuous full filling CEC-MS analysis. Positive ions are pulled out of the electrospray plume and accelerated in the electrical field directed towards the inlet to the mass spectrometer. Analyte ions enter the mass spectrometer where they are analysed, while the much more massive nanoparticles go to waste. Reprinted with permission from Ref. [51]. Copyright (2002) American Chemical Society.
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4.5.3 Limitations with stationary and pseudostationary phases In all analyses involving stationary phases, there is a risk of contamination of the stationary phase. The contamination may occur either from the analytes themselves, or from molecules present in the sample matrix. Contamination is particularly a risk during analysis of samples present in complex matrices. Such contamination of the stationary phase can result in changed retention behaviour of the analytes and an increased noise during the detection. Furthermore, there is also the risk that the column is clogged or even destroyed as a result of adsorbing contaminants. Quantification of particularly low abundant analytes may be impossible or erroneous if analytes from previous runs are remaining and eluting from the column. If, for example, biological samples, such as blood plasma or urine are to be analysed, a sample pre-treatment step may be necessary to avoid severe memory effects or even destroying the column. Contamination problems can be overcome through the use of a pseudostationary phase instead of an immobilised stationary phase. The pseudostationary phase is constantly replaced throughout the analysis, thus regenerating the column during each analysis, allowing each sample to be injected on a new and fresh pseudostationary phase. With pseudostationary phases, possible memory effects will have to be due to either contamination of the capillary walls, or be related to carry-over effects (e.g., via the electrodes and the electrolyte vials). Fortunately, the lack of an immobilized stationary phase combined with the strength of an open-tubular fused silica capillary allows for thorough and rapid rinsing between runs that can remove contaminants from the capillary wall. In addition to this, the use of pseudostationary phases avoids the need for retaining frits that otherwise may add to band broadening [59,60]. Although solving problems with memory effects, the pseudostationary phases may have negative effects on the detection. With absorbance or fluorescent detection, light scattering, fluorescence and absorbance by the pseudostationary phase will add to the background noise, often making the analytes completely undetectable. Mass spectrometry is often a desired detection technique, as it can provide both quantitative and structural information on the analytes. However, also with MS detection, the pseudostationary phase can add noise, and it may also have a suppressing effect on the ionization of the analytes [61]. The mass spectrometer can also be contaminated by the pseudostationary phase, and may not even be compatible with some phases. In one study using MEKC with sodium dodecyl sulphate (SDS), the signal from the ESI-MS detection decreased by 65% during one day of analysis and the ion source had to be cleaned on a daily basis [62]. One way to circumvent detection problems associated with the use of pseudostationary phases, is to use the partial filling technique that was developed in the early 1990s [44]. In partial filling, the pseudostationary phase is injected into the capillary as a small plug (Figure 12). The benefit with the technique is that the analytes will be separated as they are passing the plug of pseudostationary phase, but will be detected before the pseudostationary phase reaches the detector. Thus, detection interferences from the pseudostationary phase will be avoided or minimized. However, the partial
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A
B
C
D
Figure 12 Schematic diagram illustrating the use of a pseudostationary phase in the partial filling technique. The background electrolyte is illustrated by grey fields. The pseudostationary phase is illustrated by grey dots and the sample is illustrated by black bars. (A) The capillary is filled with background electrolyte. (B) A plug of pseudostationary phase is injected into the capillary. (C) The sample is injected into the capillary. (D) The sample moves faster than the pseudostationary phase and is separated as it passes the plug of pseudostationary phase. When the experimental conditions are optimized, the sample will be eluting from the capillary before the pseudostationary phase.
filling technique has some disadvantages. Time-consuming optimizations that limit the separation may be necessary in order to allow the sample to completely pass the pseudostationary phase prior to reaching the detector, i.e., the sample needs to be moving at a rate considerably faster than the pseudostationary phase, or the plug of pseudostationary phase must be kept very small. The latter may severely limit the retention factors of the analytes and therefore limit the resolution of the separation. Furthermore, the EOF is likely to be different in the plug compared to the surrounding background electrolyte, which will result in pressure differences and induced parabolic flow rate profiles that can result in band broadening and reduced separation efficiency. Partial filling has been used with several different types of pseudostationary phases including proteins [44], micelles [63], and polymeric microparticles and nanoparticles [50,51]. Some of these issues regarding detection interferences with pseudostationary phases and limitations to the partial filling technique are dealt with in the nanoparticle-based CFF-CEC technique [51]. In the CFF-CEC technique, an orthogonal ESI-MS interface (Figure 11) is used to enable efficient detection of the analytes while at the same time excluding the nanoparticles of the pseudostationary phase from entering the mass spectrometer, as was described earlier under CEC with pseudostationary phases — EKC.
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5. DETECTION TECHNIQUES IN CAPILLARY-BASED SEPARATIONS Many different types of detectors exist but the focus in this chapter will be on absorbance, fluorescence, and electrospray MS techniques. Special considerations must often be taken when choosing a detector for a capillary-based separation technique because of the often very low volumetric flow velocities, small column/capillary volumes, and small sample volumes associated with these techniques. Minimizing the dead volumes is necessary to avoid severe band broadening, as was previously discussed. Furthermore, a concentration sensitive detector is often beneficial because of the low volumetric flow rate, with its resulting low analyte mass flow. Because of the light transparent properties of the fused silica capillary (when the polyimide coating is removed by, for example, burning or scratching), optical detection can be performed directly on the column, which does not add any extra dead volumes. However, on-column detection suffers from short optical path-lengths and therefore by poor detection sensitivity. Different solutions to improve the pathlength and the detection sensitivity have been developed [64] and two of them are presented in Figure 13. A bubble cell [65–67] takes advantage of an increased internal diameter over a small portion of the capillary. The use of a bubble cell has proved to increase the detection signal-to-noise, but it does on the other hand induce band broadening. A z-cell [68,69] can increase the optical path-length even further than a bubble cell, but losses in the separation resolution have been observed [68]. Path length
P0 A P
P0
Path length
B
P
Figure 13 Schematic illustrations of (A) a bubble cell and (B) a z-cell. P0 indicates the incoming light and P the outgoing light from the capillary. The path lengths for the light are longer than that for a standard capillary.
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Electrospray mass spectrometry [70,71] is an attractive detection option for on-line coupling to capillary separation techniques, as it provides high sensitivity and is compatible with the low volumetric flow rates associated with these techniques (normal ESI can typically be used with volumetric flow rates of a few mL min1). However, CLC, CEC, and CE separations using small diameter capillaries are frequently performed in the nL min1 region, and such low flow velocities result in unstable and non-quantitative ESI-MS detection unless a sheath-flow is used. The sheath-flow is pumped coaxially to the outlet of the separation capillary mixing with its effluent at the interface to the mass spectrometer and therefore sheath-flows have the disadvantage of diluting the sample and thereby decreasing the MS signal intensity. The low flow rate version of ESI termed nanoelectrospray (nESI), or microelectrospray (mESI) [72,73] has working ranges suitable for many capillary-based separation techniques, without the need for a sheath-flow. The use of nESI often greatly increases the signal intensities, compared to standard ESI. In nESI the effluent from the separation capillary is electrosprayed from a small diameter capillary (a few micrometer) with thin walls, which results in a very efficient ionization of the analytes. ESI and nESI are often performed in positive-mode, in which the analytes are positively charged by protons. Positive ionization is therefore best performed at low pH, which may be problematic if a high pH mobile phase or electrolyte is required for the separation. In those cases, it may be best if a sheath-flow is used that has a low enough pH to break the buffered effluent from the separation capillary. If open-tubular separation capillaries are used in combination with sheath-flow assisted ESI, the eluent times for the analytes are often decreased as a result of an increased volumetric flow rate caused by the suction-force from the sheath flow. This does not just change the elution times, but it also induces a parabolic flow-profile with a resulting band broadening of the peaks. This effect can be minimized by using a separation capillary with a small internal diameter. When choosing buffer salts for the mobile phase or background electrolyte, it is important to choose salts that are easily evaporated, not to cause contamination of the mass spectrometer or disturbance of the ESI process. Buffer salts of shortchained organic acids and ammonia, for example, ammonium formate and ammonium acetate are commonly used, as well as ammonium carbonate. Together these salts buffer a pH range from approximately 3 to 10, which should be sufficient for most separation situations.
6. AUTOMATION AND ON-LINE/IN-CAPILLARY COUPLED SOLID-PHASE EXTRACTION IN CAPILLARY ELECTROPHORESIS Commercial CE and CEC instruments as well as CLC systems are highly automated and can be operated over a long time without much labour needed. Several sample and electrolyte or mobile phase vials can be loaded and automatically switched between, and pre-separation conditionings and washing steps can be programmed. However, analysis of samples present in for example
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complex matrices may also require a sample pre-treatment step to clean and concentrate the analytes, in addition to the sample separation step. Sample pretreatment techniques are often laborious and can be a bottle-neck in the analysis chain unless they are automated and coupled on-line to the separation system. When analytical methods are designed to be used with automated instruments for the analysis of perhaps hundreds of samples per day, unmonitored by man, it is of great importance that the methods perform reliably with high repeatability and reproducibility. Several different solutions have been proposed and developed for on-line couplings and in-capillary couplings of sample pretreatment to capillary electroseparation systems. SPE is a commonly used sample pre-treatment technique that is employed to remove contaminants from the sample, which may otherwise clog the separation capillary and increase the background noise during detection. In addition to this, SPE is performed to pre-concentrate the analytes to improve the limit-ofdetection. SPE relies on strong interactions between the analytes and a solidphase, and it is a special case of chromatography with very high retention factors. The retention can be based on reversed phase mechanisms, ionic interactions, or other properties of the solid-phase and the analytes. SPE is ideal for in-capillary coupling to CE, as the lack of a stationary phase in CE make cleaning and washing of the extracted sample straightforward with a minimized risk of contaminating the separation capillary. SPE-CE is an important complement to the in-capillary concentration techniques described above under CE, as SPE is a very efficient means to handle complex or contaminated sample matrices. For efficient coupling with CE, miniaturization of the SPE-equipment is important [74,75] and such equipments are often known as solid-phase micro extraction (SPME) or micro-SPE (mSPE). mSPE can be performed on a short bed of packed particles inside the CE capillary [76–80]. Also membranes [81–83] have been used as solid-phase matrices in on-line approaches of SPE-CE. Several other matrices have also been employed for SPE coupled on-line or in-capillary to CE [74,75], such as monolithic phases [84,85].
7. APPLICATIONS IN DETERMINATION OF FOOD CONTAMINANTS USING CAPILLARY ELECTROPHORESIS Determination of contaminants in food is a great challenge as the contaminants often are present in very low concentrations and in complex mixtures and sample matrices. Therefore, the high separation efficiencies that can be obtained with capillary-based separation techniques (as has been discussed in this chapter) is of great value for determination of food contaminants. When complex sample matrices are analysed, and low abundant compounds determined, sample pretreatment and sample clean-up steps may be vital to obtain accurate analysis results. The challenge is often to concentrate the analytes of interest while at the same time getting rid of the sample matrix compounds present at much higher concentrations.
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CE is a separation technique that has gained great interest and that has been widely used within the field of determination of food contaminants and food analysis [86–88]. Several different application areas have been found, including determination of amino acids, vitamins, organic acids, proteins, different food additives, toxins, antioxidants, phenols, pigments, mutagenic compounds, and pesticides, which is evident from the many reviews written within this field (e.g., Refs. [86–89]). To tackle the complex matrices of foods, different sample clean-up techniques have been employed prior to analysis by CE. For example, different microextraction techniques, including examples of interfacing with CE, have recently been reviewed in Refs. [90–92]. The capillary separation techniques have several benefits, as has been discussed in this chapter, including high separation efficiencies, low LODs, low sample consumptions, possibilities of interfacing with sample pre-treatment techniques, and possibilities for automation of the analyses. These techniques, therefore, are very useful for demanding analyses and well suited for determination of food contaminants. They will likely develop for applications in food analyses and their use will increase in the future. Miniaturized analytical techniques based on the chip format [93–95] have not been discussed in this chapter, but chip analyses have many miniaturization benefits, which make them useful for demanding separations, particularly when high speed and high through-put analyses are needed, and they have gained a great interest in the field of food analysis [96], an interest that is likely to increase.
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CHAPT ER
9 Pesticide Residues Anna Sannino
Contents
1. 2. 3. 4. 5.
Introduction Physical and Chemical Properties Health Effects Analytical Methods Instrumental Determination 5.1 Gas chromatography 5.2 Liquid chromatography 6. LC-MS-MS Applications 6.1 Chlormequat 6.2 Pesticides of new generation 7. Conclusions and Future Trends References
257 258 259 272 278 279 289 295 295 296 301 302
1. INTRODUCTION The term ‘‘pesticide’’ covers a wide range of substances that belong to many completely different chemical groups. The Food and Agriculture Organization (FAO) of the United Nations has defined a pesticide as a substance or a mixture of substances intended to prevent, destroy or control any pest [1]. Also included in the FAO definition are chemicals intended for use as plant growth regulators, defoliants or desiccants, even though they are neither normally employed as pest control agents nor usually effective as such. Pesticides include a great variety of chemicals used widely in agriculture since significant economic damage can occur when insects, nematodes, fungi and other macro- and microorganisms affect food and commodity crops [2]. Large scale use of pesticides began after World War II, when the agriculture production of food
Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00009-3
r 2008 Elsevier B.V. All rights reserved.
257
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Anna Sannino
accelerated. Currently, more than 800 active substances are formulated in pesticide products [3]. This chapter aims to describe the issues surrounding pesticide residues in food and in particular the issues associated with the analysis of pesticide residues. The state of art of the individual steps (extraction, clean-up, identification, quantitation) of methods for pesticide analysis in food is critically reviewed with emphasis laid on emerging techniques which have gained popularity. The first part of this chapter outlines some of the different types of pesticides, which can be classified by chemical structure, target pest or mode of activity. A brief introduction summarizes the properties of pesticides and the regulations that highlight its importance to meet the official requirements on analytical performance.
2. PHYSICAL AND CHEMICAL PROPERTIES Pesticides may be classified in according to the target on which they act. Examples are herbicides, which are used for weed control, insecticides that destroy insects, fungicides that control fungi, etc. They can also be grouped according to their known, or assumed, biochemical mode of action (e.g., insect growth regulators, acetylcholinesterase inhibitors). According to their chemical nature, the pesticides can be divided into two chemical groups: inorganic and organic compounds. Most pesticides used today are organic compounds. A small number are either extracted or derived directly from plants. Examples include products such as pyrethrum, rotenone and nicotine. Synthetic pesticides cover a very wide range of chemical structural types. Their use to control pests and diseases has become widespread in the 20th century. Pesticides commercially available today are characterized by such a variety of chemical structure and functional groups to make their chemical classification quite complex. The major chemical groups of the most classical categories of pesticides are given below: (i)
(ii)
(iii)
Organochlorines (OCs) are a commonly used term referring to a group of hydrocarbons with one or more chlorine atoms which include the cyclodienes, DDT and related compounds, lindane, and hexachlorocyclohexanes and toxaphene. However, their chemical stability and fat solubility were shown to give environmental hazards leading to an ability to accumulate in humans and animals. Therefore, DDT and other chemically stable OCs have been taken out of agricultural use in most countries. Organophosphorates (OPs) integrated by esters of phosphoric, phosphonic, phorothionic or related acids. They are commonly used as insecticides in a variety of crops. Some of the more common examples in this group are dichlorvos, malathion, diazinon, and phosmet. Carbamates (e.g., carbaryl and propoxur) formed by salts and esters of carbamic acid. They form an important group of insecticides. Chemicals
Pesticide Residues
(iv) (v) (vi)
259
which are structurally related to these carbamates have also been developed as fungicides, herbicides and molluscicides. Pyrethroids (e.g., D-phenothrin) which mimic the structure and action of naturally occurring pyrethrins are very widely used as insecticides. 1,3,5-triazines (e.g., atrazine and simazine) which are an important chemical class of herbicides. Substituted ureas, which comprise a large number of groups as phenylureas and sulfonylureas that are herbicides and benzoylureas that have insecticide activity.
These compounds represent the most classical chemical categories of pesticides. For the past several decades, approximately 80% of the insecticide market has been taken up by organophosphorates, carbamates and pyrethroids. However, other types of compounds are taking an increasing portion of the market. The use of pesticides has changed through the years, going from more persistent (e.g., organochlorine) to more polar, readily degradable pesticides, such as N-methyl carbamates (NMCs). Moreover, synthetic nicotinoids and neonicotinoids (related to the natural compound nicotine as the pyrethroids are related to the pyrethrum) are gaining increasing importance. Table 1 shows the chemical structure of representative compounds of the principal pesticide groups used in agriculture. Recently, a new group of pesticides has been developed for plant protection at low doses, with a reversible mode of action, less persistence, systemic action and adequate potency against crop pests. Agrochemicals belonging to carboxamide, quinazolin, phenoxypyrazol, strobilurin, pyrimidine, triazol, neonicotinoid, carbamate, morpholine classes are representative of the newly introduced molecules.
3. HEALTH EFFECTS On the negative side of the application of pesticides in agricultural praxis, many pesticides are harmful to the environment and are known or suspected to be toxic to human. Their adverse effects on human health may include acute neurologic toxicity, chronic neuro-development impairment, possibly dysfunction of the immune, reproductive and endocrine systems or cancer and many others. Toxicity studies aim to characterize the nature and extent of toxic effects caused by the pesticide and to find no adverse effects in the test animals (no observed adverse effect level, NOEL). The acceptable level of long-term dietary exposure, referred to as the acceptable daily intake (ADI) for humans may be calculated by using a safety factor (usually 100) from the NOEL (ADI ¼ NOEL/100). The most common route of exposure to pesticides is by ingestion of treated food commodities containing residues. Most pesticide residues occur in food as a result of the direct application of a pesticide to a crop or farm animal or the postharvest treatments of food commodities. Residues also occur in meat, milk and eggs from the consumption by farm animals of feed from treated crops. Residues can also occur in food from environmental contamination and spray drift. In
Table 1 Chemical structures of major classes of pesticides Typical pesticide
Insecticides Organochlorines
DDT
Chemical structure
260
Class
H C
Cl
Cl Anna Sannino
C Cl
Cl Cl Dieldrin
Cl Cl
Cl Cl
O
Cl
Endofulfan
Cl
Cl Cl
Cl
O
Cl
O S O Cl
Cl Organophosphorus
Heptenophos
CH3
O
O
Cl
P
Malathion
CH3
O
CH3
CH2 O
O
O C S CH2
C CH3
CH2
O
O
CH3
O
CH3
P S
CH
O
Carbamate
Carbaryl
H 3C
O N
C
H
Aldicarb
O
O
H3C N H
CH3
C O
N
CH
C
CH3
S CH3 Benfuracarb
O CH2
C CH3
CH2
O
H3C N
CH2
O
N CH3
S
C O
CH
O
CH3 CH3
Benzoylphenylurea
Teflubenzuron
F O O
C N F
Cl
C N H
F Cl
261
H
F
Pesticide Residues
CH3
262
Table 1 (Continued ) Class
Typical pesticide
Chemical structure Anna Sannino
Diflubenzuron
F O C
O N
F
C Cl
N
H
H Pyrethroids
Permethrin
Cl
Cl C HC
O O
C
CH2 H3C Cypermethrin
CH3
O
Cl Cl
C
O
HC
C
O
N C
HC H3C Neonicotinoid
Imidachloprid
Cl
CH3
O
N
NO2 N H2C
N
N
H
Macrocyclic lactone
Abamectin
CH3
O H O
HO
O
CH3
O
H
H3C
H3C
CH3
H O
H
O
CH3
O
H
O
H3C
H
avermectin B1a (major component)
O
O
OH
H
Cl
CH2 C H CH3
H
H
O H HO Botanical
Azadirachtin
CH3
CH3 H3C
H
O
C
C C CH3
H3C O
C O H
O OH H3C O
CH3
H
C
O O
pyrethrin [ H
OH C
O CH3
O
O CH3
C
O
H CH3
O H
O
H2C
(Z )-(S )-alcohol (1R)-trans-acid H
CH C
C H
263
CH3
O
CH2
CH3
C
OH
H
CH H3C
O O
Pesticide Residues
Pyrethrin
H
Class
Typical pesticide
Pyrazole
Fenpyroximate
264
Table 1 (Continued ) Chemical structure O O C
H3C
CH2
C
N
O
N CH3
Tebufenpyrad
CH3
CH3
C H3C
CH2 N H
O
CH3
C
N
Cl Fungicides Phthalimide
Captan
O N
Cl
S C
O Dithiocarbamate
Ziram
H3C
Cl
S N
H3C
Cl
C S
Zn
C CH3
O
N
CH3
S
CH3 C
S
N CH3
N CH2
CH3
CH3
Anna Sannino
H
Benzimidazoles
Carbendazim
H H
N N N
C O
CH3 Thiabendazole
O
H N
S
N Captafol
N O N
Cl
S
Cl
C Cl
O Imidazole
C H
Cl
Imazalil
CH2 CH
Cl Cl
CH
CH2
O
CH2 N N
Aliphatic nitrogen
Cymoxanil
N
O C
N
O
C N
C
C N
H
CH2
CH3
H Dicarboximide
Iprodione
O
O N
N O
Cl
C H
CH CH3
CH3
265
N
Cl
Pesticide Residues
O
CH3
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Table 1 (Continued ) Typical pesticide
Chemical structure
Famoxadone
CH3
O
Anna Sannino
Class
O N
O
O Triazole
N H
Tebuconazole
OH CH3 CH2
Cl
C
CH2
C
CH3
CH2 CH3 N N N Myclobutanil
Cl
C
N
C
CH2
CH2
CH2 N
N
N Pyrimidine
Mepanypirim
H H3C
C
C
N
N N
CH3
CH2
CH3
Nuarimol
N F
N C HO Cl Pyrrole
Fludioxonil
H N F O
F
C
N
O
Strobilurin
Azoxistrobin
CH3
O
H
O
C
C
C
CH3 N C
O O
O N
N Dimethomorph
O C CH
O
N
C
Cl
O O Substituted phenol
CH3 CH3
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Morpholine
2-Phenylphenol
267
OH
Typical pesticide
Herbicides Triazine
Atrazine
Chemical structure H N
Cl N
H
N
CH2
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Class
268
Table 1 (Continued )
CH3
N N
CH3
CH CH3
Simazine
H N
Cl N H Amide
CH2
N
CH3
N N
CH2
CH3
Diphenamide
O CH
C CH3
N CH3 Dinitroanaline
Pendimethalin
CH3 CH2
NO2
CH CH3
N H CH3
NO2
CH2
CH3
Chloroacetanilide
Alachlor
O Cl
CH3
C
CH2
N CH3
Phenoxyalcanoic acid
2,4-D
O
CH2
H2C
CH2
CH3
Cl O O
CH2
C OH
Cl MCPA
Cl O O
CH2
C OH
CH3 Phenylurea
Chlorotoluron
O
Cl
C N
CH3
N
CH3 H Linuron
O
Cl
C CH3
N
O
H
Cl
269
CH3
N
Pesticide Residues
CH3
270 Anna Sannino
Table 1 (Continued ) Class
Typical pesticide
Solfonylurea
Rimsulfuron
Chemical structure CH3 CH3
O N N N
Amidosulfuron
CH3
O
CH3
O N
Amitrol
O H N N
N
N
S
H
O
O
N
Triazole
C
H
N
CH3
CH2 O S O O
O
NH2
H
C
O
N
O S
N
S
N
H
O
CH3
CH3 O
Ammonium quaternarium
Chlormequat
CH3 Cl
Mepiquat
H3C
CH2
+ N
CH2
N+
Cl−
CH3 Cl−
CH3
CH3
Diquat
N+
Carbamates
Chlorprofam
N+
Cl O N H
C O
CH
CH3
CH3
Pesticide Residues
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addition, transport of residues and sediment may also contaminate drinking water sources [2]. Dietary exposure to a pesticide depends on both the actual residue in or on foods and the food consumption pattern. In determining the health risks associated with chronic intake, it is necessary to determine the quantity of the pesticides likely to be consumed over a prolonged period of time and compare this estimate with the ADI. Monitoring of pesticide residues is therefore crucial for proper assessment of human exposure to pesticides through foods. Maximum residue levels (MRLs or tolerances) of pesticides in foodstuffs and drinking waters have been set by Government agencies and the European Union Commission to guarantee consumer safety and to regulate international trade. In general, the MRLs in foods are in the range 0.01–10 mg/kg, depending on the combination commodity and pesticide, the lowest is characteristic of banned compounds because it is considered that this would be the minimum limit of detection (LOD) achievable [4,5]. Specific rules on the presence of pesticide residues in infant and follow-on formulae, as well as in processed cereal-based baby food and baby foods are set out in Commission Directives [6,7]. These Directives require that baby food contains no detectable levels of pesticide residues, meaning not more than 0.010 mg/kg of pesticide residues. In addition, Directives 2003/13/EC and 2003/14/EC [8,9] prohibit the use of highly toxic pesticides (ADIp0.5 mg/kg body weight per day) in the production of any baby food and establish MRL values between 0.003 and 0.008 mg/kg for these very toxic pesticides. All aspects commented upon earlier have an important impact on method development because all of them are interdependent on the required performance of the analytical methods. Low MRLs have promoted the development of more powerful and sensitive analytical methods to meet the requirements in food.
4. ANALYTICAL METHODS The determination of pesticide residues in foodstuffs is a requirement to support enforcement of legislation, to ensure trading compliance and in the conduct of surveillance programs to monitor residues in regional and national dietary components. As noted earlier, approximately 800 active compounds are currently used in pesticide formulations. In addition, several metabolites, degradation products and ‘‘old’’ persistent pesticides have to be considered by pesticide analysts. Approximately 500 pesticides are regulated by the EU Commission [4,5]. Residue analysis in food is probably one of the most complex fields of analytical chemistry because of the need for the isolation and determination of substances at the picogram and femtogram level. Nevertheless, the analysis of pesticides in food samples is made even more complex by the fact that the concentration of matrix components is often greater than the concentration of the target pesticide. Major progress has been made in the development of analytical methods for pesticide residue since the early days of pesticide residue regulation
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in the 1950s and 1960s. Colorimetric methods were the best methods available at that time. These methods had high limits of quantitation (LOQ), being approximately 1 mg/kg. Modern methods mostly using gas chromatography (GC) or high-performance liquid chromatography (HPLC) with very sensitive detectors routinely measure residue concentrations at 1–10 mg/kg in food commodities. Before 1960 individual procedures were used for nearly each pesticide. As the number of pesticides increased, the application of a large number of individual methods to determine them became economically impracticable. In addition, the spray history of a given sample was generally unknown; thus, the residue chemist did not know which methods to apply. The physical and chemical properties of pesticides may differ considerably. There are several acidic pesticides; others are neutral or basic. A number of compounds are very volatile, but several do not evaporate at all. Without question, the most efficient approach to pesticide analysis involves the use of multiclass, multiresidue methods (MRMs). MRMs are preferable because many pesticides can be determined in a single analysis, reducing time and costs. The basic units of pesticide residue analysis are: (1) (2) (3) (4)
Sampling Extraction of pesticides from samples Clean-up Identification and quantitative determination of pesticide residue.
The procedure employed for isolation, clean-up and analysis of food samples is dictated by the composition of the food matrix, especially the fat content. Products with fat percent above 2% are considered fatty foods. Foods with fat composition of 2–20% include milk, nuts, wheat, corn, fish, liver, meat from pork, poultry, beef, eggs and avocado. The polarity ranges of the different pesticide families in water are an important consideration in the development of a universal residue analytical method which should have the widest scope possible. A universal extraction method is considered as a procedure in which all pesticide residues are transferred from sample matrix to organic phase [10]. It is important to transfer as much of the pesticide as possible from the matrix into the organic phase and the solvent system should eliminate the major part of the sample matrix from the organic phase; thus, this step can be seen as a raw clean-up procedure. The first developed MRM used a polar water-miscible solvent able to extract all pesticides including their metabolites. This solvent is miscible with the intrinsic food water, which generally enhances the quantitative pesticide extraction. Then, for the partitioning of the pesticides, it is necessary to use a non-polar solvent, which is miscible with the polar solvent and non-miscible with water. By this procedure the pesticides are distributed from aqueous phase into organic phase. The extent of this distribution into the organic phase depends mainly on the solubility of each pesticide in both solvent layers.
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The first notable MRM was developed by Mills in the 1960 [11] when the nonpolar OC pesticides were the main focus for analysis. With this method, OC and other non-polar pesticides were extracted from non-fatty foods with acetonitrile, which was then diluted with water and the pesticides were partitioned into a non-polar solvent (petroleum ether). As a consequence, relatively polar pesticides, such as certain organophosphorus (OP) compounds were partially recovered. Later, new methods were developed to extend the analytical polarity range to cover OCs, OPs and organonitrogen pesticides (ONs) in a single procedure [12,13]. These MRMs used acetone instead of acetonitrile for the initial extraction and still a non-polar solvent (dichloromethane- or dichloromethane– petroleum ether) to remove water from the organic phase in the liquid–liquid partitioning step. Furthermore, sodium chloride was added to the water phase in both methods during the partitioning step. The basis of these extraction techniques is the use of an electrolyte (salting-out effect) for the partitioning of pesticides into organic phase. The Becker method used a NaCl solution, which only partially saturated the water phase with salt. However, in the extraction procedures developed by Luke et al. [13] and Specht and Tills [14], the water phase was saturated with solid NaCl to force more acetone into the organic layer, thus increasing its polarity and enhancing the extraction of the polar pesticides. The Becker method became official in Germany as the DFG-S8, and Specht method became the official DFG-S19 procedure [15]. The Luke method became the official PAM 302 electron ionization (EI) procedure [16], and several years later became AOAC Official Method 985.22 [17]. Taking into consideration the toxic potential of chlorinated solvents, many new developed methods use cyclohexane–ethyl acetate (1 + 1) instead of dichloromethane (or dichloromethane–petroleum ether, 1 + 1) in the liquid– liquid partitioning step whereas others use solid-phase extraction (SPE) to isolate pesticides from diluted acetone extracts [18]. Some authors [19,20] investigated the use of salts to separate water from acetone without using non-polar solvents. However, acetone is too miscible with water to be easily separated without using non-polar solvents. In the case of procedures in which acetonitrile is chosen as extraction solvent, salt alone can be used to form a satisfactory separation from water. Several multiclass MRMs based on salting-out principle with acetonitrile have been developed [21–24]. Anastassiades et al. [25] introduced the quick, easy, cheap, effective, rugged and safe (QuEChERS) method for the analysis of pesticide residues in fruit and vegetables. Lehotay et al. [26] demonstrated its effectiveness for more than 200 pesticides in lettuce and orange matrices. The procedure involves initial single-phase extraction of 15 g sample with 15 mL acetonitrile, followed by liquid–liquid partitioning formed by addition of 6 g anhydrous MgSO4 and 1.5 g NaCl. Removal of residual water and clean-up are performed simultaneously by mixing acetonitrile extract with 1.8 g of anhydrous MgSO4 and 300 mg primary secondary amine (PSAm) sorbent. The results were excellent for a wide range of pesticides, including polar and basic compounds such as methamidophos, acephate, omethoate, imazalil, thiabendazole (TBZ). The pesticides captan, chlorothalonil, dichlofuanid, folpet, tolylfluanid and pymetrozine
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are well known as being difficult for their determination using existing methods, including QuEChERS. The modification to improve the extraction and stability of the mentioned problematic pesticides has been presented by Lehotay et al. [27]; sample preparation entailed extraction with acetonitrile that contained 1% acetic acid and partitioning with a mixture of MgSO4 and sodium acetate. A third extraction solvent commonly used in MRMs is ethyl acetate (EtAc) [28,29]. EtAc has the advantage of partial immiscibility with water, which makes unnecessary the addition of non-polar solvents to separate water from organic extracts. However, a problem with EtAc is that some of the most polar pesticides are not readily extracted. To increase recoveries of polar compounds, large amounts of NaSO4-to bind the water- or polar co-solvents, such methanol and ethanol, are usually added. Another disadvantage associated with the use of EtAc is the extraction of a large amount of non-polar co-extractive, such lipids and epicuticular wax material, which must be removed before the determination step. With the demand for the faster sample preparation and reduced solvent usage, new techniques based on solvent extraction have been tested. One of these novel techniques that are emerging is pressurized liquid extraction (PLE) also known commercially as accelerated solvent extraction (ASE). The extraction process has three sequential steps. First, the analytes must diffuse from the core of the matrix to the surface. They are then transferred from the surface into the extraction fluid and, finally the analytes are eluted. Bogialli et al. [30] analysed residues of carbamate insecticides in vegetables and fruit using hot water as an effective extractant. PLE can be carried out with conventional solvents such as dichloromethane-hexane [31] or EtAc [32]. This technique requires dry samples — freeze-dried and powdered [31] or previously homogenized with a drying or dispersive agent [32]. PLE is fully automated but requires rather high-cost equipment. After the extraction step, the obtained crude extracts can be injected directly into the chromatographic system, but the majority of the sample preparation methods utilize different clean-up steps. Clean-up is still required to minimize matrix effects, to achieve better ruggedness and to decrease LOQs in pesticide residue analysis. SPE is a simple preparation technique based on the separation by liquid chromatography (LC) where the solubility and functional group interactions of sample, solvent and sorbent are optimized to affect the retention and elution. SPE is used for pesticide analysis for liquid samples, such as juices, wine and honey, and to clean-up organic solvent extracts after analyte isolation by means of liquid extraction [33]. Many MRMs include a clean-up step using adsorption columns, in particular Florisil, aluminium oxide (alumina) and silica gel. Most adsorbent columns provide a good clean-up only when they are eluted with solvent mixtures of low polarity, eluting less polar residues and leaving more polar coextractives in the column. A Sep-Pack cartridge containing 0.900 g Florisil was chosen as the SPE stationary phase by Sannino et al. [34] for the clean-up of processed fruit and vegetables. The eluate is very clean for the determination of 12 pyrethroids by GC-ECD.
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The introduction of various types of co-polymeric sorbents has helped to make SPE a more robust purification technique with a wider application range than the conventional silica-based sorbent. A polymeric sorbent frequently used for the isolation of pesticides from food samples is a hydrophilic-lipophilic balanced (HLB) copolymer of N-vinylpirrolidone and divinylbenzene [35]. A very effective way how to utilize SPE sorbents for removing co-extractants from a matrix, and concurrently reduce manual handling, published by Anastassiades et al. [25] within the development of the QuEChERS method. In this study, the authors used a very simple clean-up approach that they call ‘‘dispersive-SPE.’’ A 1 mL aliquot of the sample extract is added to a vial containing a small amount of PSAm sorbent, and the mixture is mixed in a Vortex mixer for 30 s. PSAm as a weak anion exchanger sorbent is able to remove matrix components, such as organic acids, polar pigments, sugars and some other co-extractives that form hydrogen bonds [36]. With the current trends towards miniaturization of sample preparation, matrix solid-phase dispersion (MSPD), based on the dispersion of the sample on an adsorbent is a new SPE and clean-up technique that was first reported by Barker et al. in 1989 [37]. MSPD, based on dispersion of the sample on an adsorbent, such as Florisil, C18, alumina, silica or diatomaceous earth is a technique that allows the extraction and clean-up in a single step. For solid samples, the mixture is done in a mortar and then transferred to the extraction columns [37], whereas for liquid samples the dispersion of the matrix in the adsorbent is done directly in the extraction columns [38]. The basic concept of the technique is to allow the extraction of pesticides from homogeneously dispersed food samples in a solid support. The sample matrix is placed in a column, and the polar compounds, such as pigments, are held on the support. In this manner, various pesticide residues can be rapidly eluted with organic solvents and the extract can be analysed. MSPD eliminates the traditional processes of extraction and clean-up, reduces time and costs and avoid emulsification. It has been used mainly for the analysis of pesticides in fruit and vegetables but has also been applied to the analysis of liquid samples such as milk and fruit juices. Perret et al. [39] developed a method based on MSPD for the determination of multiclass pesticide residue (omethoate, dimethoate, carbendazim, propoxur, TBZ, carbaryl pirimicarb, azinphos-methyl, metidathion and iprodione) in peach, apple, apricot and pineapple juices. A flow diagram of the method is shown in Figure 1. MSPD is also a sample strategy appropriate to extract pesticide residues in matrices with high fat content. Ferrer et al. [40] proposed a methodology based on MSPD using aminopropyl as sorbent material with a clean-up performed in the elution step with Florisil to extract pesticides from olives and olive oil (the latter with a preliminary liquid–liquid extraction). Solid-phase microextraction (SPME) is a recent developed technique, which avoids organic solvents, and, can be easily automated [41–43]. In combination with GC, SPME is able to extract and to detect volatile residues in an easy and, relatively fast and cheap approach. Because of complications in quantitation, strong dependence on matrix, and certain practical matters, some quality in the results is sacrificed for speed and ease.
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1g fruit juice mixed with 1g diatomeaceous earth
→ elution with 10 ml ethyl acetate
→ evaporation of solvent
→ residue reconstituted with 500 µL methanol- water (50+50)
→ 50 µL injected into LC/MS-MS
Figure 1 Flow diagram of MSPD procedure employed by Perret et al.
SPME is based on the partition of analytes between the matrix and a silica fibre, coated with a stationary phase, whereas the analytes are retained due to their high affinity for this phase. SPME can be applied to the analysis of liquid matrices, and pesticides can be extracted by direct immersion in aqueous samples or headspace analysis. The other important step in SPME is usually performed by thermal desorption and gas chromatographic analysis, although it can also be achieved by solvent desorption and liquid chromatographic determination. SPME application to pesticide analysis requires, typically, a previous extraction step. Fruit samples have been extracted with high-speed blending with water [44]. A variation on the SPME technique is the use of a stir bar covered with poly(dimethylsiloxane) (PDMS). This technique, called stir-bar sorptive extraction (SBSE) is performed by stirring the sample with a stir bar for a given time. Applications to pesticide analysis in beverages have been recently published [45–47]. Bicchi et al. [47] determined nine pesticides in pear pulp by extraction on PDMS-SBSE followed by recovery through thermodesorption and GC/mass spectrometry (MS) analysis. The levels of pesticides range from 10 to 100 ng/g. The most universally applicable clean-up is gel permeation chromatography (GPC). It was introduced as a clean-up technique for pesticides by Stalling et al. [48]. They found that an automated GPC system removed the lipids from fish extracts and gave good recoveries of non-ionic chloride and polychlorobiphenyls (PCBs). Separation is generally performed by using divinylbenzene-linked polystyrene gels, mostly BIO-Beads SX3 (200–400 mesh, Bio-Rad, USA). GPC has the advantage of being applicable to a wide range of structurally dissimilar compounds, unlike some adsorption techniques that are narrower in scope. It is suitable for OC, OP and nearly all other types of pesticides. The stationary phase is a porous matrix with the pores closely controlled in size. Separation results through differences in the size of the sample molecules. Lipid molecules that are too large to enter the pores are totally excluded and are therefore eluted from the
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column first. Most synthetic pesticides have molecular mass of between 200 and 400, whereas those of most lipids range from 600 to 1,500. This system could clean-up a wider range of sample types, including green plant lipids, animal feed extracts, human adipose tissue and beef tallow. Another valuable feature is that GPC can be carried out in an automatically controlled device. A GPC clean-up of vegetables, fruit and crops for the analysis of organophosphate pesticide residues was introduced by Ault et al. [49]. This GPC system used BIO-Beads SX3 with dichloromethane–cyclohexane (15:85, v/v) as the elution solvent, which is useful for the clean-up of seven carbamates and three related metabolites in fruit and vegetables [50]. The preparation of fatty samples for the determination of pesticides by chromatographic techniques requires the complete removal of the high-molecularmass fat from the sample to maintain the chromatographic system in working order. Most MRMs for the determination of pesticides in fatty matrices involve extraction of fat with an organic solvent, partition of residues in an immiscible solvent and/or clean-up by GPC and/or successive clean-up on a Florisil column [51–53]. Methods for extraction of fat are recommended which are simultaneously applicable for the extraction and determination of fat and the residue analysis in the fat portion. Sannino et al. [54,55] used a GPC procedure for determination of 24 OC pesticides, 9 PCBs and 39 OP compounds in 8 fatty preserved foods of vegetable and animal origin. Samples are extracted with dichloromethane and cleaned up by automated GPC with Bio-Beads SX3 and dichloromethane– cyclohexane (15:85, v/v) as the eluting solvent. A new method using GPC clean-up has been established for quantitative determination of 437 pesticide residues in animal tissue such as beef, mutton, pork, chicken and rabbit [56]. In this method, 10 g animal samples were mixed with 20 g sodium sulfate and extracted with 35 mL of cyclohexane–ethyl acetate (1:1, v/v). An aliquot of extract was injected into a 400 25 mm SX3 GPC column, with cyclohexane–ethyl acetate (1:1, v/v) as mobile phase at a flow rate of 5 mL/min. However, all these mentioned techniques, using large columns and flow rates, need long analysis times and large amounts of solvents. Some pesticides with high molecular mass (e.g., pyrethroids) are not sufficiently separated from wide elution band of co-extractants, which can result in lower recoveries [36]. Considering the entire analytical process, much effort is still required to reduce analysis time and cost. Recently Liu et al. [57] have proposed an online GPC-GC/MS for the analysis of residual pesticides. In this experiment, a semi-micro GPC column was employed. A low flow rate of 0.1 mL/min resulted in a 40-fold reduction in solvent consumption compared to conventional GPC column applications. This system can determine 97 pesticides in 90 min.
5. INSTRUMENTAL DETERMINATION Analytical methodologies employed should be capable of residue measurement at very low levels and must also provide unambiguous evidence to confirm both the identity and the magnitude of any residues detected.
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Chromatographic methods are most widely used for the analytical separation, identification and quantification of pesticide residues in different matrixes.
5.1 Gas chromatography The introduction of commercially available fused-silica capillary columns was a great step forward with regard to the separation power and enhanced sensitivity. Due to the reduction of peak width, capillary columns attains the separation and determination of a large number of pesticides with similar physico-chemical characteristics. The selection of columns depends upon the nature of the pesticide to be separated. In general, 5% diphenyl, 95% dimethylpolysiloxane stationary phase, normal bore columns with ID (internal diameter) of 0.25 mm (20– 30 m 0.25 mm ID 0.25 mm) were successfully utilized in multiresidual analysis. Owing to its high separation efficiency and the variety of selective and sensitive detection methods that can be used, GC is the preferred method for determination of volatile and thermally stable compounds such as OC and OP pesticides. An ideal selective detector for the analysis of pesticide residues would respond only to the target pesticides, while the co-extracted compounds remain transparent. Pesticides almost always contain heteroatoms and often have several atoms in a single molecule. The most frequently encountered heteroatoms are O, P, S, N, Cl, Br and F. Therefore, most GC methods employ element-selective detectors. Two of these detection methods are nitrogen phosphorus detection (NPD) for N- or P-containing compounds and flame photometric detection (FPD), which is better suited for the detection of P-containing compounds. For the determination of OP compounds the FPD is widely used because of its very good selectivity and good sensitivity. This makes the detector particularly suitable for the determination of OP pesticide residues in various crops without extensive clean-up of the sample [55]. For the determination of OC pesticides, electron capture detection (ECD) has been used for the past 30 years because of the extraordinary sensitivity for organic halogen compounds. Since the retention time is characteristic for each compound in a given chromatographic system, the pesticides can be tentatively identified by comparison of their individual retention times. The detectors mentioned earlier do not provide unequivocal confirmation of identity and are often subjected to matrix interferences. Confirmation of results required the use of a further chromatograph equipped with a different type of column or detector. Moreover, the limiting disadvantage in conventional GC analysis was that only a limited number of compounds could be detected in a single determination. In multiclass, MRMs, several GC injections are often required when these selective detectors are used. When fused capillary columns became feasible to couple to a mass selective detector, the routine confirmation process could be carried out. In the past, mass spectrometry was chosen as the method for confirmation of pesticides initially detected in food by other techniques. Its unique combination of specificity and sensitivity, together with the wide applicability to a diverse range of organic contaminants, makes mass spectrometry (MS) the primary
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detection method for pesticide residue analysis [58]. The MRMs are preferred, not only due to cost benefits but also due to importance of checking a large number of pesticides in one single analysis compared to methods for determination of single analyte. The need for analysing pesticides belonging to different classes requires the use of universal detectors able to determine them at residue levels. MS detection is a good option because it can be employed as a universal detector at full scan and also as a selective detector in the selected ion monitoring (SIM) mode. The most widely used GC/MS technique for pesticide residue analysis is based on single quadrupole instruments and EI. Selectivity of GC-MS can be adjusted by the selection of appropriate molecular and fragment ions (SIM mode) to avoid interferences from co-extracted sample materials. Therefore, the importance of GC with ECD, NPD or FPD detection has decreased in pesticide residue laboratories. A MRM for the quantitative determination of 39 OP compounds including the parent pesticides and their major metabolites in 7 fatty preserved foods of vegetable and animal origin is described by Sannino et al. [55]. OP compounds are quantified by capillary gas chromatography with flame photometric detection (GC/FPD) using OV-1701 and DB5 columns and two temperature programs. Determination of all 39 OP compounds by a single chromatographic run was possible by GC/MS with SIM. Figure 2 shows total ion profiles of meat sauce extract and same sample fortified with all 39 compounds obtained by the SIM program. Co-elution is usually not a problem because each pesticide has its own characteristic ion pattern. Figure 3 illustrates a typical example of how two co-eluting analytes were separated and estimated. With the assistance of a software program the single-ion chromatograms of m/z 221 and 232 (pyrazophos), 160 and 132 (azinphos-ethyl) were extracted and reconstructed from the 36 to 37 peak of Figure 6. Moreover, since most of the monitored ions were above 100, a minimum background and less spectral interferences were observed in the fragmentograms. Simultaneous determination of 24 organochloride pesticides and 9 PCB congeners [54] can be performed by a single chromatographic run following the SIM program of Table 2. Gas chromatographic conditions as capillary column, temperature program and flow rate were the same as those used in method for OP pesticide determination [55]. Then, two injections at two SIM settings were required per sample to cover 24 OC pesticides, 9 PCBs and 39 OP compounds. The main limitation of GC/MS is the relatively low sensitivity obtained for many pesticides in the full scan mode when EI is selected. The introduction of the ion-trap detector (ITD) improved the LODs. Analysis is normally carried out in the full scan mode — thus complete mass range is acquired for all GC peaks. In full scan mode, these instruments are sensitive and confirmation by library search is possible at lower concentrations. But, at trace levels, quantitative analysis is based on chromatograms of ions extracted from mass spectra and that are specific to the substance. Compared to single quad instrument running in SIM, identical pesticides are covered and the sensitivity does not differ significantly. Lehotay and Eller [59] developed a MRM using GC-ITMS for analysis of 46 pesticides in fruit and vegetables. Simultaneous determination of 19 fungicides in processed
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TIC: MS703.D Abundance (a) 4000
3000
2000
1000
0 Time-->
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
TIC: MS705.D Abundance (b)
4000
8 3
3000
1
5
2
6
7
36-37 34-35 38 39
28 15-16 19 18 21-22 23 20
10 9
2000
31
12-1314
4
11
30 24-25 26-27 29
32 33
17
1000
0 Time-->
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
Figure 2 Total ion current chromatograms of (a) unspiked meat sauce and (b) meat sauce spiked with 39 OP compounds at 0.025 mg/kg. Peaks: dichlorvos (1), mevinphos (2), thionazin (3), ethprophos (4), sulfotep (5), phorate (6), dimethoate (7), fonofos (8), diazinon (9), disulfoton (10), formothion (11), methyl-parathion (12), chlorpyrifos-methyl (13), malaoxon (14), paraoxon (15), enchlorfos (16), fenitrothion (17), pyrimifos-methyl (18), malathion (19), fenthion (20), chlorpyrifos-ethyl (21), parathion (22), bromophos (23), chlorfenphos (24), isofenphos (25), mecarbam (26), quinalphos (27), methidathion (28), bromophos-ethyl (29), tetrachlorvinphos (30), ethion (31), carbophenothion (32), EPN (33), phosalone (34), azinphos-methyl (35), pyrazophos (36), azinphos-ethyl (37), dialifos (38), coumaphos (39). Experimental conditions: 30 m 0.25 mm 0.25 mm HP MS-5 column. MS analyser: quadrupole operating in SIM mode. Reproduced from Ref. [55] with permission from AOAC International, Copyright 1995.
fruit and vegetables was carried by Sannino et al. [60]. Quantitative analysis was performed both by GC/ITMS and GC/ECD and the results compared. Figure 4 illustrates a GC/ITMS typical example of how a targeted pesticide was detected in a sample extract. The normalized ion chromatogram of m/z 173 and 175 254 (Figure 4b) was extracted and reconstructed from the total ion profile (Figure 4a).
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Abundance
Ion 132.00 (131.70 to132.30): MS704.D Ion 159.95 (159.65 to160.25): MS704.D Ion 232.05 (231.75 to232.35): MS704.D Ion 221.00 (220.70 to221.30): MS704.D
2400 2200 2000 AZINPHOS-ETHYL
1800 1600 1400 1200 1000 800 600
PYRAZOPHOS
400 200 Time--> 0 22.40 22.50
22.60
22.70 22.80 22.90 23.00 23.10
23.20 23.30
23.40
Figure 3 Extracted single-ion chromatograms of characteristic ions of pyrazophos and azinphos-ethyl from the peaks 36 to 37 in Figure 2. Reproduced from Ref. [55] with permission from AOAC International, Copyright 1995.
With the assistance of computerized graphing and data manipulation, the extracted ion data showed the possible presence of propyzamide. The above positive finding led to further examination of the full mass spectral data at 11.18 min. The exact matching with the NIST library spectrum of propyzamide (Figure 4c and d) provides the conclusive fingerprint of the presence of pesticide in the extract. An additional advantage of IT instruments is the capability of performing tandem mass spectrometry (MS-MS) by means of collision-induced dissociation (CID). The use of MS-MS improves the selectivity of the technique, sometimes with a drastic reduction in the background and without loss of identification capability. It enables analysis of pesticides at trace levels in the presence of many interfering compounds. Dissociation conditions were selected for each pesticide using methods built with the automated method development (AMD) editor. As general criteria,
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Table 2 SIM data for simultaneous determination of 24 organochloride pesticides and 9 PCB congeners Compound
Acquisition group
Start time (min)
Characteristic masses, m/z (relative abundance, %)a
a-HCH HCB b-HCH g-HCH Trans-chlordene d-HCH PCB 28 Heptachlor PCB 52 Aldrin Heptachlor epox g-Chlordane o,p DDE a-Endosulfan PCB 101 a-Chlordane Dieldrin p,pu DDE o,p DDD Endrin Perthane b-Endosulfan 118 p,pu DDD o,p DDT PCB 153 PCB 105 Endosulfan sulfate p,pu DDT 138 p,pu methoxychlor 156 180
1 2 3 4 5 6 7 8 9 10 11 12 13
8.0 8.9 9.3 9.7 10.1 10.5 11.0 11.9 12.6 13.4 15.0 16.9 17.3
14
18.6
15 16 17
19.1 19.4 19.6
18
19.9
19
20.1
20
20.5
21
21.1
22
21.6
183(100),181(95),219(92) 284(100),282(48),286(40) 181(100),183(85),219(76) 181(100),183(85),219(76) 66(100) 181(100),183(82),219(75) 256(100),258(99),260(20) 272(100),274(82),237(45) 292(100),290(74),294(50) 263(100),265(62),293(32) 353(100),355(82),237(41) 373(100),375(98),377(37) 246(100),248(64),318(32) 239(92),195(100),241(80) 326(100),324(60),328(65) 373(100),375(100),377(69) 277(100),263(76) 318(70),246(100),248((64) 235(100),237(68),165(60) 263(100),265(56),281(34) 223(100),224(15) 237(100),240(37) 326(100),324(65),328(60) 235(100),237(58) 235(100),237(63) 360(100),362(79),358(70) 326(100),324(60),328(68) 272(100),229(64),387(30) 235(100),237(67) 360(100),362(84),358(72) 227(100),228(14) 360(100),362(86),358(75) 394(100),396(98),398(53)
Source: Reproduced from Ref. [54] with permission from AOAC International, Copyright 1996. a An asterisk indicates an ion used in quantitation.
precursor ion dissociation conditions are selected in order to obtain a balance between maximum sensitivity, minimal spectral interferences and enough structural information for an unequivocal identification. So, the base peak on the spectrum is generally selected as the precursor ion with a narrow mass
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Spect 1 BP 173 (453=100%) erbi11.sms
11.198 min. Scan: 672 Chan: 1 Ion: 24999 us RIC: 2871 BC
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Figure 4 (a) Display of the total ion chromatogram from retention time 10 to 12 min of an apricot sample fortified with 10 ng/g propyzamide. (b) Extracted ion chromatograms of the most characteristic ions of propyzamide (173 + 175 + 254 m/z). (c) Mass spectrum of the eluate at 11.19 min. (d) NIST library spectrum of propyzamide.
isolation window. In the case in which the precursor ion presents a low m/z or can be confused with a matrix ion, a different fragment, with lower abundance, is selected. Ideally, the MS-MS spectrum should be optimized to maximize the signal of the highest mass product ion while obtaining at least two product ions for confirmation purposes. To perform MS-MS [21], each analyte must have its own ion preparation method (IPM), which performs the functions of ionization, isolation and precursor fragmentation. Each analyte must have its own list of
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Purity = 768 Fit = 911 RFit = 814 Average = 831 Ion Range: 43 - 329 Spect 1 BP 173 (453=100%) erbi11.sms
11.198 min. Scan: 672 Chan: 1 Ion: 24999 us RIC: 2871 BC 173
100%
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Propyzamide CAS No. 23950-58-5, C12H11Cl2NO, MW 255 173
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Spect 1 - Match 1 of 25 Normalized
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50%
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Figure 4 (Continued)
parameters that will create the desired product spectrum. Table 3 shows examples of IPM parameters for various pesticides. To analyse co-eluting or closely eluting peaks the selected reaction monitoring (SRM) methods were created [61,62]. Unlike full-scan MS, in which one set of conditions can detect all analytes in a GC run, MS-MS with an IT instrument is much like SIM, in which
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Examples of IPM parameters for various pesticides
Pesticide
Start time
Precursor ion Storage level Excitation (m/z) (m/z) voltage (V)
Product ion (m/z)
Biphenyl 2-Phenylphenol Tecnazene Diphenylamine Dicloran Quintozene Pirimetanil Metalaxyl Dichlofluanid Triadimefon Fenson Ciprodinyl Penconazole Fipronyl Mepanypirim Buprofezin
4.0 6.3 7.1 7.1 8.0 8.8 8.8 9.2 11.0 11.0 12.6 13.3 13.3 14.2 11.8 16.0
154 169 261 169 176 295 198 206 224 208 268 224 248 367 222 175
122 115 201+203 139+140 148 265 155+129+182 132+162 123 180+144 141 208 192+206+157 332+324+292 193+191+218 117+132
60 70 110 70 70 128 85 88 90 90 95 95 100 130 90 75
70 70 78 74 71 80 93 70 65 75 0.28 98 90 0.8R 2.6R 65
Note: R, resonant mode.
specific conditions are used for targeted analytes within time segments during a GC run. The IT instrument used by Lehotay [22] was capable of employing five different sets of conditions within a single segment. The GC-MS-MS analysis allows for more accurate analysis than GC-MS primarily due to the removal of matrix interference. The typical food matrix contains many compounds that are thousands of times more concentrated than the pesticide residue levels. Even relatively small ion intensities from a co-eluting matrix compound can cause a significant positive interference. Figure 5 shows an example of the advantages of GC-MS-MS over GC-MS in the detection of dichlofuanid at 20 ng/g in an olive extract. Interference from coextracted plant material can also be reduced significantly in comparison with GC-MS. The GC-MS-MS analytical method was able to identify the presence of the low amount of dichlofuanid in the complex extract, whereas the GC-MS method, obtained with the same instrument, is not capable of discriminating between the analyte and matrix signal. In recent years, some interesting applications of GC-MS-ITD in the analysis of pesticide residues in food have been published. Lehotay [22] determined 22 diverse pesticide residues in mixed apple, green bean and carrot extracts by GC-MS-MS using IT chromatography. For most pesticides the LODs in GC-MS-MS were 5–20 times lower than those in GC-MS with the same instrument. Martinez-Vidal et al. [63] developed and validated a selective procedure to determine multiclass pesticide residues in lyophilized agricultural plants by low-pressure GC coupled with MS-MS.
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Chromatogram Plots Plot 1: d:\sa\sa042.sms Ions: 224+226 all Plot 2: d:\ca\ca226.sms Ion: 123 merged kCounts
Ions: 224+226
Olive oil fortified with dichlofuanid at 0.020 mg/kg
80 70 60 50 40 30 20
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10 0 kCounts
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Ion: 123
S/N (P-P): 69
3.0 2.5
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2.0 1.5
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1.0 0.5 0.0 11.0
11.1
11.2
11.3
11.4
11.5
11.6
minutes
Figure 5 Comparison of GC-MS and GC-MS-MS chromatograms obtained by gas chromatograph/ITMS in the detection of dichlofuanid at 20 ng/g in olive oil extract. See Table 3 for MS-MS conditions.
The number of analytes that can be simultaneously determined in a time segment is limited to 5–6. The use of the triple quadrupole (QqQ) mass spectrometer can solve this technical limitation because this instrument can measure 25–30 analytes at the same time by use of the SRM mode. This scan mode is faster than the production ion scan mode available on an ITD. Within each segment, precursor ions are isolated in the first quadrupole, and then fragmented by collision-induced decomposition in the second quadrupole with the goal of generating spectra at different voltages, and in the third quadrupole the produced ions were separated before detection. The chromatographic separation is not a critical point in the analysis of pesticide residues by GC/QqQ-MS-MS because the analyser is able to monitor a high number of co-eluting compounds: the only limit was due to the shape peak and number of points per peak. Recently, GC-QqQ-MS is becoming a powerful tool for pesticide residue analysis in a variety of complex matrices, due to its inherent advantages: selectivity are notably improved, the sample pre-treatment step can be minimized and
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reliable quantitation and confirmation can be easily achieved at low concentration levels required. In the last few years, several GC-MS-MS methods have been developed for the determination of a large number of multiclass pesticides in food [64–69]. Walorczyk and Gnusowski [67] investigated the feasibility of low-pressure gas chromatography (LP-GC-MS) in conjunction with a QqQ mass spectrometer, as a route towards fast pesticide residue analysis. LP-GC is a chromatography technique that involves the use of a relatively short (10 m) large-diameter column connected with a restriction capillary (0.1–0.25 mm of appropriate length) at the inlet end. Under the optimized conditions the analysis time was reduced to 13.3 min which corresponds to an almost three-fold gain in speed versus the conventional GC (37 min). For evaluation purposes, 78 target pesticides were included in the screening method and 12 target pesticides in the quantitative methods. Martinez Vidal [68] described the validation of a MRM in cucumber matrix, able to analyse 130 multiclass pesticide residues in less than 12 min with a single chromatographic injection. A first identification of the pesticides is based on an MS-MS screening method, which monitors a single transition for each target compound. Potentially positive samples in the screening method were further analysed by the MS-MS confirmation/quantitation method which monitors two or three MS-MS transitions for each compound, also in less 12 min. The calculated LOD and LOQ were typicallyo3.2 and 9.6 mg/kg, respectively. Plaza Bolan˜os et al. [69] developed and validated a method for 151 pesticide residues in a single injection in strawberry samples. The list of target compounds included various classes of pesticides such as OC, OP, carbamates, pyrethroids, triazoles and dicarboximides. The combination of QuEChERS and GC/QqQ/ MS-MS allows the preparation of a batch of 10 samples in about 1 h, and the subsequent chromatographic analysis in less than 3.5 h. Time-of-flight (TOF) instruments have recently gained popularity in quantitative analysis. TOF mass spectrometers (TOF-MS) have advantages over quadrupole MS spectrometers because of their fast acquisition rates and highresolution capabilities. Gas chromatograms have narrow peaks and require a fast-scanning detector. TOF-MS, unlike scanning instruments, have the ability to acquire chromatograms in microseconds, depending on the acceleration potential (L.N. Williamson and M.G. Bartlett). Normally, TOF-MS have been used for accurate mass measurements for empirical formula verification. However, over the past decade, they have been used quantitatively as well. Cajka and Hajslova [70] demonstrated the high-resolution TOF-MS as a powerful tool for reliable detection and accurate quantitation of pesticide residue even at very low concentration levels. With only few exceptions, LOQ for most of pesticides involved in this study were far below 0.01 mg/kg. The combination of large volume-difficult matrix introduction (LV-DMI) with GC/TOF-MS, described by Patel et al. [71] provides the analyst with a simple but rapid alternative GC-MS technique for analysis of 98 pesticides in fruit-based baby foods at 0.010 mg/kg. Samples were extracted with EtAc in the presence of Na2SO4 and NaHCO3, and the crude extracts were analysed directly using LV-DMI/GC-TOF-MS.
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The instrumentation and basic principle have been described by De Koning et al. [72]. Depending on their volatility, analytes present in the extract, which is held in a microvial inside the injector liner of the GC, are transferred onto column as the temperature of the injector is increased. The non-volatile matrix components are retained in the DMI vial and consequently do not contaminate the chromatographic system.
5.2 Liquid chromatography One of the disadvantages of GC has always been, and in fact still is, the requirement that the compound to be determined has to be thermally stable and should have a sufficient volatility. In GC analysis, these compounds either show sign of thermal decomposition or fail to elute to column. In recent years many new pesticides have been developed that are not very well amenable to GC. Among the analytical approaches, LC is the preferred separation technique for the most polar and thermally labile pesticides. There are some classes of pesticides for which HPLC is superior to GC, such as carbamates, urea herbicides, benzoylurea insecticides and benzimidazole fungicides. HPLC methods for the determination of pesticides could employ reversedphase. As most pesticides are low-polarity compounds, they often are analysed by reversed-phase chromatography with C18 or C8 columns and aqueous mobile phase, followed by UV adsorption, UV diode array, fluorescence or mass spectrometric detection. In the determination of phenylureas, which are used extensively to protect a large number of crops against weeds, LC methods have been used in various matrices. UV detection used in many methods [73,74]) is sensitive enough in most cases but lacks selectivity, especially when trace levels in complex samples need to be assayed. Effective clean-up before LC/UV determination could be sufficient to isolate phenylureas from matrix interferences well enough to allow determination at low levels required by current regulator laws for baby and organic foods (p0.010 mg/kg). Sannino [75] described a method for quantitative determination of nine phenylurea herbicides in potatoes, carrots and mixed vegetables. Samples are extracted with acetone, partitioned with ethyl acetate–cyclohexane (50 + 50, v/v) and cleaned up by GPC with ethyl acetate–cyclohexane (50 + 50, v/v) as eluent. A further purification on Florisil cartridge was necessary to obtain an extract free from interferences when detected by LC with UV at 254 nm. The fluorescence detector has been in use for a number of years on a more or less routine basis. Fluorimetry is much more selective and in many cases much more sensitive than UV absorbance spectrometry. The better selectivity is because two wavelengths, the excitation and the emission wavelength, are involved, and by a careful selection of these two wavelengths a very selective detection is possible. The very high selectivity of fluorimetry is of course also a disadvantage because there are only a rather limited number of compounds that can be detected with good sensitivity. Some classes of compounds show a strong native fluorescence, but there are not many pesticides that show a native fluorescence.
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An example of LC method with fluorescence detector is the determination of TBZ and carbendazim, two compounds that belong to the benzimidazole class. They are systemic fungicides used as either pre-harvest or post-harvest treatment for the control of a wide range of fruit and vegetable pathogens. Carbendazim is the major metabolite and fungitoxic principle of benomyl and thiophanate methyl (TM). Sannino [76] used a reversed-phase liquid chromatographic method for determining carbendazim, TBZ and TM in fruit products (nectars, purees, concentrates and jams). The extraction and clean-up method, based on the procedure developed by Bicchi et al. [77], is schematized in Figure 6. The fungicides were separated and quantified by using an ion-pairing mobile phase with UV and fluorescence detectors in tandem, following the procedure of Gilvydis and Walters [78]. UV and fluorimetric detectors connected in series allowed the simultaneous determination of the non-fluorescent TM and fluorescent carbendazim and TBZ. Figure 7 shows the chromatograms of apricot puree extract and the sample spiked with 0.1 mg/kg of carbendazim and TBZ and 0.01 mg/kg of TBZ. For those compounds which do not possess appreciable native fluorescence, a derivatization technique can be employed. In pesticide residue analysis the main application of derivatization is the determination of NMCs. They comprise an important class of insecticides, widely used for crop protection with some of the most common ones being carbaryl, carbofuran, aldicarb, methomyl and oxamyl. Carbaryl has been included in the final list of compounds to be considered for periodic re-evaluations by the 2001 Joint FAO/WHO Meeting on the Pesticide Residues [79]. NMCs can be determined by UV detector [80]. However, the sensitivity and selectivity offered by UV detection is very poor, because the carbamates present their absorption maximum at about
Non Fatty food
Extraction with HCL/ methanol
Centrifugation- filtration
Aliquot to pH 7.5
Clean-up on a diatomaceous earth column
Analysis by HPLC/ UV/ FL
Figure 6 Scheme for simultaneous determination of benzimidazole fungicides employed by Sannino [76].
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190 nm. Post-column hydrolysis and derivatization coupled with fluorescence detection has been accepted as a standard protocol by several official organizations (US-FDA, EN 14185-2) [81,82]. The derivatization is specific for primary amines and thus adds a high degree of selectivity to the detection compared to direct UV absorbance detection of the parent compounds. The carbaryl metabolite, 1-naphthol, which is not carbamate, is unaffected by this reaction sequence, but exhibits an equally strong native fluorescence. Thus, on the basis of the pioneering work by Moye et al. [83] which was refined by Krause [84] various sensitive and selective LC methods including post-column derivatization for simultaneously determining NMCs in complex matrices such as vegetables [85,86] and meat [87] as well as water [88] have been proposed. The typical method is as follows: the pesticides are extracted from food with organic solvents, the extract is cleaned up by a method based on liquid–liquid or solid–liquid partition, and is analysed by LC with post-column derivatization. Podhorniak [89] uses an acetone extraction, followed by an aminopropyl SPE. A method is presented for the determination of 24 compounds (13 parent NMC pesticides and their metabolites, and piperonyl butoxide) in selected fruit and vegetables. The Comite´ Europe´en de Normalisation (CEN; European Committee for Standardization) Technical Committee CEN/TC 275 ‘‘Food Analysis-Horizontal Methods’’ has prepared the European Standard EN 14185-2 [82] method for the determination of NMC in fruit and vegetables based on post-column derivatization. The extract is purified on a diatomaceous earth column. A disadvantage of the post-column technique is that special equipment is needed, such as a mixer chamber, a reactor and an extra pump. In addition, coexisting substances having fluorescence from food, especially from citrus fruits, frequently interfere with the determination of the pesticides.
5.2.1 Liquid chromatography-mass spectrometry The number of compounds that cannot be determined by GC because of their poor volatility, high polarity and thermal instability has grown dramatically in the last few years. Agrochemicals belonging to carboxamide, quinazolin, phenoxypyrazol, strobilurin, pyrimidine, triazol, carbamate, neonicotinoid, morpholine classes are representative of the newly introduced molecules. The identification of pesticides in complex samples can be a problem for LC with traditional detection methods such as UV or even diode array detection (DAD), because the latter technique may not be specific enough for spectral differences that are too small. A fluorescence detector can offer a greater selectivity compared to UV detection, but if there are many different compounds to be analysed, this technique will not provide adequate results [90]. Nowadays, liquid chromatography coupled with mass spectrometry (LC/MS) is becoming one of most powerful techniques for the residue analysis of polar, ionic or low volatility pesticides in fruit and vegetables. Compared to traditional detectors electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources in combination with MS instruments have increased the sensitivity of LC detection by several orders of magnitude. Single quadrupole
Anna Sannino
1.0
UV (280nm)
-MBC
-TM
volts
0.8
0.6
(b) 0.4 (a) 1.5
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x10 min UV (305 nm) -TM
4.5
x 10-1 volts
4.0 3.5
(b)
3.0 (a)
2.5 1.0
1.5
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2.5
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Fluorescence
-MBC
(b)
4.0
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x 10-1 volts
292
3.0
(b) 2.5
(a) 1. 0
(c)
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3. 0
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was the first mass spectrometer introduced in the market for combination with LC by means of atmospheric pressure ionization (API) sources. Those interfaces have the characteristic of being designed to provide a soft ionization process that lead to a mass spectrum with only few ions (commonly only the molecular ion), without the required specific structure diagnostic ions. The poor fragmentation of molecules is translated in deficient specificity because isobaric interferences (compounds with the same m/z ratio) or multiple component spectra are frequently observed in extracts of complex matrices [91]. Then, in contrast to GC/MS, single quadrupole mass spectrometers are not used in the majority of recent studies dealing with LC/MS. A disadvantage of single quadruple instruments is the high intensity of background signals derived from sample matrix and HPLC solvent clusters. Owing to this chemical noise in real samples, very low LODs cannot be achieved even if the sensitivity of these instruments is high. The chemical background can be reduced significantly if MS-MS in combination with SRM is applied. Reaction-monitoring modes enhance the detection limits in analytical procedures. In a QqQ, Q1 and Q3 quadrupoles are both fixed at a single mass. This technique is applied when the precursor and the product ions are known before analysis. Even if the co-extracted matrix component has the same molecular mass of a pesticide it can be separated in SRM experiments because their fragmentation in the collision cell most often results in different product ions. Therefore, MS-MS offer excellent sensitivity and unsurpassed selectivity. For this reason, QqQ analysers have been the most widely used technique for the determination of pesticides in food until now. In the last 4 years, many LC-MS-MS methods have been developed for multiclass pesticides in fruit and vegetables and water [92–101]. Nowadays, a modern commercial QqQ mass spectrometer is suitable to detect approximately 100 analytes simultaneously with sensitivity sufficient for residue determination at the 0.010 mg/kg level [92]. In the case of sufficiently high concentrations, the simultaneous observations of approximately 200 SRM transitions are feasible. The use of time window programs (periods) is not necessary unless the number of analytes to be analysed within one run is significantly increased, or pesticides with very low response are determined. Because of the high sensitivity achieved by MS-MS, gradient elution on a small reversed-phase column is usually used. However for confirmation purposes at least two transitions must be recorded. Pizzutti et al. [101] developed and validated a method for the analysis of 169 Figure 7 Chromatograms of (a) apricot puree free from carbendazim, TM and TBZ, and (b) apricot puree spiked with 0.1 mg/kg of carbendazim and TM and 0.0010 mg/kg of TBZ obtained by UV (at 280 and 305 nm), and (c) fluorescence detection at excitation/emission wavelengths of 280/310 nm, immediately following carbendazim elution at 305/345 nm. Column: 5 mm Supelcosil LC-18-DB; injection volume, 25 mL; mobile phase; 35% methanol in ion pairing solution. 1 g sodium decanesulfonate dissolved in a mixture of 200 mL of water, 7 mL of phosphoric acid and 10 mL of triethylamine and diluted to 1 L with water); column temperature: 401C and flow rate: 1.5 mL/min. Reproduced from Ref. [76] with permission from Elsevier, Copyright 1995.
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pesticides in soya, without clean-up, by LC-MS-MS using positive and negative ESI. In total, 155 pesticides were analysed in the positive mode and 14 pesticides in the negative mode, each mode in one single chromatographic run. ITDs may also operate in the MS-MS mode, which reduces the background to a level known from MS-MS. When performing MS-MS, quadrupole ion trap (QIT) instruments are generally more difficult to handle than QqQ analysers but they have the advantage of working in product-ion-scan without loss of sensitivity. Moreover, QIT offers the possibility of performing multiple-stage fragmentation (MSn). However, ion collection, fragmentation, and mass analysis of fragments in a step-by-step process in traps require much more time than in QqQ instruments, which do this in parallel. Furthermore, ion traps suffer from a limited dynamic range, a smaller potential to fragment very stable ions and the inefficiency to trap low-mass fragments. Soler et al. [102] compared QIT and QqQ for determining pesticides in orange samples. The results indicated that TQ provides higher precision, better linearity, it is more robust and when the purpose of the analysis is quantitative determination, it is preferable over QIT. However, the LOQs were almost the same for both instruments. All LC-MS instruments can be equipped with at least three types of soft ionization sources, which are ESI, APCI and photoionization. Up to now, applications of photoionization to the determination of pesticides have been rarely published. ESI and APCI are used more often. The ESI coupled with MS-MS is supposed to have a high sensitivity and selectivity for a wider range of pesticides in food. There are several instrumental parameters that have drastic influence on the ionization efficiency. The instrumental optimization includes the adjustment of typical interface parameters such as the ionization voltage in ES, the pressure of the spraying/nebulizing gases, the interface temperature and the clustering potential. The mobile phase is important to obtain a good chromatographic separation, but it also affects the analyte ionization and the sensitivity of the mass spectrometry. Usually, the use of MS-MS does not require any chromatographic separation between analytes, because it is very rare to find molecules that share the same unique transition. However, the simultaneous analysis of a high number of compounds by MS-MS requires at least a sufficient chromatographic separation, in order to reduce the matrix effect. Methanol and/or acetonitrile are usually used as organic modifiers. LC separations are sometimes improved at acidic pH, using acetic acid or formic acid, as such or in combination with ammonium acetate or ammonium formate. The intensity of signal can be increased in the positive ionization (PI) mode; however the presence of H+ ions inhibited the negative ionization. A relatively new and valuable technique in the field of pesticide analysis is the TOF-MS. The accurate mass measurements of the TOF-MS (typical mass error o2 mDa) ensure an equal selectivity as that obtained with the two type of MS-MS. The high degree of selectivity is obtained by removing matrix interferences at the same nominal mass measurement (using the exact mass chromatograms). The main advantage of this type of instrument is the
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identification of non-target pesticides and unknown peaks in a sample even if analytical standards are not available. Thurman et al. [103] developed an identification scheme using a combination of LC/TOF-MS (accurate mass) to generate elemental composition of ions and LC/MS ITMS (MS-MS) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in citrus fruit extracts. This scheme has been applied to identify two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil and a non-previously reported prochloraz degradation product. The high speed and acceptable sensitivity have made TOF an attractive alternative to quadrupole or QqQ. Recently, LC/TOF-MS has been proven to be a sensitive and selective method for the determination and confirmation of pesticide residues in vegetables and fruit [104] obtaining LODs in compliance with established MRLs. Gilbert-Lopez et al. [105] developed a method based on LC/ESI-TOF-MS for the determination of 12 pesticides (MCB, TBZ, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, iprodione, diphenylamine and procymidone) in fruit-based baby foods. The confirmation of the target pesticides was based on accurate mass measurements of protonated molecules (M + H)+ and fragment ions. LODs were between 0.1 and 10 mg/kg depending on pesticide studied.
6. LC-MS-MS APPLICATIONS 6.1 Chlormequat In agriculture, the quaternary ammonium herbicides chlormequat (CQ) and mepiquat (MQ) are used as plant growth regulators either individually, as mixtures, or together with other pesticides. CQ has especially attracted the attention of enforcement laboratories and regulatory agencies in Europe, reflected by numerous publications and website notifications on violative levels of residues in fresh pears, pear juices for infants, tomatoes and cereals [106]. Before introduction of method based on LC/MS in the late 1990s, the analysis of CQ was difficult owing to its ionic character and to the absence of chromophore groups in its chemical structure. The GC method most frequently applied involved the conversion of CQ into acetylene, which was determined by flame-ionization detection [107]. However, this reaction was not specific, and an extensive clean-up was required. Recently, with wider availability of robust instruments, a number of methods based on LC/MS and LC/MS-MS for the determination of CQ [108,109] or for the determination of CQ and MQ [110,111] have been reported. The CEN Technical Committee CEN/TC 275 ‘‘Food Analysis-Horizontal Methods’’ has prepared the European Standards EN [112] 15054 and EN 15055 [113] for two methods applicable to all kinds of fruit, vegetables and cereal grains. These methods both use stable isotope internal standardization and identical extraction procedure employing MeOH–water and they do not involve any clean-up. The sample is mixed with deuterated internal standards, water and methanol and the
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Table 4
a
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Analyte MS-MS transitions established by EN 15055 method
Compound
Precursor
Firsta transition mass (m/z)
Second transition mass (m/z)
Chlormequat d4-Chlormequat Mepiquat d3-Mepiquat
M+H M+H M+H M+H
122-58 126-58 114-98 117-98
124-58 128-58 114-58 117-101
MS-MS transition used for quantitation.
homogenate is centrifuged. An aliquot is analysed by LC-MS using PI mode. The two methods differ in specifying the use of LC/MS or LC-MS-MS for the determination, the amount of internal standard used and their expected LOD. Alder and Startin [114] reported the results of an interlaboratory validation study of these methods conducted on behalf of CEN TC 275/WG 4 by the German Federal Institute for Health Protection of Consumer and Veterinary Medicine (now Federal Institute for Risk Assessment), Berlin. The method has been collaboratively studied on mushrooms, pears, wheat flour, fruit puree and on infant formula. For LC-MS the instrument operated in SIM mode for the ions 122 and 124 (CQ), 126 and 128 (d4-CQ), 114 (MQ) and 117 (d3-MQ). For LC-MS-MS the instrument operated in multiple reaction monitoring. MRM transitions established by the CEN LC/MS-MS method are listed in Table 4. Figure 8 shows LC-MS-MS chromatograms of a pear puree extract and the same sample fortified with CQ at 10 mg/kg obtained with a QqQ instrument. Under the isocratic conditions described in EN 15055 method the analyte elutes at approximately 6 min. The additional selectivity of LC-MS-MS allows measurement at 10 mg/kg or below, as required for infant foods, whereas LC-MS is satisfactory for higher levels.
6.2 Pesticides of new generation Recently, several applications have described the use of MS-MS with both QqQ and IT analysers in multiresidue analysis of pesticide [97,115–117]. Most of the methods achieve satisfactory results even without making use of clean-up treatments. Sannino et al. [117] described a sensitive and selective method for the determination of 24 new pesticide residues (azoxystrobin, trifloxystrobin, kresoxim-methyl, fenazaquin, indoxacarb, fenothiocarb, furathiocarb, benfuracarb, imidachloprid, dimethomorph, fenpyroximate, hexythiazox, tebufenpyrad, tebufenozide, defeconazole, fenbuconazole, flusilazole, paclobutrazol, tebuconazole, tetraconazole, bromuconazole, etofenprox, fenhexamid, pyridaben) in apple puree, concentrated lemon juice and tomato puree. Samples were extracted with acetone and partitioned with cyclohexane/ ethyl acetate (50 + 50, v/v). This method requires only small volumes of solvent per sample, short analysis time and does not use any chlorinated solvents.
Pesticide Residues
297
650 600 550 (a)
500 450
Intensity,
400 350 300 250
122 → 58
200 150 100 50 0
1 1
0
4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 min
650
5.6
600 550
(b)
500 450
Intensity,
400 350 300 250 200 150 100 50 0 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 min
Figure 8 LC/MS-MS chromatograms (m/z 122-58) of pear puree: (a) unfortified and (b) fortified sample with CQ at 10 mg/kg. Injection volume, 20 mL of a 0.333g/mL sample; column Shodex Rspack De 413, 150 4.6 mm; mobile phase: Methanol/water 1 + 1 (v/v) with 50 mmol ammonium acetate and acetic acid 2.5% and flow rate: 0.3 mL/min.
Analyte MS-MS transitions, retention time and instrument conditions Retention time (min)
Firsta transition Mass (m/z)
DPb (V) CEc (V)
Second transition mass (m/z)
DPb (V)
CEc (V)
Third transition mass (m/z)
DPb (V)
CEc (V)
Azoxystrobin Benfuracarb Bromuconazole Difeconazole Dimethomorph Etofenprox Fenazaquin Fenbuconazole Fenhexamide Fenothiocarb Fenpyroximate Flusilazole Furathiocarb Hexythiazox Kresoxim-methyl Imidachloprid Indoxacarb Paclobutrazol Pyridaben Tebuconazole Tebufenozide Tebufenpyrad Tetraconazole Trifloxystrobin
8.13 9.11 7.91 8.60 7.23 9.96 9.62 8.17 7.40 8.26 9.32 8.08 9.11 9.41 8.64 3.04 9.02 6.97 9.58 7.57 8.21 8.72 7.87 8.94
404-372 411-195 378-159 406-251 388-301 359-183 307-161 337-125 302-97 254-72 422-366 316-247 383-195 353-228 314-206 256-209 528-249 294-70 365-309 308-70 297-133 334-117 372-159 409-186
19 13 25 49 26 52 22 27 64 20 50 21 20 15 10 26 15 30 40 27 16 60 27 15
404-344 411-252 376-159 408-253 390-303 394-177 307-147 337-70 304-97 254-160 422-135 316-165 383-252 353-168 314-116 256-175 528-293 296-70 367-311 310-70 353-139 334-145 374-161 409-206
19 13 25 47 32 9 22 27 62 20 50 21 20 15 10 26 15 35 40 27 46 60 27 15
30 18 40 32 27 20 26 44 33 13 46 37 23 35 19 22 20 40 18 49 24 45 45 20
404-329 411-190 380-161
19 13 25
40 17 40
307-57
22
45
314-267
10
9
528-218
15
31
308-151
27
35
20 32 40 34 26 31 21 40 34 30 23 26 25 23 10 19 23 41 18 47 20 55 37 24
Source: Reproduced from Ref. [117] with permission from Elsevier, Copyright 2004. a MS-MS transition used for quantitation. b Declustering potential (similar to the cone voltage of other manufacturers). c Collision energy.
Anna Sannino
Compound
298
Table 5
8.13
1.6e4
8.64
Azoxystrobin 2μ/Kg
Intensity, cps
1.2e4
404
1.0e4
→
373
10μ/Kg
8000.0 6000.0
1500
4000.0
1000
2000.0
500
0.0
0
5.0e4 Intensity, cps
Kresoxim-methyl
2500 2000
6
8 Time, min
4.0e4
10
9.62
6.6e4 6.0e4
307
3.0e4
1.0e4 0.0
1550 1400 1200 1000
6
8 Time, min
10
3.04
Imidachloprid 10μ/Kg 256 → 209
→ 206
2000.0 6
8 Time, min
10
9.02
Indoxacarb 4μ/Kg 528 → 249
7000 6000
3000
2000
2000
1000
1000 6
2000
8 Time, min
10
6
8 Time, min
10
9.11 1.20e5
Dimethomorph 5μ/Kg 388 → 301
1.00e5
Benfuracarb 10μ/Kg 411 → 195
8.00e4 6.00e4 4.00e4
1500 1000
200
0
Fenothiocarb 4μ/Kg 25 4 → 72
2.00e4
500 5 Time, min
0
6
8 Time, min
10
0.00
6
8 Time, min
10
299
Figure 9 Typical MRM profiles of a fortified apple puree at 2–10 mg/kg. Injection volume, 40 mL of 1 g/mL sample. HPLC conditions: A 4-mm Synergy Polar-RP column (150 2.0 mm) (Phenomenex, Aschaffenburg, Germany) was operated at a flow rate of 0.250 mL/min. The following elution program was used: at the start 60% solvent A (0.1% aqueous formic acid) and 40% solvent B (acetonitrile); after 0.5 min the percentage of solvent B was linearly increased to 95% in 4.0 min; kept constant for 5.5 min; ramped to original composition in 1 min and then equilibrated for 9.0 min. MS analyser: QqQ operating in MRM mode (Table 5). Reproduced from Ref. [117] with permission from Elsevier Science, Copyright 2004.
Pesticide Residues
2500
10
8.25
7.23
4975 4500
600
8 Time, min
8000
4000
3500
6
9000
3000
3000
400
0.0
5000
4000
→ 186
6000.0
4000
800
0
4 09 8000.0
5000
0
Trifloxystrobin 2μ/Kg
8.77
6000
57
1.0e4
4000.0
7000
Fenazaquin 4μ/Kg →
314
8700 8000
2.0e4
Intensity, cps
3000
8.94
1.3e4 1.2e4
3500
1.4e4
7.87 4500
2.0e4
4000
1.4e4
Tetraconazole 4μ/Kg 372 → 159
1.5e4
3500 3000
Bromuconazole 4μ/Kg 378 → 159
Anna Sannino
Tebuconazole 4μ/Kg 308 → 70
1.2e4 Intensity, cps
7.91
300
7.57 1.6e4
1.0e4 2500 1.0e4
8000.0
2000 6000.0
1500 5000.0
4000.0
1000
2000.0
500
0.0
0
0.0 6
8 Time, min
10
6
8
8
10
9. 96
7.40
3300
2500
10
Time, min
Time, min
9.58
3450
3000 3000
Intensity, cps
2000
Etofenprox 10μ/Kg 359 → 183
Fenhexamide 10μ/Kg 302 → 97
2500
2500
2000
1500
Pyridaben 8μ/Kg 365 → 309
2000
1500
1500
1000 1000
1000
500
500
500
6
8 Time, min
Figure 9 (Continued)
0
0
0 10
6
8 Time, min
10
8 Time, min
10
Pesticide Residues
301
Extracts were analysed by LC-MS-MS without any further clean-up steps. Suitable transitions from precursor to product ions (MRM transitions) were identified for each compound. For confirmation of results a second and, for azoxystrobin, kresoxim-methyl, fenazaquin, benfuracarb, tebuconazole, bromuconazole, a third fragmentation of the selected parent ions can be used. The simultaneous measurement of 55 MRM transitions was carried out (Table 5). Thus, 24 pesticides can be screened in a single injection using only one retention time window (period). Typical chromatograms of individual MRM transitions for 24 pesticides in apple puree extract at concentrations ranging from 2 to 10 mg/kg are shown in Figure 9. Although interferences are not visible in the LC-MS-MS chromatograms, co-eluting matrix components could inhibit or enhance the analyte signal. This phenomenon is referred to as matrix effects (13). The best way to compensate for matrix effects is the use of isotope internal standards, however for most pesticides these compounds are not available. In this study, calibration was performed by external matrix matched standards to eliminate the matrix effect and to obtain a more realistic determination.
7. CONCLUSIONS AND FUTURE TRENDS The area of pesticide analysis is complicated by the large number of possible residues, which are classified in terms of functionality as well as being classified in according to their chemical nature. Nevertheless, the analysis of pesticides in food samples is made even more complex by the fact that the concentration of matrix components is often greater than the concentration of the target pesticide. The MRMs are preferred due to the importance of checking a large number of pesticides in one single analysis. Simplifying complicated and time-consuming sample pre-treatment is still a pending subject. Traditional solvent extraction remains the most widely used method for sample preparation with a tendency to suppress the clean-up procedures of this isolation step. However, with the current trends to reduce solvent usage and manual labour, several alternative extraction approaches have been introduced, for example MSPD, SPME and SBSE. GC coupled with EI-MS and LC with MS-MS using ESI are the most important detection techniques in pesticide residue analysis today. Alder et al. [118] compared the applicability and sensitivity of MS in combination with GC and LC for the determination of 500 high-priority pesticides. This chapter summarizes all typical precursor and product ions appropriate for LC-ESI-MS-MS determination and the sensitivity obtained. The applicability of GC-MS with EI-MS was evaluated for the list of pesticides. Only for one substance class, the OC pesticides, GC-MS achieves better performance. The development, optimization and implementation of recent approaches in LC-MS-MS, including linear traps, and hybrid instruments are expected to improve performance even more in the near future. During the past 6 years, the number of applications of LC-MS-MS to control pesticide residues rapidly increased.
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Moreover, important advances were made in the development and application of GC combined with QqQ mass spectrometer. The detection of pesticides by GC-tandem quadrupole mass spectrometry is now supplanting current GC-MS detection with a single quadrupole, as demonstrated by the many examples of application of GC-MS-MS to the analysis of pesticide residues that can be found in recent literature.
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CHAPT ER
10 Veterinary Drug Residues Sherri B. Turnipseed and Wendy C. Andersen
Contents
1. Introduction 1.1 Antibiotics 1.2 Anthelmintics and antifungals 1.3 Tranquilizers and anti-inflammatory drugs 2. Physical and Chemical Properties Affecting Analytical Methodology 2.1 Properties of veterinary drugs 2.2 Properties of food matrices 3. Human Health Effects from Veterinary Drug Residues 3.1 Direct effects 3.2 Antibiotic resistance 4. Analytical Methods 4.1 Types of methods 4.2 Summary of analytical methods 4.3 Specific examples of recent methodology 5. Occurrence in Food 6. Future Trends 6.1 Biosensors 6.2 Multi-class methods References
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1. INTRODUCTION The use of animal drugs in food production has been a standard practice for decades. The high density of animal populations in agricultural facilities can increase the potential for disease outbreak. This results in the need for medications to be given therapeutically to treat existing infections or prophylatically to minimize the impact of an outbreak spreading across an animal population. In addition, veterinary drugs are sometimes provided to food Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00010-X
r 2008 Elsevier B.V. All rights reserved.
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animals at subtherapeutical levels to promote growth efficiency and increase feed conversion. A 1999 study indicated that 60–80% of all animals consumed in the United States had been treated at one point with an approved drug [1]. Therefore, the possibility of drug residues remaining in the edible product and the potential human health problems associated with exposure to these residues is a concern to public safety. The following is a brief description of the types of veterinary drugs that are used in food animals. The structures of many of the representative drugs discussed are shown in Figures 1–3 [2].
1.1 Antibiotics Antibiotics are commonly administered to food animals both therapeutically and subtherapeutically. Several classes of antibiotics are used in livestock veterinary medicine. Sulfonamides are derivatives of sulfanilamide, one of the first drugs discovered to treat Streptococcal infections. Sulfamethazine and sulfadimethoxide are shown as examples of this class of drugs in Figure 1. Although not technically classified as antibiotics, these drugs are bacteriostatic agents that effectively interfere with the synthesis of folic acid in susceptible organisms and are widely used in both humans and food-producing animals to prevent infectious diseases [3]. Penicillin is one example of a b-lactam antibiotic historically used in veterinary medicine. All antibiotics in this class contain a b-lactam ring [4]. Many b-lactams approved as animal drugs are based on the penicillin molecule, including ampicillin, amoxicillin and cloxacillin. Cephapirin and ceftioflur, however, contain a cephalosporin nucleus. These drugs are effective in cattle that are fighting against mastitis as well as respiratory and intestinal infections. They are also administered to swine and allowed to be used in some countries for farm-raised finfish. Aminoglycoside drugs are antibiotics effective against most gram-positive and negative organisms. They are isolated from Streptomyces or Micromonospora species and their mode of action is to interfere with bacterial protein synthesis. These drugs consist of linked amino sugar groups; examples include streptomycin, apramycin, dihydrostreptomycin, gentamicin and neomycin (Figure 1). They are administered both therapeutically and prophylatically to treat cattle, swine and poultry [5]. Aminoglycosides are not absorbed orally and so are usually administered via intramuscular injection. Residues of these drugs tend to concentrate in the kidney as they are generally excreted through the urinary tract. Tetracyclines are broad-spectrum antibiotics that inhibit protein synthesis in bacterial cell walls. They consist of a substituted 2-napthacenecarboxamide molecule. These antibiotics were originally isolated from Streptomyces. The most common tetracyclines with animal health applications are tetracycline, oxytetracycline and chlortetracycline [6]. In the U.S., these drugs have been approved for beef cattle, calves, swine, sheep, chickens and turkeys. Oxytetracycline has been specifically approved for use in dairy cattle and aquacultured catfish, salmon and lobster. This drug has not been approved by the U.S. for shrimp aquaculture, although it is believed to be used widely in shrimp production in other parts of the world.
CH3
OCH3
N
O O
S
CH3
N N H Sulfamethazine
H2N
OCH3 N N H Sulfadimethoxine
H2N
O H
S
CH3 CH3
N
OH
O
N Ceftiofur
OH OH O
O
S
N
O
O
O
CH3
F
COOH
O2N
NH2 O
H3C H3CO
O H CH3 COOH
H3C
O
H
H O
Monensin
Cl Cl
H
CH3
O
CH3 H O
Veterinary Drug Residues
OH
H N
N Nitrofurazone
HO
H N
Chloramphenicol
NH2
H3C
CH3
HO OH
309
Figure 1 Structures of antibiotics [2].
OH
O OH
O
O2N
CH3 O
O
N
OH
Neomycin
OCH3
O Sarafloxacin
CH2 O
CH3 CH3
OH
O
HO
NH2 CH2
N 2′ O
CH3 Erythromycin
F
NH2 H2N O O
O
H3C
CH3 O HO
CH3
N
O
NH2
H2N
OH H3C O
HN
O
OCH3 O COOH
HO HO
OH
S
N H
H2C HO HO
CH3
OH
Oxytetracycline
H H N
H3C
NH2
COOH H 2N
O H3C
H3C CH3 CH3 OH N H H OH
HO
H
N H Benzylpenicillin (Penicillin G) O S
N
O O
S
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O
H N N
S
OCH3
N H
Fenbendazole
H
S
N N
Levamisole
H3C
N
CH3
N
CH3
CH3
Malachite Green
OC 3 OCH HO OCH3 O H3C
O
CH3
H O H3C
CH3
H O
O CH3
H
O H
H3C
Ivermectin
O
O
OH
H
H R
O CH3 H OH
Figure 2 Structures of anthelmintics and antifungals [2].
Another class of antibiotics originally isolated from the Streptomyces species is the macrolides [7]. Macrolides are multi-membered lactone rings with one or more sugars attached. These drugs are most effective against gram-positive organisms and some strains of Listeria and Mycoplasma. Erythromycin (Figure 1) and tylosin are the drugs most commonly given to food-producing animals. Oleandomycin, spiramycin, sedamycin and tilmicosin are also used therapeutically to treat bovine respiratory diseases and mastitis and swine dysentery, or used as a feed additive to promote growth efficiency. Macrolides can also be added to the food and water supplies for chickens and turkeys to prevent respiratory disease and enteritis, as well as to promote growth. These drugs are also effective in treating bee colonies for brood disease. Although their chemical
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N O
N N CH3
F
H N
S
Azaperone
N H3C
Xylazine
CH3 N
H N
CF3
COOH
Flunixin
O
N N
H3C O
Phenylbutazone
Figure 3 Structures of tranquilizers and anti-inflammatory drugs [2].
structure is quite different, the lincosamide antibiotics (lincomycin and pirlimycin) have similar antibacterial activity, clinical applications and cellular mechanisms as the macrolides. Quinolones are pyridone carboxylic acid derivatives that are effective against gram-negative bacteria. Examples of quinolone drugs include oxolinic, nalidixic and piromidic acids. Quinolones have been found to be effective as a prophylactic treatment for aquacultured species; they have also been investigated by the swine and poultry industry. Fluoroquinolones are the fluorinated subclass of the quinolones that also exhibit activity towards gram-positive bacteria [8]. Sarafloxacin, enrofloxacin, difloxacin, ciprofloxacin, norfloxacin and ofloxacin are just some examples of fluoroquinolones. As is discussed later, there are growing concerns with the use of these drugs in foods of animal origin due to the problem of increased antibiotic resistance associated with this subclass of antibiotics. Compounds that retard the development of a parasite in a host cell are known as coccidiostats. A common class of drugs used for this purpose is the ionophores, which include monensin (Figure 1), salinomycin and lasalocid [9]. Other drugs such as amprolium, clopidol, halofuginone, nicarbazin and even the tetracyclines can also be effective coccidiostats. While these drugs are most commonly given to poultry populations, monensin, lasalocid and amprolium can
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also be administered to cattle to treat coccidiosis. The ionophores may also be used to promote growth in cattle. Another class of drugs used in veterinary medicine is the peptide antibiotics including bacitracin, colistin, virginiamycin and avoparcin. Each of these drugs are actually a mixture of peptides produced by Bacillus and Streptomyces species and may be glycosylated in some positions. They are primarily added to animal feed to enhance growth. There are concerns regarding antibiotic resistance from glycopeptides because of their structural similarity to the human drug vancomycin [10]. Other antibiotic classes that will be discussed in further detail later in the chapter include the nitrofurans and the phenicols such as chloramphenicol and florfenicol. Representative drugs from these classes of antibiotics are also included in Figure 1.
1.2 Anthelmintics and antifungals Anthelmintic (deworming) compounds control parasitic infections. Benzimidazoles are anthelmintics that contain a common 1,2-diaminobenzene nucleus; most of them also have a carbamate functional group. Albendazole and fenbendazole are common drugs in this class. These drugs protect against roundworms and flukes by inhibiting the processes necessary for parasitic mitochondrial production of ATP [11]. The use of fenbendazole (Figure 2) can also increase the rate of weight gain in growing and finishing swine. Avermectins are anthelmintics isolated from Streptomyces avermitilis. Ivermectin, doramectin and eprinomectin are avermectin compounds that may be administered to cattle to combat parasitic infections such as roundworms, lungworms and mites, among others [12]. Approvals have been granted for injectable or oral (bolus) dosage forms for some of these drugs in cattle, but it is common for these drugs to be applied topically. Ivermectin and doramectin are not approved for lactating dairy cattle. Some avermectins, including emamectin, have also been shown to be effective for the treatment of fishfin diseases such as sea lice in farm-raised salmon. Moxidectin is a synthetic milbemycin drug with similar properties to the avermectins; it has been approved for use in dairy cattle. Levamisole (Figure 2) is another anthelmintic that is used for the control of lungworms and gastrointestinal nematodes in cattle, sheep and swine. It can be administered orally, topically, via injection or in a medicated feed. Other compounds such as the tetrahydropyrimidines (i.e., pyrantel or morantel) are administered to swine for the treatment of parasites and are considered broadspectrum anthelmintics in ruminants. Organophosphates (i.e., dichlorvos and coumaphos) can also be considered anthelmintics in food animals, as well as being an effective external pest control. Triphenylmethane dyes are effective fungicides and are used illegally in food production facilities for that purpose. Malachite green, in particular, is added to the water of aquaculture ponds to treat fungal infections. An analogous compound, crystal violet, has also been reported to have been used for aquaculture as well as in chicken feed to prevent fungal growth.
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1.3 Tranquilizers and anti-inflammatory drugs Drugs administered to food animals to treat acute conditions include tranquilizers and anti-inflammatory drugs (Figure 3). These medicines might be given to animals shortly before they are sent to slaughter, resulting in potentially high levels of residues that may persist in edible tissue. For example, tranquilizers, such as acepromazine, azaperone, chlorpromazine, propionlypromazine and xylazine, have been shown to reduce stress or aggressiveness during transport for certain species of pigs [13]. The b-blocker carazolol has also been used [14]. In the U.S., azaperone is approved for the control of aggressiveness in small pigs. In the European Union (EU), maximum residue limits have been set for carazolol and azaperon; the use of chlorpromazine in food animals is not allowed [13]. The non-steroidal anti-inflammatory drugs (NSAIDs) are effective in reducing inflammation and managing pain. Those used in veterinary medicine include flunixin, phenylbutazone, ketoprofen, aspirin, dipyrone and naproxen. In general, these drugs are only approved for pets and other non-food animals (horses). Although flunixin is approved for some therapeutic applications in cattle, there is concern that the non-approved drugs may also be administered covertly to disguise lame or sick food animals, thus allowing them to pass federal meat inspections. In 1992, a poll of nearly 1500 U.S. veterinarians revealed that over 66% of the veterinarians used dipyrone, aspirin and phenylbutazone to treat food-producing animals [15].
2. PHYSICAL AND CHEMICAL PROPERTIES AFFECTING ANALYTICAL METHODOLOGY 2.1 Properties of veterinary drugs Residue methods must be able to isolate and detect very small amounts (partper-billion or lower) of an analyte in a variety of complex matrices. The physical and chemical properties of the drug(s) of interest as well as the accompanying matrix must be evaluated to determine the optimal procedures for extraction, isolation and detection at residue levels. Specifically, molecular properties that can affect an analytical approach for any given compound include polarity, charge state, size, volatility, thermal stability and the presence or absence of a chromophore. For example, the polarity and/or charge state of an analyte can be a crucial factor in designing an effective extraction and isolation scheme. Traditionally, liquid–liquid extraction with pH adjustment (i.e., back extraction) can be used to transfer the analyte between polar and non-polar solvents, removing matrix impurities such as salts and lipids in the process. The use of reversed-phase, ion-exchange or mixed-mode solid-phase extraction (SPE) cartridges can also achieve similar results. The size of an analyte may be advantageous for sample clean-up with techniques such as gel permeation chromatography. Fedeniuk and Shand have thoroughly reviewed the relationship between the chemical properties of veterinary drugs and strategies needed for their extraction and isolation [16].
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Further isolation of an analyte is often achieved with chromatographic separation. Volatile, thermally stable compounds can be very effectively separated from the sample matrix and other sample components using gas chromatography (GC). More polar or less thermally stable analytes can also be analysed by GC after derivatization, but are more commonly analysed by reversed-phase liquid chromatographic (LC) methods. The optimal LC stationary phase (e.g., C18, C8 and phenyl) and mobile phase components (buffer, organic modifier) are determined by properties such as the pKa of any ionizable groups and the presence of other functional moieties such as a phenyl ring. The detection of any compound must also take into account the physical and chemical properties of the molecule. For example, there are element-specific detectors for nitrogen- and sulfur-containing compounds for GC. Volatile analytes containing electrophilic functional groups are very selectively detected by GC electron capture detectors (ECD). Traditional LC systems generally utilize ultraviolet (UV), visible or fluorescence detectors that take advantage of unique chromophores present (or added via derivatization) in an analyte. Recently, liquid chromatography-mass spectrometry (LC-MS) has become a predominant methodology for the detection of veterinary drug residues. One decided advantage of MS is its ability to act as a universal detector without the need for a significant chromophore. The physical and chemical properties of the analyte, however, will determine the optimum parameters for an LC-MS method. The most common ionization techniques used are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). In general, ESI works well for more polar analytes that are charged in solution, while APCI will ionize, via proton exchange in the gas phase, a larger range of less-polar compounds. Recently, no-discharge APCI has been shown to be very sensitive and selective for certain veterinary drug residues, particularly those that easily form sodium adducts [17].
2.2 Properties of food matrices Because the human diet is so diverse, the type of matrices in which animal drug residues are found is quite varied. The following is a brief description of the food commodities commonly monitored for veterinary drugs. The prevalent use of veterinary drugs in cattle, swine and poultry production has resulted in the need to monitor residues in the tissues of these animals. This includes muscle meat such as various cuts of beef and pork, as well as in poultry products. Initial sample preparation for meat products involves cutting the tissue into small portions and homogenizing the pieces with a homogenizer or similar apparatus. In some cases, residues can bind to the proteins in the muscle and hydrolysis is required to release the residues. Owing to typical animal excretion pathways, it is also quite common to find residues in organ meats such as liver and kidney. These tissues typically have a high fat content, which requires additional fat removal clean-up steps. The residues may also occur as conjugates, i.e., modified with glucoronide or acetyl groups, when present in these organ
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meats. The use and analysis of veterinary drugs in meat have been reviewed [18,19]. Dairy cows are susceptible to mastitis, a common bacterial infection that is effectively treated with antibiotics. Because milk is a commodity that is consumed in a higher proportion by children, the monitoring for residues in milk is of increased importance. Often the allowed amount of approved residues in milk is lower than that for edible bovine tissues. Sample preparation steps for veterinary drug residue methods for milk often involve defatting and protein precipitation, with acetonitrile or trichloracetic acid, prior to or concurrent with the extraction of residues. Information concerning the incidence of veterinary drug residues in other dairy products, such as dried or condensed milk or different kinds of cheeses, is fairly limited. Recent studies, however, indicate a growing interest in these types of food products [20–22]. Aquaculture, or fish farming, is a rapidly growing industry. Approximately 80% of the fish and shellfish consumed in the U.S. is imported from other countries, amounting to over 2 billion pounds of harvested seafood imported into the U.S. in 2004. A large percentage (approximately 40%) of that imported seafood is produced by aquaculture facilities [23]. The use of antibiotics and antifungal drugs in aquaculture has also increased with the expansion of the industry [24]. Fish raised in these high-density environments may experience higher stress and weakened immune systems requiring drugs to stem disease outbreaks. Methods for residue extraction and isolation in aquatic organisms are similar to those used for other animal tissues. The fat content of fish can vary significantly depending on the species and even the specific environmental growing conditions. Veterinary drug residues can also be found in foods such as eggs and honey produced as animal by-products. The drugs of concern in eggs are similar to those monitored for in poultry tissue, such as coccidiostats. Residue-extraction procedures designed for tissue can be applied to eggs, but additional steps may be required to eliminate sample components that are unique to the egg matrix [25]. Foul brood disease has become a global problem in the apiary industry and many drugs such as tetracyclines, macrolides and phenicols have been used to combat this threat to honey production. The high sugar content of honey complicates residue isolation and clean-up. Additional aqueous wash steps to eliminate the excess sugar, as well as the use of internal standards to compensate for any background interference or signal suppression, are often required [26].
3. HUMAN HEALTH EFFECTS FROM VETERINARY DRUG RESIDUES 3.1 Direct effects The public health significance of veterinary drug residues in foods of animal origin has been a topic of much discussion [1]. Potential human health problems include adverse reactions to the drug residues themselves or the possible development of resistant bacterial populations in the food supply. There are a few
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limited reports of allergic responses caused by drug residues in foods. For example, incidences of sensitization to aminoglycosides [27] and b-lactams [28,29] have been reported. Some drugs have potentially more serious acute effects. One example is chloramphenicol, a dichloroacetamide derivative of 1-phenyl-2-amino-1-propanol (Figure 1). Chloramphenicol is prescribed for stubborn bacterial infections in both humans and companion animals. It is given cautiously, however, as there have been reports that chloramphenicol can cause aplastic anemia, a fatal bleeding disorder, in susceptible individuals [30]. It is not approved for use in food-producing animals so that unintended exposure, even at residue levels, can be avoided. Monitoring food for residues that may occur as a result of inappropriate use is important because chloramphenicol has been found to be effective in treating infections in food animals. In 2002, residues of chloramphenicol were found with a relatively high frequency in shellfish (shrimp, crab and crayfish) and honey products from certain parts of the world. Florfenicol, an analog of chloramphenicol that does not cause the same potentially dangerous side effects, has been approved for the treatment of bovine respiratory disease and for the control of enteric septicemia in farmed fish. Residues of the nonsteroidal anti-inflammatory drug phenylbutazone (Figure 3) may also be a human health concern as this drug has been shown to induce tumors in some species and exposure to this drug has also been reported to cause aplastic anemia and gastrointestinal bleeding in susceptible people [31]. Other drugs are of concern due to possible carcinogenicity. One example is the nitrofuran compounds, which have been used as antibiotics in the poultry industry as well as for the treatment of cattle and pigs. The structure of nitrofurazone is shown in Figure 1. Residues of these drugs also have been found in farm-raised shrimp and honey. Some data suggest that nitrofurans may be carcinogenic [32]; therefore, these drugs are not approved for use in any food animal. Another example is malachite green, a triphenylmethane dye used to control fungi in aquaculture ponds (Figure 2). Malachite green undergoes a metabolic reduction to the leuco form in fish tissue. Leucomalachite green is lipophilic and can remain in fatty tissue for extended periods of time. Studies have shown that these compounds may be mutagenic and teratogenic [33].
3.2 Antibiotic resistance One of the main issues associated with the use of antibiotics in food animals is the concern of developing antibiotic resistance. Animals fed with subtherapeutic levels of antibiotics may harbor bacterial populations that have evolved to be immune to these drugs. Humans may be exposed to these resistant bacterial populations during the preparation or consumption of food. There is general support for the fact that the use of these drugs can cause resistant bacteria to develop in animals and contaminate the resulting meat products [34–37]. There is evidence that this may translate into increased resistance to these drugs in the human population [37], although this remains an area of debate [34]. Many of these resistant organisms are non-pathogenic and do not cause infection when
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ingested by humans. The appearance of resistance among pathogenic organisms, such as Salmonella DT-104 and Campylobacter, is of more concern [38–40]. For example, there appears to be a correlation between the use of fluoroquinolone antibiotics in chickens and an increase in resistant Campylobacter infections in humans. As a result, the use of fluoroquinolones, such as enrofloxacin and sarafloxacin, for the treatment of poultry populations is no longer allowed in the U.S. The link between enterococci resistance to glycopeptides and their use in animal feeds has also been investigated [10,37,41]. In the late 1990s, the National Research Council convened a group to evaluate the benefits and risks of using drugs in the food animal industry [1]. This group identified antibiotic resistance as the most serious risk associated with the continued use of drugs in food animals. They recommended that the animal health, medicine, consumer advocate and academic communities work together with the various government agencies to formulate integrated research plans and policy development to address this issue. The group recommended continued reevaluation of the animal drug approval process to expand the number of drugs that are available. The investigation of possible alternatives to the use of drugs in food animals, such as reducing overall animal population density, was also discussed. With the intent of mitigating antibiotic resistance from the treatment of food animals, the U.S. Food and Drugs Administration (FDA) recently posted guidance that outlines how a sponsor of a new animal drug application should evaluate their product for potential effects on non-target bacteria. This FDA guidance suggests that the drug sponsor provide information such as known resistance mechanisms and genetics, occurrence and rate of transfer of resistance determinants and possible resistance selection pressure. These factors, along with the knowledge concerning how the drug, or drug class, is used in human medicine, will be considered as part of the approval process [42].
4. ANALYTICAL METHODS Because of the potential health risk from exposure to some animal drugs and the low levels at which they are found in edible products, monitoring the food supply for these residues is an important analytical challenge. Tolerances or maximum residue levels are established for drugs that are allowed for use in food animals. For these drugs, analytical methods need to be able to accurately determine if a residue is present above these set limits. For some compounds (e.g., chloramphenicol, nitrofurans and malachite green) the health concerns are such that no amount of residue is allowed. In these cases, analytical methods need to be as sensitive and selective as possible to monitor for any residue that might be present.
4.1 Types of methods Several broad types of analytical methods can be described; these include screening, determinative and confirmatory procedures. Screening methods are
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designed to be rapid, easy to use tests that will give a positive or negative response for a drug at a given concentration level in a matrix. Historically, microbial inhibition tests (MITs) have been used to screen large numbers of samples for antibiotics. All MITs are based on the inhibition of bacterial growth by antibacterial residues present in a biological fluid that results in zones of inhibition on assay plates. MITs are relatively simple to perform and can detect many classes of antibacterial compounds. Selective sensitivity for specific classes of antibacterials can be obtained by changing the culture medium, indicator bacteria or pH. Although these tests are still widely used today, they often lack the specificity and sensitivity required for residue detection at the required levels, may be affected by non-specific inhibitors, do not detect microbiologically inactive metabolites and often have a 20–24 h incubation time. In addition to MITs, new rapid test kits are being used to screen samples for specific drugs. A database of available antibiotic and veterinary drug residue test kits is maintained by the AOAC International [43]. The information integrated in the database includes the analyte, the manufacturer, the kit name, primary matrices and whether or not the kit has undergone performance testing (validation) by the AOAC. Several types of kits are listed, including bacterial receptor cell assay, enzyme-linked immunoassay (ELISA) and modified rapid MITs. Alternative screening methods for veterinary drug residues have also recently been reviewed [44]. Recent advances in analytical technology have led to other types of rapid tests, including biosensors and multi-class MS methods, which will be discussed later in the chapter. Determinative methods are designed to separate, quantify and perhaps provide some qualitative information on the analyte of interest. For many of the drugs used in animal husbandry, the determinative method of choice is LC with UV detection using a variable wavelength or diode array detection system. LC using fluorescence, chemiluminescence, or post-column reaction detectors are also available and have been successfully used to determine drug residues in animal tissue. Volatile compounds can also be measured by GC with flame ionization, ECD or element-specific detectors. The increased availability and reliability of GC-MS and especially LC-MS instruments has led to an increased number of quantitative residue methods that use these technologies. Certain considerations for issues such as matrix effects and variable ionization must be taken into account when developing determinative methods with MS detection. Matrix-based standards may be needed; deuterated internal standards are commonly used and are highly recommended. Quantitative methods must be validated prior to widespread implementation for accuracy, precision, limits of detection and quantification, linearity and ruggedness. Several guidelines have been published outlining the rigorous validation procedures [45–47]. In addition to determining how much of a drug residue is present, confirmation of identity is also required for a complete analytical regulatory package. MS is ideally suited for this qualitative analysis due to its inherent selectivity and sensitivity. Certain criteria, in terms of the number and quality of ions monitored for any given compound, need to be met to definitively determine if a residue has been positively identified. These criteria have been
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discussed [48–50] and guidelines have been issued by several regulatory bodies [45,51]. MS methods are also advantageous in that it is possible to design a procedure that can simultaneously screen, quantify and confirm multiple drug residues in one sample analysis.
4.2 Summary of analytical methods There have been several other general review articles or texts recently published for the analysis of veterinary drug residues in food of animal origin [18,19,44,52–59]. Table 1 provides an updated list of representative veterinary drug residue methods categorized by drug class. This table is certainly not exhaustive and, in general, only methods published since 2000 are included. This is done primarily for the sake of brevity and is not meant to diminish the importance of earlier-published methods. In fact, many of the more recent methods are based on work done previously and the interested reader should be able to refer to the citations within the given references for a complete history of these analyses. In addition, review articles specific to each class of drugs are listed in Table 1 and should be of help in finding a more complete list of relevant methods.
4.3 Specific examples of recent methodology In the next section, a few specific important examples are examined in more detail. These include methods for the analysis of sulfonamides in milk and dairy products, chloramphenicol in shellfish, nitrofurans in various foods and triphenylmethane dyes in farmed fish and shrimp.
4.3.1 Sulfonamides in dairy products Sulfonamide residues in dairy products have been a regulatory issue for a long time. These drugs effectively treat bovine mastitis and can then be transferred into the cow’s milk. While some of these drugs are approved for such use, appropriate withdrawal times need to be observed to prevent the occurrence of residues in the milk and other dairy products. Several reviews for the analysis of sulfonamide residues in food have been published [3,73]. Many innovations in analytical chemistry have been applied to sulfonamide residue methods. The detection of these drugs is fairly straightforward since the compounds have a strong chromophore of around 270 nm. The primary amine group on these compounds can also be derivatized with fluorescent groups to provide even greater sensitivity and selectivity for LC detection. There have been several examples utilizing unique extraction and isolation techniques for sulfonamide residues in food. Supercritical fluid (CO2) along with in-line adsorption on alumina has been used for the extraction of sulfonamides from chicken tissue [184]. A hot-water matrix solid-phase dispersion method has also been used to extract these drugs from milk and eggs [66]. Roybal et al. illustrated the use of size-exclusion chromatography for the isolation of sulfonamides from shrimp [68]. Van Rhijn et al. isolated these drugs from small quantities of milk after a simple clean-up using only ultrafiltration with molecular weight cutoff
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Table 1 Selected veterinary drug residue methods Function (used to treat)
Sulfonamides
Examples of compounds
Typical matrices
Detection
Pneumonia, Sulfamethazine, other respiratory sulfadimethoxine, diseases, mastitis, sulfamerazine, diphtheria, diarrhea sulfathiazole
Milk, bovine, swine and poultry tissue, fish, eggs, shrimp, honey
LC with UV Multi-residue (270 nm), liquid methods chromatographyavailable fluorimetric detector (LC-FLD) after derivatization, LC-MS
[21,60–72] Review: [73]
b-Lactams
Colibacillosis, bacterial enteritis, salmonellosis, mastitis
Milk, bovine, swine and fish tissue
LC-UV penicillins: 210–230 nm, cephalsosporins: 260–295 nm, LC-MS
[74–83] Review: [4]
Tetracyclines
Enteritis, pneumonia, Tetracycline, anaplasmosis, oxytetracycline, growth efficiency chlortetracycline, doxycycline
Milk, bovine, poultry and swine tissue, shrimp, fish, eggs, honey
LC or ion Chelating chromatography agents used with UV at 270/ in extraction 350 nm; LC-FLD of and LC mobile metal complexes, phase LC-MS
[84–92] Reviews: [6,93]
Aminoglycosides
Broad-spectrum Streptomycin, antibiotics, growth gentamicin, efficiency neomycin
Milk, bovine, poultry and swine tissue (kidney)
LC-FLD w/ derivatization; GC-ECD, LC-MS
[94–99] Reviews: [5,100,101]
Macrolides
For gram-positive organisms and strains of Listeria and Mycoplasma, growth efficiency
Milk, bovine, swine and fish tissue, eggs, honey
LC-UV, LC-FLD Weak chromophores after derivatization, LC-MS
Penicillin, ampicillin, amoxicillin, cloxacillin, cephapirin, ceftioflur
Erythromycin, tylosin, oleandomycin, spiramycin, tilmicosin
Comments
May use tungstic or trichloroacetic acid in extraction
Poor chromophore, may need ion pair reagents for LC
References
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Drug class
Quinolones
Broad spectrum effective against gram positive, fluoroquinolones are effective for gram-negative species
Oxolinic acid, nalidixic acid, sarafloxacin, enrofloxacin, ciprofloxacin, flumequine
Milk, bovine, poultry, swine and fish tissue, eggs
LC-FLD and/or UV detection, LC-MS
Concerns regarding antibiotic resistance to these drugs
Phenicols
Infections, bovine respiratory disease
Chloramphenicol, florfenicol, thiamphenicol
Milk, bovine, poultry, swine and fish tissue, shrimp, eggs
LC-UV, GC-ECD or GC-MS after derivatization, negative ion LC-MS
No tolerance or [25,26,115, maximum 121–132] residue limit (MRL) for chloramphenicol residues
Ionophores and other coccidiostats
Parasites, growth efficiency
Monensin, lasalocid, salinomycin, halofuginone,
Bovine and poultry tissue, eggs
LC with visible or fluorescence detection after derivatization, LC-MS
Ionophores form sodium adducts that are detected by LC-MS
Nitrofurans
Broad spectrum antibiotics
Furazolidone, furaltadone, nitrofurazone, nitrofurantion
Poultry and swine tissue, shrimp, honey
LC-MS after Need to hydrolyze [32,138–147] derivatization with bound nitrobenzaldehyde metabolites, no tolerance
Peptides
Growth efficiency
Bacitracin, avoparcin, virginiamycin, colistin
Bovine, swine and poultry tissue, milk,
LC-MS, LC-UV, LC with electrochemical detection
[9,104,133–137]
[148–151]
Veterinary Drug Residues
Concerns regarding antibiotic resistance to these drugs
[110–120] Reviews: [8,100]
321
322
Table 1 (Continued ) Function (used to treat)
Examples of compounds
Typical matrices
Detection
Comments
References
Benzimidazoles
Anthelmintics, growth efficiency
Albendazole, fenbendazole, oxfendazole, thiabendazole
Bovine and swine tissue, milk
LC-UV (290 nm), LC-MS
May form sulfone metabolites
[152–156] Review: [11]
Avermectins and milbemycins
Anthelmintics
Ivermectin, eprinomectin, doramectin, moxidectin
Milk, bovine, swine and fish tissue
LC-fluorescence Form sodium after derivatization, adducts that LC-MS are detected by LC-MS
[20,157–161] Review: [12]
Triphenylmethane dyes
Fungus and parasite infections
Malachite green, crystal violet, brilliant green
Fish, shrimp
LC-visible, LC-MS
Convert leuco metabolites to chromic form for detection, no tolerance
[162–172] Review: [30]
Tranquilizers
Reduce stress and aggression transport aid preanesthetic
Acepromazine, azaperone, chlorpromazine, propionlypromazine, xylazine, carazolol
Tissue
LC-UV, LC-FLD, LC-MS
Multi-residue methods available
[13,14, 173–176]
Antiinflammatory
Reduce inflammation
Flunixin, phenylbutazone, ketoprofen
Milk, bovine tissue (kidney)
LC-UV, LC-MS
5-Hydroxyflunixin [177–183] is flunixin metabolite
Sherri B. Turnipseed and Wendy C. Andersen
Drug class
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filters [64]. In-tube solid-phase microextraction (SPME) was used to isolate five sulfonamides from milk; low levels (ng/mL) were detected by LC-UV after chromatographic separation on a capillary monolithic LC column [72]. Because maximum or safe levels for these drugs have been established (10 ng/mL in the U.S. and 100 ng/mL according to EU regulations), it has not been necessary to develop methods with lower detection limits. Instead, many of these innovative methods have been able to reduce the sample size needed, down from a 10–20 mL portion to 1 mL or less and still successfully detect and measure the drugs at the required concentrations. The emergence of MS has had an influence in this area of analysis with early methods utilizing GC-MS [185] and many more recent methods taking advantage of the capability of LC-MS [64,66,67,186]. For example, a detection and confirmation LC-MS-MS method for sulfonamides in milk was developed that can detect 14 residues at levels below 10 ng/mL [67]. Another paper describes the analysis of these drugs in processed dairy products such as condensed milk and soft cheeses using LC-MS-MS [21]. An example of the analysis of three sulfonamides in condensed milk using this method is shown in Figure 4. The development of rapid screening tests for sulfonamides has also been of interest for many years. These drugs do not respond very well to traditional microbial inhibition testing, so effective alternative screening methods are important. Early chemical methods for the screening of milk for sulfonamide residues included thin layer chromatography methods [187]. There have also been rapid test kits available for quite some time [43]. The detection of 1.49
100
3.85
7.95E2 SRM ms2 251@-25 3.68E4 SRM ms2 256@-25
4.52
100
Sulfathiazole
100
4.36E2 SRM ms2 250@-25
100
2.79E2 SRM ms2 265@-25 1.09E5 SRM ms2 279@-25
6.38
100
Sulfamethazine
5.20E2 SRM ms2 285@-25
100
1.39E5 SRM ms 2 311@-25
9.02
100 Sulfadimethoxine
1.06E2 SRM ms2 301@-25
100
0
5 Time (min)
10
Figure 4 LC-MS-MS combined ion chromatograms for sulfathiazole, sulfamethazine and sulfadimethoxine in control condensed milk fortified at 5 ng/mL. Reprinted with permission from Ref. [21]. Copyright 2005 by AOAC International.
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sulfonamides in milk was one of the earliest applications of the use of biosensors in animal drug residue methodology [188] and is still in use today [189].
4.3.2 Chloramphenicol Residues in Seafood and Other Products As mentioned earlier, chloramphenicol residues are of concern because of the drug’s unique capability to produce aplastic anemia in susceptible individuals. A review of the analytical methodology for chloramphenicol is also a good illustration of how the field of veterinary drug residue methodology has changed over the last several years. Initially, methods to detect residues of chloramphenicol or analogous compounds in food used either LC-UV, or GC with (ECD) or MS after derivatization. For example, one of the first methods for chloramphenicol in shrimp involved GC-ECD analysis of a silyl derivative after liquid–liquid extraction and isolation [190]. In an extension of that method, chloramphenicol, florfenicol, florfenicol amine and thiamphenicol were silylated and analysed by GC-ECD after liquid–liquid extraction and isolation from shrimp by a series of SPE columns; the quantification limit for this method was 5 mg/kg [121]. In 2001 and 2002, chloramphenicol residues in food became an urgent problem, specifically in seafood and honey imported to EU countries, Canada and the U.S. from China. More sensitive methods were needed to adequately monitor for these residues. With the advent of widely available LC-MS instrumentation, the detection limits for chloramphenicol were decreased significantly to below 0.5 mg/kg. For this reason, there were many methods published in 2003–2004 describing the analysis of chloramphenicol in these food products using LC-MS [115,122–127,132]. Most of these methods utilized negative ion ESI to monitor the deprotonated molecular ion of chloramphenicol at m/z 321. A triple quadrupole LC-MS-MS instrument was most commonly utilized and the methods were capable of both quantification and confirmation of the residue. A few methods described the use of a single quadrupole or ion trap instrumentation. Regardless of the MS instrumentation used, the chloramphenicol molecule breaks apart into consistent fragment or product ions that can be used for confirmation. In some methods, the chlorine isotope ratio was also used for identification purposes. Internal standards were often used for quantification including meta-chloramphenicol, deuterated chloramphenicol or an analogous compound such as thiamphenicol. The extraction procedure for many of these methods was still fairly extensive with liquid–liquid extraction and SPE isolations. More recently published methods for chloramphenicol residue analysis generally utilize simplified generic or novel extraction methods and have expanded the analysis to additional matrices such as crab, honey, milk and eggs [26,128,131]. LC-MS-MS analysis of chloramphenicol in honey by electrospray with negative ion detection and quantification with meta-chloramphenicol as an internal standard is illustrated in Figure 5 [26]. Biosensor-based assay test kits have expanded the possibility of rapidly screening food matrices for chloramphenicol residues at or below a concentration of 0.07 mg/kg [191].
325
Veterinary Drug Residues
(a) RT: 4.00 - 9.00 SM: 5G
200
2000
100
80 60 40 20 0 40
1000 500
1500
Intensity
Intensity
RT: 6.70
1000 0
RT: 6.70
3000 2000 1000
2000
321 > 176 500 RT: 5.37 0
30 20 10 0
0
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1500 RT: 5.98 RT: 8.15
0
RT: 5.56
RT: 6.80
1000
0 2000
RT: 5.62
200
1500
RT: 6.78
1000
400
RT: 5.58
321 > 152 RT: 7.40
0
RT: 5.44 RT: 5.33 RT: 5.59
600
1000
RT: 6.79
4000
RT: 5.95 RT: 5.61
0
RT: 6.73
0
RT: 4.00 - 9.00 SM: RT: 7.41
4000
0 1500
(c)
Intensity
RT: 4.00 - 9.00 SM: 5G RT: 6.71
(b)
RT: 5.60
1000
1000
500
500
321 > 257
RT: 5.65
1000 500 0
RT: 5.64
1000 500
RT: 6.74
0
0
0 4
5
6 7 8 Time (min)
9
321 > 207
4
5
6 7 8 Time (min)
9
4
5
6 7 8 Time (min)
9
Figure 5 LC-MS-MS ion chromatograms for chloramphenicol of (a) 0.5 ng/g standard, (b) control honey with internal standard meta-chloramphenicol and (c) a chloramphenicolpositive honey sample. Ions monitored are (from top to bottom) m/z 152, 176, 194, 257 and 207. Reprinted with permission from Ref. [26]. Copyright 2006 by AOAC International.
4.3.3 Nitrofuran residues in seafood, poultry and honey Nitrofuran antibiotics are broad-spectrum agents effective against infections common to poultry, swine and aquacultured shrimp. Nitrofurans may also be used to treat honey bees. The most common nitrofurans associated with food production are furazolidone, furaltadone, nitrofurazone and nitrofurantion. The drugs metabolize rapidly so the parent compounds are not detected as residues. The metabolites of these drugs, which correspond to a side chain of the original molecule bound to proteins, do persist in tissue for a significant amount of time. The metabolites generated are 3-amino-2-oxazolidinone, 3-amino-5-morpholinomethyl-2-oxazolidinone, semicarbazide and 1-aminohydantion from furazolidone, furaltadone, nitrofurazone and nitrofurantion, respectively. There are concerns regarding the carcinogenicity and mutagenicity of these metabolites, making monitoring for these compounds both important and a significant analytical challenge [32]. Because of the human health concerns, there are no tolerances or maximum residue limits set for nitrofuran metabolite residues. The EU has established a minimum residue performance level of 1 mg/kg.
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Methods have been developed for nitrofuran metabolite residues in tissue [139,147], shrimp [140,141], eggs [139] and honey [145]. In general, these methods have several attributes in common. A hydrolysis step, usually incubation with dilute hydrochloric acid, is needed to release the protein-bound metabolites. The molecules are then derivatized with nitrobenzaldehyde. While LC detection by UV is possible at this point, LC-MS is usually utilized to obtain the necessary detection limits as well as residue confirmation. There are some variations in this basic protocol. The clean-up and isolation of the residues after hydrolysis, but before derivatization, can be accomplished with liquid–liquid extraction or with a SPE cartridge, or by using a combination of both. In some methods, the matrix is first washed to remove any unbound residue, so that only compounds covalently attached to proteins are measured in the final extract; other procedures do not incorporate this step. One particular concern is for the residues of semicarbazide. While the metabolism of nitrofurazone is one source of semicarbazide in food, there are other possible explanations for the presence of this residue. Axodicarbonamide is an industrial chemical used as a blowing agent for rubber gaskets on jars (such as baby food) and in some cases as a food additive in flour. This chemical can also thermally degrade to form semicarbazide. It is also possible that semicarbazide could originate from the treatment of nitrogen-rich food with hypochlorite, a procedure that occurs when seaweed products are processed into gelling agents. It was originally thought that any protein-bound semicarbazide would necessarily originate from nitrofurazone exposure and that washing unbound residue prior to hydrolysis could reduce the amount of semicarbazide residues in the matrix from these other sources. However, a more extensive study of this issue is still needed [32]. The quantification of these metabolites can be complicated. In most cases, selective reaction monitoring using a triple quadrupole LC-MS-MS system is used to quantify the residues. Additional ion transitions can be monitored for confirmatory purposes. The fragmentation patterns of these derivatized metabolites have been established [192]. Ion trap and single quadrupole instruments can be used as well, but the results may not be as reliable for quantitative purposes as those obtained with a triple quadrupole procedure [140]. Matrix effects, i.e., ion suppression, can be an issue in obtaining good quantitative LC-MS methods [193]. For these methods, matrix effects can be overcome with matrix-based standards; but more often deuterated internal standards of one or more of the analytes are added to compensate for any ion suppression as well as for lack of recovery through the extraction procedure. The results of a proficiency study for nitrofuran metabolites in shrimp highlight some of the issues with these methods [140].
4.3.4 Triphenylmethane dyes in fish Triphenylmethane dyes are used as inexpensive and effective water bath treatments for fungal and parasite infections in fish. Malachite green is the most commonly used treatment within this class of compounds. The suspected carcinogenicity and teratogenicity of malachite green has rendered its use inappropriate for fish raised for human consumption [33]. Crystal violet (also
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known as gentian violet) and brilliant green are structurally similar dyes with similar antifungal and toxicity properties [33,194,195]. Regardless of the international restrictions on the use of malachite green, illegal residues are regularly found in fish tissue. Reports of crystal violet abuse have also been noted [196]. The use of these therapeutic dyes in fish has been reviewed [30,195,197]. Methods to detect triphenylmethane dyes and their reduced leuco metabolites in fish tissue often include extraction by mixing the ground fish tissue with an acidic buffer and acetonitrile. This is followed by defatting and sample clean-up and residue isolation with liquid–liquid partition and/or SPE. The cationic triphenylmethane dyes have strong chromophores in the visible region of the spectrum, yet their reduced leuco metabolites are colorless. Since it is the leuco moiety that is found in greatest concentration in tissues, methods must emphasize the detection of this metabolite. Many traditional malachite green analytical methods have taken advantage of the sensitivity offered by LC with detection in the visible region by including a lead oxide, post-LC column reactor to oxidize the leuco residues to their chromic analogs [198–203]. The lead oxide reactor, however, is often difficult to prepare and can lead to rapid depletion and peak broadening resulting in a decrease in method sensitivity for regulatory use. Because all of the triphenylmethane dyes are banned, regulatory action can be taken in many countries if any level of residue is found in fish tissue. Method detection limits therefore drive regulatory action and should be as sensitive as possible. Recent methodology for the detection of malachite green in fish tissues has included several different approaches to meet minimum performance limits (2.0 mg/kg and lower) of international agencies [204]. In the first approach, leucomalachite green residues were oxidized to malachite green using 2,2,7dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) prior to chromatographic analysis, enabling the measurement of the sum of malachite green and leucomalachite green residues by LC with visible detection at 1.0 mg/kg [163,168]. In another approach, residues at the 0.5 mg/kg level were determined using LC separation followed by the visible detection of malachite green at 620 nm and florescence detection of leucomalachite green at 360 nm (265 nm excitation) [171]. Both of these methods are advantageous for screening numerous samples with less expensive instrumentation. Mass spectrometric analysis of malachite green and leucomalachite green residues are divided by those that use an oxidation procedure to detect leucomalachite green as malachite green and those that detect leucomalachite green directly. Malachite green is cationic and more sensitively detected than leucomalachite green by LC-MS. In the former case, DDQ has been used prior to sample analysis with no-discharge LC-MSn to detect the sum of malachite green and leucomalachite green at 0.25 mg/kg [165,168]. This method was used to detect and confirm malachite green residues in store-bought basa fish (Figure 6). The lead oxide reactor has also been used to detect leucomalachite green as low as 0.1 mg/kg [164,166,172]. Several other methods rely on detection of malachite green and leucomalachite green individually using triple quadrupole LC-MS-MS, which is more sensitive for the quantification of leucomalachite green [162,167,170,205]. These methods offer excellent sensitivity with limits of detection
Relative Abundance
328
Sherri B. Turnipseed and Wendy C. Andersen
100
5.11 Area: 256,730,573 SN: 16,141
1.76E7 m/z= 328.5-329.5 ms2
[email protected] [150.00-350.00]
100
5.06 Area: 187,769,285 SN: 15,042
1.16E7 m/z= 313.0-315.0 ms2
[email protected] [150.00-350.00]
100
5.11 Area: 73,898,029 SN: 9,767
4.90E6 m/z= 284.0-286.0 ms2
[email protected] [150.00-350.00]
100
5.05 Area: 83,353,865 SN: 8,411
5.80E6 m/z= 250.5-251.5 ms2
[email protected] [150.00-350.00] 9.48E6 m/z= 207.5-208.5 ms2
[email protected] [150.00-350.00]
5.07 Area: 132,887,237
100
SN: 10,484
0
1
2
3
4
5 6 Time (min)
7
8
9 1.41E7
Relative Abundance
[email protected] [ 150.00-350.00]
329.3
100 80 60
208.2
40 20 0
251.2 193.1
160
200
237.2 241.1
240
270.3
285.2 284.2
280
313.3 314.2
320
m/z
Figure 6 LC-MSn extracted ion chromatograms and product ion spectrum from the malachite green (m/z 329) product ion trace in an extract from retail basa (diluted 1:5). Extracted ion ranges (from top to bottom) are m/z 329, 313–315, 284–286, 251 and 208. Reprinted with permission from Ref. [168]. Copyright 2006 American Chemical Society.
below 0.5 mg/kg. Some of these newer methods have also been applied to the concurrent determination of crystal violet and brilliant green residues [170,206]. Finally, an ELISA test kit based on DDQ oxidation for the detection of malachite green at 0.5 mg/kg in fish tissue has recently been commercialized [207].
5. OCCURRENCE IN FOOD It is not possible to obtain an absolute accounting of the rate of occurrence of violative veterinary drug residues in human food. The violation rate can depend greatly on not only the specific residue and matrix, but also on the number of samples analysed and the type of methodology utilized. Nevertheless, the results from several monitoring programs can be informative in evaluating the overall scope of the issue. In the U.S., one monitoring program for veterinary drug residues in meat products is administered by the Food Safety Inspection Service of the U.S. Department of Agriculture (FSIS/USDA). Tissue from many production classes of cattle, swine, lambs, goats and poultry are tested for a variety of residues. The
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results from this program are published in the FSIS/USDA Red Book [208]. In 2004, over 3,000 tests were performed for antibiotic residues and 38 violations were recorded. Violative residues of penicillin (6), tilmicosin (3), neomycin (25) and gentamicin (4) were found; many of these occurred in veal products. In addition to these tests, a similar number of tests were performed for sulfonamide residues. Five sulfonamide violations were found in hogs, veal and turkey. Over 1,000 samples were analysed for chloramphenicol in cows, veal and poultry, but no residues were found. The National Milk Drug Residue Database is maintained by the U.S. FDA’s Center for Food Safety and Nutrition in cooperation with state governments in the U.S. This database reports the results from a voluntary industry-reporting program. Samples are obtained from milk pick-up tanks, pasteurized fluid milk and milk products and analysed for drug residues using rapid test kits. In the 2003 report [209], over 4 million samples were analysed. Many of these tests were for b-lactam residues and the most violations, approximately 3,000 (o0.1%), were found for these residues. Sulfonamide residue tests were performed over 66,000 times and 23 positive samples were found. A few non-compliant residues of tetracyclines and aminoglycosides were also detected in a limited number of samples. A Canadian total diet study monitoring veterinary drug residues in fish and seafood was conducted over a period of more than 10 years [210]. This study examined 30 samples of shrimp, marine fish, fresh fish and canned fish. The products were analysed for 39 different drug residues and 9 positive samples were found. The most common violations were for quinolone and nitrofuran metabolite residues in shrimp; leucomalachite green and chloramphenicol residues were also present. The EU conducts an extensive residue-monitoring program; the results from the 2004 program have been posted [211]. This report includes residuemonitoring data from 25 countries (member states). In 2004, approximately 807,000 targeted samples and 64,000 suspect samples were tested. In addition to antibiotics testing, samples were also monitored for hormones and other veterinary drugs. Many types of food matrices were part of this program, including milk, eggs, honey, aquaculture and meat products. Residues of prohibited substances such as chloramphenicol and nitrofurans were found to be violative at a rate of approximately 0.11%. Residues of malachite green were found in aquaculture products from many of the member states. The violation rate for antibiotics was 0.22%. There were a few violative results for other drugs such as tranquilizers and NSAIDs and the incidence of coccidiostat residues in eggs was highlighted as a continuing problem.
6. FUTURE TRENDS 6.1 Biosensors In the past 10 years, biosensor technology increasingly has been relied on for the sensitive and reliable monitoring of veterinary drug residues in food [212–215]. This technique typically relies on contacting the food sample or extract with a
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Table 2
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Biosensor veterinary drug residue methods
Drug class
Examples of compounds
Matrices
Sensitivity (limit of detection (LOD))
References
Sulfonamides
Sulfamethazine, sulfadiazine, sulfamethoxazole
Milk, pig bile 1–23 mg/kg and muscle, chicken serum and plasma
[188,189, 217,218]
b-Lactams
Benzylpenicillin, ampicillin, amoxicillin, cephapirin, ceftiofur, etc.
Milk
1.2–100 ng/mL
[219,220]
Aminoglycosides Gentamicin, neomycin, kanamycin, streptomycin, dihydrostreptomycin
Milk powder, milk, honey, pig muscle and kidney
15–70 mg/kg
[221,222]
Macrolides
Tylosin
Honey
2.5 mg/kg
[223]
Quinolones
Enrofloxacin, ciprofloxacin
Milk
2–3 ng/mL
[224]
Phenicols
Chloramphenicol, florfenicol, thiamphenicol, florfenicol amine
Milk, honey, chicken muscle, shrimp
0.02–250 mg/kg
[191,225]
Coccidiostats
Nicarbazin
Eggs, liver
17–19 mg/kg
[226]
Anthelmintics
Ivermectin, levamisole
Milk, liver
0.5–16.2 mg/kg
[227]
residue-specific antibody to form a drug–antibody binding pair. The binding reaction generates a signal that can be detected by various methods, e.g., optical, piezoelectric, etc. The inherent specificity of the binding reaction allows biosensors to offer many advantages over traditional analytical methods. Food matrices require only minimal sample preparation and the analytical signal is typically absent of background interference. As a result, biosensor methods are easy to use, fast and offer excellent sensitivity for high-throughput screening. For veterinary drug residue analysis, most of the literature methods have been developed using optical biosensor instrumentation based on surface plasmon resonance. Methods have been developed for a broad range of the drug classes described previously in foods. A selection of recent methodology is summarized in Table 2. In addition, binding assay test kits are commercially available for sulfonamides, chloramphenicol, streptomycin and others [216].
Table 3
Multi-class veterinary drug residue methods Extraction
Detection
Number of residues
References
Eggs
Ionophores, macrolides
Silica SPE
Ion trap LC-MS electrospray
7
[104]
Eggs
Fluoroquinolones, sulfonamides, tetracyclines, b-lactams Sulfonamides quinolones tetracyclines, dyes
Succinate buffer extraction, polymeric SPE
Ion trap LC-MS electrospray
29
[232]
Trichloroacetic acid extraction, polymeric SPE
Ion trap LC-MS APCI/no-dischargeAPCI
19
[228]
Tissue, Sulfonamides, benzimidazoles, fish, eggs tranquilizers, fluoroquinolones, dyes
Acetonitrile extraction, cation exchange SPE
LC-UV/Fluorescence, LC-MS (APCI)
30
[233]
Milk
b-Lactams, tetracyclines, aminoglycosides, macrolides, quinolones, sulfonamides
Trichloroacetic acid or acetonitrile LC-MS-MS triple quad, extraction electrospray
44
[234]
Tissue
Macrolides, fluoroquinolones, etc.
Acetonitrile extraction, on line polymer SPE
LC-MS-MS triple quad, electrospray
13
[229]
Tissue
Quinolones, sulfonamides, tetracyclines, macrolides, benzimidazoles, b-lactams, etc.
Acetonitrile/methanol (95:5 extract), hexane wash
LC-MS-MS triple quad, electrospray
130
[231]
Fish
Quinolones, sulfonamides, tetracyclines, macrolides, dyes, imidazoles, b-lactams, etc.
Acetonitrile/hexane extraction, hexane wash
Ion trap LC-MS electrospray
35
[235]
Tissue
Tetracyclines, sulfonamides, 70% methanol extract diluted with LC-MS-MS triple quad, macrolides, quinolones, b-lactams water electrospray
19
[236]
Milk
Tetracyclines, sulfonamides, pyrimethamine
10
[230]
Shrimp
Trichloroacetic acid, polymeric SPE
LC-MS (single quad) electrospray
331
Drug classes
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Matrix
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6.2 Multi-class methods An emerging trend in veterinary drug residue analysis is the development of methods that are capable of monitoring a wide variety of residues, regardless of drug class, in a single sample. Often this involves a generic extraction and cleanup, designed to be as inclusive as possible, combined with the sensitivity and selectivity of LC-MS detection. For example, several methods utilize a polymeric SPE clean-up, such as OASISs, to isolate many of the drug residues from the initial extract [104,228–230]. A hexane wash is sometimes required to remove lipid materials, but in general, extensive liquid–liquid extractions are avoided to maximize throughput. Screening and confirmatory data are collected by MS; some methods also provide quantification of residues [231]. Both ion trap and triple quadrupole MS-MS instruments have been successfully utilized. Several multi-class veterinary drug residues methods have been reported in recent years and these are summarized in Table 3. Multi-class multi-residue LC-MS methods are an effective way to monitor a wide variety of drug classes in a single sample, maximizing laboratory resources and allowing for flexibility in a risk-based regulatory program.
REFERENCES 1 National Research Council, The Use of Drugs in Food Animals: Benefits and Risks, National Academy Press, Washington, DC, 1999. 2 The Merck Index on CD Rom., Version 12:3, Merck & Co., Whitehouse Station, NJ, 2000. 3 C.C. Walker and S.B. Turnipseed, In: S.B. Turnipseed and A.R. Long (Eds.), Analytical Procedures for Drug Residues in Food of Animal Origin: Sulfonamides, Science Technology System, West Sacramento, CA, 1998. 4 W.A. Moats, In: S.B. Turnipseed and A.R. Long (Eds.), Analytical Procedures for Drug Residues in Food of Animal Origin: Beta-Lactams, Science Technology System, West Sacramento, CA, 1998. 5 R. Gehring, S.R. Haskell, M.A. Payne, A.L. Craigmill, A.I. Webb and J.E. Riviere, J. Am. Vet. Med. Assoc., 227 (2005) 63. 6 H. Oka, Y. Ito and H. Matsumoto, J. Chromatogr. A, 882 (2000) 109. 7 S.B. Turnipseed, In: S.B. Turnipseed and A.R. Long (Eds.), Analytical Procedures for Drug Residues in Food of Animal Origin: Macrolides and Lincosamides, Science Technology System, West Sacramento, CA, 1998. 8 J.A. Herna´ndez-Arteseros, J. Barbosa, R. Compan˜o and M.D. Prat, J. Chromatogr. A, 945 (2002) 1. 9 L. Mortier, E. Daeseleire and C. Van Peteghem, Rapid Commun. Mass Spectrom., 19 (2005) 533. 10 N. Woodford, J. Med. Microbiol., 47 (1998) 849. 11 M. Danaher, H. De Ruyck, S.R.H. Crooks, G. Dowling and M. O’Keeffe, J. Chromatogr. B, 845 (2007) 1. 12 M. Danaher, L.C. Howells, S.R.H. Crooks, V. Cerkvenik-Flajs and M. O’Keeffe, J. Chromatogr. B, 844 (2006) 175. 13 Y. Govaert, P. Batjoens, K. Tsilikas, J. Degroodt and S. Srebrnik, Analyst, 123 (1998) 2507. 14 A. Kaufmann and B. Ryser, Rapid Commun. Mass Spectrom., 15 (2001) 1747. 15 M. Kopcha, J.B. Kaneene, M.E. Shea, R. Miller and A.S. Ahl, J. Am. Vet. Med. Assoc., 201 (1992) 1868. 16 R.W. Fedeniuk and P.J. Shand, J. Chromatogr. A, 812 (1998) 3. 17 S.B. Turnipseed, W.C. Andersen, C.M. Karbiwnyk, J.E. Roybal and K.E. Miller, Rapid Commun. Mass Spectrom., 20 (2006) 1231.
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208 2004 Red Book. United States Department of Agriculture, Food Safety Inspection Service, 2004. Available at http://www.fsis.usda.gov/Science/2004_Red_Book/index.asp 209 National Milk Drug Residue Data Base. Food and Drug Administration, Center for Food Safety and Nutrition, 2004. Available at http://www.cfsan.fda.gov/Bear/milkrp03.html 210 S.A. Tittlemier, J. van de Riet, G. Burns, R. Potter, C. Murphy, W. Rourke, H. Pearce and G. Dufresne, Food Addit. Contam., 24 (2007) 14. 211 European Union Report for 2004 on the Results of Residue Monitoring in Food of Animal Origin in the Member States SANCO/3400/2005, Annex I, 2005. Available at http://europa.eu.int/ comm/food/food/chemicalsafety/residues/workdoc_2004_en.pdf 212 P.D. Patel, Trends Anal. Chem., 21 (2002) 96. 213 G. Cacciatore and M. Petz, Deut. Lebensm.-Rundsch., 101 (2005) 501. 214 S.A. Haughey and G.A. Baxter, J. AOAC Int., 89 (2006) 862. 215 U. Bilitewski, Anal. Chem., 72 (2000) 692A. 216 Biacore, Uppsala, Sweden. Available at http://www.biacore.com 217 P. Bjurling, G.A. Baxter, M. Caselunghe, C. Jonson, M. O’Connor, B. Persson and C.T. Elliott, Analyst, 125 (2000) 1771. 218 S.R. Crooks, G.A. Baxter, M.C. O’Connor and C.T. Elliot, Analyst, 123 (1998) 2755. 219 E. Gustavsson, J. Degelaen, P. Bjurling and A. Sternesjo¨, J. Agric. Food Chem., 52 (2004) 2791. 220 G. Cacciatore, M. Petz, S. Rachid, R. Hakenbeck and A.A. Bergwerff, Anal. Chim. Acta, 520 (2004) 105. 221 J.P. Ferguson, G.A. Baxter, J.D.G. McEvoy, S. Stead, E. Rawlings and M. Sharman, Analyst, 127 (2002) 951. 222 W. Haasnoot, G. Cazemier, M. Koets and A. van Amerongen, Anal. Chim. Acta, 488 (2003) 53. 223 M. Caldow, S.L. Stead, J. Day, M. Sharman, C. Situ and C. Elliot, J. Agric. Food Chem., 53 (2005) 7367. 224 C. Mellgren and A. Sternesjo¨, J. AOAC Int., 81 (1998) 394. 225 V. Dumont, A.C. Huet, I. Traynor, C. Elliott and P. Delahaut, Anal. Chim. Acta, 567 (2006) 179. 226 B. McCarney, I.M. Traynor, T.L. Fodey, S.R.H. Crooks and C.T. Elliott, Anal. Chim. Acta, 483 (2003) 165. 227 J.V. Samsonova, G.A. Baxter, S.R.H. Crooks and C.T. Elliot, J. AOAC Int., 85 (2002) 879. 228 H. Li, P.J. Kijak, S.B. Turnipseed and W. Cui, J. Chromatogr. B, 836 (2006) 22. 229 H.P.O. Tang, C. Ho and S.S. Lai, Rapid Commun. Mass Spectrom., 20 (2006) 2565. 230 U. Koesukwiwat, S. Jayanta and N. Leepipatpiboon, J. Chromatogr. A, 1140 (2007) 147. 231 R. Yamada, M. Kozono, T. Ohmori, F. Morimatsu and M. Kitayama, Biosci. Biotechnol. Biochem., 70 (2006) 54. 232 D.N. Heller, C.B. Nochetto, N.G. Rummel and M.H. Thomas, J. Agric. Food Chem., 54 (2006) 5267. 233 G. Stubbings, J.A. Tarbin, A. Cooper, M. Sharman, T. Bigwood and P. Robb, Anal. Chim. Acta, 547 (2005) 262. 234 B. Delepine and J.P. Moretain, Antibiotic Residues in Milk: LC-MS-MS Confirmation (Identification and Quantification) of Positive Results Obtained with Microbial Inhibitor Screening Tests, Proceedings of the Euroresidue V Conference, Noordwijkerhout, The Netherlands, May 10–12, 2004. 235 S. Smith, C. Gieseker, R. Reimschuessel and M.C. Carson, Multiclass Confirmation of Veterinary Drug Residues in Fish by Liquid Chromatography-Ion Trap Mass Spectrometry, FDA Science Forum, Washington, DC, April 27–28, 2005. 236 K. Granelli and C. Branzell, Anal. Chim. Acta, 586 (2007) 289.
CHAPT ER
11 Analytical Strategies to Control the Illegal Use of Banned Growth Promoters in Meat Producing Animals Bruno Le Bizec, Jean-Philippe Antignac and Gaud Pinel
Contents
1. Introduction 2. Chemical Properties, Health effects 2.1 Stilbenes 2.2 Antithyroid agents 2.3 Steroids 2.4 Resorcylic acid lactones (RALs) 2.5 b-Agonists 3. Analytical Methods 3.1 Steroids 3.2 Corticosteroids 3.3 b-Agonists 3.4 Thyrostats 3.5 Growth hormone 4. Occurrence in Foods 5. Future Trends References
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1. INTRODUCTION Since 1 January 1989, according to Directive 88/146/EEC replaced later by Directive 96/22/EC [1], the European Commission (EC) prohibits the administering to a farm animal by any means whatsoever of, inter alia, substances having a thyrostatic, oestrogenic or gestagenic action for growth promotion purposes. Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00011-1
r 2008 Elsevier B.V. All rights reserved.
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As a result, the use of the hormones oestradiol-17b-testosterone, progesterone, zeranol, trenbolone acetate and melengestrol acetate alone or in combinations for growth promotion purposes in meat production is prohibited. The prohibition covers both the use of these hormones for domestic production and imports from third countries of meat from animals treated with these hormones. The prohibition reflects the fact that the EC chose a level of sanitary protection of accepting no or ‘‘zero’’ additional risk to human health from the residues in meat and meat products of these hormones when used for growth promotion purposes. Even if banned substances are sometimes reduced and summarized to ‘‘hormones’’, the reality is different. In Europe, five classes of compounds are identified in the regulation, as listed in community Directive 96/23/EC: stilbenes (group A1), thyrostats (A2), steroids (A3) including estrogenic, androgenic and progestagenic substances, resorcylic acid lactones (A4) and b-agonistic drugs (A5). The control of growth promoters in meat producing animals is probably one of the most challenging areas in the field of chemical residue control in food, when considering the wide number of target substances, the variability of their chemical structures, their concentration level, as well as the variability of the biological matrix. Main compound families are anabolic steroids, stilbenes, resorcylic acid lactones, corticosteroids, b-agonists and thyrostats, even if some additional groups can be supposed to be already used (ecdysteroids, somatotropine, IGF-I). The complexity of target biological matrices as well as the trace level of residues obliges the Analyst to rely his strategy on analytical methods combining both specificity and sensitivity, respectively. Whereas, Enzyme Linked Immuno Sorbent Assay (ELISA) and Radio Immuno Assay (RIA) are still sometimes used for screening purpose, mass spectrometric methods constitute the obliged strategy during confirmatory processes, technically and strategically speaking. We will focus hereafter on the main strategies followed in the official network of reference laboratories in Europe, and we will develop especially the mass spectrometric approaches used from benchtop technologies to more hyphenated ones for the diagnostic absence/presence, and for the chemical structure determination of metabolites and new unknown compounds introduced onto the black market. Future of the control will be discussed.
2. CHEMICAL PROPERTIES, HEALTH EFFECTS 2.1 Stilbenes The bad reputation of hormones is mainly caused by the harmfulness of diethylstilbestrol (DES). The properties of DES as a growth promoter were discovered in 1954 in the USA. It has been widely used as a feed additive or as an implant into the ear to promote growth and increase feed efficiency in cattle and sheep (until 1979 in the USA). DES is a non-steroidal estrogenic agent which does not occur naturally. The estrogenic effect is mainly ascribed to the same spatial distance of the two OH-groups as for estradiol. Hexestrol and dienestrol are
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synthetic non-steroidal estrogens that are structurally related to DES. Based on their carcinogenity, the EC banned their use in food producing and no residues of these anabolics should be present in animal products imported or produced within the EC (Directive 96/22/EC).
2.2 Antithyroid agents Action of these compounds is characterized by the inhibition of the thyroid gland function causing a decreased production of thyroid hormones, i.e., L-thyroxine (T4) and L-triiodothyronine (T3). Administration of thyrostatic drugs to cattle results in a significant increase in live weight-gain mainly consisting in the development of the gastro-intestinal tract and higher water retention in animal tissue. As a consequence, the meat is characterized by an inferior quality, mostly wet and colourless. Thyrostats can be divided into two main groups: Natural thyrostats. A series of natural products with oxazolidine-2-thiones chemical structures found in plant material in the form of glucosinolates. The 5-vinyl-oxazolidine-2-thione derivative is present in large amounts (up to 1%) in the seeds of Cruciferae (e.g., rapeseed). (2) Synthetic thyrostats. The first group contains thiouracil analogues such as thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracil (PhTU). The second group consists of the mercaptoimidazole analogues of which tapazol (TAP) is the most important. All are cheap drugs and readily available on the black market. Classified 2A and 2B by the International Agency for Research on Cancer (IARC), the use of thyrostatic drugs for promoting animal growth is prohibited in all EU member states (Directive 2003/74/EC amending Directive 96/22/EC). (1)
2.3 Steroids Anabolic steroids can be distinguished according to their chemical structure (C18, C19 or C21) and origin (endogenous or xenobiotic). Apart from their hormonal activity and depending on the compound applied or combination used, anabolic steroids stimulate growth, leading to gain in protein deposition based on an improved food conversion rate. Estrone (E1), estradiol (E2) and estriol (E3) are the main natural estrogens. Only 17b-estradiol is used in anabolic preparations. By esterification of the OH-groups (positions 3 and 17) of 17b-estradiol, compounds with more efficient activity are obtained. Main synthetic estrogens are 17a-ethynylestradiol and mestranol. Testosterone, etiocholanolone, androsterone and 5a-dihydrotestosterone are among the main natural androgens. Esters of 17b-testosterone have been found in anabolic preparations used in meat production. Nandrolone (19-nortestosterone), norethandrolone, trenbolone, 4-chlorotestosterone, fluoxymesterone, 17a-methyltestosterone, stanozolol, boldenone (1-dehydrotestosterone), dianabol (17a-methylboldenone) are some examples of androgenic steroids discovered in the past in Europe for cattle production. Progesterone is the main natural progestagen; semi-synthetic derivatives, such as
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acetate or caproate esters of 17a-hydroxyprogesterone, have already been demonstrated to be used. The most popular synthetic progestagens are medroxyprogesterone acetate, megestrol acetate, melengestrol acetate and chlormadinone acetate. Throughout the EC the use of xenobiotic anabolic agents is prohibited in food producing animals and no residues of these anabolics should be present in animal products imported into or produced within the EC (Directive 96/22/EC).
2.4 Resorcylic acid lactones (RALs) Zeranol has been widely adopted as a growth stimulant with estrogenic activity and is also employed to reduce stress in cattle. Zeranol may also be formed in vivo from the mycotoxins, zearalenone and a-zearalenol, that can be carried over from Fusarium contaminated feedstuff (maize, wheat, barley and oats) to animals. Application of zeranol to cattle has been banned in the European Union since 1985.
2.5 b-Agonists The control of b-agonists misuse received extra attention after outbreaks of food poisoning in 1990 in Spain caused by consumption of bovine liver. This was the first time that pharmacological residues found in slaughtered cattle were found to have caused acute intoxication in consumers. As therapeutic drugs, they have been first used as a bronchodilator for the treatment of pulmonary diseases in humans and animals. b-Agonists act specifically by binding to cell membrane b-receptors, and therefore, by activating the adenylate cyclase-cyclic AMP system. Physiological responses to stimulation of the b-receptors are increased lipolysis and reduced lipogenesis in adipose cells, increased glycogenolysis, increased protein synthesis and reduced proteolysis in striated muscle fibres, and vasodilatation, bronchodilatation and relaxation of smooth muscle fibres. Although clenbuterol is still probably the most popular b-agonist illegally used on farms, other b-agonists have been pointed out on the ‘‘black market’’. b-Agonists belong to the group of arylethanolamines, and can be catalogued in three chemical classes according to their chemical structure: the anilines (e.g., clenbuterol), resorcinols (e.g., terbutaline) and phenols (e.g., salbutamol). Zilpaterol ((7)-trans-4,5,6,7-tetrahydro-7-hydroxy-6-(isopropylamino)imidazo [1jk,4,5]-[1]benzazepin-2(1H)-one) is a new approved b-agonistic drugs (USA, Mexico, South Africa) characterized by a cyclic chemical structure.
3. ANALYTICAL METHODS 3.1 Steroids Monitoring programs to control their use are frequently based on the initial screening of animals for steroid abuse using chemical or immunochemical
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methods [2,3], followed by the complete chemical confirmation of steroids in suspect samples by mass spectrometry (MS) [4–12]. Urine and hair samples are normally ad hoc matrices since they are readily available at both abattoir and farm. Until recently, the standard technique for steroid analysis has been gas chromatography (GC)-MS. This required the derivatization of the steroids using silylation or acylation reactions, depending on the individual properties of the steroid. Liquid chromatography (LC)-MS provides a universal detector, since steroids may be analysed without derivatization. The rules governing screening and confirmation of analytical methods for veterinary drug residues and their validation have been revised by the EU [13]. The rules have been extended to include a number of MS techniques, which have gained in popularity over the past decade, e.g., LC-MS, and acquisition techniques such as high resolution selected ion monitoring (HR-SIM) and selected reaction monitoring (SRM). Today, minimum required performance levels (MRPL) are lower and lower so that competent methods must be more sensitive (pg level), more robust whatever the biological matrix (hair, faeces, urine, tissue, etc.), more specific (HR-MS and tandem MS (MS/MS)) and relevant to challenging analytes such as ecdysteroids, or endogenous steroids. Analytical protocols dedicated to the extraction and purification of anabolic steroids in biological fluids and tissues are generally based on at least two stages. Single use solid-phase extraction (SPE) columns are generally utilized to extract and purify steroids from biological matrices. C18, C8, NH2 and SiOH are amongst the most popular stationary phases; SAX (strong anion exchange) columns are sometimes used for the isolation of steroid conjugates. Solid samples (faeces, hair, tissues, feeding stuff) are preliminary extracted either by accelerated solvent extraction (ASE), soxhlet or liquid–liquid extraction before further purification step(s). Conjugates are usually hydrolyzed either by enzymatic (glucuronidase) or chemical approaches (solvolysis or methanolysis). A digestive juice from Helix pomatia was used in the past for its b-glucuronidase and arylsulfatase activity. However, because a 3b-hydroxysteroid oxidoreductase and a 3-oxosteroid-5,4-isomerase activity have been observed in this formulation, use of H. pomatia is usually avoided in analytical procedures. Bacterial (Escherichia coli) preparations with a pure b-glucuronidase activity and no arylsulfatase activity are now generally accepted for hydrolysis. When GC-MS is used, steroids are quite always derivatized, the most popular approach being the silylation, acylation (acetylation, perfluoroacylation) or oxime formation. N-Methyl-N-(trimethylsilyl)-trifluoro-acetamide (MSTFA) is often used as silylating agent in combination with ammonium iodine (NH4I) or trimethyliodosilane (TMIS) as catalyst, and dithiothreitol (DTE) as antioxidant. When LC-MS is used, derivatization is not mandatory but is sometimes used to enhance the signal or improve the specificity. Standard reference steroids are generally available in Sigma (St Louis, MO, USA), Steraloids (Wilton, NY, USA) and Research Plus (Bayonne, NJ, USA). GC-MS is routinely used for steroid analysis; mass analysers are mainly quadrupoles, but ion trap technologies can be found as well. Single MS is progressively replaced by multidimensional approaches, either by triple
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29 081205004 Sm(Mn, 1x1) 100
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Figure 1 GC-QqQ-MS. Application to the detection of low pg.mL-1 (ppt) of gonadic steroids in children’s blood (2 mL). 17b-estradiol (0.9 ppt) eluted at 19.86 min, 17b-estradiol (1.3 ppt) at 20.60 min (PFB derivatization, negative chemical ionization, SRM acquisition, 343W343).
quadrupole (QqQ) and ion trap (external sources), for steroid analysis. One illustration of the ability of GC-MS/MS (QqQ) approach to bring ad hoc information is given in Figure 1. In this example, gonadic steroid concentrations as low as 0.9 ng L1 were made possible to quantify in limited sample size of blood (2 mL). High resolution-mass spectrometers (HR-MS) are mainly electromagnetic instruments operated at resolution equal or better than 10,000 (10% valley); they are used in confirmatory processes (control laboratories) or more generally when both sensitivity and specificity are needed. HR-MS approaches are possible nowadays on orbital ion trap systems as well at higher resolution values. One illustration of the signal specificity offer by this kind of analyser is given in Figure 2. Two isotope ions of boldenone sulphate are shown on a mass spectrum recorded after negative electrospray ionization (ESI) and full scan acquisition at a resolution better than 70,000. A tremendous separation of C19H25O5S and C13 18C1H25O5S species was made possible. In recent years, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) has been developed to trace the origin of steroids (endogenous versus exogenous). This technique made it possible to discriminate exogenous testosterone/estradiol metabolites from endogenous ones, by measuring the 13C/12C ratio of the steroids [14–15]. Eluted compounds from the GC are combusted in a catalytic furnace to N2 and CO2. For the carbon isotope ratio determination, masses 44 and 45 are determined with great precision and accuracy. The measured carbon isotope ratio (13C/12C sample) is related to an international fossil carbonate standard ‘‘Pee Dee belemnite’’ or ‘‘PDB’’. This approach is already used in a few National Reference Laboratories in Europe for official controls. In all cases (low resolution-mass spectrometry (LR-MS), HR-MS, C-IRMS) capillary columns are preferred; typical dimensions are 30 m 0.25-mm i.d., 0.25-mm film thickness. Stationary phases are non-polar to medium polar. Helium (n55) is used as carrier gas at 1 mL min1. Injector and transfer line temperatures are set at high values, 2501C and 2801C, respectively.
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041006002 #8 RT: 0.11 AV: 1 NL: 9.49E4 T: FTMS-p ESI Full ms [350.00-370.00] 365.14282 R=69901 z=1 32 C19H25O5 S1 100 95 90 85 80 75 70 Relative Abundance
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Figure 2 LC-HR-MS. Application to determination of the elemental composition of steroid conjugates (the example of boldenone sulphate is given). The ionization was performed in the negative ESI mode, and full scan acquisition mode was used at RW70,000. m/z 365.1428 corresponds to C19H25O5S and 366.1461 to C13 18C1H25O5S.
LC is either coupled to QqQ analyser or ion trap detectors (but at least in the MS/MS or multiple stages mass spectrometry (MSn) mode) operated in the positive or negative (steroid conjugates) ESI mode. Atmospheric pressure chemical ionization (APCI) or atmospheric pressure photon ionization (APPI) is preferred for non-polar steroids such as progestagen esters or estrogens, respectively. Reversed-phase LC is generally performed on octadecyl (or octyl) grafted silica stationary phase (e.g., 50 2 mm, 5 mm) equipped with a guard column (e.g., 10 2 mm, 5 mm). Elution solvents are based on methanol or acetonitrile and 0.5% (v/v) acetic acid in water. N2 is used as nebulization and desolvation gas. Potentials applied onto the capillary (from 3 to 4 kV), cone (from 15 to 35 V) and collision cell (from 5 to 30 V) are significantly different from one steroid group to another; the parameters have to be optimized for each class of compounds. One illustration is given on steroid conjugates (Figure 3); reversed-phase LC permitted the separation of 17b-boldenone sulphate and 17b-boldenone sulphate. Negative ESI offered a good specificity regarding boldenone metabolite spectrometric signals combined with a good sensitivity, thus authorizing the low ppt level detection of these two steroid sulphates in the urine sample.
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Figure 3 LC-QqQ-MS. Application to the direct detection of steroid conjugates (the example of boldenone sulphate is given). The ionization was performed in the negative ESI mode; the SRM acquisition mode was selected. 17b-boldenone eluted at 9.02 min, and 17s-boldenone at 9.23 min.
3.2 Corticosteroids Fifty years after the discovery of natural corticosteroid hormones and their antiinflammatory properties, many synthetic derivatives of these molecules are today available. Their legal utilization in human and veterinary medicine is strictly regulated, with withdrawal periods between treatment and slaughtering and maximal residue levels in edible biological matrices for some compounds. These substances have been also illegally used as growth promoters in cattle, whereas these practices are not allowed in Europe. Indeed, low concentrations of glucocorticosteroids are known to increase weight-gain, reduce feed conversion ratio, and have a synergetic effect with other molecules such as b-agonists or anabolic steroids [16–18]. For many years, various analytical methods have been proposed for the identification of corticosteroid residues in various biological matrices. During the 1980s and early 1990s, the main detection methods used for corticosteroids were radioimmunology [19,20], fluorimetry [21,22] or LC with UV detection [23–27]. Nowadays, MS coupled to GC or LC is the method of choice for the unambiguous identification of these compounds at trace level in biological matrices. GC-MS with electronic impact ionization (EI) was the first MS technique to be applied for these compounds [28–38]. GC-C-IRMS was also used to distinguish natural endogenous corticosteroids from their exogenous analogues [39]. But the further development of LC-MS [40–43] and LC-MSn [44–53] with atmospheric pressure ionization (API) techniques permitted significant improvements in terms of sensitivity and specificity, as well as kinetic and metabolism studies [54]. The recently introduced ultra performance/fast LC systems should be an ultimate reason for considering now LC-MSn as the actual standard for corticosteroid analysis. Due to the moderate polarity of corticosteroids, their gas-chromatographic analysis requires preliminary derivatization step in order to increase their
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volatility and avoid adsorption phenomenon. The most currently used techniques remain chemical oxidation followed by negative chemical ionization in MS, or silylation followed by positive electron ionization. Mixtures such as MSTFA/TMIS/DTE lead to mixtures of tri- to penta-TMS derivatives, unstable under EI conditions, and then leading to weak molecular ions and to a high number of fragment ions, and finally to a limited sensitivity. Alternatives to this derivatization strategy have been proposed, as for instance mixed oxime-TMS derivatives [55]. Another route is the use of boronic esters. This technique was widely used for a-g diol steroids, and consists of the action of methylboronic acid/ethylacetate followed by silylation. The derivative is stable and intense molecular ions for a-g diol compounds are observed even under EI conditions. However, its main disadvantage remains once again an insufficient sensitivity for the analysis of trace residue levels in complex biological matrices. Amongst the most popular approaches, the elimination of the polar C17 side chain by chemical oxidation and subsequent oxidation of residual hydroxyl made indeed corticosteroid analysis possible and efficient by GC approaches. This technique was the most currently used before the development of LC-MS systems. When LC is used, direct measurement of corticosteroids is then possible. The LC separation is significantly worse than the one observed in GC. However, when MS/MS is used, co-elution of corticosteroids is not a problem anymore. The recent development in chromatography (resolutive and fast) increased the number of theoretical plates, and thus authorized the chromatographic separation of corticosteroids with very close chemical structure. ESI as well as APCI is clearly well adapted for corticosteroids. Under slightly acidic conditions, these relatively polar compounds give an intense pseudomolecular ion [M+H]+ in ESI+ and an adduct with the conjugated base of the used organic acid [M+base] in ESI [56]. These diagnostic ions are suitable to be selected as precursor ions for further fragmentation in MS2. In ESI+, some numerous but not very specific fragment ions can be observed, which correspond to losses of water molecules and/or halogen atoms, as well as some other minor cleavages within the B and C rings. In ESI, the fragmentation is reduced to two ions; the pseudo-molecular ion [M–H] and the fragment corresponding to a cleavage of the side chain with loss of formaldehyde [M–CH2O–H]. This fragmentation pathway appears extremely efficient for measurement of a large number of corticosteroids at trace residue levels in biological matrices. An illustration is given in Figure 4 for liver extracts spiked with 1.5 ng g1 of fluocinolone acetonide; one can appreciate the great improvement both in terms of sensitivity and specificity when using MS/MS approach (QqQ). Moreover, if additional diagnostic ions are required, a high cone potential inducing the in-source fragmentation of the [M+base] ion can be used to select the resulting [M–CH2O–H] fragment as precursor; this ion can be further fragmented in the collision cell. Finally, the APCI techniques operating in the negative mode represent today the technique of choice for the ionization and fragmentation of corticosteroids because of its better sensitivity and specificity compared to all other ionization techniques. The chromatographic separation of isomers such as dexamethasone and dexamethasone may remain an ultimate analytical
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110902012 100 m/z=511
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Figure 4 Comparison of the spectrometric signal specificity after LC-MS and LC-MS/MS analysis [liver sample spiked with 1.5 ng g1 fluocinolone acetonide (RT ¼ 8.28 min)]. Negative ESI was used as ionization mode and SRM was used as acquisition technique.
challenge. Some specific stationary phases are today available permitting this separation. But the relative intensities of diagnostic ions produced in ESI can be another way to distinguish these two isomers, using conventional model [57] of multidimensional statistical approach [58].
3.3 b-Agonists b-agonists, the so-called b-sympathomimetics, are characterized by their structural and pharmacological properties, which are very close to those of catecholamines. These drugs are used as bronchodilators, tocolytics or heart tonics in human and veterinary medicine. During the past 20 years, several studies focused on the effects of such synthetic molecules on growth rate and performances, when administered per os, mixed with feedingstuffs. From 1984, it was pointed out that a specific b-agonist, the so-called clenbuterol, was able to increase notably the protein-to-fat ratio [59–62] in athletes and meat producing
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animals. In the following years, these properties were also emphasized for other b-agonists such as cimaterol, ractopamine and fenoterol. Because of their ability to shift nutrients towards protein instead of lipid anabolism, such molecules were gathered under the generic name of ‘‘repartitioning agents’’ [63]. Basic knowledge on b-agonists concerns mainly the previously mentioned substances [64]. Although b-agonistic drugs have never been licensed for growth promoting purposes, their use as growth promoters appeared to spread to a large extent, especially in livestock. Since toxicological data on such pharmacological manipulations are lacking, their addition to animal feed has been forbidden within the European Union Member States. Furthermore, the illegal use of b-agonists was revealed to be of major concern for public health and some food poisoning cases were reported [65]. Faced with such problems, analytical techniques designed for the unambiguous detection and identification of b-agonists in biological matrices has been developed. Among the available techniques (e.g., thin-layer chromatography (TLC), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), high-performance LC (HPLC), infrared (IR) spectrometry), MS appeared to be the most efficient tool to provide results which are sensitive and specific enough to allow enforcement action. Consequently, several methods based on MS instrumentation have been set up, among which GC-MS and LC-MS often reinforced by MS/MS or HR-MS configurations are recognised today as the most powerful approaches. Concerning GC-MS techniques, trimethylsilylation or tert-butyldimethylsilylation are commonly used derivatization methods. However, when analysed under electron ionization conditions, these derivatives are well known to give very poor informative data (especially for clenbuterol-like compounds) and their use usually does not permit the identification of residues (according to official required quality criteria) below the 1 ng mL1 or ng g1 level in biological matrices. Consequently, other analytical tools based on different derivatives and/or ionization modes have been set up, notably for confirmatory purposes. Cyclic boronic derivatives [66–68] were shown to give far more informative data when analysed by means of GC-MS after EI ionization. In addition, 2-dimethylsilamorpholine, the so-called cyclic dimethylsilylmethylene derivatives [69] also proved to be of major interest for the confirmation step. Unfortunately, these cyclic derivatizations are useful for clenbuterol-like compounds but not easily feasible for b-agonists containing additional hydroxy substituent(s). Therefore, silylated (or cyclic) derivatives may be analysed as well in the positive chemical ionization (PCI) mode. If GC-MS instruments were historically the more widely used for various classes of residues, LC-MS today appears as the method of choice and the major actual investment for many laboratories, especially for the analysis of polar compounds. Undoubtedly, reversed-phase LC and positive ESI is nowadays the method of choice for most b-agonistic drugs as shown on Figure 5, where an incurred retina sample (100 mg) collected in an animal treated with ractopamine is shown. Even seven days after the end of the treatment, ractopamine can be detected and quantified on the basis of at least four transitions.
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Figure 5 LC-MS/MS ion chromatogram of an incurred pig retina sample, seven days after the end of the animal treatment by ractopamine. Positive ESI was used as ionization mode and SRM as acquisition technique. Retention time of ractopamine was 8.64 min, isoxsupinrine was used as internal standard (RT ¼ 10.45 min).
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3.4 Thyrostats Most of known thyrostats or thyroid-inhibiting compounds are thionamides which are small molecules derived from TU or mercaptoimidazole. These substances show a goitrogen activity, thanks to their thiocarbamide group which inhibits the normal metabolism of the thyroid gland and causes a decreased production of thyroid hormones. The consequent hypothyroidism increases the growth rate of the animal although the observed weight-gain is mainly the result of water absorption and retention within edible tissue as well as filling of the gastro-intestinal tract [70–71]. Consequently, for several years during the 1980s, these substances have been employed as additives in animal feeds to increase the weight of cattle prior to slaughter; especially the use of MTU with a 5-g/day/animal dose for 30 days was widespread. The consequence of such abuse is not only the production of inferior meat quality (meat may be exudative), but overall the potential risk to human health of drug residues. Consumption of meat contaminated with thyrostats has caused an increased incidence in Spain of aplasia cutis, a characteristic scalp defect [72]. For these particular reasons, thyrostats have been banned within the European Union since 1981 (Directive 81/602/EC) [73] for animal production. These compounds have indeed been classified by IARC as belonging to group 2B that represents carcinogenic and teratogenic compounds. Different techniques have been developed in order to control the illegal use of such compounds in meat producing animals. Thyrostats are challenging compounds to analyse for several reasons linked to their chemistry. The first difficulty concerns their extraction from biological matrices in which they occur through six different tautomeric forms due to the delocalization of the p electrons on the ring structure. The second difficulty, when separated onto reversed-phase LC, is due to their high polarity that prevents a good retention and separation of these compounds on the stationary phase. Finally, the analysis by MS of these small molecular weight analytes (100–200 u) is not satisfactory in term of sensitivity (signal-to-noise ratio) since the signals are usually disturbed by the chemical noise, as illustrated by Blanchflower and co-workers [74]. These difficulties might be overcome thanks to derivatization procedures placed at the beginning of the protocol and occurring as nucleophilic substitution with halogenated compounds. The aims of the derivatization are numerous and include the stability of the compounds, the reduction of their polarity to improve their separation as well as the increase of their molecular weight to allow lower detection capabilities and limits of decision. Different derivative reagents have been studied and compared, such as pentafluorobenzylbromide (PFBBr), iodobenzylbromide (IBBr) and bromobenzylbromide (BrBBr). Thyrostats were first studied with colorimetric methods based on the reactivity of thiol or thione groups, with for example 2,6-dichloroquinonechloroimide or 7-chloro-4-nitrobenzo-2-oxal-1,3-diazole (NBD-Cl) [75,76]. Various techniques have been described in the literature for the detection of administration of thyrostats to animals. These range from weight measurement
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of the thyroid gland [77] to various methods based on TLC [78–80], GC using nitrogen–phosphorus detection, flame photometric detection, or MS detection [81–82] and HPLC using either UV [83] or electrochemical detection [84]. More recently, methods based on LC coupled to MS have been published. Detection performances of the different published methods were 50 mg/L in urine using GC-MS, 20 mg kg–1, for six different thyrostats, in thyroid samples using LC-MS/MS [85] and 25 mg kg–1 in muscle with a GC-MS/MS detection [86]. More recently, a detection protocol performed in MS2 by LC-MS/MS (QqQ) after negative ESI was set up [87]. The protocol has been successfully applied to biological matrices (urine, tissues, faeces and hair) (Figure 6) and the multiresidue method was validated according to the EU criteria (2002/657/EC Decision) leading to improved performances of identification and quantification (mg L1 range). Thyrostats are also easily analysed in MS2 by GC tandem quadrupole MS/MS after electron ionization (Figure 7) [88,89]. Reliable, sensitive and specific methods, mandatory based on MS, have been set up to ensure efficient control of their illegal use. The newly developed protocols exhibit performances allowing detection and identification of the thyrostatic compounds in biological fluids and edible tissues in the mg kg1 or mg L1 range which is in accordance with the requirements of the European
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Union regarding the provisional MRPL suggested at 100 mg L1. Since high urine concentrations of residues (WW 100 mg L1) are generated upon drug administration aimed at increasing animal weight, methods currently running in laboratories enable efficient control of the illegal use of thyrostats. However, the occurrence of TU in bovine urines from national monitoring plans with quantifications in the range 1–10 mg L1 occasionally rises the question of its origin which might either be the consequence of an illegal administration or the result of an ‘‘endogenous’’ production. Until now, the identification of TU in animal biological matrices is systematically interpreted as the consequence of an illegal administration. Recent studies however [90,91] achieved an independent and confident identification (LC(ESI)MSMS, GC(EI+)MSMS and HR-MS (EI and NCI) measurements) of TU in bovine urine samples collected on animals never treated with any thyrostatic drugs and the demonstration of a relationship between a diet based on cruciferous and the occurrence of TU in urine. This result is of prime importance for laboratories and risk managers involved in the field of forbidden growth promoters control as evidence for presence of TU residue has to be carefully considered as a non-systematic proof of illegal administration.
3.5 Growth hormone Growth hormone (GH), also known as somatotropin (ST), is a 22-kDa protein naturally produced by the anterior pituitary gland. STs are important factors influencing metabolic and somatogenic processes including growth, immune function, reproduction and lactation in mammals [92–95]. It was known as early as the 1930s that the injection of dairy cows with pituitary extracts increased milk yield [96]. GHs are widely used outside Europe to stimulate milk production in dairy cows and as a general growth promoter in pigs [97]. Human GH is also thought to be widely abused in human sports since the increase in muscle size and strength makes them a viable alternative to anabolic steroids [98,99]. Recombinant DNA techniques allow the production of large quantities of recombinant GHs which may exhibit slightly different chemical structures from the pituitary ST, by adding a number of amino acids on the N-terminal side. Indeed, various forms of recombinant somatotropins (rSTs) with slightly different N-terminal sequences have been produced by several companies and described in literature [100]. Recombinant bovine and porcine STs, legally used in the USA since the approval by the Food and Drug Administration (FDA) in 1994 and in other countries, are banned in the European Union [101,102], for animal welfare and food safety reasons. Nevertheless, and because of widespread application, the illegal distribution and use cannot be excluded within the EC [103,104], putting a strong demand on the availability of screening and confirmatory analysis methods for rSTs. Until now, the detection of GH in plasma, milk and tissues has been based on RIAs and ELISA procedures allowing for quantification in the range 10–20 mg L1 [105–111], biosensor based methods have also been reported recently for the detection of STs in concentrated injection preparations [112]; however, none of
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these methods allowed distinguishing between the endogenous and the rSTs since one of the challenging aspects in the detection of rST abuse is the high level of similarity between the endogenous, native hormone, and exogenous, synthetic recombinant hormone. New strategies intended to discriminate between endogenous and recombinant bovine ST have been set up. In this context, MS measurements with soft ionization modes such as ESI and matrix-assisted laser desorption (MALDI) adapted to proteins have been successfully reported for pure ST solutions. STs identification is either performed after accurate mass determination of the entire molecule (Figure 8) or after tryptic digestion and identification of the characteristic N-terminal end from the recombinant form (Figure 9) [113,114]. However, GH extraction and analysis from biological samples in order to investigate the potential misuse of rSTs encountered difficulties linked to the trace level (mg L1) at which the ST is present within matrices such as milk and blood [115]. Recently, however, a method based on appropriate equine plasma sample preparation, tryptic digestion and subsequent N-terminal MS measurements allowed for the detection of recombinant equine ST in horses treated with the molecule (Figure 10) [116]. This study is the first ever reported in the field and is very promising for the future and its application in the anti-doping and food safety controls. Indirect approaches to prove rST treatments have also been reported. They exploit circulating molecules that can be considered as analytical targets and indirect indicators of the treatment. Therefore, biomarkers of GH action are being investigated as potential test for GH abuse. Both insulin-like growth factor A17;1287.8 A16;1368.1
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Figure 10 HPLC-ESI ion chromatograms of N-term peptide fragments of recombinant equine somatotropin (reST) in equine plasma 3 h after reST administration.
I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) have been reported as suitable markers as they are notably under GH control and their concentrations increase after rST administration [117,118]. Their quantification has already been reported either by the use of specific immunoassays [119,120] or MS [121]. In horses, a threshold value was set at 800 ng mL1 for IGF-I in plasma above which a sample is considered as suspicious for GH abuse.
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Detection in serum of antibodies raised against GHs as a consequence of their administration to animals has also been successfully reported as a long-term approach for the detection of GH abuse [122–126]. Immunological techniques such as ELISA [127], Western-Blot [128] or biosensor based immunoassays [127] have allowed for screening of administrated animals over several months (Figure 11).
4. OCCURRENCE IN FOODS As laid down in Article 8 of Directive 96/23/EC, the Commission shall report to Member States within the Standing Committee on the Food Chain and Animal Health on the outcome of the checks carried out, in particular on the implementation of the national plans and on the developments in the situation in the various regions of the Community. The aim of the annual report is to summarize the results of the national residue monitoring plans in the Member States. Report for 2005 on the results of residue monitoring in food of animal origin in the Member States includes around 780,163 (871,000 in 2004) samples for residue control in all food commodities. For hormones (steroids and zeranol derivatives), 0.13% (0.12% in 2004 and 2003) of the samples taken in bovines were found to be non-compliant and 0.44% in pigs (0.3% in 2004), mainly due to the presence of presumed endogenous nandrolone and contamination with the metabolite zearalenone. Whereas, no non-compliant results for antithyroid agents were reported from 2000 to 2004, 8 non-compliant were reported for thyrostatic agents in bovine in 2005 (mainly TU). The finding was enabled by improving the performance of the analytical methods used for the analysis lowering the limit of detection from 50 to 1 ppb, and caution should be taken regarding the origin of these thyrostat residues as explained before in this chapter. No non-compliant samples for stilbenes have been found in 2004 and 2005 in Europe. The number of non-compliant results for corticosteroids in bovines varied over the years from
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186 in 2005 to 64 in 2004, and 130 in 2003; dexamethasone was the most frequently found substance for corticosteroids. For b-agonists, the incidence of non-compliant results increased from 0.02% of the bovines analysed in 2003 to 0.06% in 2004 and 0.08% in 2005.
5. FUTURE TRENDS Since the beginning of the implementation of the control laboratory network in Europe, chromatography coupled to MS was implemented increasingly especially in the field of growth promoters in food producing animals. Noncompliant samples are not disputed nowadays, the risk of false positives being extremely low especially when MS/MS or HR-MS techniques are used. Moreover, because of their sensitivity, MS techniques authorize the demonstration of an illegal use far away from the treatment. The remaining challenge is rather linked to the fishing of unknown anabolic compounds potentially used on the black market. The current tendency consists of on-line measurement of biological extracts by biological tests (estrogenic or androgenic receptors for instance) and parallel mass spectrometric characterization of the suspected anabolic steroid by accurate mass determination and fragmentation study on orbital ion trap or hybrid quadrupole-time of flight instruments (QqTOF).
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CHAPT ER
12 Mycotoxins Carlo Brera, Barbara De Santis, Francesca Debegnach and Marina Miraglia
Contents
1. Introduction 2. Classes of Mycotoxins 2.1 Major mycotoxins 2.2 Minor mycotoxins 3. Health Effects 3.1 Cases of acute mycotoxicosis in humans 3.2 Effects of low doses of mycotoxins 3.3 Quantitative evaluation of human exposure to mycotoxins 3.4 Tolerable maximum limit for mycotoxins in food 4. Sampling 5. Analytical Methods 5.1 Introduction 5.2 Sample preparation 5.3 Determination and detection 5.4 Other methods 6. Occurrence in Food and Feed 6.1 Aflatoxins 6.2 Ochratoxin A 6.3 Fusarium toxins 6.4 Patulin 6.5 Multi-Fusarium toxins 6.6 Other mycotoxins 7. Decontamination Procedures 8. Future Needs Acknowledgement References
Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00012-3
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1. INTRODUCTION Microscopic filamentous fungi, commonly known as moulds, can develop on food commodities of plant origin (maize, wheat, etc.) and in some cases also on commodities of animal origin (meat products, sausages). These moulds can, in suitable environmental conditions, produce chemical toxic compounds, known as mycotoxins. The word mycotoxin stems from the Greek word ‘‘mykes’’, meaning mould, and ‘‘toxicum’’ meaning poison. Mycotoxins are naturally occurring secondary metabolites of some fungal species mainly belonging to the genera Aspergillus, Penicillium and Fusarium. Even if it is reasonable to argue that mycotoxins could have played a significant role in some of the human and animal diseases since the Roman era, the scientific interest for their implication for human and animal health began in the 1960s. Ergotism, for instance, that hit various populations of Central and North Europe in the Middle Age as a consequence of the high consumption rates of rye and other cereals containing some alkaloids produced by the Claviceps purpurea fungus, can be considered as the first recognized disease, known as S. Anthony’s Fire, to be related to mycotoxins [1,2]. Essential factors for mycotoxin production are both the stress of the plant, as derived by the extreme soil dryness or the lack of a balanced nutrient absorption and environmental factors such as climate changes, temperature, humidity, water activity, mechanical damage of kernels as well as insects and pest attack [3]. Currently more than 300 mycotoxins are known, but the scientific attention has been so far focused only to approximately 10 compounds presenting a known toxicological impact on human and animal health. Mycotoxins are characterized by differentiated chemical structures as a result of the multiple varieties of moulds responsible for their production. Among them, aflatoxins present a highly oxygenated heterocyclic structure, ochratoxins consist of a polyketidederived dihydroiso-coumarin moiety linked through the 12-carboxy group to phenylalanine, and trichothecenes (deoxynivalenol (DON), NIV, T-2 and HT-2 toxin, diacetoxyscirpenol (DAS)) are characterized by a quite similar chemical structure presenting a double bond between C9 and C10 and an epoxidic group with functional alcoholic and ester groups at the C12–C13 position responsible of their toxicity. Other relevant mycotoxin are zearalenone (ZEA) and its metabolites, that are estrogenic resorcylic acid lactone compounds, fumonisins (FBs) presenting a long linear carbon chain with ester, carboxylic- and aminosubstituting functional groups and patulin, a polyketide lactone. The toxic effects of mycotoxins are mainly related to genotoxicity, carcinogenicity, immunotoxicity, mutagenicity, nephrotoxicity and teratogenicity. Crops can be directly infested by toxic moulds and contaminated by mycotoxins: it has been calculated that, during the various cycles of the agrifood chain including production, processing, transport and storage, about 25% of crops are affected each year with ‘‘unacceptable’’ levels of mycotoxins. However, the percentage of crops affected by mycotoxins is much higher.
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According to a 1993 World Bank report, ‘‘Investing in Health’’, approximately 40% of disability-adjusted life years lost to premature adult death in developing countries are due to diseases linked to mycotoxin-contaminated food consumption. The individuation of the intrinsic properties of a food commodity favourable to mycotoxin contamination is quite multifaceted. In general, substrates rich of carbohydrates and lipids are more exposed to mould attack and potential production of mycotoxins. The presence of mycotoxins in a food commodity can occur even in the absence of a visible mould infestation due to a ceased vital cycle of the microorganism or by the effect of a removal of the mould due to technological processing of the food commodity. Nevertheless, the presence of a visible mould on the surface of a food product does not represent a clear indication of the presence of a mycotoxin. In addition, one specific mycotoxin can be produced from different genera and species of moulds and one specific mould can produce more than one mycotoxin. Generally, plant-origin commodities directly contaminated by mycotoxins are cereals, with maize as the most risky crop, dried fruit, spices, grape, coffee, cocoa, fruit juices especially apple-based and, in minor extent, meat products and liquorice. During the storage cycle, mycotoxins can directly contaminate also cheeses and sausages. In addition, food products can become contaminated as a consequence of a carry over from contaminated feeds and be present in food of animal origin such as milk, eggs and, in a minor extent, meat. Even if some technological processing is able to reduce the level of mycotoxins of the raw commodity, some processed food such as wine and beer can result in contamination by mycotoxins as a result of the use of contaminated raw material. It has to be noted that mycotoxins resist high temperatures and the common domestic cooking procedures are not able to destroy them. In addition to food diet, humans and animals can also be exposed to mycotoxins by inhalation of heavily contaminated dusts. This phenomenon is particularly observed in certain working places such as harbours and warehouses and also in the houses as a result of an indoor contamination caused by wallpapers and mouldiness of domestic environments. The industrial processing is in some cases effective in reducing or at least modifying the pattern of contamination. For instance, milling of cereals determines a distribution of some mycotoxins present in raw kernels that is variable throughout the various milled fractions, with concentration factors in germ, bran and flour for feed preparations and dilution factors in grits and flour for human-intended use; roasting of coffee beans is another process able to largely destroy Ochratoxins A (OTA) (up to 90%). Apart from all the above-mentioned decontamination strategies, it should be considered that an integrated approach based upon the implementation of preventive actions starting from the seeding and modulated along the whole agri-food chain, is still the most effective strategy to eliminate or at least reduce the mycotoxin contamination in food and feed commodities.
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Mycotoxins can occur at pre-harvest, harvest and post-harvest stage. Therefore, in any of these phases preventive actions can be performed. Prevention through pre-harvest management is the best method for controlling mycotoxin contamination. The major effective actions are (i) a correct management of insect infestation, since insect-damaged kernels provide infection routes and allow kernel drying to moisture levels more favourable for growth of mould species, (ii) a suitable management of crop residues and crop rotation, (iii) a proper irrigation control and treatment of soil to avoid extreme conditions of either drought or excessive moisture, (iv) a development of naturally resistant plant varieties to fungal infection, since host resistance may present a promising strategy for the pre-harvest prevention of mycotoxin contamination. Among the strategies for enhancing host resistance, the genetic engineering research, aimed at the addition or enhancement of antifungal genes present in many endogenous low molecular weight compounds and biomacromolecules, is one of the most modern and debated issues involved in some controversial economic, ethic, safety and environmental aspects. During harvesting and post-harvest phases, it is important to control factors such as the avoidance of damaging kernels during the mechanical or manual harvesting procedures, and the use of appropriate and complete drying procedures of the agricultural products. This control is essential for preventing mycotoxin formation during storage. As soon as the crop is fully grown and the crop cycle is completed, harvesting should take place. Some studies have reported that crops left on the field for longer periods of time may present higher levels of toxin contamination. Decontamination procedures for reducing or eliminating the presence of mycotoxins in food products have not yet been standardized worldwide due to the related high costs and, in some cases, the scarce feasibility of effective results. However, chemical, biological and physical treatments are known to be effective somehow in reducing the mycotoxin content. This issue will be treated in Section 7.
2. CLASSES OF MYCOTOXINS Mycotoxins represent a quite wide spectrum of chemical compounds characterized by different molecular structures as a result of the numerous species of fungi responsible for their production. Major and minor classes of mycotoxins can be distinguished: among major class aflatoxins, ochratoxins, trichothecenes, FBs, patulin and zearalenone represent the most concern for human and animal health, while in the minor class, ergot alkaloids, citrinin, cyclopiazonic acid, sterigmatocystin, moniliformin, gliotoxin, citreoviridin, tremorgenic mycotoxins, penicillic acid, roquefortine, 3-nitropropionic acid, and fusaproliferin are noteworthy.
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2.1 Major mycotoxins 2.1.1 Aflatoxins On the basis of their potent toxic effects aflatoxins, in particular aflatoxin B1 (AFB1) [CAS number: 1162-65-8], are historically the main toxins of concern since their discovery occurred in 1958. These toxins are mainly produced by two species of Aspergillus fungi genus, namely Aspergillus flavus and Aspergillus parasiticus, particularly in hot and humid areas, together with Aspergillus nomius and Aspergillus pseudotamarii [4,5]. Moisture higher than 85% and temperatures above 25% are favourable to the growth of aflatoxin-producing fungi during storage [6]. Aflatoxin B-hydroxilated metabolites, aflatoxins M1 and M2 [CAS number: 6885-57-0], are two toxins occurring in milk and derived products, as an effect of the fast metabolism of aflatoxins B1 and B2 (AFB2) in animals fed with aflatoxinscontaminated feeds. Aflatoxins (Figure 1) are substances that are chemically related to difuranocoumarin and classified in two broad groups according to their chemical
O
O
O
O
O
O O
O
OCH3
AFLATOXIN B1
OCH3
AFLATOXIN B2
O
O
O
OH
O
OH O
O O
O
OCH3
AFLATOXIN M2
AFLATOXIN M1 O
OCH3
O
O
O
O
O O
O O
OCH3
AFLATOXIN G1
Figure 1 Molecular structure of aflatoxins.
O
OCH3
AFLATOXIN G2
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structure; the difurocoumarocyclopentenone series (including AFB1 and AFB2 [CAS number: 7220-81-7]) and the difurocoumarolactone series (including aflatoxin G1 (AFG1) [CAS number: 1165-39-5], aflatoxin G2 (AFG2) [CAS number: 7241-98-7] and aflatoxin M1 (AFM1) [CAS number: 6795-23-9]). The G series contains a D-lactone ring, while the B series contains a cyclopentenone ring, which is responsible for the major toxicity of the B series. The aflatoxins fluoresce strongly in UV light (ca. 365 nm); AFB1 and AFB2 produce a blue fluorescence whereas AFG1 and AFG2 produce green fluorescence. Aflatoxins are crystalline substances, soluble in moderately polar organic solvents, such as chloroform, methanol, dimethysulfoxide, scarcely soluble in water (10–30 mg/mL) and insoluble in non-polar organic solvents. Aflatoxins in dry state are very stable to heat up to their melting point. Pure aflatoxins are destroyed by UV radiations, unstable at pHo3 and W10 and in the presence of oxidizing components.
2.1.2 Ochratoxins Ochratoxins A (OTA) [CAS number: 303-47-9], B and C (Figure 2) are compounds with phenylalanine containing dihydroisocoumarin. OTA, the toxin of most concern, also contains a chlorine atom on the aromatic ring, which accounts for its toxicity. Ochratoxin is produced by both Aspergillus ochraceus and Penicillium viridicatum (among others), with OTA being the most relevant toxin [7]. OTA is a colourless crystalline compound exhibiting blue fluorescence under UV light. Its sodium salt is soluble in water, as an acid, it is moderately soluble in polar organic solvents such as chloroform and methanol and dissolves in dilute aqueous sodium bicarbonate. On acid hydrolysis, OTA yields phenylalanine and an optically active lactone acid, ochratoxin a. Reaction in methanol and chloridric acid yields the OTA methyl ester, which can be used as a confirmatory reaction in high-performance liquid chromatography (HPLC) determination.
2.1.3 Trichothecenes Trichothecenes (Figure 3) are a cluster group of about 150 mycotoxins [8] produced by various species of different fungi of genera: Fusarium (in particular Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides), Myrothecium, Stachybotrys, Trichoderma, Cephalosporium, Trichothecium and Verticimonosporium [9,10]. Updated information on the chemical diversity of Fusarium species producing thricothecenes has also been published by Thrane [11]. In addition, an exhaustive COOH
O
OH
O
N H CH3 Cl
Figure 2 Molecular structure of OTA.
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Figure 3 Molecular structure of the main trichothecenes. R ¼ OH or acyloxy group (most often acetoxy). For class B trichothecene DON, R1 ¼ OH, R2 ¼ H, R3 ¼ OH, and R4 ¼ OH.
review on taxonomy and clinical aspects of Fusarium species has been published by Nelson [12]. These toxins are chemically strictly related and are characterized by a basic group consisting of a common tetracyclic, sesquiterpenoid 12,13-epoxytrichothec-9-ene ring, the epoxide group known as responsible for causing the toxicity [13]. Depending on the presence on C7 of a side chain, trichothecenes are distinguished as type A and B. Group A includes toxin T-2 and HT-2 and DAS, also known as anguidine. Main trichothecenes of group B include DON, commonly known as vomitoxin, nivalenol (NIV), 3- and 15-acetoxy NIV and fusarenon X. Trichothecenes are chemically stable and can persist for long periods once formed. Group A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, MAS and DAS) are highly soluble in ethyl acetate, acetone, chloroform, dichloromethane and diethyl ether. The Group B trichothecenes (DON, NIV, 3-acetylDON, 15acetylDON, fusarenone-X, scirpentriol and T-2 tetraol) are highly hydroxylated and relatively polar being soluble in methanol, acetonitrile and ethanol. DON [CAS number: 51481-10-8], contains one primary and two secondary hydroxyl groups and is soluble in water and polar solvents such as methanol and acetonitrile. Unlike many of the other trichothecenes, the molecule contains a conjugated carbonyl system and this results in some UV absorbance that assists its detection by TLC or HPLC methods. T-2 toxin [CAS number: 21259-20-1] and HT-2 toxin [CAS number: 26934-87-2] are mycotoxins produced by F. acuminatum, F. poae and F. sporotrichioides, which are commonly found in various cereal crops (wheat, maize, barley, oats and rye) and processed grains (malt, beer and bread). T-2-and HT-2 toxin often occurs together in infected cereals. Recently, a new Fusarium species, namely Fusarium langsethiae, was discovered as producing T-2 toxin [14].
2.1.4 Zearalenone ZEA (Figure 4) [CAS number: 17924-92-4] is a non-steroidal, phenolic resorcyclic acid lactone estrogenic toxin [15] produced mainly by Fusarium graminearum, Fusarium culmorum and Fusarium sacchari. ZEA is a white crystalline compound, which exhibits blue-green fluorescence when excited by long wavelength UV
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light (360 nm) and a more intense green fluorescence when excited with short wavelength UV light (260 nm). It is soluble in water, slightly soluble in hexane and progressively more soluble in benzene, acetonitrile, dichloromethane, methanol, ethanol and acetone. It is also soluble in aqueous alkali.
2.1.5 Fumonisins In 1988, Bezuidenhout [16] characterized the structures of FBs, a new group of mycotoxins that had been purified from cultures of Fusarium moniliforme. FBs are a family of 28 toxins. The mycotoxins are mainly produced by Fusarium verticillioides (Sacc.) Nirenberg (former F. moniliforme Sheldon) and Fusarium proliferatum. The most abundant and toxic is fumonisin B1 (FB1) [CAS number: 116355-83-0]. FB1 is chemically described as a diester of propane-1,2,3tricarboxylic acid and 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyeicosane [17,18]. The structure of FB1 is shown in Figure 5. Fumonisin B2 (FB2) [CAS number: 116355-84-1] is a deoxy-analogue of FB1 in which the corresponding epimeric units on the eicosane backbone have the same configuration. The full stereochemistry of fumonisins B3 (FB3) and B4 is unknown yet, but the amino terminal of FB3 has the same absolute configuration as that of FB1 [19,20]. FB1 typically accounts for 70–80% of the total FBs produced, while FB2 usually makes up 15–25% and FB3 usually makes up from 3% to 8% when cultured on corn or rice or in liquid medium [21]. From a structural point of view, FBs are correlated to the sphyngoid bases. The pure substance is a white hydroscopic powder that is soluble in water, acetonitrile–water or methanol. FBs are soluble in polar solvents because of the four free carboxyl groups, the hydroxyl groups and the amino group. Their insolubility in many organic solvents such as chloroform and hexane commonly used in mycotoxin analysis partly explains the difficulty
Figure 4 Molecular structure of ZEA.
COOH
HOOC
NH2 H3C
O OH OH
CH3 CH3
O
OH OH HOOC
HOOC
Figure 5
O
Molecular structure of FB1.
O
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OH
O O
Figure 6 Molecular structure of patulin.
in their original identification. Fumonisins B1 and B2 are reported to be stable over a 6-month period at 251C in acetonitrile–water (1:1).
2.1.6 Patulin Patulin (Figure 6) [CAS number: 149-29-1] was first isolated in the 1940s but is now known to occur worldwide in apple and apple products. Patulin is produced by different species of the genera Penicillium, Aspergillus and Byssochlamys moulds that may grow on a variety of foods including fruit, grains and cheese. Penicillium expansum is the most productive species. Patulin is reported to be destroyed by alcoholic fermentation and thus is not found in either alcoholic fruit beverages or vinegars produced from fruit juices [22,23]. Thermal processing appears to cause only moderate reductions in patulin levels, thus patulin present in apple juice will survive the pasteurization process [24–26]. The level of contamination is generally strictly related to the mouldiness and rot degree of food products even if the toxin is concentrated only in mouldy parts. Its presence in fruit juices, particularly apple juices, can be regarded as the main potential risk for humans, since rot or mouldy parts of the fruit could be employed in manufacturing of these products. From a chemical point of view it is a lactone. Patulin can be isolated as colourless to white crystals from ethereal extracts that have no optical activity. It melts at about 1101C. It is soluble in water, methanol, ethanol, acetone and ethyl or amyl acetate and less soluble in diethyl ether and benzene. It undergoes all the reactions expected from a secondary alcohol, reduces warm Fehlings solution and decolourizes potassium permanganate. It is stable in acid solutions but is susceptible to alkaline hydrolysis.
2.2 Minor mycotoxins 2.2.1 Ergot alkaloids The first documented epidemic of ergotism likely occurred in 944-945 AD, when some 20,000 people of the Aquitane region of France (about half of the population) died of the effects of ergot poisoning. Ergoline or ergot alkaloids were first isolated from ergot, a black tuber-like non-endophytic fungus that infects grains and causes the disease known as ergotism. More specifically, the ergot alkaloids are produced by several species of Claviceps, a genus of fungi that invades the female portion of the host plant and replaces the ovary with a mass of fungal tissue called sclerotium. The isolation by G. Barger and F.H. Carrin of ergotoxine, so named since it appeared to have more toxic effects than the
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therapeutic properties of ergot, was the starting point, followed by the isolation of ergotamine, the first ergot alkaloid used in therapeutic, in 1918 by A. Stoll. With the determination of the basic chemical structure of the ergot alkaloids in the early 1930s, an era of intensive exploration of synthetic derivatives began. The ergot alkaloids are indole compounds that are biosynthetically derived from L-tryptophan and represent the largest group of nitrogenous fungal metabolites found in nature. Over 80 different ergot alkaloids have been isolated, mainly from various Claviceps species (over 70 alkaloids), but also from other fungi and higher plants. In addition to the naturally occurring ergonovine (used as an oxytocic) and ergotamine (an analgesic used to control migraine), synthetic derivatives of continuing importance today are the oxytocic methergine, the antimigraine drugs dihydroergotamine and methysergide, the anti-senility nootropic (smart drug) Hyderginet and bromocriptine, used for numerous purposes including treatment of Parkinson’s disease. Newer synthetic ergolines used for Parkinson’s disease include pergolide and lisuride. Perhaps the most famous ergoline derivative of all is the psychedelic drug lysergide (LSD). To sum up, the ergot alkaloids comprise the largest group of nitrogen-containing fungal compounds and are generally divided into four major groups based on their chemical similarities: (1) the clavines (agroclavine, elymoclavine and lysergol), (2) the lysergic acids, (3) the lysergic acid amides (ergine, ergonovine, methergine, methysergide and LSD and (4) the ergopeptines (ergotamine, ergovaline, aergosine and a-ergocryptine) [27]. Selected members of these groups of compounds are involved in either nervous or gangrenous syndromes in humans and animals that consume grains or grain products contaminated with the sclerotia of the fungus.
2.2.2 Citrinin Citrinin [CAS number: 518-75-2] was first isolated as a pure compound from a culture of Penicillium citrinum in 1931 [28]. Later, yellowish coloured rice imported from Thailand to Japan in 1951 was found to be contaminated with P. citrinum and subsequent investigations showed that isolates of the fungus produced citrinin. Citrinin is a mycotoxin, which is produced by A. ochraceus, P. citrinum, and related species, which contaminate grain. It causes nephropathy in livestock and has been implicated as a cause of Balkan nephropathy in humans. It frequently co-occurs with OTA with which it showed a documented synergistic effect [29,30].
2.2.3 Cyclopiazonic acid Cyclopiazonic acid [CAS number: 18172-33-3] is mainly produced by Penicillium cyclopium and other fungi species including A. flavus. Because of this peculiar characteristic, it has the potential to co-occur with aflatoxins in a range of commodities. Cyclopiazonic acid is soluble in chloroform and dimethyl sulfoxide. It is a specific inhibitor of Ca2+-ATPase in the intracellular Ca2+ storage sites [31]. It is considered as a potent neurotoxin that may be fatal if swallowed or inhaled.
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However, cyclopiazonic acid only appears to be toxic when present in high concentrations.
2.2.4 Sterigmatocystin Sterigmatocystin [CAS number: 10048-13-2] is mainly produced by the fungi Aspergillus nidulans and Aspergillus versicolor. It has been reported in mouldy grain, green coffee beans and cheese, although information on its occurrence in food is limited. It seems to occur much less frequently than the aflatoxins, even if no validated analytical methods have been carried out at microgram per kilogram (mg/kg) levels. Sterigmatocystin contains a xanthone nucleus and is considered as a precursor of AFB1 in biological pathway. Although it is a potent liver carcinogen similar to AFB1, current knowledge suggests that it is nowhere near as widespread in its occurrence. A number of closely related compounds such o-methyl sterigmatocystin are known and some may also occur naturally.
2.2.5 Moniliformin Moniliformin [CAS number: 71376-34-6] was discovered by Cole et al. [32] in 1973, while screening for toxic products of a North American isolate of F. moniliforme Sheldon (F. verticillioides [Sacc.] Nirenberg) cultured on corn. More recent studies demonstrated that previous ones that ascribed moniliformin production to F. moniliforme, actually tested an aggregate consisting of more than one species. Moniliformin is formed in cereals by a number of Fusarium species that include F. avenaceum, F. subglutinans and F. proliferatum and it occurs as the sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione. It is an ionic compound forming sodium and potassium salts and is soluble in water and polar solvents. This mycotoxin has not yet received much attention because it does not appear to be carcinogenic and relatively high amounts appear to be necessary to cause significant toxicological effects.
2.2.6 Gliotoxin Gliotoxin [CAS number: 67-99-2] was discovered by Weindling and Emerson in 1936 [33] from culture filtrates of Gliocladium fimbriatum. It is a highly immunosuppressive toxin produced by a wide variety of widespread moulds. Interestingly, gliotoxin is produced by the organism Aspergillus fumigatus during its pathogenic state as a causative agent of the respiratory disease known as aspergillosis in turkeys. This compound belongs to a group of fungal metabolites, some of them toxic, called epipolythiodioxopiperazines. This toxin exhibits immunosuppressive activity against certain cells of the immune system. Intoxication of camels is reported due to the ingestion of this toxin and it seems to be involved in human infections caused by Candida albicans.
2.2.7 Citreoviridin Citreoviridin [CAS number: 77-92-9] a potent neurotoxin, originally was isolated from cultures of moulds obtained from rice associated with a disease called cardiac beriberi that has occurred for three centuries in Japan.
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Citreoviridin was obtained from Penicillium citreonigrum Biourge (synonyms: Penicillium citreoviride and Penicillium toxicarium), one of the so-called ‘‘yellowed rice’’ fungi [34] and subsequently from a number of other Penicillium species [35]. The toxin is also produced by P. ochrosalmoneum. Its structure was first discovered in 1964 by means of chemical degradation and 1 H NMR spectroscopy [36]. The fungus is favoured by the lower temperatures and shorter hours of daylight occurring in the more temperate rice-growing areas. Citreoviridin has been found in un-harvested corn in the United States. Citreoviridin is an unusual molecule consisting of a lactone ring conjugated to a furan ring, with a molecular weight of 402. Citreoviridin and aflatoxin have been found to occur simultaneously in maize, which indicates their possible interaction in producing animal disease [37]. Citreoviridin causes paralysis, dyspnea, cardiovascular disturbances and loss of eyesight in experimental animals [38]. Citreoviridin is structurally closely related to aurovertin B and to asteltoxin [39].
2.2.8 Tremorgenic mycotoxins These toxins are produced by a wide spectrum of fungi belonging to the genera Penicillium, Aspergillus, Claviceps and Acremonium [40]. On the basis of chemical similarity, at least 20 mycotoxins have been identified as tremorgens (compounds capable of inducing serious muscle tremor in one or more vertebrates). These toxins can be separated into four groups: the penitrems, the fumitremorgen verruculogen group, the paspalines and the tryptoquivaline group. Penicillium species produce the most tremorgenic mycotoxins [41]. The penitrems, produced by Penicillium species such as Penicillium crustosum, have been the most widely studied in laboratory animals [42] and in livestock [43]. Penitrem A [CAS number: 12627-35-9] has become recognised and used pharmacologically as a selective blocker of high conductance calcium-activated potassium channels [44–48]. Intoxication with these mycotoxins causing trembling syndromes has been documented in many animals, including dogs, cattle, sheep, rabbits, poultry and rodents [49,50]. Clinical signs include muscle tremor, uncoordinated movements, general weakness in the hind legs, and stiff, stilted movements of the forelegs. Other intoxications involving fungal tremorgens, most notably the penitrem mycotoxins, have been reported from mouldy cream cheese, hamburger buns (bread) and walnuts consumed by dogs [51]. Other noteworthy tremorgenic mycotoxins are lolitrem A–F. Lolitrem B [CAS number: 81771-19-9] is a potent neurotoxic indole-diterpen, generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass and responsible for ryegrass staggers in livestock [52]. Paxilline [CAS number: 57186-25-1] produced both by Penicillium species of fungi [40] and Acremonium lolii [53] also blocks high conductance
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calcium-activated potassium channels [54] and is thought to be a precursor in the biosynthesis of lolitrem B by A. lolii [55,56].
2.2.9 Penicillic acid Penicillic acid [CAS number: 90-65-3] is a polyketide mycotoxin produced by several species of Aspergillus and Penicillium with particular emphasis on P. cyclopium and P. canescens. This mycotoxin is toxic in experimental animals and has been reported to be hepatocarcinogen in some animal species, and has also been reported to affect the heart.
2.2.10 Roquefortine Roquefortines A [CAS number: 58735-64-1], B and C are neurotoxic metabolites of Penicillium roqueforti [57]. P. roqueforti is also responsible for the so-called Penicillium roqueforti (PR) toxin, one of the most potent mycotoxins, that being unstable and rapidly perishable, does not pose a health problem. A study reported that roquefortine is also produced by cultures of Penicillium commune in combination with penitrem A [58].
2.2.11 3-Nitropropionic acid 3-Nitropropionic acid [CAS number: 504-88-1] is a known neurotoxic secondary metabolite produced by many species of Astragalus of the Leguminosae family. In 1992, Liu et al. [59] reported that some species of Arthrinium were the etiological fungi of mouldy sugarcane poisoning (MSP) occurring in China. The toxic metabolite 3-nitropropionic acid (NPA) produced by Arthrinium was the main causative agent. NPA is a plant fungal toxin, which inactivates mitochondrial succinate dehydrogenase [60].
2.2.12 Fusaproliferin Fusaproliferin is a toxic sesterterpene isolated from F. proliferatum, a widespread pathogen of cereals. In 1995, researchers in Italy reported a previously unknown toxin, fusaproliferin, produced by F. proliferatum and F. subglutinans [61–63]. It is a sesquiterterpene compound that is toxic to brine shrimp (Artemia salina), insects and human cell cultures in laboratory assays. It also has teratogenic effects on chicken embryos [64]. Fusaproliferin has been detected in livestock feed samples associated with feed refusal [65], but there is no direct evidence that it causes feed refusal symptoms. No mammalian feeding studies have been conducted to date. Although the concern of this compound in animal and human health has not been to be determined, its acute toxicity to brine shrimp exceeds that of more familiar compounds such as the FBs.
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3. HEALTH EFFECTS The impact on human health derived from the interaction between mycotoxins and human biosystems can range from acute to chronic effects and can derive from various sources of exposure namely dermal contact, inhalation and consumption of contaminated food, the latter being the more relevant and investigated route. As for most xenobiotics, the effects of mycotoxins are dependent on the dose, length of the exposure and possibly interaction with other toxic or non-toxic agents. For mycotoxins, as well as for other toxic agents, the systematic evaluation of the impact on human health is performed through the risk-assessment process that consists of the hazard identification, implying the toxicological and epidemiological assessment, the hazard characterization, involving the assessment of the dose response and the extrapolation to humans, the exposure assessment and the risk characterization. This latter step relies on the previous evaluations and as defined by the EC Scientific Steering Committee in 2000 [66] allows for ‘‘the quantitative or semiquantitative estimation, including attendant uncertainties, of the probability of occurrence and severity of adverse effect(s)/ event(s) in a given population’’. Basically risk characterization correlates the outputs of the hazard characterization with different levels of exposure and it should represent the basis for decision-making processes including the issue of defining maximum tolerable limits in foodstuff. The topic for mycotoxins has been thoroughly and extensively reviewed by Kuiper-Goodman in 2004 [67].
3.1 Cases of acute mycotoxicosis in humans Over past centuries the presence of mycotoxin, mainly aflatoxin and trichothecenes in food, has been reported to recurrently cause outbreak of acute intoxication even resulting in death. The most recent was reported in 2004 in eastern Kenya, where, due to aflatoxin contamination of home-grown maize, 317 cases of acute hepatic failure were identified with 125 cases occurring in persons who subsequently died. The case-control study conducted by AzzizBaumgartner et al. in 2005 [68] proved that aflatoxin concentration in maize, serum AFB1–lysine adducts concentrations and positive hepatitis B surface antigen titres were all associated with the case status. In addition, it was found that male mortality due to aflatoxicosis was higher than female mortality, similarly to that found for the 1974 outbreak of aflatoxicosis in India [69]. From an outbreak of hepatitis associated with consumption of corn contaminated with aflatoxins, over 100 deaths occurred in India in 1976, the ingested dose causative of the event at that time estimated by local authorities was 2–6 mg/day over a month [70]. Among other relatively recent cases of human aflatoxicosis, the death of 13 Chinese children due to acute hepatic encephalopathy has been reported by Lye et al. [71]. The above researches reported the acute toxicity in family clusters
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probably due not only to common food eaten, but also to genetic polymorphism of cytocrome P450 enzymes [72]. Early symptoms of hepatoxicity from acute aflatoxicosis include anorexia, malaise and low-grade fever possibly turning into vomiting, abdominal pain, jaundice, hepatic failure and death. Fatty degenerative changes in the liver as centrolobular necrosis and fatty infiltration have also been reported [3]. Several outbreaks attributable to trichothecenes-contaminated food have been reported. In addition to the historical (1931–47) outbreaks of alimentary toxic aleukia (ATA) in the former USSR, several epidemic incidents attributable to trichothecenes have been recently reported in India, China and Japan [73–75]. Trichothecenes are often regarded as radiomimetic toxins since some related effects such as the effect on bone marrow are similar to those seen in radiation poisoning. T-2 and DAS were placed by the Centers for Disease Control and Prevention of USA on the ‘‘Selected Agents List’’ in relation to agents of biowarfare or terrorism [76].
3.2 Effects of low doses of mycotoxins By considering the overall low frequency of outbreaks of acute intoxication and the worldwide reported high rate of occurrence of mycotoxins at low concentration in food, it can be concluded that it is likely that the population is worldwide generally exposed to low doses of mycotoxins over long periods and consequently that most of the toxic effects exerted by mycotoxins are chronic. According to Kuiper-Goodman [67] and as far as chronic disease is concerned, the risk attributable to the presence of mycotoxins in food should be considered much more severe than other sources of risk such as anthropogenic contaminants, food additives and microbiological agents. This has prompted the most relevant international organizations such as WHO, IARC, ILSI, JECFA, SCF, EFSA to issue the topic of mycotoxin health impact, a considerable number of detailed reports being now available under the auspices of the above organizations. The wide variety of chemical compounds grouped under the common term of mycotoxins makes them responsible for a broad spectrum of chronic toxic effects and they range from the well-documented carcinogenic to the less-investigated immunosuppressive action. The most important and studied effects are genotoxicity and carcinogenicity. From the viewpoint of hazard characterization, genotoxic, carcinogenic and noncarcinogenic mycotoxins are considered separately. In this respect, a crucial and often controversial point is the appropriateness of the definition of a threshold dose based on the mechanism of action of the toxin. By definition, genotoxic compounds have a probability of inducing an effect at any small dose and their absence from food is strongly recommended. No threshold dose should, therefore, be established for genotoxic mycotoxins. Among mycotoxins, genotoxicity is so far fully established only for AFB1 and is still matter of debate for OTA. Due to its genotoxicity no safe dose can be
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postulated for AFB1 and the principle of ALARA (as low as reasonably achievable) is applied at European level in Regulation (CE) 1881/2006 [77]. Recently, it was however recognized that the principle of ALARA could be misleading since it does not provide the risk managers with the necessary basis for priorities; in addition, the approach of deriving the ‘‘safe dose’’ through extrapolation methods has been criticized in favour of approaches aimed at the establishment of a ‘‘margin of exposure’’. Therefore, the Scientific Committee of the EFSA recently put forward an opinion addressing the risk assessment of substances that are both genotoxic and carcinogen [78]. It is desirable that the case of AFB1 could be re-evaluated under this new perspective. As for carcinogenic mycotoxins, the IARC evaluated in 1993 the available studies on OTA, fusarin C, ZEA, DON, NIV, fusarenone X, T-2 [79] and in 1993 and again in 2002 the available studies for aflatoxins and fumonisin B1 [80]. The outputs of the evaluations were the following: AFB1 was assigned in Group 1 (with sufficient evidence of carcinogenicity in humans resulting in the evaluation of carcinogen to humans); AFM1, OTA and F. moniliforme (including FB1) toxins in group 2B (insufficient evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals resulting in the evaluation of possibly carcinogenic to humans), ZEA and patulin in group 3 (for ZEA limited evidence in animals and for patulin negative evidence in humans and insufficient evidence for animals). A threshold dose should theoretically not be defined for carcinogenic DNAreactive mycotoxins, but only for mycotoxins with an indirect carcinogenic mechanism and for non-carcinogenic mycotoxins. For the two latter cases a ‘‘safe dose’’ is estimated usually as provisionally tolerable daily intake (PTDI) or acute reference dose (RfD), the latter being less used in mycotoxin evaluation since it is more appropriate in cases of high short-term dose or single exposure. The TDIs for the main mycotoxins have been obtained and reported in Table 1. For some mycotoxins the risk assessment process has been performed by various organizations under diverse toxicological basis and extrapolation methods, therefore resulting in different values for the proposed TDI or in other ‘‘safe dose unit measurements’’ such as provisional tolerable weekly intake (PTWI) as in the case of OTA. For this toxin a TWI was recently put forward by EFSA [81]. In addition, it is questionable if the impact on human health attributable to the combined toxic effect of mycotoxins could be higher than the impact attributable to the mycotoxins alone. This consideration is obviously valid for other classes of toxic compound, but for mycotoxins it could imply profound consequences due to the largely proven coexistence of several mycotoxins in food commodities. The issue was recently addressed by Speijers and Speijers [82] with particular reference to trichothecenes. The toxicology of mycotoxins can be reviewed both through their impact on biological systems/organs as reviewed by Richard [83] and Fung et al. [84] and by considering the toxic effects of the single mycotoxins. Most of the toxicological evaluation is based on studies/observations on experimental/farm animals, the epidemiology of toxic effect of mycotoxins is still scarcely developed, although
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TDI/TWI for the major mycotoxins
Mycotoxin
TDI (ng/kg bw per day)
Organization
OTA
4 5 5 14 120 (TWI) 2,000 2,000 400 3,000 1,000 1,000 1,000 1,000 100 100 500 200 400 400 400
Health Canada (1989, 1996) Nordic Council (1991) EU (1998) JECFA (1996, 2001) EFSA (2006 [81]) EU (2000) JECFA (2000) Health Canada (2001) Health Canada (1985) Health Canada (2001) Nordic Council (1998) EU (1999) JECFA (2000) Health Canada (1987) Nordic Council (1998) JECFA (2000) EU (2000) JECFA (1996) EU (2000) Health Canada (1996)
FBs
DON
ZEA
Patulin
Source: Adapted from Ref. [67].
some results are available for some toxins. The JECFA evaluated the potential risks for aflatoxins and OTA in 1987, 1997 and 1999 [85–88]. In the last report the evaluation for fumonisins B1, B2 and B3, DON and T-2 and HT-2 toxins have also been performed. The herein reported information are related to the major mycotoxins most of them being derived from those sources. For OTA, the reference report has been the EFSA opinion [81]. Information on the toxic effect of mycotoxins is given in the Table 2.
3.3 Quantitative evaluation of human exposure to mycotoxins In the frame of soundly evaluating the impact on human health due to mycotoxins, as usually performed in risk characterization, both the level of exposure of population and the sensitivity of particular groups shall be taken into account. For threshold mycotoxins, risk characterization matches the level of daily exposure lifetime with the TDI of the toxins. Whenever data are insufficient to obtain a TDI or for non-threshold mycotoxins, the exposure can usefully be compared with the NOEL (no observed adverse effect level) or the LOAEL (lowest observed adverse effect level) of the various toxins [67]. Therefore, uncertainties in risk characterization of mycotoxins reflect both hazard
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The toxic effect of mycotoxins
Mycotoxin
Toxic effect
References
Aflatoxins
In animals: hepatoxicity and hepatocellular carcinoma. Aflatoxins intake seems to be able to reduce the resistance to bacterial, fungal and parasitic infections, suppressing the cell-mediated immune response In humans: the chronic toxicity of AFB1 is usually linked to hepatocellular carcinoma (studies performed before 1980 in population from Thailand, Kenya, Mozambique and Swaziland). AFB1 is considered the most potent carcinogen of the aflatoxins The carcinogenicity of AFB1 is higher in people carrier of hepatitis B virus In animals: carcinogenic to rodents; nephrotoxic, neurotoxic, teratogenic and immunosuppressive activity Genotoxicity is in debate. In humans: BEN and the development of urinary tract tumours are supported by epidemiological data Kidney is the target organ for OTA in all mammalian species, with the exception of mature ruminants, with wide differences reported within species and sex EFSA reported the recent toxicological data indicating that the site-specific renal toxicity as well as the DNA damage and genotoxic effects of OTA are most likely attributable to cellular oxidative damage In animals: affects the reproductive system of animals causing vulvovaginitis and estrogenic responses in swine In humans: suspected to be, together with its metabolites, an aetiological agent for premature thelarche, pubarche and breast enlargement in young children or fetuses exposed to this estrogenic compound In animals: rodents, non-human primates and other animal species fed with FB1-contaminated diets demonstrated the toxic effect in the liver and kidney. In horses and other equids, equine leukoencephalomalacia (ELEM) In humans: the consumption of FBs contaminated corn has been correlated with oesophageal cancer in regions of South Africa and China even if it does not subsist yet a confirmation of a direct implication
[89,90]
OTA
ZEA
FBs
[3]
[78]
[91,92]
[93,94]
[95,96]
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Table 2 (Continued ) Mycotoxin
Patulin
Trichothecenes
Toxic effect
References
The consumption of contaminated corn was also considered as a cofactor in the high incidence of liver cancer in China Risk factor for neural tube defects in South Africa, China and in Southwestern USA Embryotoxicity accompanied by maternal toxicity and no reproductive or teratogenic effects Cytotoxicity and of some of the genotoxic and immunotoxic effects arose from the reaction of the patulin with the sulfhydryl group Antibiotic properties In animals: symptoms of acute/subacute toxicity of DON include vomiting, feed refusal, weight loss and diarrhea. Emesis and anorexia are mediated by the serotonergic system in the central nervous system (CNS) or via peripheral actions on serotonin receptors. In humans: DON, NIV, T-2 and HT-2 are considered the most important for human health although the paucity of available occurrence data on the last three toxins does not allow the EU Commission to consider the possibility of imposing maximum toleration for their presence in food
[97]
[94] [98] [82]
[99] [100]
[101]
Note: BEN, Balkan endemic nephropathy.
identification and exposure-related uncertainties. An opinion related to the uncertainties in dietary exposure assessment has been recently issued by the EFSA [102]. Exposure assessment for the more relevant mycotoxins has been carried out in Europe for many years. The mycotoxins so far considered in the framework of the Scientific Cooperation on Questions relating to Food (SCOOP) project tasks were aflatoxins in 1995 [103], OTA in 1996 and 2002 [104,105], patulin in 2003 [106] and Fusarium toxins also in 2003 [107]. The evaluations have been performed with a point estimate approach on the basis of the occurrence and consumption data available at the time of the exercise. In conclusion for OTA, FBs and patulin seem to be rather reassuring since the intakes of the toxins as derived from the SCOOP reports are generally below the TDIs. Any sound conclusion could be derived from the other Fusarium toxins, since the available occurrence data were not sufficient for deriving scientifically based intake values. However, some critical points and limitations of the SCOOP exposure assessments need to be highlighted.
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Among the factors implying the underestimation of the intake can be mentioned: (i) the accuracy of the sampling methodologies employed in the less recent occurrence data, (ii) the lack of data on mycotoxins occurrence for all the food matrices susceptible to contamination; as a consequence, in the total intake data the contribution of a certain number of food matrices were missing. This could be particularly relevant for OTA that is known to be eventually present in a large number of food commodities. Additional factors leading to underestimation/overestimation of the intake data from the SCOOP tasks are the evaluation of the data under the limit of detection (LOD) in the final estimate, the effect of technological procedures and the questionable compatibility of the food categorization systems between occurrence and consumption data. The intake of mycotoxins has also been evaluated in some countries by adopting methodologies other than the point estimate through occurrence and consumption data. Among them, the total diet approach has been recently followed by several countries, including France and Italy. In the French study [108], the intakes of OTA, aflatoxins including AFM1, patulin and trichothecens, FBs and ZEA have been evaluated. The intake results were as follows: for OTA the estimated average intake is 2.2 ng/kg bw/day for normo-reporters adults, 4.1 ng/kg bw/day for children (aged 3–14 years), while the 95th percentile exposure is 3.6 ng/kg bw/day and 7.8 ng/kg bw/day for the above groups of population. For vegetarian populations, the average intake was between 2.2 and 3.7 ng/kg bw/day depending on the group studied (ovolattovegetarian, lattovegetarian and vegan/macrobiotic) and the respective 95th percentiles values are 3.7 ng/kg bw/day for the first two groups and 8.5 ng/kg b.w./day for the third. The main food contributors to the intake were cereals and cereal products (70%), while the many other vectors (mainly grape-derived products, coffee, nuts and oilseed) contributed for less than 5% each). For vectors contributing similar results were found in the SCOOP task for OTA. However, the total diet methodology is more complete in terms of comprehensiveness of all the susceptible matrices. For aflatoxins the proportion of population exceeding the threshold of 1 ng/kg bw/day suggested by SCF/JECFA is in the range 0.01–3.4% for adults and children (3–14 years), and in the range 2.6–23% for the various classes of vegetarians. In the same study and for population other than vegetarians the intake of AFM1, patulin, trichothecens and FBs is very low as compared with the provisional daily intake proposed by JEFCA or SCF. Vegetarians resulted in more exposure, a proportion of them ranging from 3.8% (for DON and NIV) to 31% for ZEA exceeding the TDIs proposed by JEFCA or SCF. The explanation for this latter finding relies on the high percentage of susceptible cereals in the diet of this group of population. The Italian total diet study [109] on the exposure of Italian population to FB1 concluded that the intake was below the TDI value (2 mg/kg bw/day) suggested by the EU Scientific Committee for food, however being higher in the North West of Italy during winter.
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A more modern methodology for the evaluation of risk characterization of mycotoxins is under development under the umbrella of the SAFEFOODS project (http://www.safefoods.nl) and is based on the probabilistic evaluation of both the hazard and exposure characterization.
3.4 Tolerable maximum limit for mycotoxins in food During the last few decades the knowledge of the health effect of mycotoxins in humans and animals has prompted many countries to establish maximum tolerable levels in food and feed. A recent enquiry from FAO [110] reviewed the topic. The main factors driving the establishment of maximum limits are the consideration of health effects and the economic interest of producers and traders. Other factors influencing decisions are the availability of information on the occurrence and exposure as well as that of sampling and analysis methods. According to the FAO report, up to 2003, at least 99 countries had mycotoxin regulation in a large variety of food and feed. The total population covered by mycotoxins legislation represents the 90% of the world’s inhabitants. Limits have been established for aflatoxins, OTA, patulin, ZEA and Fusarium toxins in most of the regulated countries. Depending on local needs minor mycotoxins are regulated such as agaric acid and phomopsin in Australia and New Zealand. Still a quite large range of limits can be observed among regulated countries: AFB1 (1–20 mg/kg in 56 countries), total aflatoxins (0–35 mg/kg in 75 countries), OTA (2.5–50 mg/kg in 37 countries), DON (300–2,000 mg/kg in 37 countries), ZEA (50–1,000 mg/kg in 16 countries), FBs (1,000–3,000 mg/kg in 6 countries), patulin (5–100 mg/kg in 33 countries). Europe has the most complete and detailed mycotoxin regulation, providing limits for a large variety of mycotoxins and foodstuff in also establishing limits in baby foods and food for medical purpose. Mycotoxin regulation in Europe is still evolving and Regulation 1881 of 2006 [77], setting maximum levels for certain contaminants in foodstuffs, provides a collection of all the limits established so far.
4. SAMPLING To make available reliable samples consists of (i) the decision of ‘‘why, where and when’’ to take samples, in other words, the process of statistically individuating the populations (sites) from which food samples should be taken; (ii) the process of ‘‘how’’ to take representative samples from the lot under investigation. While the first point has features common to other analytes, the second one presents almost unique characteristics for mycotoxins since only a few units (approximately 0.1% for a wide variety of agricultural commodities) are likely to be highly contaminated, resulting in an overall significant contamination of the lot [111,112]. Due to this heterogeneous distribution, the mycotoxin concentration in a bulk can be assumed the
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same as in a small sampled and analysed portion of the lot, only if an appropriate sampling plan is applied. The topic has been recently reviewed by Miraglia et al. [113]. Without the implementation of a good sampling plan a misclassification of the lot can easily occur, implying negative impact on the ultimate scope of the sampling: for monitoring/surveillance purposes a poorly developed sampling plan will result in false information on the level of contamination of the lot (usually false negative). In turn, a wrong contribution to the monitoring/ surveillance database to be used by risk assessors/managers will be given. For quality assurance purposes, incorrect sampling can likely imply undesirable economic and trade impact. Among the steps usually employed in the evaluation of the mycotoxin level in a lot (sampling, sample preparation and analysis), sampling step is by far the largest contributor to the total error, the associated variability being largely dependent on the level of the toxin. Since 1993, it was recognized that a sampling plan is a function of the employed testing procedures and of the acceptance/ rejection limit; the limit can or cannot be the legal depending on the ultimate scope of the sampling [114]. The variability associated with the sample preparation has so far been described for aflatoxin in several crops, for FBs in corn and for DON in wheat. In all cases, for a given size of sub-sample a decrease of the variance with a decrease of the particle size has been demonstrated. The slurry procedure can contribute to reduce the sample preparation variability with respect to the dry grinding [115]. Due to the variance associated with each step of the mycotoxin evaluation, a 100% level of certainty is far from being achievable; an overestimation leads to a risk for the seller/producer that a good lot could be wrongly rejected, while an underestimation can result in the buyer/ consumer risk of acceptance of a ‘‘bad’’ lot that, when processed, will produce food or feed not acceptable from the health or the trade point of view. The aim of any good-sampling plan is to reduce the above risks. Currently the most complete legislative reference for the issue has been given by the EU that since 2001 put in force a package of directives concerning sampling procedures for the most prominent mycotoxins, namely aflatoxins, patulin, Fusarium toxins and OTA. Recently the Commission Regulation 401/2006 [116] gives provisions for the sampling of the regulated mycotoxins in food (cereals and derived products, dried fruits, groundnuts, nuts, spices, milk and derived products, coffee and derived products, fruit juice and solid apple products). The basic principle underlying the provided sampling plan is the sampling of an usually high number of incremental samples evenly distributed in the lot, the gathering of the collected incremental samples in a number of aggregate (generally one) samples, the grinding or slurring of the aggregate(s) samples and the deriving of the laboratory samples from the aggregate sample(s). The number and weight of the incremental samples and the aggregate sample(s) are dependent from the matrix, and from the size of the lot under consideration.
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5. ANALYTICAL METHODS 5.1 Introduction Notwithstanding the relevance of the sampling step in the mycotoxin evaluation, the quali/quantitative analysis represents the core of the whole process, huge efforts have been devoted in previous decades to the optimization of this step. Actually, before the implementation of the practical analysis, the analyst has to face many decisions, the first one being the selection of the fit-for-purpose method. Monitoring, exposure assessment, official control issue, quality control at production and research are all activities that require appropriate methods, the parameters to be weighed as the required limit of detection/quantification, achievable costs, required time of analysis, number of samples/time and need of rapid answer. In this section an overview of the methods for mycotoxin analysis is given. The classification of methods can be performed on different bases. With respect to the reliability and accountability of the results, methods can be:
Official Reference Confirmatory Screening.
Classification can be also usefully done according to the type of the chemical/ biological/physical principle used and the time of analysis: Chromatographic methods (HPLC, gas chromatography (GC), thin layer chromatography (TLC), liquid chromatography tandem mass spectrometry (LC-MS/MS)) Rapid methods (enzyme-linked immunosorbent assay (ELISA), lateral flow device (LFD), dip-sticks) Alternative methods (Biosensor, near- and mid-infrared spectroscopy (NIR/ MIR), capillary electrophoresis (CE), fluorescence polarization immunoassay (FPIA)). As a general rule, each laboratory involved in a loop leading to a final measurement of an analyte, such as mycotoxins, should address any effort to reach a high degree of analytical quality in terms of reliability of its measurements. More and more stringent is this need if applied to laboratories involved in official control activities. To stress this simple assumption, it should be reminded that the European legislation laid down in 2000s rendered mandatory, for laboratories, the existence of a quality assurance/control system and the requirement of being accredited. Some of the key elements to be satisfied for accreditation purposes are the use of validated methods (from inter or singlelaboratory trials), the participation to proficiency testing, the use of (certified) reference materials and the adequate training of the personnel.
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5.2 Sample preparation Mostly, the typical steps in mycotoxin analysis are represented by the extraction, clean-up and detection. In a few cases extraction and/or clean-up are not necessary.
5.2.1 Extraction step The very first step for food and feed analysis is usually represented by the extraction of mycotoxin from the matrix. The conventional procedures are liquid/ liquid extraction, employing a mixture of aqueous organic solvents depending on the mycotoxins (Table 3), and performed by stirring or by homogenization. Non-conventional extraction includes ultrasonic system, microwaves, accelerated solvent extraction (ASEs, Dionex Corporation, 1228 Titan Way, Sunnyvale, CA 94085, USA) [117–119], and supercritical fluid extraction (SFE). The ASE is an automated extraction technique that aims to reduce time and organic solvent volume required to perform traditional sample preparation. ASE uses a combination of elevated pressure and temperature with common liquid solvents to increase the efficiency of the extraction process. Supercritical fluids can be used to extract analytes from samples [120]. The main advantages of using supercritical fluids for extraction is that they are inexpensive, contaminant-free and less costly to dispose of safely with respect to organic solvents.
5.2.2 Clean-up step The main clean-up procedures are liquid/liquid partition, solid phase extraction (SPE) and multifunctional solid phase extraction (Mycoseps SPE). Polar, nonpolar and ion exchange-based SPE are commercially available. Mycosep cartridge is packed with different adsorbent material with the aim to retain particular groups of compounds that may create interferences in analytical methods. Multifunctional SPE columns allow mycotoxins to pass through aiming to remove the interferences from the sample. A number of analytical methods for multimycotoxin analysis include the use of SPE also for minor mycotoxins analysis [121–123]. The use of multimycotoxin cartridge OASIS HLB (Waters) [124], allows the simultaneous determination of DON, NIV, HT-2 and T-2 toxins. Table 3
Solvent for liquid/liquid extraction of mycotoxin analysis
Mycotoxin
Solvents
Aflatoxins OTA ZEA Trichothecenes FBs
Methanol/water, acetonitrile/water, acetone/water Methanol/NaHCO3 3%, acetonitrile/water Acetonitrile/water, methanol, acetonitrile Acetonitrile/water, water Methanol/water, acetonitrile/water, acetonitrile/methanol/ water Ethyl acetate
Patulin
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The immunoaffinity columns (IAC), containing specific antibodies, have the purpose to specifically bind the mycotoxin as first step, followed by mycotoxin elution because of the antibody denaturation. IAC improvements are represented by the availability of multifunctional columns for the simultaneous determination of different mycotoxins and by the setting up of reusable IACs. The multimycotoxins now available are for aflatoxins and OTA and for aflatoxins/ OTA/ZEA [125]; new IAC columns for aflatoxins/FBs/OTA/ZEA/DON and aflatoxins /FBs /OTA/ZEA/DON/T-2 will be finalized in the year 2008. As for the advantage/disadvantage evaluation, SPE is rapid and offers the possibility of a multimycotoxins analysis; however, it is not really selective. The IAC is a good fit for the mycotoxin analysis in complex matrices as food and feed, since it is highly selective and allows high accuracy and sensitivity. The major disadvantage of IAC is represented by the fact that columns can only be used once and their costs are high. SPE and IAC are commercially available for almost all the major mycotoxins. The molecularly imprinted polymers (MIPs) are artificially synthesized polymeric matrices, highly stable, that show the capacity to recognize and specifically interact with particular substances. Polymeric stationary phases obtained by the technique of molecular imprinting, are valid alternatives to the immunoaffinity phases in terms of limited costs, column stability and reproducibility [126–128]. An MIP which recognized OTA, was prepared in a co-polymerizing reaction where a basic functional monomer, a cross-linking agent and a template miming OTA (a chloro-naphthoylamide) [129,130], makes a three-dimensional polymer structure with a high-binding capacity capable of recognizing the mycotoxin from various biologically relevant matrices. The molecular imprinted molecules have demonstrated the potential as a technique for the separation of analytes with a high degree of selectivity both for clean-up as an SPE adsorbent [131] and for packaging HPLC columns with polymer particles as in the case of molecularly imprinted molecules for DON and ZEA [132].
5.3 Determination and detection 5.3.1 Chromatographic methods 5.3.1.1 High-performance liquid chromatography. A wide variety of methods for most mycotoxins are based on HPLC, which is the methodology of choice for aflatoxins, OTA, ZEA, FBs and patulin. The HPLC separation is followed by fluorescence detection for aflatoxins, OTA, ZEA and FBs, usually as derivative compounds. UV detection is used for DON and patulin determination. Advantages of HPLC determination include excellent performance characteristics, low detection levels (as low as 0.1 ng/g) and safety for the operator. For aflatoxins, a large number of HPLC-based methods are available. The AOAC method 49.2.19A includes a Mycoseps clean-up procedure, followed by fluorescence detection of the trifluoroacetic acid (TFA) derivative, for aflatoxins in corn, almonds, Brazil nuts, peanuts and pistachio nuts [133]. The multifunctional column contains a mixture of reversed-phase, ion exclusion and ion exchange
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adsorbents. Packing retains interferences such as fats, proteinaceous compounds, pigment and carbohydrates extracted from the matrices. Methods including IAC and post-column derivatization with pyridinium bromide perbromide (PBPB) are reported for the determination of AFB1 and total aflatoxins in peanut butter, pistachio paste, fig paste and paprika powder [134], and for AFB1 determination in baby food (infant formula) [135]. Other derivatization reactions for aflatoxin analysis include post-column derivatization with iodine [136], electrochemically generated bromide (Kobra Cell) [137,138], photolytic derivatization [139] and pre-column derivatization with TFA [140]. HPLC methods, without any derivatization step, are also reported for the determination of AFM1 in milk and cheese [141,142], since this aflatoxin shows native fluorescence that allows this simplification in the analytical procedure. Methods for OTA determination by HPLC, mainly based on fluorescence detection, are available for different matrices. Official methods include OTA determination in barley, roasted coffee [143], baby food, and wine. Since OTA shows native fluorescence usually no derivatization step is required for the detection of this toxin. For confirmation purposes three methods are usually adopted, based on methylation [144,145], ammonia-derivative formation [146] or direct determination by LC-MS [147,148]. Notwithstanding, trichothecenes are frequently analysed by GC, HPLC methods are also available, such as that developed by Trucksess et al. [149] for DON in wheat-finished products. The purification step is performed by passing the extract through an SPE cartridge, and the HPLC separation is followed by UV determination. The LC determination of ZEA and a-zearalenol (a-ZOL) in corn was collaboratively studied by Bennett et al. [150]. The determination of ZEA and a-ZOL using UV or fluorescence detection has been adopted as an official method by AOAC [126]. The extraction is based on liquid/liquid partition between CHCl3 and water with the aid of diatomaceous earth. Other methods based on HPLC and fluorescence detection are reported by Tanaka et al. [151] and Seidel et al. [152]. FB analysis is usually performed by reversed-phase LC with fluorometric detection after pre-column derivatization with o-phtaldialdehide (OPA)/ 2-mercaptoethanol [153,154]. An AOAC-IUPAC collaborative study develops an LC method, based on the previous use of SPE cartridges for the determination of fumonisins B1, B2 and B3. This method has been adopted by AOAC International as an official first action method for corn analysis [133]; however, the method could not be successfully applied for the analysis of corn-based food products such as corn bran flour, corn bran breakfast cereals, mixed baby cereals and cornflakes due to low recoveries and inadequate clean-up. The use of IACs for the clean-up step has been reported to produce higher FB recovery [155]. LC with fluorescence detection is still the method of choice for the determination of FBs [153,154]. An AOAC-IUPAC collaborative study developed an LC method, based on the use of SPE cartridges and OPA as pre-column derivative agent for the determination of FB1, FB2 and FB3. This method was accepted as an official method of analysis [133]. A collaborative study was also
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preformed using IAC clean-up and pre-column derivatization with OPA. The method is suitable for the determination of FB1 and FB2 [156]. The use of SAX (strong anion exchange)+C18 in the clean-up step for FBs analysis was presented by Moller and Gustavsson [157]. Also for patulin, HPLC determination is preferred. Since there is no IAC for patulin, the sample preparation step is different. According to the method reported by Mac Donald [158], the sample is extracted with ethyl acetate, employing the enzyme pectinase for ameliorating the extraction performance on fruit matrix, dried under a stream of nitrogen, redissolved in mobile phase and determined with HPLC. Patulin detection and quantification is then performed with UV detection (276 nm). A combination of Extrelut and Florisil cartridges for the patulin determination in apple cider [159] by LC method is used for extraction and clean-up steps. The use of SPE was also reported by Trucksess [160]. Minor mycotoxins analysis is also performed by HPLC, methods with fluorescence detection are reported for citrinin determination [161,162]. A method for the determination of cyclopiazonic acid in corn and rice by HPLC and UV with a photodiode array detector is reported [163]. The determination of cyclopiazonic acid in cheese is also performed by HPLC anticipated by an SPE clean-up step [123]. A wide number of HPLC methods for the analysis of various mycotoxins, tested by collaborative studies to evaluate their effectiveness, are periodically reported on Methods Committee Reports published in Journal of AOAC International. Validated methods for mycotoxins determination are also collected by CEN within WG5 Biotoxins of TC275. An overview of the official methods available for mycotoxins analysis is reported in Table 4. Mycotoxins HPLC chromatograms are shown in Figures 7 and 8.
5.3.1.2 Gas chromatography. Since trichothecenes do not strongly absorb in the UV-Visible range and are non-fluorescent, GC-based methods represent the most widely employed technique for their determination. In particular, the analytical techniques employing GC and capillary column are useful for the simultaneous determination of different trichothecenes. For the detection step heptafluorobutyryl (HFB), trimethylsilyl (TMS) and trifluoroacetyl derivatives are frequently used coupled with electron capture detection (ECD) [164,165]. However, MS is strongly recommended for peaks confirmation. A GC method for trichothecenes determination in barley and malt [166] has been validated and accepted by the American Society of Brewing Chemists in its Methods of Analysis, 8th ed. A method, for trichothecenes determination in barley and malt, has also been developed by supercritical fluid chromatography (SFC) [167]. Supercritical fluids can be used as the mobile phase to separate analytes with either a packed or capillary GC column. Compared with GC, capillary SFC can provide high-resolution chromatography at much lower temperatures. A GC method for the determination of DON in wheat is reported by AOAC Official methods [168].
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List of Official AOAC and CEN methods
Mycotoxins
Matrix
Reference
Method
Aflatoxins (AFB1, AFB2, AFG1, AFG2)
Peanuts, pistachios, figs, and paprika powder
AOAC-999.07 CEN–EN 14123:2003
Aflatoxins
Nuts and nut products
AOAC–2005.08
Aflatoxins
Almonds, peanuts, pistachio nuts, brazil nuts Peanut butter Cereals, shell fruits and derived products
AOAC-994.08
IAC-HPLC with post column derivatization and fluorescence detection HPLC with postcolumn photochemical derivatization Mycosep-HPLC
AOAC-991.45 AOAC-991.31 CEN–EN 12955:1999
Aflatoxins Aflatoxins (AFB1 and AFs)
Aflatoxins (AFB1, AFB2, AFG1) AFB1
Corn
AOAC-993.16
Feeding stuffs
AOAC–2003.02 CEN–EN ISO 17375:2006
AFB1
Baby foods
AOAC–2000.16
Aflatoxins AFM1
Peanuts and corn Milk and milk powder
AFM1
Milk and milk powder Milk and cheese Foods for infants and young children Corn and barley Cereals and derived products
AOAC-993.17 AOAC–2000.08 CEN–EN ISO 14501:1999 CEN–EN ISO 14675:2003 AOAC-980.21 CEN-New itema
AFM1 AFB1
OTA OTA
AOAC–991.44 CEN–EN ISO 15141-1:1998
ELISA IAC-HPLC with post column derivatization and fluorescence detection ELISA IAC-HPLC with post column derivatization and fluorescence detection IAC-HPLC with post column derivatization and fluorescence detection TLC IAC-HPLC with fluorescence detection ELISA TLC –
HPLC Silica gel clean-up– HPLC with fluorescence detection
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Table 4 (Continued ) Mycotoxins
Matrix
Reference
Method
OTA
Cereals and derived products
CEN–EN ISO 15141-2:1998
OTA
Barley
AOAC–2000.03
OTA
Roasted coffee
AOAC–2000.09
OTA
Barley and Roasted coffee
CEN–EN 14132:2003
OTA
Wine and beer
OTA OTA
Green coffee Barley and green coffee Foods for infants and young children Dried (vine) fruit
AOAC–2001.01 CEN – EN 14133:2003 AOAC–2004.10 AOAC–975.38
Bicarbonate cleanup–HPLC with fluorescence detection IAC-HPLC with fluorescence detection IAC-HPLC with fluorescence detection IAC-HPLC with fluorescence detection IAC-HPLC with fluorescence detection HPLC TLC
OTA
OTA
DON DON
DON
DON ZEA ZEA
ZEA
ZEA ZEA Fumonisins (FB1, FB2, FB3)
Wheat Cereals (including maize) and cereal products Foods for infants and young children Wheat Corn Cereals (including maize) and cereal products Foods for infants and young children Wheat and animal feed Corn Corn
CEN-New itema
CEN-New itema
AOAC–986.18 CEN-New itema
IAC–HPLC with fluorescence detection IAC–HPLC with fluorescence detection GC IAC–HPLC with UV detection
CEN-New itema
–
AOAC-986.17 AOAC–985.18 CEN-New itema
CEN-New itema
TLC HPLC IAC–HPLC with fluorescence detection –
AOAC-994.01
ELISA (qualitative)
AOAC-976.22 AOAC – 995.15
TLC HPLC
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Table 4 (Continued ) Mycotoxins
Matrix
Reference
Method
Fumonisins (FB1, FB2)
Maize based foods
AOAC–2001.04 CEN–EN 14352:2005
Fumonisins (FB1, FB2)
Maize
CEN-EN 13585:2002
FBs Fumonisins (FB1, FB2)
Corn Maize based foods for infants and young children Clear and cloudy apple juice and puree
AOAC-2001.06 CEN-New itema
IAC-HPLC with pre column derivatization and fluorescence detection SPE-HPLC with pre column derivatization and fluorescence detection ELISA -
Patulin
Clear and cloudy apple juice and puree
CEN-EN 14177:2004
Patulin Patulin
Apple juice Clear and cloudy apple juice and puree for infant and young children
AOAC-974.18 CEN-New itema
Patulin
AOAC–2000.02
Liquid/liquid partition cleanup–HPLC with UV detection Liquid/liquid partition cleanup–HPLC with UV detection TLC Liquid/liquid partition cleanup and SPE– HPLC with UV detection
a
Methods of analysis ready to be mandated according to the Mandate for standardization addressed to CEN in the field of methods of analysis for mycotoxins in food. Deadline 12/2008.
GC coupled with MS could be used for the confirmation of patulin in apple juice [155]. GC/MS methods are also reported operating both without [169] and with various derivatizing agents [170,171]. Limitations such as the need to derivatize the samples before analysis have led to a prevalence of LC-MS over GC-MS approaches [172]. Figure 9 shows a chromatogram obtained after separation and detection with GC-ECD after trimethylslylation of five B trichothecenes.
5.3.1.3 Thin-layer chromatography. The employment of TLC-based methods has decreased steadily over the past few years, but, especially in developing countries, this analytical technique is still routinely used. TLC methods allow for
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Figure 7 HPLC chromatogram of a corn sample naturally contaminated with aflatoxins. Level of contamination: AFB1 and AFG1, each at 6.03 mg/kg, and AFB2 and AFG2, each at 1.49 mg/kg.
Figure 8 HPLC chromatogram of a wheat sample naturally contaminated with OTA. Level of contamination: 4.53 mg/kg.
a precise determination at levels not lower than 2 ng/g; their disadvantages however include the use of solvents that are considered to be ecological hazard. TLC techniques have been reviewed and updated for the analysis of selected Fusarium toxins by Krska [120]. A large number of TLC-based methods have been
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Figure 9 Gas chromatogram for wheat spiked with DON, 3-AcDON, 15 Ac-DON, Fus-X, and NIV at a concentration of about 1,000 ng/g. Source: International Programme Chemical Safety (IPCS) Inchem, www.inchem.org DON.
accepted by the AOAC as official methods including the methods for the determination of DON in barley and wheat, for aflatoxins in peanuts and corn, for AFM1 in milk and cheeses, for OTA in barley and in green coffee and for ZEA in corn. All the above-mentioned methods are reported in Table 4. The proposed use of TLC in combination with IACs represents a promising application that can improve the performance characteristics of the TLC-based methods [173]. An improvement in the technique includes the use of a microcomputer interfaced with a fluorodensitometer to simplify data handling [174]. New detection techniques for TLC have also been developed as alternatives to traditional TLC scanners; the employed principles are based on the use of a semiconductorbased detection cell (SeBaDeC) or of a modified office scanner. As the cost of commercial office scanners is very low, these are of special interest for the quantification of mycotoxins whenever other instruments are not available, such as in developing countries, in which this technique still represents the most economic method for the analysis of trichothecenes.
5.3.1.4 Liquid chromatography-mass spectrometry. LC coupled with MS and especially, LC-MS-MS have become very popular in recent years, because of the universal, selective and sensitive detection. Combining HPLC with MS/MS results in a powerful tool for characterization and identification especially for
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detection of the so-called conjugated mycotoxins, in which the toxin is usually bound to a more polar substance such as glucose or other sugars. These conjugated are referred to as masked mycotoxins, as these substances escape routine detection methods but can release their toxic precursors after hydrolysis [175]. Also with LC-MS there is currently a strong trend toward multimycotoxin methods for the simultaneous determination, particularly for mycotoxins belonging to different chemical families [176]. This approach has the advantage of enabling the reduction or possibly the omission of sample clean-up. Determination of DON and NIV in corn [177] and a rapid determination of FB1 are performed without any clean-up or purification step by a liquid chromatographic/tandem mass spectrometrer using pneumatically assisted electrospray ionization (HPLC-ESI-MS), the mass analyser being a triple-stage quadrupole equipped with a standard ESI interface [178]. Other methods described in the literature include a clean-up step, for example, graphitised carbon black for extracts from corn meal samples before LC-ESI-MS-MS multiresidue analysis [179]. An HPLC-ESI-MS method was developed for the determination of 18 mycotoxins and metabolites, OTA, ZEA, a-ZOL, b-ZOL, a-zearalanol (zeranol), b-zearalanol (taleranol), FB1, FB2, T-2 toxin, HT-2 toxin, T-2 triol, DAS, 15-MAS, DON, 3-acetylDON, 15-acetylDON, deepoxy-DON and AFM1, in milk [180]. Results confirm that, especially for multimycotoxin methods expanding over a broad range of classes, sample pretreatment is a critical step, because even within a single class of mycotoxins significant losses may occur during extraction or clean-up. A method for the determination of 39 mycotoxins in wheat and maize using a single extraction step followed by LC-ESI-triple-quadrupole MS without the need for any clean-up, has recently been developed and validated in-house [181]. The analytes included type-A and type-B trichothecenes and related derivatives including conjugated DON, FBs, ochratoxins, ZEA, aflatoxins, moniliformin, enniatins and ergot alkaloids; the limits of detection ranged from 0.05 to 220 mg/kg. The extraction solvent mixture, diluted 1:2, and injection without any clean-up, was chosen as a compromise for the extraction of the 39 analytes from both wheat and maize. In addition to the multianalyte requirement, multisubstrate methods are even more demanding. This approach, nevertheless, has the advantages of speed and ease of application with one method for multiple mycotoxin-matrix combinations over a broad range of mycotoxin classes, which is of special interest for high-throughput routine analysis. LC-MS instruments using atmospheric pressure chemical ionization (APCI) interfaces have recently been employed for the determination and identification of ZEA, trichothecenes, including DON and NIV, at trace levels [118,119,182,183]. Figure 10 shows a total ion LC-MS-MS chromatogram obtained after clean-up of a spiked maize sample containing 100 mg/kg of each mycotoxin.
5.3.2 Capillary electrophoresis CE is an analytical technique more and more extensively used in food analysis. It is generally used as a chromatographic technique, where mycotoxins are separated from one another and from matrix components using electrical
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Figure 10 Total ion chromatogram obtained after clean-up with MycoSeps #226 columns and LC-MS-MS analysis of a spiked maize sample containing 100 mg/kg of each mycotoxin. Vertical lines illustrate change of ionization polarities from negative to positive (5.4 min) and back to negative (6.4 min). Source: Berthiller et al. Ref. [172].
potential [184]. Its main advantages are reduction of organic solvents, small sample volume, increased efficiency and resolution. However, due to the rather high detection limit, the technique is not generally suitable for the determination of low concentrations of mycotoxins. A method for patulin determination in apple juice by CE is available and presents the advantage of a relatively low limit of detection (3.8 mg/L) [185]. A method allowing the simultaneous determination of ochratoxins A and B and four aflatoxins is also reported [186]. Micellar electrokinetic capillary chromatography (MEKC) is particularly useful for detecting neutral compounds such as aflatoxins [187]. Recent advances in mycotoxin detection with CE include the use of cyclodextrins. Since some cyclodextrins are capable of significantly enhancing the native fluorescence of the estrogenic mycotoxin ZEA, a CE method for ZEA determination involving cyclodextrins, with a limit of quantification of 5 ng/g, is reported [188], while b-cyclodextrins combined with multiphoton excitation are used for aflatoxins detection [189].
5.3.3 Rapid methods Rapid methods, or screening methods, should be simple and easy-to-use, the method should be fast and capable of testing also mycotoxins in field. Rapid methods allow the testing of a large number of samples, but the confirmation of positive ones is recommended. A high reliability of the screening methods is
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based on a low number of false positive, as well as on a low percentage of false negatives (less than 5%). These techniques meet one of the needs of the food industry production, represented by the availability of rapid methods allowing the ‘‘on-line control’’ of mycotoxins in food. Biosensors and kits based on dipstick techniques are promising methodologies based on immunochemical reactions [190].
5.3.3.1 Fluorescence polarization immunoassay. Several FPIA formats were studied using agrochemicals, toxins and a food pathogen to determine whether this technology was quicker to perform yet equivalent in sensitivity to ELISA technology. The sensitivity of FPIA was similar to ELISA for detecting DON in cereal products. FPIA, however, was the least labour-intensive and required the lowest time to be performed. ELISAs required approximately 3 h to perform following incubation overnight with coating conjugate. In contrast, the FPIAs required less than 90 min to be performed and no coating of plates was required. A FPIA based on a monoclonal antibody for the determination of OTA was developed [191]. Fluorescein-labelled OTA derivative (tracer) was synthesized and purified by thin-layer chromatography. The optimized OTA-FPIA had a dynamic range from 5 to 200 ng/mL with IC50 value of 30 ng/mL and a detection limit of 3 ng/mL. The method developed was characterized by high specificity and reproducibility. Cross-reactivity with other mycotoxins (ZEA, aflatoxins, patulin and T-2 toxin) was negligible (o0.1%). The results of OTA determination in barley were compared with those determined by indirect competitive ELISA. Recoveries for the samples spiked at 50, 100 and 500 ng/g levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean-up. The principle of FPIA was also used for the determination of DON in wheat and for aflatoxin determination in grains [192–195]. A number of instrument- and antibody-based methods, including ELISAs, have been developed to detect ZEA and related toxins in commodities and food [196]. Although convenient, the commercial ELISAs for small molecules such as ZEN require a washing step to separate bound and unbound enzyme labels before detection. In FPIAs, separation of bound and unbound labels is not required, a property that reduces the time needed to perform the assays. An FPIA for ZEN in maize was developed, combined with a rapid extraction technique; the assay could be used to detect as little as 0.11 mg/g of ZEN in maize within 10 min. The assay showed cross-reactivity to the ZEN analogs such as zearalanone, a-zearalanol, a-ZOL, b-ZOL, and b-zearalanol of 195, 139, 102, 71 and 20%, respectively, relative to ZEN (100%). Recovery of ZEN from spiked maize samples over the range 0.5–5 mg/g averaged 100.2%. The FPIA results were comparable to those obtained with a liquid chromatographic method. The FPIA provides a rapid method for screening of maize for ZEN.
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5.3.3.2 Enzyme-linked immunosorbent assay. The technology of ELISA is based on the ability of a specific antibody to distinguish the three-dimensional structure of a specific mycotoxin. Steps to perform an ELISA analysis are herein described: after the solvent extraction of the mycotoxin from the ground sample, a portion of the extract and a conjugate of an enzyme-coupled mycotoxin are mixed and added to the antibody-coated microtitre wells. Any mycotoxin in the sample extract is allowed to compete with the enzyme-conjugated mycotoxin for the antibody-binding sites. ELISA-based methods represent user-friendly methods at relatively low cost, also having the advantage to reduce the amount of organic solvent employed for the analysis with respect to LC analytical methods. ELISA tests are recommended for screening analysis because simultaneous determination of a high number of samples is allowed. However, the disadvantages of this technique are represented by the possibility of obtaining false positives when a cross-reaction of the antibodies with other mycotoxins may happen, as well as false negatives, when low levels of contamination, often close to the legal limit, are not detected. Interferences of the matrices can also occur during ELISA analysis. Therefore, the ELISA methods are suggested as low-cost screening methods prior to confirmatory analysis, in order to considerably lower the costs, when a high number of samples have to be analysed [190]. The WHO provides the criteria for the acceptance/validation of immunoassay-based kits and of other protein-binding systems [197]. An ELISA method for the detection of aflatoxins and ZEA in corn, wheat and feed (Agri-screen method) has been tested by an interlaboratory study and accepted as AOAC official method [133]. Different brands of ELISA kit are available for almost all the major mycotoxins. For OTA analysis, the available ELISA test kits have been widely applied to cereals, cereal products, dried fruits, coffee, cocoa, wine and tea, beans, potatoes, maize, wheat, flour, barley and beer [198–200]. Many immunochemical methods are given for trichothecenes [201–203]. As for patulin, no ELISA kit is commercially available since no specific antibodies have yet been developed. The detection limits associated with these methods greatly depend on the mycotoxin being evaluated and on the specific kit employed. 5.3.3.3 On-line control methods. Based on the same principle of ELISA test, other and even more faster screening methods are available. These techniques are designed for on-line mycotoxin determination. Membrane-based immunoassay. This method is based on a principle of direct competitive ELISA, in this case the anti-mycotoxin antibody is coated on a membrane surface. The membrane-based immunoassay methods are rapid, easy-to-use, suitable for testing mycotoxins in the field, and they do not require any equipment. However, interpretation of results may be difficult when the toxin concentration in the sample is close to the method cut-off. A membrane immunoassay method for OTA determination in roasted and green coffee was developed [204,205]. The obtained results are consistent with those obtained employing LC determination. Test kits based on this
Mycotoxins
399
immunoassay have been validated for OTA determination in wheat, rye, maize and barley [206]. LFD test/card/dip-stick. The LFD, also called strip test, is an immunochromatographic test, fundamentally similar to a pregnancy test kit. A typical test strip is composed of a sample pad, a conjugated pad, a membrane, an absorbent pad and an adhesive support. A sample extract is simply added onto the sample pad. Any mycotoxin present binds to the anti-mycotoxin antibody gold particle complex in the conjugate pad and migrate together with the anti2nd antibody gold particle complex along the membrane. The membrane contains a test zone and a control zone, onto which a mycotoxin–protein conjugate and a 2nd antibody are dried, respectively. The mycotoxin–protein conjugate in the test zone can capture any free anti-mycotoxin antibody gold particle complex, allowing colour particles to concentrate and form a visible line. Hence, a positive sample with a mycotoxin concentration greater than or equal to the assay cut-off level will result in no visible line in the test zone. Conversely, a negative sample with a mycotoxin concentration less than the cut-off level will form a visible line in the test zone. The control zone will always be visible regardless of the presence or absence of mycotoxin because the 2nd antibody always captures the anti-2nd antibody gold particle complex indicating the validity of the performed test. These immunochromatographic tests are user-friendly, very rapid and are particularly suitable for testing mycotoxins in the field, since they are semi-quantitative methods; for any positive samples, confirmation by a reference method, such as HPLC, is required. A dipstick enzyme immunoassay for the rapid detection of Fusarium T-2 toxin in wheat was developed [207], and kit from Romer, AgraStrips Lateral Flow Kit, qualitatively determines the presence of aflatoxins B1, B2, G1 and G2, and can be performed in 5 min. Additionally dipstick tests have been developed, which claim a detection limit of FB1 as low as 0.04–0.06 mg/g in corn-based foods. These techniques couple the rapidity with the possibility to detect many mycotoxins simultaneously [208]. R-Biopharm Roˆhne Ltd. produces cards for the rapid determination of AFB1, total aflatoxins and OTA, while other tests are available by Neogen Corporation for the determination of aflatoxins, OTA, DON, ZEA and FBs. Charm Science Inc. produces the Rosas tests (Rapid One Step Assay) for AFB1 in cereals, DON in wheat and barley, OTA in wine and ZEA in corn. These latter tests provide positive/negative response and no equipment is required to read the distinct visual results. However, an optional digital reflectance strip reader has also been approved to verify results and electronically record test data. The threshold of the test is based on set legal limits for the different mycotoxins. A collaborative study for AFM1 determination in milk was recently performed for a Rosa test [209]. Biosensors are compact analytical devices that use biological components (nucleic acids, enzymes, antibodies, cells), associated with a transduction system that process the signal produced by the interaction between the target molecule and the biological component. The advantages of this technology may include
400
Carlo Brera et al.
more rapid assays, reusable and long-life sensor elements, the capacity for continuous monitoring and industrially low-cost devices [210]. The use of biosensors for the detection of aflatoxins is rapidly increasing, including disposable DNA electrochemical biosensors [211], handled immunoaffinity fluorimetric biosensors [212] and biosensors based on lipid films and liposomes [213]. Potential application of biosensor devices in combination with MIPs in mycotoxin analysis has been reported for the analysis of OTA in a linear range of 0.05–0.5 ng/g demonstrating a novel combination of biosensor device and MIP technique [214]. Biosensors used for the analysis of mycotoxin are those based on the interaction of a specific antibody, or an antibody fragment, and the mycotoxin: Optical immunosensor. In optical immunosensors monoclonal antibodies are covalently coupled with an optical fibre, studies of the detection of FB1 in a quantitation range of 10–1000 ng/mL with a limit of detection of 10 ng/mL has been carried out [215]; an antibody-bound glass prism surface uses SPR for the direct detection of FB1, AFB1, DON and multimycotoxin in general [216–220]. The evanescent wave-based fibre optic immunosensor is another optical immunosensor that was applied for detection of FB and aflatoxin in maize in a rapid effective screening assay but it requires a clean-up step to increase sensitivity (0.4 mg/g) [221–223]. Piezoelectric immunosensor. An alternative biosensor is the quartz crystal microbalance (QCM) where the transduction measures the mass change at the surface resulting from interaction of an analyte with the receptor immobilized at the surface of the crystal. Hauck reported the determination of OTA in liquid food products; the method was applied in a linear range of 2–100 ng/mL [224]. Electrochemical immunosensor (screen-printed electrodes). These immunochemically based assays are used for detecting mycotoxins in food and have seen rapid development for their simplicity, adaptability, sensitivity and selectivity. Screen-printed electrodes are (antibody-based) sensors, with electrochemistry as the basis of the technique. A screen-printed electrode system as transducer for chronoamperometry and a monoclonal antibody for molecular recognition, was applied for the detection of AFM1, the high selectivity of immunoanalysis with the convenience of electrochemical probes, have shown that AFM1 can be measured with a detection limit of 0.025 mg/kg with a working range between 0.030 and 0.160 mg/kg. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection [225]. Pemberton showed the development of a screen-printed carbon electrochemical immunosensor array for mycotoxins that have a detection limit as low as 0.15 ng/mL for AFB1 analysis [226]. Being this value compatible with the requirements of the food industry, further studies using real samples from food extracts are essential for the application of the array. Sensitive immunosensors, using an indirect competitive ELISA format, have been used for the determination of AFB1 in barley. The high selectivity and
Mycotoxins
401
simplicity of an immunoassay combines with high sensitivity, rapidity and low cost of electrochemical measurements. This immunosensor (coated SPCE) can be stored for up to one month, to be ready for in situ determination. The immunosensor exhibited a linearity range that is comparable to that for conventional methods (HPLC) and had also a detection limit suitable for on-site monitoring [227].
5.4 Other methods 5.4.1 Fourier transform infrared photo-acoustic spectroscopy (FTIR-PAS) This technique has been in the past few years desirable as a confirmatory alternative, especially for control and monitoring purposes, in which a high number of analyses with high reliability in a short time are requested. This possibility would also be useful to obtain information on the exposure to many mycotoxins and on the effect of combined exposure to human health. Two techniques, PAS and diffuse reflectance spectroscopy (DRS), were coupled to an FTIR spectrometer to provide information about the MIR absorption spectra of corn infected with F. moniliforme and A. flavus, two mycotoxin producers [228].
5.4.2 Near- and mid-infrared spectroscopy NIR, 700–2,500 nm, and MIR, 2,500–30,000 nm, spectroscopy is a practical spectroscopic procedure for the detection of organic compounds, and has been used routinely for many years in the field for determination of protein and moisture in cereals. The technique is useful because of its non-destructiveness, accuracy, rapid response and easy operation, and has the potential for determination of a number of compounds. The NIR methodology can be applied for monitoring mould contamination in post-harvest maize, to distinguish contaminated lots from clean ones and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply. Pattersson and Aberg [229] investigated the use of NIR transmittance instrumentation in the wavelength range 570–1,100 nm for finding out the mycotoxin DON in wheat kernel. Principal component analysis and partial least square regression calculations were used to formulate the best models. The wavelength range 670–1,100 nm gave the best regression model with a slope of 0.949 having a correlation coefficient of 0.984 with a standard error of 381 mg mycotoxin DON/kg of wheat. All spectra (570–1,100 nm) were collected using the FOSS Infratec 1241 Grain Analyser with the added colour module, and reference analysis using HPLC or GC methods. Based on current findings, it appears that NIR could also be used as a screening tool to measure DON in cereals. The applicability of NIR for the rapid identification of mycotoxigenic fungi and their toxic metabolites [230] produced in naturally and artificially contaminated products, indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the
402
Carlo Brera et al.
quantity of ergosterol and FB1 in meal. The statistics of the calibration and of the cross-validation for mould infection and for ergosterol and F B1 contents were significant. A method based on mid-infrared/attenuated total reflection (ATR), which enables the determination of fungal infection with F. graminearum on corn within minutes, has been developed [231]. After the spectra were recorded, they were subjected to principal component analysis (PCA) and classified using cluster analysis. Observed changes in the spectra reflected changes in protein, carbohydrate and lipid contents. DON served as reference parameter. The developed method enabled the separation of samples with DON content as low as 310 mg/kg from non-contaminated (blank) samples. The investigated concentration range was 310–2,596 mg/kg for DON. The percentage of correctly classified samples was up to 100% for individual samples compared with a number of blank samples. For both, the MIR and the NIR spectroscopic approach further work on calibration development and the expansion of the calibration databases will reveal the true potential of IR spectroscopy for this kind of application.
6. OCCURRENCE IN FOOD AND FEED As already discussedin this chapter, two opposite but related factors contribute to the persistence of mycotoxins in crops and commodities, that are natural and man-made causes much often coexisting. The presence of mycotoxins in food and feed is, in fact, due to several multifaceted factors including environmental conditions, mainly climate and insects as fungi spores vectors, improper agricultural practices, wrong postharvest activities, no specific HACCP plans and scarce implementation of decontamination procedures. For the above, only an integrated and systematic approach from the farm to the consumer aimed at the reduction of these potent toxic compounds can be put into practice in the future. Data available on worldwide mycotoxins occurrence are characterized by an extreme degree of non-homogeneity, in terms of number of samples analysed, quality of the analytical methodology used, adopted sampling procedures, finalities and objectives of the monitoring or survey programs, and representativity at national level of the performed surveys. For this reason, quite recently the European Commission, according to Council Directive 93/5/EEC ‘‘on the assistance to the Commission and co-operation by the Member States in the scientific examination of questions relating to food’’, member states of the EU co-operated on problems facing the Commission in the area of food, performing five specific tasks on mycotoxins. The rationale for each task was to provide harmonized and reliable informative databases to be used by the Commission for the management of
Mycotoxins
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problems related to food. All the information provided in the most recent tasks is listed in the specific databases available at the following Internet sites: Aflatoxins-SCOOP Task 3.2.1; not available on Internet OTA-SCOOP Tasks 3.2.7; http://www.ec.europa.eu/food/fs/scoop/3.2.7_en.pdf Patulin-SCOOP Task 3.2.8; http://www.ec.europa.eu/food/fs/scoop/3.2.8_ en.pdf Fusarium toxins (trichothecenes, ZEA and FBs)-SCOOP Task 3.2.10; http:// www.ec.europa.eu/food/fs/scoop/task3210.pdf Furthermore, it should be kept in due consideration that reliable occurrence databases are one of the key elements for a correct exposure assessment and constitution of worldwide regulations.
6.1 Aflatoxins As mentioned, the first European complete database on occurrence of aflatoxins in raw commodities and processed food has been concluded in 1991 within SCOOP Task 3.2.1 and published in 1997 [103]. That database, reflecting the situation at that time, reported unreliable or even missing information on various features such as the type and target of adopted sampling procedures, the implementation of quality assurance systems in the reported surveys, the use of validated methods and the representativity of the survey for the member state; for these reasons that source of information is now highly outdated. Aflatoxins can occur in various raw commodities and processed food products. Among raw commodities aflatoxins are mainly present in cereals, spices, cottonseed, copra, dried fruits and nuts; among processed food, eggs, pasta, bread and milk and cheese (AFM1) resulted naturally contaminated with aflatoxins. A list of some of the most interesting worldwide surveys on aflatoxin occurrence in food products is reported in Table 5.
6.2 Ochratoxin A As for OTA, the most recent European database is provided by the report of the SCOOP Task 3.2.7 [232]. Basically, participants were asked to provide information on the exposure of the population to OTA in their country through the elaboration of the following categories of data: 1 2 3 4 5
Occurrence data in food and beverages, including those from different methodological approaches (i.e., total diet, duplicate test portion) Consumption data Best estimate of dietary intake Occurrence data in biological fluids and intake as derived from data on serum Data of intake for lactating babies through the ingestion of breast milk.
404
Table 5
Occurrence of mycotoxins in food matrices Food matrix
Concentration levels (mg/kg or mg/l)
Analytical technique
Country
Year
Reference
Aflatoxins Aflatoxins Aflatoxins Aflatoxins Aflatoxins
Nut products Spices Corn products Sultanas, dried figs Miscellaneous foods at retail Maize-based food products Cereal, spices
9.9–710 0.2–14.6 1.0–23.3 0.3–1500.0 0.10–2.59 (peanut butter) 2–59 (popcorn)
HPLC-FL HPLC-FL HPLC-FL IAC-HPLC-FL IAC-TFA-HPLC-FL
UK UK Brazil Brazil Japan
2003–2004 2003–2004 1999–2001 2002–2003 2004–2005
[233] [234] [235] [236] [237]
TLC
Brazil
2002–2003
[238]
o0.35 (Cereal products) 0.02–9.10 (ginger) 0.15–1.13 0.002–0.996 (ricebased infant food) 0.5–5.0
IAC-HPLC-FL
Morocco
2002
[239]
IAC-HPLC-FL IAC-HPLC-FL
Greece Canada
2004 2002–2005
[240] [241]
Liq-liq partition/ HPLC-FL IAC-HPLC-FL
Morocco
Unknown
[242]
Italy
2002–2003
[243]
IAC-HPLC-FL IAC-HPLC-FL IAC/HPLC-FL IAC/HPLC-FL
Turkey Italy Hungary Qatar
2003–2004 1995–1999 2004 2002
[244] [245] [246] [247]
ELISA
Cote d’Ivoire
2002
[248]
ELISA IAC/HPLC-FL
Turkey Brazil
2005 2004–2005
[249] [250]
Aflatoxins Aflatoxins
Aflatoxins Aflatoxins Aflatoxins
Olives Breakfast and infant cereals Olives
Aflatoxins
Oil and Olives
Aflatoxins Aflatoxins Aflatoxins Aflatoxins
Dried Figs Maize Spices Miscellaneous food products Cereals, peanuts
Aflatoxins AFM1 AFM1
UHT milk Pasteurized, and UHT Goat milk
0.006–0.04 (oil) 0.03–1.5 (olives) 0.1–35.1 0.05–158 0.14–15.7 (red pepper) 0.14–81.64 (pistachio nuts) 1.5–10 (rice) 1.5–10 0.01– 0.54 0.011–0.161
Carlo Brera et al.
Mycotoxin
AFM1 AFM1
AFM1 AFM1 AFM1 AFM1 AFM1 AFM1 AFM1 AFM1 OTA OTA OTA
Raw, Pasteurizedand UHT milk Pasteurized homogenized commercial milk Cheese Ewe’s milk Cow milk Cheese Yoghurt Parmesan cheese Cow, ewe and goat raw milk Cow, ewe and goat cheese Spices
Oil and Olives
OTA OTA OTA OTA OTA
Dried Figs Infant foods Beer Breakfast cereals Cereal-based infant foods
Brazil
2002–2003
[251]
0.010–0.289
IAC/LC-FL
Colombia
2004–2005
[252]
0.19–2.05 0.002–0.108 0.03–3.13 0.11–0.52 0.010–0.098 0.001–0.406 0.005–0.170
TLC HPLC/FL HPLC HPLC IAC/HPLC IAC/HPLC-FL HPLC/FL
Iran Italy Libya Libya Portugal Italy Greece
2003–2004 2000 2002 2002 2001 1993–1999 1999–2000
[253] [254] [255] [255] [256] [257] [258]
0.05–0.250
HPLC/FL
Italy
1997–1999
[259]
0.2–152.2 (ground chilli) o 10 0.1–12.5 (raisin)
HPLC-FL
UK
2003–2004
[234]
TLC IAC/HPLC-FL
Brazil Japan
1999–2001 2004–2005
[235] [237]
6.4–64 (corn flour)
TLC
Brazil
2002–2003
[238]
0.01–7.22 (Corn) 0.41–1.86 0.2–1.02
IAC-HPLC-FL IAC-HPLC-FL Liq-liq partition/ HPLC-FL IAC-HPLC-FL
Morocco Greece Morocco
2002 2004 Unknown
[239] [240] [242]
Italy
2002–2003
[243]
IAC-HPLC-FL HPLC HPLC HPLC HPLC
Turkey Spain Spain Spain Spain
2003–2004
[244] [260] [260] [260] [261]
0.1–0.234 (oil) 0.1–8.0 (olives) 0.1–26.3 0.035–0.740 0.012–0.205 0.066–0.975 1.67 (max value)
Unknown
405
OTA
OTA
IAC/TLC
Mycotoxins
OTA OTA OTA
Corn products Miscellaneous foods at retail Maize-based food products Cereal, Olives Olives
o0.02–0.26
406
Table 5 (Continued ) Food matrix
Concentration levels (mg/kg or mg/l)
Analytical technique
Country
Year
Reference
OTA
Cereal-based baby foods Grape
IAC/HPLC-FL
Italy
Unknown
[262]
IAC/HPLC-FL
Portugal
2001–2003
[263]
IAC/HPLC-FL
South America
IAC/HPLC-FL LC-MS/MS (ESI-MRM)
Lebanon Different geographical regions Different geographical regions Different geographical regions Italy Italy Italy Italy Hungary Hungary Italy Spain Qatar
OTA
Grape based products, wine
OTA OTA
Red wine Red wine
0.06–0.74 (organic rice) 0.100–1.637 (pea berry) 0.021–0.100 (grape juices) 0.021–0.071 (red wine) 0.001–14 (dried black vine fruits) 0.012–0.126 0.05–0.75
OTA
Rose´ wine
0.05–0.09
LC-MS/MS (ESI-MRM)
OTA
White wine
0.05–0.22
LC-MS/MS (ESI-MRM)
OTA OTA OTA OTA OTA OTA OTA OTA OTA
Red wine Dessert wine Rose´ wine White wine Wine Spices Pork products Rice and rice products Miscellaneous food products
0.01–4.00 0.01–1.64 0.01–1.04 0.01–0.21 o 0.01 10.6–66.2 (red pepper) 0.01–28.42 4.3–27.3 0.20–4.91
IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL HPLC-FL IAC/HPLC-FL
OTA
[264]
2004 2001–2004
[265] [266]
2001–2004
[266]
2001–2004
[266]
1997–2002 1997–2002 1997–2002 1997–2002 1997–2002 2004 2001–2002 Unknown 2002
[267] [267] [267] [267] [267] [246] [268] [269] [247]
Carlo Brera et al.
Mycotoxin
OTA
Coffee
OTA OTA
Beer Cocoa products and chocolate Raisins and currants Dried vine fruits Dried vine fruits Cereals, peanuts
OTA OTA OTA OTA OTA
OTA Citrinin
Grains Olives
Citrinin ZEA
Breakfast cereals Maize-based food products Cereals Maize Miscellaneous food products Cereals, peanuts
ZEA ZEA ZEA ZEA ZEA
Brazil
1998–1999
[270]
IAC/HPLC-FL IAC/HPLC-FL
Belgium Spain
1998–2001 Unknown
[271] [272]
IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL; ELISA
Sweden Canada UK Cote d’Ivoire
1999–2002 1998–2000 Unknown 2002
[273] [274] [275] [248]
IAC/HPLC-FL
Germany
2004
[276]
IAC/HPLC-FL IAC/HPLC-FL HPLC/FL
Croatia Croatia France
2002 1996–1997 2003
[277] [278] [279]
UK Morocco
1999–2000 Unknown
[280] [242]
1.5–42 76.8–448 (popcorn)
IAC/HPLC-UV Liq-liq partition/ HPLC-FL HPLC/FL TLC
France Brazil
2003 2002–2003
[279] [238]
10–17 (corn) 50– 2531 0.18–6.81 (cornflakes)
IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL
Morocco Italy Qatar
2002 1995–1999 2002
[239] [245] [247]
50–200 (rice) 50-200 4–5072 (rye)
ELISA
Cote d’Ivoire
2002
[278]
IAC/HPLC-FL
Germany
2004
[276]
0.43–39.12 1–2 214 (flour)
IAC/HPLC-FL IAC/HPLC-FL IAC/HPLC-FL
Croatia Denmark Germany
2002 1998–2001 Unknown
[277] [281] [282]
0.9–2.54 0.26–613.7 0.2–8.8 (dry fruit and bran) 0.3–231 (feed-wheat) 0.5– 0.52
407
ZEA ZEA ZEA
Cereals and cereal products Maize Wheat and rye flour Soy
IAC/HPLC-FL
Mycotoxins
OTA OTA OTA
Cereals and cereal products Maize Maize Breakfast cereals
0.31–5.87 (roasted ground) 0.003–0.185 0.1–23.1 (roasted cocoa shell) 0.1–34.6 0.1–26.6 0.2–53.6 0.14–0.91 (rice) 0.20–0.64 0.1–1.7
408
Table 5 (Continued ) Food matrix
Concentration levels (mg/kg or mg/l)
Analytical technique
Country
Year
Reference
ZEA ZEA
Grains Miscellaneous food products Corn products Miscellaneous foods at retail Cereals Maize Cereals, peanuts
1–1790 2–67 (Bran)
IAC/HPLC-FL IAC/HPLC-FL
Germany Germany
2000–2001 2000–2001
[283] [284]
20–8600 10–354 (popcorn grain) 30–5960 (corn) 53–47078 300–1500 300–6000 1190–12.950 200–110.000 5–6617
HPLC/FL SAX/LC-MS
Brazil Japan
1999–2001 2004–2005
[235] [237]
IAC-HPLC-FL IAC-HPLC-FL ELISA
Morocco Italy Cote d’Ivoire
2002 1995–1999 2002
[239] [245] [248]
IAC/HPLC-FL ELISA IAC/HPLC-FL
Iran Nigeria Germany
2000 2003 2004
[285] [286] [276]
113–2.026
IAC/HPLC-FL
Portugal
2005
[287]
50–160 43–1329 20–464 142.2–1377 (FB1) 68.4–3084 (FB2) 12–11661 1–1110 10–2870 (wheat) 10–2870 (wholemeal flour) 25–1642
HPLC/FL HPLC/FL HPLC/FL IAC/HPLC-FL
Taiwan South Africa Belgium Croatia
Unknown 2001–2004 2003–2004 2002
[288] [289] [290] [279]
IAC/HPLC-FL HPLC/FL LC-MS LC-MS
Croatia France Italy Italy
1996–1997 2003 Unknown 2001–2002
[278] [279] [291] [292]
HPLC
USA
1998
[287]
GC-ECD
Italy
1995–1999
[243]
FBs FBs FBs FBs FBs FBs FBs FBs
FBs FBs FBs FBs
Maize Maize Cereals and cereal products Maize and maizebased products Corn products Beer Cornflakes Maize
FBs FBs FBs FBs
Maize Breakfast cereals Cereal based products Cereal based products
FBs
Corn and corn-based products Maize
FBs
DON
117–9357
Carlo Brera et al.
Mycotoxin
DON
Wheat and rye flour
DON DON DON DON DON
Patulin Patulin Patulin
Corn Corn-based products Soy Grains Miscellaneous food products Cereal products Cereals Cereals Apple juices Apple-based foods Miscellaneous applebased products Apple juice concentrates Apple-based drinks Apple products Fruit products
Patulin
Apple products
T-2
Cereals and pulses
DON DON DON Patulin Patulin Patulin Patulin
IAC/HPLC-UV
Qatar
2002
[247]
50–445
IAC/HPLC-DAD
Germany
2004
[276]
4.0–56.7 20–600 (feed) 7–930 (organic mix) 7–930 (wholemeal flour 20–2591 (durum wheat) 102–542 o20 260 (crisp) 7–6682 319–389 (Bran)
ELISA IAC/HPLC-UV GC-ECD GC-ECD
European countries UK Italy Italy
2000–2002 1999–2000 Unknown 2001–2002
[293] [280] [291] [292]
GC-ECD
Denmark
1998–2001
[281]
UV GC-MS GC-MS GC-MS GC-MS
Brazil Brazil Germany Germany Germany
1994–1995 2001–2002 Unknown 2000–2001 2000–2001
[294] [295] [282] [282] [284]
5–111 20–1440 (barley) 100–8600 8.1–122.6 (cloudy) 0.05–1166 W25
GC-MS GC-MS TLC, HPLC HPLC-UV SPE/HPLC-UV SPE/HPLC-DAD
Finland Norway Russia Belgium Italy The Netherlands
1998 1996–1998 1989–2001 Unknown Unknown Unknown
[296] [297] [298] [299] [300] [301]
7–376
HPLC-UV
Turkey
1996–1997
[302]
2.5–38.8 1.4–74.2 0.5–69.3 (organic apple juice) 5–45 (carbonated apple juice) 450–1900
HPLC-UV HPLC-UV HPLC-DAD
Belgium Italy Italy
2001 Unknown 2003–2004
[303] [304] [305]
HPLC-UV
South Africa
1996–1998
[306]
HPLC-UV
Turkey
Unknown
[307]
409
DON
DON
86.43–182.94
Mycotoxins
DON DON DON DON
Miscellaneous food products Cereals and cereal products Beer Grains Cereal based products Cereal based products
410
Table 5 (Continued ) Food matrix
Concentration levels (mg/kg or mg/l)
Analytical technique
Country
Year
Reference
T-2 T-2 T-2 T-2 T-2 T-2
Corn Wheat and rye flour Corn-based products Grains Cereal grains Miscellaneous food products Oat Cereals Cereal grains Wheat and rye flour Miscellaneous food products Oat Cereals Cereal grains Wheat and rye flour Corn-based products Soy Grains Miscellaneous food products Oat Cereal products Cereals
50–104 10–193 (rye) 50–767 (corn grits) 4–1810 o12 6–12(Flakes)
ELISA GC-ECD GC-MS GC-MS GC-MS GC-MS
Brazil Denmark Brazil Germany Italy Germany
1994–1995 1998–2001 2001–2002 2000–2001 Unknown 2000–2001
[294] [281] [295] [283] [308] [284]
10–703 20–380 (oat) 10–71 10–70 (durum wheat) 6–12(Flakes)
GC-MS GC-MS GC-MS GC-ECD GC-MS
Poland Norway Italy Denmark Germany
1997 1996–1998 Unknown 1998–2001 2000–2001
[309] [297] [308] [281] [284]
10–703 20–380 (oat) 10–71 10–70 (durum wheat) 50–555 (corn grits) 11 (flour) 3–1469 5–33 (Bran)
GC-MS GC-MS GC-MS GC-ECD GC-MS GC-MS GC-MS GC-MS
Poland Norway Italy Denmark Brazil Germany Germany Germany
1997 1996–1998 Unknown 1998–2001 2001–2002 Unknown 2000–2001 2000–2001
[309] [297] [308] [281] [295] [282] [283] [284]
10–47 10–20 20–880 (oat)
GC-MS GC-MS GC-MS
Poland Finland Norway
1997 1998 1996–1998
[309] [296] [297]
T-2 T-2 HT-2 HT-2 T-2 T-2 T-2 HT-2 HT-2 HT-2 HT-2 HT-2 HT-2 HT-2 HT-2 HT-2
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Additional information was also asked, among others and whenever possible, on the following issues: – – – – –
Sampling procedures employed Methods of analysis Quality assurance of analytical data Data on OTA occurrence in working places Regulations related to the toxin (maximum limits, sampling plans, others). The main conclusions from the report were the following:
Cereals represented the main source of intake in almost all the European countries. Rye, among cereal grains, generally seemed to be the most frequently and heavily contaminated. Coffee was the main contributor in Greece. Beer contributed to the total intake to a low extent. Wine represented the main source of intake in Italy and, as far as consumers only are concerned, in the Netherlands. Cocoa including cocoa powder contained within chocolate, provided a considerable contribution to the total intake in the United Kingdom; such contribution for 1.5–4.5 years population being similar to that attributed to cereals. Dried fruits contributed generally to a low extent to the total intake, except for the young population in the United Kingdom. Meat and spices contribute to a low extent to the total intake. Other potential contributors such as fruit juice seemed to be the most susceptible commodity. Breast milk represents a relevant source of exposure for lactating babies. As for OTA occurrence in grapes and wine two interesting reviews are available in literature [310,311]. Other surveys related to OTA occurrence in foods are reported in Table 5.
6.3 Fusarium toxins Similarly to OTA, SCOOP Task 3.2.10. report includes information on the intake of Fusarium toxins. The report is structured in a general section (part I), the three subtasks (trichothecenes, ZEA and FBs) and an annex part (II). Among trichothecenes both type A and B are reported for information on occurrence and in particular the following are included type A trichothecenes (T-2 and HT-2 Toxin, T-2 triol, neosolaniol, DAS, MAS) and type B trichothecenes: (3-acetylDON, 15-acetylDON, fusarenon X). An exhaustive summary of the outputs of the task has also been published where data on the occurrence of trichothecenes in food and dietary intake by the population of EU member states are reported only for DON, NIV, T-2 and HT-2 toxin. Results showed that corn had the highest level of trichothecene contamination, although DON was most often found in wheat. Intake data indicated that the main source of trichothecene consumption was through wheat
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and wheat products, such as bread and pasta; however, there was a lack of consumption data in some countries, particularly relating to children and infant foods. Although the average intake for DON was found to be below the TDI, high intake levels were found to exceed it, especially in young children. Both average and high NIV intake levels were found to be far below the TDI, while most of the average and high intake levels for T-2 and HT-2 toxins were above the temporary TDI [312]. A quite recent review on worldwide occurrence of FBs in foods and agricultural crops has been published in 2004. Aspects considered include disease cycles of Fusarium spp., which are responsible for the majority of FB synthesis; the chemical structure of FBs; diseases with which FBs are associated; occurrence of fumonisins B1 and B2 in foods and crops from Africa, America, Europe and Oceania and fumonisins B1–B3 in foods and crops from Asia; factors affecting the incidence of crop infection with Fusarium spp. (climatic conditions and latitude, genotype, spoilage, infection with other fungal diseases) and aspects of food preparation that affect FB occurrence (temperature, use of alkaline solutions and water) [313]. Similarly to FBs, an updated review of the most recent databases on ZEA occurrence in foods has been recently reported. Data from Europe, Africa, North and South America, Asia and Oceania are duly reported [314]. A review of the most recent noteworthy publications on Fusarium toxin occurrence in foods are reported in Table 5.
6.4 Patulin Correspondingly to the other mycotoxins, the European Commission launched in 2000 a SCOOP Task to collect information on the intake of patulin by the European population. The results of the study have been described in the report of SCOOP Task 3.2.8. Basically, the following conclusions are reported: Apple juice/apple nectar represented the main source of intake in Austria, Belgium, France, Germany, Portugal UK and Norway (apple nectar). Pear juice contributed to the total intake to a low extent in Germany and Portugal. Grape juice contributed to the total intake to a low extent. Fruit juice represented the main source of intake in Italy, Sweden and Spain. In Italy and Spain the main fruit juice was apple juice. Cider including drinks based on cider, provided a considerable contribution to the total intake of male adults consumers in France. Purees contributed to a low extent to the total intake, with the exception of female adults and adults in France where it was the main source for patulin intake. Baby foods had a higher level for intake for 1–3 years old children in Germany than in Italy, with the reservation that Italy only took into account pap (baby food with or without milk powder), but Germany pap and juice.
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Tomatoes, canned and concentrated, contributed generally to a low extent to the total intake. Among the category ‘‘Others’’, jellies and tomato products might be susceptible commodities. Fresh fruit represented the main source of intake in Italy. Other recent surveys related to patulin occurrence are reported in Table 5.
6.5 Multi-Fusarium toxins With reference to the new EC Regulation 1881/2006 that will be amended by the introduction of new maximum tolerable limits for Fusarium toxins, all the information available regarding the co-occurrence of Fusarium toxins in cereals represents very useful information both for the potential synergistic effect of these mycotoxins and for the development of new analytical methods able to co-extract, co-elute and co-detect these toxins in a single sample. Information about the co-occurrence of Fusarium toxins is schematized in Table 5. In particular, co-occurrence of AFs and OTA [234–246]; AFs, OTA and ZEA [238]; AFs, OTA, ZEA and FBs [235]; AFs, OTA and FBs [237]; AFs, OTA, ZEA and DON [248]; AFs, ZEA, FBs and DON [245]; AFs, OTA and CIT [242]; OTA, ZEA and FBs [239,277]; OTA, ZEA, FBs and DON [276]; OTA and FBs [278]; OTA, FBs and CIT [279]; OTA and DON [280]; FBs and DON [291]; ZEA, DON, NIV, T-2 and HT-2 [281]; DON and T-2 [294]; DON, NIV, T-2 and HT-2 [295] are reported. Surveys including a miscellaneous of Fusarium toxins are also available in literature. A total of 45 samples of soy food including whole beans, roasted soy nuts, flour and flakes, textured soy protein, tofu, protein isolate including infant formulas and fermented products (soy sauce) were randomly collected in food and health food stores and analysed for Fusarium toxins. A spectrum of 13 trichothecenes of the A-type as well as of the B-type was determined by GC/MS. Detection limits ranged between 1 and 19 mg/kg. At least one of the toxins investigated was detected in 11 out of a total of 45 samples of soy food belonging to different commodities. Scirpentriol, 15-MAS, 4,15-DAS, T-2 tetraol, HT-2 toxin, DON, 15- and 3-acetylDON, were detected in at least one sample, T-2 triol, T-2, neosolaniol, NIV and FUS-X were not detected in any sample. Five out of eleven samples were positive for one toxin, one sample for two, three, six or seven toxins, two samples for five toxins, demonstrating the possibility of a contamination of soy food with a spectrum of Fusarium toxins. Scirpentriol, DON and ZEA were found up to 108, 260 and 214 mg/kg, the other toxins did not exceed 61 mg/kg [282]. Samples of cereals, cereal by-products, corn plants and corn silage as well as non-grain-based feedstuffs (n ¼ 220) were collected during 2000 and 2001 in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol, MAS, DAS, T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol, DON, 3-acetylDON (3-ADON), 15-acetyldeoxynivealenol (15-ADON), NIV and fusarenon-X were determined by GC/MS. ZEA and a- and b-ZOL were analysed
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by HPLC with fluorescence and UV detection. Detection limits were in the range 1–19 mg/kg. Results showed that, of 125 samples of wheat, oats, corn, corn by-products, corn plants and corn silage, only 2 wheat samples did not contain any of the toxins analysed. Mean levels of positive samples were between 6 and 758 mg/kg. Of 95 samples of hay, lupins, peas, soy meal, rapeseed meal and other oilseed meals, 64 samples were toxin negative. DAS, T-2 triol, neosolaniol and FUS-X were not detected in any sample, while mean levels of positive samples were between 5 and 95 mg/kg [283]. Occurrence of mycotoxins was investigated in 219 food samples of plant origin collected in German retail outlets during 2000 and 2001. Samples, including cereal products, fruits, oilseeds and nuts, were tested for the presence of Fusarium toxins by GC-MS and HPLC with fluorescence and UV detection. Of 84 samples of cereal products, 60 were positive for Z1 of the following toxins: DON, 15-acetyl-DON, 3-acetyl-DON, NIV, T-2 toxin, HT-2 toxin, T-2 tetraol and ZEA, at incidences of 57, 13, 1, 10, 12, 37, 4 and 38%, respectively; however, none of the cereal samples contained detectable levels of scirpentriol, MAS, DAS, T-2 triol, fusarenon-X, a-ZOL or b-ZOL. Contents of DON were in the range 8–389 mg/kg, whereas concentration of other toxins were o100 mg/kg. The pseudocereals tested, amaranth, quinoa and buckwheat, were not contaminated by any of the toxins investigated. Of 85 fruit and vegetable samples, 10 were toxin positive; ZEA and the type A trichothecenes, MAS, scirpentriol, DAS and HT-2 toxin were detected in 7, 3, 2, 1 and 1 samples, respectively. Of 35 samples of oilseeds and nuts, 7 were toxin positive, with HT-2, T-2 and ZEA being detected in 4, 3 and 4 samples, respectively. None of the B-type trichothecenes were found in vegetables, fruit, oilseeds or nuts, and levels of the detected toxins were all o50 mg/kg [284]. A total of 449 grain samples (102 barley, 169 wheat and 178 oat), collected from different regions of Norway from 1996 to 1998, mainly from grain loads and silos, were analysed for type A and B trichothecenes by GC-MS. DON and HT-2 toxin were the trichothecenes most frequently detected, followed by T-2 toxin, NIV and scirpentriol. Scirpentriol was detected in only seven samples (W20 mg/kg). Oats were the most heavily contaminated grain species with an incidence and mean concentration of positive samples of 70% and 115 mg/kg for HT-2 toxin, 30% and 60 mg/kg for T-2 toxin, 57% and 104 mg/kg for DON and 10% and 56 mg/kg for NIV; corresponding values for barley were 22% and 73 mg/kg, 5% and 85 mg/kg, 17% and 155 mg/kg and 6% and 30 mg/kg, respectively, and for wheat they were 1.2% and 20 mg/kg, 0.6% and 20 mg/kg, 14% and 53 mg/kg and 0%, respectively. Norwegian oats were found to contain HT-2 and T-2 toxin in concentrations that might pose a threat to human health for high consumers of oats. The amount of DON detected was found to be significantly lower than in the crop from previous years [297].
6.6 Other mycotoxins Fusarium mycotoxins, beauvericin, enniatins (A, A1, B, B1) and moniliformin, were analysed in 38 Finnish grain samples (22 barley, 14 wheat, 1 oat, 1 rye)
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harvested in 2001–2002. The contaminating Fusarium spp. were identified using primer-specific PCR as well as morphological studies. All studied mycotoxins were found in the samples. Enniatins B and B1 were detected in all samples, and enniatin A, enniatin A1, beauvericin and moniliformin were found in 74, 95, 95 and 74% of the samples, respectively. Higher mycotoxin concentrations were detected in cereal grains harvested in 2001 than in those harvested in 2002. Mycotoxin levels were highest in samples harvested late in the autumn after a long rainy period. F. avenaceum, which infected up to 29.5% of kernels, was the most abundant Fusarium sp. in both years. A correlation (po 0.001) was found between F. avenaceum contamination level and the concentration of moniliformin, enniatin B and enniatin B1. It is suggested that the prevalence of beauvericin, enniatins and moniliformin in Finnish grain may pose a health risk to Finnish people and farm animals [315]. Norwegian grain samples (73 oats, 75 barley, 83 wheat) from the 2000–2002 growing seasons were examined for contamination with moniliformin, and the association between this fungal metabolite and the number of kernels infected with common Fusarium spp. was investigated. Before quantification of moniliformin using ion pairing RP-HPLC with diode array UV light detection, all samples were extracted using acetonitrile/water (84/16) and disposable strong anion exchange columns were used for clean up. The detection limit was 40 mg/kg. Moniliformin was found in 25, 32 and 76% of the barley, oats and wheat samples, respectively. Maximum concentrations of moniliformin in barley, oats and wheat were 380, 210 and 950 mg/kg, respectively. Prevalence of the moniliformin-producing F. avenaceum/arthrosporioides was as high as 100%, and on average, the infection level was W53%. Moniliformin concentrations were significantly correlated with grain species, growing season and infection with F. avenaceum/arthrosporioides and F. culmorum. Results indicate that the prevalence of moniliformin in Norwegian grain is high, especially in wheat, although it is thought that field conditions do not favour high levels of moniliformin contamination of grain [316]. Food-grade corn and corn-based foods intended for human consumption were analysed for the incidence and levels of FB1, FB2, moniliformin, and Fusarium fungi. One hundred food-grade commercial corn samples were obtained from two corn-processing companies at five different locations in the United States; 71% of samples contained FB1 at concentration of 43–1,642 mg/kg. None of the samples contained FB2; 50% of samples contained moniliformin (26–774 mg/kg). All samples were infected by Fusarium fungi at an infection rate of 8–88%. Of 34 samples of corn-based foods, purchased from supermarkets in Arizona, California, Nebraska and Ohio, 65% of samples contained FB1 (28–2,679 mg/kg). FB2 was detected in 29% of samples (30–797 mg/kg); 68% of samples contained moniliformin (31–858 mg/kg) and 62% of samples contained viable Fusarium mold propagules ranging from 9.5 10(1) to 5.5 10(5)/g). Simultaneous occurrence of FB1 and moniliformin was observed in 34% of corn samples and 53% of cornbased foods. It is concluded that the co-occurrence of FBs and moniliformin in food-grade corn and corn-based foods, shown in this study, indicates a risk of simultaneous exposure of consumers to both toxins [317].
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The occurrence of fusaproliferin was investigated in 22 samples of pre-harvest maize ears Fusarium rot, collected in 1994 in Italy. The most frequently recovered Fusarium species were F. moniliforme and F. proliferatum. Chemical analysis by high-perfomance LC and GC/MS identified FP (up to 500 mg/kg) in nine samples mostly infected by F. proliferatum. FP was often found to be associated with FB1 (up to 300 mg/kg) and beauvericin (up to 520 mg/kg). The occurrence of FP as a natural maize contaminant and its co-occurrence with FB1 and bauvericin is reported here for the first time [318]. Numerous samples (W2,000), representing 73 different kinds of Turkish foods and agricultural products, were analysed for total fungal flora. Samples were collected from 9 different agricultural geographical regions and from 34 different cities in Turkey. A total of 1977 isolates, representing 1,317 species and 40 different genera, were scanned for mycotoxin-producing activities, using 31 different mycotoxin standards. Qualitative screening, using TLC, specific solvent systems and standard methods, indicated 32.5% of the cultures were able to produce mycotoxin, with 19 different types of mycotoxins identified. The dominant mycotoxins, respectively, were roquefortin C and sterigmatocystin. Dominant fungal isolates varied by region. The extent of contamination was also determined and a mycotoxin risk profile was constructed for each agricultural region of Turkey [319].
7. DECONTAMINATION PROCEDURES An exhaustive review of the most used decontamination procedures is available on the Internet site of the European Mycotoxin Awareness Network (EMAN), http://www.mycotoxins.org. Typical biological detoxification can be defined as the enzymatic degradation or biotransformation of mycotoxins that can be obtained by either the whole cell or an enzyme system. Other approaches that can be regarded as biological include the use of biocompetitive agents and genetically engineered plants for reducing mycotoxin contamination. Lactic acid bacteria and yeasts expressing mycotoxin-degrading enzymes, may offer a natural way of providing these activities in fermentation processes. Biological degradation of aflatoxins has been reported for a number of microorganisms including lactic acid bacteria. Black yeast fungus and a bacterium strain have been found to hydrolyze FB1 to aminopentol and tricarballylic acid. The same microorganisms seem to be able to further detoxify aminopentol with release of CO2. Microbial strains (yeasts, fungi or bacteria) have been proposed for detoxification of DON, T-2 toxin, ZEA and other trichothecenes, but their practical use has not been shown. Among physical methods, mechanical separation, density segregation, colour sorting, removal of the fines or screenings from the bulk shipments of grains and nuts significantly reduce the mycotoxin content of grains. Simple washing procedures, using water or sodium carbonate solution, result in some reduction in concentrations of DON, ZEA and FBs in grains or corn cultures. Other
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methodologies are irradiation either with gamma-rays, microwaves and UV light, and solvent extraction. Another approach to detoxification of mycotoxins involves the use of adsorbent materials with the capacity to tightly bind and immobilize mycotoxins in the gastrointestinal tract of animals, thus reducing the bioavailability of the toxin. Alluminosilicates (HSCAS) in aqueous suspensions have been reported to tightly bind aflatoxins in aqueous suspensions; markedly diminish aflatoxin uptake by the blood and distribution to target organs; prevent aflatoxicoses in farm animals; decrease the level of AFM1 in milk from lactating dairy cattle. Divinylbenzene-styrene polymers (anion-exchange resins) bentonite and activated carbon exhibited beneficial effects when added to T-2 contaminated diet fed to rats. The addition of the resin to ZEA-contaminated diet resulted in a major decrease in urinary excretion of conjugated ZEA and its metabolites by rats. Cholestyramine has been reported to reduce the bioavailability of FBs, ZEA and OTA in mice and/or rats. As for chemical methods, it should be noted that chemical treatment is not allowed within the EC for commodities destined for human food. The chemicals used fall into the categories of acids, bases (e.g., ammonia, sodium hydroxide), oxidizing reagents (e.g., hydrogen peroxide, ozone), reducing agents (e.g., bisulfite, sugars), chlorinating agents (e.g., chlorine), salts and miscellaneous reagents such as formaldehyde. Often chemical treatments have been used in combination with physical treatments to increase the efficacy of decontamination. A wide variety of chemicals, including calcium hydroxide monomethylamine, sodium bisulfite, moist and dry ozone, chlorine gas, hydrogen peroxide, ascorbic acid, hydrochloric acid, sulfur dioxide, formaldehyde, ammonia and ammonium hydroxide, have been found to be effective (at different extents) against several mycotoxins, including DON, ZEA, T-2 toxin, aflatoxins and FBs. Ammoniation is the method that has received the most attention for detoxification of aflatoxincontaminated feeds and has been successfully used in the United States, France, the UK and Africa. Ammoniation did not show the same efficacy against FBs since no reduction in toxicity was found when ammoniated FB1 was fed to rats despite reduction in FB1 content. The reaction with sodium bisulfite is one of the most effective against DON in corn, although the treatment is not suitable for direct application to human foods. In addition to the above-mentioned preventive actions, a further key contributing component of an integrated approach to food safety is represented from the adoption of HACCP principles. This approach is intensely used to control or reduce mycotoxin levels before industrial processing revealing, so far, as a useful tool to keep under control mycotoxin presence within acceptable ‘‘safe’’ levels and to prevent the persistence of mycotoxins in finished products. The most effective mycotoxin control measures are to dry the commodity such that the water activity (Aw) is too low to support mould growth and/or prevent mycotoxin production. To prevent the growth of most moulds the Aw needs to be around 0.70, which translates to a moisture content of approximately 14% for maize and 7.0% for groundnuts at 201C.
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Another important key critical control point for avoiding the processing of materials at unacceptable mycotoxin levels is the acceptance of the raw material, by the implementation of periodical testing performance including the adoption of reliable sampling procedures and validated methods of analysis.
8. FUTURE NEEDS Despite the huge amount of data available on the major mycotoxins, most of them mainly related to toxicological effect, occurrence and methods of analysis, the global view on the real impact of those toxic to human beings is still not fully clear. Many gaps in the available status of art and many factors contribute to the above uncertainty. A preliminary consideration is that the scenario of mycotoxin impact on human beings is unfortunately very differentiated among the different areas of the world, thus implicating the need of different approaches depending on the dimension of the problem. Toxicology on experimental animals is rather well developed; however, the transposition to human biological systems, even taking into account the safety factors, can represent a source of ambiguity as well as the lack of toxicological studies on the combined effects of the very likely coexistent toxins. In addition, a substantial lack of toxicological information still exists for the effects on immunological systems. This effect is well known as far as farm animals are concerned: the hypothesis of similar effects on human beings could incredibly increase the scenario of diseases attributable to mycotoxins. Epidemiological studies could represent a valuable approach parallel and complementary to toxicological studies, but in this respect there is a substantial lack of investigation, some studies, many of them inconclusive, being available in the more affected areas where the acute effects are prevalent on chronic effects. One of the main outputs of the toxicological studies is represented by the possibility to compare the derived TDIs with the actual intake of the population whatever the methodology for this latter evaluation could be (point estimation or probabilistic approach). Actually, at least at European level, in the past decades valuable evaluations of dietary intake have been performed for the main mycotoxins with the data at that time available. However, the reliability of those data could currently be questionable under several respects: (i) the adopted consumption database were not fully suitable to the mycotoxin problem; (ii) the occurrence data could suffer from poor sampling procedures that could generally lead to the underestimation of the data; (iii) at the time of the evaluations several food products had not yet been recognised as susceptible to contamination, their contribution therefore missing in the total dietary intake. An additional gap both in the TDIs and in the actual total intake of the population is the consideration of vulnerable groups of population such as youngest and eldest people as well as people affected by peculiar diseases such as coeliac, renal, hepatic and immunological disorders.
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The above source of uncertainties suggests the need for new data for the total dietary intakes based on novel sets of data as obtained from fit for purpose plans. This need is consistent with the new vision of the risk analysis that recommends an iterative process in the acquisition of risk-related data. A corollary of the above future needs is related to the methods of analysis: the sector of reliable rapid methods should be further developed since they could certainly simplify the attainment of occurrence data as well as the implementation of prevention actions.
ACKNOWLEDGEMENT The authors wish to thank Mrs. Viviana Renzi for her valuable technical assistance in the editing of the text.
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CHAPT ER
13 Phycotoxins Kevin J. James, Daniel O’Driscoll, Javier Garcı´a Ferna´ndez and Ambrose Furey
Contents
1. 2. 3. 4.
Introduction Seafood-Poisoning Toxins Paralytic Shellfish Poisoning (PSP) Toxins Diarrhetic Shellfish Poisoning (DSP) Toxins 4.1 Toxicity of DSP toxins 4.2 Analysis of DSP toxins 5. Azaspiracid Poisoning (AZP) Toxins 5.1 Determination of azaspiracids using LC-MS and LC-MSn methods 6. Amnesic Shellfish Poisoning (ASP) Toxins 7. Yessotoxins (YTXs) 7.1 Rapid screening methods for YTXs 7.2 Determination of yessotoxins using LC-MS and LC-MSn methods 8. Neurotoxin Shellfish Poisoning (NSP) Toxins References
429 430 430 432 435 435 439 440 442 445 446 447 448 452
1. INTRODUCTION Phytoplankton are microalgae and are the main contributor to the marine food web as well as oxygen on earth. The beneficial role of algae in the food chain arises from the fact that they are the only organisms that can readily make long chain polyunsaturated fatty acids (PUFAs). Shellfish feed on phytoplankton and the potential beneficial role of shellfish in the human diet has been attributed to the presence of oils that are rich in PUFAs. Shellfish are also a rich source of protein, essential minerals, especially iron, vitamins A and D. However, some marine microalgae are known to produce bioactive compounds that negatively impact on human health through their accumulation in shellfish [1,2]. Many of these toxins Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00013-5
r 2008 Elsevier B.V. All rights reserved.
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are known to be highly potent [3]. The proliferation of toxin-bearing algae, mainly diatoms and dinoflagellates, called harmful algal blooms (HABs), has been a major cause of concern in recent years [4]. Bivalve molluscs filter large volumes of water when grazing on microalgae, and can concentrate both bacterial pathogens and phycotoxins [5]. These shellfish, especially mussels, scallops, oysters and clams, are major vectors for toxins that are implicated in several human toxic syndromes. These include amnesic shellfish poisoning (ASP) [6], diarrhetic shellfish poisoning (DSP) [7], paralytic shellfish poisoning (PSP) [8], neurotoxic shellfish poisoning (NSP) [9] and azaspiracid poisoning (AZP) [10]. ASP first came to attention in Canada in 1987 when human fatalities occurred from eating mussels (Mytilus edulis) [11]. Domoic acid (DA), which is produced by marine diatoms of the Pseudo-nitzschia spp., was identified as the causative toxic agent [12,13]. ASP causes gastric upset, headache and dizziness but the syndrome was named due to the persistent short-term memory impairment experienced by some patients [11]. Toxins produced mainly by marine dinoflagellates are some of the most potent poisons known and have a major impact on human health [1]. The accumulation of toxins in shellfish and fish has led to serious human toxicosis as well as animal and bird deaths. Table 1 shows the toxins produced by marine phytoplankton.
2. SEAFOOD-POISONING TOXINS Six main classes that cause poisoning from the consumption of seafood are discussed, with an emphasis on the methods for their detection and determination. The phycotoxin poisonings are widely known by their acronyms and include: Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Azaspiracid Poisoning (AZP), Amnesic Shellfish Poisoning (ASP), Yessotoxins (YTX) and Neurotoxic Shellfish Poisoning (NSP). Other less common seafood-poisoning toxins that are not discussed in this review include ciguatoxins [14], tetrodotoxins [15] and palytoxins [16].
3. PARALYTIC SHELLFISH POISONING (PSP) TOXINS The PSP toxins are basic, water soluble compounds, which are extremely sensitive to alkaline pH and air-oxidation. PSP toxins are potent marine neurotoxins, which specifically block the excitation current in the nerve and muscle cells, and this finally results in signs of paralysis. To date, there are over 21 known saxitoxin congeners. The structures of saxitoxin (Figure 1) congeners vary by differing combinations of hydroxyl and sulphate substituents at four sites on the molecule (R1–R4). Based on the substitutions at R4, the saxitoxins can be subdivided into four sub-groups, the carbamate, sulfocarbamoyl, decarbamoyl and deoxydecarbamoyl toxins. Substitutions at R4 result in substantial changes in toxicity, with the carbamate toxins being the most potent [17].
Phycotoxins
Table 1
Seafood toxic syndromes and toxin-producing phytoplankton
Toxic syndrome
Toxins
Affected seafood
Source of toxins
Paralytic shellfish poisoning (PSP)
Saxitoxin (STX), Neosaxitoxin (NEO), Gonyautoxin (GTX) (21 analogues) Okadaic acid (OA), Dinophysistoxins (DTXs), Pectenotoxins (PTXs) Yessotoxins (YTXs)a
Shellfish, crustacions
Alexandrium spp. [136], Gymnodinium spp. [137]
Shellfish
Dinophysis spp. [35,38], Prorocentrum spp. [37,138]
Shellfish
Protoceratium reticulatum [108], Lingulodinium polyedrum [139] Karenia brevis (Ptychodiscus brevis) [140,9] Pseudo-nitzschia spp. [12,141] Protoperidinium crassipes [76] Gambierdiscus toxicus [142] Ostreopsis siamensis [143]
Diarrhetic shellfish poisoning (DSP)
a
431
Neurotoxin shellfish poisoning (NSP)
Brevetoxins (PbTx)
Shellfish, finfish
Amnesic shellfish poisoning (ASP) Azaspiracid poisoning (AZP) Ciguatera poisoning
Domoic acid (DA) and analogues Azaspiracids (AZAs) Ciguatoxins (CTXs)
Shellfish, finfish
Palytoxin poisoning
Palytoxin
Shellfish Finfish Seaweed, crabs, finfish
YTXs were originally included in the DSP category.
Human symptoms of PSP vary depending on the dose and the individual. In the case of a mild intoxication, a sensation of tingling or numbness of the lips is experienced, gradually progressing to the face and neck; pins and needles in the extremities; headache, vertigo, nausea, vomiting and diarrhoea occur in tandem. Extreme intoxication results in muscular paralysis, choking and extreme respiratory difficulty often culminating in respiratory failure [18]. Although marine dinoflagellates have been identified as the progenitors of PSP toxins that contaminate bivalve shellfish, these toxins are also produced in freshwaters by cyanobacteria (Aphanizomenon flos-aquae) [19] and have been responsible for mass mortalities of cattle and sheep [20]. Several analytical techniques have been developed to determine the PSP toxins including: receptor binding assay [21], mouse bioassay [22], liquid chromatography with fluorimetric detection (LC-FLD) [23], liquid chromatography with mass spectrometric detection (LC-MS) [24] and capillary electrophoresis [25].
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R4 NH
O O R1
NH
N
NH2 H2N
N R3
Saxitoxins STX GTX II GTX III NEO GTX I
R1 H H H OH OH
NH OH OH R2
R2 H OSO −3 H H − OSO 3
R3 H H − OSO 3 H H
Figure 1 Structure of the most common PSP toxins; R4 ¼ H; Carbamoyl.
Hydrophilic interaction LC with MS detection has also been used for the analysis of PSP toxins [26]. The most frequently used methods for PSP determination involve LC-FLD and these are based on the oxidation of PSP toxins to fluorescent products. These sensitive determinations can be performed post-column, as first developed by Oshima [27], and these remain the definitive methods for the determination of a wide range of PSP analogues. This derivatization procedure has also been used for the determination of PSP toxins in body fluids in the forensic investigation of fatal intoxications following shellfish consumption [18]. However, the pre-column oxidation method, developed by Lawrence et al., is more convenient for the routine monitoring of PSP toxicity and has recently been adopted as an AOAC International reference method [28–30]. PSP toxins are basic and can be readily extracted from a shellfish homogenate using dilute hydrochloric acid. The oxidation reaction can be carried out using either periodate or peroxide and the fluorescent products can be separated using either reversed-phase or ion exchange columns. Figure 2 shows the chromatograms that were obtained from contaminated mussel tissue [29]. The main disadvantage of this method is that not all PSP analogues can be identified as the oxidation step can produce the same fluorescent product from more than one PSP toxin.
4. DIARRHETIC SHELLFISH POISONING (DSP) TOXINS DSP is a widely distributed seafood contamination. Three classes of toxins were initially included in the DSP group (a) dinophysistoxins, (b) pectenotoxins (PTXs)
Phycotoxins
433
Figure 2 Sample chromatograms from the inter-laboratory study of determination of PSP toxins in various bivalve shellfish, using the Lawrence LC-FLD method that involves pre-column oxidation. (A) Analysis of mussels containing PSP toxins; (B) Analysis of clams containing PSP toxins. Adapted with permission from Ref. [29]. Copyright 2005 by AOAC International.
and (c) yessotoxins (YTXs) [7,31,32]. However, YTX is not diarrhetic and is no longer classified as a DSP toxin [33,34]. DSP was first reported in Japan in 1978 but the illness is now recognized as an important threat to public health throughout the world and outbreaks have resulted in prolonged closures of shellfish culturing industries. Dinophysistoxin-1 (DTX1) was identified in Japan as the causative toxin and was accumulated in bivalve shellfish through filter feeding on the dinoflagellate, Dinophysis fortii [35]. However, most incidents of DSP have involved the demethyl analogue, okadaic
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acid (OA), as the responsible toxin arising from a variety of Dinophysis sp. [36–38]. DTX2, an isomer of OA (Figure 3), was first isolated from Irish mussels and this was the predominant toxin during major DSP events in 1991 and 1994 [39,40]. DTX2 was subsequently identified in shellfish along the western coastline of Europe [25]. The PTXs were first isolated from toxic shellfish in Japan and frequently cooccur with OA and DTXs [7]. It is often observed that the toxin profiles in shellfish are more complex than in the marine phytoplankton where toxins originate. Thus, although PTX2 (Figure 4A, R ¼ CH3) is the main PTX in Dinophysis spp., several PTX analogues are found in shellfish tissues that are formed from PTX2 by repeated oxidation; PTX1 (R ¼ CH2OH), PTX3 (R ¼ CHO) and PTX6 (R ¼ COOH) as well as other PTX analogues that are formed by epimerization at C-7 [32]. Pectenotoxin-2 seco acids (PTX2SAs) have also been isolated and identified in dinoflagellates and shellfish [41,42]. PTX2SAs have an open chain structure and not a lactone ring like the rest of the PTXs (Figure 4B).
O
HO
HO
O
OH
R1
O
O
O
O
OH
H
O
H OH 1
O R2 2
Figure 3 Structures of dinophysistoxins; Okadaic acid (OA); R ¼ Me, R ¼ H; dinophysistoxin-1 (DTX1); R1 ¼ R2 ¼ Me, dinophysistoxin-2 (DTX2); R1 ¼ H, R2 ¼ Me.
O
CH3 O
A)
O
O H3C
O OH CH3 O
OH OH
O
O
O
R CH3
O
O
B)
HO
CH3
O
CH3
H 3C
O
OH CH3 O
OH OH O O
O
O
OH H 3C
H 3C
O
O
O CH3
CH3
CH3
Figure 4 Structure of (A) Pectenotoxins; PTX2 (R ¼ CH3), PTX1 (R ¼ CH2OH), PTX3 (R ¼ CHO), PTX6 (R ¼ COOH) and (B) Pectenotoxin-2 seco acids (PTX2SAs).
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4.1 Toxicity of DSP toxins Serious diarrhetic effects have only been proven for DTXs. Hamano et al. first investigated the diarrhetic effects caused by OA [43]. The three major DTXs, OA, DTX2 and DTX1, are potent inhibitors of protein phosphatases (PP1 and PP2A) [44] and they are tumour promoters [45]. This phosphatase inhibition may be linked to degenerative changes in absorptive epithelium cells of the small intestine thus producing diarrhoea. The EU regulatory limit for total DTXs in shellfish is 0.16 mg/g edible tissues. Although PTX2 can induce diarrhetic symptoms, this only occurs at relatively high levels when compared with the DTXs [46]. The PTX2SAs exhibit substantially less toxicity than PTXs, indicating that the cyclic structure of PTX2 is important for toxicity [41].
4.2 Analysis of DSP toxins Live animal bioassays were the first methods that were used for the detection of DSP toxins, but problems due to the lack of sensitivity, false positives, lack of method validation and the prohibition of such testing in many countries on ethical considerations, has led to an examination of a variety of alternative analytical methods [47,48].
4.2.1 Rapid screening methods for DSP toxins Rapid screening methods, based on immunoassays provide convenient alternatives to live animal bioassays for screening shellfish tissues but these methods cannot be used for the precise quantitative determination of the DSP toxins [49–51]. A very sensitive method utilized LC linked with a protein phosphatase assay that used a 32P-labeled substrate but there are problems associated with using radioactive materials in regulatory laboratories [52]. More convenient colourimetric phosphatase inhibition assays have been developed in which the ability of PP2A to dephosphorylate a colourless substrate, p-nitrophenyl phosphate (p-NPP), to a coloured substrate, p-nitrophenol, was used to determine OA and DTXs [53,54]. A fluorescent inhibition assay for these toxins provides enhanced sensitivity for DTX detection [55].
4.2.2 Determination of DSP toxins using LC-FLD LC-FLD using the carboxylic acid derivatizing reagent, 9-anthryldiazomethane (ADAM), has been the most widely used method for the determination of DTXs and PTX2SAs [38,56]. Other fluorimetric reagents have been successfully employed for the determination of DSP toxins and have been discussed in a previous review [48]. Unfortunately, PTX2 and most other PTXs cannot be determined using LC-FLD as they lack a carboxylic acid moiety. Although these LC-FLD methods are sensitive, sample clean-up, especially with shellfish samples, can be prolonged and relatively complicated [57,58]. The high sensitivity of LC-FLD was demonstrated when this method was used to show that the non-culturable phytoplankton, Dinophysis acuta, was the progenitor of DTX2 by the analysis of unialgal samples (22–100 cells), collected manually from microscope slides [38].
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4.2.3 Determination of DSP toxins using LC-MS and LC-MSn methods One of the earliest applications of LC-MS for the analysis of food contaminants was the determination of DSP toxins in shellfish [59]. LC-MS allows the determination of DTXs and PTXs without recourse to derivatization and multiple tandem MS methods do not require rigorous sample clean-up [60]. Table 2 summarizes the LC-MS methods that have been used for the analysis of DSP toxins (DTXs and PTXs). All of the MS methods used were carried out using an API source with an ionspray/electrospray interface, coupled to a single quadrupole or a triple quadrupole mass analyser. A major problem with LC-MS methods for the analysis of toxins in shellfish tissues is ion suppression and this is evident when using exhaustive extraction procedures, especially alcohols without sample clean-up. The consequence is that multi-toxin methods that encompass a wide range of analyte polarities are very difficult to validate for quantitative analysis but may play a role as screening methods for toxins. High performance size exclusion chromatography has been applied for the clean-up of raw extracts from mussel tissue. The proposed protocol can be performed automatically to enable the fast and sensitive analysis of a large number of samples. The recovery using this method was approximately 70% with good repeatability [61]. One method to determine the potential impact of ion suppression is to analyse an extract from a toxin-free shellfish sample with simultaneous infusion of a toxin standard. OA was used as a representative toxin and the signal for this toxin was acceptably consistent in the regions where toxins are expected to elute, thus showing that ion suppression did not significantly vary [62]. Different MS scans events have been applied for the identification and quantification of DSP toxins in shellfish and phytoplankton samples. Single scan MS experiments and selected ion monitoring (SIM) were used in most LC-MS applications. SIM is useful for the identification of toxins and screening samples but the lower selectivity when compared with multiple tandem MS does present difficulties, especially with shellfish samples whenever there is limited sample clean-up [63,64]. Both positive and negative modes have been used for the LC-MS determination of DSP toxins (Table 2). In positive ion mode, MS is notorious for generating multiple adduct ions for DSP toxins, with [M+NH4]+, [M+Na]+and [M+H]+ ions being observed together in the full scan spectra [64,65]. This has a detrimental effect on quantitation due to division of signal. It is prudent therefore to use an additive, for example, ammonium or sodium acetate, to assist in the formation of a predominant ion type. However, some adduct ions, especially the sodiated ions, are very stable and are difficult to fragment. The first LC-MS/MS method for polyether toxins was applied to the determination of four DTXs in shellfish using selected reaction monitoring (SRM). Three of these toxins are isomers with identical CID spectra and therefore chromatographic separation is essential [66]. Negative polarity does not exhibit the problems of multiple ion formation but in some instances the spectra may not be rich in product ions. Draisci et al. proposed a negative mode method for the different DTXs and PTXs but poor resolution was achieved for isomers [60]. Suzuki et al. developed another negative polarity method using LC-MS (SIM) for OA, DTX1 and PTX6 with reasonable detection limits [65].
Table 2
LC-MS methods for the determination of DSP toxins
Compound
Matrix
OA, DTX-1, DTX-2, DTX-3
Mussel (M. edulis)
OA, DTX1, DTX2 OA, DTX1, DTX2, DTX2B OA, PTX2SAs
PTX2, PTX2SAs
OA, DTX1, PTX6, PTX2SAs PTX1, PTX2
Stationary phase
5 mm Vydac 214TP (C4) column (250 2.1 mm) Phytoplankton Vydac201TP C-18 Mussels (M. edulis) (250 2.1 mm, 5 mm) Mussel (M. edulis), Supelcosil LC18DB Mussel (300 1 mm, (M. galloprovincialis) 5 mm) Mussel (M. edulis), Supelcosil LC18DB (M. galloprovincialis) (300 1 mm, 5 mm)
Mobile phase
Ionization MS experiments source
Detection limit
References
ACN:H2O (90:10, v/v) 0.1%TFA
ISP
SIM; [M+H]+
0.4 mg/ml
[144] ([145])
70–80% MeOH in H2O 0.1% TFA
ISP
SIM; [M+H]+
8 ng/g HP
[146]
ACN:H2O (80:20, v/v, 0.1% TFA)
ISP
0.03 mg/g HP
[66] ([64])
ACN:H2O (80:20, v/v) 0.1% TFA (positive) and 2mM NH4OH (negative) ACN:H2O (7:3, v/v) containing 0.1% AcOH
ISP
SRM; Product ion scan; [M+H]+ SIM; [M+H]+; [M-H]
nr
[60] ([42])
ESI
SIM; [M-H]
400 pg per injection
[65] ([147])
ESI
SIM; [M-H]
0.01–0.08 mg/g HP
[63] ([61,65,148,149])
Scallop Luna-C18(2) (P. yessoensis) (150 2 mm, 5 mm) Mussels (M. galloprovincialis) ACN:0.05% AcOH Scallop Symmetry C18 (70:30, v/v). (P. yessoensis) (150 2.1 mm, 3.5 mm) Develosil ODS-MG- ACN:0.05% AcOH (70:30, v/v). 5 (150 2 mm, 5 mm)
SIM; [M+NH4]+
Table 2 (Continued ) Compound
Matrix
Stationary phase
Mobile phase
Ionization MS experiments source
PTX6, PTX2, PTX2SAs, PTX1i, PTX1SAs OA, DTX1, DTX2, PTX2, PTX2SAs OA, DTX1, DTX2, PTX1, PTX2
Greenshell Mussel (P. cancliculus)
Luna-C18(2) (150 2 mm, 5 mm)
ESI
SRM; [M+NH4]+ 0.01 mg/g
[150] ([151])
Mussel (M. edulis) Phytoplankton cells (D. acuta) Mussel (M. edulis) Oysters (C. gigas) Scallop (P. maximus)
Luna-C18(2) (150 2.2 mm, 5 mm) C8 (50 2.1 mm, 3 mm)
ESI
SRM; [M-H]
0.5 ng/g
[62] ([67])
ESI
SIM; [M-H]; [M+H]+; [M+Na]+
nr
[152]
Mussels (M. edulis)
Hypersil-BDS-C8 (50 2 mm, 3 mm)
ACN:H2O 4mM NH4OH and 50 mM formic acid. ACN:H2O both containing 1 mM NH4OAc 5mM ammonium acetate (pH 6.8) in water and ACN (95% v/v) A=water and B=ACN–water (95:5), both with 50 mM formic acid and 2 mM ammonium formate.
ESI
SRM or Product Ion Scanning
nr
[153] ([154])
OA, DTX1, PTX2, PTX2SAs
ESI, electrospray; ISP, ionspray; SSI, sonic spray; TSI, turbospray; HP, hepatopancreas tissues; SRM, selected reaction monitoring; nr, not reported.
Detection limit
References
439
Phycotoxins
x 17
Intensity %
100
2
x 136
3
5
50 4
1
x 23
x 233
B) 100 1 Intensity %
A)
6
5 x 28
50 x 14
4
3 0
1
2
3
4
5 6
7 8 (min)
9 10 11
0
1
2 3
4
5
6 7 (min)
8
9 10 11
Figure 5 LC-MS/MS of DSP toxins. (A) Irish mussels (M. edulis), (B) Norwegian mussels (M. edulis). (1) OA (2.8 min), (2) DTX2 (3.5 min), (3) PTX2SAi (4.9 min), (4) 7-epi-PTX2SA (6.5 min), (5) PTX2 (9.8 min), (6) DTX1 (6.65 min). (Signal amplification, if any, is shown above each peak.). LC conditions: a gradient of acetonitrile-water containing 1 mM ammonium acetate was used and the column was a Luna C-18(2) (150 2.1 mm, 5 mm, Phenomenex). Adapted with permission from Ref. [62]. Copyright 2004 by Elsevier.
The identification of DSP toxins in marine biological material is seriously hampered by the lack of commercial availability of many standard toxins and the isolation of toxins from shellfish tissues to use as standards is a very protracted process. Using LC-MS/MS, characteristic product ion spectra can be obtained for each component in a mixture without interference from the other components. However, for most applications where sensitivity of determination using LC-MS/MS is important, the monitoring of specific precursor/product ions with Q1 and Q3, respectively, whilst fragmentation occurs in Q2 (SRM/MRM) is necessary. Triple quadrupole MS offers excellent quantitative analysis with high selectivity and a wide calibration range. The polarity difference between neutral toxins, such as PTX2, and the acidic toxins has implications both for efficient LC separations and MS ionization efficiency. In Figure 5, the SRM negative mode (Q1/Q3 pairs) were: 803/255 (OA and DTX2), 876/137 (PTX2SAs), 817/255 (DTX1), 857/137 and (PTX2). The optimized LC gradient gave excellent resolution of the OA isomers, the PTX2SA isomers and PTX2 in relatively short run-times (10 min) [62,67]. The case with which toxins can be determine when present in widely different concentrations is demonstrated in Figure 5B. Thus, PTX2 (peak 5) has a signal that is 233 times less than DTXI (peak 6).
5. AZASPIRACID POISONING (AZP) TOXINS AZP is the most recently discovered toxic syndrome from shellfish consumption [10]. The first confirmed event was in 1995 in the Netherlands and resulted from
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the consumption of mussels (M. edulis) from Killary Harbour in Ireland. Azaspiracids have also been found in other European countries, including the UK, Norway, France and Spain [68–70] and throughout the western coastline of Ireland [71–73]. Azaspiracids possess a polyether backbone, that include a trispiro ring assembly, an azaspiro ring fused with a 2,9-dioxabicyclo (3.3.1) nonane, and a terminal carboxylic group and co-occurs with an isomer, AZA6, and demethyl and methyl analogues, AZA1 and AZA3, respectively. Symptoms of AZP include nausea, vomiting, diarrhoea and abdominal cramps; similar to DSP. However, AZA1 is distinctly different from DSP toxins as it target organs include liver, spleen, the small intestine and it has also been shown to be carcinogenic [74,75]. The dinoflagellate, Protoperidinium crassipes, was identified as the responsible organism for the production of azaspiracids. This was achieved with LC-MS3 using only 200 cells that were manually collected from a microscope slide [76]. Protoperidinium spp. are difficult to culture in a laboratory environment and extensive sampling may need to obtain wild phytoplankton. P. crassipes is distributed in temperate to tropical waters and may even form blooms in warm water estuaries [77]. Following the total synthesis by Nicolaou et al., the structure of AZA1 (and consequently the structures of other AZAs) has been revised [78]. Figure 6 shows the revised structures of azaspiracids.
5.1 Determination of azaspiracids using LC-MS and LC-MSn methods LC-MS quantitative analysis of a complex matrix, such as a shellfish tissue extract, can be problematic and interferences, including ion suppression, are to be expected. The first LC-MS method that was employed to determine AZA1–AZA3 involved a preliminary diol solid phase extraction (SPE) clean-up step [79]. A comprehensive study of the clean-up and toxin recoveries using various types of SPE showed that C-18 and diol phases were preferred for azaspiracids [80]. Sample clean-up using SPE should also be applicable for incorporation into less specific and less sensitive analytical methods for the determination of azaspiracids in seafood. LC-MS(/MS) is a universal method for marine toxins, and has been successfully applied to the simultaneous determination of various groups of polyether toxins [60]. The first LC-MS/MS method was developed by Draisci et al. for the determination of the predominant azaspiracid, AZA1, in mussels using a triple quadrupole instrument [81]. This was later extended to the analysis of other azaspiracids using a monolithic column [82]. Although monolithic columns produce high chromatographic resolution, the high flow rates involved mean that these columns are rarely adopted for use with MS instruments. Using an ion-trap detector (ITD) MS, LC-MS3 methods have been developed for a wide range of azaspiracids [83–86], that included the full chromatographic resolution of eleven azaspiracids using reversed-phase LC. The elucidation of the fragmentation pathways of azaspiracids was especially important for the development of analytical methods. To this end, the powerful complementary roles of hybrid quadrupole time-of-flight (QqTOF) MS that produces high mass accuracy data and ITD MS that yields MSn data (Figure 7, Table 3), was
Phycotoxins
441
R1 O HO
8
R3
A 10 3
1
O
H
O
B
O H 14
C
D 19 O
H
H 20
HO 21 22 O E 23 H 39
NH
OH R2 R4
H
I H
37
O
O G O
27 28
F 30
H
Toxin
R1
R2
R3
R4
AZA1
H
CH3
H
H
AZA2
CH3
CH3
H
H
AZA3
H
H
H
H
AZA4
H
H
OH
H
AZA5
H
H
H
OH
AZA6
CH3
H
H
H
AZA7
H
CH3
OH
H
AZA8
H
CH3
H
OH
AZA9
CH3
H
OH
H
AZA10
CH3
H
H
OH
AZA11
CH3
CH3
OH
H
Figure 6 Structures of azaspiracids.
demonstrated in a study of AZA1–AZA3 fragmentations [87]. These toxins exhibit charge-remote fragmentation in positive mode following the initial water loss from the geminal diol moiety. A significant fragmentation is the loss of the A-ring in azaspiracids which was observed at the MS3 stage. This results in the loss of the C1-C9 portion of azaspiracids that contains the R1 and R3 substituents, leaving a residual ion [M+H-H2O-C9H10O2R1R3]+, that retains R2 and R4 [87]. It was possible to select unique product ions for each azaspiracid isomer (Table 3), thus eliminating the requirement for complete chromatographic separation for the determination of isomers [86]. Figure 8 shows the chromatograms and spectra from the analysis of the isomers, AZA4 and AZA5, which have the same mass but were conveniently discriminated by the selection of characteristic
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Kevin J. James et al.
R1 A-ring cleavage C-ring cleavage
A O H C H
HO O NH
10 100
H O
R2
A-ring
E
O
E-ring cleavage
G O
672.6
F
H G-ring cleavage
C27-C28 cleavage
-H2O [C27H44NO5]+
C19-C20
60
362.4
-H2O
770.6
300
-H2O
C-ring
444.5
-H2O
-H2O
512.4 530.3
344.3
350
-H2O
806.6 462.4
0 250
-H2O
-H2O
-H2O
752.5
426.5 262.2 301.1
788.6
400
450
[M+H-5H2O]+
20
[C22H36NO3]+
40
E-ring
654.5
[C31H48NO6]+
C27-C28 [C16H26NO3-H2O]+
Relative Abundance (%)
C19-C20 cleavage OH
H
I
80
H
D O
[M+H-3H2O]
O
[M+H-2H2O]+
O
H
[M+H-4H2O]+
HO
B
[M+H-H2OC9H12O2]+
O
500
55
636.5 588.1
600 m/z
650
700
750
800
850
900
Figure 7 Multiple tandem mass spectrum (MS3) of AZA1, produced by positive electrospray ionization using a quadrupole ion-trap (ITD) instrument. The inset shows the main chargeremote fragmentation processes. (The G-ring fragmentation is not observed in ITD.) Adapted with permission from Ref. [86]. Copyright 2004 by Elsevier.
product ions formed by A-ring fragmentation [85]. Although LC-MSn using a ITD MS instrument is less sensitive than MRM methods using a triple quadrupole MS instrument, the ability to acquire spectra in MS2 and MS3 modes without a diminution in the sensitivity of detection is an important advantage of ITD MS.
6. AMNESIC SHELLFISH POISONING (ASP) TOXINS DA (Figure 9) is an excitatory amino acid that has been implicated as responsible for a toxic outbreak in Canada in 1987 [13]. The intoxicated patients exhibited
Phycotoxins
443
Table 3 Azaspiracid structural assignments, R1–R4 (see Figure 7), and the molecule-related and product ion masses using positive electrospray mass spectrometry Name
R1
R2
R3
R4
[M+H]+
[M+H–H2O]+
[M+H–H2O–C9H10O2R1R3]+
AZA1 AZA2 AZA3 AZA4 AZA5 AZA6 AZA7 AZA8 AZA9 AZA10 AZA11
H CH3 H H H CH3 H H CH3 CH3 CH3
CH3 CH3 H H H H CH3 CH3 H H CH3
H H H OH H H OH H OH H OH
H H H H OH H H OH H OH H
842.5 856.5 828.5 844.5 844.5 842.5 858.5 858.5 858.5 858.5 872.5
824.5 838.5 810.5 826.5 826.5 824.5 840.5 840.5 840.5 840.5 854.5
672.4 672.4 658.4 658.4 674.4 658.4 672.4 688.4 658.4 674.4 672.4
gastrointestinal disorders, including nausea, vomiting, abdominal cramps and diarrhoea, manifesting about 24 h after the consumption of mussels. This was followed by the neurological symptoms of headache, confusion, disorientation, seizures and coma within 48–72 h. However, the permanent loss of short-term memory in some of the survivors led this toxic syndrome to be named ASP. The toxin in mussels causing the intoxication was identified as DA, which is a tricarboxylic amino acid (Figure 9). It was later shown that diatoms of the PseudoNitzschia spp. were the causative organisms for producing DA [12]. Following this outbreak of ASP, there have been worldwide reports of DA contamination of seafood [88–91]. Several methods have been developed for the detection of DA in shellfish, including radioimmunoassay and enzyme immunoassay [92,93]. However, LC-UV is used by most regulatory agencies for the quantitative determination of DA in shellfish and a limit of 20 mg DA/g has been generally adopted [94,95]. Analysis is complicated somewhat by the presence of isomers of DA, as well as tryptophan, in naturally contaminated samples. The DA analogues differ from DA by isomerism involving diene side-chain and they are not always chromatographically resolved. A rapid and sensitive LC-UV method has been developed for analysis of DA in shellfish extracts without the need for SPE cleanup. Isocratic reversed-phase LC separation of DA and its isomers from shellfish matrix interferences and from the prevalent amino acid, tryptophan, was achieved by using an optimized mobile phase pH of 2.5 [96]. Fluorimetric derivatization of DA was used in the application of three LC-FLD methods and the improved sensitivity has allowed the determination of DA at much lower concentrations in seawater and marine phytoplankton. Derivatizing reagents used include fluorenylmethoxycarbonyl chloride (FMOC) [97], 6-aminoquinoloyl-N-hydroxysuccinimidyl carbamate [98] and 4-fluoro7-nitro-2,1,3-benzoxadiazole (NBD-F) [99]. However, only the NBD-F method could be readily applied to the analysis of DA in shellfish without post-reaction
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A
95
90
Relative Abundance
4.63
90 85 80 75
B (RT=3.73 min) min
80 70 658.5
60 50 40 30
790.4826.6
20
65 60
10
55
0
50 45
100
40
90
35
80
Relative Abundance
Relative Abundance
70
808.5
x5
100
3.73
100
30 25 20 15 10 5
764.6 448.6 374.1 251.3 380.4 467. 640.4 851.8 545.1 4 400 600 800 m/z
C (RT=4.63 min)
70 60 674.4
50 40 30
4
6
8
10
Time (min)
790.4 826.4
446.0 362.5 326.2 438.1 509.5 587.1
10 2
808.5
x5
20
0
1000
0
400
600 m/z
736.8 800
1000
Figure 8 (A) Chromatogram showing the separation of the isomers, AZA4 & AZA5, which was obtained using LC-MS3; AZA4 (3.73 min) and AZA5 (4.63 min). (B) and (C) are the mass spectra corresponding to AZA4 and AZA5, respectively. Reproduced with permission from Ref. [84]. Copyright 2002 by Elsevier. CH3 COOH H
COOH CH3
N
COOH
H
Figure 9 Structure of domoic acid.
clean-up using SPE. Figure 10 shows the LC-FLD chromatograms for extracts from scallops (Pecten maximus), following derivatization with NBD-F, in which DA and two DA isomers (arrowed) are resolved as well as tryptophan (Try).
Phycotoxins
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Fluorescence Intensity
DA
Try
0
2
4
6
8
10
12 14 Time (min)
Figure 10 LC-FLD of domoic acid in an extract from scallops, contaminated with DA. DA isomers (arrowed) are resolved from DA and tryptophan (Try) does not interfere.
Other methods that have been employed for the determination of DA include capillary electrophoresis with UV detection [100] and derivatization followed by GC-MS [101]. Several LC-MS methods for DA have been developed [102,103], but the benefits of using multiple tandem MS, including improved selectivity and sensitivity, have been demonstrated [104,105].
7. YESSOTOXINS (YTXS) YTXs originally were isolated from the digestive glands of scallops (Patinopecten yessoensis) [106]. Originally classified as DSP toxins as they do not induce diarrhoea YTXs have now been removed from this category [33,34]. The YTXs are sulphonated polyether toxins (Figure 11) and have been reported in shellfish in several countries including, Japan [106], Norway [107], New Zealand [108] and Italy [109]. Structurally, YTX is unusual as it is a sulphonated polyether with eleven fused rings [106,110]. Homoyessotoxin (homoYTX) has an additional methylene in the sulphated side-chain and together with their 45-hydroxy analogues, these constitute the main YTXs that have been found in shellfish. Several low-abundant analogues of YTX have also been reported [111,112]. Toxicological studies carried out in rodents show that YTX is highly toxic towards mice after intraperitoneal (i.p.) administration but it is virtually non-toxic when ingested orally [34]. The dinoflagellate, Protoceratium reticulatum, was found to produce high levels of YTX in New Zealand and Japan [113] and Lingulodinium polyedrum (Gonyaulax polyedra) has been identified as the producer of homoYTX as well as YTX in shellfish collected in the Adriatic Sea [114]. The mouse bioassay that is used for regulatory control of shellfish is very sensitive to YTX and false positives assay results are common for DSP assays. This is an unfortunate scenario as this bioassay, which has the greatest sensitivity for the least toxic shellfish contaminant, is still the official EU method.
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Kevin J. James et al.
OH C 502
O
439
O
467
O OH 541†/319
186 -O3S O
O
2-
[C55H80O21S2]
[C26H37O10S]-
[C46H68O20S2]2-
[C26H38O14S2]2-
396.1352
[C42H62O19S2]2-
467.1639
O
-
O
[C39H58O18S2]2-
O
[C11H16O10S2]2
96.9519 [HSO4]-
Intensity (counts)
O
O
2.0e4
1.0e4
396
O
[C35H52O16S2]2
-O3S
3.0e4
O
O
570.2317
-
233.1829 319.085 439.1582 502.1835 186.014 541.2050 284.0664 7 9 0.0
100
200
300 400 m/z (amu)
500
600
Figure 11 Nano ESI QqTOF MS spectrum of YTX in negative mode and the main fragmentation processes. Adapted with permission from Ref. [124]. Copyright 2004 by Elsevier.
A sensitive LC-FLD method has been developed using the highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxaliny)ethyl]-1,2, 4-triazoline-3,5-dione (DMEQ-TAD) for the determination of YTX and two of its analogues 45-hydroxyYTX and 45,46,47-trinorYTX in mussels and scallops [115]. These YTXs possess a diene moiety and DMEQ-TAD undergoes a Diels-Alder reaction with the diene producing two epimers. Unfortunately, this means that a chromatogram will show two poorly resolved peaks for each YTX.
7.1 Rapid screening methods for YTXs There are various ELISA methods that have been developed for a wide range of marine biotoxins, such as ASP, NSP, DSP and PSP [116]. A competitive indirect ELISA for screening the total amounts of YTXs in shellfish samples has been developed recently by Briggs et al. [117]. This has a working range (I20–I80) of
Phycotoxins
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50–1100 pg/mL, and is suitable for monitoring YTXs in shellfish as well as in algae and seawater [116,117]. The polyclonal antibodies (produced by different cells) used in the YTX-ELISA recognize multiple analogues of YTX and this is why results of YTX analysis by ELISA are expected to be higher than those by LC-FLD or LC-MS. Improvements in the extraction and clean-up of YTXs from mussels have been proposed [118] as well as a slot blot procedure as a functional assay for YTXs [119].
7.2 Determination of yessotoxins using LC-MS and LC-MSn methods LC-MS and LC-MSn are incomparable tools for unambiguous confirmation of known toxins and for the identification of novel compounds. They are particularly useful in analysis of toxins when their reference standards are unavailable or very limited and the discovery of a wide range of YTX analogues has been made possible due to the increasing availability of multiple tandem MS instruments. The first MS studies on YTX were performed by Murata et al. [106] using negative Fast Atom Bombardment (FAB) MS. YTX exhibited two molecule-related ions, the desodiated ion [M-Na] (m/z ¼ 1163) and [M-SO3Na+H-Na] (m/z ¼ 1061) and the latter was used for identification of YTXs [106,120,121]. The applicability of tandem MS to the structural elucidation of polyether compounds was well demonstrated by Naoki et al. for YTX [110]. Extensive fragmentation of YTX in negative mode produced spectra that were particularly informative, especially the repetitive cleavage of each ring of the toxin giving a regular fragmentation pattern by a process known as charge-remote fragmentation. Thus, on the basis of MS/MS data alone, the authors succeeded in assigning the number and location of 11 rings of YTX, confirming previous NMR data [110]. The first LC-MS and LC-MS/MS methods developed for direct identification of YTX in shellfish samples [122] used an API source with an ionspray interface. Analytically useful molecular-related ions were observed, including the [M-2Na+H] ion, which was the most abundant, and was therefore selected as a precursor ion for CID MS/MS experiments. LC-MS/MS, using SRM, was developed. This method included an acetone extraction of YTX from shellfish, followed by reversed-phase LC, with isocratic elution using aqueous acetonitrile containing ammonium acetate 4 mM as the mobile phase. An instrumental detection limit of ca. 50 pg was estimated [66,114]. A reversed-phase SIM LC-MS analysis in negative ion mode was used to determine eleven DSP toxins, DTXs, PTXs as well as YTXs in the same chromatographic run with a total time of 20 min. Chromatographic separation is performed isocratically with a mobile phase of acetonitrile–water (80:20 v/v) containing 2 mM ammonium acetate [60]. Detection limits for the investigated toxins ranged from 100 to 400 pg of injected material and this method was proposed as a screening method. The simultaneous determination of ten toxins associated with DSP by LC-MS in scallops was reported [63]. Mass spectrometric measurements were performed using an API source with an electrospray ionization (ESI) interface. Chromatographic separation was carried out using a combination of different columns and mobile phases. Carboxylic acid toxins were determined using SIM of the
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% Relative Intensity
100
50
14.0
15.0
16.0
17.0
18.0
19.0 20.0 Time (min)
21.0
22.0
23.0
24.0
Figure 12 Chromatogram of an extract of in picked cells of Protoceratium sp. using nano LC – QqTOF MS to determine YTX. Adapted with permission from Ref. [124]. Copyright 2004 by Elsevier.
deprotonated [M-H] ions, neutral toxins were using their [M+NH4]+ ions, while for YTXs were determined using their [M-Na] ions. Limits of detection in shellfish tissues were in the range, 40–80 ng/g. LC-MSn was used for the determination of YTX and 45-OHYTX in shellfish using an ESI source with an ion-trap mass spectrometer, in negative mode [123]. The molecule-related ion at m/z 1141 [M-2Na+H] was used as the precursor ion for multiple MS experiments. MS2 and MS3 gave major fragment ions at m/z 1061 [1141SO3H] and m/z 924 [1061-C9H13O]. Predominant ions, due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX, using acetonitrile–water (60:40) with 0.5% ammonium acetate as mobile phase. The detection limit corresponded to 3 ng/g in shellfish tissue [123]. The first application of nano LC-QqTOF MS for the analysis of phycotoxins has been reported. Figure 11 shows the QqTOF MS spectrum which produces high mass accuracy MS data and the main fragmentation processes in negative mode are shown. The high mass accuracy data of precursor and product ions are particularly valuable for confirmation of toxin identity. The high sensitivity of this method allows the analysis of micro-samples and was applied to determine YTX in Protoceratium cells that were manually collected from a microscope slide. Figure 12 shows the chromatogram from the determination of YTX in an extract from cells of P. reticulatum, picked from a microscope slide. The injection volume (1 ml) was the equivalent of one cell. The MS range was m/z 500–1200 and the chromatogram was produced by selecting the m/z 570, [M-2Na]2, ion [124].
8. NEUROTOXIN SHELLFISH POISONING (NSP) TOXINS The polycyclic ether toxins, brevetoxins (PbTx), are responsible for NSP cause massive kills of marine animals, including endangered species, and threaten
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Figure 13 MS/MS spectrum of the ammonium adduct of brevetoxin-3 using nanoelectrospray ionization. Reproduced with permission from Ref. [163]. Copyright 2006 by Wiley.
human health (Figure 13). Brevetoxins are depolarizing substances that open voltage gated sodium ion channels in cell membranes, leading to uncontrolled sodium ion influx into the cell. The main route of human intoxication is from the consumption of shellfish [9,125]. The effects of NSP are felt within 30–60 min and include chills, headache, diarrhoea, muscle weakness, muscle and joint pain, nausea and vomiting. In extreme cases, altered perception of hot and cold, difficulty in breathing, double vision and difficulty in swallowing. These toxins also cause respiratory distress by inhalation of sea spray [126]. NSP has not been known to be fatal, with symptoms generally resolving within a few days. Many brevetoxins have been identified as being products of the red tide dinoflagellate, Karenia brevis (formerly Ptychodiscus brevis) [127,128]. Studies into the metabolic products produced in shellfish, especially mussels, have revealed multiple brevetoxin analogues [129,130]. Various approaches to the detection of brevetoxins have been used, including mouse bioassay which is based on the time until death of mice injected intraperitoneally with crude toxin extracts from shellfish [131]. Cell bioassays that detect brevetoxins due to their sodium channel enhancing effects [132,133] and receptor-based assays, and radioimmunoassays, provide good detection sensitivity and have been widely used as screening tests [134,135]. LC-MS methods for the identification and quantitation of brevetoxins have been important, especially in studies of bioconversion and production of analogues in fish and marine animals following intoxication by brevetoxins (Table 4). One of the problems in the use of MS methods for brevetoxins is the formation of sodium adducts that resist fragmentation. Early approaches involved the optimization of acidic media for flow injection electrospray MS/MS to improve
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Table 4 LC-MS methods for the determination of NSP toxins Compound
Matrix
Stationary phase
PbTx-2, PbTx-3
Oyster; (C. virginica)
YMC J’Sphere ODS L80 S-4, (250 2 mm)
BTX-B1, PbTx-3
Cockle; (Austrovenus stutchburyi), Oyster; (Crassostrea gigas)
Glycine-cysteine, gOyster (Crassostrea glutamyl-cysteine virginica) and glutathione analogues of PbTx-A and PbTx-B PbTx-3, BTX-B5, BTX-B1, PbTx-2, PbTx-3, BTX-B1
Cockle (Austrovenus stutchburyi), Oyster (C. gigas), Mussel (Perna canaliculus)
Mobile phase
(A) 0.1% acetic acid in water and (B) 0.1% glacial acetic acid in ACN Cadenza CD-C18 (A) 0.1% formic acid – (150 3 mm, acetonitrile (45:55) 3 mm) and (B) ACN for PbTx-3 YMC J’Sphere Water-acetonitrile ODS-L80 S-4 gradient with 0.1% (150 2 mm) or acetic acid Luna C8(2), (250 2 mm, 5 mm) Cadenza CD-C18 0.1% formic acid(150 3 mm, MeOH, 0.1% formic 3 mm) acid-acetonitrile, 0.2 M ammonium acetate-MeOH (1:1)
Ionization MS experiments source
Detection limit Reference
ESI
SIM; [M+H]+
nr
[156]
ESI
SRM; [M+H]+ and [M–Na]
0.4 and 2 ng/g for BTX-B1 and PbTx-3
[157]
ESI
SIM; MH+
nr
[158]
ESI
SRM; [M+H]+; [M–H]; [M–Na]
0.2–2 ng/g
[159]
PbTx-2, PbTx-3 BTX-B5, BTX-B1
Cockle (A. stutchburyi), Mussel (Perna canaliculus)
PbTx-3, PbTx-9, PbTx-7, PbTx-10
Oyster (C. virginica)
PbTx-3
Clams (Chione cancellata, C. mercenaria), Whelk (Busycon contrarium)
Cadenza CD-C18 (150 3 mm, 3 mm)
(A) 0.1% formic acid– ESI acetonitrile (45:55); (B) 0.1% formic acid– acetonitrile (25:75); (C) 0.2 M ammonium acetate-MeOH (3:7) and (D) MeOH YMC J’Sphere water (A) and ESI ODS-L80 S-4 acetonitrile (B), (150 2) or Luna with 0.1% acetic C8(2) 100 A acid (YMC column) (250 2 mm, or 0.5% acetic acid 5 mm) (LUNA column) 20% ACN to 65% YMC ODS-L80 ESI or (250 2 mm). ACN in 45 min APCI +0.1% formic acid
SRM; [M+H]+-895 [M+H]+-897 [MNa]-1016, [MH]-909
nr
[160]
SIM; [M+H]+
0.15 ng PbTx-3
[161]
SIM; [M+H]+
25 ng PbTx-3
[162]
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the production of protonated species [127,128,155]. Another recent approach used by Wang and Cole was to use ammonium fluoride in the eluent which led to the formation of [M+NH4]+ adducts that readily converted to protonated species, followed by further fragmentation as shown in Figure 13 [163].
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CHAPT ER
14 Persistent Organochlorine Pollutants, Dioxins and Polychlorinated Biphenyls Marie-Louise Scippo, Gauthier Eppe, Claude Saegerman, Georges Scholl, Edwin De Pauw, Guy Maghuin-Rogister and Jean-Franc- ois Focant
Contents
1. Introduction 2. Physical and Chemical Properties and Use of POPs 3. Health Effects 3.1 Effects on reproduction 3.2 Carcinogenicity 3.3 Other effects 3.4 Mode of action of dioxins and toxicity equivalence 4. Analytical Methods 4.1 Extraction 4.2 Clean-up 4.3 Determination by gas chromatography (GC) 4.4 Determination by biological techniques 5. Occurrence in Food 6. Future Trends References
457 460 462 462 462 463 463 467 467 468 469 489 495 496 498
1. INTRODUCTION Persistent organochlorine pollutants (POPs) are chemical substances that have long half-lives in air, soil, sediments or biota and thus persist in the environment. Because of their high lipophilicity, they can contaminate all levels of the food chain and bioaccumulate up to higher trophic levels. They undergo long-range transport and are found in remote places such as the Artic. They are toxic and cause adverse health effects to human and wild life. Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00014-7
r 2008 Elsevier B.V. All rights reserved.
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The POP group includes 12 substances (the dirty dozen). Among the chemical compounds that have been identified as POPs, nine are agrochemicals (aldrin, chlordane, 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT), dieldrin, endrin, heptachlor, hexachlorobenzene (HCB), mirex and toxaphene), and three are industrial substances (polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)) (Table 1) [1,2]. From the latter group, PCDD/PCDFs have never been produced deliberatively but are released as accidental by-products from combustion processes or industrial synthesis of other chlorinated chemicals. Although neither dioxins nor furans have ever had any commercial applications, PCBs have been heavily synthesized since 1930 (global estimated production of 106 t [1]) for a variety of industrial uses, such as dielectrics in transformers and capacitors, heat exchange fluids, paint additives and in plastics [3]. PCBs, which were supposed to be confined in industrial settings can however, like dioxins and furans, be virtually found in all global ecosystems [4]. The international community is taking action to reduce the background POP contamination level. For example, the UN-ECE (Convention on Long-Range Transboundary Air Pollution of the United Nations-Economic Commission for Europe) protocol on POPs, [5,6] and the Stockholm POP convention [7,8] entered in force, in October 2003 and May 2004, respectively. Both texts focus on the prohibition or restriction of production, restriction on export and import, handling of existing stocks, safe disposal of wastes and reduction of emissions for unintentionally produced POPs. The European Union has adopted both the UN-ECE protocol and the Stockholm convention in its Council Regulation (EC) No 850/2004 [9]. As the top predators, humans are exposed to significant levels of POPs because of biomagnification. Human exposure to POPs can be estimated by biomonitoring of blood, milk and other (adipose) tissues. The primary matrix used for biomonitoring of persistent toxicants is blood. Blood sampling is still an invasive procedure, but less invasive than adipose tissue collection, which requires surgery. Breast milk is also valuable because it is relatively easily collected and not only represents a biomonitoring matrix, but is also a source of nutrition for a segment of the population [10]. Because only a part of the population can be sampled, and due to the depuration rates that have to be taken into account while breast-feeding is taking place, human milk analysis is usually restricted to infant exposure studies rather than general population studies. Lots of biomonitoring data are available for POPs till date. They all point out a general decrease of the human body burden for the listed POPs. Exposure to other emerging persistent contaminants, such as brominated flame retardants, is increasing but is out of the scope of the present chapter [11,12]. Except for particular occupational exposures (e.g., people working in a pesticide plants), the consumption of food of animal origin is the principal source of human exposure to POPs [13–20]. It is well-recognized that foodstuffs from animal origin account for more than 90% of the human exposition, with meats, eggs, dairy and fish products as the main contributors [21–25]. The proportion of the exposure for background subjects from inhalation or direct contact with dioxins in the environment is estimated to be no more than a few percent [26].
Persistent Organochlorine Pollutants, Dioxins and Polychlorinated Biphenyls
Table 1
Structure of the 12 POPs of the Stockholm convention [7,8]
Name of the POP
Structure
Use
Aldrin
Insecticide
Chlordane
Insecticide
Chlordecone
Insecticide
DDT
Insecticide
Dieldrin
Insecticide
Endrin
Insecticide
Heptachlor
Insecticide
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Table 1 (Continued ) Name of the POP
Structure
Use
Hexachlorobenzene
Fungicide, byproduct of industrial process
Mirex
Insecticide
Toxaphene
Insecticide
PCB
3
2
4
Clw 5 PCDD
2' 1 1'
6
1 2
4'
6' 5' Clz
O
9 8
3 4
Clx PCDF
O
1
7 Cly
6 9
2
8
3 Clx
Industrial uses
3'
4
O
7 6
By-product of industrial or natural process
By-product of industrial or natural process
Cly
Consequently, in an attempt to maintain the declining exposure trend, a careful monitoring of our foodstuffs is mandatory. International regulation offices have therefore defined control levels to facilitate the screening and the decision concerning the consumability of foodstuffs. This chapter discusses some of the aspects of the analysis and occurrence of POPs in food of animal origin, with emphasis on PCBs and dioxins.
2. PHYSICAL AND CHEMICAL PROPERTIES AND USE OF POPS POPs are highly stable organic compounds, resistant to thermal degradation and biological metabolism. They are all chlorinated compounds, the higher the
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461
chlorination level, the better it resists degradation [27]. POPs are virtually nonsoluble in water, their high Kow values allow them to penetrate the phospholipidic membrane of cells and accumulate in fat tissue of living organisms. POPs have long half-lives (e.g., 10–12 years in soil for dioxin or endrin). They are semivolatile, so that they are subject to long-range transport by atmospheric currents. The nine organochlorine pesticides (OCPs) were introduced from 1920 to 1950 mainly for agricultural use. By 1990, the production and use of these substances were banned in western countries due to their human toxicity. Some of them are still in use as intermediates for other chemical synthesis or in developing countries against malaria [28,29]. For aldrin and DTT, metabolites (dieldrin and DDE, respectively) can be more persistent than the original compound [30]. PCDDs and PCDFs are two groups of planar tricyclic compounds that have similar chemical structures and properties. Dioxins are made of two six carbon atom aromatic rings connected together by two bridging oxygen atoms, whereas only one oxygen atom ensures the connection for furans. PCBs can be defined as bicyclic compounds since the two aromatic rings are connected together with no intervention of oxygen atoms (Table 1). Numbers on aromatic rings represent potential positions for chlorine atoms, these being in maximum 8 (0 p x,y p 4) in case of PCDDs and PCDFs and 10 (0 p w,z p 5) for PCBs. All together, dioxins (75), furans (135) and PCBs (209) represent 419 molecules (congeners), depending on the number and position of chlorine atoms on aromatic rings. In the case of PCBs, the planarity of molecules depends on the chlorine substitution pattern. If no chlorine atoms are present in positions 2,6,2u,6u (ortho positions), the molecule adopts a planar geometry, similar to the one observed for dioxins and furans. Once chlorine atoms start to be connected in ortho positions, steric hindrance drives the molecule to evolve to a non-planar configuration. These substitutional and geometrical parameters are of prime interest since they are closely related to the toxicity of these compounds. They are only very slightly volatile, sparingly soluble in non-polar solvents and almost insoluble in water. They are also persistent in environment and prevalent in biota due to extreme stability in environment (t1/2 environment ¼ 10–12 years) and due to bioavailability–bioaccumulation (mainly 2,3,7,8-substituted congeners) in humans (t1/2 human ¼ 5–10 years in adults) [32]. Their dispersion in the atmosphere is likely to occur mainly in particulate aerosols [33]. PCDDs and PCDFs are not produced intentionally but occur frequently as unwanted by-products in chemical processes, such as the synthesis of pesticides or PCBs [34]. Combustion processes are recognized as the major sources of PCDDs and PCDFs. Most thermal reactions, which involve the burning of chlorinated organic or inorganic compounds, appear to result in the formation of these substances. PCDDs and PCDFs have been detected in emissions from the incineration of various types of wastes, particularly municipal, medical and hazardous wastes, from the production of iron and steel and other metals, from fossil fuel plants, domestic coal and wood fires, especially those involving chlorine-containing materials, such as polyvinyl chloride (PVC) and PCBs [35]. Dioxins released into the environment many years ago continue to contribute to contemporary exposure [36].
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Individual PCB congeners are often crystalline but commercial mixtures exist as viscous liquids or resins. They have low chemical reactivity and rather high density. They have low electrical resistance, which, in combination with their heat stability, makes them very suitable as cooling liquids in electrical equipment [37]. They were produced between 1930 and 1970 and commercialized in relative large quantities for use as dielectrics, hydraulic fluids, plastic and paint additives [38]. The universal distribution of PCBs throughout the world suggests that PCBs undergo long-range transport by air [39].
3. HEALTH EFFECTS Numerous adverse health effects are attributed to POPs. OCPs are suspected to act as endocrine disruptors, causing perturbations at the reproductive stage [40–44], but this is still controversial and not formally demonstrated [45–47]. Various effects have been reported in animals exposed to PCDDs, PCDFs and PCBs [48]. Many of the toxic effects of dioxins were high dose effects [49,50]. The most commonly reported pathologies are endometriosis, immunotoxic effects, cancer, birth defects, effects on the reproductive and the neuro-endocrineimmune systems, altered metabolism and specific organ dysfunction [35,51–56].
3.1 Effects on reproduction Evidence of POPs effects on reproduction comes from wild life, with numerous examples of accidental (or not) contamination having caused perturbation of the reproductive system of the fauna (e.g., the accidental contamination of the lake Apopka in Florida with DDT, where alligators display altered reproductive functions [57], development and reproductive defects in fish-eating birds [58], other examples are reviewed in Ref. [59]). Effects on human male reproduction have been described for some POPs, such as PCBs and DTT (the latter acts through its active metabolite 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene or p,pu-DDE) [60–62]. These substances alter sperm motility [63–65] or sperm chromatin integrity [66,67], but seem to have no effect on sperm concentration [68]. They also may be the cause of an increased time to pregnancy [69]. OCPs act as endocrine disruptors by interfering with the estrogen or the androgen receptor or by disturbing the hormonal function (reviewed in Ref. [70]).
3.2 Carcinogenicity First classified as probably carcinogen for humans (class B2) by the US EPA in 1987 [71], aldrin and dieldrin were then described as ‘‘not likely human carcinogen’’ [72,73], since reported epidemiologic studies showed no correlation between pesticide exposure and cancer [74,75]. POPs and PCBs contamination of the environment is suspected to be the cause of the increasing incidence of breast cancer in European countries [76]. DDT is classified by International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (group 2B) [30].
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Dioxins are carcinogenic in several animal species [77] and humans, and increased risk of cancer has been demonstrated at exposure levels more than 100 times the normal intake of the general population [78]. Dioxins are classified as human carcinogenic by the IARC [79–81] and either ‘‘carcinogenic to humans’’ or ‘‘likely to be a human carcinogen’’ by EPA [35]. This is consistent with the observations of Bertazzi et al. [82,83], showing an increase of rectal and lung cancer cases, 20 years after the 1976 Seveso accident. However, there is no consensus in the scientific community on the carcinogenicity of tetrachlorodibenzo-p-dioxion (TCDD), controversial for some [84] and confirmed for others [80].
3.3 Other effects TCDD is known to cause chloracne [82,85–87] and POPs, in general, are suspected to cause diabetes in humans [88,89]. More particularly, TCDD could be one of the multiple factors causing atherosclerosis, hypertension, vascular ocular changes and signs of neural system damage [90].
3.4 Mode of action of dioxins and toxicity equivalence Toxicological properties of dioxins, furans and PCBs are directly related to their chemical structure (number and position of chlorine atoms) [91]. Over the 419 possible congeners that can theoretically be released in the environment, 17 congeners substituted in position 2,3,7,8 (seven PCDDs and ten PCDFs) as well as 12 ‘‘dioxin-like’’ PCBs (Table 1) present such a sterical conformation that they can efficiently bind to an intracellular receptor, with which a stable complex can be formed [92]. This dioxin-specific receptor is called arylhydrocarbon receptor (AhR) [93,94]. Briefly, once the dioxin–AhR complex is formed, it can enter the nucleus, bind to another regulatory protein (AhR nuclear translocator, Arnt), access the genetic material of the cell and activate the transcription of target genes such as a cytochrome P450 detoxifying enzyme, the CYP1A1, via binding to a specific sequence of the DNA referred as the Dioxin Responsive Element (DRE) (Figure 1) [95–97]. As the dioxin molecules are very stable in the cell and not metabolized, AhR is abnormally maintained under its activated form, with adverse effects on physiologic functions [55,97–101]. The involvement of AhR in many cellular- and tissue-specific functions could explain most effects of dioxins, such as chloracne, teratogenic effect (e.g., cleft palate), immunosuppression, carcinogenesis [101] and/or oxidative stress [102]. Structure–activity requirements based on AhR binding characteristics can be used to predict the relative potencies (REP) of different agonists for producing toxic effects [103]. In other words, toxic potency of congeners correlates with their ability to bind the AhR. It appears that the lateral 2,3,7,8 position of the dioxin molecule has to be fully chlorinated to induce toxicity. Removal of any of these substituents or moving them to an apical (1,4,6 or 9) position dramatically decreases the toxicity [104]. Among dioxins, 2,3,7,8-TCDD has the greatest
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Cytoplasm
Dioxin
Nucleus
Hsp 90 AhR
Arnt
TGCGTG
DNA
DRE
Figure 1 Simplified mechanism of action of dioxins.
affinity for binding to the AhR and is often described as the most potent animal teratogen [105,106]. The concept of toxic equivalence factors (TEFs) has been established for evaluating congeners other than TCDD (the most toxic, with a TEF of 1) and mixtures of dioxins [107,108]. TEFs values (called I-TEFs) for humans were first proposed by NATO for 10 dioxin and 7 furan congeners [109], then, 12 dioxin-like PCBs congeners were added. TEFs were re-evaluated by the WHO in 1998 [108] and in 2006 [110]. During the first re-evaluation, the TEF for PCDD was increased from 0.5 to 1, while during the second re-evaluation, dioxin-like PCBs TEFs were decreased (Table 2, columns 2–5). These TEFs are used to calculate toxic equivalent (TEQ) content of feed and food samples, assuming that the effect of the 29 targeted congeners are additive. This facilitates risk assessment and regulatory control of exposure to these mixtures [111]. In practice, when an analytically measured congener concentration is multiplied by its TEF, it is converted into TEQs, which indicate how much 2,3,7,8-TCDD would be needed to produce the same toxic effect as the dose in question [112]. The global toxicity for a sample containing several different congeners can be obtained using the formula depicted in the following equation: TEQ ¼
X X X ðPCDDi TEFi Þ þ ðPCDDFi TEFi Þ þ ðPCBi TEFi Þ n1
n2
n3
Using the available toxicological database on dioxins in 1998, the WHO established a tolerable daily intake (TDI) in humans between 1 and 4 pg TEQ/kg bw/day [113]. This TDI is based on the range of lowest observed adverse effect level (LOAEL) for the most sensitive experimental animals and could be transformed into a range of estimated long-term human daily intake (EDI) of 14–37 pg TCDD/kg bw/day when applying an uncertainty factor of 10 [53].
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Table 2 List of 29 congeners of dioxins, furans and dioxin-like PCBs to which a TEF has been assigned. I-TEFs, 1988 [109]; WHO-TEFs, 1998 [108]; WHO-TEFs, 2005 [110]; DR-CALUX REP, relative potencies measured with the DR-CALUX assay [215] Congener
I-TEFs (1988)
WHO TEFs (1998)
WHO TEFs (2005)
DR-CALUX REP
Dioxins 2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,4,6,7,8-HpCDD OCDD
1 0.5 0.1 0.1 0.1 0.01 0.001
1 1 0.1 0.1 0.1 0.01 0.0001
1 1 0.1 0.1 0.1 0.01 0.0003
1 0.5 0.1 0.06 0.06 0.03 0.0005
Furans 2,3,7,8-TCDF 2,3,4,7,8-PeCDF 1,2,3,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF OCDF
0.1 0.5 0.05 0.1 0.1 0.1 0.1 0.01 0.01 0.001
0.1 0.5 0.05 0.1 0.1 0.1 0.1 0.01 0.01 0.0001
0.1 0.3 0.03 0.1 0.1 0.1 0.1 0.01 0.01 0.0003
0.4 0.4 0.1 0.07 0.08 0.1 0.1 0.01 0.04 0.004
Coplanar or non-ortho substituted PCBs 3,4,4u,5-TrCB (PCB 81) – 3,3u,4,4u-TrCB (PCB 77) – 3,3u,4,4u,5-PeCB (PCB 126) – 3,3u,4,4u,5,5u-HxCB (PCB 169) –
0.0001 0.0001 0.1 0.01
0.0003 0.0001 0.1 0.03
0.002 0.0004 0.04 0.0008
Mono-ortho substituted PCBs 2,3,3u,4,4u-PeCB (PCB 105) 2,3,4,4u,5-PeCB (PCB 114) 2,3u,4,4u,5-PeCB (PCB 118) 2u,3,4,4u,5-PeCB (PCB 123) 2,3,3u,4,4u,5-HxCB (PCB 156) 2,3,3u,4,4u,5u-HxCB (PCB 157) 2,3u,4,4u,5,5u-HxCB (PCB 167) 2,3,3u,4,4u,5,5u-HpCB (PCB 189)
0.0001 0.0005 0.0001 0.0001 0.0005 0.0005 0.00001 0.0001
0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003
NR 0.00002 NR NR 0.00002 NR NR NR
Note: NR: no response observed.
– – – – – – – –
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The upper limit of the TDI should be considered as the maximum tolerable intake on a provisional basis with the ultimate goal to reduce human intake levels below 1 pg TEQ/kg bw/day. More recently a tolerable weekly intake (TWI) of 14 pg/kg bw/week [49] and a tolerable monthly intake (TMI) of 70 pg/kg bw/month [50,107] were also determined respectively to reflect the long half-life of TCDD. The above three methodologies assume a threshold for human cancer risks. Another recent international evaluation based on a non-threshold effect [114,115] estimates the lifetime risk for all cancers to be 1 103 risk/pg TEQ/kg bw/day. According to the TWI of 14 pg WHO-TEQ/kg bw/week, maximum tolerable concentrations have been fixed in feed and food in Europe [116] (Table 3). With the aim to reduce the dioxin contamination of feed and food, Europe has proposed alert levels [117] at which it is recommended to member states to investigate to find and eliminate the source of the contamination. These actions
Table 3 Maximum levels [116] and action thresholds [117] for dioxins, furans and dioxin-like PCBs, for some food matrices Maximum levels (pg WHOTEQ/g fat or product)
Action threshold (pg WHO-TEQ/g fat or product)
Sum of dioxins and furans
Sum of dioxins, furans and dioxinlike PCBs
Sum of dioxins and furans
Dioxinlike PCBs
3
4.5
1.5
1
2 1
4 1.5
1.5 0.6
1.5 0.5
Milk and milk products, including butter fat, hen eggs and egg products
3
6
2
2
Marine oil (fish body oil, fish liver oil and oils of other marine organisms intended for human consumption)
2
10
1.5
6
Mixed animal fats
2
3
1.5
0.75
Muscle meat of fish and fishery products and products thereof with the exception of eel
4
8
3
3
Meat, meat products and fat Ruminants (bovine animals and sheep) Poultry and farmed game Pigs
pg WHO-TEQ/g product.
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would be helpful to decrease the background contamination of feed and food, and Europe would like to propose lower maximum tolerable levels by 31 December 2008.
4. ANALYTICAL METHODS PCDDs, PCDFs and dioxin-like PCBs are found at levels as low as pico- or femtogram per gram of matrix depending on the investigated food sample. In addition, matrix-related interferences are present in concentrations at orders of magnitude higher than the analytes of interest. For those reasons, a complex multi-step approach is required to (1) extract the analytes from the matrix core, (2) separate undesirable interferences and (3) finally isolate, separate and quantify analytes of interest. In terms of cost per sample and sample throughput, it is not only the final measurement of the analyte concentration, but — maybe even more importantly — the complex sample preparation procedure, which makes this measurement possible. For PCBs, a minimum number of seven essential PCBs (IUPAC number 28, 52, 101, 118, 138, 153, 180) are screened for the monitoring of food, fish and environmental samples. They are found at higher levels compared to dioxins (i.e., low nanogram to microgram per gram of matrix depending on the investigated biological sample). For this reason, the analytical methodologies are less complex. Several review papers on analytical methodologies for OCPs and PCBs are available in the literature. Keith [118] summarized US EPA methods for PCBs and OCP in sediment and biological materials. Wells and Hess [119,120] reviewed and recommended methods for the separation, isolation and recovery of OCPs and PCBs from soils, sediment and biological matrices. De Boer and Law [121] provided a useful overview of current analytical methodology for OCPs and PCBs. More recently, Muir and Sverko [122] outlined analytical methods to measure PCBs and OCPs in environmental and biological samples.
4.1 Extraction The extraction device presented by Professor von Soxhlet in 1879, which re-circulates the extraction solvent while accumulating extracted analytes in a heated flask, is still used in the dioxin field and is often considered as the reference extraction method, at least for solid samples, such as soils, sediments and fly ashes [123,124]. For liquid samples, liquid–liquid extraction (LLE) has been used for a long time, mainly for biological fluids [125,126]. Although easy to set up, several critical drawbacks such as phase emulsions, required quantities of solvent and intensive handling make it unappealing to use. Extractions of biological tissues using supercritical fluid extraction (SFE) have also been reported using supercritical CO2 [127]. During an extraction cycle, static (fluid immobilized with the sample in the closed vessel) and dynamic (percolation of the fluid through the cell) modes can be used together to ensure both penetration of the matrix by the fluid and to avoid saturation of the fluid
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[128]. Co-extracted lipids may be eliminated in part by directly adding adsorbents inside the cell [129–132]. SFE may be coupled with various analytical methods like gas chromatography (GC) but very fine-tuning is then required [133,134]. Since first reports on the use of domestic microwave ovens to carry out extraction of organic compounds, microwave-assisted extraction (MASE) has been applied to OCPs and PCBs present in solid matrices [135–137]. Selected extraction solvents usually have high dielectric constant to absorb the microwave energy efficiently. In pressurized liquid extraction (PLE), also named PFE (pressurized fluid extraction), organic solvents are used in the liquid phase at temperatures above their boiling point. The stainless steel extraction cell can generally be heated up to 2001C and pressurized up to 3,000 psi. A minimum extraction time of 10 min is usually required to ensure efficient transfer of analytes out of the sample matrix [138]. For extraction from high-fat content biological samples, quantitative recovery rates can be obtained using classical organic solvents but hexane is usually preferred to solvent mixtures because less matrix-related interferences are co-extracted under mild temperature conditions [139]. Drying of the sample can be carried out using lyophilization prior to extraction. This reduces the risk of extracting traces of water, which may lead to over estimation of the lipid content of the sample. The pre-lyophilization freezing process can be accelerated by cryohomogenization of the samples using liquid nitrogen [140]. The dry material can then be easily homogenized before extraction. Among potential alternative techniques, solid phase extraction (SPE) constitutes the alternative of choice for extraction of dioxins from liquid samples. Bonded-silicas [141,142] and styrene-divinyl benzene synthetic polymer [143,144] are well suited for isolating groups of compounds from sample matrix components. The use of vacuum manifold and automated SPE workstation easily regulates flow rates through the cartridges. In the case of dioxins and related compounds, the use of C18 cartridges has been reported for water, serum and milk [145–151]. Good recovery was achieved when fat globules were efficiently disrupted in order to release the pollutants from the lipoproteins. It has successfully been applied in the dioxin field by different laboratories [152,153]. Solid phase dispersion on diatomaceous earth can also be used in SPE cartridge format. This is a valuable SPE method for the extraction of milk samples without the inconvenience of losing part of the lipids during the extraction step. Lipids are therefore quantitatively extracted from the milk matrix and isolated for gravimetric determination prior to further clean-up.
4.2 Clean-up Highly efficient clean-up procedures are required to purify samples issued from the extraction step prior to the final analysis and quantification. Classical solid– liquid adsorption chromatographic separations based on sorbents, such as silica, alumina and Florisil, have long been regarded as important in the field [154]. For biological samples, sulfuric acid silica or gel permeation chromatography (GPC) columns are used to remove the bulk of the lipids and other oxidizable
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components. Basic alumina columns are then used to separate dioxins from pesticides and PCBs [155]. Activated carbon sorbent can join the column set as a complementary fractionation tool to alumina [156–158]. Due to its affinity for certain planar aromatic systems, especially those with adjacent aromatic rings and electronegative substituents, carbon-based sorbent can fractionate the planar dioxins, furans and PCBs from other classes of aromatic compounds, improving sample clean-up. The multi-sorbent clean-up procedure can be automated. The fractionation procedure can be tuned to allow the collection of different fractions [159]. To accommodate foodstuffs analysis, generally characterized by low levels of POPs and high lipid content, high capacity disposable silica columns have been developed to accommodate 4–5 g of lipids on-column [140]. Coupling the extraction and clean-up steps can further be done to reduce sample and extracts handling [160]. Recent reports proposed on-line PLE and clean-up for PCB analysis in solid food and feedingstuffs by directly adding sorbents such as acidic silica and activated carbon inside the extraction cell [161,162]. Another approach for the sample preparation of solid samples is the use of a modified version of the automated clean-up system described earlier. Either an SPE or a PLE system is directly connected to the clean-up instrument and the resulting integrated system is fully automated [160]. This system was studied for the on-line extraction and clean-up of different foodstuff types.
4.3 Determination by gas chromatography (GC) Once extracted and purified from matrix interferences, the chromatographic separation of the target compounds has to take place before analysis. Due to the semi-volatility of dioxins and related compounds, GC is used to separate the different congeners and to allow non-ambiguous identifications.
4.3.1 Injection This paragraph summarizes different injection techniques that have been reported for POPs analysis. Splitless injection is the most frequently used technique for detecting ultra-traces of dioxins, PCBs and OCPs [163]. It is a robust and reliable injection technique. Generally, a volume of maximum 2 mL is injected into a hot injector left at a temperature between 2501C and 3001C. On-column injection may be used as an alternative for OCPs [164] in particular for DDT that is easily degraded in hot injector above 1501C. Large-volume injectors (LVI) are becoming more popular taken into account that the low concentrations of dioxins and furans occur in biological materials required a high sensitive technique. There are three ways of injecting large volume onto the GC column: on-column injection, loop-type injection and Programmable Temperature Vaporizer (PTV) injection. On-column LVI is a less successful way of injection for dioxins compared to PTV injector [165–167], particularly because of the strong influence of solvent-impurities in the chromatogram and contamination of the column and detector. These techniques enable injection of volume as large as 100–200 mL, but most dioxin
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applications by PTV injection use 5–30 mL. The main drawback of PTV is the number of parameters to optimize compared to simple split/splitless technique.
4.3.2 GC Separation techniques 4.3.2.1 Classical gas chromatography (GC). Capillary high-resolution GC (HRGC) columns ensure the required selectivity, especially for congeners of the same chlorination level. The separation characteristics and elution profiles on GC columns have been studied. Ryan et al. [168] published a comprehensive study on separation characteristics and elution profiles of different GC columns. An appropriate combination of column length, internal diameter and stationary phase polarity is needed. For the analysis of biological samples, non-polar GC columns are usually used. They allow separation between homologue groups and can separate 2,3,7,8-substituted congeners from each other [169], showing the efficiency of a non-polar (methyl polysiloxane with 5% phenyl) column to separate the 2,3,7,8 toxic congeners from the rest, especially for 2,3,7, 8-tetrachlorodibenzofuran (TCDF). The apolar GC column is the most widely used column for 2,3,7,8-PCDD/Fs and non-ortho PCBs analysis in foodstuffs, feedingstuffs and human samples. Usually 40–60 m columns with 0.18–0.25 mm internal diameter and 0.15–0.25 mm film thickness are selected. With polar phase (cyanopropyl stationary phases), the resolving power specially improves for the separation of the 22 TCDD congeners, but separation of 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF still remains incomplete [170]. This column is used for environmental matrices (e.g., fly ash, air, soils, sediments and biota) characterized by the presence of many PCDD/Fs congeners. The major drawback of the polar column is the non-linked phase and its low stability at elevated temperature (withstand temperature up to 2751C), which tends to produce significant bleed. For PCB fractions, the 8% phenyl polycarborane-siloxane is often used. The carborane group has a high affinity for PCBs with a low degree of ortho-substitution. Although this phase does not allow the separation of all the 209 PCB congeners, it separates some critical pairs of co-elutions present with other phases [171]. For example, indicator trichlorinated PCB-28 and PCB-31 (not followed), pentachlorinated mono-ortho PCB-123 and PCB-118, as well as hexachlorinated PCB-163 (not followed) and the indicator hexachlorinated PCB-138 are separated. A 25 m 0.25 mm 0.2 mm HT-8 seems to be a good compromise between the required resolving power and the GC run time of roughly 30 min. It should be mentioned that till date, none of the existing stationary phases is capable of the separation of all the PCB congeners. Even emerging hyphenated methods such as comprehensive two-dimensional GC (GC GC) coupled to time-of-flight mass spectrometry (TOF-MS) can at the most separate 192 congeners [172].
4.3.2.2 Comprehensive Two-dimensional Gas Chromatography (GC GC). GC GC has been developed to meet an increasing need for complex sample analysis and to address limitations, such as peak capacity, dynamic range and restricted specificity of mono-dimensional (classical) GC systems (i.e., to improve the global efficiency of
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the separation). It is a chromatographic technique where the entirety of a sample is subjected to two orthogonal separation processes. This is the case when two independent separation mechanisms are used (orthogonality rule), and when the separation obtained in the first dimension is maintained in the second dimension (conservation rule) [173]. This technique is based on the fast sampling and transfer of the sample by a cryogenic modulator located between the first dimension (1D) column and the second dimension (2D) column connected in series. The entire 1D chromatogram is continuously sampled following a modulation period (PM) of a few seconds and sent into 2D for a fast GC-type separation (Figure 2A). Because modulation occurs during the 1D separation, the total GC run time of a GC GC separation is the same as in classical GC. As in classical GC, a trace is monitored continuously at the detector located at the end of 2D. In reality, series of high-speed secondary chromatograms of a length equal to PM (3–10 s) are recorded one after another (Figure 2B). They can be computerized by specific software and combined to describe the multi-dimensional elution pattern by means of contour plots in the chromatographic separation space (Figure 2C). In GC GC the peak capacity (the maximum number of peaks that can be placed next to each other in the available chromatographic separation space at a given resolution) is equal to the product of the peak capacities of the two coupled systems maki
