Reference Materials for Chemical Analysis: Certification, Availability, and Proper Usage

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Reference Materials for Chemical Analysis Edited by Markus Stoeppler, Wayne R. Wolf; PeterJ. Jenks

Reference Materials for Chemical Analysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler, Wayne R. Wolf; Peter]. Jenks

@WILEY-VCH Weinheim - New-York - Chichester - Brisbane - Singapore - Toronto

The Editors ofthis Volume

Or. Markus Staeppler Mariengarten Str. ra 52428 Julich Germany Wayne R. WOK Ph.D.

Human Nutrition Research Center US Department of Agridture 10300 Baltimore Blvd. Beltsville, MD 20899, USA Peterj. Jenks, B.Sc

Laboratory ofthe Gouvernment Chemist Queens Road, Teddington Middlesex TWII oLY, England Sections 3.4 (Authors Steven A. Wise and Jiirgen Jacob)and 5.4 (Authors Barbara C. Levin and Dennis J. Reeder) are contributions ofthe US National Institute of Standards and Technology (NIST) and as such are not subject to copyright. Certain commercial equipment, instrnments, materials or companies are identified in these contributions to specify the experimental procedure. Such identification does not imply recommendation or endorsement by NIST. nor does it imply that the materials or equipment identified are the best available for this purpose.

.

This book was carefully produced. Nevertheless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.:

applied for British LibraryCataloguingin-Publication Data

A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek - CIP Cataloguingin-Publication Data

A catalogue record for this publication is available from Die Deutsche Bibliothek

0 Wiley-VCHVerlag GmbH

69469 Weinheim (Federal Republic of Germany),2001 AU rights reserved (includingthose of translation in other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means -nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Composition Kuhn & Weyh,

Freiburg, Germany Printing Strauss Offsetdruck

Morlenbach, Germany Bookbinding J. Schaffer GmbH Griinstadt, Germany ISBN 3-527-30162-3

I"

Foreword No one would deny that the works of great philosophers of the 18th Century are very important. Using the names of Kant or Rousseau demonstrates a certain level of cultural education. Yet these works are hardly ever read. The Foreword of a text-book has this in common with the works of the philosophers. But this gives the person who writes the Foreword the opportunity to propose some more philosophical thoughts. He can even be daring, as the Foreword is not considered a part of the book and therefore it has hardly any importance. With this excuse I can use the opportunity this book gives me as an occasion for thinking, on paper! This book appears at a moment when one of the major developments of the last century in analytical chemistry, measurement science, is coming to its full maturity. The past hundred years have shown an enormous expansion in measurement activities: what is measured, the purpose of the measurements, the use of measured data, and the demands placed upon these data. From the initial, almost exclusive, use of chemical reactions to make measurement the field became wider. Introducing physical and biological reactions and sensors has enormously extended the scope of analytical chemistry. Some 15 years ago the US National Institute of Science and Technology, better known as NIST, estimated the economic impact of measurements and arrived at a figure of some G % of the GDP of a developed country. Even if this estimate is wrong by 50 %, which is unlikely, then this figure is still impressive. However, once we realize where the requests for data come from, then we may better understand the magnitude of this figure. Measurement data are being used for the diagnosis of illness in people and domestic animals, for the control of food and animal feed quality, to establish transfer of dangerous substances through the environment and ecocompartments, to find correlations that may lead to a better understanding of causeeffect relationships, to control raw materials and finished products, and for many other purposes. The rg70's demonstrated a trend: "chemistry is going out of analytical chemistry". However, what was not used anymore up-front for analysis came back in the form of sample preparation techniques. For example, IUPAC devoted as much attention as ever before, but now to the chemistry needed to prepare the sample for measurement and to avoid losses and contamination.

VI

I

Foreword

A growing tendency to take measurements away from the specialized laboratory and place them in the hands of the end-users made paramount the need for good control of produced and used results. The risk of wrong results and biased data increases rapidly in such situations. Consequently, there came about a general tendency to formalize or codify procedures to arrive at data which are sufficiently accurate for the intended purpose. The end-user of the data became increasingly aware of the risk that conclusions could be based on wrong analytical results. The increasing complexity of society (where more and more people, over greater distances, were attributing characteristics or properties to a final product) meant that often the “good reputations” of the past were no longer seen to give the certainty needed for the increasing volume of data. As everywhere in society, relationships were formalized. What started in medieval times with the standard weights and sizes in every town with market rights, was developed in the last half of the past century to a globally elaborated standards system. Standards are now used everywhere to demonstrate reliability. Standards have replaced good trust in reputations. Psychology, the relation of human understanding, has been replaced by the science of measurements. Standards are being used in the context of Analytical Quality Assurance, which is the demonstration to the end-user that the delivered product, the measurement data, are reliable and have been made according to the best practices currently possible. Standards facilitate, some would say are essential to, living and trading in the global village. Standards have become standard in everyday life. This development has taken place remarkably quickly over the past thirty years. In that period the first attempts to arrive at a proven analytical accuracy were made. Those who led the move were often considered as people with hobbies, obsessive even. Some eye-opening publications demonstrated clearly that generating analytical results could be compared with the generation of numbers in a lottery (G. Tolg), but were received skeptically by the scientific establishment. Even as recently as the late 1970’s even the most highly respected universities still had to be made aware that a result was not necessarily an accurate result. But over the past thirty years or so, with increasing awareness of analytical error, the situation changed. Global trade, environmental modelling, etc. caused a drastic change in attitude. Quality Control and Assurance have become integral parts of a scientific curriculum. It is commonly accepted now that 10-15 % of all measurement costs are Quality Assurance costs. This means that the subject of this book covers some I % of a developed country’s GDP. Such an amount is substantial, knowing that the GDP of agriculture in many developed countries does not exceed 10% of the total GDP. This book marks the conclusion of this strong period of development and is therefore a milestone in measurement science. As such, the field already has a history. But as history makes no sense without a future, the last Chapter of this book deals with expected further developments in terms of organization and needs. Between history and future the book presents, as a snap shot, the application of standards in analytical chemistry. The perspective of Quality Assurance is never forgotten.

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After so many developments (often worked out by an in-crowd of devoted, enthusiastic scientists), the time has come to transfer the results of these scientific developments to a wider audience. Not every scientist producing measurement data is interested in the theories worked out in so many vigorous discussions by leading groups. But every scientist using or producing data should be aware of the facts presented herein. The information, however, is of importance for an even wider audience than just the scientific circles. For example, if ever we want to understand whether global warming to a substantial degree is, or is not, caused by human activities, then we should first of all know and understand the performance of our models and the accuracy of data we put into these models. If ever we want to know whether all efforts to improve the quality of the sea for biota are effective, then we need to know the accuracy and uncertainty of all our data. If we do not want to take Quality Assurance into account, we will never be able to lay the solid scientific foundations for our research, political and legal actions etc. In other words: we will remain only believers. I do hope that this book will not only find a wide audience, but that it will also contribute something to progress in the world. This may sound somewhat grand and optimistic, but is it forbidden to dream that next century will indeed bring real progress to mankind? Can this progress be based on anything else except scientific developments? If this is to be so, then what other than sound measurement data can be the basis? Sound measurements mean that data are accurate for the intended purpose and that the uncertain is known and taken into full account. In that case we may have walked along with Kant, Rousseau and so many others. Bernard Griepink, Brussels, Belgium

30. December 1999

I

Contents Foreword

XV

Preface XVIl 1

Introduction

1.1 1.1.1 1.1.2 1.1.3 1.2 1.3 1.4

Historical 1 Early Developments 1 Growth and Maturity 4 Milestones and The Future G The Theoretical Basis 7 Technical Requirements 11 References 16

1

2

From Planning to Production

2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3

Material Collection and Preparation 20 Introduction 20 General Collection and Preparation Principles 22 Specific Examples 26 Concluding Remarks and Recommendations 30 Control of Material Properties 31 Particle Size and Particle Size Distribution 31 Homogeneity/Heterogeneity 33 Humidity (Water Content) 37 Degradation Studies/Shelf Life 40 References 43

3

Certification

3.1 3.1.1 3.1.2 3.1.2.1 3.1.2.2 3.1.2.3

20

49

Certification Philosophy of RM Producers 49 Introduction 49 Approaches to the Characterization/Certification of Reference Materials 50 General Principles of Certification 50 Classification of Characterization/Certification Schemes 52 Specific Examples 58

IX

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Contents

Conclusions Go Certification of Elements Go Methods Used for the Certification of RMs for Elements Go Multi-Method Elemental RM Certification 64 River Sediment 64 Lichen 65 Examples of Selected RMs Certified for Elements 66 Certification of Element Contents by Neutron Activation Analysis GG General Features GG Internal Cross-Checking(Self-verification)in NAA 68 Applications in Certification and Analysis Gg NAA for the Detection of Errors 73 Summary 74 Certification of Organometallic and Other Species 75 Introduction 75 Potential Sources of Error in Speciation Analysis 76 Restricted List of Chemical Species for Trace Elements and Their Compounds 77 3.3.3.1 Aluminum 77 3.3.3.2 Antimony 77 3.3.3.3 Arsenic 77 3.3.3.4 Bromine 78 3.3.3.5 Chromium 78 3.3.3.6 Mercury 79 3.3.3.7 Lead 80 3.3.3.8 Selenium 81 3.3.3.9 Tin 81 3.3.3.10 Metallothionein 82 3.3.4 Fractionation 82 3.3.5 Conclusions 82 3.4 Certification of Organic Substances 83 3.4.1 Introduction 83 3.4.2 CRMs Available for Organic Constituents 84 3.4.2.1 Pure Substances 84 3.4.2.2 Calibration Solution CRMs 85 3.4.2.3 Natural Matrix SRMs 85 3.4.3 Certification Approach for Organic Constituents 88 3.4.3.1 NISTApproach for Certification 89 3.4.3.2 NIST Analytical Approach for the Certification of Organic Constituents in Natural Matrix SRMs 91 3.4.3.3 BCR Approach to Certification 97 3.5 References IOI

3.1.3 3.2 3.2.1 3.2.2 3.2.2.1 3.2.2.2 3.2.2.3 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.3.5 3.3 3.3.1 3.3.2 3.3.3

4 4.1

Particular Developments

111

RMs in Quality Control and Quality Assessment

111

4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.5.1 4.1.5.2 4.1.5.3 4.1.5.4 4.1.5.5 4.1.5.6 4.1.6 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.4.1 4.3.4.2 4.3.4.3 4.3.4.4 4.3.5 4.4 4.4.1 4.4.2 4.4.3 4.4.3.1 4.4.3.2 4.4.3.3 4.4.4 4.5 4.5.1 4.5.1.1 4.5.1.2 4.5.1.3 4.5.2 4.5.2.1

Introduction 111 Proper Usage 113 Characterization of Methods 113 Internal Quality Control 115 External Quality Assurance 117 State of the Art 118 Performance of Individual Laboratories 119 Supplement Internal Quality Control 119 To Obtain Consensus Values 119 Investigate Factors Contributing to Performance 120 To Act as an Educational Stimulus To License Laboratories? 120 Conclusions 121 Fresh Materials 121 Introduction 121 Packing Materials 122 Preparation 124 Homogeneity 125 Stability 126 Certified Reference Materials for Microanalytical Methods 127 Homogeneity of Components in CRMs 129 A Priori and a Posteriori Homogeneity of Materials 130 Aspects of Homogeneity Determination 131 Examples 132 Homogeneity Determinations with Solid Sampling Atomic Absorption Spectrometry 133 Homogeneity Determinations with Instrumental Neutron Activation Analysis (INAA) 134 Uncertainty Budget of INAA 135 Homogeneity Factors in Test Materials Determined with INAA 136 Conclusion 137 CRMs as calibrants 138 Principles 138 Calibration Techniques 139 Examples 140 Biological Materials 140 Environmental and Geological Materials 141 Technical Materials ip Conclusion 143 RMs for Radioisotopes, Stable Isotopes and Radiopharmaceuticals 143 Radioisotopes 143 Requirements and uses 143 Available Reference Materials 144 Future Developments 146 Stable Isotopes I& Requirements and uses q G

XI1

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Contents

4.5.2.2 Future Developments 147 Radiopharmaceuticals 147 4.5.3 References 148 4.6 5

5.1 5.1.1 5.1.2 5.2 5.3 5.3.1 5.3.2 5.3.2.k 5.3.3 5.3.3.1 5.3.3.2 5.3.4 5.4 5.5 5.5.3 5.5.2 5.5.3 5.5.3.1 5.5.3.2 5.5.3.3 5.5.2.4 5.5.4 5.5.5 5.5.6 5.5.6.1 5.5.6.2 5.5.6.3 5.5.6.4 5.5.7 5.5.8 5.5.9 5.5.10 5.5.11 5.6 6 6.1 6.1.1 6.1.2 6.1.3

Reference Materials for “Life” Analysis 154 Standard Reference Materials for Microbiological Assays 154 Standards for Official Assays and Tests 155 Quality Management of Biological RMs 156 Certified Microbiological Culture Materials 158 Reference Materials for DNA Analysis 160 SRMzjgo 160 S R M Z ~ ~161 I Recertifiration of SRM 2391 162 SRMz392 163 DNA Source 163 Inter-Laboratory Evaluation of SRM 2392 164 Summary 164 Future Developments in Molecular Reference Materials 171 Reference Substances and Spectra for Pharmaceutical Analysis Intsductiorr 173 Definitions and Guidelines 174 Uses of Pharmacopoeia1Reference Substances 175 Reference Substances Used for Identification 175 R d a m c e Substances Used for Related Substance Tests 176 Refereme Substances Used for Assay 180 Minimizing the Use of Reference Substances 180 Procurement of Candidate Reference Substances 181 Requirements for Candidate Reference Substances 182 EFahation 182 Reheace Substances Used for Identification 182 Reference Substances Used for Related Substance Tests 183 Reference Substances Used for Assay 183 CRS as Calibrators 189 ng Programme 189 Packaging and Filling 190 Certificatesof Analysis / Expiry Date / Catalogue i g i Storage and Distribution 192 International Harmonization 192 References 193 Generd Application Fie€& igG Workplace Air Monitoring 196 Introduction 196 Solvents 197 Elements and Inorganic Compounds

198

172

6.1.4 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.2.2.3 6.2.2.4 6.2.3 6.2.3.1 6.2.3.2 6.2.3.3 6.2.3.4 6.2.4 6.2.5 6.2.6 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.4.1 6.3.4.2 6.3.4.3 6.3.4.4 6.3.4.5 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5

Asbestos igg Clinical Application Fields igg Introduction igy Elements 201 Essential Electrolytes 202 Essential Trace Elements 202 Elements Therapeutically Used 203 Non-Essential Elements 204 Organic Compounds 206 Solvents 206 Polyaromatic Hydrocarbons (PAH) 207 Pesticides 207 Persistent Compounds 207 Proteins and Enzymes 207 Lipids 209 Other Compounds 209 Food/Biological 210 Introduction 210 Food Matrix Triangle 211 Available Reference Materials 214 Mode of Application and Application Examples 214 Procedures for Reference Material Selection and Use 217 Procedures for Reference Material Utilization 217 Performance Interpretation and Corrective Action 217 Examples of Application of RMs to Certification of other RMs 218 Examples of Applying RMs in Analyses 218 Applications of Reference Materials in the Geological Sciences 220 Introduction 220 Producers of Geochemical Reference Materials 222 General Application: Calibration of Instrumental Measurements 223 General Application: Method Development and Validation 224 General Application: Quality Control in Multilaboratory, or Long-Term Within Laboratory, Studies 224 6.4.6 Specific Application: Geochemical Exploration 22j 6.4.7 SpecificApplication: Petrogenic Modelling Based on Bulk Rock Analysis 227 6.4.8 SpecificApplication: Petrogenic Modelling Based on Microanalysis 228 6.4.9 Application: Studies of Paleoclimates 228 6.4.10 Summary 229 6.5 References 229 Proper Usage o f Reference Materials 236 7.1 Selection, Use, and Abuse of RMs 236 7.1.1 Conventional "Proper" Uses of RMs 237 7.1.1.1 Method Development and Evaluation 237

7

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XIV

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Contents

7.1.1.2 7.1.1.3 7.1.2 7.1.2.1 7.1.2.2 7.1.2.3 7.1.2.4 7.1.2.5 7.1.2.6 7.1.2.7 7.1.3 7.1.4 7.1.5 7.1.6 7.2 7.2.1 7.2.2 7.3 7.3.1 7.3.2 7.4 7.5 7.6 8

8.1 8.1.1 8.1.2 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.3 8.3.1 8.3.1.2 8.3.1.3 8.3.1.4 8.3.2 8.3.2.1 8.3.2.2 8.4 8.4.1

Assurance of Measurement Compatibility 237 Establishment of Measurement Traceability 237 Mis-Useand Other Causes of Errors 238 Documentation Errors 238 Selection Errors 239 Handling and Use Errors 241 Storage 241 Shelf Life and Expiration Dates 241 Sampling and Preparation of RMs for Analysis 242 Sample Characteristics 243 Continuity 244 Artifacts 244 Data Interpretation Errors 245 Reporting Errors 246 Statistical Consequences in the Assurance of Measurement Compatibility 247 Uncertainty 247 Indicative Approach to Quantifying Uncertainty 248 Traceability 249 Definition 249 Practical Aspects 250 Conclusions 252 References 253 Further Reading 254 Availability and Sources of Information 256 Introduction 256 Printed Publications 257 Catalogs, Lists, and Directories 257 Journals 259 Electronic Sources and the Internet 262 The “COMAR Database 262 The IAEA Database 264 WinRefPro Database of Elements in Metals 265 Website Addresses 266 Organizations and Symposia 267 organizations 267 AOAC International 267 EURACHEM 268 VAM 269 Conferences and Meetings 269 BERM 269 Other Meetings 272 The Pharmacopoeia 273 United States Pharmacopoeia 273

Contents

8.4.2 8.4.3 8.5 8.6

The European Pharmacopoeia 273 The British Pharmacopoeia 274 The Movement of Reference Materials 274 References 277

9

Future Trends for Reference Material Activity 279 Introduction 279 Overview of General Issues 280 Review of Trends 280 Projection of Challenges 280 Analysis of Driving Forces and Construction of Scenarios 281 Thinking “Outside the Box” 282 Future Projections 283 Selecting Strategies 285 Needs for Specific Reference Materials 286 Reference Material Needs for Regulatory Nutrient Analysis 287 Perspectives from Distributors of Certified Reference Materials 289 RM Needs in Developing Countries 290 References 291

9.1 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 9.1.6 9.2 9.3 9.4 9.5 9.6

About the genesis ofthis book

Subject Index 295

293

Ixv

Preface It is usual for the editors to set the scene for the writing of such a book as this. But there is a long story to tell, for the genesis of the book goes back to the early I ~ ~ o ’ s , and the story is much about us, so we invited our publisher to pull together our story: you will find his tale, should you be interested, at the end of the book. More importantly we editors want to place on record our very great gratitude to all the authors who contributed to this work, our colleagues and friends, many of whom have accompanied us for years. Without their work the history of RMs and also the RM Symposia would be less interesting and colorful. In particular we want to acknowledge Herbert Muntau working from the Joint Research Center at Ispra, Italy, who was a major influence in the evolution of RM preparation and use and did great work from the early beginnings. We deeply regret that he was not able to join the project due to his enormous workload. Many thanks go also to Robert Parr, formely IAEA, Vienna, as a contributor to many symposia and also to this book, to Milan Ihnat from Argiculture Canada for his fine texts and editorial helps, to Steve Wise and Rolf Zeisler from NIST for their valuable chapters, to Jean Pauwels, IRMM, Philippe Quevauviller, SM&T (formely BCR), and Harry Klich, BAM, Germany, for their always ready support. The names of all contributors cannot be listed here, they are acknowledged in the chapters that they wrote: even so we thank them all for t y n g to be as concise and as informative as possible in their delivery of their most instructive sections, sometimes, it must be said, under “mild pressure from the editors, on time and most importantly for the benefit of the readers. We also want to thank the team at WileyVCH for all their advice and technical help during the production of this book. Julich, Germany, Beltsville USA and Teddington, England July 2000

Markus Stoeppler, Wayne Wolf, Peter Jenlts

I

List o f Contributors Dr. Agnes Artiges

Dr. Vanessa Dekou

Director European Pharmacopoeia Commission B.P. 907 F-67029 Strasbourg Cedex, France E-mail: [email protected]

Laboratory of the Government Chemist Queens Road, Teddington Middlesex TWII oLY, England E-mail: [email protected] Dr. Vincent EgloB

Dr. Humphryj.M. Bowen

West Down, West Street Winterborne Kingston Blandford, Dorset DTII 9AT, England Dr. Anthony R. Byrne

Joief Stefan Institute Jamova 39 Slo-1001 Ljubljana, Slovenia E-mail: [email protected]

European Pharmacopoeia Commission B.P. 907 F-67029 Strasbourg Cedex, France Dr. Bernard Criepink

Commission of the European Communities 200 rue de la Loi B-1049 Brussels, Belgium E-mail: [email protected] Dr. Karl-Heinz Grobecker

European Pharmacopoeia Commission B.P. 907 F-67029 Strasbourg Cedex, France

Institute for Reference Materials and Measurements (IRMM) Retieseweg B-2240 Geel, Belgium E-mail: karl-heinz.grobeclter@i~m.jrc.be

Dr. Rita Corneh

Proj Dr. Robert F.M. Herber

Laboratory of Analytical Chemistry Rijksuniversiteit Gent Institute for Nuclear Sciences Proeftuinstraat 86 B-9000 Gent, Belgium E-mail: [email protected]

Coronel Institute for Occupational and Environmental Health Academic Medical Center P.O. Box 22700 NL-1100 DE Amsterdam, The Netherlands E-mail: [email protected]

Dr.jacob de Boer

Dr. Milena Horvat

DLO-Netherlands Institute for Fisheries Research (RIVO-DLO) P.O. Box 68 1970 AB Ijmuiden. The Netherlands E-mail: j [email protected]

Joief Stefan Institute Jamova 39 Slo-1001 Ljubljana, Slovenia E-mail: [email protected]

Dr. Emmanuelle Charton

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List ofcontributors

Milan /hnat, Ph.D. Pacific Agri-Food Research Centre - Summerland Agriculture and Agri-Food Canada Summerland, British Columbia, Canada VOH 120, E-mail: [email protected]

Prof: Dr. Ulrich Kufirst Fachhochschule Fulda, Fachbereich HSrE. Marquardstrage 35 36039 Fulda, Germany E-mail: [email protected]

Prof: Dr. Heinz-Dieter lsengard Universitat Hohenheim Institut fur Lebensmitteltechnologie Garbenstrage 25

Barbara C. Leuin, Ph.D. 100 Bureau Drive, Stop 8311 National Bureau of Standards and Technology (NIST) Gaithersburg, M D 20899-8311,USA E-mail: [email protected]

70593 S t U W f l E-mail: [email protected] Prof: Dr.JiirgenJacob Biochemisches Institut f i r Umweltcarcinogene Prof Dr. Gernot Grimmer Stiftung Lump 4 zag27 GroEhansdofi, Germany E-mail: [email protected] PeterJJenks, B.Sc. Laboratory of the Government Chemist Queens Road, Teddington Middlesex TWII oLY, England E-mail: pjj @lgc.co.uk Shung-ChangJong. Ph.D. American Type Culture Collection (ATCC) 10801 University Blvd. Manassas, VA 22715, USA E-mail: [email protected] Ms.Jean 5. Kane

Robert T. Kane Associates Inc. HCR 4 Box zjr Brightwood, VA 22715, USA E-mail: [email protected] Dipl. Ing, Harry Klich Bundesanstalt fiir Materialforschung und Prifung (BAM),Referat 1.01 Rudower Chaussee 5 12489 Berlin, Germany E-mail: [email protected]

Dr. Jan Kucera Nuclear Physics Institute CZ-2jo68 Re? near Prague, Czech Republic E-mai1: [email protected]

Dr. John H. McB. Miller European Pharmacopoeia Commission B.P. 907 F-67029 Strasbourg Cedex, France E-mail: [email protected] Pro$ Dr. Ing. Knut Ohls Bungerstrage 7 44267 Dortmund, Germany E-mail: [email protected] Dr. Robert M. Pan Langackergasse 28a A-1190 Vienna, Austria E-mail: [email protected]

Dr.Jean Pauwels Institute for Reference Materials and Measurements (IRMM) Retieseweg B-2240 Geel, Belgium E-mail: [email protected] Dr. Philippe Queuauuiller

European Commission DG Research (MO7j 3jg) zoo rue de la Loi B-1049 Brussels, Belgium E-mail: Philippequevauviller@ cec.eu.int DennisJ. Reeder, Ph.D. IOO Bureau Drive, Stop 8311 National Bureau of Standards and Technology (NIST) Gaithersburg, MD 20899-8311, USA E mail: [email protected] Dr. Ulrich Rose European Pharmacopoela Commission B.P- 907 F-67029 Strasbourg Cedex, France

List ofContributors Dr. Matthias Rossbach

Dr. Adriaan M.H. van der Veen

International Atomic Energy Agency (IAEA) Chemistry and Industrial Applications P.O. Box I O O A-1400 Vienna, Austria E-mail: [email protected]

Korte Slagen NL-4823 LN Breda, The Netherlands E-mail:[email protected]

Stephan Riickold, Dip/.-Lebensmittel-lng.

Institute for Reference Materials and Measurements (IRMM) Retieseweg B-2240 Geel, Belgium E-mail: [email protected]

Stephen A. Wise, Ph.D.

National Bureau of Standards and Technology (NIST) Analytical Chemistry Division, Bldg. 2 2 2 , Rm. B 208 Gaithersburg, M D 20899, USA E-mail: [email protected] Wayne R. Wolf; Ph.D.

Dr. Markus Stoeppler Mariengartenstr. Ia

52428 Jdich, Germany E-mail: Markus. [email protected] Jan Straub, M A .

Coronel Institute for Occupational and Environmental Health Academic Medical Center P.O. Box 22700 NL-1100 DE Amsterdam. The Netherlands Jane Tang, Ph.D.

American Type Culture Collection (ATCC) 10801University Blvd. Manassas, VA 22715, USA E-mail: [email protected] Dr. Andrew Taylor

Centre for Clinical Science and Measurements School of Biological Sciences University of Surrey Guildford, G U gXH, UK E-mail:[email protected] Dr. Yngvar Thomassen

National Institute of Occupational Health P.O. Box 8149 DEP N-oojj Oslo, Norway E-mail:[email protected] Dr. Barry Tylee Health & Safety Laboratory Broad Lane Sh&ieId, Sy 7HQ, UK E-maiI: barry.t/lee@hsl,gov.uk

Human Nutrition Research Center US Department of Agriculture 10300 Baltimore Blvd. Beltsville, MD 20899, USA E-mail:[email protected] Dr. RolfZeisler

National Bureau of Standards and Technology (NIST) Analytical Chemistry Division, Nuclear Methods Group, Bld. 235, Rm. B - I ~ Gaithersburg, MD 20899, USA E-mail: [email protected]

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Abbreviations General

AOAC ASTM ATTC BAM BAS BP BCR B ERM/B RM BGS BNM BRPs BRSs CBNM

CITI CRM CRS DGKC DUREM EP EURACHEM FBI GBW GMO IAEA IFCC IPCRSs IRMM

Association of Official Analytical Chemists International American Society for Testing and Materials American Type Culture Collection Federal Institute of Materials Research, Germany (Bundesanstalt fur Materialforschungund -priifung) Bureau of Analytical Samples LTD., UK British Pharmacopoeia Community Bureau of Reference International Symposia on Biological and Environmental RMs British Geological Survey Bureau National de Mktrologie, France Biological Reference Preparations Pharmaceutical Reference Substances Central Bureau for Nuclear Measurements, Geel, Belgium, now IRMM Chemicals and Inspection Testing Institute, Japan Certified Reference Material Chemical Reference Substances Deutsche Gesellschaft fur lclinische Chemie National Workshop on Development and Use of Reference Materials (India) European Pharmacopoeia Association of European Chemical Laboratories US Federal Bureau of Investigation CRMs of NRCCRM, China Genetically modified organism International Atomic Energy Agency International Federation of Clinical Chemistry International Pharmacopoeia Chemical Reference Substances Institute for Reference Materials and Measurements, Belgium, formerly CBNM

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Abbreviations

IRSID IS0 ISO-REMCO IUPAC LGC NBS NCCLS NIBSC NIES NIOH NIST

NPL NRCC NRCCRM NTRM NWRI PAHs PCBs Ph. Eur. CRSs PTB REMTAF RIVM RM SMELT SRM TPhT TBT TDRM USDOD USFDA USGS USP VAM WHO

Institut de Recherches de la Siderurgie, France International Organization for Standardization I S 0 Council Committee on Reference Materials International Union for Pure and Applied Chemistry Laboratory of the Government Chemist, UIC, formerly NPL National Bureau of Standards, USA, now: NIST National Committee for Clinical Laboratory Standards, USA National Institute for Biological Standards and Control, UK Japanese National Institute for Environmental Studies National Institute of Occup. Health, Oslo, Norway National Institute of Standards and Technology, USA, formerly NBS, National Physical Laboratory, UIC National Research Council Canada Chinese National Research Center for CRM NIST Traceable RM National Water Research Institute, Canada Polycyclic Aromatic Hydrocarbons Polychlorinated Biphenyls European Pharmacopoeia1Chemical Reference Substances Physiltalisch-TechnischeBundesanstalt, Germany National Task Force on RMs, India National Institute of Public Health and Environmental Protection, NL Reference Material Standards, Measurements and Testing Programme, EU, formerly BCR Standard Reference Material, NBS, NIST, Trade Mark Triphenyltin Tributyltin AOAC Technical Division on RMs United States Department of Defense United States Food and Drug Administration US Geological Survey United States Pharmacopoeia Valid Analytical Measurement, UK World Health Organization

Abbreviations

Analytical Methods

AAS AES AF S ASV

csv

CVAAS DCPAES DPP ENAA ET-AAS FAAS FLU GC GC-FID GC-MS G F-AAS HGAAS HGICPAES HPLC HPLC-MS ICP-AES ICP-MS IDMS INAA I PAA LAS LC-F L NAA NPLC PAA PFE PGNAA PIXE RNAA SPE ss-AAS S S-ETV-ICP-AE S SS-GFAAS SSMS ss-ZAAS XRF

Atomic Absorption Spectrometry Atomic Emission Spectrometry Atomic Fluorescence Spectrometry Anodic Stripping Voltammetry Cathodic Stripping Voltammetry Cold Vapor AAS Direct Current Plasma AES Differential Pulse Polarography Epithermal NAA Electrothermal AAS, also: GF-AAS Flame-AAS Fluorometry Gas Chromatography GC with Flame Ionization Detector GC-Mass Spectrometry Graphite Furnace-AAS, also: ET-AAS Hydride Generation AAS Hydride Generation ICP-AES IHigh Performance Liquid Chromatography High Performance Liquid Chromatography-MS Inductively Coupled Plasma-AES Inductively Coupled-Plasma MS Isotope Dilution MS Instrumental NAA Instrumental Photon Activation Analysis Molecular Light Absorption Spectrometry Liquid Chromatography with Fluorescence Detection Neutron Activation Analysis Normal Phase Liquid Chromatography Proton Activation Analysis Pressurized Fluid Extraction Prompt Gamma NAA Particle Induced X-Ray Emission Radiochemical NAA Solid Phase Extraction Solid Sampling AAS Solid Sampling-Electrothermal Vaporization-ICP-AES Solid Sampling Graphite Furnace AAS Spark Source MS Solid Sampling Zecman AAS X-Ray Fluorescence

I

XXV

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

I’ 1

Introduction Edited by Markus Stoeppler 1.1

Historical Markus Stoeppler and HumphtyJM Bowen

This Chapter reviews some selected historical examples of the development, production and use of reference materials (RMs), from the past century up to the present. It is, of necessity, a rather short and incomplete review describing international efforts in this area. From the references given at the end of this Chapter and at the end of Chapter 3, the reader may investigate further into the past. 1.1.1

Early Developments

The history of reference materials is closely linked with the development of analytical chemistry. In the 19th Century all chemicals were, in comparison with those of today, of poor purity. Thus, for volumetric analysis suitable purified materials as primary standards had to be specified. One of the first examples was the recommendation of As(II1) oxide by Gay-Lussac (1824) for this purpose. Somewhat later, Sorensen (1887) proposed criteria for the selection of primary chemical standards. These were further elaborated by Wagner (1903)at the turn of the last century. It is worthwhile mentioning that their criteria were quite similar to those used today. One of the first attempts to use a biological RM was for the analysis of the fat content of milk. This was carried out in London in the late 1880’s by a number of analpcal chemists who were trying to identify adulterated milk. At that time milk was sold unpackaged and at least 20 % of the milk sold in London was adulterated by dilution with water. This work appears to be the first empirical round-robin approach for characterization of a RM. In medicine the need for standards was just as acute. Beal (1951)mentioned that the U.S. Pharmacopoeia VI, issued in 1880,took a big step forward by adding tests for purity and quality of the materials described in it, but the use of reference materials as an integral part of the pharmaceutical monographs for drugs did not start until the 1950’s. Another example of activities at the end of the 19th Century was associated with the introduction, by Ehrlich, of the first diphtheria antitoxin and his

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7 Introduction

discovery of Salvarsan (arsphenamine), the first effective cure for syphilis. Whilst these were by no means the first such medicines, Ehrlich was the first to calibrate the purity of the preparations of these substances by bio-assay against an arbitrary standard kept at low temperature (Ehrlich et al. 1894; Ehrlich 1910).Others followed his lead, from 1921the State Serum Institution of Copenhagen, Denmark, collected a set of international standards for therapeutic agents including diphteria antitoxin as well as arsphenamines. These standards were increased in number after the discovery of vitamins and sex hormones in the 1930’s (e.g. Dale 1939).A main source of these clinical standards was the National Institute of Medical Research in London. In 1948 a total of 35 International Standards were held at this institute (Miles 1948). In the 1960’s the need for reliable quality control of clinical chemistry determinations had been highlighted in the USA and England following the introduction of the first reliable automated analytical systems, the Technicon Auto-Analyzer Whiteheads work at the University of Birmingham led to the introduction of routine inter-laboratoryperformance testing schemes and the regular use of thoroughly validated reference materials (Radin 1967; Meinke 1971; Booth et al. 1974; Whitehead 1976).Today the UK-EQAS scheme and the CAP scheme in the USA share a common philosophy of continuous quality improvement through repeated use of inter-laboratory studies - a concept far from the “pass-fail” mentality common in other disciplines, see also Sections 4.1 and 6.2. Other scientific disciplines required standards. When the American Type Culture Collection (ATCC) was founded in 1925,one of its chief roles was to be a source of standards for the rapidly developing public health laboratory activity in the USA. In this context we mean standard organisms, rather than standard materials or chemicals, but their use was analogous, they helped produce better analpcal data. In 1901, the U.S. National Bureau of Standards (NBS) - now the National Institute of Standards and Technology (NIST) - was founded because of the increasing demand for various kinds of standards in the rapidly developing engineering industries. The early history of the NBS reference material program started in 1905 with a cooperative effort within the iron and steel industry whereby industrial analysts helped characterize the individual reference materials. Cooperation with NB S was recognized as a mark of achievement for the laboratory, so this effort served a dual purpose. It both helped the laboratory develop its measurement skills and also helped NI ST understand the measurement problems associated with a given matrix. The analysis of irons and steels is mostly about the measurement of inorganic elements in an inorganic matrix. Hence a variety of separation and measurement techniques could be used, compared and evaluated to arrive at the best answer. Problems were usually solved by looking for complete dissolution and understanding the interferences affecting the various methods that were employed. Since most methods used were related to gravirnetric quantities (i.e. weighed quantities of pure inorganic substances), traceability to the mole was not an issue and comparability and compatibility of measurements is what was sought. NIST’s first four certified reference materials were steel samples, and these were followed by many others. The program supplied analytically well characterized homogeneous materials. This program included, from the beginning, homogeneity

7 . 7 Historical

evaluation, cooperative multi-laboratory characterization and an evaluation of the measurement process to assure accurate values (Cochrane 1966; Uriano and Gravatt 1977).The success of this work initiated many requests from other industries for the development of appropriate materials and led to a rapid growth of this part of the NBS program and the adoption of the name “Standard Reference Material” or “SRM”; later these terms were registered as Trade Marks of NIST. By 1951NBS was advertizing 541 SRMs, of which 2 0 0 were alloys, ores or ceramics and 204 were hydrocarbons or oils (Bright 1951). Similar work for RMs in the fields of metallurgy and ceramics was performed by many U.S. commercial sources and in other countries as well. Examples of early suppliers of appropriate RMs include in the United Kingdom the Bureau of Analyzed Samples Ltd. (BAS), who issued RMs from 1916; in Germany the Federal Institute of Materials Research and Testing (BAM), founded 1904 as the “Royal Bureau of Materials Testing” and who issued RMs from 1912, and the Physikalisch-Technische Bundesanstalt (PTB), the Japanese Iron and Steel Institute, the French BNM (Bureau National de Mktrologie) and IRSID (Institut de Recherches de la Siderurgie FranGaise) and the Polish Committee on Standardization and Measures. All of these organizations still carry out their work; and their RMs have developed and evolved as the demands of the metallurgy industry have increased. In geochemistry, the introduction of RMs did not take place until 1951but, once RM usage became a regular part of geochemical analysis, the consequences were not far short of amazing. For many years geochemical analysts had been concerned about the accuracy of their determinations of major elements in rocks, but it was the potential of emission spectrometry for the determination of trace elements which set off the production of the first rock Certified Reference Materials (CRMs), G-I and W-I by the U.S. geological Survey (USGS) (Ahrens 1951). Geochemical CRMs characterized by a number of different institutes, including NBS, were distributed in increasing numbers by the USGS. This led in the following years to remarkable improvements in resolving major disagreements between analysts using similar or distinct techniques in geochemical analysis. From about 1965 many other international organizations were supplying geochemical RMs, (e.g. Richardson 1995; Imai et al. 1996;Potts 1997). For details see Section 6.4. Until the 1950’s the only “biological” reference materials available were a few commercial sera produced by Seronorm A/S, the Welcome Foundation and others, as a result of the U.K. and U.S. clinical chemists’ initiatives. As many elements are found in biological matrices at much lower levels than in industrial and geological samples, improvement of the quality of elemental analysis in agricultural applications was the aim of a committee convened at Michigan State University in November 1950. About g kg of leaves from four orchard trees, apple, cherry, peach, and orange were homogenized and distributed to 16 U.S. laboratories in a round-robin exercise. The results showed good agreement for the major elements Ca, K, Mg, N, and P, but were much less precise for essential trace elements (Kenworthyet al. 1956).A similar collaborative study using nine vegetable RMs sent out to 13 Canadian laboratories was reported somewhat later (Ward and Heeney

1960).

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Humphry Bowen did pioneering work for the development and use of appropriate biological matrix reference materials. He was at that time developing techniques for radiochemical neutron activation analysis (RNAA) and realized that there were no standards or RMs to check the accuracy of results in this work. He had some geochemical CRMs but these were unsuitable because biological materials have a different matrix and contain orders of magnitude lower concentrations of most trace elements. Thus he prepared in 1960 IOO kg of kale (Brassica oleracea) powder, a large amount for the time (Bowen 1965);for details of the preparation see Section 2.1. Indeed, Bowen’s kale served for more than two decades as a reference and valuable aid for many analysts (Wainerdi 1979; Bowen 1984). Thus his work and the wide demand for his kale RM was pivotal to the future direction of biological reference material and stimulated the planning, preparation, distribution and analysis of further materials of similar kind. 1.1.2 Growth and Maturity

The production of biological matrix RMs for elemental analysis began in national and international institutions in the late 1g60’s.The National Bureau of Standards, now NIST, announced its intention to produce biological RMs in 1967. Orchard leaves, its first botanical reference material, was certified and distributed in 1971 (Meinke 1971).Workers at NIST were also the first to see the need to introduce sterilization of CRMs using y-radiation for longer shelf life. In 1911 NBS offered just 23 certified reference materials, by 1975 more than 1000 SRMs were available with approximately 700 of these intended for use in chemical analysis (Cali and Stanley 1975). At the end of the last century ( ~ g g g )about , the same number of NIST SRMs were available, with many now including a considerable percentage certified for organic constituents and other analytes never dreamed of by Bowen (Trahey 1998).As we enter the zIst Century, attention turns to the measurement of DNA in plant material as the arbiter of genetic identity, and RMs will be required, see Section 5.4. Another important part of the history of reference materials has been the contribution of the International Atomic Energy Agency (IAEA) in Vienna. The IAEA began an analytical quality control service (AQCS) aiming at assisting its Member states to maintain and improve the quality of analytical data obtained in their laboratories. Indeed in the 1960’sAQCS was concerned primarily with radioactive measurements. Later it became involved with the reliability of nuclear methods in elemental analysis and practically all IAEA AQCS RMs started out as intercomparison materials. The first five IAEA biological reference materials were issued in 1970. They included a marine RM (mussel shells) from the IAEA laboratory at Monaco, and by 1983 the IAEA had issued 28 CRMs, but most of them were quickly exhausted because of their popularity (Parr 1984). The 1998199 IAEA AQCS catalogue contains more than 90 CRMs of environmental and biological origin for a wide range of determinedness, encompassing radionuclides, trace elements, petroleum hydrocarbons, pesticides, and PCBs (International Atomic Energy Agency 1998).

1. I Historical

In the 1970’s and 1980’s,a number of other organizations started programs designed to provide biological, environmental, and food RMs and CRMs. Projects for the development and certification of food matrices were initiated by the U.S. Department of Agriculture, Agriculture Canada and the U.S. Food and Drug Administration (Wolf and Ihnat 1984; Ihnat and Wolf 1984; Tanner 1984) in co-operationwith NIST. Examples are a total diet SRM (Wolf et al. 1990) and a series of agricultural/ food RMs (Ihnat and Wolynetz 1993). Early in the 1970’s the then European Economic Community (EEC) established a community-wide CRM program in order to pull together many of the diverse and widespread RM activities under the direction of the Community Bureau of Reference (BCR) (van der Eijk 1979). BCR made first a compilation that covered the major producers, both governmental and private, not only in Europe but on a worldwide basis (Commission of the European Communities 1973; Cali and Stanley 1975).The BCR of the EEC, Brussels, now the 5th framework program of the European Comissions DG Research, initiated in 1970 a program to make available a broad range of CRMs (Griepink et al. 1991);the first biological CRMs were issued in 1983. A large number of candidate materials were initially prepared within that program at the Joint Research Centre Ispra (Rossi and Colombo 1979). It should be mentioned that the continuous efforts of Herbert Muntau at the Ispra Laboratories were the basis for many new environmental and biological CRMs (Muntau 1979, 1980, 1984). BCR started also the production and certification of food CRMs in cooperation with several qualified European laboratories (Wagstaffe 1984). In 1984 the Institute for Reference Materials and Measurements (IRMM), previously called Central Bureau for Nuclear Measurements (CBNM), initially purely nuclear, was given a major role in the storage and distribution of BCR RMs. At the same time a series of major investments were started at the IRMM to set up facilities for the preparation of highest quality candidate RMs in economically attractive conditions (Kramer et al. 1998);see also Section 2.2. From 1995 IRMM had complete responsibility for stock management, sales policy, and renewal of sold-out materials. The increase in production and certification is significant: in 1984 BCR issued about IOO individual CRMs, in 1999 this number had increased to 570, including nuclear and isotopic materials (IRMM 1999). Valuable contributions were made by two Canadian agencies, particularly by the National Research Council Canada (NRCC) who, from about 1976, provided marine and marine biological CRMs certified for metals, metal species and organic constituents (Berman 1984; Willie 1997). More recently their Halifax laboratories have issued a highly respected range of CRMs for the determination of shellfish toxins. Another Canadian producer, the National Water Research Institute (NWRI) specialized in marine (water and sedimentary) CRMs, and from the late 1980’s their matrix materials certified also for organic compounds (Chau et al. 1979; Lee and Chau 1987). In the United Kingdom chemical RMs were first produced some time in the late 1960’s at the National Physical Laboratory (NPL) Division of Chemical Standards at Teddington. This Division was transferred to the Laboratory of the Government Chemist LGC in November 1978. Early work was based on the development of highly

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1 Introduction

purified and certified pesticides, issued as CRMs. Further developments continued under LGC and the range expanded steadily. In the 1980’sthe Validity of Analytical Measurement (VAM) programme started to consider the role CRMs play in the production of valid results. Following a number of consultation exercises a list of needed matrix CRMs was developed and during the 1990’s a wide range of matrix CRMs were produced and certified. At present it offers about 300 CRMs. Further afield, in 1978 the Japanese National Institute for Environmental Studies (NIES) started the production of a series of biological and environmental matrix CRMs, certified for a number of trace elements (Okamoto and Fuwa 1985).Recently also the certification of metal species in some materials was reported (Okamoto and Yoshinaga 1999). A remarkable level of activity can be seen in China. The National Research Center for CRM (NRCCRM) was founded in 1980 and the certification and accreditation program for “ G B W RMs started in 1983 by co-operation with many Chinese Institutions. In 1993 around Go RMs and CRMs were available (Chai Chifang 1993) and in 1999 the availability of about 1000 CRMs was reported, around 30 of them clinical, IOO environmental, zoo geological, and 300 metallic matrix materials (Rong and Min 1999). Increasing activities for the production and certification of biological, environmental and geological CRMs from the late 1980’s have been also reported from Poland (Dybczyhslti 1995) and the Czech Republic (ICutera et al. 1995,1998). From 1983 to 1997, seven international symposia on biological and environmental reference materials have been alternately held in the USA and Europe. The proceedings mirror the global developments and problems that were discussed during these meetings including also the needs of developing countries. These symposia are described together with other RM meetings in some detail in Chapter 8. 1.1.3

Milestones and The Future

Finally some additional milestones in organization, research and development of RMs need to be mentioned. The large increase in the number of reference materials being produced led in 1975 to the formation of an I S 0 Council Committee on Reference Materials (ISO-REMCO) charged with the establishment of international guidelines on principles of certification, methods of use, needs, availability and nomenclature (Klich 1999). see also Sections 1.2 and 1.3. Significant improvements in analytical methodology and better controlled sampling procedures for environmental and biological materials led in the 1980’sto the recognition that older data describing the analysis of trace metals in, for example, natural waters and biological fluids were erroneously high, sometimes by orders of magnitude. The causes have been found to be due to inadequate methods and contamination from sampling and analytical tools. This reality must be reflected in the preparation of a new class of, as far as possible, contamination-free reference materials prepared with utmost care and reflecting the state of the art at the end of the 20th Century. Examples for this are the procedures applied at NRCC for the prep-

7.2 The Theoretical Basis 17

aration of natural water CRMs (Berman et al. 1983)and the efforts to produce similarly contamination-free bovine (Veillon et al. 1984) and human serum CRMs. The latter were prepared under rigid quality control by a group of expert laboratories and called “second generation biological RMs” (Versieck et al. 1988);for more details of preparation of this material see Section 2.1. Another step forward was the introduction of milling of solid, mainly biological, materials at liquid nitrogen (cryogenic) temperatures. These techniques allowed homogenization under “contamination minimized” conditions, previously thought impossible to achieve. Cryogenic techniques were used to prepare materials like hair. Another advantage of the cryogenic procedures is that smaller particle sizes can be obtained than with most conventional procedures (Zeisler et al. 1983; Schladot and Backhaus 1988;Kramer et al. 1993).The technique was successfully applied and tested in the preparation of Specimen Bank materials for long-term stored under cryogenic temperatures without any change of chemical composition. From the experience gained during these programs, cryogenic milling and long-term cryogenic storage offers unique possibilities to prepare RMs with practically indefinitely long shelf-lives (Stoeppler and Zeisler 1993; Emons 1997). Two challenging, but very difficult tasks have been tackled mainly or increasingly during the last two decades: the certification of organometallic species and valency states of elements (see Section 3.3), and organic compounds (see Section 3.4). But doubtless this was just the beginning and a wealth of work waits in the future to serve all needs of the analytical community (Quevauvillerand Maier 1999). From the mid 1980’s the rise of Quality Standards, Total Quality Management and Accreditation schemes created a booming demand for RMs and CRMs. Thus, the use and production of matrix RMs rapidly increased the new IAEA database lists 56 producers from 22 countries and about 1640 RMs. The 1998 Comar database, which covers a much wider scope, lists more than 200 producers and around 10ooo RMs; see Chapter 8 for more details. The demand for RMs and CRMs continues to grow. As traditional chemical analysis moves into biochemistry and molecular biology the demand for RMs does not abate: the only question is “what is next?” Chapter g considers these, and other future issues critically.

1.2

The Theoretical Basis Adriaan van der Veen

The basis for the preparation and use of reference materials (RMs) is given in I S 0 Guides 30-35. These documents deal with the following aspects of the preparation and use of RMs 30. Terms and definitions 31. Certificates, reports, and labels 32. Calibration using RMs 33. Other uses of RMs

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I Introduction

34. Quality systems of RM producers 35. General and statistical principles of the preparation of RMs Each of these documents will be reviewed briefly in this Section. The definition of a certified reference material (CRM) is given in I S 0 Guide 30 (1992)and it forms the root of all other I S 0 Guides: Reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to a n accurate realization Ofthe unit in which the property values are expressed, andfou which each certij?ed value is accompanied by a n uncertainty at a stated level of conjidence. Additionally,there is also a definition for a RM:

Material or substance, one or more of whose property values are suflciently homogeneous and well established to be used for the calibration of a n apparatus, the assessment Ofa measurement method, or f o r assigning values to materials. The key difference between a CRM and an RM is the traceability. In order to play any role at all in metrology, traceability is a key property. “Traceability” refers to a property value of the CRM, and thus to the underlying measurements. Insufficient traceability of these measurement results will eventually lead to a RM that cannot be certified, as the property value cannot be related to other standards. In the ideal case, traceability is realized up to the International System of Units, SI, but this is only feasible for a very small number of CRMs. Most CRMs are so-called “matrix-CRMs”,identifying that they have been made from material sampled in nature. For these materials, it is impossible to come up with property values traceable to SI, as preparation steps cannot directly be related to that. At best, a comparison can be made among methods, from which usually one is the technically best established method, and the results of such a comparison may flow into the establishment of the property values and their respective uncertainties. The most important document, accompanying a CRM is its certificate. I S 0 Guide 31 (1981)provides guidance for the establishment of certificates, labeling of CRMs, and certification reports. The certificate contains among other information the certified values and their respective uncertainties. As important as this information is the traceability statement, which defines to what references the CRM is traceable. Ideally, a CRM is traceable to a suitable (combination) of SI units. This is not always possible, so other “stated references” may appear here. Especially when certifying matrix reference materials, malting the measurements traceable to SI does not imply that the CRM is traceable to SI as well. The steps necessary to transform the sample into a state that can be measured may have a serious impact on the traceability of the values, and thus on the traceability statement. Although labels and certificates are mandatory, certification reports are not. It also depend on the kind of RM, whether such a report is of any relevance. For instance, for the certification of gas mixtures, a certification report would not usually

7.2 The Theoretical Basis

contain more information than is already presented on the certificate. In other cases, a certification report might be of interest, but for other reasons (economical, political) is not feasible. For many matrix RMs, brief or extensive certification reports are made available, containing for instance the results of the homogeneity and stability studies, as well as the results from the characterization measurements. This allows the user to gain some extra insight in the properties of the RM, and possibly about problems in measuring the CRM with certain methods. There are t w o main uses of a RM: calibration and method performance checking. I S 0 Guide 32 (1997)deals with the use of RMs for calibration purposes. RMs used for calibration purposes are usually RMs prepared by synthetic means. Commonly, the property values of these RMs are known from preparation, and verified by some kind of suitable measurement technique. This can be a technique directly providing a value for a property of interest, or a technique that allows the comparison of the new material against older measurement standards. I S 0 Guide 32 provides guidance in two ways. Apart from the guidance on using RMs for calibration purposes, it also provides information on the preparation and use of calibrants in a laboratory, and checking them against other RMs or measurement standards. I S 0 Guide 33 (1998)deals with other uses of RMs. It elaborates on various uses of RMs, excluding calibration, which is the subject of I S 0 Guide 32. In most cases, RMs are used as a quality control measure, i.e. to assess the performance of a measurement method. Most matrix RMs are produced with this purpose in mind. Other purposes of RMs are the maintenance of conventional scales, such as the octane number and the pH scale. I S 0 Guide 33 provides guidance on the proper use of RMs, and therefore it is together with I S 0 Guide 32 the most important document for users of CRMs. The assessment of the performance of a method is commonly checked by means of a (C)RM. In those cases where there is no RM available, considerable effort is requested from the laboratory to assess the performance of their own methods. The aspect of traceability of the certified value(s) is also of great importance: whenever necessary, the laboratory will make modifications in its procedures if the result of a measurement using the RM appears to be unsatisfactory. If the traceability of the values to other references is not fully established, then this judgement may be clouded by doubts about the certified value(s). Another question to be addressed by the laboratory is the portability of the measurement results on the RM to their common test samples. The behaviour of matrices that are named the same may still widely differ. There are also examples known where, for selected parameters, it is very well possible to transfer the results on the RM directly to the daily practice. As a rule, this is not possible. The conchsions drawn from a measurement on a RM should be translated with care to the measurement practice. The result from a measurement on a RM is commonly a difference between the observed value and the certified value. This difference is called measurement bias, and can, appreciating both the uncertainty on the RM as well as the uncertainty added during the measurement, be tested for (statistical)significance. I S 0 Guide 33

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I Introduction

provides the uncertainty calculations necessary to carry out such an assessment. Commonly, if a measurement bias appears to be significant, the laboratory attempts to improve the measurement procedure, effectively reducing the measurement bias. It should be noted that a measurement bias smaller than the expanded combined uncertainty from the RM and measurement is not meaningful, unless a history record exists that shows trends. This kind of trend analysis may be important for the laboratory, but falls outside the scope of I S 0 Guide 33. Another important use of RMs is the maintenance of conventional scales. The octane number of gasoline is an example of such a scale. The scale is defined through chemicals. This definition can be realized through RMs. Another example is the pH scale, which is defined by buffers with pH = 4, pH = 7, and pH = 10. These buffers are defined as mixtures of salts, dissolved in water. These define the pH scale can be used by laboratories for the purpose of calibrating their pH meters. By now, it should be clear what role RMs play in measurement science. This puts great responsibility on the producers of RMs, as they must see how to satisfy the requirements set implicitly or explicitly by the users regarding matrix, parameters, uncertainty, and traceability. Laboratories use RMs often as a quality control measure, but it this obviously only valid if the RM is produced under proper conditions. In order to ensure the quality of RMs, it is highly recommended that producers work under a quality system. I S 0 Guide 34 (1996) provides guidance on how to set up a quality system for the production of RMs. It builds on I S 0 Guide 25 ( ~ g g o ) , setting apart from the same requirements a series of extra requirements related to material and sample management, homogeneity and stability testing, and the traceability of the property values. In the first edition, I S 0 Guide 34 was a document to be used on top of I S 0 Guide 25. In the voting draft of I S 0 Guide 34:1ggg, the document has become stand-alone, thus fully discussing the requirements for a quality system. Demonstrating competence in this field is of particular interest. Field laboratories are required to demonstrate their traceability through using RMs where possible and appropriate. Obviously, the producers of these RMs must also be able to demonstrate quality and traceability, as otherwise the international measurement infrastructure becomes a set of isolated smaller networks, rather than one big network. So, providing traceability is one of the key issues in the production of RMs. Whereas I S 0 Guide 34 sets requirements for the quality system of a CRM producer, I S 0 Guide 35 (1989) provides guidance on how to implement many of these requirements. Among these, the document also provides a general and statistical outline of the process that leads to CRMs. The current edition of I S 0 Guide 35 is a little outdated, but still most of the contents are valid. The preparation of RMs is possibly the most complex subject dealt with in the I S 0 Guides 30-35. In I S 0 Guide 35, the general requirements as well as the statistical frame for setting up certifications of RMs are discussed. As RMs cover a very wide area, ranging from high-quality gas mixtures with very tight uncertainties, via soils certified for organic contaminants up to microbiological RMs, the exact implementation of these requirements is as manifold as there are RMs.

7.3 Technical Requirements

I S 0 Guide 35 provides details on how to implement homogeneity testing, stability testing, and different ways of characterizing RMs. The document also provides a statistical framework on how to evaluate the results of these measurements and how to establish the certified value and its uncertainty. For the producer of a RM, this guide is probably the most important one, as it details a possible implementation of the production of traceable RMs. The next Section gives an overview of the technical requirements when producing RMs, along the lines of I S 0 Guide 35.

1.3 Technical Requirements Adriaan van der k e n

The preparation of a reference material requires a great deal of planning prior to undertaking any actual activity in the project. A substantial part of the planning deals with the amounts of material needed, as well as with the design of the homogeneity, stability, and characterization studies. The design also includes the choice of appropriate measurement methods for these studies. The number of samples to be produced is also a very important variable in the planning process. With a basic outline of the items mentioned, the amount of raw material to be sampled can be estimated. The planning of a project starts with the definition of what reference material is to be produced. Usually, this definition just say is something like: 8

“preparation of a soil reference material containing a series of trace elements at relevant concentration levels for environmental analytical chemistry”

Although this definition might need some further specification, it is satisfactory to start the design of the project. The first task in such a project is to obtain a sufficient amount of raw material with the desired properties. The amount of material needed is dictated by the following parts of the projects:

8

the number of samples of reference material needed the need for a feasibility study the number of samples needed for the homogeneity study the number of samples needed for the stability study the number of samples needed for a characterization of the reference rnaterial

Each of these aspects will be addressed briefly in this Section. The number of samples of reference material needed is a commercial issue in the first place. An important variable is the number of samples likely to be sold during the lifetime (“shelf life”) of the reference material. As the lifetime is a function of the intrinsic stability of the material, this variable also affects the amount of raw material is needed. For instance, microbiological materials have limited intrinsic stability, and therefore their lifetime is expected to be shorter than for a dry sediment certified for trace elements. So, under the assumption of an equal number of sam-

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7 Introduction

ples to be dispatched per year, the number of samples needed for the rnicrobiological material is greater than for the dry sediment. In those cases where there are any doubts about the feasibility of producing a sufficiently homogeneous and stable reference material, a feasibility study might be needed. For this study, an extra amount of material is needed. Questions regarding the best way of preparing the sample, the stability of the material, or the fitness for purpose might justify the inclusion of a feasibility study in the project. In the BCR projects, it is common practice to have a feasibility study, which usually has as the sole purpose of assessing the performance of the laboratories in the collaborative study in relation to the certification of the reference material. The feasibility study allows the participants to fine-tune their equipment, their methods, and their procedures in view of the characterization measurements. In each of these cases, a considerable extra number of samples is needed. The design of a project aiming to produce a new reference material may also include aspects of blending of materials. In several areas, like for instance in environmental chemistry, reference materials are needed with a very wide range of parameters at appropriate (concentration) levels. Often, it is impossible to find all parameters in one material. In those cases, blending two or a few similar matrices may lead to a batch of raw material suitable for the project. The problems associated with this practice are potentially greater regarding homogeneity and stability of the reference material. The extra problems with obtaining sufficient homogeneity are obvious. Extra problems regarding stability usually come from differences in the matrix, allowing for physical and/or chemical processes that would not take place otherwise. Adaptation of the preparation procedures may very well solve these (potential)problems. Another issue in the preparation of reference material is the required shelf life. The shelf life of reference material is the time that it remains stable under proper storage conditions. Depending on the nature of the mechanisms affecting the stability of the material, various actions can be taken to improve the shelf life. Reduction of the moisture content is one of the first options to be considered. In many cases, moisture plays a key role in mechanisms leading to instability of the matrix and/or parameters. In other cases, sterilization or pasteurization of the material might be considered in order to stop bacterial activity. When preparing solutions, additives may increase the shelf life. Obviously, the shelf life of material is also a function of the storage conditions. In the preparation of many solid state reference materials, reduction of the grain size plays an important role. Usually this reduction is required because of the measurement methods to be used both in the projects and later by the users of the reference material, as well as to come to an acceptable minimum sample intake. The minimum sample intake can be defined as the minimum amount of material needed, so that the heterogeneity of the material does not affect the repeatability of the measurement method. The reduction of the grain size is usually implemented by crushing and/or grinding techniques. The techniques employed and the equipment used must be suitable for the purpose of processing the material. Potential problems of contamination, loss of volatile components, and/or other physical and

1.3 Technical Requirements

chemical changes during grinding must be addressed in advance and evaluated during the project whenever necessary. After reduction of the grain size, the material can be divided into portions appropriate for use as a reference material in a laboratory. The size of these portions depends on the expectations of the users, and what is technically possible. In some cases, these test portions are intended for use as such, but in most cases some subsampling is needed. Both approaches have their advantages and disadvantages.The need for sub-sampling can be both: on one hand if no sub-sampling by the user is needed, the producer will have less concern about possible complaints from customers with respect to the homogeneity of the material. On the other hand however, many written standards and other procedures involve some kind of sub-sampling, and having a reference material that does not require sub-sampling implies that the user does not have the option of verifying this step with the reference material. In some cases, providing samples for single-use is a necessity: after opening, the material has so little stability that it is unlikely that it could be used again. In those cases, the desired size of the sample is a portion suitable for single-use only. There is a wide choice of method and equipment for dividing the material into portions. Again, the choice of the method as well as the equipment depends on the nature of the material. A basic requirement however is that the method used guarantees within certain limits that it may be expected that the first sample produced will have the same properties as the last one. The use of dynamic riffling techniques is only one option. Automated subdivision techniques and equipment is becoming increasingly popular, as these systems allow producing batches of a few thousands of samples; and they can also be linked to a labelling system. Especially in those projects, where portions for single use are produced the number of samples is usually large. The whole process so far dealt with obtaining the raw material and the production of sub-samples from it. The issue of homogeneity and stability, as well as the characterization of the material under proper conditions now needs full attention. In the past, homogeneity and stability testing were primarily intended to see whether the sample preparation was completed successfully. This is perfectly reflected in I SO Guide 35 (1989).In recent years, complete new categories of reference materials have come to existence which cannot be dealt with that way. Despite all the effort put in the preparation and conservation of the material, there still may be some heterogeneity and instability left. Just testing for significance with respect to the repeatability of the test method is incorrect in two ways. It does not answer the question of how this remaining heterogeneity and instability affects the uncertainty of the reference material. Furthermore, it leaves open the option of selecting measurement method with a poor repeatability, so that the homogeneity study will not demonstrate any heterogeneity. In cases where test pieces (or items) are prepared, the issue of obtaining a homogeneous batch of items is even more complex. Here the preparation procedure sets limits, in combination with the properties to be certified. The uncertainty of the property values should appreciate this fact, as otherwise the uncertainty of the reference material is only valid for the batch, not for a single item from the batch. This

14

I is an essential requirement, which obviously may also have an impact 7 Introduction

on the design of the project. In doubtful cases, a feasibility study might be needed to investigate whether a reference material can be obtained with an appropriate level of uncertainty. For the issue of stability, the same reasoning holds. In heavy metals analysis, limitations of stability are only an issue for solutions and for some highly unstable matrices. In most matrices, heavy metals are fairly stable and a lifetime of these materials typically exceeds ten years. In organic analysis, as well as in microbiological analysis, materials usually have limited stability. These judgements can obviously only be made when comparing data from the stability study with the uncertainty from the characterization of the reference material. For most proper reference materials in these categories, some uncertainty from limited stability should be taken into consideration. Obviously, if a material shows a slow but steady degradation, the data should be analyzed critically in order to find out whether material could be certified. An additional problem is that usually the stability study is also accompanied by a considerable uncertainty, which should be taken into account somehow. After the verification of homogeneity and stability,the characterization of material can take place. Frequently, this step is named certification rather than characterization, which is wrong in view of the discussion about homogeneity and stability and their impact on the reference material. The certification of our reference material is more than the characterization of the material. However, for most people working on the development of measurement methods, the characterization is the most interesting part of the project. This probably explains the huge amount of literature available. The characterization of a reference material can take place in different ways. Depending on the source cited, there are three or four mainstream approaches; I S 0 Guide 35 (1989)distinguishes between three:

characterization by a single method characterization by multiple methods characterization by means of an inter-laboratory study The third approach is also known as a collaborative study or a collaborative trial. Both names underpin the joint effort of the coordinator and participants to characterize the reference material. In any case, the measurement methods used in the characterization should be traceable to what is called “stated references”,and preferably to SI. The aspect of traceability of measurement results goes well beyond the actual measurements; it also includes the transformation of the sample from the state of the reference material to the state in which it can be measured. An example of such a transformation is the destruction of the sample. Traceability of measurement results is essential in the establishment of a certified reference material. As stipulated in I S 0 Guides 3 0 and 35, a certified reference material can only be certified if there is an uncertainty statement with a traceability statement. Basically, traceability means anchoring. In classical analytical chemistry, that SI system is often the best choice as a reference (= “anchoring point”). However, there is a wide range of parameters either defined by a method or defined by the

7.3 Technical Requirements

conditions under which the measurements take place; and for these measurements another reference might be more appropriate. The choice of the references is primarily the responsibility of the producer. However, he has to look at the measurement practice in the particular area. Another aspect of traceability of the results is the linkage of data from the homogeneity study, the stability study, and the characterization study of the reference material. In order to establish this link, the coordinator must be in the position to demonstrate that the results of these three studies have a common reference. Such a reference can be a calibrant, reference material, or possibly some realization by means of a suitable method. If such a common reference is not available, it is impossible to link the data sets, and therefore it is impossible to translate the results from the homogeneity and stability studies to the characterization of the material. This is also an aspect that should be addressed in the design of the project. In addition to the requirements regarding traceability of measurement results, the measurement methods employed should represent “state-ofithe-arY‘ in the particular field. Failing to do so would lead to a reference material with an uncertainty that has become too large to serve as a quality control. The better the methods perform in terms of uncertainty and traceability, the better the reference material will serve the interests of the (potential)users. The measurement method used for the homogeneity study should have a very good repeatability. For a stability study, where often samples are measured at different days, the reproducibility of the measurement method is of primary importance. So, the methods for homogeneity and stability studies are not necessarily the same. This is not a problem, as long as this common reference already mentioned is available. For the characterization of the reference material, especially in the case of matrix reference materials, it is often desirable to use multiple methods, and often also multiple laboratories. Under these conditions, it is easier to arrive at an uncertainty that represents the “state-of-the-art”of the laboratories. In summary, the preparation of reference material involves the following steps: Definition of the reference material, i.e. the matrix, the properties to be certified, and their desired levels Design of a sampling procedure Design of a sample preparation procedure Selection of method appropriate for homogeneity and stability testing Design of the characterization of the reference material Sampling Sample preparation Homogeneity testing Stability testing Characterization of the reference material Combination of the results from homogeneity testing, stability testing, and characterization and assembling an uncertainty statement Set-up of a certificate and, if appropriate, a certification report

115

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I

1 Introduction

It should always be kept in mind that certified reference materials are used by laboratories, authorities, and regulating bodies for quality control purposes. The primary objective of a reference material is therefore the anchoring of measurements. This anchoring of the measurements of a laboratory using the reference material under ideal conditions i s as good as the anchoring of the measurements used for establishing the reference material. So, there i s a great responsibility for all persons involved in certification projects to work under proper “traceable” conditions, because otherwise the resulting reference material is useless. A reference material with a lack of traceability to stated and acceptable references cannot be used as such. Moreover, the users of reference materials expect to buy traceability, and their interests are only served if the producers paid sufficient attention to these aspects.

1.4 References

AHRENSLH (1951)A story of two rocks. Geostds Newslett 1x57-161. BEAL(1951) The basic philosophy of standards. Anal Chem 23:1528-1531. BERMANSS, STURGEON RE, DESAULNIERS JAH and MYKYTIUKAP (1983) Preparation of the sea water reference material for trace metals, NASS-I. Mar Pollution Bull 14:69-73. BERMANSS (1984) Marine biological reference materials for trace metals. In: Wolf WR, ed. Biological Reference Materials, pp79-88. John Wiley & Sons. BOOTH E, CROFTON P and ROBERTSLB (1974)The influence of standards on interlaboratory quality control programmes. Clin Chim Acta 55:367-375. BOWENHJM (1965) A standard biological material for elementary analysis. In: SHALLISPW, ed. Proc of the SAC Conference, Nottingham, pp 25-31, W Heffer and Sons, Cambridge. BOWENHJM (1984) Kale as a reference material. In: WOLFWR, ed. Biological reference materials. Availability, uses and need for validation of nutrient measurement, pp3-17. John Wiley Sr Sons. BRIGHT HA (1951) Standard sample program of the National Bureau of Standards. Anal Chem 23344-1547. CALIJP and Stanley CL (1975) Measurement compatibility and standard reference materials. Annu Rev Mat Sci 5:3zg-343. CALIJP, MEARSTW,MICHAELISRE, REEDWP, SEWARDRW, STANLEYCL, YOLKENHT, and Ku HH (1975) The role of standard reference materials in measurement systems. NBS Monograph 148, Washington DC. CHAICHIFANG (1993) Present status and future trends in biological and environmental reference materials in China. Fresenius J Anal Chem 345:93-98. J and LEE H-B (1979) Analytical reference materials. TI. Preparation and samCHAUASY, CARRON ple integrity of homogeneous fortified wet sediment for polychlorinated biphenyl quality control studies. J Assoc Off Anal Chem Gz:1312-1314. COCHRANE RC (1966) Measures for progress - a history of the National Bureau of Standards. US Library of congress catalog card 65-62472, p 93. Commission of the European Communities (1973) Reference Materials (Provisional) RM-19730001 or ISP-1973-01,Community Reference Bureau. DALEH (1939) Biological standarchsation. Analyst 64:554-567. R (1995) The contribution of various analytical techniques to the certification of refDYBCZYNSKI erence materials. Fresenius J Anal Chem 352:120-124. EHRLICH P (1910) (Lecturewithout title) Deutsche Med Wochenschr 36:1893-1896.

1.4 References I 1 7

EHRLICH P, Kossel H and von Wassermann A (1894) Ueber Gewinnung und Verwendung des Diphterieheilserums.Deutsche Med Wochenschr zo:y,3-255. EMONS H, ed. (1997) Biological Environmental Specimen Banking (besb 2 ) 2nd International Symposium and Workshop held at Stockholm, Sweden 20-23 May 1996. Chemosphere Vol34 Nos. 9 and 10. FAJCELJA and PARKANY M, eds.(Iggg)The Use of Matrix Reference Materials in Environmental Analytical Processes. The Royal Society of Chemistry. GAY-LUSSAC JL (1824)Instruction sur 1’Essaidu Chlorure de chaux. Ann Chim Phys 26:162-175. GRIEPINK B, MAIEREA, QUEVAUVILLER P and MUNTAUH (1991)Certified reference materials for the quality control of analysis in the environment. Fresenius J Anal Chem 339:599-603. IHNAT, WOLFWR (1984) Maize and beef muscle agricultural and biological reference materials. In: WOLF WR, ed. Biological Reference Materials, pp 141-165. John Wiley & Sons. IHNAT M, WOLYNETZ MS (1993) Summary of an interlaboratory characterization (certification) campaign to establish the elemental composition of a new series of agriculture/food reference materials. Fresenius J Anal Chem 345:185-187. IMAIN, TERASHIMA S, ITOH S, ANDOA (1996) 1996 compilation of analytical data on nine GSJ geochemical reference samples “SedimentaryRock Series”.Geostds Newslett ~0:165-216. International Atomic Energy Agency (1998) IAEA AQCS catalogue for reference materials and intercomparison exercises 1998/1999. IRMM (1999) BCR Reference Materials. Institute for Reference Materials and Measurements, (IRMM) Reference Materials Unit, European Commission Joint Research Centre, Retieseweeg, 244.0 Geel, Belgium. I S 0 Guide 25 (1990) Guidelines for assessing the competence of calibration and testing laboratories. International Organizationfor Standardization,Geneva. I S 0 Guide 30 (1992)Terms and definitions used in connectionwith reference Materials. International Organization for Standardization,Geneva. I S 0 Guide 31 (1981) Contents of certificates of reference materials. (Revised April 1996 as ISO/ REMCO document N 382. Actual update 20 July 2000.) International Organization for Standardization, Geneva. I S 0 Guide 32 (1997) Calibration in analytical chemistry and use of certified reference materials. International Organization for Standardization,Geneva. I S 0 Guide 33 (1998) Uses of certified reference materials. Actual update 20 July 2000. International Organization for Standardization,Geneva. I S 0 Guide 34 (1996) Quality system guidelines for the production of reference materials. (Revised March 1998 as ISO/REMCO document No 464 “Generalrequirements for the competence of reference material producers”.The revised Guide 34 will appear early zooo.) International Organization for Standardization,Geneva. I S 0 Guide 35 (1989) Certification of reference Materials-General and statistical principles. International Organization for Standardization,Geneva. KENWORTHYAL, MILLEREj and MATHIS WT (1956) Nutrient-element analysis of h i t tree and leaf samples by several laboratories.Proc Amer SOCHortic Sci 67x6-21. KLICH H (1999) Overview on the activities of ISO/REMCO. In: FAJGELJ A and PARKANY M, eds. The use of matrix reference materials in environmental analytical processes, pp 188-195. The Royal Society of Chemistry, Cambridge. KRAMERGN, MUNTAUH, MAIERE, PAUWELSJ (1998) The production of powdered candidate biological and environmental reference materials in the laboratories of the Joint Research Centre. Fresenius J Anal Chem 360: 299-301. KRAMERGN, PAUWELSJ and BELLIARDOJJ (1993) Preparation of biological and environmental reference materials at CBNM. Fresenius J Anal Chem 345:133-136. K U ~ E R J,A MADERP, M I H O L O VD, ~ CIBULKA J, FALTETSEK J and KORDIKD (1995)Preparation of the bovine kidney and bovine muscle reference materials and the certificationof element contents from interlaboratorycomparisons. Fresenius J Anal Chem 35~:66-72.

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7 introduction

KUEERAJ, SYCHRAV and KOUBEKJ (1998) A set of four soil reference materials with certified values of total element contents and their extractable fractions. Fresenius J Anal Chem 360: 402-405. LEEH-B and CHAUASY (1987)Analytical reference materials Part VII. development and certification of a sediment reference material for total polychlorinated biphenyls. Analyst 112:37-40. MEINKEWW (1971) Standard reference materials for clinical measurements. Anal Chem 43(6):28A-47A. MILESAA (1948) Some observations on biological standards. Analyst 73:530-538. MUNTAUH (1979) Five years of environmental candidate reference material production at the Joint Research Centre Ispra. In: Proc of the First International Symposium on Production and Use of Reference Materials, Berlin, pp 185-218. MUNTAUH (1980) Measurement quality improvements by application of reference materials. In: BRLTTERP and SCHRAMEL P, eds. Trace Element Analytical Chemistry in Medicine and Biology, pp707-726. Walter de Gruyter & Co Berlin New York. MUNTAUH (1984) Ispra activities in the production of candidate biological reference materials. In: WOLFWR, ed. Biological Reference Materials, pp. 109-140. John Wiley & Sons New York. K and FUWA K (1985)Certified reference material program at the National Institute for OKAMOTO Environmental Studies. Anal Sci 1:206-207. OKAMOTO K and YOSHINAGAJ (1999) Proper use of reference materials for elemental speciation M, eds. The use of matrix reference materials in environstudies. In: FAIGELIA and PARKANY mental analytical processes, pp 46-56. Royal Society of Chemistry, Cambridge. PARRRM (1984) IAEA biological reference materials. In: WOLFWR, ed. Biological Reference Materials, pp 45-62, John Wiley & Sons. P o n s PJ (1997) Geoanalysis: Past, present and future. Analyst 122:1179-1186. QUEVAUVILLER Ph (1995) Certified reference materials for specific chemical forms of elements. Analyst 120:597-602. QUEVAUVILLER Ph (1999) The BCR framework: 25 years of quality measurements within the European Union. Trends Anal Chem 18(5):302-311. QUEVAUVILLER Ph and MAIEREA (1999). Interlaboratory Studies and Certified Reference Materials for EnvironmentalAnalysis. Elsevier Publishers B.V., Amsterdam. RADIN N (1967)What is a standard? Clin Chem 13:55-76. RICHARDSON J M (1995) Certified reference materials programme at the Geoscience Laboratories, Sudbury, Ontario, Canada. Analyst 120:1513-1518. RONG PX and MIN 2 (1999) China GBW reference materials. In: FAJGELJA and PARKANY, eds. The use of matrix reference materials in environmental analytical processes, pp 1-30. Royal Society of Chemistry, Cambridge. ROSSI G and COLOMBO A (1979) Reference materials for chemical analysis. Highlights on the activity of JRC-Ispralaboratories. Fresenius 2 Anal Chem .zg7:13-17. JD and BACKHAUSFW (1988) Preparation of sample material for Environmental SpeciSCHLADOT men Banking purposes-Milling and homogenization at cryogenic temperatures. In: WISESA, ZEISLER R and GOLDSTEINGM, eds. Progress in Environmental Specimen Banking, pp 184193. NBS Special Publication 740. U.S. Government Printing Office, Washington. SL (1897) Ueber die Anwendung des Natriumoxdats in der Titrieranalyse. 2 Anal SORENSEN Chem 36:639-648 STOEPPLERM and ZEISLERR, eds. (1993) Biological environmental specimen banking. A collection of papers presented at the 1st International Symposium on Biological Environmental Specimen Banking, Vienna, Austria, 22-25 September 1991. Sci Total Environ, Vols. 139 and 140. JT (1984) The FDA-IFC infant formula methods study and standards for organic nutriTANNER ents. In: Wolf WR, ed. Biological Reference Materials, pp 197-205. John Wiley & Sons. TRAHEY NM (1998) NIST Standard Reference Materials Catalog 1998-99. NIST Special Publication 260. National Institute of Standards and Technology, Gaithersburg. URIANOGA and GRAVATICC (1977) The role of reference materials and reference methods in chemical analysis. CRC Crit Rev Anal Chem 6:361-411.

VAN der EIJK W (1979) The activities of the European Community Bureau of Reference - BCR. Fresenius 2 Anal Chem 297:10-12. VEILLONC, PATTERSON KY and REAMERDC (1984) Preparation of a bovine serum pool for trace element analysis. In: WOLFWR, ed. Biological Reference Materials, pp167-177. John Wiley &

Sons. VERSIECKJ , VANBALLENBERGHE L, de KESELA, BAECICN, STEYART H, BYRNEAR and SUNDERMAN FW Jr. (1988) Certification of a second-generation biological reference material (freeze dried human serum) for trace element determinations. Anal Chim Acta 204:63-75. J (1903)Proc 5th Internat Congr Appl Chem 5314. WAGNER P J (1984) Development of food-oriented reference materials by the Community WAGSTAFFE Bureau of Reference (BCR) In: Wolf WR, ed. Biological Reference Materials, pp 63-78. Wiley & Sons. WAINERDI (1979) Reference material for trace analysis by radioanalytical methods: Bowen’s Kale. Pure Appl Chem 51:1183-1193. WARDGM and HEENEY HB (1960) A collaborative study of methods for the determination of potassium, calcium and magnesium in plant materials. Canad J Plant Sci 40:589-595. TP (1976) Quality Control in Clinical Chemistry. Wiley, Chichester. WHITEHEAD WILLIESN (1997) The preparation of National Research Council Certified Reference Materials. In: CLEMENT RE, KEITH LH and SIUKWM, eds. Reference Materials for Environmental Analysis, pp 43-59. CRC Press Inc. WOLFWR and IHNATM (1984) Evaluation of available certified biological reference materials for inorganic nutrient analysis. In: WOLFWR, ed. Biological Reference Materials, pp 89-105. John Wiley & Sons. WOLFWR, IYENGAR GC and TANNER JT (1990) Mixed diet reference materials for nutrient analysis of foods: preparation of SRM-1545Total diet. Fresenius J Anal Chem 338:473-475. R, LANGJAND J K and HARRISON SH (1983) Cryogenic homogenization procedure for bioZEISLER logical tissues. Anal Chem 55:2431-2434.

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

*O

I 2

From Planning to Production Edited by Markus Stoeppler 2.1

Material Collection and Preparation Milan Ihnat')

2.1.1

Introduction

A large number of considerations and factors must be entertained for the conception, development, preparation, assessment, characterization, and certification of RMs, including (a) end use requirements, (b) selection of materials, (c) preparation, (d) physical characterization, (e) chemical characterization, (f) certification, (g) documentation, and (h) distribution. Most of these have an overwhelming impact on the finally developed RM and on its credibility. This section deals with the steps, collectively denoted as collection and preparation, occurring early in the scheme of RM development. It treats general collection and preparation principles, and provides specific examples of preparative procedures. No. 2069 from Pacific Agri-Food Research Centre - Summerland

1) Contribution

2. 7 Material Collection and Preparation

Table 2.1

Classes o f biological and environmental RMs for chemical analysis with examples

Main class

Subclass

Examples

Biological

Animal tissues (see also foods) Plant tissues (see also foods) Foods and agricultural products

Animal bone, ground whole carp, cod muscle, tuna, dogfish liver Chlorella, aquatic plant, grass, hay, spruce twigs and needles, olive leaves, peach leaves, tobacco leaves Bovine muscle, bovine liver, pig kidney, milk powder, cereals, single cell protein, butterfat, fish oil, animal feedstuffs, textiles Whole blood, blood serum. Urine, human hair, blood plasma and serum proteins, enzymes Spray-driedmilk matrix with stabilized micro-organisms

Clinical tissues and fluids Microbiological materials Environmental Ashes and dusts

Wastes and sludges Waters

Coal ash, coal fly ash, power station fly ash, incinerator ash, vehicle exhaust particulates, urban dust, atmospheric dust, metal smelter dust, welding dust, diesel particulates, particulates on filter media Sewage sludge, wastewater Seawater, rainwater, river water, estuarine water, open ocean water, fresh water, ground water, drinking water

Rocks and ores

Geological

Basalt, granite, manganese nodules, shale, flint clay, iron formation materials, phosphate rock, fertilizers Soils Calcareous loam soil, loess, polluted farmland soil, sand soil Sediments Marine sediments, estuarine sediments, freshwater pond sediments, harbour sediments, stream sediments, lake sediments Blast furnace slag, concentrates, ore mill tailings, plating Mineral processing products sludge Ceramics and glasses Silicon carbide, high boron borosilicate glass, trace elements in glass Fossil fuels Coal, coke, petroleum crude oil, residual fuel oil

Other

Synthetic and spiked materials Pure elements and compounds

Instrument performance Stable isotope RMs RMs for determination of radionuclides

Gelatin, trace elements in glass, glass fdters, graphite, synthetic materials High purity elements and compounds, organic compounds, organo-metalliccompounds, high purity compounds for microchemical and microanalyticaltechniques, pharmaceuticals Pure element and compound solutions for calibration and instrument performance, trace elements in glass, glasses, glass filters Water, pure compounds, biological materials, minerals Animal tissues, plant tissues, sediments, soils, radiopharmaceuticals

I

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22

I

2 From Planning to Production

At the outset, it may be useful to list the major classes of RMs, and measurands therein, of interest to this book (generally biological, environmental, and geological; and generally for chemical analysis). Table 2.1 presents the major RM classes with subclasses; Table 2.2 lists some of the most prevalent examples of measurands (analytes) for which RMs have been produced. Table2.z

Examples of measurands (analytes)for which RMs have been produced

Measurand class

Examples

Elements

Major, minor and trace elements of nutritional, environmental, clinical, toxicological significance: Al, As, Ba, Be, Br, Ca, Cd, Cs, Cu, F, Hg, I, Li, Mn, Mo, N,P, S, Sb, Se, Sn, Th, Tl, U, V, W, rare earth elements. Stable isotopes: 'H,I3C,I5N, l80.Radionuclides: 401<, 60Co,"Sr, 137Cs,*!'%I, 238Pu,241Am

Speciated elements

Elemental species: inorganic moieties such as nitrate, ammonium, phosphate, methylmercury, monobutyltin, dibutyltin, tributyltin, trimethyl lead, Cr(III),Cr(VI),Se(IV), Se(V1)

Organic compounds

Toxic compounds: polychlorinated biphenyls, polycyclic aromatic hydrocarbons, organochlorine pesticides, chlorinated pesticides, dioxins, veterinary drug residues, hormone residues, aflatoxins, toxic compounds in shellfish. Compounds of nutritional significance in foods: vitamins, fat, lipids, carbohydrates, protein, energycalorific value, proximates, dietary fibre, ash. Other compounds: hormones in blood serum

Microbiological organisms

Enterococcusfaecium, Salmonella fyphiwturium,Enterobacter cloacea,

Escherichia coEi, Listeria monocytogenes

References: Ihnat (1995),International Atomic Energy Agency and United Nations Environment Programme (1995), National Oceanic and Atmospheric Administration (1995),Analytical Quality Control Senices (1998),Quevauviller et al. (1998a),Trahey (1998), European Commission, Joint Research Centre, IRMM (1999). 2.1.2 General Collection and Preparation Principles

Collection/preparation refers to all of the physical (and chemical) steps necessary to bring the starting material to RM status. It constitutes a major and important phase of RM development and entails many considerations in the many required steps for proper execution of RM development. Major considerations include: (a) planning, (b) material selection, (c) collection, (d) preparation, (d) characterization, (e) storage, and (f) documentation. Each of these can be further subdivided. Attention to detail in all collection/preparation steps is mandatory. The flowchart (Figure 2.1)presents a general summary of steps applied to the collection and preparation of biological, environmental, clinical and geological RMs based on collective descriptions in sever-

2.7 Material Coilection and Preparation

I

23

a1 reports (Berman 1985; European Commission 1994; Gardiner 1993; Ihnat 1988a, 1988b, 1988c, 1994 [and unpublished data]; Ihnat and Wolf 1985; Ihnat et al. 1987; IO-arner GN et al. 1993, 1995, 1998; Kramer KJM et al. 1999; Quevauviller et al. 1998a; Roper et al. 1999; Schladot et al. 1993; Zeisler and Wise 1985). A wealth of preparative and physical characterization information may also be found in internal reports of the Institute of Reference Materials and Measurements, Geel. This comprehensive listing attempts to cover all steps possibly required in the preparative scheme and it should be recognized that not all steps are required in every instance and that the sequence and details will vary.

PLANNING

Overall planning and development of framework

I

MATERIAL SELECTION

I

COLLECTION

Selection of collection location Selection of collection equipment and materials Collection and acquisition Interim transport and storage

I

PREPARATION

Selection of processing equipment and materials Production procedure investigation Development and organization of processing facilities Preliminary processing Preliminary drying Preliminary crushing/coarse grinding Particle size fractionation(sieving) Homogenization Interim collection of processed material Interim physical characterization Interim chemical characterization Stability testing Packaging

I

CHARACTERIZATION

Final physical characterization Chemical characterization (homogeneity)

I STORAGE

I

DOCUMENTATION Fig. 2.1

Flowchart of general reference material collection and preparation steps

24

I A logical first item is overall planning and development of the framework for the 2 From Planning to Production

RM project. It includes consideration of end-use requirements of the material and provision for scientific and technical management and execution of the project by trained, dedicated, critical scientists and technologists. End-use requirement dictates an assessment of the nature of the problem to be addressed by the product, the market requirements, the analytical methods to be served, the measurands, forms and concentrations to be certified, the level of certification required, and the quantities of starting and final product needed, leading to overall definition of the undertaliing. Material selection is based on the nature and physical form of the matrix desired (which should have a good measure of identity to real-world samples and be representative of the population of materials in production, commerce and undergoing analysis) and an evaluation of measurand and matrix compositions desired. Raw materials for the production of natural-matrix, natural-measurand RMs can come from commercial sources, can be commercial or in-house custom-produced products, or can result from specific collection ventures. Commercial sources are convenient for products such as foodstuffs, whereas in-house efforts are pursued for biological fluids, microbiological or other products of high quality. Materials collected and archived under environmental specimen banking activities are good guides to the choice of required control materials; in fact surpluses of such carefully collected and processed materials can serve as candidate RMs. A survey of RM availability is an obvious component prior to material selection; lack of correspondence, with respect to matrix/measurand, between available RMs and commodities analyzed is a guide to the choice of additional, appropriate control materials. The process of collection includes selection of the collection location, considering biological species, material desired, matrix, and measurand levels. Selection of collection equipment and materials leads to appropriate metallic, non-metallic, ceramic or plastic sampling and collection implements, and materials; and it defines requirements for their cleaning, conditioning, and storage. Collection and acquisition includes growing specific crops, animals or micro-organisms under selected conditions, physical removal of sufficient quantity of starting material from collection sites, collection of correct materials / species / tissues, and acquisition of commercially available materials. The final step in collection is interim transport and storage whereby the collected material is transported to a processing facility and put into interim storage, under freezing or other storage conditions as required. The major activity is preparation. One of the first important considerations is again selection of processing equipment and materials as described for collection. Production procedure investigation deals with the feasibility study of the proposed preparation procedure: selection, testing, and trial/pilot runs of proposed procedures and selection of final large scale preparative protocols, preparation and homogeneity, and stability-testing of test batches, and control of contamination/losses. Selection and control of environments for all processing steps, preparation of laboratory and pilot plant processing room(s), and development of production and maintenance protocols form part of the development and organization of processing facilities. Collected products may require preliminary processing: cleaning, washing,

2.7 Material Collection and Preparation

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25

separation into component structures/tissues, trimming and removal of extraneous matter and tissues such as bone, cartilage, fat, connective tissue, and other components judged to be extraneous to or expected to compromise final RM quality. Preliminary drying by air-drying, heat-drying, spray-drying,vacuum-drying, freeze-drying, or centrifugation-drying may be indicated to facilitate transport, storage, and further processing. Preliminary crushing/coarse grinding by breakinglcrushing, manual or grinder cutting, jaw crushing, chopping, cryogenic crushing, pre-crushing using mortarlpestle, or hammermill milling may be carried out to facilitate fine milling/grinding. For the latter, cryogenic grinding, cryogenic milling, ball milling, hammermill milling, impact milling, fluidized bed jet milling, planetary disc milling, or rotor beater milling may be selected. Particle size fractionation (sieving) for isolation of suitable particle size fractions using various sieving or air classification techniques is carried out prior to homogenization which entails mixing and blending. Considerations for proper execution are: nature and number of phases; particle sizes, distribution, shape and density, mixing technique and duration, and spiking if required (addition of measurand to pool of material for production of spiked RMs). Interim collection of processed material, including interim packaging and storage (e.g. vacuum packing, freezer storage) awaiting final processing may be needed. Interim physical characterization, needed for planning homogenization, demixing and moisture stability studies, uses macroscopic (visual)and microscopic examination to assess appearance, color and flow characteristics. Particle size distribution, shape and density are measured by test sieving, light and electron microscopy, particle counting, and light scattering techniques. Moisture loss/pickup characteristics and moisture measurement techniques are evaluated. Stabilization is effected by defatting (extraction of oil and fat), addition of antioxidants or preservatives, packaging under inert atmosphere, hermetic packaging, deep freezing, drying by heat-drying, air-drying, spray-drying, vacuum-drying, or freeze-drying, sterilization by electron beam irradiation, gamma-ray irradiation, heat, pasteurization, or chemical treatment. Measurement of selected measurands for the assessment of contamination and loss control and the determination of the effect of particle size on chemical composition is termed interim chemical characterization to segregate it from final characterization/certification. For stability testing, stability problems to be addressed are microbiological decay, phase separation, oxidation, and other chemical transformation of matrices and measurands, and losses of volatile measurands. Assessment of stability expected during long-term storage is evaluated by accelerated high temperature degradation tests as is settling and demixing during storage and transport. The final step in preparation is packaging, for which appropriate storage containers of glass, quartz, metal, plastic, or metallplastic foil are selected and inert atmosphere/hermetic packaging is considered. Units are numbered and labelled and those required for testing and certification are set aside during the packaging operation according to a predetermined statistical design. Characterization of the finally-produced material includes final physical characterization along the lines described under interim physical characterization and chemical characterization (homogeneity). The latter includes: selection, development, assessment, and validation of methodologies for homogeneity testing includ-

26

I ing physical, chemical, and natural radioactivity techniques as may be required, 2 From Planning to Production

adaptation of statistical protocols for homogeneity testing and data analysis, analysis of the final material, and monitoring of contamination. Note that certification is a separate operation outside collection/preparation. Selection of appropriate/convenient storage conditions such as ambient and low temperature storage including storage at liquid nitrogen temperatures, evaluation of settling and demixing of material during storage and transport and stability of matrix and measurand by periodic examination and testing constitutes storage. Finally, documentation of all collection and processing steps and characterization procedures, material and measurand descriptions and results of homogeneity testing completes the RM collection/preparation phase. 2.1.3 Specific Examples

A number of reports have appeared in the literature describing the preparation of RMs. One of the earliest detailed accounts (Bowen 1965) described the growing, collection, drying, grinding, sieving, and blending of leaf tissues from marrow stem kale. Since then, steady advances in the development of a wide variety of RMs have led to many scientific and technical articles describing, to varying degrees, the preparation of many materials. A number of such publications are listed in Table 2.3. Extracts of descriptions from several selected publications are given below to provide insight into some of the variety of details followed in RM collection/preparation schemes. The reader is encouraged to consult the original publications for perusal of in-depth, descriptions. For a PCDD- and PCDF-contaminated carp RM, CARP-I (Fraser et al. 1995),the carp was harvested near the warm water discharge of a power plant on Saginaw Bay, ground whole and stored at -zo"C. The thawed carp was comminuted in a food cutter equipped with a fine Type K 28 column 010-020 cutter on the first pass and a finer 010-OIOcutter on subsequent passes. An antioxidant, ethoxyquin, and water were added, the resulting slurry was processed in a homogenizer, divided, and bottled under nitrogen by means of an ampouling machine. To stabilize the homogenate, the ampoules were heated in a steam retort and individually heat-sealed in trilaminate retort pouches. For homogeneity testing, random samples were analyzed for nine major PCDD and PCDF congeners. To test stability, random samples were analyzed on a regular basis over a 13-month period. Animal Tissues

For preparation of one of the earliest botanical RMs, Kale (Bowen 1965).about 0.2 ha were sown with 10 kg of Marrow Stem Kale seed (Brassica oleracea). Harvesting yielded 1.6 tonnes of fresh leaf. Drying was effected by air- and oven-drying, yielding 160 kg of dry material. Care was taken to exclude much of the petiole when collecting the leaves and the lamina midrib was separated out after drying. The dried leaves were crushed between oak rollers and a coarse fraction was rejected by initial sifting. A Perspex grinding mill, to avoid metallic contamination, Plant Tissues

2.7 Maten'al Collection and Preparation Tab. 2.3

Examples of publications describing preparation o f RMs

RM classification (subclass)

References

Berman and Sturgeon (1988);Pauwels et al. (1990); Hardstaff et al. (1990); Schladot et al. (1993);Fraser et al. (1995);Quevauviller et al, 1998d Kramer et al. (1999);Morabito et al. (1999) Plant tissues Bowen (1965);Ihnat and Wolf (1985);Becker and Gills; Quevauviller et al. (1998b);Zeisler et al. (1998) Foods/agricultural products Ihnat et al. (1987);Ihnat (1988);Jerome (1993);Zeisler et al. (1993); Quevauviller et a1 ( 1 9 9 8 1998f, ~ 1998g);Sharpless et al. (1999) Yeoman et al. (1985);Versieck et al. (1987);Quevauviller et a1 Clinicaljuids and tissues (1992a,1998e);Stone et al. (1995);Yoshinaga et al. (1997) Microbiological materials Mooijrnaii et al. (1992) Heller-Zeisleret al. (1998);Quevauviller et al. (1998, 1999) Ashes and dusts Wastes and sludges Griepink et al. (1991);Vercoutere et al. (1995);Quevauviller et al. (1998h 19981~) National Research Council Canada (1992);Quevauviller et al. Waters ( ~ y p b1996); , Merry (1995);Benolie1 et al. (1997);Quevauviller et al. (1998h, 1998k) Lister (1978) Rocks and ores Soils Bowman et a]. (1979);LaBrecque (1990);Vercoutere et al. (1995); Quevauviller et al. (1998m, 1998k) Sediments Olcamoto and Iwata (1982);Berkovits and Lukashin (1884);Epstein et al. (1989);Schantz et al. (1990);Bowman (1994) Fossil Fuels Gonska et al. (1984) Synthetic and spiked Zaichick (1995);Bogershausen et al. (1997) Animal tissues

materials Pure elements and compounds

Griepink et al. (1981);Marchandise and Vandendriessche (1985)

was used to grind the dry leaves. The ground tissue was passed through a 220-pm nylon sieve with the fine fraction collected in polythene bags. Mixing of the final 91kg of powder was carried out with a polythene shovel on a polythene sheet and the material was stored in polythene bins at 2°C. Physical characterization was particle size, moisture and ash. Material homogeneity was established by physical and chemical testing including: natural radioactivity of 40K, loss in weight upon extraction with acetone, optical densities of acetone extracts, ash, Na, and K contents. Foods and Agricultural Products For preparation of a bovine muscle powder RM (Ihnat et al. 1987),beef cattle (Bos taurus) were raised to market weight and slaughtered at a commercial slaughter facility (as part of another experiment). About goo kg of boneless hip (grade A round steak) was trimmed, using stainless steel knives and plastic cutting boards, of all visible fat and connective tissues, cut into small pieces, and frozen. Frozen cubes were ground, with dry ice, in a stainless steel comminuting machine equipped with ca. I mm stainless steel screen. The fine powder was collected in food grade polyethylene pails and stored at -30°C to allow dissipa-

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2 From Planning to Production

tion of the dry ice. The resultant finely milled frozen product was then freeze-dried, bulk-sterilized with 2.0 Mrad of ‘OCo y-radiation and stored prior to additional processing. All subsequent processing was carried out in a specially constructed clean room. The room was equipped with preparative equipment (sieve shaker, blender, sieves, plastic utensils), microscope, and laboratory furniture, constructed where possible from wood or plastic materials and sheathed with polyethylene sheeting to minimize metallic and dust contamination of materials being processed. Workers were clothed in polypropylene coveralls, polyethylene shoe and head covers, and nylon gloves. Plastic and glass utensils, and materials coming in contact with the RMs were cleaned according to rigorous procedures using hot I % solution of Triton X-100, I M HC1, and distilled water, dried in the clean room, and stored in polyethylene bags. Glass bottles for packaging were water-rinsed in a commercial laboratory washer and handled with nylon-gloved hands. Sieves were constructed from high density white polyethylene pails and nylon monofilament sieve cloths. A sieve set consisted of a pair of sieves, typically 253/53 pm and 2531 go pm, to provide suitable middle cuts, placed on a custom wood holder affixed to an oscillating table. Additional particle size reduction was effected by ball milling with Teflon-TFE balls in a Teflon-PFA (perfluoroalkyoxy-polymer)screw-cap jar. In the typical operation, coarse material from the sieving of the original bovine muscle preparation was ball-milled and sieved and remilledlresieved to provide a final material with a relatively narrow particle size distribution not exceeding 250 pm. Homogenization was by mixing in a custom-designed 420-L polymethylmethacrylate (PMM) V blender. The two ends were fitted with PMM lids to permit access for loading and cleaning and the bottom of the chamber was fitted with an outlet for removal of material. The maximum speed of rotation was 5.5 rpm about the steel shaft attached along the axis of rotation. Eight separate blending operations over seven days yielded the final product. Packaging was done manually by mass into nominally 120-mL clear glass bottles with pulp-lined black plastic screw-caps to give a total of 3412 units. Random units were set aside for physical and chemical characterization and certification; later all units were hermetically packaged in aluminium-nylon sacs. Physical characterization was by visual and microscopic examination. Material homogeneity was established by high precision / high accuracy atomic absorption spectrometric determinations following acid dissolution and additionally from analytical information obtained from the certification campaign. In their work on the collection and preparation of a second generation biological RM, Versieck et al. (1987) described the collection and preparation of a human blood serum with trace element levels approximating those in uncontaminated, real human blood plasma or serum. Following a feasibility study, blood collection was via a polypropylene catheter from donors selected from those undergoing specific treatment. High purity quartz blood collection tubes, low density polyethylene screw-cap storage and processing containers, and quartz Clinical Tissues and Fluids

2.1 Material Collection and Preparation

spoons were cleaned with extreme care under dean laboratory conditions, with hydrogen peroxide, nitric and sulfuric acid mixtures, deionized water, quartz-bidistilled water, and steam. Serum was separated by centrifugation into quartz tubes and finally into polyethylene screw cap containers and stored at -25'C in a clean deep freezer until homogenization. The pooled material was homogenized by mixing in a polyethylene container and transferred into low density polyethylene screw-capcontainers, frozen and lyophilized. An urban dust material described by Quevauviller et al. (1999) originated from 15 kg of road dust collected by sweeping a lay-by in the central section of the Queensway tunnel in Birmingham (UK), a major traffic route. The dust was passed through a 5oo-pm sieve to remove large particles of debris and air dried in a well ventilated and dark room. It was ground in a ballmill and sieved through a 125-prn sieve. The material was homogenized by shaking, in portions, in a 5-L darkglass bottle. The recombined material was then freeze-dried and immediately bottled in 15 g portions into Goo 30-mL amber-glass bottles and stored at 4'C. A separate batch of material was spiked with trimethyllead chloride. The slurry was stirred manually, freeze-dried and ground in a porcelain mortar. It was homogenized by mechanical shaking in a I-L glass bottle and distributed into bottles to produce a road dust containing 50 pg Pb/kg. Ashes and Dusts

Wastes and Sludges According to Vercoutere et al. (1995) a sewage sludge was

collected from a concrete basin at a water purification plant in Italy. Extraneous matter, such as pieces of plastic, aluminium, or wood was removed manually. The sludge material was air-dried,lumps were pulverized, the material passed through a 2-mm sieve discarding the fraction > 2 mm and was y-irradiated to a dose of 25 kGy. Subsequent processing consisted of grinding in a hammer mill, sieving (
Waters

Soils Four soil samples (Bowman et al. 1979) were selected to represent major kinds of soil materials and to include a wide range of properties. Bulk materials were taken by soil scientists from four different regions of Canada. For example, the

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I sampling site for soil

2 From Planning t o Production

SO-I was 23 km northwest of Hull, Quebec at 45” 30’ 50’’ N, 75’’ 58’ 40” W. The material is a somewhat weathered Champlain Sea clay from a depth of 35-75 cm from the surface in an upland position. It is of the C horizon of Rideau clay, a Regosok soil, containing about 80 % clay of mixed mineralogy. At the laboratory, each soil was dried in 70-kg batches at 120°C for 17 h. Stones and

gravel were removed and each batch was ball-milled to pass through a zoo mesh (74 pm) sieve. All batches of each soil were combined and homogenized by tumbling in a rotating conical mixer. Bottling was in 200 g units. Homogeneity measurements on each RM were conducted on six randomly selected units for four elements by energy dispersive X-ray fluorescence spectroscopy. Sediments Technical considerations associated with the preparation of a pond sediment RM (Okamoto and Iwata 1982) included speedy processing and care to prevent contamination. During cleaning operations in May 1977, about 500 kg fresh weight of sediment was dredged from the bottom surface at the centre of Sanshiro pond at the University of Tokyo, to a depth of I m. The wet material was sieved through a nylon sieve to remove gravel and leaves, filtered by suction to remove interstitial water, and air-dried on filter paper at room temperature for two weeks. The dry material was ground in an alumina ball mill conditioned by pregrinding a small portion of sediment. Sieving was through a mechanically vibrated set of sieves arranged in order: 50 mesh, IOO mesh, and 200 mesh. Material passing 200 mesh (about40 kg) was divided into two with a riffle sampler constructedofpolyvinylchloride. The final product was bottled in lots of 20 g in zoo0 acid-washedglass bottles and sterilized by “Co radiation to 2 Mrads to minimize deterioration due to biological activity. For assessment of homogeneity, acid-dissolutionflame atomic absorption spectrometry was used to determine several elements in 11 randomly selectedbottles. 2.1.4

Concluding Remarks and Recommendations Raw and processed biological, environmental and geological materials in worldwide utilization constitute an extremely wide range of matrix and measurand chemical composition. For optimum quality control, a close correspondence between RMs and test materials is required, with respect to measurand and matrix; and thus a wide variety of RMs must be available for matching. Collection and preparation of starting materials constitutes a major and important phase of RM development. Fairly critical is the need to maintain material integrity, i.e. not seriously altering its physical and chemical properties, so that the final RM product closely represents the native, starting material and in fact is very similar to materials submitted to the laboratory for analysis. This need extends to maintaining measurand integrity, including speciation, and avoiding or minimizing external contamination during the multitude of collection and preparation steps. Close attention to details, feasibility studies and participation of critical scientists will go a long way to the production of highly relevant and acceptable RMs.

2.2 Control ofhlraterial Properties

2.2 Control of Material Properties Antony R. Byrne, Karl Heinz-Crobecker, Heinz-Dieter kengard, Jan Kuc‘era, Jean Pauwels, Stephan Riickold, Markus Stoeppler

Important steps during RM production are the control of properties that determine the quality of the final product and its long-term stability (see the overview of techniques in Table 2.4). As some of the methods discussed below, for example determination of homogeneity, stability and shelf life (expiry date) are also mentioned in other Chapters; and in the interest of brevity the reader is referred to them for further details. 2.2.1 Particle Size and Particle Size Distribution

Particle sizes and their distribution affect the chemical composition of the material as far as homogeneity and the representability of test portions taken from it are concerned. The need to determine particle size is linked with the formal requirements for RM preparation, such as uniformity and the uncertainty assigned to the s u b Tab. 2.4

Methods generally used for the determination of RM properties

RM Property

Method(s)

Remarks

Particle size/particle size distribution

Sieving analysis Microscopicalinspection

Very effective, also for particle size distribution

Homogeneity/ heterogeneity

“Classical”methods based on decomposition for elements and extraction for organometallic and organic compounds Methods capable of directly analyzing solid samples (e.g. NAA,XRF,

Due to combination of several analytical steps often significant errors Small methodological error, small subsamples, but only applicable to elements

Humidity (water)

Freeze dryinglweighing Oven dryinglweighing Karl Fischer titration

Not completely selective Not completely selective Selective

Degradation

Long-term studies at different temperatures with suitable methods for elements and compounds

For reliable results duration of up to two years needed Best approach: “isochronous measu. rements”

Shelflife/expiry

Long-term high quality trend analyses with graphical evaluation of measured data

Three to four years and continued during use of CRM; very reliable approach: “isochronous measurements”

ss-AAS)

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I sampling of RMs, and the 2 From Planning t o Production

concordance/correspondence of the RM composition and structure with the intended purpose (Fajgelj and Zeisler 1998).These properties are of increasing importance not only for the overall quality of RMs but also for the development and application of analytical methods that require small sample masses of the order of 5 10mg; see Section 4.3. The classical approach for particle size determination, or more correctly for particle size selection - which is still used for solids like soils, sediments and other technical materials like coal, and also for biological materials - is sieving analysis. The raw material is milled, generally after drying, see Section 2.1,and if the required particle size is obtained, typically ranging from 50.1to a few mm, it is allowed to pass sieves with different apertures to discard coarse particles and remaining materials. For materials consisting of numerous different particles microscopical inspection is used. Today laser beam instruments, available from several manufacturers, are increasingly used for particle size determination, predominantly of biological reference materials (Kramer et al. 1995; Koglin et al. 1997; Fajgelj and Zeisler 1998). This technique, which is mainly applied after milling and/or sieving, is presently the most advanced and promising approach for particle size measurements. Generally in all devices a laser beam is focused through a series of lenses onto the measuring cell. A dispersing unit supplies the sample, suspended in an appropriate solvent or dispersant, to this cell, often after ultrasonic treatment. The hardware and software that make up the equipment allow the measurement of particles 0.1-600 km in size,without disturbing the studied material. The theory and operation oflaser partide sizing is described in detail elsewhere (Miiller and Schuhmann 1996).Results obtained in this way are relative, dependent on the laser sizing system and the dispersant used. Figure 2.2 shows an example ofa material prepared at IRMM, Geel, Belgium.

particle size I pm Fig. 2.2 Particle size distribution: Candidate CRM 669Tuna muscle, Helos device, dispersant: 2-propanol (Kramer et al. 1997)

2.2 Control of Material Properties

2.2.2

Hornogeneity/Heterogeneity

Homogeneity testing is of prime importance for the certification and use of RMs. It should demonstrate the validity of the certified values and their uncertainties in the analysis of individual units or sub-samples thereof (Pauwels et al. IggSa). A determination of the analyte homogeneity in RMs is generally performed prior to certification in the basic material as a preliminary test for homogeneity, and later in the subsampled (that is bottled) material. It is usually defined as between-bottle and withinbottle (or ampoule) homogeneity. The between-bottlehomogeneity is verified by the determination of the analyte(s) on appropriate sample intakes ranging from a few mg for metals and methods consuming small sample amounts to 2 I g for organic compounds. Generally 10-30 sub-samples are taken from a number of bottles which were set aside at regular intervals during the whole bottling process; see Section 2.1.The within-bottle homogeneity is assessed by, for example, ten replicate determinations on the content of one bottle. Sometimes RM producers require that bottles are homogenized before use. If so it is important that the instructions are followed as to fail to do so is a common source of error; see Section 7.1. The homogeneity determinations are performed to confirm that the variations between units (bottles, ampoules) are neither statistically nor practically significant compared to the certified uncertainty so that an appropriate result about the level of homogeneity is obtained. Micro methods requiring no sample digestion for homogeneity determination of elements have the advantage that they can produce results with high precision. Homogeneity studies using these methods showed that for a number of CRMs the minimum sample mass recommended by the producer, typically 2100mg, were overestimated. Thus it can be argued that the certified values given on the Certificates for these CRMs remain valid down to sub-sample amounts of 20 mg or less, which allows in many cases their use as calibrants (Pauwelset al. 1991; Pauwels and Vandecasteele 1993); see also Section 4.4. Because of their growing importance during RM production, methods that need only milligram samples, like NAA and solid-sampling GFAAS, will be subsequently discussed in some detail; see also Section 4.3. A well suited method for homogeneity determination in micro samples requiring only milligram and even sub-milligram test samples and no decomposition procedure is Neutron Activation Analysis (NAA). For the special position of this method in the preparation of RMs, i.e. in homogeneity testing and certification analyses; see Sections 3.2 and 4.3. In homogeneity testing, the crucial problem is the differentiation of measurement uncertainty from the uncertainty due to sample inhomogeneity. In NAA, especially in its non-destructive mode, so-called instrumental NAA (INAA),this problem can be solved much more easily than in most other analytical techniques. The special suitability of INAA for homogeneity assessment is due to the following:

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the technique's non-destructive character multi-elemental capability high sensitivity for many elements the sources of uncertainty in the analysis are well understood and can be derived in advance, modelled and assessed experimentally Unlike non-radiometric methods of analysis, uncertainty modelling in NAA is facilitated by the existence of counting statistics, although in principle an additional source of uncertainty, because this parameter is instantly available from each measurement. If the method is in a state of statistical control, and the counting statistics are small, the major source of variability additional to analytical uncertainty can be attributed to sample inhomogeneity (Bedter 1993). In other words, in Equation (2.1): where sh is the degree of inhomogeneity expressed as the standard deviation due to inhomogeneity, sf is the overall observed variance, and :s is the total analytical variance. The last parameter can be assessed very accurately in NAA, because the corresponding standard deviation may be considered to arise from only three major terms, as estimated by (Equation 2.2):

si

where :s is the variance due to irradiation positioning, is the variance due to geometrical positioning in counting, and s: is the variance due to counting statistics, which in y-ray spectrometry is given by: sz = S + z B where S is the signal (peak area) and B is the background under the peak. Obviously, a comparison of the counting statistics for a set of samples with the overall analytical uncertainty can provide insight into other sources of variability of NAA results, including the homogeneity of the material analyzed. The standard deviation due to the sum of the variances sf and sg" can be well controlled under appropriately designed experimental conditions (or even directly determined by repeated analyses of an identical sample portion using shorttime activation), and kept well below 1.0% (ICuEera et al. 1997). The variance s: which is known from each measurement, is much more variable compared with the previous two parameters, and depends largely on the detection limit of a particular element. Since, however, INAA has sufficiently low detection limits for many elements, the counting statistics can also be very small (up to 0.1-0.2%) for both major and (very sensitive) trace elements, especially in geological and environmental matrices. Thus, element levels differing by 7-8 orders of magnitude can frequently be determined with about the same and very high precision, and inhomogeneities as low as 1% can be detected. For instance, the homogeneity of 25-mg sub-samples of a coal fly ash were tested by determining Al, Dy, Mn, and V by INAA, with concentrations from the mg/kg-' up to the% level attaining quite good precision ranging over 0.9-2.9 % (KuEera and Soultal 1988). This demonstrates the major advantage of INAA over X-ray fluorescence analysis (XRF). The latter is also very suitable for homogeneity testing but, due

2.2 Control of Material Properties

to limited sensitivity, is not capable of determining such widely different element levels. Examples of the use of INAA in homogeneity testing of RMs are numerous and therefore only a few illustrative examples can be mentioned. More than ten elements can frequently be used for the accurate estimation of homogeneity of several types of biological materials (Tu and Lieser 1984),while a much higher number of elements can usually be precisely assayed in geochemical and environmental RMs. For instance, the homogeneity of a set of soil RMs was studied by determining 25 elements by INAA in 150-mgsub-samples (Kutera et al. 1997). Evaluation of homogeneity of sub-samples over IOO mg is the current practice of major RM producers. However, since the advent of micro-analytical techniques, such as particle-induced X-ray emission (PIXE), micro-PIXE, solid sampling AAS, electron- and laser-microprobe techniques, etc. which analyze (or consume) sample masses from 1-2 mg down to I pg, there is an increasing demand for RMs certified for correspondingly low sample masses (Zeisler 1998); see also Section 4.3. This results in more stringent homogeneity requirements and INAA again plays a significant role in homogeneity testing of RMs for microanalysis, at least for sample masses above 0.1 mg, as was demonstrated by several authors. INAA was employed for e.g. homogeneity testing of ten elements in I-mg and larger sub-samples in a new Polish CRM for inorganic trace analysis (Dybczynski et al. 1998); and in a study on even smaller sample portions (from 0.1mg to I mg) of clay RMs, homogeneity of ten elements could be assessed using INAA (Filby et al. 1987). It is worth noting that microscale homogeneity is likely to be better in materials which are intrinsicaIly homogeneous, e.g. spores, pollen, single-cell algae, etc. Using various AAS modes by the 1980’s, there were many more or less successful approaches reported for metal analysis in milligram samples without prior decomposition, mainly graphite furnace techniques (Kurfurst 1998). In 1979, a GFAAS system with direct Zeeman effect background correction (SS-ZAAS, i.e. Zeeman magnet around the light source), specially designed for elemental analysis of solid samples was commercially introduced (Kurfurst and Grobecker 1981).It used small sample amounts at the milligram level, or even less, placed on a graphite boat inside the graphite furnace. Because of the low sample amount and its quick and easy operation, this system soon became an excellent tool, not only for direct metal determinationin a broad variety of materials,but also for evaluationof their distribution in the matrix, i.e. their homogeneity (KurfLirstet al. 1984);for details see Section 4.3. In the meantime, SS-ZAAS has gained in popularity in numerous applications, and has become of increasing importance for analyte homogeneity determination in the production and use of reference materials. Examples are: Pb, Cd, Hg, Zn, and Fe in codfish candidate RM, Hg in copper metal, Zn in mussel tissue, Cd, Pb, Hg, Fe, and Zn in cod muscle, Cd in plastic CRMs, and Cu, Cd, and Pb in various IAEA CRMs (Pauwels et al. 1991, 1993, 1994; Sonntag and Rossbach 1997). Figure 2.3 shows results of Cd measurements in a biological CRM with a SS-ZAAS system. Thus the method allowed precise information on the micro-homogeneity of formerly and more recently prepared materials that could not be obtained with most classical analytical approaches. The limitation of SS-ZAAS, however, because of its

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0.0

\

0.0 Fig. 2.3

0.5

1.o

1.5

2.0

2.5

Mass (rng)

Heterogeneity evaluation o f Cu in Bovine muscle, SM

30 SS-ZAAS system (Crobecker, unpublished data)

design requiring special light sources, is that only about 20 elements can be measured. Recently a new GF-AAS system was introduced. It is equipped with tools for semi-automatic analysis of solids using the quite sophisticated approach of a transversely heated graphite atomizer with inserted graphite platform, deuterium background correction and a new generation of software. It allows the measurement of all elements accessible to conventional GFAAS and will certainly improve and expand the performance of homogeneity tests (Nowka and Miiller 19g7), also in comparison with NAA (Rossbach and Grobeclcer 1999). This system is now also available with Zeeman background correction, which might further widen its application to matrices hitherto quite difficult to analyze with deuterium background correction AAS systems. Another improvement that will allow quick simultaneous oligoelement homogeneity determinations in milligram samples can be expected by the use of solid Sampling ETV-ICP-MS/AESand laser ablation ICP-MS which are now being studied in detail (Moens et al. 1995; Schiffer and Krivan 1999; Dobney et al. 2000). Homogeneity was, and still is, determined for elements in RMs by various modes of e.g. NAA, XRF, AAS, ICP-AES, ICP-MS and electrochemical methods after decomposition; see Section 3.2; and for organometallic and other compounds by combination of chromatographic techniques with these methods, see Section 3.3. Extraction procedures combined with GC, GC-MS, HPLC, and HPLC-MS etc. are commonly applied for homogeneity determination of PAHs, PCBs and other organic compounds; see Section 3.4. For these approaches sample sizes up to 10g and even more are often needed for reliable results (Schantz et al. 1995). For most of the methods mentioned above the method uncertainty - important for the expression of material homogeneity, better inhomogeneity - is influenced by different analytical steps such as digestion, and/or extraction, derivatization, separation etc., that are often required to achieve the final result. This consequently increases the method uncertainty.

2.2 Control ofMateria1 Properties

Finally it should be emphasized that a homogeneous distribution of selected elements, even if they are considered for various reasons to play the role of “inhomogeneity tracers”, does not guarantee the homogeneous distribution of other components. In otherwise homogeneous RMs, inhomogeneity for certain analytes has indeed been reported (Byrne 1993). 2.2.3 Humidity (Water Content)

To achieve the desired long-term stability avoiding biological, physical and chemical changes, removal of moisture is indispensable for most solid reference materials. Thus it is common practice to apply different drying techniques for biological matrices containing organic and organometallic analytes. The drying procedure is often followed by y-irradiation to protect against microbial attack. More robust materials such as plants and soils for metal analysis were dried at various temperatures; see Section 2.1. For dry mass correction the residual moisture (i.e. water) in RMs in most cases is still determined by weighing the difference of mass after drying, and less frequently but more effectively by Karl Fischer titration. Drying to constant weight, however, does not apply to many materials; so an arbitrary procedure, e.g. drying at elevated temperature for a distinct time followed by weighing is often recommended. The result of such a procedure is rather a weight loss under defined conditions and not necessarily the true water content. This points to the inherent problems of moisture determination due to the large variety of materials with differing properties and behaviors. Effects like significant hygroscopicity and the volatilization of compounds other than water make the situation quite complicated. In fact correct water determination by drying techniques is often difficult or cannot be achieved with reliable results. This significantly influences the analytical data obtained for other compounds. Therefore, it appears necessary, based on recent studies at IRMM and relevant literature, to explain the principles of water content, its control, reliable determination and influence on RM properties (Pyper 1985; Isengard 1995; Ruckold et al. 2000). Water taken up by solid materials is generally classified as water bound by physical forces or water bound by chemical bonds. Physically bound water includes adsorbed water, trapped or liquid-inclusion water, and absorbed water. The physical adsorption of water occurs when water condenses or is held on the surface; the surface includes the cracks, crevices, etc. of real materials. Liquid inclusion occurs during the crystallization process when bubbles of water are trapped. Water absorption is the process of taking up and retaining water uniformly throughout the structure of the host, e.g. on the surface of macromolecules, particularly those with a membrane or emulsifier function. Removal in this case is only possible with the application of much energy; and results at least in a change of functional properties of the substances under consideration. Chemically bound water, or water of crystallization, is divided into two main categories, bound water and zeolite water. They are distinguished by the effect upon

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I water removal; the crystal structure is altered if it is bound water, but the original 2 From Planning t o Production

skeletal structure remains if it is zeolite water. Water can also be a molecular part of the substance, e.g. a fixed, stabilizing component in the structure of proteins. The most difficult removal of such water molecules destroys the spatial structure, the biological activity, and mostly also the molecule itself. The availability of water, i.e. the water activity, in a material is of great importance for its biological and biochemical properties. It depends both on the water content, and significantly on the nature of the structural bond of water molecules, in other words, how strongly they are retained by the matrix. Thus, for similar water contents, when determined by Karl Fischer titration, quite different water activities may be obtained for different materials. This is of paramount importance for RM stability. Figure 2.4 shows the equilibrium relationships of biological materials between the water content and the water activity, am at constant temperatures and pressures. These data were first published in 1971, but did not find much attention in the RM field until now. At equilibrium the water activity is related to the relative humidity 9 of the surrounding atmosphere (Equation 2.3) where p is the equilibrium water vapor pressure exerted by the biological material and p o the equilibrium vapor pressure of pure water at the same temperature.

a, - p”0 - Av

(“A)

(2.3)

100

water content

E22F lloldds

n Fig. 2.4

10

20

30

10

50

60

70

80

rel. equilibrium humidity p

Scheme o f different possibilities o f deterioration with respect t o the equilibrium cp at T = const., t = const. The water content is with respect to the sorption isotherm (Heiss and Eichner 1971)

90

loo

2.2 Control of Material Properties

Water sorption isotherms are usually described by a plot of the water content as a function of the equilibrium water activity or the equilibrium relative humidity, giving rise in most, but not in all cases, to curves of sigmoid shape. The sorption isotherm of a distinct material is a useful tool for evaluating the amounts of water that can be taken up by this material under equilibrium conditions, and the state of water in biological materials in general. Hence, the determination of water activity is important for the characterization of biological materials concerning the availability of water for biological (e.g. microbial growth), physical, and chemical changes. Microbial growth, enzymatic reactions, non-enzymatic browning (reaction between carbonyl and amino compounds), and lipid oxidation are the major deterioration mechanisms that limit the stability of low moisture (a, <0.60) and intermediate moisture foods (0.60 < a, <0.85)and biological materials. Thus, in the observation of the drying process of biological materials, the water activity is at least as valuable as the water content of a material. As water activity measurements are complete in about 3 min, they represent a useful tool for control of the drying process. The sorption isotherm in Figure 2.4 is a typical example for an isotherm of biological materials. The slope of this isotherm is at a minimum at a relative equilibrium humidity of 20-45 %. This means that the tendency to pick up water is low under the moderate ambient conditions usually found in laboratories. Drying to a relative equilibrium humidity (< 20 %) increases the hygroscopic behavior of the material. Drying off the sorbed monolayer of water from the material increases the danger of lipid oxidation and hence leads to an unnecessarily high amount of deterioration. Drying to water activities higher than those mentioned increases the danger of quality changes caused by microbiological metabolism or enzyme activity. To fulfill the needs for long term stability of a biological material, the optimum range ofwater activity lies between 0.20 and 0.35. This area represents the best compromise between lipid oxidation and non-enzymatic browning. Enzymatic browning is suppressed under these conditions, and growth of microorganisms is impossible. In this area the change of water content, Aw.~.,as a function of the change of relative equilibrium humidity, A q , as a function of water activity: (a, 100 = q), Aw.c./Acp, is at a minimum. This also minimizes the potential error in a certified value by water taken up from the surrounding area. Based on these findings, it appears absolutely necessary that during the preparation of each material, water activity as well as water content must be determined and adjusted to achieve optimal stability and thus also a long shelf life of the final product. The practical measurement of water activity nowadays is usually performed by dew point determination or a change of electrical properties of the material depending on the relative humidity. The water content should be and is increasingly determined during RM preparation by Karl Fischer titration. The principle of this method is that it quantifies water selectively by measuring the consumption of iodine. During the titration, iodine oxidizes methylsulfite, formed from methanol and sulfur dioxide in a first step, to methylsulfate under stoichiometric involvement of water. Complete reac-

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2 From Planning t o Production

tions are obtained in the presence of a base. The form of the titration curve, the plot of volume of titration solution versus time, gives, to a certain extent, information of how free the water in the sample is (Isengard 1995). For routine use, this method is commercially available in automated form at moderate price. The use of Karl Fischer Titration is therefore strongly recommended for all laboratories preparing or working with RMs. A few examples are given below from the application of both methods described above and based on the relationships shown in Figure 2.4 (Riickoldet al. 2000). BCR CRM 402 Trace Elements in White Clover: a, = 0.456 at 21.r"C; W.C. = 7.52 + 0.04%. Despite its relatively high value, the water activity is still in the range for long-term stability and major differences in the water content as a consequence of sorption processes are not to be expected. This is supported by the fact that no spoilage has been observed, even though the production was in 1987. BCR CRM 422 Cod Muscle: a, = 0.495 at 20.4"C; W.C. = 9.60 + 0.05 %. Though the material has a relatively high a, and production took place in 1989, no deterioration was observed and noticeable sorption of water is not to be expected under normal storage and laboratory conditions. BCR RM 405 Wholemeal Wheat Flour: a,<0.03; W.C. = 1.70 k 0.04 %. It is important to note that this material is rikely to have handling problems due to its hygroscopic behavior. The water activity is in the range where the monomolecular surface layer of water may be removed. Thus, deteriorationofthe lipids is likely to occur during storage. These examples illustrate that, for many materials, the water content can be relatively high without leading to any material instability during shelf life, which depends on the water-bonding capacity of the material. When water is strongly retained, the water activity and thus the amount of free water will be low. It must be mentioned that this is quite contradictory to some former and still presently reported assumptions that the water content of CRMs should generally not exceed a rather low value, e.g. around 3 % for long-term stability of RMs. 2.2.4

Degradation Studies/Shelf Life

Stability testing and monitoring are extremely important as essential parts of the process of certifying RMs (Pauwels et al. 1998a) and explicitly required in various guidelines, including I S 0 Guide 35 (1989) and I S 0 Guide 34 (1996). But at present the results of these studies need only to be assessed in a qualitative way as shortterm and long-term degradation studies. Short term degradation studies have to be carried out to simulate degradation during transport and to decide under which conditions the material, once certified, should be dispatched. They allow the decision whether the material is stable enough to become a CRM. Long-term stability studies of an actual CRM upon storage are generally an integral part of the certification project. They typically cover a storage period of at least two years. Schemes to carry out stability testing of CRMs prior to release have been recently described and have been included in certification programs from the late 1980's. They are, in general, based on multiple temperature

2.2 Control ofMateria1 Properties

measurements, comparing either time ratios or temperature ratios. Common practice is storage at different temperatures and measurement of the analyte at predetermined time intervals. In 1990 the notion of combined uncertainty (UT) on these ratios was introduced (Griepink et al. 1990) taking into account the coefficients of variation (CV) resulting from the measurements, the studied temperature (T) and a reference temperature (RTj.This uncertainty is expressed as (Equation 2.4):

+

UT = (CVT2 CVRT2)"2x RT/100

Stability is then considered as known and defined when RT f UT is not significantly different from one. However the uncertainty calculated for the ratio RT based on the sum of CVs of two measurements carried out at two temperatures is a W a n d not a confidence interval. In fact it does not consider the number of measurements carried out at the two temperatures; and the use of this combined CV is not correct. In many cases it is an underestimation, as usually only two or three replicates are made. However, stability should be determined on the basis of a trend analysis, which is of importance also for any shelf life quantification; see below. One example, a candidate matrix material of organotin species in marine water, had stability determined by storage for 120 days at 4°C in the dark, at ambient temperature, and exposed to daylight (Quevauviller and Donard 1991).Frequently storage at different temperatures over at least a I-yearperiod are reported. Examples include organochlorine pesticides (OCPs) in BCR CRM 430,where pork fat was stored at -2o"C, +zo"C, and +37'C (vander Paauw et al. 1992). Storage at -zo"C, +zo"C,and + 4 0 T was performed for total and methyl Hg in BCR CRMs 4G3 and 464, tuna fish (Quevauviller et al. 1994). and metals in BCR CRM Goo, EDTA and DTPA-extractable trace metal contents in calcareous soil (Quevauvilleret al. 1998m). Certified reference materials are still not required to have clearly defined expiry dates set when the material is produced, so long-term stability studies are followed by long-term stability monitoring, lasting the whole lifetime of the CRM. However, this practice may change in the future due to the pressure of accreditation bodies, as plans exist to incorporate in the revised version of I S 0 Guide 31 the obligation to mention expiry dates "for all CRMs where instability has been demonstrated or is considered possible". This demand, although understandable from a theoretical perspective, might cause potential difficulty. The cost of a properly conducted stability study during the certification of CRMs would add significantly to the overall production cost, which would have to be reflected in the selling price. Already most CRMs are produced with substantial financial subsidy (Jenkset a1.1998). In practice, unless highly repeatable and reproducible methods such as isotope dilution mass spectrometry carried out under metrological conditions are used, the only way to fulfil these requirements is to carry out isochronous measurements as is now performed in detail at IRMM, Belgium, in different recertification projects of sold-out BCR RMs (Lamberty et al. 1998).The principle of this alternative approach to hitherto performed stability studies is based on a storage design with the samples at different temperatures for different time intervals, allowing all measurements to be done at the same time, i.e. at the end of the study. The storage design for a typical long term study is shown in Figure 2.5.

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2 From Planning to Production

Fig. 2.5 Typical storage design for isochronous measure ments-long term study (Lamberty et al. 1998)

Samples stored at a given temperature ( K ) are stored for various times, and either before or afterwards they are stored at a very low (reference) temperature at which their stability is supposed to be good. At the beginning (t = o), all samples reserved for the stability study are transferred to a very low storage temperature (e.g. <-zo”C). For each of the storage temperatures studied, samples are moved from this very low reference temperature to the corresponding storage temperature at different times (e.g. up to 18 months for the long term and up to 5 months for the short term study). At the defined end time the samples are immediately analyzed or put back (for a short time) at the reference temperatures before analysis. The samples that remained at the reference temperature for the entire study give the starting value for t = 0. All samples are then analyzed under repeatable conditions in a period of time as short as possible. Thus the risk of deviations in the response of the analytical equipment, the analytical method, and/or the calibration is minimized. The advantages of this method are: stability studies can be performed under repeatability conditions similar to those needed for homogeneity studies; this largely facilitates their execution as long-term reproducibility is no longer required, (2) the evaluation can be made temperature by temperature, starting with the samples stored at the highest temperature. If instability is detected it would appear necessary to analyze at the next lower temperature. If, on the other hand, stability is demonstrated for the full period at a given temperature, no further analyses of samples stored at lower temperature are required and (3) contrary to “classical” stability testing on the basis of RTas function oft, it is not required to analyze samples stored at two (or more) different temperatures including the reference temperature. (I)

A possible disadvantage could be that a larger number of samples has to be measured within a relatively short period of time. This can however be avoided by “freezing-in” the status of those samples by short-term storage at the reference temperature until the analysis can be carried out. Another disadvantage may be that the results of the long-term study are only available after two years, which may possibly

2.3 References

cause delay in the certification of the CRM. Thus it is advisable to extend the shortterm scheme from three to six months so that sufficient pre-information is available. Moreover, the bulk of the CRM should be stored at a sufficiently “safe”temperature until the long-term storage temperature is decided. If this approach is used correct strategic planning before initiation of a CRM project is necessary. The consequence of this would be that to establish shelflije quantijication, a long-term stability study must be carried out over a period of at least 3-4 years. It must be designed on the basis of a trend analysis, considering all measurements carried out over time, and should last until just before the start of sales of the CRM. This is because the determination of an expiry date for the validity of the certificate requires long-term high quality testing described in detail elsewhere (Pauwels et al. 1998b); see also Section 5.5 for pharmaceutical substances. Following such a protocol is a much more stringent control of quality of CRMs than is generally done at present.

2.3

References

Analytical Quality Control Services (1998) AQCS Intercomparison Runs Reference Materials 1998-1999. International Atomic Energy Agency, Vienna. BECKERDA (1993)Unique quality assurance aspects of INAA for reference material homogeneity and certification. Fresenius J Anal Chem 345:298-301. BECKERDA, Gills TE (1995) Recent developments in NIST botanical SRMs. Fresenius J Anal Chem 352:163-165. BENOLIELMJ, QUEVAUVILLER P, RODRIGUESE, ANDRADEME, CAVACO MA, CORTEZ L (1997) Certification of reference materials for quality control of major element determinations in groundwater Part I: Feasibility study. Fresenius J Anal Chem 358:574-580. BERKOVITSLA, LUKASHIN VN (1984) Three marine sediment reference samples: SDO-I, SDO-2 and SDO-3. Geostds Newsl 8:51-56. BERMANSS (1985) Marine biological reference materials for trace metals. In: WOLFWR, ed. Biological Reference Materials: Availability, Uses and Need for Validation of Nutrient Measurement, pp 79-88. John Wiley and Sons, New York BERMANSS, STURGEON RE (1988) A new approach to the preparation of biological reference materials for trace metals. Fresenius 2 Anal Chem 332:546-548. BOGERSHAUSENW, CICCIARELLI R, GERCKEN B, KONIG E, KRIVANV, M U L L E R - ~ F ER,RPAVEL J, SELTNER H, SCHELCHER J (1997) Pure graphite as a reference material for the determination of trace elements - an interlaboratorycollaborative study. Fresenius J Anal Chem 357:266-273. BOWENHJM (1965) A standard biological material for elementary analysis. In: SHALLIS PW, ed. Proceedings of the SAC Conference, Nottingham, pp 25-31. W. Heffer and Sons, Cambridge. BOWMANWS (1994) Stream sediment reference materials STSD-I to STSD-4. In: Catalogue of Certified Reference Materials, CCRMP 94-1E. Natural Resources Canada, Ottawa. BOWMANWS, FAYEGH, SUTARNO R, MCKEAGUEJA, KODAMA H (1979) New CCRMP reference soils SO-I to SO-4. Geostds Newsl 3:1og-113. BYRNEAR (1993) Review of neutron activation analysis in the standardization and study of reference materials including its application to radionuclide reference materials. Fresenius J Anal Chem 345:144-151. DOBNEY AM. MANKAJG, CONNEELY P, GROBECKER K-H, DE KOSTER CG (2000) Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)as a tool for studying heterogeneity within polymers. Submitted for publication

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2 From Planning to Production DYBCZYNSKI R, POLKOWSKA-MOTRENKO H, SAMCZYNSKI Z and SZOPAZ (1998) Virginia tobacco

leaves (CTA-VTL-2)- new Polish CRM for inorganic trace analysis including microanalysis. Fresenius J Anal Chem 360:384-387. EPSTEIN MS, DIAMONDSTONE BI, GILLSTE (1989)A new river sediment standard reference material. Talanta 36:141-150. European Commission (1994) Guidelines for the Production and Certification of BCR Reference Materials. European Commission Doc. BCR/48/gj, Brussels. European Commission, Joint Research Centre, IRMM (1999) BCR Reference Materials 1999. Institute for Reference Materials and Measurements,Geel. FAJGELJA and ZEISLER R (1998)Particle size determination of some IAEA and NIST environmental and biological reference materials. Fresenius J Anal Chem 360:442-445. FILBYRH, NGUYENS, CAMPBELL S, BRAGCA and GRIMMCA (1987)Evaluation of sample homogeneity for geochemical standard reference materials used in microanalysis.J Radioanal Nucl Chem Articles 110:147-158. FRASERCA, GARDNER GJ, MAXWELL PS, KUBWABO C, GUEVREMONT R, SIU KWM, BERMANSS (1995) Preparation and certification of a biological reference material (CARP-I) for polychlorinated dibenzo-p-dioxinand dibenzofuran congeners. Fresenius J Anal Chem 3p~43-147. GARDINERPE (1993) Consideration in the preparation of biological and environmental reference materials for use in the study of the chemical speciation of trace elements. Fresenius J Anal Chem 345:287-190. GONSKAH, GRIEPINKB, COLOMBO A, MUNTAUH (1984) The Certification of the Contents of Arsenic, Cadmium, Chromium, Cobalt, Fluorine, Manganese, Mercury, Nickel, Lead and Zinc in a Coal BCR No. 40. European Commission Report EUR 9473 EN, Community Bureau of Reference, Brussels. GRIEPINKB, HAMERS L, LE DUIGOUY (1981) Certification of the Elemental Composition of the BCR Reference Materials No. 71, Containing Carbon, Hydrogen, Bromine, Chlorine, Nitrogen, Oxygen and Sulphur, and No. 73 Containing Carbon, Hydrogen, Bromine, Chlorine, Fluorine and Nitrogen. European Commission Report EUR 7352 EN, Community Bureau of Reference, Brussels. GRIEPINKB, MAIEREA, MUNTAUH, WELLS DE (1991) Certified reference Materials chlorobiphenyls (IUPACnos 28, 52, 101,118,153 and 180)in dried sewage sludge - CRM 392. Fresenius J Anal Chem 339x73-180. MA, SIM PG (1990) Reference materials HARDSTAFF WR, JAMIESON WD, MILLEYJE, QUILLIAM for domoic acid, a marine neurotoxin. Fresenius J Anal Chem 338:520-525. HEISSR, EICHNERK (1971) In: HEISSR, EICHNERI<, eds. Haltbarmachen von Lebensmitteln Chemische, physikalische und mikrobiologische Grundlagen der Verfahren. 3. uberarbeitete und erweiterte Auflage, Springer 1994. SF, FAJGELJA, BERNASCONIG, TAJANI A, ZEISLER R (1998) Examination of a HELLER-ZEISLER procedure for the production of a simulated filter-based air particulate matter reference material. Fresenius J Anal Chem 360:435-438. IHNATM (Ig88a) Preparation of twelve candidate agricultural reference materials. Fresenius 2 Anal Chem 332:539-545. IHNATM (1988b)Criteria for the development of biological reference materials. Fresenius 2 Anal Chem 332:568-572. IHNATM (1988~) Biological reference materials for quality control. In: MCKENZIEHA, SMYTHE LE, eds. QuantitativeTrace Analysis of Biological Materials, pp 331-351. Elsevier Science Publishers, Amsterdam. IHNATM (1994) Development of a new series of agricultural/food reference materials for analytical quality control of elemental determinations. J AOAC Intl77:1Gog-1Gz7. IHNATM (1995) Key analytes and matrices lacking in the CRM-System and future needs for CRMs. Fresenius J Anal Chem 352: 5-6.

2.3 References

IHNATM, CLOUTIER R, WOODD (1987) Reference materials for agricultural and food analyses:

preparation and physical characterization of a bovine muscle powder candidate reference material. Fresenius 2 Anal Chem 326:627-633. IHNAT M, WOLFWR (1985)Maize and beef muscle agricultural and biological research materials. In: WOLF, WR, ed. Biological Reference Materials: Availability, Uses and Need for Validation of Nutrient Measurement, pp 141-165. John Wiley and Sons, New York. International Atomic Energy Agency and United Nations Environment Programme (1995)Survey of Reference Materials Volume I and Volume 2. IAEA-TECDOC-854, International Atomic Energy Agency, Vienna. ISENGARD, H-D (1995) Rapid Water Determination in Foodstuffs. Trends in Food Sci Techno1 5:155-162. I S 0 GUIDE 34 (1996) Quality system guidelines for the production of reference materials. (RevisedMarch 1998 as ISO/REMCO document No 464 “General requirements for the competence of reference material producers”. The revised Guide 34 will appear early 2000.) International Standards Organization, Geneva. I S 0 GUIDE 35 (1989) Certification of reference materials - General and statistical principles. International Standards Organization, Geneva. JENKS P, BOEKHOLTA, MAASKANT J, RUCINSKIR (1998) Are Certified Reference Materials a victim of Quality Systems? Fresenius J Anal Chem 360:366-369 JEROME SM (1993)Production ofa spiked milk reference material. Sci Total Environ 130/131:355-358. KLICH H, WALKER R (1993) COMAR-the international database for certified reference materials. Fresenius J Anal Chem 345104-106. KOGLIN D, BACKHAUSF, SCHLADOTJD (1997) Particle size distribution in ground biological samples. Chemosphere 34:2041-2047. KRAMERGN, GROBECKER KH, PAUWELS J (1993)Comparison of methods used for the preparation of biological CRMs. Fresenius J Anal Chem 352:125-130. KRAMERGN, GROBECKER KH, PAUWELS J (199s) Comparison of methods used for the preparation of biological CRMs. Fresenius J Anal Chem 352:125-130. KRAMERGN, MUNTAUH, MAIERE, PAUWELS J (1998) The production of powdered candidate biological and environmental reference materials in the laboratories of the Joint Research Centre. Fresenius J Anal Chem 360:299-303. KRAMERGN, OOSTRA A, D E Vos P, CONNEELY P (1997) The preparation of lyophilized tuna muscle samples for a feasibility study and for BCR candidate reference material CRM 669 rare earths. JRC-IRMM Report GE/R/MRM/q/g7. KRAMERGN, PAUWELS J, BELLIARDOJJ (1993) Preparation of biological and environmental reference materials at CBNM. Fresenius J Anal Chem 345x33-136. KRAMERKJM, DORTEN WS, GROENEWOUD H VAN HET,DE HAANE, KRAMER GN, MONTEIROL, MUNTAUH, QUEVAUVILLER PH (1999) Collaborative study to improve the quality control of rare earth element determinations in environmental matrices. J Environ Monit 1:83-89. K U ~ E R J,A SOUKAL L (1988) Homogeneity tests and certification analyses of coal fly ash reference materials by instrumental neutron activation analysis. J Radioanal Nucl Chem Articles 121:245-259. K U ~ E R J,A SYCHRA V, HORAI~OVA J, SOUKAL L (1997) Use of INAA in the preparation of a set of soil reference materials with certified values of total element contents. J Radioanal Nucl Chem Articles 215:147-155. KURFURST U (1998) Solid Sample Analysis - Direct and Slurry Sampling using GF-AAS and ETV-ICP. Springer, Berlin, Heidelberg, New York. KURFURST U, GROBECKER KH (1981)Feststoffanalytilc mit der Zeeman-AAS. Laborpraxis 5:28-31. KURFURST U, GROBECKER KH, STOEPPLER M (1984) Homogeneity studies in biological reference and control materials with solid sampling and direct Zeeman-AAS. In: SCHRAMEL P, B R ~ T T E R P, eds. Trace Element Analytical Chemistry in Medicine and Biology, Vol. 3, pp 591-601. de Giuyter, Berlin.

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LABRECQUEJ J (1990) The comparison of the results of two independent intercomparison studies (BAK-I and SLB-I) from the same bulk material of a lateritic soil. Fresenius J Anal Chem 338:498-500. LAMBERTY A, SCHIMMEL H, PAUWELS J (1998) The study of the stability of reference materials by isochronous measurements. Fresenius J Anal Chem 360:359-361. LISTERB (1978)The preparation of twenty one ore standards, IGS 20-39, preliminary work and assessment of analytical data. Geostds News1 2x57-186. MARCHANDISE H, VANDENDRIESSCHE S (1985)The Certification of the Impurity Contents (Ag,As, Bi, Cd, Ni, Sb, Se, Sn, Te, TI and Zn) in Three Grades of Lead, Electrolytically Refined Lead BCR No 286, Thermally Refined Lead BCR No 287, Lead with Added Impurities BCR No 288. European Commission Report EUR 9665 EN. Community Bureau of Reference, Brussels. MERRYJ (1995) Reference materials for monitoring nutrients in sea water environment, approach, preparation, certification and their use in environmental laboratories. Fi-esenius J Anal Chem 35~148-151. MOENSL, VERREPTP, BOONENS, VANHAECKE F and DAMSR (1995) Solid sampling electrothermal vaporization for sample introduction in inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. Spectrochim Acta 50B:463-475. MOOIJMANI U , IN? VELD PH, HOEKSTRA JA, HEISTERUMP SH, HAVELAAR AH, NOTERMANS SHW, ROBERTSD, GRIEPINKB, MAIERE (1992) Development of Microbiological Reference Materials. European Commission Report EUR 14375 EN, Community Bureau of Reference, Brussels. MORABITOR, MUNTAUH, COFINO W, QUEVAUVILLER PH (1999)A new mussel certified reference material (CRM 477) for the quality control of butyltin determination in the marine environment. J Environ Monit 1:75-82. MULLER RH, SCHUHMANNR (1996) Teilchengrofienmessung in der Laborpraxis. Wiss.Verlagsges, mbH Stuttgart. National Oceanic and Atmospheric Administration (1995) Standard and Reference Materials for Environmental Science. NOAA technical Memorandum NOS ORCA 94, Silver Spring. National Research Council Canada (1992) Certified Reference Material NASS-4 Open Ocean Seawater Reference Material for Trace Metals. National Research Council of Canada, Ottawa. National Research Council Canada (1995) Certified Reference Material CARP-I Ground Whole Carp Reference Material for Organochlorine Compounds. National Research Council of Canada, Ottawa. NOWKAR, MULLERH (1997) Direct analysis of solid samples by graphite furnace atomic absorption spectrometry with a transversely heated graphite atomizer and D,-background correction system (SS GF-AAS).Fresenius J Anal Chem 359:132-137. K, IWATA Y (1982) Preparation of pond sediment. In: OKAMOTO K, ed. Preparation, OKAMOTO Analysis and Certification of Pond Sediment Certified Reference Material. Research Report No. 38, pp 13-22, National Institute for Environmental Studies, Ibaraki. J, VANDECASTEELE C (1991) Determination of the minimum sample mass of a solid PAUWELS CRM to be used in chemical analysis. Fresenius J Anal Chem y5:121-123. PAUWELS J, DE ANGELISL, GROBECKER KH (1991) Solid sampling Zeeman atomic absorption spectrometry in production and use of certified reference materials. Pure Appl Chem 63:11991204. J, HOFMANN C, VANDECASTEELE C (1994) On the usefulness of SS-ZAAS for the microPAUWELS homogeneity control of CRMs. Fresenius J Anal Chem 348:418-421. J, KRAMERGN, DE ANGELISL, GROBECKER KH (1990) The preparation of codfish candiPAUWELS date reference material to be certified for Pb, Cd, Hg, Fe and Zn. Fresenius J Anal Chem

338:515-5 19. J, KURFURST U, GROBECKER KH, QUEVAWILLER P (1993) Microhomogeneity study of PAUWELS BCR candidatereference material CRM-422- cod muscle. Fresenius J Anal Chem 345:478-481. J, LAMBERTY A, SCHIMMEL H (1998a) Homogeneity testing of reference materials. PAUWELS Accred Qua1 Assur y51-55.

2.3 References

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PAUWELS J, LAMBERTYA, SCHIMMEL H (199%) Quantification of the expected shelf life of certified reference materials. Fresenius J Anal Chem 361395-399. PYPER JW (1985) The determination of moisture in solids - a selected review. Anal Chim Acta 170:159-175. QUEVAUVILLER PH, DONARDOFX (1991) Organotin stability during storage of marine waters and sediments. Fresenius J Anal Chem 339:6-14. I, MUNTAUH, GRIEPINK B (1994) The certification of the contents QUEVAUVILLER PH, DRABAEK (mass fractions) of total and methyl mercury in two tuna fish materials CRMs 463 and 464. EUR 15902 EN. QUEVAUVILLER PH, EBDON L, HARRISON RM, WANGY (1999) Certification of trimethyl-leadin an urban dust reference material (CRM 605). Appl Organomet Chem 13:1-7. QUEVAUVILLER PH, KRAMERKJM, VINHAST (1996) Certified reference material for the quality control of cadmium, copper, nickel and zinc determination in estuarine water (CRM 505). Fresenius J Anal Chem 354:3g7-404. PH, LACHICA M, BARAHONAE, GOMEZ A, RAURETG, UREA, MUNTAUH ( 1 9 9 8 4 QUEVAUVILLER Certified reference material for the quality control of EDTA- and DTPA-extractable trace metal contents in calcareous soil (CRM 600). Fresenius J Anal Chem 360:505-511. PH, MAIEREA, KRAMERKJM (1998a) Production of Certified Reference Materials QUEVAUVILLER for Pollutants in Environmental Matrices. European Commission Report EUR 18157 EN, CCF Academic Press, Tarbes. QUEVAUVILLER PH, MAIEREA, KRAMERKJM (199%) Trace elements in aquatic plants (CRMs 60, 61 & 62) pp 55-58; (1998~)CBs in fish oil (CRMs 349 & 350) pp 103-107; (1998d) Organotin compounds in mussel tissue (CRM 477) pp 116-120; (1998e) Trace elements in human hair (CRM 397) pp 127-131; (1998f) Pesticides in animal fat (CRM 430) pp 134-136; (1998g) Dioxins in milk (CRM 607) pp 146-149; (1998h) Major elements in freshwater (CRMs 398 & 399) pp 154-158; (1998i) Trace elements in groundwater (CRMs 609-612) pp 181-187; (1998j) ChlorobiTrace elements in soils and sludges (CRMs 141R-146R & 597) pp 225-232; (19981~) phenyls in sewage sludge (CRM 392) pp 240-243; (19981)Trimethyl-leadin urban dust (CRM 605) pp 274-278; In: Production of Certified Reference Materials for Pollutants in Environmental Matrices. European Commission Report EUR 18157 EN, CCF Academic Press, Tarbes. I<, MUNTAUH, GRIEPINKB (1g92a) Certified refQUEVAUVILLER PH, MAIER EA, VERCOUTERE erence material (CRM 397) for the quality control of trace element analysis of human hair. Fresenius J Anal Chem 343:335-338. I<, GRIEPINK B (1992b) Certified reference materials (CRMs 398 QUEVAUVILLER PH, VERCOUTERE and 399) for the quality control of major element determination in freshwater. Mikrochim Acta 108:195-204. ROPERP, BURKES, LAWNR, BARWICKV, ELLISONS, WALKER R (1999) Applications of Reference Materials in Analytical Chemistry. LGC/VAM/r99g/oj6, Laboratory of the Government Chemist, Teddington. ROSSBACHM, GROBECKER KH (1999) Homogeneity studies of reference materials by solid sampling-AAS and INAA. Accred Qua1 Assur 4:498-503. RUCKOLDS, GROBECKER KH, ISENGARDH-D ( 2 0 0 0 ) Water as a source of errors in reference materials. Fresenius J Anal Chem (in preparation). MM, BENNERBA JR,CHESLER SN, KOSTERBJ, HEHNKE, STONE SF, KELLYWR, ZEISLER SCHANTZ R, WISESA (1990) Preparation and analysis of a marine sediment reference material for the determination of trace organic constituents. Fresenius J Anal Chem 338:501-514. R, GREENBERG RR, SCHANTZ MM, BENNERBA J R , HAYSMJ, KELLYRW, VOCKERD J R , DEMIRALP SCHILLERBS, LAUENSTEINGG, WISESA (1995) Certification of standard reference material (SRM) 1941a, organics in marine sediment. Fresenius J Anal Chem 352:166-173. U, KRIVAN V (1999) A graphite furnace electrothermal vaporization system for inducSCHIFFER tively coupled plasma atomic emission spectrometry, Anal Chem 70:482-490.

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SCHLADOT JD, BACKHAUSF, BuRow M, FRONING M, MOHLC, OSTAPCZUK P, ROSSBACH M (1993) Collection, preparation and characterization of fresh, marine candidate reference materials of the German Environmental Specimen Bank. Fresenius J Anal Chern 345:137-139. KE, GILLLM, MARGOLISSA, WISESA, ELKINSE (1999) Preparation of standard refSHARPLESS erence material 2383 (baby food composite) and use of an interlaboratory comparison exercise for value assignment of its nutrient concentrations. J AOAC Intl82276-287. SONNTAGTM, ROSSBACH M (1997) Micro-homogeneity of candidate RMs characterized by particle size and homogeneity factor determination. Analyst 122:27-31. S, HORVAT M, KRATZER I(, PARRRM, STONESF, BACKHAUSFW, BYRNE AR, GANGADAHRAN SCHLADOTJD, ZEISLERR (1995)Production of hair intercomparison materials for use in population monitoring programmes for mercury and methylmercury exposure. Fresenius J Anal Chem 352:184-187 J K (1993) Standard Reference Materials: Handbook for SRM Users. NBS Special PublicaTAYLOR tion 260-100, National Bureau of Standards, Gaithersburg. NM (1998) NIST Standard Reference Materials Catalog 1998-99. NIST Special PublicaTRAHEY tion 260, National Institute of Standards and Technology, Gaithersburg. Tu SHU-DE,LIESERKH (1984) Homogeneity test of Chinese biological standard reference materials by means of instrumental neutron activation analysis. J Radioanal Nucl Chem Articles 84301-306. VAN DER PAAUW CG, RIETVELDAMJ, MAARSEH, GRIEPINK B, MAIEREA (1992) Development of pork fat reference material for OCPs BCR-CRM 430. Part I. Preparation, homogeneity and stability. Fresenius J Anal Chem 344:297-300. VERCOUTERE K, FORTUNATIU, MUNTAUH, GRIEPINK B, MAIEREA (1995)The certified reference materials CRM 142R Light sandy soil, CRM 143R sewage sludge-amended soil and CRM 145R sewage sludge for quality control in monitoring environmental and soil pollution. Fresenius J Anal Chem 352:1g7-202. VERSIECK J, HOSTEJ, VANBALLENBERGHE L, DE KESELA, VAN RENTERGHEMDJ (1987) Collection and preparation of a second generation biological reference material for trace element analysis. J Radioanal Nucl Chem 113:2gg-304. YEOMANWB, COLINET E, GRIEPENKB (1985) the Certification of Lead and Cadmium in Three Lyophilized Blood Materials CRM No 194, 195, 196. European Commission Report EUR 10380 EN, Community Bureau of Reference, Brussels. YOSHINAGAJ, MORITAM, OKAMOTO K (1997) New human hair certified reference material for methylmercury and trace elements. Fresenius J Anal Chem 357279-283. WE (1995) Application of synthetic reference materials in the Medical Radiological ZAICHICK, Research Centre. Fresenius J Anal Chem 35x219-223. R (1998) Reference materials for small-sample analysis, Fresenius J Anal Chem ZEISLER 360376379, R, DEKNER R, ZEILLER E, DOUCHAJ, MADERP, K U ~ E R JA(1998) Single cell green algae ZEISLER reference materials with managed levels of heavy metals. Fresenius J Anal Chem 360:429432. V, PERSCHKE H, DEKNER R (1993)The preparation of a cabbage candidate ZEISLER R, STRACHNOV reference material to be certified for residues of agrochemicals. Fresenius J Anal Chem 345:202-206. R, WISESA (1985)Quality assurance and protocols in sampling and sample preparation ZEISLER of biological samples. In: WOLFWR, ed. Biological Reference Materials: Availability, Uses and Need for Validation of Nutrient Measurement, pp. 257-279. John Wiley and Sons, New York.

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

I 3 Certification

Edited by Milan lhnat

3.1 Certification Philosophy o f RM Producers Milan Ihnatl, 3.1.1

introduction

All steps in RM development require appropriate and critical care in their execution and pose varying degrees of difficulty. The task of certification,defined as the assignment of concentration data which approaches as closely as possible the “true value”, together with uncertainty limits, is one of the most, if not the most, demanding challenges. Strictly,certification implies the reliable assignment of a value to a property of a material by a legally-mandated, standards-producing, national or international agency. It encompasses selection of measurands, suitable analytical methodologies, analysts and the certification protocol. Importantly, it relies on an accuracybased measurement system, that “...produces precise numerical values of the property under test or analysis that are free of, or corrected for, all known systematic errors...and are also related to the “true value” of the property under test or analysis” (Cali and Reed 1976). Legally, the certification process indicates that a certified RM carries with it the full weight and legal authority of the legally mandated national or international organization. Scientifically, certification deals with the establishment of “true values’’, with the provisos (Cali and Reed 1976; Cali et al. 1975) that: (I) systematic errors in the measurement process leading to certification are always investigated, but it should be realized that advances in the state-of-the-artmay uncover additional systematic errors that were unsuspected at the time of the original work; therefore, a cautious, conservative estimate of residual and unknown systematic error is the rule, and this should always be reflected in the final stated uncertainty, (2) every material is inherently unstable and property values will change with time, and ( 3 ) certified values are only valid when the RM is used in the manner for which it is intended and with all stated precautions followed by the user. 2068 from Pacific Agri-Food Research Centre - Summerland

I ) Contribution No.

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A variety of terms have been used by producers of RMs to describe the type of the numerical value reported (Ihnat 1988a). The International Organization for Standardization ( I S 0 Guide 30 1992) officially recognizes certijied value (...the value having been certified by a technically valid procedure), uncertijed value (...the value obtained by inter-laboratorytesting but which is not certified...), consensus value (...the value obtained by inter-laboratory testing, or by agreement between appropriate bodies or experts...), and best estimate or recommended (an estimate of the value that is optimized by taking into account both metrological and technical judgement and statistical factors). However the concentration value is presented, the credibility associated with the RM is of course directly related to the degree of confidence placed in the certifying organization. A review is presented here of certification approaches, followed by several of the major agencies and individual developers of RMs for chemical composition, addressing some of the many associated scientific aspects that significantly impinge on the conduct and outcome of the analytical characterization exercises. These include: definition of analytical methods; selection of analytical methodologies, analysts and laboratories; in-house characterization and cooperative inter-laboratory characterization. 3.1.2

Approaches to the Characterization/Certification of Reference Materials 3.1.2.1

General Principles of Certification

The overall certification strategy comprises many components which have a profound bearing on certification including the following: (I) nomenclature and definitions of RM and control materials, ( 2 ) nomenclature and definitions of certified values and related concentration terms, (3) overall measurement system, (4) material homogeneity, (5) material stability, ( 6 )definition of analytical methods, (7)selection of measurands for certification, (8) performance of analytical methods, (9) selection of analytical methodologies, (10) analyst parameters: considerations, selection of analystsIlaboratories, (11) selection of statistical protocols, uncertainty statements, (12)in-house characterization, (13) cooperative inter-laboratoryanalytical characterization campaign, (14)data quality control of the inter-laboratorycampaign, (15) critical evaluation of the methods used by cooperators, (IG) evaluation of data on technical merits, (17) evaluation and selection of multi-laboratory/multi-methodanalytical data, (18)statistical treatment of data, (19) calculation of concentration values and associated uncertainties, ( 2 0 ) reporting of results and information, (21) publication of approaches/protocols followed, and (22) future status of certified and informational values. The literature on RM certification indicates that there are two broad types of approaches for the characterization of RMs: (I)statistical, and (2) measurement. The statistical approach relies on the in-depth application of statistical calculations to a body of analytical results obtained from diverse exercises, often widely scattered and discordant. The approach based on measurement emphasizes laboratory measurement aspects and deals more in detail with various diverse analytical measure-

3.7 Certification Philosophy of RM Producers

ment possibilities to generate a coherent dataset, followed by necessary minimal calculations. The thrust of this Section is the general approach focusing on measurement-based laboratory considerations. The key characteristic of a RM is that the properties of interest are measured and certified on the basis of accuracy. The means of attaining the true value are varied, and several different philosophies have been utilized in the quest for the best estimate of the true value. The goal of all approaches is arrival at the best possible estimate of the true value: a reliable and unassailable numerical value of the concentration of the chemical constituent, under constraints of economics, state-of-the-art analytical technologies, availability of (new and old) methods, analyst competence, availability of analysts and RM end-use requirement. The basic requirement for producing reliable data is appropriate methodology, adequately calibrated and properly used. It is generally accepted that a property can be certified when the value is confirmed by several analysts/laboratories working independently using either one definitive method, or more likely, two or more methods of appropriate and equivalent accuracy. This premise then lays the ground rules for two commonly used general approaches to assuring technically valid RM certification: (I) the use of a single method of the highest accuracy (referred to as a “definitive”or “absolute”method) and usually employed by a single laboratory, and ( 2 ) the use of an inter-laboratory testing approach to RM certification, which involves more than one method. These approaches require development of a statistical plan for sampling and measurement, selection of reliable methodology of known and demonstrable accuracy, maintenance of statistical control of the measurement process, and quality assessment of the data by concurrent measurements of suitable RMs. These measurements require that the systematic and random errors of the procedures used to determine the particular constituent be sufficiently well known to state the concentration of that measurand within a required uncertainty level. A meaningful measurement process must yield numerical values that are (I) specific, reflecting only the property under test, ( 2 ) precise, and ( 3 ) free of systematic error (or bias) within the agreed on or practical limits required for the end use; the resulting numerical value can be equated to the “true value”. This section lists and describes a composite synthesis of the major certification approaches followed by the many agencies and individuals involved in RM development, based on many publications (Abbey 1983; Cali and Reed 1976; Cali et al. 1975; Commission of the European Communities 1981; Dams 1983; Dybczynslti et al. 1996, 1997; European Commission 1994, 1999; Fraser et al. 1995; Gladney 1980; Griepink et al. 1983, 1984; Ihnat Ig88a, 1g88b, 1993, 1994, 1gg5a, 1gg5b, Ihnat and Wolynetz 1994; I S 0 1985, 1996; Leaver and Bowman 1gg4a, 1gg4b, 1gg4c; Marchandise 1985, 1987; Mavrodineanu 1977; McLaren et al. 1990; Morrison 1979; Quevauviller 1998; Sim et al. 1987, 1988; Taylor 1993; Uriano and Gravatt 1977); a synopsis has appeared previously (Ihnat 1998). There are advantages and disadvantages to each certification approach. Usage is not necessarily clear-cut, and in many cases various combinations of these approaches have been used in certification. The most important consideration in any scheme is that systematic errors inherent in the methods

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errors, method precision, material variability and material stability must all be understood and taken into account when deriving the uncertainty statements for certified properties. 3.1.2.2

Classification of Characterization/Certification Schemes

All of the major RM characterization/certification approaches, for total concentrations of constituents, can be classified in one of four categories. A fifth approach deals with method-specific schemes in which characterization is by a defined method giving a method-specific assessed property value. The following is a classification based on the author’s interpretation and adaptation of descriptions of RM certification procedures in the literature (see summary in Table 3.1). Tab. 3.1

Classification o f typical characterization/certification approaches Methodological approach

Organizational aspect

7

Definitive method

z

Independent reference methods

3 4

Independent reference and validated methods Volunteer analysts, various methods

5

One specific method

One organization, several laboratories and analysts, One organization, several laboratories and analysts Multiple organizations, laboratories and analysts Multiple organizations, laboratories and analysts Multiple organizations, laboratories and analysts

(1)

Definitive method - one organization: a single definitive method used by a single organization, of the highest reputational quality, preferably applied in replicate by two or more highly skilled analysts, in more than one separate laboratories, working totally independently, preferably using different experimental facilities, with equipment and expertise to ensure traceability to the SI system. An accurately characterized, independently different, back-up method, independently applied, is employed to provide additional assurance that the data are correct.

The term “definitive method is applied to an analytical or measurement method that has a valid and well described theoretical foundation, is based on sound theoretical principles (“first principles”), and has been experimentally demonstrated to have negligible systematic errors and a high level of precision. While a technique may be conceptually definitive, a complete method based on such a technique must be properly applied and must be demonstrated to deserve such a status for each individual application. A definitive method is one in which all major significant parameters have been related by a direct chain of evidence to the base or derived SI units. The property in question is either directly measured in terms of base units of

3.1 Ceitifcation Philosophy of RM Producers

measurement or indirectly related to the base units through physical or chemical theory expressed in exact mathematical equations. The written protocol indicates how each of these critical parameters in the measurement process has been controlled, how traceability to the base units has been accomplished, and what the bounds are to the limits of systematic error and thus uncertainty. Such methods, applied with high reliability, give “true values” and provide the fundamental basis for accuracy in chemical analysis. Examples of definitive methods are: isotope dilution mass spectrometry, gravimetry (including fire assay analysis), coulometry, and calorimetry. Limitations of time, technical skills, specialized equipment, and resources preclude the widespread use of the definitive methods. Furthermore, most analytical methods cannot be classified as definitive methods, usually because there is no straightforward theory relating all the experimental variables to the final result (e.g. the common techniques of atomic absorption and emission spectroscopies), or because effects, including matrix effects, are too complex to handle by theory. The certification of an RM by one measurement method requires a method of high scientific status, laboratories of the highest quality, and skilled analysts. The method must be sufficiently accurate to stand alone and reported results must have negligible systematic errors relative to end-use requirements of the data. The acceptance of an RM certified in this way depends on the user community’s confidence in the ability of the certifying agency to carry out the definitive method. Independence of analysts and analyses in one organization is a fundamental question. It is important to have, even for the most reliable methods, more than one analyst/laboratory involved to avoid possible analystllaboratory-specificbiases. Certification by a single laboratory, without confirmation by another laboratory or method is risky. Measurement by a single definitive method is usually performed by two or more analysts worlcing independently to minimize possible biases. Frequently, an accurately characterized back-up method is employed to corroborate the data. Some agencies feel that a certification campaign should not be based on a single measurement procedure and therefore do not normally certify values on the basis of a definitive method applied in one laboratory. (2)

Independent refrence methods - one organization: Two or more independent reference methods, each based on an entirely conceptually different principle of measurement, independent in theory and experimental procedure, applied in replicate, within a single organization, of the highest reputational quality, by two or more expert analysts, working independently. The methods used can, naturally, include definitive methods, and the results should be corroborated by a third or additional, independently different, accurately characterized, well established, thoroughly validated, definitive, reference, or other methods.

A “reference method” is defined as a method of known and proven accuracy, thoroughly validated, and experimentally demonstrated to have negligible systematic errors and a high level of precision. Its development involves removing the principal systematic errors of the process, reducing them to tolerable levels, or when actual

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physical elimination is impossible, applying correction factors. The meaning of the term “independent” is that the basic theoretical and experimental principles on which one method rests, are entirely different from the principles of the other method(s). Reference methods are generally arrived at by consensus and fairly extensive testing by a number of laboratories. For example, the flame atomic absorption method for Ca in serum developed under the leadership of the agency fondly remembered as NBS, now NIST (Cali et al. 1972),was established after several inter-laboratory comparison exercises. The results were evaluated after each exercise and the procedure was changed as necessary. After five exercises, it was felt that the state-of-the-arthad been reached, with the reference method being capable of measuring Ca in serum with an accuracy of z % of the true value determined by IDMS (note that attainment of high accuracy and precision is not only a matter of the method, but is a function of both the method and analyst expertise). Since definitive methods are often unavailable, the multi-method approach is more frequently used in certification. A necessary condition for the certification of constituent concentrations is that determinations must be made by at least two independent, complementary, valid, reliable methods to avoid systematic errors associated with any one particular method or technique. Such measurements must agree within reasonable limits to permit certification. If significant discrepancies among analytical results from the different methods occur, additional work is carried out to reconcile them; otherwise the property values cannot be certified. Every effort should be made to use methods based on more than one principle of measurement (three independent principles being desirable) and to engage trained and experienced analysts. The independent reference methods approach is based on the rationale that the likelihood of two independent methods being biased by the same amount and in the same direction is small. Therefore, when the results from two, three or more independent reference methods agree, one can have a high degree of assurance that they are likely to be accurate. The philosophy of basing RM results on determinations by at least two independent methods of analysis or on determinations by a definitive method is often referred to as “the National Bureau of Standards approach”. It may be noted that NBS originally relied largely on approach I, using isotope dilution mass spectrometry. The concern of the independence of analysts in one organization still remains a fundamental question. Again it is important to have more than one analyst/laboratory involved to avoid possible analyst-specific and laboratory-specific biases. The characterization should be corroborated by additional methods or laboratories to provide additional assurance that the data are correct.

(3) Independent reference and validated methods by selected expert analysts - multiple organizations and laboratories: Two or preferably three or more independent reference and/or validated methods, each based on an entirely conceptually different principle of measurement, independent in theory and experimental procedure, applied in replicate, by selected expert analysts, of high reputational quality and recognized competence working independently in an ad

3.7 Cert9cation Philosophy of RM Producers

hoc network of laboratories participating in the collaborative inter-laboratory characterization campaign under very carefully prescribed and controlled conditions. The methods used can, naturally, include definitive methods. All analytical methods are well characterized and established, thoroughly validated, of acceptable demonstrated accuracy and uncertainty, and the exercise is planned to incorporate widely different methods, based on different physical or chemical principles. The analysts, are carefully selected, on the basis of their established capabilities for the consistent production of precise and accurate results, reputation, expertise and experience in the specific field of analysis, familiarity with the matrix investigated, appreciation for the RM development concept, and a sense of healthy scepticism, and participate, on invitation,in the analytical campaign. For the wide variety of materials and constituents in RMs, reference methods and definitive methods as well as in-house, single organization competencies are often not available. Thus the certifying agency cannot utilize certification approaches I or z but must resort to this approach relying on independent analysts and laboratories, using different (validated) methods. In this, a combination of definitive and reference methodologies applied by a single organization (approaches I and 2) is augmented by input from external analysts. This characterization philosophy is a variation of the two or more independent and reliable method approaches and can be briefly denoted as the expert analyst - different independent method approach. This characterization strategy is viable as long as the selection process selects analytical chemists with the requisite expertise (specifictype of measurements and materials) and proven track records of performance, using definitive, reference or validated methods of analysis. Technical discussions with all participants before and after the exercise, as practiced by BCR, is beneficial. The general premise behind this concept of certification by inter-laboratorymeasurement is based on at least two assumptions: (I) There exists a population of laboratories that is equally capable in determining the characteristics of the RM to provide results with acceptable accuracy, (2) the differences between individual results, both within- and between-laboratories,are statistical in nature regardless of the causes (i.e. variation in measurement procedures, personnel, equipment, etc.). Each laboratory mean is considered to be an unbiased estimate of the characteristic of the material. The inter-laboratory comparison mode, has been widely used by national and other laboratories for the certification of RMs. The following guidelines, enunciated by NIST are instructive. According to NIST (Cali and Reed I976), this is a mode that must be used with the greatest restraint and under very carefully prescribed and controlled conditions. At that agency, this approach is used only when the following circumstances apply: (I) The RM under study is in a technical area that is well established and one where many good, reliable methods exist, (2) each of the laboratories in the network are of very high quality and are known to produce very reliable results, (3) each laboratory agrees to the conditions set forth by NIST, (4) NIST controls the experimental design and evaluation of data, and (5) a previously issued RM, having similar properties to the RM candidate is used by each

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I laboratory as an internal quality control check. When these conditions are met and 3 Certification

maintained, this mode may be used with assurance to produce RMs of high accuracy and integrity. It must be assured that a wide range of reliable independent methods is covered with an absolute minimum of two but preferably three or more; it is furthermore beneficial and advocated that each method is used by at least two but preferably three independent analysts/laboratories. With entirely different basic principles used for the analysis, possible interferences or other systematic errors can reasonably be expected to be different. Each analyst should use well established method(s), which demonstrate adequate performance in terms of trueness (no significant bias) and reproducibility (standard deviation consistent and explicable on the basis of random experimental errors). If definitive and reference methods exist for the particular constituent/matrix combination, they should be targeted for inclusion in the repertoire of methodologies sought for certification. Although sophisticated methods may constitute the core methods for certification it is useful to include good, well executed routine methods. In order to further minimize systematic error, a conscious purposeful attempt should be made to get methods and procedures with wide-ranging and different sample preparation steps, including no decomposition as in instrumental neutron activation analysis and particle induced X-ray emission spectrometry. An overriding criterion regarding selection of methods and laboratories is the reputation, expertise and dedication of the scientist, analytical chemist, analyst or technologist conducting and responsible for the analyses. The choice i s made on the basis of analyst, not laboratory, although the laboratory should have an acceptable reputation and environment conducive to good work. The laboratories, collaborating in the analytical campaign should be carefully selected, without political, regional, administrative, or other constraints on the basis of reputation, experience in the specific field of analysis, familiarity with the matrix to be investigated, and the availability of the required analytical technique. Participation is by invitation. It is not necessary that participating laboratories be formally recognized, accredited or certified. Measurement of the property of interest should be completed by, or under the supervision of a technically competent manager qualified either in terms of suitable academic qualifications or relevant work experience. The participating laboratory should consider the analysis as a very special one, to be performed with special attention and all possible care, and not have it performed as part of its regular routine.

(4) Volunteer analysts, various methods - multiple organizations and laboratories: A “round robin” exercise with the participation of volunteer analysts in many laboratories, volunteering freely to participate, or chosen either fairly completely at random or selectively according to some selection criteria, based on political, regional, administrative, or other constraints, which may or may not be based on expertise or competence, due to an obligation to involve laboratories from a defined population of countries, regions or other groupings. Analybcal methods used are vaned, generally self-selected, and include reference, validated, invalidated, routine, as well as definitive methods, and this

3.7 Certification Philosophy of RM Producers

inter-laboratorycharacterization exercise is carried out without imposition of prescribed conditions and controls. More in-depth statistical treatment is needed to deal with the wide range, and likely, discordant nature, of analytical results received. Calculations and reassessment of data reported in the literature, to arrive at estimates of concentrations and uncertainties, can be considered a component of this approach. It is not always possible to have certification based on analyses done in-house or by selected laboratories according to strategies I, 2, or 3 defined above. One must then resort to the last, and in the author’s opinion, least preferred, mode of method-independent characterization, based on analysts, freely volunteering to participate, or selected without necessarily solid regard for expertise or competence, utilizing various methods. This approach, based on volunteer analysts and various methods in multiple organizations and laboratories, represents a round-robin type approach. Since no controls have been imposed upon the investigators, the limitations of such an approach and the data therefrom must be recognized. It must be appreciated that no mathematical processing can prove the validity of a concentration value derived from a mass of widely scattered data, the typical outcome of an exercise involving contributing analysts of varied backgrounds. Excellent insight into the problems associated with this approach has been provided by several experienced practitioners (Abbey 1970, 1977,1980,1983;Flanagan 1974; Gladney et al. 1979; Parr 1980; Von Lehmden et al. 1974).Ingamells (1978)in fact suggested that the “round robin collaborative analysis” approach was a waste of time and effort and proposed instead a strategy involving only two mutually independent analysts, working in different laboratories and presumably using mutually independent methods. Parr (1980) felt that one of the criticisms that can be levelled against this type of certification procedure is that the participating laboratories are self-selected and some may have very little experience. He postulated that there could be considerable improvement in the analytical results if data were accepted only from experienced laboratories (e.g. approach 3 above). While some improvements in the confidence intervals associated with the certified values can sometimes be achieved in this way, the problem (of scattered data) certainly cannot be made to disappear; selection of laboratories can only be made on rather subjective grounds by the person responsible for certification. With reference to analysis of standard rocks and minerals, Abbey (1980)clearly and forcefully observes that: “Given a highly incoherent set of results for the determination of each constituent of a proposed reference sample, the originator is faced with the difficult problem of estimating the “true” concentration. No known test can prove the validity of a concentration value derived from a mass of incoherent data”. These observations apply equally well to all analytical endeavors.

(5) Method-spec@: characterization by a spec@, validated method by selected expert or experienced analysts - multiple organizations and laboratories: One specified analytical method applied in replicate, by selected expert or experienced analysts, of high reputational quality and recognized competence working independently in an ad hoc network of laboratories, participating under carefully prescribed and controlled conditions, giving a method-specific assessed prop-

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erty value. The analysts are selected, on the basis of their established reputation, expertise and experience with the method and the specific field of analysis and familiarity with the matrix investigated, and participate, on invitation, in the analytical collaborative inter-laboratory characterization carnpaign. In a few instances, RMs are certified for the value of a constituent or property that is method-dependent because existing technology or technical or scientific applications require this. In analytical chemistry, examples of this are the Kjeldahl technique for nitrogen, EPA mandated and other extraction procedures for leacheable toxic constituents, extraction procedures for soil nutrients and toxicants in agronomy and soil science, and various enzyme-based, or enzyme-measuring methods in clinical science. In such cases, demonstration of statistical control of the rneasurernent process and agreement of results by independent analysts are the requirements for certification. As usually viewed by the RM scientist, the philosophy of certification rests on the concept of application of independent methodology to generate concordant results leading to one reliable value for the property. Such values are thus method-independent.Extractable and other concentrations generated by specific procedures are method-dependent,an idea which has to be rationalized with the fundamental method-independent concept in RM Certification. 3.1.2.3

Specific Examples

Examples of approaches followed by a small selection of the major RM developers are provided below and summarized in Table 3.2. It must be emphasized that these assignments are based on the author’s interpretation of approaches described in the literature: that placing into one of the approaches defined in this paper is not always feasible due to cross-over between different modes, fusion of ideas from one and another, elimination of steps, selective choices of parameters, modification of parameters, and streamlining of the overall procedure. Variations on a theme are unavoidable. In assigning certification approaches, the numbers refer to the approaches defined above; multiple numbers indicate a blending of two or more modes with a asterisk indicatingthe author’sassignment ofthe dominant mode of certification. The European Commission (1994) has prepared a detailed and most useful guide for the production and certification of RMs. The following is a summary of items relating to certification:

The participants: The range of participants should, whenever possible, be chosen in such a manner that widely different methods (based on different physical or chemical principles) can be used. The number of participants (recommended IS) should be sufficient to allow meaningful statistical processing of the results. When the laboratories feel the need for a CRM, either because the available calibrants are not comparable and a primary calibrant appears necessary for traceability, or because a reliable certified control material is needed but not available, then it is recommended that these laboratories do not plan a certification project entirely on their own, but that they involve laboratories having a background in traceability.

3. I Certification Philosophy of RM Producers Tab. 3.2

Assignment of certification approaches to various RM-developing organizations'

Approach Organization Standard Reference Materials Program, National Institute of Standards and Technology European Commission, Measurement and Testing Programme European Commission, Institute for Reference Materials and Measurements Marine Analytical Standards Program, National Research Council of Canada International Atomic Energy Agency' Canadian Certified Reference Materials Project National Institute for Environmental Studies, Japan Agriculture and Agri-Food Canada (M Ihnat, via NIST) Institute of Nudear Chemistry and Technology, Poland (R Dybczynski) US Department ofAgriculture (WR Wolf, C Veillon) HJM Bowen, United Kingdom 1)Asterisk indicates the dominant mode of certification, listed in Table 3.1 and described in detail in Section 3.1.2.2,based on this author's

assessment of published work.

2) Dominant mode was 4; some recent work follows mode 3 .

Quality and traceability: It is not required that participating laboratories are formally recognized, accredited or certified, provided that quality and traceability requirements are met. The methods: It is advised that, prior to the certification measurements, the participants discuss their methods so that all participants have confidence in each others methods and there is a good level of agreement between laboratories. As it i s preferred to certify on the basis of the agreement between different methods applied in different laboratories, a proposal should include, where relevant and possible, a group of laboratories offering a range of widely different measurement methods. Each laboratory should use well established method(s), with which it can demonstrate adequate performance in terms of trueness and in terms of reproducibility. Evaluation of the results: Evaluation of the results consists of (I) technical scrutiny of the consistency and of the quality of the data; the acceptance, on technical (not statistical) grounds, of data to be used to calculate the certified value and its uncertainty, (2) the calculation (using the appropriate statistical techniques) of the certified value and its uncertainty. The approach includes technical discussion of the results among all cooperators, rejection o f outliers, statistical evaluation, and calculation of the certified value and uncertainties.

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3 Certification 3.1.3

Conclusions

The ultimate goal of the chemical certification strategy is to arrive at reliable, unassailable assigned numerical values for the concentrations of chemical constituents in RMs. All certification approaches comprise several components and the careful and critical implementation accorded the exercise by the RM developer and coordinator of the characterization campaign. The foregoing represents the author’s interpretation and composite presentation of the different approaches followed by several of the major RM producers based on information available in the literature. It is this author’s view and contention that, in certification, painstaking care is needed in the selection of the cooperating analysts, laboratories and analytical methods and the main eflort is in the generation of a n excellent, tight dataset which is then subjected to minor mathematical manipulations to arrive at final property measures. The analytical determinations should be made with extra care to yield small uncertainties in the results. Throughout the overall task of RM development, there is an overwhelming requirement for a critical approach by critical analytical and measurement scientists and the involvement of national or international government agencies. Judging from the emphasis placed on analyst expertise and experience, it is clear that the author considers the role of the analyst to be of paramount importance in the certification exercise; good analysis and a good analyst go hand in hand. It is an opinion that it matters little as to how the analysis was performed as to who did it (Abbey, personal communication); all that is required for proper analysis is the selection of a good analyst. Analyst training, experience, familiarity with the problem on hand, skill, attitude, motivation and judgement are necessary prerequisites with which satisfactory solution of analytical problems is possible. Patterson and Settle (1976) state: “Most present analytical practices for lead cannot reliably determine lead concentrations at the I ng/g level because of a universal lack of familiarity with lead contamination during sample collection, handling and analysis. Consequently, the great mass of published data on lead in plant and animal tissue and in water is associated with gross positive errors which obscure the meaning of most work dealing with lead at the few pg/g level.”

3.2 Certification of Elements Milan /hnat, Anthony R Byrne, Jan Kubra and Philippe Quevauviller 3.2.1

Methods Used for the Certification o f RMs for Elements

An extremely wide variety of analytical methods are used by RM producers and developers in the certification of RMs for inorganic elemental content. These methods range from the classical, through current instrument based methods to highly specialized definitive methods.

In general, a typical method contains three basic stages: sample pretreatment and treatment, measurand preconcentration or isolation/separation (Table 3.3), and (3) its quantitative measurement (Table 3.4). (I)

(2)

Methods for the certification of RMs for elemental content: Initial stages: pretreatment, preconcentration, isolation and separation

Tab. 3.3

7:

Sample pretreatment, treatment procedures

No pretreatment or treatment Minimal pretreatment such as pelletizing, briquetting, grinding Acid digestion/dissolution in open vessels (atmospheric pressure) Acid digestion/dissolution in closed vessels (under pressure) Kjeldahl acid digestion Dry ashing, Eschka digestion Alkaline digestion, fusion, borates Combustion, bomb/tube combustion, ignition, pyrolysis Extraction, leaching 2: Preconcentration,

isolation, separation

No preconcentration or isolation/ separation procedures applied Physico-chemical separation/preconcentration Hydride generation, cold vapor generation (Hg) Solvent extraction, including complexation Precipitation, co-precipitation Chromatography including extraction,ion exchange, adsorption Distillation,volatilization Electrolysis, electrodeposition

The latter table presents a compilation, of necessity not comprehensive, of specific methods and elements determined by the major producers and based on work done under the auspices of agencies such as NIST, NIES, and BCR. The table may serve as an indication of the general status of method-element relations in R M certification. These stages are shown with the understanding that the typical complete method (often but not always) has a contribution from each of these three. For example, the method of acid digestion solvent extraction flame atomic absorption spectrometry has acid digestion as the sample treatment step, solvent extraction as the measurand preconcentration and isolation step and finally flame atomic absorption spectrometry as the quantitative determinative step. Under the techniques for quantitative determination some examples of specific determinative techniques are included, defining complete methods of analysis. The large number of elements and element/material combinations for which concentration values are desired and the important requirement in certification of confirming data by several independent analytical techniques, necessitates application of a large number of analflcal methods to the certification exercise. The large variety of sample treatment, ashing and digestion approaches as well as detection and measurement schemes leads to

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3 CertFcation Tab. 3.4

Methods for the certification of RMs for elemental content: determination methods used by various R M producers for biological, environmental, clinical, agricultural, food, geological, occupational materials, and pure compounds'

Analytical method

Elements determined

Atomic Absorption Spectrometry (AAS)

Flame AAS (FAAS) Electrothermal AAS (ETAAS) Hydride generation AAS (HGAAS)

A1 Ca Cd Co Cr Cu Fe K Mg Mn Na Ni Pb Sr V Zn A1 As Cd Co Cr Cu Fe Hg Mg Mn Ni Pb Ma Sb Se V Zn As Sb Se

Cold vapor AAS (CVAAS)

Hg

Atomic Emission Spectrometry (AES)

Flame AES (FAES) Inductively coupled plasma AES (ICP-AES) Hydride generation ICPAES (HGICPAES) Direct current plasma AES (DCPAES)

Ca I< Na Rb Sr A1 As B Ba Be Ca Cd Co Cr Cu Fe Hg I< Li Mg Mn Mo Na Ni P Pb S Sb Se Si Sr T i V Y Zn Zr As Hg Sb Se A1 Ca Cr Cu Fe I< Mg Mn Mo Na Ni Pb Zn

Atomic fluorescence spectrometry (AFS)

Cold vapor atomic fluorescence AFS (CVAFS)

Hg

Mass spectrometry (MS)

Mass spectrometry Isotope dilution MS (IDMS) Spark source MS (SSMS) Neutron activation MS (NAMS) Inductively coupled plasma MS (ICP-MS)

B Ca Cd Cu Fe Ma Pb Se Sr Zn Ag Ba Ca Cd Cr Cu Fe Hg K Pb Mg Mn Na Ni Pb Rb S Se Tl Zn Cu Fe Sr Zn B A1 As B Ba Ca Cd C1 Co Cr Cu Fe Hg I Mg Mn Ma Na Ni Pb Sb Se Sr Zn

Nuclear methods

Instrumental photon activation analysis As Ba Ca Co Cs Fe Mg Mn Na Ni Pb Rb Sb Sr Ti Y Zn Zr (IPAA) Proton activation analysis (PAA) Instrumental neutron activation analysis (INAA) including thermal, epithermal and prompt y techniques NAA with radiochemical separation (RNAA)

As Cd Co Cr Fe Hg Mn Na Ni Pb Zn

Al As Au B Ba Br Ca Cd Ce C1 Co Cr Cs Cu Eu Fe Ge Hf Hg I In I< La Lu Mg Mn Ma Na Ni P Rb Sb Sc Se Sm Sr Ta Tb Th Ti U V W Y ~ IZn Zr As Cd Cl Co Cr Cu Fe H g I M n Ma P Sb Se V Zn

X-ray emission (Fluorescence)

Energy dispersive, wavelength dispersive and particle induced techniques

As Br Ca Cd Cu Fe K Mn Ma Ni Pb Rb Se Sr Ti Zn

Tab. 3.4 (Continued)

Analytical method

Elements determined

(Molecular) light absorption spectrometry Spectrophotometry, colorimetry (LAS)

A1 As B C1 Co Cr Cu F Fe I Mn Ma Na Ni P S Sb Ti

(Molecular) fluorometry (FLU)

Eu Se S m

Electrochemistry voltammetry, anodic stripping (ASV), cathodic stripping (CSV),polarography, differential pulse polarography (DPP), ion selective electrode (ISE)

Br Cd C1 Co Cr Cu F Fe I Mo Ni P Pb Se V Zn

Other methods Kjeldahl method Combustion elemental analysis Volumetry (titrimetry) Chromatography Gravimetry

N CHNS Al As Ca C1 Fe Mg N P S Ca C1 Fe KMg Na S Al C Ca H P S Si

1) Different authors may have different nomenclature for methods. References: (Dybczynskiet al. 1997; Ihnat 1994, 2000; Mavrodineanu 1977; Okamoto 1980,1982; Pszonicki and Hanna 1985; Que. vauviller et al. 1998; Quevauviller and Maier 1999).

useful, multiple variants of each method. An attempt is made to get wide-ranging techniques and procedures including different sample preparation steps, including the absence of sample treatment as in instrumental neutron activation analysis and particle induced X-ray emission spectrometry, as well as different detection and measurement techniques. Such techniques, which do not require sample decomposition, provide most useful analytical contributions and are major contributors to certification as sample decomposition, which can be a major source of systematic and random error, is no longer needed. The determination of the typical element is usually associated with some half dozen methods (including variants of basic techniques), but can range from two methods for some elements to one dozen or more for other elements. This section contains further two typical certification applications and a table presenting selected examples for environmental and biological RMs from major producers. This is followed by a more detailed treatment of the use of neutron activation analysis methods.

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3 Certification Tab. 3.5

Certified values for

Element

A1 Ca C

Fe Mg

Sb As Ba Cd Cr

co cu

25

%

6.11 i 0.16 2.60 i 0.03 3.348 ? 0.0016 4.11 f 0.10 1.20 i 0.02 3.79 i 0.15 23.4 f 0.8 414 2 12 3.45 i 0.22 135 f 5 14.0 i 0.6 98.6 f 5.0

constituent elements in NIST SRM 2704, River sediment Element

P I< Si Na

Ti Pb Mn Hg Ni

T1 U V

Zn

% 0.0998 f 0.0028 2.00 f 0.04 29.08 i 0.13 0.547 * 0.014 0.457 i 0.018 mg/kg 161 f 17 555 i 19 1.44 f 0.07 44.1 2 3.0 1.2 t 0.2 3.13 f 0.13 95i4 43%i 12

3.2.2 Multi-Method Elemental RM Certification 3.2.2.1

River Sediment

Multi-methodcertificationof 25 elements with information provided on another 22 elements was performed in NIST SRM 2704 (Buffalo River sediment) by NIST laboratories with four co-operatinglaboratories from USA and Canada (Epsteinet al. 1989). Except for INAA and inert gas fusion (IGF), which can be used to analyze material directly, most techniques required a decomposition step before analysis. In order to maintain the independence of analytical methods various different decomposition procedures were specified for similar methods. For example a lithium metaborate fusion was selected for DCP analysis, since this method commonly uses Li as an interference buffer. Various open and closed vessel acid digestion (HF, HC104, HN03, H2S04)as well as oxygen bomb procedures were chosen prior to a number of methods applied by the cooperating laboratories. These methods covered a broad range including the already mentioned techniques, all modes of AAS, XRF, Ion Chromatography, IDMS and Polarography. Table 3.5 summarizes the values for the 25 certified constituent elements. Each certified value is a weighted mean of results from two or more independent analytical techniques. When only NIST laboratory results were used to determine the certified values, the weights for the weighted means were computed according to an iterative procedure. When co-operatinglaboratories results were included in calculation of the certified values, all results were weighted equally. Compared to formerly prepared sediment SRMs the confidence limits for certified values at all concentration levels decreased by a factor of almost two. This was attributed to experience gained in collection and processing as well as to improved quality control and analytical methodology.

3.2 Certijcation of Elements 6.2

6.6

7.0

7.4

7.8

+........+.......+........+.......+.........+.......+........+....*...+

07 R N M 09 I N M

03 ICP-MS

02 OW-AES 02 DPASV

I

<----*-----, I

<------*------> I I

<-------M------->

/MEANS:

Fig. 3.1 Data sets for Cu in CRM 482 Lichen accepted after the technical and statistical evaluation. The laboratory codes are indicated with the methods used (Quevauviller et al. 1996b).

Uncertified values are given for 22 constituent elements (Cl, S, Br, Ce, Cs, Dy, Eu, Ga, Hf, I, La, Li, Lu, Rb, Sc, Se, Sm, Sn, Sr, Th, M,and Zr). The element concentration could not be certified, if a bias was suspected in one or more of the methods required for certification, or if two independent methods were not available. 3.2.2.2

Lichen

Lichens accumulate trace elements from the atmosphere and thus are frequently used instead of air filters for pollution monitoring. Certification of nine elements (Al, As, Cd, Cr, Cu, Hg, Mo, Ni, Pb, and Zn) by a group of 11 selected laboratories was performed in BCR CRM 482 (Lichen) after an inter-comparison on trace elements in samples of this material (Quevauviller et al. 1996b). The applied pretreatment techniques were digestion with a combination of acids in the pressurized or atmospheric mode, programmed dry ashing, microwave digestion and irradiation with thermal neutrons. The analyhcal methods of final determination, at least four different for each element, covered all modern plasma techniques, various AAS modes, voltammetry, instrumental and radiochemical neutron activation analysis and isotope dilution MS. Each participating laboratory was requested to make a minimum of five independent replicate determinations of each element on at least two different bottles on different days. Moreover, a series of different steps was undertaken in order to ensure that no substantial systematic errors were left undetected. The sets of technically and statistically acceptable results are represented in the form of “bar-charts”of which an example is given for Cu in Figure 3.1. The length of a bar corresponds to the 95 % confidence interval of the mean. The certified values

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were calculated as the arithmetic means of the laboratory means, taking into account the number of sets accepted for certification. They appear as vertical dotted lines in the bar-graphs. The uncertainty is given by the half-width of the gs % confidence interval of the mean of the laboratory means. During the course of the certification, some laboratories individually determined and reported values for a total of 14 additional elements (Ba, Br, Ca, Co, Fe, K, Mn, Mo, P, S, As, Se, Sn, and V). The obtained indicative values were not certified, as the data did not meet the already mentioned requirements for certification, but is presented as information values in the publication quoted above. 3.2.2.3

Examples o f Selected RMs Certified for Elements

Table 3.6 presents examples for environmental and biological natural matrix CRMs from BCR, NISTand NRCC certified for elements using the above described analytical multi-method approaches. 3.2.3 Certification o f Element Contents by Neutron Activation Analysis

3.2.3.1

General Features

Neutron activation analysis ( N U ) is based on the measurement of characteristic radiation, most frequently gamma-rays, of radionuclides formed by irradiation of the material with neutrons (Heydorn 1984). The highest sensitivity for most elements is obtained when irradiation is carried out in a nuclear reactor. Due to a higher potential for accuracy compared with other methods of elemental analysis, NAA is recognized as one of the most favorable techniques for the certification of element contents, and moreover, its multi-elemental capability and the possibility to perform analysis non-destructively is especially useful for homogeneity testing; see Sections 2.2 and 4.3. The low uncertainty of results, even at very low element levels, required for certificationcan be achieved due to the following advantageous features of the technique (Byme 1993):its sensitivity and applicability to minor and trace elements in a wide range of matrices, the virtual absence of an analytical blank, its relative freedom from matrix and interference effects, the possibility to perform analysis nondestructively using so-called instrumental neutron activation analysis (INAA), its high specificity based on the individual characteristics of the induced radionudides, such as y-ray energies and half-lives, the capability of INAA for multi-element determination, often allowing 30-40 elements to be determined in many matrices, an inherent potential for accuracy compared to other analyixal techniques, since the theoretical basis of NAA is simple and well understood, so the sources of uncertainty can be modelled and well estimated, its totally independent principle as a nuclear-basedproperty in contrast to the electronicnature of most other analwcal techniques, and the isotopic basis which often offers a choice of analyhcally independent routes for element determination and forms the most important part of the self-verification principle in NAA (Byme 1993; Byrne and ICutera 1997);see Section 3.2.3.2 below. In cases where the induced radionuclides of trace elements are masked by matrix activity, radiochemical separation provides interference-free detection limits close to

3.2 Certification of Elements Tab. 3.6

Examples for BCR, NlSTand NRCC natural biological and environmental matrix CRMs/ SRMs certified for elements CRMISRM No.

Certified

Uncertijied

Producer

Material

values

values

BCR

281

Rye grass

13

2

101 060 279 414 184 063R 189 143R 320

Spruce needles Lagarosiphon major Ulva lactuca Plankton Bovine musde Slim milk powder (natural) Whole meal flour Sewage sludge amended soil River sediment

9 7 6 10 8 12

10

7 7 1 6 9 3 9 5 25

NIST

1547 1573a 157% 1633b 16341, 1643d 1944 2586 2781 2711

Peach leaves Tomato leaves Bovine liver Coal fly ash Trace elements in fuel oil Trace elements in water NY/N J Waterway sediment Trace elements in soil Domestic sludge Montana soil No 2

20 22 18 22 9 26 9 4 10 24

21 16 6 21 7 4 19 18 11 26

NRCC

DORM-1 DOLT-2 LUTS-1 BCSS-1 SLEW-1 SLRS CASS-2 NASS-4

Dogfish muscle Dogfish liver Lobster hepatopancreas Estuarine sediment Estuarine water Riverine water Nearshore seawater Open ocean seawater

16;t 15" 24" 14/12;t;t

-

10 21 11 12

-

7 8

I

3 -

1

Remarks: t includes methylmercury as mercury, **matrix and minor elements

the theoretical ones. Thus, in the radiochemical mode of NAA (RNAA), the technique has two other advantageous features: trace and ultra-trace (radio)chemistry can be performed under controlled conditions by inactive carrier additions, and the chemical yield (recovery) of the separation can be obtained simply by using carrier budgeting or the radiotracer method. It should be pointed out that no other analytical technique has the capability to provide multi-element data non-destructively, often with good detection limits and

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I in a virtually matrix-independent manner. This is largely based on the penetrating 3 Cert9cation

nature of neutrons and of the y-rays emitted by the induced nuclides. Of the analytical techniques capable of multi-element trace analysis of bulk samples, the virtual absence of an analytical blank is unique to NAA. In addition to the relative freedom from matrix and interference effects, it should be emphasized that the analytical signal in NAA is linear over a very wide range of element concentrations. Both the inherent potential for accuracy and the totally independent principle as a nuclearbased property make NAA invaluable in RM certification. 3.2.3.2 Internal Cross-Checking (Self-Verification) in NAA

The above features and advantages of NAA have long been appreciated and utilized. However, only relatively recently has it been fully realized that the possibility to determine a particular element using different neutron induced reactions of its isotopes, together with the ability to perform the analysis both by INAA and RNAA, with additional options for internally cross-checkingthe results obtained by various methodological approaches in NAA, forms the basis for a unique ability to verify analytical data (Byrne 1993; Byme and KuEera 1997). The primary source of the independence of the analytical data is the essentially isotopic nature of NAA, i.e. activation of different isotopes of the same element, and the possibility of using different, again essentially independent, isotopic nuclear reactions, such as (n, y), (n, p), (n, a), (n, 2n), (n, n’), (n, f ) in NAA. It has been shown (Byme and Kutera 1997) that for example the elements Hf, Se and Sn can be determined by employing up to five fully independent nuclear reactions leading to different products by NAA, and more than 25 of the elements most frequently assayed in biological and environmental matrices can be determined by at least two independent reactions giving y-emitting radionuclides. In addition to the undoubted independence of analybcal data provided by NAA using different isotopic reactions, other internally independent routes exist in NAA: (I) using the different modes of NAA, i.e. INAA, RNAA or NAAwith a pre-irradiation separation, frequently termed “chemical” or “molecular”activation analysis (MAA - this method is also capable of determining various element species),(2) using selective activation (with thermal, epithermal or fast neutrons), and induction of different nuclear reactions on the same isotope, (3) using selective measurement techniques (a,y and X-ray spectrometry, Compton suppression counting in y-ray or p-y coincidencecounting, fi and Cherenlcov counting, and delayed neutron or fission track counting for uranium determination, and (4)using Combinations ofthe above. Use of the four strategies (1)-(4)(and different isotopic routes) provides assurance that the errors to which NAA is liable, like all analytical techniques, will be reduced or at least altered in the alternative route. The more elements of independence are combined, the less the likelihood of repeating the same error. Of the four strategies given above, the best condition for obtaining independent data for quality control (QC) are satisfied when INAA and RNAA results are compared, because the use of RNAA dramatically improves the selectivity of signal measurement and eliminates or greatly reduces the measurement uncertainty sources, such as spectral interferences. A variety of radiochemical separations and

3.2 Certification ofElements Tab. 3.7

Results for NIST SRM 1633a Coal Fly Ash using the self-verification principle of NAA (Byrne and Kutera 1997; I
Nuclear Reaction

NAA mode

This work

RNAA, c2 RNAA, c3 RNAA, c4 INAA RNAA INAA-p RNAA, s l RNAA, s2

5.70 i 0.57 5.58 i 0.25 5.41 i 0.67 5.80 +_ 1.14 10.4 +_ 0.3 (6) 10.3 i 0.4 (4) 0.47 (2) 0.441 * 0.043 (4) 0.446 +_ 0.025 (4)

NfSTvalue

5.7 f 0.2

10.2 f 0.3

counting of y-rays of " 'n c2: counting of Hg X-rays of *'?'I c3: liquid scintillation counting of 20% c 4 counting of Cerenkov radiation of "?1 INAA-p: pre-irradiation separation RNAA, sl: KOH fusion RNAA, s 2 O2 combustion CI:

counting techniques can be employed in RNAA, so that this method itself can be internally verified. The usefulness of such an approach was recently demonstrated in the low level determination of T l in biological and environmental RMs (ICuCera et al. 1997). There are also several possibilities to perform internal self-verification using a purely instrumental approach, e.g. by comparing INAA results with those obtained by application of epithermal neutron activation analysis (ENAA) followed by Compton suppression counting in y-ray spectrometry. It has been shown that this instrumental mode may also lead to a drastic reduction of background and spectral interferences compared to INAA for several sample types (Landsberger et al. 1990; Landsberger and Peshev 1996).The selectivity of analysis can also be significantly enhanced if a pre-irradiation separation is carried out. However, in this case sample contamination must be avoided or minimized, since one of the most valuable advantages of NAA (its blank-free nature) is being lost. Examples of several approaches to self-verificationof NAA results in analysis of NIST SRM-16jja Coal Fly Ash are shown in Table 3.7. So far, in practice few NAA laboratories are fully exploiting all these possibilities for internal QC. 3.2.3.3

Applications in Certification and Analysis

Depending on the neutron fluence rate and efficiency of the semiconductor (mostly high purity Ge-HPGe) y-ray detector available, usually the following elements can be determined by INAA in a variety of environmental and geological matrices at the mg/kg level (elements in parentheses only at somewhat elevated levels):Ag, Al, As, Au, Ba, Br, Ca, (Cd),Ce, C1, Co, Cr, Cs, (Cu),Dy, Eu, Fe, (Ga),Hf, (Hg),(I), In, IC, La,

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3 Certijcation

Lu, Mn, (Mo), Na, Nd, (Ni), Rb, (S), Sb, Sc, (Se), (Si), Sm, Sr, Ta, Tb,Ti, Th,U, Yb, V, W, Zn, and (Zr). In biological materials, where many important essential and/or toxic trace elements are contained at lower levels, and the characteristic y-rays of their radionuclides are masked by the matrix activity (mainly due to the radionuclides s2Br, 38Cl, 421(, 24Na,and 32P), the possibilities of INAA are less favorable, especially for animal and human tissues and/or body fluids. Thus only about a half of the above elements can be determined by INAA in biological materials; see Table 3.8. However, numerous reliable RNAA procedures have been developed that allow high accuracy determination of elements in biological materials at sub-pg/kg levels by separation of the relevant radionuclides in groups (mostly applicable for medium- and long-lived radionuclides), or in single fractions (mostly required for shortlived radionuclides). For example, RNAA played a key role in the certification of a second generation, human serum reference material (Versieck et al. 1988).The successful applications of RNAA in the determination of trace elements in biological RMs at the nanogram level were reviewed (Byme et al. 1984; Delfanti et al. 1984; Cornelis et al. 1990) and numerous examples of RNAA procedures for these matrices and other types of matrix can be found in textbooks and reviews (the biannual reviews published in Analytical Chemistry by Ehmann and coworkers until 1994 are particularly valuable). Since RNAA is mostly employed for the certification of biological and environmental RMs, it can be mentioned that reliable procedures are available at the major RM producers and co-operating laboratories for determining very low levels of the elements As, Cd, Co, Cu, Cr, Hg, I, Mn, Mo, Ni, Sb, Se, Sn, Th, Tl, U, V, and Zn in such materials. As an example of the capabilities of a combination of INAA and RNAA using a two group separation scheme for the simultaneous determination of As, Cd, Cu, Mo, Sb, Hg, and Se (ICutera and Soultal1993), and a single separation procedure for vanadium determination (Byme and Kurera 1991),Table 3.8 shows results for NISTSRM-1573a Tomato Leaves that were obtained prior to certification of this material (Icutera 19951, and later on taken into consideration for deriving the NIST certified and information values (Becker 1995, personal communication). The advantageous features of NAA are well recognized by the major producers of RMs and result in the frequent, even dominant, use of the technique, as can be inferred from RM certificates. Table 3.9 shows that the percentage of elements certified in selected environmental and biological RMs produced by the US National Institute of Standards and Technology (NIST, formerly National Bureau of Standards - NBS) and by the Standards, Measurements and Testing Programme of the European Commission (SM&T, formerly Community Bureau of Reference-BCR) with the aid of INAA and RNAA methods is in the range of 50-86 %. This clearly exceeds the share of any other trace element analytical technique. The share would even be higher if non-certified elements are considered, and if prompt gamma neutron activation analysis (PGNAA) capable of measuring matrix elements, such as C, C1, H, K, N, Na, and traces of elements like B and Cd, were also included (Lindstrom 1998). It may also be inferred from Table 3.9 that the use of NAA slightly increased in the last two decades even though several other trace element analytical techniques improved dramatically and became highly competitive during this period. Examples

3.2 Cert$cotion of Elements

Tab. 3.8

INAA and RNAA results for NlST SRM-iyja Tomato Leaves (dry weight) (Kutera 1995)

Ba, mgjkg

Zn,mglkg

Method

Mean f uncertainty

INAA, 13’Ba INAA, I3lBa INAA INAA RNAA INAA INAA INAA INAA RNAA INAA INAA INAA RNAA INAA INAA INAA RNAA INAA INAA RNAA, 122Sb INAA, IZ4Sb INAA RNAA INAA INAA INAA INAA RNAA INAA

109.6 i 4.1 57.1 i 12.2 63.0 i 3.8 1189 * 33 4.970 f 0.143 1.55 * 0.06 6647 i 102 0.561 i0.021 1.95 * 0.03 51.6 i 5.1 4.64 * 0.14 36 * 3 369 * 17 128 i 4 33.4 i 1.2 2.681 t0.064 2.26 t0.06 247.1 t4.7 463 I 33 133t4 14.6 I 0.3 61.9 * 8.8 59.9 1 9 . 7 101 * 3 57.6 I 5.8 0.186 i- 0.015 80.4 t6.9 116 t6 0.81 t0.20 0.783 t0.057 30.1 I 1.1

11214 (63) (1300) 5.05 I 0.09 1.52 I 0.04 -

0.57 I 0.02 1.99 * 0.06 (53) 4.70 I 0.14 368 I 7 (‘140) 34t4 2.70 f 0.05 (2.3) 246 I 8 (460) 136 I 4 14.89 i 0.27 63 I 6 ( 100) 54+3 (0.19)

(85) (120) 0.835 f 0.01

30.9 I 0.7

are various methods of atomic absorption spectrometry (AAS), including solid sampling AAS, inductively coupled plasma optical emission spectrometry (ICP-OES), and ICP-mass spectrometry (ICP-MS), etc. From another point of view it may be noticed that the number of elements certified increased in newer RMs, especially in those prepared at NIST, and it may be considered that with the increasing number of certified elements, the share of NAA utilization in the certification process is also expanding.

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NIST SRM 1632 Coal NIST SRM 1633 Coal fly ash NIST SRM 1571 Orchard leaves NIST SRM 1577 Bovine liver NIST SRM 1632b Coal NIST SRM 16331, Coal fly ash NIST SRM 1570a Spinach leaves NIST SRM 1573a Tomato leaves NIST SRM 1577b Bovine liver BCR CRM 038 Coal fly ash BCR CRM 277 Estuarine sediment BCR CRM 101 Spruce needles 23 19 21 18 13 10 9

24

14 20 23 12

Number of elements certified

Yo) 57 50 78 58 67 65 79 86 72 85 60 78

As, Cd, Cr, Cu, Hg, Mn, V, Z n As, Cd, Cr, Cu, Fe, Hg, Na, Rb, Se, Th As, Cd, Cr, Cu, Fe, Hg, I<, Mg, Mn, Mo, Na, Ni, D, Rb, Sb, Se, U, Z n Fe, Hg, K, Mn, Na, Se, Zn Al, As, Ba, Ca, Co, Fe, K, Mg, Mn, Na, Rb, Se, Th, Ti, U, Zn Al,As, Ba, Ca, Cr, Fe, K, Mn, Na, Se, Sr, Th, Ti, U, V A], As, Ca, Co, Cu, Hg, I<, Mn, Na, Ni, Se, Sr, Th, V, Zn Al, As, Ca, Cd, Co, Cu, Cr, Hg, Fe, K, Mn, Na, Ni, Rb, Sb, Se, V, Zn Ag, Cd, C1, Cu, Fe, I<, Mg, Mn, Mo, Na, Rb, Se, Zn As, Cd, Co, Cr, Cu, F, Fe, Hg, Mn, Na, Z n As, Cr, Hg, Sc, Se, Zn Al, Ca, C1, Mg, Mn, P, Zn

Share of NAA in the certification Elements certified using INAA and RNAA

Use o f NAA in the certification ofelement contents in various environmental and biological RMs

Reference material

Tab. 3.9

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3.2 Cert$cation ofElemevrts

3.2.3.4 NAA for the Detection o f Errors

To illustrate the special position of NAA in RM certification, several examples are given where the consistent application of the technique’s advantages and internal QC features helped in resolving discrepant analytical data, even by retrospective evaluation of NAA results, as was shown in the certification of platinum in two automobile catalyst SRMs (Becker 1gg5), or even lead to corrections of originally (erroneously) certified values. Thus using both INAA and RNAA it has been proved (Damsgaard and Heydorn 1973; Heydorn 1980)that the originally certified value of As in NBS sRM-1571 Orchard Leaves of 14 + z mg/kg (US NBS 1971) was positively biased. This error was also noticed by other authors using NAA (Heydorn 1980, p. 200). Later a new certified value of 10 z mg/kg was issued (US NBS 1976). Recommended values of Cu and Mn in RM Milk Powder A-11 prepared by the International Atomic Energy Agency (IAEA) at the beginning of the 1980’s from an open inter-comparison were questioned (De Goeij et al. 1983),because there was disagreement among the mean values of two major techniques, namely NAA and AAS. The purely statistical approach to inter-comparison data was also criticized, the necessity for considering analyixal factors emphasized, and the special role which NAA can play in validating such data brought forward (Kosta and Byrne 1982). As a result, a follow-up study was organized in selected laboratories, again largely using NAA. New recommended values were derived in this study for the above elements with the mean values being 45 % and 68 % of the original ones (Byme et al. 1987). Even larger differences were found between the “old and “new” values for other elements, namely Cd, Cr, I, Mo, Ni, and Pb in IAEA RM A-11 Milk Powder (Table 3.10). and in IAEA RM H-4 Animal Musde, also included in the above follow-up study (Byme et al. 1987).Recently the Mn and V values in NIST SRM 1648 Urban Particulate Matter were updated (US NIST 1999) based on significantly lower values of both elements determined by INAA (Kurera and Soulal 1998) compared with the previous values (US NISTIggI). The determination of chromium in various matrices is frequently associated with severe difficulties attributed to contamination problems at very low element levels and to problems of complete dissolution, especially in environmental and geological materials. Thus destructive analytical methods may underestimate the total chromium values if inadequate dissolution techniques are employed (e.g. conventional wet-ashing procedures without HF), as was noticed when comparing INAA results with those obtained by destructive techniques (including RNAA) for chromium in NBS sRM-1569 Brewer’s Yeast and IAEA Calcinated Animal Bone A-3/1 (De Goeij et al. 1978), in three leaf US NIST SRMs (Lindstrom et al. ~ g g o )and , in IAEA Soil7 and SD/M/2/TM Marine Sediment inter-comparisons (Byrne 1993). However, even if HF is used for decomposition of botanical materials this may not be sufficient for the complete dissolution of chromium and other elements contained in siliceous or calcareous mineral matter (‘‘grif‘) (Lindstrom et al. 1990; Becker and Gills ~ g g s )and , moreover some trace elements may precipitate or co-precipitate, e.g. rare earth elements (Greenberg et al. 1990). Comparison of INAA and RNAA results (and the other QC options mentioned above) can often resolve these problems. Unlike most other analytical techniques, RNAA is also capable of providing

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3 Certijication Tab. 3.10 Comparison of old and new data for selected elements in IAEA A-11 Milk Powder (results in ng/g dry weight; number ofaccepted laboratory averages in brackets) (Byrne et al. 1987)

Element

Original intercomparison (Dybczynski et a/. 7980)" Overall mean t S D

New results Overall mean r SD

48 * 116 (8)

Cd co cr cu

Hg I

Mn Mo Ni Pb

526 f 683 (8) (Information value only) 5 k l(6) (recommended with acceptable confidence) 257+ 262 (11) (information value only) 838 f 450 (31) (recommended with acceptable confidence) 2.5 t 0.7 (6) (recommended with acceptable confidence) 1,469 t 1,548 (4) (information value only) 377 i 177 (21) (recommended with satisfactory confidence) 1,266 t 1,649 (information value only) 931 f 920 (8) (information value only) 267 -c 255 (8) (information value only)

1.7 + 0.2 ( 3 ) 4.5 f 0.8 (6) 17.7 + 3.8 (6) 378 t 31 (9) 3.2 2 0.6 (4) 87 i 6 (3) 257 f 6 92 f 9 (5) [54 f 28 ( 3 ) ] 54 * 10 ( 3 )

reliability of the quoted value according to the original evaluation

accurate results for extremely low levels of vanadium, such as in human serum (Byrne and Versieck 1990; Byrne and Kurera 19g1),and iodine in various biological and environmental matrices, e.g. RMs and diet (Dermelj et al. 1990; Poltorn et al. 199g), milk (Parr et al. ~ g g ~urine ) , (Dermelj et al. ~ g g z )etc. , where it provides reference values. 3.2.3.5 Summary

It can be concluded that, in the field of RM certification, NAA represents a major analytical technique contributing significantly to the certification of element contents in environmental and biological RMs, as was also pointed out earlier (Dybczynski 1980 1995).and also provides the bulk of the literature data on NIST SRMs (Gladney et al. 1987,1993). NAA also has a similar position in the field of geological RMs (Roelandts 1991). It possesses unique quality assurance and self-verification aspects (Bedter 1993; Byrne 1993; Byrne and ICuEera 1gg7), though these in-built features are rarely exploited in full at present. It should be realized that the advanta-

3.3 Certification of Organornetallic and Other Species

geous features of NAA can be employed and sufficiently accurate results obtained by NAA only if the technique, like any other analytical technique, is correctly and rigorously applied in a state of statistical control. This especially concerns RNAA (determination of the chemical yield in each measurement decreases the uncertainty of analytical results), which however requires sufficient skill and experience, especially when working with short-lived radionuclides. When a state of statistical control is reached, NAA can be regarded as a reference method (Heydorn 1991, 1995).Limitations of the technique include insufficient sensitivity for some important elements, especially Pb and Be, time restrictions dependent on half-lives of the radionuclides measured, the radiation burden of the personnel, and the necessity of access to a nuclear reactor. This latter aspect is becoming a serious concern in view of the ageing and shut-down of research reactors.

3.3

Certification of Organometallic and Other Species Rita Cornelis, Milena Horvat and Philippe Queuauuiller 3.3.1

Introduction

Today it has become clear that the effect of trace elements in living systems, in food, and in the environment depends on the chemical form in which the element enters the system and the final form in which it is present. The form, or species, clearly governs its biochemical and geochemical behaviour. IUPAC (the International Union for Pure and Applied Chemistry) has recently set guidelines for terms related to chemical speciation of trace elements (Templeton et al. 2000). Speciation, or the analytical activity of measuring the chemical species, is a relatively new scientific field. The procedures usually consist of two consecutive steps: (I) the separation of the species, and ( 2 ) their measurement. An evident handicap in speciation analysis is that the concentration of the individual species is far lower than the total elemental concentration so that an enrichment step is indispensable in many cases. Such a proliferation of steps in analytical procedure not only increases the danger of losses due to incomplete recovery, chemical instability of the species and adsorption to laboratory ware, but may also enhance the risk of contamination from reagents and equipment. For the high accuracy needed in the quantitative measurement of the species, quality assurance of the analytical procedures is of prime importance. This can only be achieved by using representative RMs, certified for the relevant species. Up to now the number of existing certified reference materials is very limited. This section will give a survey of the main species that are presently determined routinely or for research purposes.

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3 Certijication 3.3.2 Potential Sources o f Error in Speciation Analysis

These can be mainly at the level of: sampling of the specimens, separation of the species, including sample pre-treatment, and (3) measurement (Quevauvilleret al. 1995). (I)

(2)

Collection of a representative sample with preservation of the species is the first challenge. General guidelines fashioned to the matrix and their trace element species would be most welcome to the scientific community. The next step, that of the sample pretreatment, envisages the isolation of the species from the interfering matrix. For example, organometallic species, such as TBT (tributyltin) compounds, have to be quantitatively extracted from a sediment without loss or contamination, without any change in composition, and without interfering agents. This extract may then be concentrated and submitted to chromatographic separations. Derivatization is often used to separate some trace element species from their matrix, e.g. by the generation of volatile species which can be separated by gas chromatography. The method is based on the addition of a simple group (pentyl, ethyl, butyl, e.g. with Grignard reagents). Separation of the species is mainly by chromatographic and electrophoretic systems. In this way, tributyltin is separated from triphenyltin, dibutyltin, and inorganic arsenic species are differentiated from the methylated forms. The identification of the species is based on its retention time. At the end of the chain comes the element specific detector, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS),or radioactivity measurement when using radiotracers. A most interesting method to obtain accurate analyses for the “real time” concentrations of the chromatographic peaks is isotope dilution mass spectrometry (IDMS) (Heumann et al. 1998; Kingston et al. 1998). Two different spiking modes are possible, one using species-specific and another one using a species-unspecificspike solution of isotope-enriched labelled compounds. The speciesspecific mode is only possible for element species well defined in their structure and composition, e.g. bromate, chromate, and selenite, whereas the species-unspecific mode must be applied in all cases where the structure and the composition are unknown, for example for metal complexes with proteins or humic acids. For a number of well-known compounds, such as TBT, non-specific detectors such as FID, FDD, and ECD are used after LC or GS separation. It is evident that huge errors may occur during all these consecutive steps. The only way to validate the methodology is to have at hand suitable RMs certified for the trace element species content in the relevant matrix, together with suitable calibrants. Few of these are yet commercially available.

3.3 Certijication oforganometallic and Other Species

3.3.3 Restricted List of Chemical Species for Trace Elements and Their Compounds 3.3.3.1

Aluminum

In aquatic systems dissolved aluminum species are toxic to fish. There exists a vast number of aluminum species, ranging from inorganic monomeric to complex colloidal, polymeric and organic complexes. A major problem, when studying aluminum species in water is that the species quickly convert one into the other (Fairman and Sanz-Medel1gg5). Separation methods The methodology for their separation often consists of fractionation methods (see Section 3.3.4) and chromatographicmethods of the soluble species. Common detection methods are AAS, ICP-OES,and fluoride electrodemethods. Existing CRMs Although aluminum speciation in water has been studied extensively during the last decades, many problems have to be solved before the preparation of a water certified for aluminum species can be considered. First of all the species of aluminum in water need to be defined unequivocally. Then sampling and sample storage procedures have to be prescribed for the unknown samples to ensure the preservation of the species. As various ligands present in the RM are liable to interact with the aluminum species during the specific speciation procedure under investigation, the concentrations of the ligands in any future RM will have to be very precisely documented.

3.3.3.2

Antimony

Four species of antimony have been identified in natural waters: Sb(III), Sb(V), monomethylstibonic acid, and dimethylstibinic acid (the latter two are due to microbial activity)(DeLa Calle-Guntifias et al. 1995). In view of the high toxicity of antimony, a very low maximal admissible level of Sb in dnnking water is imposed (the EC maximum admissible level of Sb in drinking and surface water is 10pg/L). As Sb(II1) is more toxic than Sb(V) and inorganic species are more toxic than organic ones, a distinction between the different species becomes mandatory. Separation and detection methods There exists an array of methods for the separation of the Sb species, ranging from liquid-liquid extraction to the formation of complexes with immobilized reagents, followed by HPLC or reversed phase chromatography, and eletroanalytical methods. Detection is based on spectrophotometric measurement, AAS, ICP-OES and ICP-MS. ExistingCRMs

3.3.3.3

RMs or CRMs for antimony species are not available yet.

Arsenic

The arsenic compounds most commonly found in environmental and biological materials, and in working places, are arsenite and arsenate ions [As(III)and As(V)], monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsine, di- and tri-

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I methylarsine, and organoarsenicals such as arsenobetaine (AsB), arsenocholine 3 Cert9cation

(AsC), arsenolipids, and arsenosugars. The toxicity depends on the chemical form (Edmonds and Francesconi 1993).Only As(III), As(V),and arsine are considered to be violent poisons. The others are far less toxic or not toxic at all. In the case of liquid samples, e.g. water and serum, the separation of the species is relatively straightfonvard. The high molecular mass compounds are eliminated with the aid of a guard filter, and the low molecular arsenic species are separated by HPLC (Zhang et al. 1996). The analysis is more cumbersome when analyzing seafood or sediments, because the species have to be extracted and purified prior to separation. Besides liquid chromatography, capillary electrophoresis has become an interesting on-line option. Ideally the separation device is coupled to an element-specific detector for arsenic (AAS, after online transformation of the arsenic species through hydride generation, with prior digestion of the organo-arsenic compounds; OES and ICP-MS). Separation and detection methods

Currently available CRMs Presently available are CRM matrices (fish) with certified values for arsenobetaine and DMA, and calibrants for those compounds. The offer is, however, very limited. Future needs include CRMs certified for arsenosugars and calibrants for those compounds.

3.3.3.4

Bromine

Bromate in drinking water derives from ozonation treatment of bromide containing waters. Bromate, along with chlorite and chlorate, pose significant health risks even at very low levels (pg/l).Judging by the number of publications on the measurement of bromate in drinking water, there is a fast-growing interest in developing reliable methods. Separation and detection methods Ion chromatography is routinely used for the isolation of bromate. Diverse detection methods are mentioned in the literature, including isotope dilution analysis (Creed and Broclchoff 19g g), conductivity measurement (Jackson et al. 1gg8), fluorimetric determination (Gahr et al. 1gg8),ICP-MS (Seubert and Nowak 1998)and spectrophotometry (Achiliiand Romele 1999). Currently available CRMs Calibrants for bromate are easy to come by, but a drinking water certified for very low bromate concentrations is not yet available.

3.3.3.5

Chromium

The different toxicity and bioavailability of Cr(II1)and Cr(V1)are a public health concern and therefore require strict control. Cr(V1)is considered to be toxic and carcinogenic, especially for the respiratory tract. In occupational health, the OEL (Occupational Exposure Limits) for water soluble and certain water insoluble compounds in indoor air is set at 0.5 mg/m3 for Cr, 0.5 mg/m3 for Cr(III), and 0.05 mg/m3 for Cr(VI),reflecting the different toxicities of both species.

3.3 Certification ofOrgunornetullic and Other Species

The common methods used to separate the Cr(III)/(VI)species are solvent extraction, chromatography and coprecipitation. In case of Cr(V1) from welding fumes trapped on a filter, a suitable leaching of the Cr(V1)from the sample matrix is needed, without reducing the Cr(V1) species. The most used detection methods for chromium are graphite furnace AAS, chemiluminescence, electrochemical methods, ICP-MS, thermal ionization isotope dilution mass spectrometry and spectrophotometry (Vercoutere and Cornelis 1995). The separation of the two species is the most delicate part of the procedure. Separation and detection methods

Currently available CRMs for Cr(lll)/(VI) species There exist a lyophilized water certified for Cr(III)/Cr(VI)and a binder-free glass fiber filter loaded with welding dust certified for Cr(V1) and total Cr (Vercoutere et al. 1998; Christensen et al. 1999) issued by the BCR. They consist of a set of specimens for single use. There is a need for more CRMs, such as a Cr(V1) in industrial effluents and in river water containing, e.g. humic substances. 3.3.3.6

Mercury

In order to assess the impact of mercury on ecosystems and human health it is important to understand the overall mercury cycle in the environment (Fitzgerald and Clarkson 1991).Monomethylmercury (MeHg) is the most toxic Hg compound. MeHg it is mainly formed in the aquatic environment by biotic and/or abiotic processes. The accumulation of Hg in biota, and its biomagnification in aquatic food chains are of particular concern due to the element’s extreme toxicity and its ability to bioaccumulate in fish tissues. Therefore it is routinely measured in fish samples to control contamination levels prior to market sales. Recently, monitoring of MeHg contents in sediments started in research laboratories for the purpose of monitoring pollution and study Hg geochemistry (e.g. methylation/demethylation processes). Separation and detection methods The techniques developed for the determination of MeHg in air, water, biological and sediment materials involve, in most cases, a succession of analytical steps which may all be prone to systematic errors (Bloom et al. 1997; Horvat 1996). Ten years ago, these methods were poorly validated due to lack of evaluation programs and of certified RMs. The situation improved recently thanks to the development of more sensitive and specific analyixal techniques, the organization of inter-laboratory studies and the availability of CRMs from various producers. Separation and isolation of MeHg are normally performed by one or more of the following techniques: distillation, acid leaching followed by solvent extraction and/or ion-exchange,supercritical fluid extraction,alldine dissolution followed by solvent extraction,microwave digestion,derivatization,high performance liquid chromatography,and gas chromatography.A number of detection techniques are also employed such as CV (cold vapor) AAS, CV (atomic fluorescence spectrometny) (CVAFS), ICP-MS, gas chromatographyelectron capture detection (GC-ECD),etc. Currently available CRMs for MeHg There are a number of CRMs for total mercury in biological, sediment, and water samples (International Atomic Energy Agency and

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I United Nations Environment Programme 1995.1996).Unfortunately,there are only 3 Cert$cation

a

few biological samples from BCR, IAEA, NIST and NRCC and two sediment samples from BCRand IAEA-MEL certifiedfor MeHg compounds available (Horvat1999). It is well understood that these materials are not covering present needs, as most of them are of marine origin, while many laboratories are conducting research and monitoring in terrestrial ecosystems and fresh water environments. In addition, CRMs for human exposure assessment, such as blood, urine, and hair at several levels of concentrations are still lacking. In the absence of CRMs, many other actions should be undertaken to achieve, improve and/or maintain quality of data, including participation in inter-laboratory studies, proficiency testing, and production of laboratory RMs. Many of the trials have been organized to scrutinize the current MeHg techniques and the detection of the most obvious sources of systematic error, e.g. insufficient extraction, low distillation recovery, insufficient peak resolution, etc. (Donais et al. 1997; Horvat et al. 1994, 1997; Mee et al. 1992; Quevauviller 1997; Stone et al. 1995;Yoshinaga et al. 1997). So far, most of the RMs certified for MeHg were prepared by inter-comparisonof the results obtained by various analyticalprocedures employedby differentlaboratories. In the case of soluble samples such as fish, mussels, etc. the speciation analysis has been achieved most successfully (e.g. Quevauviller et al. Igg6a). In the case of solids, however, techniques to remove or solubilize MeHg are difficult to validate. When applying spiking or tracer approaches there is no proof that true exchange or equilibrium of the added species with the endogenous compounds occurred and hence it is difficult to prove that complete extraction/separation has been achieved (Byme 1992). A classical example of this difficulty is speciation of MeHg in sediments and soils. The only feasible strategy to be adopted in certification of MeHg was to use different analytical methodologies, i.e. various extraction/ separation schemes and detection methods. Good agreement between the results provides sufficient assurance that the data are meaningful. The possibility of artifacts, i.e. too high certified concentrations for MeHg is discussed in Chapter 7. However, from the present stage of knowledge it is obvious that artifacts are not too serious or even non-existent for most available CRMs (Quevauviller and Horvat 1999; Chemosphere 1999). 3.3.3.7 Lead The most important group of lead species is the alkyllead compounds, with tetraethyl lead, used as an antiknock agent in petrol, being the most prominent one (Lobinski et al. 1995).The tetraethyllead compounds degrade to tri-, di- and monoalkyllead compounds and to Pb2+.The harmful effects of organolead compounds are considered substantially exceed those of inorganic lead. Matrices that have been predominantly analyzed are air, water, dust, sediments and soils. Recently Pyrzynska (1996) published an interesting review on organolead speciation in environmental samples.

Very interesting hyphenated techniques have been developed for the separation and measurement of organic and inorganic lead spe-

Separation and detection methods

3.3 Certificationof Orgavlornetallic and Other Species

cies. They are essentially a combination of gas chromatography or HPLC of the derivatized compounds, coupled to an element sensitive detector, such as AAS, OES, or ICP-MS. Besides these techniques, there is differential pulse anodic stripping voltammetry that proved, however, to be of a limited use especially when applied to real samples. All methods appear very cumbersome, requiring a large amount of sample and tedious extraction and pre-concentration steps. The recovery of the organolead compounds is the most delicate step of the procedure. Currently available CRMs A number of organolead (tetra-and triallyllead) compounds are commercially available to be used as calibrants. There are also a number of interesting CRMs: an urban dust certified for trimethyllead and more appear to be in development. The interest in organolead compounds is, however, on the decline due to the gradual disappearance of the tetraethyl anti-knockagent from petrol.

3.3.3.8

Selenium

The selenium species that are drawing most attention are Se(1V)and Se(V1)in water and sediments, and the biomethylated products (dimethylselenide and dimethyldiselenide) that are spread into the environment (Camara et al. 1995). Se-species in food (including Se-cysteine and other species in yeast) are in the limelight (Crews 1998)because of their beneficial effect on human health and their increasing use as nutraceuticals. Separation and detection methods The analytical procedures to separate and measure the inorganic selenium species have been studied into great detail. Olivas et al. (1994) have published an interesting review on the analpcal techniques applied to the speciation of selenium in environmental matrices. The stability of the compounds is a major challenge in this speciation endeavor. The pre-concentration and separation methods can be very tedious (Camara et al. 1995).The element detection methods, following chromatographic separation,are AAS, hydride generation-AAS,and ICP-MS. Currently available CRMs Calibrants for inorganic selenium are available and also for some organic selenium compounds. As more selenium containing compounds appear to be important for their essentiality to man, there is a need for a larger choice of organic selenium containing species. CRMs certified for selenium species are not yet available, even not for inorganic selenium species in water. This is essentially due to the instability (conversion of one species into the other) in environmental samples, including water.

3.3.3.9

Tin

Butyl- and phenyltin compounds, particularly the trialkylated forms are very toxic to marine life. The antifouling paints, mainly tributyltin (TBT) but also triphenyltin (TPhT) caused and continue to cause substantial damage because of their slow biodegradation and their accumulation in the biota, notwithstanding a substantial reduction in application through multi-national regulation).

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3 Cert9cation Separation and detection methods A survey on determination of tin species in environmental samples has been published by Leroy et al. (1998).A more detailed overview of GS-MS methodology has been published by Morabito et al. (1995) and on sample preparation using supercritical fluid extraction has been described by Bayona (1995).The techniques are now under control, so that routine procedures are available at a relatively low cost (Leroy et al. 1998). Currently available CRMs Calibrants are available for TBT (very evident, as TBT is an anthropogenic contaminant) and for a number of other organic tin compounds. There is also an interesting choice of environmental CRMs (sediment and mussel tissue) certified for TBT content. 3.3.3.10

Metallothionein

Metallothioneins are a group of non-enzymatic, low-molecular mass (6-7 kDa) metal-binding proteins. They play an important role in the detoxification of a number (Zn, Cu, Cd, and Hg) of trace metals (Chassaigne and Lobinski 1998). Very refined chromatographic and electrophoretic separation techniques have been developed for metallothioneins. The detection is commonly based on the retention time and UV detection. Other researchers measured the element with e.g. ICP-MS to quantift. the compound. Combination with electrospray MS-MS leads to the unequivocal identification of the species. Separation and detection methods

Currently available CRMs

There exist a limited number of commercial calibrants of

animal metallothioneins. 3.3.4 Fractionation

When the matrix is very difficult to study and the separate species cannot be identified, scientists use fractionation procedures. These are processes of separation of an analyte or a group of analytes from a certain matrix according to physical (e.g. size, solubility) or chemical (e.g. bonding, reactivity) processes (Templeton et al. 2000). For pragmatic reasons the scientific community came to consensus on extraction schemes aiming at sequential solubility of metals bound to the specific substrate making up sediments and known to undergo changes in the yearly life cycle of lakes or rivers. Today the first materials have been certified and are available. More are due in the future (Quevauvilleret al. 1997). 3.3.5 Conclusions

It is clear that the available RMs certified for trace element species are not sufficient to cover present needs. As it is quite unlikely that producers of CRMs will ever be able to meet all demands, users are encouraged to produce their own RMs to guar-

3.4 Certification of Organic Substances

antee in-house quality assurance. It is understandable that protocols should be based on technically valid procedures, accompanied by proper documentation. Problems that need to be addressed in particular are connected to the question of the reliability of data for measurement in “difficulf‘samples (such as sediments, soils, plants, and organic rich water samples). A major share of elemental analysis will eventually evolve into speciation analysis. This point was clearly outlined in a special issue of Spectrochimica Acta (edited by Donard and Caruso 1998). The list of elements and their species listed above is not exhaustive. It is limited to the relatively simple compounds that have been determined by an important number of laboratories specializing in speciation analysis. Considering the economic importance of the results, time has come to invest in adequate CRMs. There is a steadily increasing interest in trace element species in food and in the gastrointestinal tract where the chemical form is the determinant factor for their bioavailability (Crews 1998).In clinical chemistry the relevance of trace elements will only be fully elucidated when the species and transformation of species in the living system have been measured (Cornelis 1996; Cornelis et al. 1998). Ultimately there will be a need for adequate RMs certified for the trace element species bound to large molecules, such as proteins. New developments are, however, needed to make a major step forward in the field of speciation analysis. The first part, isolation and separation of species, may be the easiest one to tackle. For the second part, the measurement of the trace element, a major improvement in sensitivity is needed. As the concentration of the different species lies far below that of the total concentration (species often occur at a mere ng/l level and below), it loolts like existing methods will never be able to cope with the new demands. A new physical principle will have to be explored, away from absorption spectrometry, emission spectrometry, mass spectrometry, and/or more powerful excitation sources than flame, arc or plasma will have to be developed. The goal is to develop ‘routine’ analytical set-ups with sensitivities that are three to six orders of magnitude lower than achieved hitherto.

3.4 Certification of Organic Substances Stephen A Wise andjurgenjacob

3.4.1 introduction

Certified reference materials (CRMs) to validate measurements of organic constituents were introduced in the early 1980’s, more than a decade after the development of the first natural matrix CRMs for inorganic constituents. There are three types of CRMs to support measurements of organic constituents:

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pure substances, calibration solutions, and (3) natural matrix materials with natural levels of organic constituents or fortified (i.e. spiked) with the analytes of interest.

(I)

(2)

The pure substances are provided with a statement of the purity and serve as authentic reference compounds to prepare calibration solutions for measurement of trace levels of the compound in natural matrix samples. The calibration solutions, which typically contain a number of analytes at known concentrations, are useful for validating the chromatographic separation step (e.g. retention times and analyte detector response). The natural matrix materials, which are similar to the actual environmental, clinical, food, or agricultural samples analyzed, are used to validate the complete analytical measurement process including extraction, cleanup and isolation procedures, and the final chromatographic separation, detection, and quantification. This Section will address the certification of CRMs for organic constituents based on the current practice of two CRM producers: (I) the National Institute of Standards and Technology (NIST) and (2) the Standards, Measurement and Testing program, formerly known as the Community Bureau of Reference (BCR) of the Commission of the European Union (EU) (Maier et al. 1997; Quevauviller 1997). This Section will focus on the certification approach used for natural matrix CRMs for the determination of organic contaminants in environmental samples (Wise 1993; Wise and Schantz 1997; Quevauviller and Maier 1998). The same general philosophy and approach applies also for the certification of trace constituents in clinical (Cohen et al. 1980; Welch et al. 1984),food (see e.g. Boenke 1997; Sharpless et al. 1999; Welch et al. zooo), and agricultural matrices; however, due to the limitations of space, examples in these areas will not be presented. In this Section the CRMs available from these two producers for measurements of environmental contaminants will be summarized briefly, and the analytical process used by each of these producers for development and certification of trace organic constituents in their CRMs will be described. 3.4.2 CRMs Available for Organic Constituents 3.4.2.1

Pure Substances

By far the largest number of pure substance CRMs are available from the BCR with the focus on environmentally relevant compounds. A total of 74 compounds with certified purities of greater than 99 % (the majority are 99.5 % or better) including polycyclic aromatic hydrocarbons (36 compounds), heterocyclic aromatic compounds (aza-, thio-, 0x0-arenes; 18 compounds), nitro-arenes (8 compounds), polychlorinated biphenyls (7 compounds), and aldehydes as 2,4-dinitrophenyl hydrazones (5 compounds). These compounds were selected on the basis of the following criteria: (I) environmental and occupational importance, ( 2 ) biological significance (carcinogenic and/or mutagenic potential), ( 3 ) analytical needs, and (4) non-avail-

3.4 Certification of Organic Substances

ability in high purity from commercial companies. Their preparation, properties, and toxicological data have been reviewed (e.g. see Jacob et al. 1984, 1986, 1991, 1994) and the certification of the purity documented in BCR reports (e.g. Karcher et al. 1980; Jacob et al. 1985; Belliardo et al. 1988). The approach of the BCR to the certification of purity is described in detail in these reports. A limited number of pure substances are available from NIST, primarily c h i cally-relevant compounds such as cholesterol, urea, uric acid, creatinine, glucose, cortisol, tripalmitin, and bilirubin (NIST SRM website). These compounds are certified for purity (greater than 9 9 %) and are used as primary calibrants in definitive methods for these clinical analytes (see below). Several additional pure substances are available for specific applications such as microchemistry, i.e. elemental composition (acetanilide, anisic acid, cystine nicotinic acid, o-bromobenzoic acid, p-fluorobenzoic acid, Mz-chlorobenzoic acid), polarimetric standards (sucrose and dextrose), acidimetric standard (benzoic acid and boric acid). Only three pure substance NIST RMs are available for environmental contaminants, namely the chlorinated pesticides, lindane, 4,4’-DDT, and 4,4’-DDE. 3.4.2.2 Calibration Solution CRMs

The calibration solution CRMs are useful for several purposes including: (I) calibration of chromatographic instrumentation for retention times and detector response factors for quantification, (2)spiking or fortifying samples, and (3) analyte recovery studies. NIST currently offers approximately 20 calibration solution SRMs for organic constituents, which are primarily for environmental contaminants (Wise et al. 2000). These solution SRMs typically contain 5-20 compounds and include the following classes of organic contaminants: polycyclic aromatic hydrocarbons (PAHs), mono-,and dinitro-substituted PAHs, polychlorinated biphenyl (PCB) congeners (including three coplanar PCB congeners), chlorinated pesticides, aliphatic hydrocarbons, phenols, halocarbons, and perdeuterated PAHs (for use as internal standards). As part of a new program in support of the externalization of the U.S. Environmental Protection Agency’s (EPA) Water Proficiency Testing Studies, NI ST is preparing a number of new calibration solution SRMs, which will be available in late 2000 and early 2001. These solution SRMs will include six different aroclors in methanol and transformer oil, toxaphene and total chlordane in methanol, chlorinated herbicides in methanol, chlorinated pesticides in acetone, haloacetic acids in methyl-t-butylether, 2,3,7,8-tetrachlorodibenzo-p-dioxin in methanol, endothall, glyphosphate, and diquat dibromide in water, chloral hydrate in methanol, carbarnates in acetonitrile, and adipate and phthalates in methanol. The BCR currently offers a limited number of calibration solutions including CRM 365 Polychlorinated biphenyls in iso-octane (ten congeners) and CRM 551 and CRM 552 Aldehydes as z,4-dinitrophenyl hydrazones in acetonitrile (four compounds). 3.4.2.3

Natural Matrix SRMs

Validation of the complete analytical procedure (including solvent extraction, cleanup of the extracts, isolation of the analytes of interest, and chromatographic separation and detection) requires the use of CRMs with matrices similar to those

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typically encountered in the analysis of actual samples. Thus, the natural matrix CRMs are the most suitable materials for this purpose. A wide variety of natural matrix CRMs are available from both NIST and BCR for measurements of contaminants in environmental matrices, clinical analytes in human serum and urine, and vitamins and other nutrients in food matrices. CRMs for Contaminants in Environmental Matrices For nearly two decades NIST has been involved in the development of SRMs for the determination of organic contaminants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs),and chlorinated pesticides in natural environmental matrices such as fossil fuels (Hertz et al.1980; Kline et al. 1985), air and diesel particulate material (May and Wise 1984; Wise et al. zooo), coal tar (Wise et al. 1988a), sediment (Schantz et al. 1990, 1g95a; Wise et al. 1995). mussel tissue (Wise et al. 1991; Schantz et al. 1997a), fish oil, and whale blubber (Schantz et al. 1995b). Several papers have reviewed and summarized the development of these environmental matrix SRMs (Wise et al. 1988b; Wise 1993; Wise and Schantz 1997; Wise et al. 2000). Seventeen natural matrix SRMs for the determination of organic contaminants are currently available from NIST with certified and reference concentrations primarily for PAHs, PCBs, chlorinated pesticides, polychlorinated dibenzo-p-dioxins (PCDDs),and polychlorinated dibenzofurans (PCDFs);see Table 3.11. The BCR has also produced a large number of environmental matrix CRMs for PAHs, PCBs, pesticides, and PCDDs/PCDFs as shown in Table 3.12. These matrices include both natural contaminant level matrices as well as natural matrices spiked with low and high levels of contaminants. When viewed together the NIST and BCR CRMs provide a wide range of environmentalmatrices in which a considerablenumber of analytes have been assigned certified and reference values.

see also Section 6.2. NIST and BCR provide a number of CRMs for trace level clinical analytes in human serum and urine. NIST has issued several human serum SRMs with certified values for cholesterol (SRM 1951a, SRM 1952a, and SRM 909b); glucose (SRM 965 and SRM 909b); creatinine, urea, and uric acid (SRM 909b); fat-solublevitamins and carotenoids (SRM 968c). The BCR also has CRMs for some of these same clinical analytes, e.g. creatinine (CRMs 573-575). as well as additional analytes such as the hormones cortisol (CRMs 192-193), progesterone (CRM 347), and 170-estradiol (CRMs 576-578). NIST has four SRMs for drugs of abuse in urine (SRM 1508 Cocaine and Metabolites; SRM 1511 Multi-Drugs of Abuse; SRM 2381 Morphine and Codeine; and SRM 2382 Morphine Glucuronide) and Cotinine in Urine (RM 8444). Clinical Analytes in Serum and Urine

see also Section 6.3. Food matrices are available with values assigned for vitamins, carotenoids, fatty acids, cholesterol, natural toxins, veterinary drugs, and hormone residues. The NIST food matrix SRMs for vitamins include coconut oil (SRM 1563), infant formula (SRM 1846), and baby food composite (SRM 2383) (particularly for carotenoids). Fatty acids and cholesterol are the primary analytes of interest in meat homogenate (SRM 1546) and diet

Vitamins and Other Nutrients in Food Matrices

3.4 Certijcation af Organic Substances 3.11 NlSTnatural matrix SRMs for the determination of organic contaminants in environmental samples

Tab.

SRM No.

Title

1580 Organics in Shale Oil 1582 Petroleum Crude Oil I588a Organics in Cod Liver Oil I58ga PCBs in Human Seium

Certified Valuesa

Reference (Noncertified] Values”

PAHs (5); Phenols (3) PAHs (6) PCBs (24);Pesticides(14) PCBs (16) Pesticides (5)

Phenols (6) PAHs (5); Phenols (2) PCBs (34); Pesticides ( 3 ) PCBs (13); Pesticides (5); PCDDs/PCDFs (IS) PAHs (18)

1597

Complex Mixture of PAHs PAHs (12) from Coal Tar PAHs (22). PCBs (35) 1649a Urban Dust Pesticides (8) 1650a Diesel Particulate Matter PAHs (16);Nitro-PAHs (I) PCBs (20);Pesticides (3) 1939a PCB Congeners in River Sediment PAHs (23) PCBs (21) 1941a Organics in Marine Pesticides (6) Sediment 1944 NY/NJ Waterway Sediment PAHs (24); PCBs (35) Pesticides (4) 1945 Organics in Whale Blubber PCBs (27); Pesticides (IS) 1974a Organics in Mussel Tissue PAHs (IS);PCBs (20) Pesticides (7) 1975 Diesel Particulate Extract PAHs (8) 2974 Organics in Freeze-Dried PAHs (14);PCBs (20) Mussel Tissue Pesticides (7) 2975 Diesel Particulate Matter PAHs (11) (Industrial Forklift) PAHs (14);PCBs (25) 2977 Mussel Tissue (Organic Pesticides (7) Contaminants and Trace Elements) 2978 Mussel Tissue (Organic PAHs (7),PCBs (22) Contaminants Raritan Pesticides (12) Bay, NJ)

PAHs (22); Pesticide (I) PCDDs/PCDFs (17) PAHs (25); Nitro-PAHs (3) PCBs (4) PAHs (14);PCBs(7) Pesticides (4) PAHs (32); PCDDs/PCDFs (17) Pesticides (7) PCBs (2);Pesticides (2) PAHs (IS);PCBs (4) Pesticides (4) PAH (23); Nitro-PAHs (18) PAHs (17);PCBs (4) Pesticides (4) PAHs (28) PAHs (16)

PAHs (zo),PCBs

(2)

a) Numbers in parentheses indicate the number of analytes in each

class for which certified or reference values are provided.

composite (SRM 1544).BCR CRMs for vitamins and carotenoids include wholemeal flour (CRM I ~ I )margarine , (CRM 122), milk powder (CRM 421), brussel sprouts (CRM 431), mixed vegetables (CRM 485), and pig’s liver (CRM 487). Values for fatty acids are provided for four BCR matrices including anhydrous milk fat (CRM 164), soya-maize oil blend (SRM 162),beef-pig fat oil blend (CRM 163). and anhydrous butter fat (CRM 519), BCR CRMs are also available for veterinary drugs and hormone residues in bovine urine (CRMs 386-391), chloramphenicol in porcine muscle (CRM 444-445), aflatoxin MI in whole milk (CRMs 282-285), and aflatoxin B1 in peanut meal (CRMs 262-264) and compound feed (CRMs 375-376).

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3 Certijkation BCR natural matrix CRMs for the determination of organic contaminants in environmental and biological samples

Tab. 3.12

CRM No.

088 1'5 187 I88 349 350 392 420 430 449

450 481 490 458 459 524 532 533 534 535 536

598 607

Material

Certijied values"

Noncertifed values"

Sewage sludge Organochlorine pesticides in animal feed Organochlorine pesticides in milk powder (low) Organochlorine pesticides in milk powder (high) Cod liver oil

PAHs (8) Pesticides (10) Pesticides (4) Pesticides (9) PCBs (6)

Pesticides

Mackerel oil Sewage sludge Waste mineral oil (lower level) Organochlorines in pork fat Waste mineral oil (high level) Milk powder Industrial soil Fly ash Coconut oil (spiked) Coconut oil (natural) Contaminated industrial soil Milk powder (natural, unspiked) Milk powder (spiked, lower level) Milk powder (spiked, higher level) Freshwater harbour sediment Freshwater harbour sediment Organochlorine pesticides in cod liver oil Natural spray dried milk powder

PCBs (6) PCBs (6) PCBs (5) Pesticides (9) PCBs (10) PCBs (6) PCBs (8) PCDD/PCDFs PAHs (6)

(2)

PCBs (4) PCBs (3) Pesticides

(I)

(12)

PAHs (6) PAHs (9)

PCDD/PCDFs (11) PCDD/PCDFs (11) PCDD/PCDFs (11)

PAHs (7) PCBs (13) Pesticides (13) PCDD/PCDFs (11)

a) Numbers in parentheses indicate the number of analytes in each

class for which certified or reference values are provided.

3.4.3

Certification Approach for Organic Constituents

There are many similarities in the certification approaches used by NIST and BCR as well as major differences. Both the NIST and BCR approaches are based on results from multiple independent methods. For the NIST SRMs the measurements are generally all performed at NIST with emphasis on the independence of the methods, whereas the BCR relies on results from a large number of laboratories that may use different methods. The certification approaches used by NIST and BCR are described below using as an example the measurement of PAHs in an environmental matrix.

3.4 Cert@catron of Organic Substances

3.4.3.1

NISTApproach for Certification

Historically, NIST has used three basic modes for “certification”of chemical composition SRMs: (I) measurements using a “definitive or primary” method, i.e. a method of high precision for which all sources of bias have been rigorously investigated (see e.g. Cohen et al. 1980;Moody and Epstein r g g ~ ) (, 2 ) measurements using two or more independent and reliable methods, and (3) measurements from a number of laboratories participating in a multi-laboratorycomparison exercise, i.e. a round robin study. The basic approach of these modes as specifically applied to the value assignment for inorganic constituents in natural matrices has been described previously (Epstein 1991). Recently NIST re-examined the various approaches used for value assignment of chemical composition in SRMs and clearly defined the terms used to describe the composition values assigned to the SRMs (May et al. 2000). Three types of assigned values were defined certified, reference, and information (May et al. 2000):

Certijed value: A value reported on an SRM Certificate of Analysis for which NIST has the highest confidence in its accuracy in that all known or suspected sources of bias have been fully investigated or accounted for by NIST. The value has an associated uncertainty that generally specifies a range within which the true value is expected to lie at a level of confidence of approximately 95;% if the sample is homogeneous or the uncertainty represents a prediction interval within which the true values of 95 % of all samples are expected to lie at a stated level of confidence if significant sample heterogeneity is included. Reference value: A value reported on an SRM Certificate of Analysis and/or a Reference Material (RM) Report of Investigation that represents the best estimate of the true value where all known or suspected sources of bias have not been fully investigated by NIST. The value has an associated uncertainty that may not include all sources of uncertainty and may represent only a measure of the precision of the measurement method(s). Information value: A value that will be of use to the SRM/RM user, but insufficient information is available to NIST to assess adequately the uncertainty associated with the value. Typically the value will be provided with no uncertainty listed. The three historical approaches to certification mentioned above were recently expanded to identify seven modes that are used at NIST for value assignment for chemical composition (May et al. 2000). These seven modes and the resulting values are summarized in Table 3.13. The basic principles of value assignment remain unchanged however, these modes now provide a well-defined link between the process used for value assignment and the definition of the assigned value (i.e. certified, reference, or information value). The terms described above provide a clear indication of the level of confidence that NIST has in the accuracy of the assigned value. The definition of a certified value implies that NIST must be involved in the measurement process for the value to be classified as a NIST certified value (see modes 1-3 in Table 3.13). Thus, modes 4 and 7, which do not involve NIST measure-

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3 CertFcation Tab. 3.13

Modes used at NlSTfor value assignment o f R M s for chemical composition Certified value

Mode of value assignment

Reference value

Information value

I.

Certificationat NISTusing a primary method with confmation by other method(s)

X

2.

Certification at NISTusing two independent, critically-evaluatedmethods

X

X

X

X

X

4. Value assignment based on measurement of two or more outside collaborating laboratories using different methods

X

X

5. Value assignment based on a method dependent

X

X

X

X

X

X

3. Certificationjvalueassignment using one method at NISTand different methods by outside collaborating laboratories

(procedurally-defined technique)

6. Value assignment based on a NISTmeasurement by a single method (but does not meet the criteria for certification) and/or outside collaboratinglaboratory measurements using a single method

7. Value assignment based on selected data from interlaboratory studies

ments, result in reference values, even though these modes may involve independent analytical methods. In some cases the reference values may be of the same quality relative to the accuracy as a certified value; however, because all sources of bias have not been thoroughly investigated by NIST, the value is designated as a reference value. The BCR (see discussion below), as well as several other organizations that provide CRMs, use results from inter-laboratoryexercises to assign certified values to their natural matrix materials; see also Section 3.1. NISTalso occasionally uses results from inter-laboratorystudies to assign values (mode 7 in Table 3.13); however, in the NIST definition of terms such values are denoted as reference values in NIST SRMs to indicate that NISTwas not involved in malting any measurements that contributed to the value assignment. The use of a definitive method for certification of organic constituents (mode I in Table 3.13) has only been used at NIST for the determination of a limited number of clinically-importantanalytes in serum including cholesterol, glucose, creatinine, urea, uric acid, and triglycerides (Cohen et al. 1980; Ellerbe et al. 1989, 1990, 1995; Welch et al. 1984, 1986; White et al. 1982).These definitive methods are based on the use of gas chromatography-isotope dilution mass spectrometry (GC-IDMS) using 13C-labelledinternal standards for each of the analytes measured. The development and use of these definitive methods for these clinical ana-

3.4 Certification oforganic Substances

lytes are described in detail elsewhere (Cohen et al. 1980; Ellerbe et al. 1989, 1990, 1995; Welch et al. 1984, 1986; White et al. 1982). Similar approaches using isotopically labelled internal standards for GC/MS analyses are used for value assignment of a large number of organic contaminants in environmental matrices (see discussion below); however these methods have not been considered as definitive methods because there is not an isotopically-labelled analog used for each analyte, resulting in potential variability in recovery that is unmeasureable. The principal mode used at NIST for certification of natural matrix SRMs for organic constituents has been the analysis of the material using two or more “chemically independent? analytical techniques (mode 2 in Table 3.13). The results of these multiple technique analyses, if in agreement, are used to determine the certified concentrations for the measured analytes. The requirement for using two or more analytical techniques is based on the assumption that the agreement of the results from the independent methods minimizes the possibility of biases within the analytical methods. When results are obtained from only one analytical technique (or multiple techniques that are not sufficiently independent), the concentrations are typically reported as reference values (mode 6 in Table 3.13) and are considered as a best estimate of the true value where all known or suspected sources of bias have not been fully investigated by NIST. As the need increases for more natural matrix SRMs with values assigned for a wider variety of analytes, NIST will need to rely more on data provided by outside laboratories (modes 3, 4, and 7 in Table 3.13) to maximize its resources. Recent certifications of natural matrix SRMs have included the use of selected results from multi-laboratory comparison exercises as an additional data set in the assignment of the certified and reference values. 3.4.3.2

NET Analytical Approach for the Certification of Organic Constituents in Natural Matrix SRMs

As stated above the approach to value assignment for organic constituents in environmental matrix SRMs at NIST is generally based on the use of two or more independent analytical techniques. The independence of the analytical techniques should be reflected in all aspects of the procedure including sample extraction; cleanup of the extract; and finally the separation, detection, and quantification of the analytes of interest. The first environmental matrix SRMs with certified values for PAHs were issued in the early 1980’susing this basic approach with gas chromatography with flame ionization detection (GC-FID)or gas chromatography with mass spectrometric detection (GC/MS) and reversed-phase liquid chromatography with fluorescence detection (LC-FL) as the two principal analytical techniques (Hertz et al. 1980; Kline et al. 1985; May and Wise 1985; Schantz et al. 1990; Wise et al. 1991).The first natural matrix SRMs with certified values assigned for individual PCB congeners and pesticides were not issued until 1989.The improvement of the analytical techniques and the evolution of the analytical approach for value assignment for PAHs, PCBs, and chlorinated pesticides from 1980 to 1993 have been summarized previously (Wise 1993;Wise and Schantz 1997).In both of these publications, the basis of the independent techniques approach for PAHs, PCBs, and pesticides was discussed in detail. In this section the recent certification of SRM

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I 1649a Urban Dust is described as an example of the current analytical approach 3 Certification

used for the value assignment of PAHs in an environmental matrix. Analytical Approach for the Determination of PAHs For the determination of PAHs in the natural matrix SRMs listed in Table 3.11,various combinations of reversed-phase LC-FL and GC/MS have been used to provide the necessary two or more independent analybcal techniques. For the natural matrix SRMs issued through the 1980’s, NIST typically provided certified concentrations for only 5-12 PAHs; an additional 5-25 PAHs were often listed as non-certified (now denoted as reference) concentrations primarily because of the lack of measurements by the required second independent analytical technique (Hertz et al. 1980; Kline et al. 1985; May and Wise 1984; Schantz et al. 1990; Wise et al. 1991). These SRMs represented the “first generation” of natural matrix SRMs for organic contaminants developed at NIST. In the mid-1990’s two renewal SRMs, SRM 1941a Organics in Marine Sediment (Wise et al. 1995) and SRM 1g74a Organics in Mussel Tissue (Mytilus edulis) (Schantz et al. 1997a) were issued with a greater number of certified and reference values for PAHs. To provide value assignment for this greater number of PAHs, four different analytical techniques were implemented: (I) reversed-phase LC-FL analysis of the total PAH fraction, (2) reversed-phase LC-FL analysis of isomeric PAH fractions isolated by normal-phase LC (i.e. multi-dimensional LC), (3) GC/MS analysis of the PAH fraction on a 5 % phenyl methylpolysiloxane stationary phase, which is the typical GC stationary phase used for PAH analyses, and (4) GC/MS of the PAH fraction on a smectic liquid crystalline stationary phase which provides excellent selectivity for the separation of PAH isomers. A detailed discussion of this approach has been reported for the certification of PAHs in SRM 1941a (Wise et al. 1995). SRMs 1941a and SRM 1g74a were the first materials that could be considered as the second generation of natural matrix SRMs for organic contaminants at NIST. The basic analytical approach for the value assignment of these two SRMs was expanded recently for the recertification of SRMs 1649a (Wise et al. zooo), with the addition of a new extraction procedure, i.e. pressurized fluid extraction (PFE),and the use of a new GC stationary phase with different separation selectivity (50 % phenyl methylpolysiloxane) compared to the 5 % phenyl phase and the smectic liquid crystalline phase. The certification of SRM 164ga is described below in detail as an example of the current approach for the determination of PAHs in environmental matrix SRMs. The analytical scheme used for the certification measurements for PAHs in SRM 164ga is illustrated in Figure 3.2. A total of seven sets of results from different methods for the determination of the PAHs were used for assignment of the certified values. (Typically only two to three data sets are available for value assignment.) These seven data sets for SRM 1649a are not completely independent of each other, but are a combination of several independent procedures in each of the steps in the analytical process, i.e. extraction, cleanup, separation, and detection. The assumption is made that each independent procedure has different sources of bias (if biases exist); therefore, if the final results are in agreement, it suggests that none of the procedures have significant biases.

SPE

SPE

Q C M S (11)

5% phenyl methyl polyslloxane

Total PAH

NPLC (amlnopropylsllane)

GCMS (111) 50% phenyl methyl polysiloxam

NPLC (aminopropylsllane) Total PAH

SPE

=ms (W

!i% phenyl methyl polyshxane

NPLC (amlnopropyloilane) Total PAH

SPE

1

PFE DCM, HexlAce,CHjCN

1

PFE DCY

-

4

6 bottles l g

*

6 bottles 0.4 Ig

Fig. 3.2 Analytical scheme used for the certification of PAHs in NlST SRM 1649a Urban Dust.

GCMS (I) Q C W (sm) 5% phenyl methyl smectlc liquid uystalll~ polyslloxane

NPLC (amlnopropylsllane) Total PAH

1

1

Soxhlet DCH

1

SOxMet DCY

.c

6 bottles 1g

10 bottles (duplicates) l g

*

SRM 1649a, Urban Dust

LC-FL (Total)

1

LC-FL (Fractions)

+I

Y

3

5 2 -

v,

0

ss.

9

a'

9 ;5 3 f?

Isomer Fractlons

1

2

(amlnopropyhilane)

SPE

i

1

Soxhlet 50% Hexand 50% Acetone

1

38

1

6 bottles

94

I Two different solvent extraction procedures (Soxhlet extraction and 3 Certification

PFE) with three different solvent systems (i.e. dichloromethane, 50 % n-hexane/50% acetone, and acetonitrile) were used to extract the air particulate material (see Figure 3.2). The traditional Soxhlet extraction procedure was used for five of the data sets and PFE, a recently developed alternative extraction procedure using high temperature ( IOO~ 0 0 ° C )and high pressure (IOOO-2200 psi; 6.90-15.2 MPa), was used for the remaining two sets of results. Prior to the incorporation of PFE as part of the certification measurements, (Schantz et al. 1gg7b) evaluated and validated the use of PFE for the extraction of PAHs, PCBs, and pesticides from environmental matrices using several SRMs and CRMs including marine sediment, air and diesel particulate matter, mussel tissue, and fish tissue. In this study they verified that the results obtained with PFE were comparable to those obtained using the traditional Soxhlet extraction procedure. Sample cleanup of the solvent extracts of SRM 1649a consisted of solid phase extraction (SPE) on an aminopropylsilane cartridge followed by either direct analysis [in the case of LC-FL (Total)]or further cleanup using normalphase liquid chromatography (NPLC) on a semi-preparative aminopropylsilane column to isolate either a total PAH fraction for GC/MS analysis or isomer fractions for LC-FL analysis (see Figure 3.2). The most significant differences (i.e. independence) in the analytical methods are provided in the final chromatographic separation and detection step using GC/ MS and LC-FL. GC and reversed-phase LC provide significantly different separation mechanisms for PAHs and thus provide the independence required in the separation. The use of mass spectrometry (MS) for the GC detection and fluorescence spectroscopy for the LC detection provide further independence in the methods, e.g. MS can not differentiate among PAH isomers whereas fluorescence spectroscopy often can. For the GC/MS analyses the 5 % phenyl methylpolysiloxane phase has been a commonly used phase for the separation of PAHs; however, several important PAH isomers are not completely resolved on this phase, i.e. chrysene and triphenylene, benzo[b]fluoranthene and benzoulfluoranthene, and dibenz[a,h]anthracene and dibenz[a,c]anthracene.To achieve separation of these isomers, GC/MS analyses were also performed using two other phases with different selectivity, a 5 0 % phenyl methylpolysiloxane phase and a smectic liquid crystalline phase. In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1gg3a).To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977; May and Wise 1984; Wise et al. 1gg3a, ~ g g j b )consists , of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their allyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers.

3.4 Certijcation of Organic Substances

In both the GC/MS and the LC-FL analyses selected perdeuterated PAHs were added to the samples prior to extraction for use as internal standards for quantification. The results of the analyses of SRM 1649a using the different methods illustrated in Figure 3.2 are summarized in Table 3.14 for selected PAHs. These results were then combined to obtain a weighted mean using the approach of Schiller and Eberhardt (1991).The uncertainty associated with the certified value is an expanded uncertainty at the 95 % level of confidence which includes random sources of uncertainty within each analytical method as well as uncertainty due to the drying study. The results in Table 3.14 are typical of the agreement of results from different methods for the determination of PAHs in environmental matrix SRMs. The relative uncertainties of the certified values for PAHs in SRM 1649a range from 2 % to 24 % with the majority of the uncertainties in the 5 % to 10% range, which is typical for most of the certified values for organic constituents in natural matrix SRMs. Statistical Evaluation of Results

Homogeneity Assessment As part of the certification measurements, a homogeneity study is incorporated as one of the analytical data sets. For a natural environmental matrix SRM the homogeneity assessment typically consists of analyses of duplicate sub-samples from 8-10 bottles of the material, which have been selected from throughout the bottling sequence. If the sample is found to be homogeneous then the uncertainty of certified value is expressed as a 95 % confidence interval. If significant heterogeneity is observed then the uncertainty is expressed as a 95 % prediction interval. Stability Assessment In general there is no formal stability study prior to the certification of a natural matrix SRM. However, the stability of the certified analytesis monitored on a regular basis, typically every 1-3 years depending on the analytes, as the SRMs are analyzed as control samples during the analyses of similar matrix samples. A recent study of PAHs in frozen mussel tissue over nearly 10years found no significant changes in the concentrationsofthe measured PAHs (Schantzet al. 2000). Use of Other Modes of Certification As described above the primary mode of value assignment at NIST for organic constituents in natural matrices has been the use of two or more independent analytical methods (mode 2 in Table 3.13). However, as the need for more SRM matrices and/or SRMs with values assigned for different analyte classes increases, NIST will have to rely on the expertise of other laboratories to assist in the value assignment process by providing measurements. The recent value assignment of concentrations for the seventeen 2,3,7&substituted polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners and total tetra-, penta-, hexa-, and hepta-substituted congeners of PCDDs and PCDFs in SRM 1944 and SRM 1649a is an excellent example of the use of results from outside collaborating laboratories. Because NIST did not have the expertise necessary to provide measurements for PCDDs and PCDFs, NIST and Environment Canada coordi-

I

95

6.45 (0.27) 5.38 (0.08) 2.25 (0.11)

1.87 (0.04) 2.39 (0.01) 3.21 (0.13) 3.72 (0.13) 0.303 (0.004)

0.331 (0.005) 0.432 (0.007)

6.32 (0.05) 5.05 (0.04) 2.14 (0.03)

1.90 (0.04) 2.49 (0.04) 3.17 (0.06) 4.07 (0.04) 0.324 (0.018)

0.325 (0.027) 0.416 (0.018)

2.21 (0.05)

1.89 (0.06) 2.44 (0.03) 2.46 (0.14) 3.10 (0.16) 0.277 (0.013)

0.306 (0.017) 0.421 (0.024)

(DB-51

CC/MS (ill)

6.59 (0.11) 5.50 (0.28)

CC/MS (If) (DB-5)

6.48 0.25) 1.94 (0.04) 2.57 (0.06) 3.44 (0.11) 4.36 (0.11) 0.321 (0.008) 0.181 (0,010) 0.265 (0.013) 0.320 (0.006) 0.437 (0.008)

2.42 (0.03)

6.39 (0.08) 5.25 (0.05)

CC/MS (IV) (DB-771

(0.08) (0.07) (0.05) (0.03) (0.09) (0.08) (0.03) (0.07)

0.320 (0.012) 0.224 (0.014) 0.300 (0.027) 0.307 (0.026) 0.413 (0.034)

6.46 5.33 3.03 2.24 1.32 5.83 1.92 2.56

CC/MS (Sm) (SB-Smectic)

a) The value in parentheses is the standard deviation of a single measurement. b) Each certified value is the equally-weightedmean ofthe means from two or more independent analytical methods. Each uncertainty, computed according to the CIPM approach as described in the I S 0 (1993)Guide, is an expanded uncertainty at the 95 % level of confidence, which includes random sources of uncertainty within each analytical method as well as uncertainty due to the drying study. The expanded uncertainty defines a range of values for the certified value within which the true value is believed to lie, at a level of uncertainty of approximately 95 %

Phenanthrene Fluoranthene Pyrene Chrysene Benz[alanthracene Triphenylene Benzo[b]fluoranthene Benzo[klfluoranthene Benzo[a]pyrene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene Dibenz[aj]anthracene Dibenz[a,c]anthracene Dibenz[a,h]anthracene Benzo[b]chrysene Picene

CC/MS (I) (DB-5)

7.04 (0.31) 1.92 (0.03) 2.55 (0.12)

6.42 (0.33) 5.29 (0.42)

LC-FL potal)

Concentration (mg/kg dry-mass basisja

Summary of analytical results from different methods for the determination of PAHs in SRM 1649a, Urban dust

Compound

Tab. 3.14

3.62 (0.13) 4.79 (0.16) 0.308 (0.011) 0.195 (0.004) 0.298 (0.008) 0.315 (0.009) 0.445 (0.013)

3.07 (0.02) 2.25 (0.02) 1.40 (0.01)

LC-FI (Fractions)

4.14 i 0 . 3 7 6.45 f 0.18 5.29 i 0.25 3.05 iO.06 2.21 f 0 . 0 7 1.36 iO.05 6.45 f 0.64 1.91 f 0.03 2.51 i 0.09 3.18 i 0 . 7 2 4.01 f 0.91 0.310 i 0.034 0.200 * 0.025 0.288 f 0.023 0.315 f 0.013 0.426 i- 0.022

Certified valueb

m

3

cu

W

3.4 Certfication of Organic Substances

nated an inter-laboratory study among 14 laboratories that routinely measure PCDDs and PCDFs to analyze two existing natural matrix SRMs. The results from this exercise were used to provide reference values (mode 7 in Table 3.13) for these compounds. NIST has also used results obtained from inter-laboratory studies as an additional set of results in the two or more methods approach (mode 2 in Table 3.13). For example for the recent value assignment for PCBs and pesticides in SRM 1944,the mean of results from 19 laboratories participating in an inter-laboratory comparison exercise was used as an additional set of data in the determination of the certified values. Similar inter-laboratorystudy results were also included in the value assignment of PAHs, PCBs, and pesticides for two recently issued mussel tissue materials, SRM 2.977 and SRM 2978. 3.4.3.3

BCR Approach to Certification

The general scheme for the certification of CRMs supplied by the Commission of European Communities is illustrated in Figure 3.3. The general certification approach of the BCR is the use of results obtained from inter-laboratory studies among Iaboratories that have demonstrated their measurement capabilities for the analytes of interest. The certification procedure applied by the BCR is outlined below briefly based on the experience obtained with a sewage sludge (CRM 088) certified for PAH content (Maier et al. 1994).A similar approach has been used for other natural matrix CRMs listed in Table 3.12 with certified values for PAH content (see e.g. de Voogt et al. 1996; Luther 1997). From a large number of laboratories

(a) original batch

Selection of qualified laboratories for the celtification (10.20 labs)

two different analytical techniques, e.g. (1) HPLC with various detection methods

r

1

Statistical evaluation of the data

I

Preparationofthe ce~ficateand the Certificationreport

1

Delivery of the CRM and handling instructions

Fig. 3.3

General scheme used by the BCR for the certification of natural matrix C R M s

I

97

98

I (generally more than 3 Certifcation

3 0 ) , typically 10-20laboratories (preferably not less than ten) are selected for the final certification inter-laboratorystudy on the basis of their successful participation in preliminary inter-laboratory exercises. These preliminary exercises are carried out with simple solutions containing the analytes of interest, extracts of natural samples, or natural matrices spiked with known concentrations of the analytes to be determined. Participants in the inter-laboratory studies who deliver incomplete or unacceptable results or withdraw selected data, indicating that the methods applied were not under control in their laboratory, are not allowed to participate in the final certification measurements.

Homogeneity Assessment Prior to the certification of the candidate CRM, the homogeneity of the material before bottling is checked by analyzing I-g sub-samples from the original batch. In addition, the between- and within-bottle homogeneity is verified by repeated determinations of the analytes to be certified. For example in the case of CRM 088, 20 bottles from a total of 1000bottles were selected during the bottling procedure (every 50th bottle). After shaking the bottle for I min, a I-g subsample was removed and analyzed. For the determination of the within-bottle homogeneity, five sub-samples were removed from one bottle and analyzed. The coefficients of variation (CV) of the results obtained for the homogeneity assessment of CRM 088 (sewage sludge) are presented in Table 3.15. The CVs for the withinbottle and between-bottle homogeneity are similar indicating that the material is homogeneous. In some cases radiometry is applied for the homogeneity studies. For example, I4C-9-phenanthrenehas been used for spiking CRM 458 (spiked coconut oil), and the results of the between- and within-bottle homogeneity study using beta counting (test portions of 2 g) were not significantly different (Fo.95-test) from those obtained with chemical analyses (test portion of 15 g). No inhomogeneity was observed at these test portion amounts, and no substantial trends in either the bottle filling sequence or the analytical method were observed (Win et al. 1998).

Tab. 3.15 Coefficients o f variation (CV) for nine PAHs analyzed in a within- and between-bottle homogeneity study o f sewage sludge BCR CRM 088 i n % (Maier et al. 1994)

Within-bottle homogeneity PAH

(n = 51

Pyrene Benz[a]anthracene Benzo[u]pyrene Benzo[e]pyrene

1.2

Benzo[b+j+k]fluoranthene Indeno[1,2,3-cd]pyrene

Benzo[b]naphtho[2,1-d]thiophene

1.G

1.4 1.2

1.6 1.5 3.0

Eetween-bottle homogeneity (n = zoj

1.4 1.1 1.8 1.5 1.8

1.7 5.1

I

3.4 Certijkation of Organic Substances

A requirement for the usefulness of CRMs is their long-term stability under the storage conditions; see Section 2.2. For most CRMs the stability of the analytes of interest is tested over a period of one year at various storage temperatures (-2o"C, +zo"C and +40°C). In the case of the sewage sludge (CRM 088), a total of 65 bottles were randomly selected during the bottling procedure for the stability study, and 2 0 bottles were stored at the above temperatures for I, 3, 6, and 12 months. Five bottles from each batch stored at the different temperatures and the initial five bottles at the beginning of the study were analyzed for the nine PAH to be certified. Results of this study indicated that the material exhibited sufficient stability at storage temperatures of -zo"C and +zo"C , whereas a slight decrease in the concentrations of benz[a]anthracene, indeno[~,z,j-cdlpyreneand benzo[a]pyrenewas observed after a period of 12 months at +40°C. Based on these studies, it was recommended that the sewage sludge CRM be stored at a temperature of 4°C or below. Stability Assessment

I

BCR Analytical Approach for the Certification o f PAHs in Natural Matrix CRMs Prior to the certificationanalyses for the CRM, each participating laboratoryhas to prepare standard solutions of the analytes to be determined from certified reference compounds (purity 199.0 %) to calibrate their instruments for response and response linearity (multiple point calibration), detection limit, and reproducibility. In the case of PAH measurements, reference compounds of certified purity are used as internal standards, which are not present at a detectable concentration in the matrix to be analyzed (e.g. inden0[1,2,3-~d]fluoranthene (CRM 267),5-methylchrysene(CRM o81R), benzo[b]chrysene (CRM 046),picene (CRM 168),and/or phenanthrene-dlo). The analpcal procedure is checked by analyses of method blanlts to assure that secondary contamination by the analytes to be determined is avoided or minimized. Because the water content of the CRM matrix to be analyzed may vary from one laboratory to another (dependent on the local humidity and temperature), the water content has to be determined. Accordingly, at least three independent samples are kept at 1og0C for 2 h, then allowed to cool to ambient temperature in a desiccator and the water loss is determined. The certified values are generallyreported on a dry mass basis. The first step for the determination of PAHs is removal from the matrix by solvent extraction,which preferably is performed with boiling toluene or benzene (hot solvent extraction by refluxing; see Jacoband Grimmer 1gg4), although other solvents (e.g. toluene/acetone, acetone, and dichloromethane)and other extraction procedures (ultrasonic treatment, Soxhlet extraction,and accelerated solvent extraction)can also be applied. To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between N,N-dimethylformamide/water/cyclohexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. For the final separation, detection, and quantification of the PAHs, capillary GC and/or LC are used. A variety of GC columns, which often exhibit different selectivity for the various PAHs, are used by the different laboratories for the analyses. The

99

100

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3 Certification

particpating laboratories use a variety of GC parameters including injection devices (on column and split/splitless injection), which adds to the independence of the measurements. Both flame-ionization detection (FID) and mass spectrometric detection (quadrupole as well as magnetic instruments with low to medium resolution) are used for quantification. In contrast to FID, which is a universal detector, mass spectrometry provides selective detection and allows for possible identification of the analytes by their specific fragmentation patterns. Alternatively, LC is used for the separation and quantification of PAHs using both UV and fluorescence detection. The analytes are identified based on their relative retention times and UV and/or fluorescence emission spectra. For UV detection an efficient cleanup is a prerequisite since this detection method is not very selective (almost universal for PAHs), and hence it also responds to many coeluting compounds. Due to the high specificity of fluorescence detection for most PAHs, this LC detection method is less susceptible to potential interferences. As in the case of GC the application of internal standard(s) is mandatory since solvents have to be evaporated during the cleanup, which may result in partial losses of some of the more volatile analytes. To validate the analytical procedure recovery experiments are performed. To this end, the CRM is spiked with a known mass of the analytes at a variety of concentration levels (at least three different levels) and the concentrations measured are compared to the expected concentrations in at least three separate experiments. The extraction step has been shown to be a critical step in the analybcal procedure and it may be responsible for poor recoveries. The efficiency of this step can be assessed either by repetitive extraction of the sample or by the addition of internal standards prior to the extraction step with the assumption that the latter actually represent the behavior of the analytes of interest.

9W 1.ooo 1.100 1.200 ............... 1................. I................. 1.................I................. J..................I................._.I1.....

Lab. Method used 01 HPLC/UV 02 03 04

GC/FID GC/MS HPLClFLU

05 06

GC/MS GCIMS GClFlD GC/FID GC/FID

07 08 09

Means

1.300

1. N O

.................1.................1.............

<.......... *........... > ............................... a * >

<................................................................... > <................. ...................> i <............. .................*..............i!.......... > <...*......> i <...........*...............> ; <............ *.......................... f

~

<............. p>

Fig. 3.4 Results from the interlaboratory study used for certification o f benzo[b]fluoranthene in BCR CRM 088 Sewage Sludge

b

3.5 References

Prior to calculation of the gs % confidence interval of the mean of the means found by various laboratories, it has to be ensured that the set of results used for certification has a normal distribution (no outliers according to the Nalimov test; normal distribution of the mean values for all analytes according to Kolmogorov-Smirnov-Lilliefors test). A typical set of results considered for the certification in the sewage sludge (CRM 088) is presented Figure 3.4 illustrating the laboratory means and the 95% confidential intervals for the determination of benzo[b]fluoranthene.The data set satisfy the precision criterion that the standard error of the mean of the set is less than the standard deviation of the distribution of all means. Based on these data the content of benzo[b]fluoranthenein CRM 088 has been certified to be 1.17+. 0.08 yglg. Statistical Evaluation of Results

3.5 References

ABBEYS (1970) US Geological Survey Standards - a critical study of published analytical data. Can Spectrosc 15:10-16. ABBEYS (1977) “Standardsamples”:how “standard”are they? Geostds News1 1:39-45. ABBEYS (1980) Studies in “Standard Samples” for use in the general analysis of Silicate Rocks and Minerals, Part 6:1g79 Edition of ’Usable’Values. Pap - Geol Sum Can Paper 80-14. ABBEYS (1983) Studies in “Standard Samples“ of Silicate Rocks and Minerals, 1969-1982. Pap Geol Sum Can Paper 83-15. ACHILII M, and ROMELEL (1999) Ion chromatographic determination of bromate in drinking water by post-column reaction with hchsin. J Chrom 847A271-277. BAYONAJM (1995) Development of supercriticalfluid extraction procedures for the determination PH, MAIEREA, and GRIEPINKB, eds. of organotin compounds in sediment. In: QUEVAUVILLER Quality assurance for environmental analysis,pp 465-487. Elsevier, Amsterdam. BECKERDA (1993)Unique quality assurance aspects of INAA for reference material homogeneity and certification.Fresenius J Anal Chem 345298-301. BECKERDA (1995) Resolution of discrepant analytical data in the certification of platinum in two automobile catalyst SRMs. Fresenius J Anal Chem 35x224-226. BECKERDA, and GILLSTE (1995) Recent developments in NIST botanical SRMs. Fresenius J Anal Chem 352:163-165. BELLIARDO JJ, JACOB J, and LINDSEYAS (1988)The certification ofthe purity of seven nitro-polycydic aromatic compounds. BCR Information,Reference materials, Report EUR 11254 EN, 53 pp. BLOOM NS, COLMAN JA, and BARBERL (1997)Artifact formation of methyl mercury during aqueous distillation and alternative techniques for the extraction of methylmercury from environmental samples. Fresenius J Anal Chem 358371377. BOENKEA (1997)Activities and current research from the EC Standards,Measurement and Testing Programme (SMT)in the area offood contact materials. Food Addit Contamin 14:561-569. BYRNEAR (1992) Some consideration regarding reference materials and their role in environmental monitoring. Analyst 117:251-258. BYRNEAR (1993) Review of neutron activation analysis in the standardization and study of reference materials including its application to radionudide reference materials. Fresenius J Anal Chem 345:144-151. BYRNEAR, and KUEERAJ (1991)Radiochemical neutron activation analysis of traces of vanadium in biological samples: A comparison of prior dry ashing with post-irradiation wet ashing. Fresenius J Anal Chem 340:48-52.

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BYRNEAR, and KUEERAJ (1997) Role of the self-validationprinciple of NAA in the quality assur-

ance of bioenvironmental studies and in the certification of reference materials. Proc. Int. Symp. Harmonization of Health Related Environmental Measurements Using Nuclear and Isotopic Techniques, Hyderabad, India, pp 223-238. IAEA Vienna. BYRNEAR, and VERSIECK1 (1990) Vanadium determination at the ultratrace level in biological reference materials and serum by radiochemical neutron activation analysis. Biol Trace Elem Res 27:52g-540. BYRNEAR, CAMARA-RICA C, CORNELIS R, DE GOEIJJJMzIYENGAR GV, KIRKBRIGHTG, KNAPP G, PARRRM, and STOEPPLERM (1987)Results of a co-ordinatedprogramme to improve the certification of IAEA milk powder A-11 and animal muscle H-4 for eleven "difficult" trace elements. Fresenius ZAnal Chem 326:723-729. BYRNEAR, DERMELJ M, I
3.5 References I103 DAMSGAARD E, and HEYDORN K (1973) Arsenic in Standard Reference Material 1571 Orchard Leaves. RIS-R-M-1633,pp 1-7. Roskilde, Denmark. DE GOEIJJJM, ICOSTA L, BYRNEAR, and KUEERAJ (1983) Problems in current procedures for

establishing recommended values of trace-element levels in biological reference materials, illustrated by IAEA Milk Powder A-11. Anal Chim Acta 146:161-169. DE GOEIJJJM, VOLKERS ICJ, TJIOEPS, and KROON J J (1978) NBS SRM 1569 Brewer’s yeast: Is it an adequate standard reference material for testing chromium determination in biological materials. Radiochem Radioanal Letters 35:13g-146. DE LA CALLE-GUNTI~AS MB, MADRIDY, and CAMARA C (1995) Antimony speciation in water. In: QUEVAUVILLER PH, MAIEREA and GRIEPINK B, eds. Quality assurance for environmental analysis, pp 264-283. Elsevier, Amsterdam. DELFANTIR, DI CASAM, GALLORINI M, and ORVINIE (1984) Five years activity in determining trace elements for the certification of standard reference materials by neutron activation analysis. Mikrochim Acta [Wien]I:239-250. DERMELJ M, SLEJKOVEC 2 , BYRNEAR, STECNARP, HOJKER S, PORENTA M, and SESTAKOVG (1992) Rapid RNAA for iodine in urine by different separationtechniques.Analyst 117:251-258. M, SLEJKOVEC 2, BYRNEAR, STEGNAR P, STIBILJV, and ROSSBACHM (1990) Iodine in DERMELJ different food artides and standard reference materials. Fresenius J Anal Chem 338359-561. DE VOOGTP, HINSCHBERGERJ, MAIEREA, GRIEPINKB, MUNTAUH, and JACOB J (1996) Improvements in the determination of eight polycyclic aromatic hydrocarbons through a stepwise interlaboratory study approach. Fresenius J Anal Chem 356:41-48. DONAISMK, SARASWATI R, MACKEYE, VANGELMG, LEVENSONMS, MANDICV, AZEMARD S, HORVAT M, BUROWM, EMONSH, OSTAPCZUK P, and WISESA (1997) Certification of three mussel tissue Standard Reference Materials (SRMs)for MeHg and total mercury content. Fresenius J Anal Chem 358:424-430. DONARDOFX, and CARUSO JA (1998) Trace metal and metalloid species determination: evolution and trend. Spectrochim Acta 53B:157-163. DYBCZYNSKI R (1980) NAA as viewed from the perspective of International Atomic Energy Agency intercomparison tests. J Radioanal Chem 60:45-54. DYBCZYNSKI R (1995) The contribution of various analytical techniques to the certification of reference materials. Fresenius J Anal Chem 352:120-1~4. R, POLKOWSKA-MOTRENKO H, SAMCZYNSKI 2, and SZOPA 2 (1996) Preparation and DYBCZYNSKI Certification of the Polish Reference Material “Oriental Tobacco Leaves” (CTA-OTL-I)for Inorganic Trace Analysis. Raporty IChTJ Seria A nr 1/96. Institute of Nuclear Chemistry and Technology, Warsawa. DYBCZYNSKI R, POLKOWSKA-MOTRENKO H, SAMCZYNSKI 2, and SZOPA2 (1937) Preparation and Certification of the Polish Reference Material Virginia Tobacco Leaves (CTA-VTL-2)for Inorganic Trace Analysis Including Microanalysis. Raporty IChTJ Seria A nr 3/97. Instilute of Nuclear Chemistry and Technology, Warsawa. DYBCZYNSKI R, VECLIAA, and SUSCHNY 0 (1980) Report on intercomparison run A-11. IAEA/Rl/ 68, IAEA, Vienna. EDMONDSJS, and FRANCESCONIKA (1993) Arsenic in seafoods -human health-aspects and regulations. Mar Poll Bull 26:665-674. ELLERBEP, COHENA, WELCHMJ, and WHITEV E (1990) Determination of serum uric acid by isotope dilution mass spectrometryas a candidate definitive method. Anal. Chem 62:2173-2177. ELLERBEPM, SNIEGOSKI LT, and WELCHMJ (1995) Isotope dilution mass spectrometry as a candidate definitive method for determining total glycerides and triglycerides in serum. Clin Chem 41397-404. ELLERBE,P, MEISELMAN S, SNIEGOSKI LT, WELCHMJ, and WHITEE V (1989) Determination of serum cholesterol by a modification of the isotope dilution mass spectrometric definitive method. Anal Chem 61:1718-1723. EPSTEIN MS (1991) The independent method concept for certifying chemical-composition reference materials. Spectrochim Acta 46B:1583-1591.

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EPSTEIN MS, DIAMONDSTONE BI, and GILLSTE (1989) A new river sediment standard reference material. Talanta 36:141-150 European Commission (1994) Guidelines for the Production and Certificationof BCR Reference Materials. Doc BCR/48/gj. Standards, Measurement and Testing Program, Brussels. European Commission (1999) BCR Reference Materials 1999. Institute for Reference Materials and Measurements,Joint Research Centre, Geel. FAIRMANB, and SANZ-MEDEL A (1995) Determination of aluminium species in natural waters. PH, MAIEREA and GRIEPINKB, eds. Quality Assurance for Environmental In: QUEVAUVILLER Analysis, pp 216-233. Elsevier, Amsterdam. FITZGERALD WF, and CLARKSON T W (1991)Mercury and monomethylmercury:present and future concerns. Environ Hlth Perspect 96:159-166. FLANAGANFJ (1974) Reference samples for the earth sciences. Geochim Cosmochim Acta 38:173 1-1744. FRASERCA, GARDNER GJ, MAXWELL PS, KUBWABOC, GUEVREMONT R, SIU KWM, and BERMANSS (1995) Preparation and certification of a biological reference material (CARP-I) for polychlorinated dibenzo-p-dioxin and dibenzofuran congeners. Fresenius J Anal Chem j52:143-147. GAHRA, HUBERN, and NIESSNERR (1998) Fluorimetric determination of bromate by ionexchange separation and post-columnderivatization.Mikrochim Acta 129:281-290. GLADNEY ES (1980) Elemental concentrations in NBS biological materials and environmental standard reference materials. Anal Chim Acta 118:385-396. I, and GILLSTE (1987, 1991 update) Standard Reference GLADNEY ES, O’MALLEY BT, ROELANDTS Materials: Compilation of Elemental Concentration Data for NBS Chemical, Biological Geological and Environmental SRMs. NBS Spec Pub 260-111, NET, Gaithersburg,MD. GLADNEY ES, PERRIN DR, OWENS JW, and KNABD (1979)Elemental concentrations in the United States Geological Survey’s geochemical exploration reference samples - a review. Anal Chem 51:1557-1569. GREENBERG RR, KINGSTON HM, WATTERS J R RL, and PRATTKW (1990) Dissolution problems with botanical reference materials. Fresenius J Anal Chem 338394-398. GRIEPINKB, GONSKA H, MUNTAUH (1983)The Certificationof the Contents (Mass Fractions) of Nitrogen, Phosphorus, Chloride, Sodium, Potassium, Magnesium and Calcium and of the Kjeldahl-Nitrogen Content of a Skim Milk Powder, BCR No. Gj. Report EUR 9138 EN. Commission of the European Communities, Brussels. B, MUNTAUH, and COLINET E (1984) Certificationof the contents of some heavy metGRIEPINK als (Cd, Co, Cu, Mn, Hg, Ni, Pb and Zn) in three types of sewage sludge. Fresenius 2 Anal Chem 318:490-494. FR, HILPERT LR, MAY WE, PARRISRM, and HERTZHS, BROWN JM, CHESLERSN, GUENTHER WISESA (1980) Determination of individual organic compounds in shale oil. Anal Chem 5x1650-1657. SM, KDLINGER G, and VOGLJ (1998)Accurate determination of element HEUMANN KG, GALLUS species by on-line coupling of chromatographic systems with ICP-MS using isotope dilution technique. SpectrochimActa 53B:273-287. K (1980)Aspects of Precision and Accuracy in Neutron Activation Analysis. RIS-R-419, HEYDORN Roskilde, Denmark. K (1984) Neutron Activation Analysis for Chemical Trace Element Research. CRC HEYDORN Press, Boca Raton, Florida. K (1991) Quality Control in activation analysis. J Radioanal Nucl Chem 151:139-148. HEYDORN K (1995) Validation of neutron activation techniques. In: QUEVAUVILLER PH, MAIEREA, HEYDORN B eds., QualityAssurancefor Environmental Analysis,pp 89-110. Elsevier, Amsterdam. GRIEPINK M (1996) Mercury speciation and analysis. In: Global and Regional Mercury Cycles: HORVAT Sources, Fluxes and Mass Balances, BAEYENSW, EBINGHAUSR and VASILIEV0, eds. pp 1-31. Kluwer Academic Publishers,The Netherlands. M (1999) Current status and future needs for biological and environmental reference HORVAT materials certified for methylmercury compounds. Chemosphere j9:1167-1179.

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Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

4

Particular Developments Edited by Markus Stoeppler

4.1

RMs in Quality Control and Quality Assessment Andrew Taylor

4.1.1 Introduction

This Chapter is concerned with quality and chemical analysis. While it can be shown that the required quality as expressed in terms of sensitivity, speed of analysis etc. is, to some extent, dependent on the particular application for which the analysis is used, in all situations these factors need to be known and features such as accuracy and precision should be as good as can be achieved. Thus, how can the quality of an analysis be measured, how can good quality be achieved, and how can good quality be maintained? Reference materials play an essential role in answering those aspects of these quality issues that relate particularly to accuracy and precision although they are also relevant to related concepts such as traceability, trueness and uncertainty ( I S 0 1993, Kristiansen and Christensen 1998).In the use of reference materials (RMs) for these purposes, a clear distinction is made between certified and non-certified reference materials. Highly characterized certified (or standard) reference materials (CRMs, SRMs), with analyte concentrations carefully defined using techniques which afford the very best accuracy, in laboratories with experience and expertise in this type of work, are extremely valuable items. They are also extremely costly. Much of the work to be described here is achieved with well prepared, stable, homogeneous materials where the analyte concentrations are unknown or are given by less rigorous protocols, and are not claimed to be certified. Nevertheless, as shown below, for those applications where such materials are readily available, either from commercial suppliers or as home-produced reagents, they have equally important €unctionswhen looking at analyhcal quality. A further distinction is made between pure solutions and matrix-based RMs. The former are available for many organic and inorganic analytes, with certified concentrations, but their role in quality control and assessment is limited. They may be used for the preparation of calibration solutions for a particular measurement but more usually these materials represent the base for traceability, through secondary

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standards to matrix-matched materials (Kessler and Sieltmann 1999).These issues of traceability and uncertainty are further discussed in Chapter 7 and elsewhere (e.g. IOistiansen and Christensen 1998). Much of the early work with certified reference materials was linked to the derivation of reference methods and there was a period in which primary or definitive (i.e. very accurate but usually very complex) and secondary (or usable) methods were reported e.g. steroid hormones (Sieltmann 1g7g), creatinine (Siekmann 1985). urea (Welch et al. 1984) and nickel (Brown et al. 1981).Although there are some application areas, such as checking the concentrations of preparations listed in a pharmacopoeia, where a prescribed, defined method has to be used, in practice such work is limited. However, this approach to chemical analysis is no longer widely used and will not be further discussed. The emphasis now is placed on using RMs to demonstrate that a method in use meets analytical criteria or targets deemed to be appropriate for the application and to develop figures of merit (Delves1984). The concepts of quality control and quality assessment are most highly developed and extensively employed for measurements involving clinical and biological specimens. The analytes determined range from simple organic compounds (e.g. urea or glucose) and inorganic elements at relatively high (millimolar) concentration, to complex steroids and proteins, and to compounds and trace elements at nanomolar or lower concentrations. An extensive range of analytical techniques is regularly employed - e.g. UV-visible spectrometry, immunoassays, chromatography, atomic absorption and mass spectrometry. Instrumentation is often highly automated to include much of the sample preparation, although more complex methods requiring greater analyst expertise are necessary for many investigations. The large number of specimens, the clinical significance of small changes in concentration (Taylor 1995) and the “quality culture” that has developed within the clinical environment since the late 1940’s has engendered total reliance on reference materials. Thus most of the illustrations used in this discussion are taken from clinical/biological areas. Because of the greater availability of RMs certified for inorganic elements, applications involving these analytes will tend to be featured. However the concepts apply equally to other analytes and to quality in other fields of chemical analysis. It must be remarked that terminology is not consistent and there are many widely used synonyms. Quality control in this Chapter refers to practices best described as internal quality control. Quality assessment is often referred to as external quality control, proficiency testing, interlaboratory comparisons, round robins or other terms. Internal Quality Control and External Quality Assessment are preferred because they best describe the objectives for which the RMs are being used, i.e. the immediate and active control of the results being reported from an analytical run or event, and an objective, retrospective assessment of the quality of those results.

4.1.2

Proper Usage

Certification and availability of reference materials are dealt with elsewhere in this book but in keeping with the title it is appropriate to discuss the proper usage of RMs for quality control and quality assessment. The primary purposes for which reference materials are employed are encompassed within the laboratory Quality Assurance Procedures. Quality assurance comprises a number of management responsibilities which focus on how the laboratory is organized, how it deals with situations, how it interacts with users, together with analytical responsibilities re internal quality control and external quality assessment (Sargent 1995; Bumett 1996). Ideally each component follows a documented protocol and written records of all activities are maintained. Within this context of quality assurance, RMs are used for: characterization of methods internal quality control external quality assessment and these applications, with examples, are described in detail in the following Sections. 4.1.3

Characterization of Methods

Glancing through journals which publish new developments in analytical chemistry, the reader may wonder why it appears to be so important to repeatedly analyze specimen types such as dogfish muscle, bovine liver or pine needles, which feature in the titles of many papers. Reading with a little more purpose will reveal that the authors propose that their work represents a general advance and, to prove that it is an effective innovation, it is necessary to analyze specimens with known concentrations, i.e. CRMs, and to present the results for scrutiny by the scientific community. Having done so, it is anticipated that those with other interests may use this new procedure for their own applications. Although the skeptic might opine that the new method has no obvious application in the real world and that the authors simply analyzed the RMs to ensure that the paper was published, this type of work represents an important, and one of the more visual, uses for CRMs; see, for example, the annual reviews of analytica1 developments in atomic spectroscopy (Cave et al. 1999; Fairman et al. 1999; Taylor et al. 1999). There are also RMs which are prepared for a specific application and are used for validation of relevant methods. Cobbaert et al. (1999) made use of Ion Selective Electrode (1SE)-protein-basedmaterials when evaluating a procedure which used an electrode with an enzyme-linked biosensor to determine glucose and lactate in blood. Chance et al. (1999)are involved with the diagnosis of inherited disorders in newborn children and they prepared a series of reference materials consisting of blood spotted onto filter paper and dried, from which amino-acids can be eluted and

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diagnose prostate cancer. The serum contains some PSA which is bound to protein and some unbound-, or free-PSA. Both fractions are usually determined as the “total PSA”. Problems associated with methods to measure total PSA were revealed in a study using RMs prepared by adding seminal fluid (mainly free-PSA) to a serum matrix (Fox et al. 1999). It was found that the accuracy of some methods became very poor as the proportion of free-PSA increased. Since the ratio of free- to boundPSA is quite different in specimens from different subjects, the reliability of many methods is now recognized to be uncertain. Apart from innovative work, RMs are essential during exercises such as the introduction to a laboratory of a method from elsewhere or the transfer of an established method onto new instrumentation. Even where the conditions for the analysis have been standardized by the manufacturer of a reagent kit, some validation work should still be undertaken so as to have documented data for quality assurance purposes, e.g. accreditation, as a basis for IQC, for later reference when problems which may be related to equipment, reagents or staff etc. need to be investigated. Validation work includes the determination of within-batch and between-batch imprecision, linearity, sensitivity and limit of detection - which can be carried off with any suitable specimen. However, to demonstrate the accuracy of the procedure, to investigate possible interferences and to establish reference ranges, it is necessary to analyze RMs with known concentrations, ideally using certified materials. Where none are available, accuracy may be assessed using other approaches, but these are less reliable and are more complicated to perform (Taylor 1995). It is important that the analytical characteristics, or figures of merit, for a procedure are systematically determined so that the user may have confidence in any results elaborated from that method. It is equally important to compare the performance of the procedure against the requirements for which it is being used. Concentrations of lead in environmental air are very low and reasonably constant, whereas ozone levels fluctuate considerably. Within a monitoring programme, analytical targets to determine significant changes or trends in the exposure to these two agents are therefore very different. Similarly, we have proposed analytical targets necessary for the measurement of aluminium in serum during the monitoring of patients with chronic renal failure, who are at risk of developing aluminium toxicity. These targets were audited by renal physicians and found to meet their surveillance criteria (Taylor and Goldie unpublished). Fraser has extensively discussed this relationshipbetween laboratorywork and clinical needs (Fraserand Hyltoft Peterson 1993)and has recently addressed the role ofdocumented analytical quality as derived from measurements of RMs (Fraser and Hyltoft Peterson 1999).Among the concepts proposed by Fraser and his colleagues, it is suggested that analpcal imprecision should be ~ 0 . 5 0CV1 and bias should be i0.25 (CV: + CV?)’’’ where CV1is the within-subjectbiological variation (i.e.changes from day to day within an individual) and CV2 is the between-subjectvariation (i.e. the reference range). These may be challenging targets for analytes present in samples at concentrations close to the detection limit of the methods. However the concept provides a valuable spur to developing improved methods.

Evaluation of a new method may include comparison of results obtained by the analysis of reference materials using established procedures. The purpose is to demonstrate that the novel approach provides results that are at least as reliable as an accepted technique. This same approach may also be used to discover which, of a number of techniques, are preferred. Comparison of analytical techniques and/or methods is considered further in the discussion of external quality assessment, below. As laboratory accreditation becomes more established, the requirements to demonstrate traceability and to determine uncertainty will inevitably feature as part of method validation (Christensen 1996).The fundamental role which reference materials play in these steps has already been alluded to in the Introduction. 4.1.4

internal Quality Control

The inherent reproducibility or imprecision of the method will have been determined as part of the validation procedure. This information can then be applied to the internal quality control programme which is designed to identify the intrusion of a bias (inaccuracy) and/or an alteration in the reproducibility of the assay. Programs for internal quality control are most extensively developed for clinical laboratories because of the availability of suitable RMs in large batches and at an affordable cost although some level of IQC is appropriate to all work carried out at a continuing basis; see Section 6.2. An internal quality control program can comprise several elements (Taylor 1gg5), but the most usual is to include a specimen(s) with a known (or target) concentration within each batch and to take that specimen(s) from a large stable pool so that it can be used in this way over a period of several months, i.e. a RM. For non-certified RMs, the target concentration will be determined by the user and, as noted above, clinical RMs are readily available from commercial suppliers. Some in fact do have recommended concentrations for many analytes, but the rigor with which these have been established rarely matches that associated with true certified RMs. Outside of the clinical and related laboratories, those involved with regular analytical procedures will either have to use CRMs for the internal quality control, or manufacture their own. With careful planning and attention to detail, extremely useful RMs may be prepared without access to the special facilities associated with the recognized providers of CRMs. Braithwaite and Girling (1988) described materials for blood lead and cadmium assays, while we have prepared serum-based materials for Al, Se, Cu and Zn (Taylor 1988,1996). Many other examples of laboratory-manufactured RMs are available, especially in publications or at conferences which concentrate on specialist analytical applications. The reproducibilityofthe method, or the confidenceinterval attributed to a CRM, will show the analyst the allowable range ofresults to anticipate around the target concentration. An acceptable range of c 2 standard deviations is usually applied and the practical approach is to draw a Levey-Jenningschart, see Figure 4.1. A series of decision criteria or “controlrules” have been developed by Westgard and his colleagues as part of the program (Westgard et al. 1981).Westgard rules advise as to the number and concentration

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Fig. 4.1 Internal quality control results displayed on a Levis-Jennings chart. This example presents four analytical situations (a) good control, (b) sudden shift in accuracy- perhaps the calibration material has become contaminated, (c) a gradual shift in accuracyperhaps a reagent has exceeded its expiry date, (d) very poor precision - perhaps an autosampler is working erratically.

of RMs to be included, the frequency of inclusionwithin a test run and how their results may be applied to decisions as to whether a runshould be accepted or rejected. However, rather than formally adopting the Westgard rules, many laboratories develop their own criteria particularly where they do not have access to the large number of non-certified RMs that are found for clinicallaboratories. The consequence of a successful IQC program is minimal analytical variation from one measurement episode to another. With analytical stability in place, temporal trends such as diurnal or seasonal patterns may be identified; similarly good control or lack of variation may be confirmed. Therefore, even small changes in concentration will be identified and crucial aberrations, e.g. disruption to a manufacturing process or effects of disease in a patient will be diagnosed (Cotlove et al. 1970). As has been seen with the decrease in blood lead concentrations during the last two decades, when accuracy control is included into the programme by using a CRM or some other mechanism to identify bias (Braithwaiteand Girling 1988; Taylor 1988) not only may temporal trends be demonstrated (Delves et al. 1996) but results from different laboratories may be directly compared. An internal quality control program pioneered by Yeoman in the late 1970’s where collaborating participants used the same RMs showed that remarkably consistent inter-laboratorydata may be produced in large national and international (EU, WHO etc.) surveys by laboratories with differing backgrounds, experience and expertise (Vahter 1982; Department of the Environment 1983; Taylor 1996).

4.1.5 External Quality Assurance

External quality assessment schemes involve the distribution by the organizer, to participating laboratories, of aliquots from the same sample. Participants analyze the received specimen and send the results back to the organizer who prepares a report to summarize all data. Schemes vary enormously in the: number of participants; from two or three in an informal exchange of specimens to several hundreds in the major schemes number of samples; usually between one and five. Recommendations are given in an internationally developed protocol for the organization of EQA programs (Thompson and Wood 1993) number of analytes that are included; may be up to 2 0 or more in the large clinical chemistry schemes frequency at which specimens are distributed to participants; there may be a simple once-only exercise, but the frequency usually ranges from weekly to once or twice a year and is associated with how often an analytical procedure is likely to be carried out in the laboratory (Thompson and Wood 1993) complexity of the statistical procedures used to evaluate the results: - for the formal identification of outliers - calculations of consensus mean, standard deviation etc. using the complete set of results and, in some schemes, according to the analFcal techniques or procedures employed by sub-groups of participants - individual results may then be compared against the “target value”. Protocols to identify the target value include the certified concentration (when CRMs are used), the consensus mean or median given by the results from a pre-selected group of reference laboratories, the consensus mean or median of all participants. These comparisons may simply be the numerical difference between a result and the target value or may be much more complex to include factors such as the standard deviation and the concentration of the analyte in the specimen (Thompson and Wood 1993) further calculations carried out from these comparisons to provide a judgment on the performance of the individual participants; this may involve the determination of a “performance score”, possibly taking into account results from specimens previously distributed over a specified time period, and some decision as to what represents poor, acceptable and good scores (Whitehead 1976;Thompson and Wood 1993; Morisi et al. 1996) anonymity The common feature of all EQAS is the use of RMs as the distributed specimens for unless a stable, homogeneous, controlled material is used the objectives cannot be guaranteed.

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In 1978 the International Federation of Clinical Chemistry produced a comprehensive summary of the objectives of External Quality Assessment (IFCC 1978). The first six points of Table 4.1 summarize the IFCC objectives; the seventh has developed since that time and is of considerable importance in many countries where strict control over laboratories undertaking particular types of work is required by legislation or by appropriate professional organizations. Tab. 4.1

Objectives o f external quality assessment schemes

1.

To provide a measure of the "state of the art" for a test. The standard deviation is a measure of the ability of different laboratories to obtain the same result. Where the true concentration is known the results show overall accuracy.

2.

To provide a measure of the quality of performance of individual laboratories. By regular testing, changes in performance can be studied.

3.

To supplement internal quality control within a laboratory.

4.

To obtain consensus values for specimens which, when certified values are not known may be used as surrogate CRMs.

5.

To investigate factors within laboratories which may contribute to its performance, e.g. methods used, size of laboratory,workload etc.

6.

To act as an educational stimulus.

7.

For the licensing of laboratories for particular work.

4.1.5.1 Stateofthe Art The questions being asked are, how well can a measurement be made, what level of confidence can be attributed to a result, have measurements improved compared with some time point in the past? In addition to being of interest to the analyst in the laboratory, these questions are crucial to those who apply the results to real life problems, as discussed by Fraser and Hyltoft Peterson (1999) - see above. It is often a salutary experience for laboratories that have invested considerable effort in providing a new analytical procedure, to then compare their results for the analysis of an EQA reference material with those of colleagues who have similar interests. The variation is likely to be much greater than had been anticipated - but this is a true reflection of the "state of the art" at that time. For example, in recent years it has been recognized that measurements of methylmalonic acid and homocysteine in serum are useful in the investigation of vitamin B12 deficiency and vascular disease, respectively. Results from a 1998 EQA scheme for these analytes showed that minimum imprecision goals were met by eight of the 15 participants in the methylmalonic acid program and by only nine of the 34 participants for homocysteine (Moller et al. 1999). Experience from other schemes, however, indicates that assays subject to regular external quality assessment improve until a degree of maturity is attained, at which point there is no further change in markers like the between-laboratory stand-

ard deviation or coefficient of variation (CV). In a scheme we organize for specialist laboratories in the United Icingdom, we have seen the between-laboratory variation for blood lead measurements improve with typical CVs of around I O - I ~ % in the I ~ ~ o falling ’ s , to 2-5 % in the late 1990’s (unpublished) and others have seen similar changes (Bullock et al. 1986). In our work with shared RMs for internal quality control and other techniques, we have shown that improvements in performance can be positively stimulated. These approaches were applied to UK hospital laboratories measuring A1 in serum and, within our international external quality assessment scheme, there was a greater improvement in the “state of the art” for this cohort compared with the non-UK participants (Taylor 1996). 4.1.5.2

Performance of Individual Laboratories

Measurements of individual laboratory performance provides for comparisons between laboratories. It then follows to ask why some laboratories report data that are more accurate and precise than do their peers, and a well designed external quality assessment scheme allows investigation of some of the important factors (see below). A comparison of performance between individual laboratories also helps to stimulate those who are not so successful to improve (or abandon the assay) and those who do well to continue with their expertise. Finally, changes of performance may be monitored as a consequence of some new factor, e.g. purchase of a new piece of equipment, work carried out by a different analyst, change to the methodology etc. 4.1 S . 3

Supplement Internal Quality Control

Theoretically IQC should be the front-line approach to quality. If a method has been adequatelyvalidated and shown to meet the requirements of the user and kept in analytical control with IQC to detect intrusion ofbias or imprecision,then the EQA needs to provide the occasional, independent, objective reassurance. In practice however, the EQA is likely to play an equal role with IQC, both in confirming problems brought to the attention ofthe analystby the IQC and in stimulating further action. An unexpected outcome from EQA observations appeared when unsatisfactory material was unknowingly used at one time for the manufacture of atomic absorption graphite furnaces. Several laboratories were aware of unsatisfactory IQC for their blood lead methods and strived to find an explanation; but it was only when the FQA data revealed a widespread problem that a group of participants compared their recent experience and were able to confirm with the suppliers that inferior graphite had been employed. 4.1.5.4

To Obtain Consensus Values

For many analytical methods there are no CRMs. It may also be that there is no primary standard, e.g. for determinations of enzyme activity, or that reliable methods for accurate determination do not exist. However, there is a requirement for RMs of some type. Samples which have previously been used within an FQA scheme may fulfil this purpose. When a large enough number of independent observations are made the mean is a good approximation to the true value (Sutton et

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to help sort

out a problem, help compare one method with another etc. 4.1.5.5

Investigate Factors Contributing to Performance

It has been recognized that many factors may influence performance - methods, instrumentation, features relating to the laboratory such as size, worltload and how often a method is in use. Where an EQA programme has sufficient number of participants, the importance of each of these factors can be determined. When we have adopted this approach to investigate the determination of trace elements in biological samples, we have found that specialist skills and analytical expertise are more important to achieving good performance than are the instrumentation or methodology (Borella et al. 1998; Sheehan and Halls 1999). 4.1 S.6

To Act as an Educational Stimulus -To License Laboratories?

Data derived from analysis of RMs in EQA schemes may be informative and, as discussed above, help to direct and stimulate the laboratory towards providing results in the day to day work, which are accurate, precise and appropriate to the needs for which they were requested. Alternatively a scheme may operate simply to identify poor performance, so as to prohibit laboratories from carrying out certain tasks. The disadvantage of just an educational objective is that there are no sanctions to ensure that poor performance does not continue without being corrected.With licensing, if a laboratory is seen to fail, they will lose income and, to avoid controversy, the analwcal standards may be set at a very low level so that only the grossly incompetent are eliminated. These two objectives are not exclusive and a scheme may be used for either or both purposes. Educational schemes are usually organized or supervised by appropriate professional organizations who can provide impartial comment on criteria for acceptable performance and similar aspects. Licensing of laboratories tends to be a feature of analytical work prescribed by legislation (Germany 1979). Mechanisms for issuing a license may be the responsibility of a government agency but are often transferred to a professional organization such as the College of American Pathologists for clinical work in North America or the Federal Chamber of Physicians for occupational medicine in Germany. Dual intent is best seen in application areas where laboratories are subject to accreditation by some independent organization (Burnett 1gg6), when participation in a recognized EQA scheme, with demonstration of satisfactory performance, is one of the conditions which have to be fulfilled in order to qualify for full accreditation status. Thus, to further the goals of quality and good analytical practice for which RMs are intended, EQA schemes should combine some aspects of both objectives according to the “political” purposes for which the scheme is being organized. Whether used for educational or licensing purposes, the ultimate intention is to ensure a certain standard of analysis is achieved and maintained in order that the user of results may be protected against errors which could be costly, in financial or human terms.

4.2 Fresh Materials

4.1.6

Conclusions

Quality, unlike beauty is not simply in the eye of the beholder. Quality may be assessed, measured and evaluated, to be expressed in terms such as good-poor, acceptable-unacceptable. Quality may also improve or deteriorate depending on the efforts, enthusiasm and expertise of the analysts and the environment in which the work is carried out. Analysis of RMs enables assessment of laboratory factors (equipment, staff etc.) on quality, but it is the awareness of the importance of reference materials within the quality culture of the laboratory (recognizing crucial steps in method validation, ambitions to have good IQC and ambitions to have better performance scores in EQA) - that they are used to greatest effect.

4.2

Fresh Materials Jacob de Boer 4.2.1

Introduction

Since the IgGo’s there has been a need for good biological reference materials for environmental contaminant analysis (Wells 1988).This concerns reference materials for short term use in inter-laboratory studies, for mid-term use as internal reference materials (IRMs) and for long-term use as certified reference materials (CRMs).Until the early 1990’smainly freeze-dried materials and fish oils have been used for these purposes, because they had the closest possible resemblance to the natural samples which were analyzed daily in laboratories (Law and de Boer 1395). However, these materials have clear disadvantages. Freeze-dried materials, often used as reference materials for trace metal analysis, have different physical characteristics from the natural samples and normally require a different treatment for destruction, which makes them less well comparable with the natural samples (Topping 1982; Berman and Boyko 1992). In addition, it may be expected that freezedried materials, unless very well kept in an extremely dry environment, are not stable in the long term because uptake of moisture may take place and this may finally lead to a change in the concentration of the contaminants (de Boer and Smedes 1997). Fish oils or fish fat have often been used as reference materials for organic contaminants such as chlorobiphenyls (CBs) and organochlorine pesticides (Musial and Uthe 1983;Tuinstra et al. 1985;Uthe et al. 1988;Wells et al. 1988).The main disadvantage of these oils is that they can directly be dissolved in an organic solvent, whereas the natural samples normally need to undergo an extraction. This means that it is not possible to control the entire analytical method with the oils. In addition, the fat content is obviously different than that of natural samples and, in relation with that, the contaminant concentrations are normally much higher than

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oils mimic natural samples as much as would be desirable; and they cannot be used as controls for the entire analytical method. Frozen reference materials have been produced by NIST (Wise et al. 1993). These materials do not have the disadvantages of the oils or freeze-dried materials, but are more difficult to transport. Obviously they have to be kept deep-frozen during transport, which makes their use rather expensive. Since the early 1990’sa new approach in this field has been introduced. This concerned the use of wet, sterilized fish and shellfish samples. These samples, packed in glass jars or in tins, were firstly used in the QUASIMEME program as reference materials for inter-laboratory studies (de Boer 1997). Later, when it appeared that the stability was maintained for longer periods, tests for organic contaminants based on this principle were also prepared. 4.2.2

Packing Materials

One of the main selection criteria for packing materials containing reference materials is the absence of interference. Obviously, reference materials for trace metals should not be kept in metal or metal-containing packings. Similarly, paclings for organic contaminant reference materials should not contain organic interference. Plastic or glass would be candidates for trace metal reference material paclings, whereas tins or glass would be suitable for organic contaminant reference materials. Glass would be relatively ideal for both types of reference materials, but it is more vulnerable during transport. However, the only full glass pacltings are ampoules, which are fine for fish oils, but cannot be used for the packing of a relatively solid matrix such as fish. Glass jars are an alternative, but immediately a disadvantage appears: the lids may contain metal or plastic materials, which may cause interference in the trace metal or organic contaminant analysis, respectively. Another disadvantage is the possible unscrewing of screw-caplids due to temperature differences. The latter difficulty can be solved by using so-called twist-off lids. Small jars with a volume of ca. 5 0 rnl, which are used in the food industry for packing caviar, are in principle suitable for the packing of trace metal reference materials. The twist-off system guarantees a completely closed packing, even during the sterilization process. However, the lids used should be carefully tested for possible contamination. These lids normally have a metal base which is covered inside with an epoxyphenolj epoxyphenyl lacquer. These lacquers may contain zinc carbonate. The concentration of the zinc carbonate should be less than 0.5 mglkg. A contribution of 0.2 % Zn to the content of the jar is considered acceptable. The presence of other trace metals such as Cu, Hg and Pb should be checked as well, but until now these metals have not been found at detectable levels. The closure of the twist-off jars is rather labor-intensive.In addition there is also a risk of leakage, even when using the twist-off lid system. Particularly with fatty materials, the jars can become fatty and slippery on their outer surface. The consequence can be that the dosing machine cannot fix the jar in one position and the jar start to rotate during closure, resulting in a poorly closed lid, which can start to leak

4.2 Fresh Materials

after sterilization or after high- temperature storage for long terms. Thus, careful inspection of the jars before closure and clean working conditions are important. In the food industry, a little steam is added just before closure. This is done to lengthen the shelf-life of the product and to tighten the lid after cooling down. However, there is no strict control on the amount of steam added and lot-to-lot variations can be introduced in this way. In addition, trace metals can possibly be present in the added steam. Alternatively, a small addition of nitrogen is used to expel the oxygen present and thus prevent any deterioration in the sample during storage. This small change in the technique makes it necessary to warm the sample shortly before it is transferred to the jar to ca. 50"C, in order to prevent leakage in a later stage. Because of these complications and to avoid any possible trace metal interference from the lids, full plastic packings have been studied as an alternative. Such an alternative was also found in the food industry. This concerned a plastic foil, which can be vacuum-packed by a vacuum-packing machine. Among other applications, this material is used for packing salmon slices. Such material was tested extensively in the QUASIMEME program (de Boer 1997). Despite the guarantee of the producer that the plastic foil would be waterproof and fat-resistant, problems were encountered with lipid-rich fat tissues such as cod liver (ca. 5 0 % fat). It appeared that after a period of storage of two months at 37T and 3-4 months at room temperature, visible leakages were observed. For other, less fatty materials no leakages were observed. However, stability tests (see below) showed that after storage of plaice and cod muscle tissue and mussels for six months at j7OC a loss of moisture was found, which resulted in a small concentration effect of ca. I %. No such effect was found after storage at room temperature. However, the possible risk of moisture losses during storage for longer periods, even at lower temperatures, was considered undesirable; and the alternative of the glass jars was preferred. Possibly, other plastics may offer an alternative, but they should be tested extensively prior to use. An additional disadvantage of the plastic foil is that it is rather difficult to get the entire sample out of the foil. This is necessary because during sterilization some separation of moisture, oil and more solid substances may take place. Re-homogenization is therefore always recommended. Due to the folding of the foil during the sterilization process, it is considerably more difficult to recover the entire sample from the foil than from a glass jar. Obviously, the lacquers containing organic interferences make the glass jars less suitable for reference materials for organic contaminants. For similar reasons, the plastic foils are not suitable for packing reference materials for organic contaminants. Therefore, tins were considered as an alternative for packing biological reference materials for organic contaminant analysis. Tins are produced for the food industry in various sizes, with 70-100 ml tins, with a diameter of 52 mm, as the smallest. They are available in minimum quantities of 15 ooo lots, which is rather large for the purpose of reference material production. However, a more serious problem is that tins made for food preservation are all coated inside to prevent corrosion. Tins without a coating can be made on request and have been used for some reference materials (de Boer 1997). The quality of some reference materials after short-term (ca. 3 months) storage was sufficient to enable a reliable inter-laboratory

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I study (de Boer et al. 1996; de Boer 1997;de Boer and Wells 1997). However, after 4 Particu/ar Developments

longer storage times, a blackish layer between the tin and the fish matrix was observed. Although significant effects on the results of inter-laboratory studies have not yet been demonstrated (de Boer and Wells 1997),this phenomenon was considered too risky to continue with the use of uncoated tins. Coated tins have been used as an alternative, although it was demonstrated that the coating could release interferences (de Boer 1997).Extraction of coated tins with pentane resulted in GC/ECD chromatograms with a number of negative peaks in the area where normally PCBs and organochlorine pesticides would elute. Despite this undesirable result, coated tins have been used for packing biological reference materials for organic contaminant analysis. Pentane has much stronger searching properties than lean fish tissue. Extraction of the tins distilled water containing 30 g/L NaC1, followed by extraction of the water with pentane, resulted in an absence of interferences in the GC/ECD chromatograms. This showed that the coated tins could be used for lean fish tissues with fat percentages of ca. I-3%. For more fatty tissues, particularly for cod liver with a fat percentage of ca. 50 %, transfer of interferences to the fish matrix is still possible. However, these interferences appear not to be able to pass a normal cleanup for a PCB/pesticides analysis, such as aluminalsilica column chromatography or sulfuric acid treatment. This means that coated tins can be used to pack reference materials for PCB and organochlorine pesticides analysis. They may also be used for materials meant for other organic contaminant analysis, but the possible transfer of interferences from the coating should always be carefully tested prior to use. The tins are normally filled to the brim with cold material to reduce the oxygen present as much as possible. They can be sealed by a sealing machine. 4.2.3 Preparation

The more solid biological materials, such as fish tissue, are thoroughly homogenized in a cutter in order to obtain an homogeneous mince. During homogenization an antioxidant, normally butylhydroxytoluene (0.02 % w/w), is added. During the preparation, contact with plastic materials is avoided as much as possible when materials for organic contaminant analysis are being made. More liquid materials, such as fish liver and shellfish, can be homogenized in a mincer, sometimes in combination with a mill equipped with toothed rotary knives, followed by cuttering. Several batches can be mixed by stirring in a stirring kettle. Following these processes, the material can be transferred to a roundbottomed flask provided with a tap at the bottom and a stirring device. The more solid materials are transferred to tins or jars by hand, using an icespoon. The more liquid materials are transferred to the jars or tins by opening the tap of the roundbottomed flask. This flask can be heated if necessary when filling the jars. Shortly after closing the twist-off lids or sealing the tins, at least on the same day of filling, the jars or tins are sterilized by autoclaving for 30 min at 120"C, whilst maintaining a pressure of 2 bar. The cooling time is 3 0 min. A disadvantage of the

4.2 Fresh Materjals

sterilization process is that a separation process may take place in the tins or jars, during which the more solid parts of the materials separate from water and oil. Particularly for tissues with a high moisture content, such as mussels and lean fish muscle tissue, this process may result in reference materials which appear rather inhomogeneous. Of course, the content of each tin is the same, but the withinhomogeneity is poor. It is always recommended to re-homogenize the materials after they have been sterilized, but in some cases this may be a difficult task for the analyst. Some alternative techniques have been developed to improve the withinhomogeneity of the tins after sterilization. Mussels for example can be heated for a few seconds or minutes before opening the shells. This can be done mechanically in the shellfish industry, but may also be done in the laboratory by coolting them very briefly. This will on the one hand assist in removing the tissue from the shell very cost effectively, and on the other hand it will reduce the water content of the tissue substantially, which results in a better within-homogeneity after sterilization. Obviously, the drawback is that the material moves away to some extent from the natural material which has to be analyzed in the laboratory.Another option is to add a few percent of starch to the material. This can very effectively bind the moisture, so that a nice and homogeneous material is obtained. It is important that the starch is tested for interference, because otherwise contamination can easily occur. 4.2.4

Homogeneity

The heterogeneity of a reference material is usually obtained by analyzing the determinant in several different batches and within a single batch followed by a significance test on the resulting data for that specific sample mass. There are various models for these studies (European Commission 1993). Occasionally, in cases where the analysis of the target determinant is relatively expensive, for cost-effective reasons a surrogate determinant, e.g. total lipid instead of PCBs, or sodium and potassium instead of trace metals, has been determined (de Boer 1997).Although such surrogate determinants may give a good indication of the homogeneity of the material, the determination of the target analytes themselves is preferred. Stability tests should obviously always be carried out with the target analytes. A recently prepared candidate CRM PCBs in mussels (BCR no. 682) gives a good indication of which degree of homogeneity can be obtained in these fresh materials (Table 4.2) (de Boer et al. 2000). These mussels were shortly heated before removing them from the shell. The relative standard deviation (Rsd) for the inhomogeneity is determined by the Rsd of the within-homogeneity and the between-homogeneity, according to the formula: RsdZinhom = RsdZbemeen - RsdZwithin The between-homogeneity was determined by analyzing the CBs 52, 101, 118, 153 and CB 180 in 15 lots of the candidate CRM. The within-homogeneity was determined by five analyses of the same CBs in one lot of mussels. In addition the Rsd values of a ten-fold analysis of a standard solution and of a five-fold analysis of a fat

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4 Particular Developments Tab. 4.2 Relative standard deviations measured in homogeneity tests of a candidate CRM PCBs in mussels (BCR 682) (de Boer et al. 2000)

CB52

Rsdst RSddean-up RSd-thm RSdbetween

1.7 3.3 3.5 G.9

CBlOl

CB118

2.1 2.9 3.7 5.2

2.9 4.1 2.4 6.0

CB180

2.3 2.5 2.2 5.9

3.5 4.3 3.6 6.9

I

extract of the mussels were determined. Table 4.2 shows that there is a small inhomogeneity in the samples in the order of 5 %. Given the nature of these wet materials, resembling as much as possible a natural fish or, in this case, shellfish tissue, this small inhomogeneity is acceptable. The variation coefficients obtained in the certification varied between 10% and 30 %, which means that the contribution of the small inhomogeneity of the candidate CRM will be negligible compared to the overall error in the measurement. Mussels are more difficult to homogenize then most fish muscle tissue. Minor remainders of the contractor of the mussels can have a negative influence on the homogeneity. Most fish muscle tissue materials will normally show a better homogeneity than that given in this example. 4.2.5

Stability

The stability of candidate CRMs is normally tested over a period of ca. two years. The candidate CRM is stored at a range of temperatures and the target analytes are analyzed at regular intervals.The candidate CRM PCBs in mussels discussed above was stored at -2o"C, 20"C, 37°C and 5 0 C (for six months only);see Figure 4.2. De Boer et al. (2000) concluded that for the CBs 28,52,101,118,14g,153,170 and 180,over the entire testing period for the temperatures 20°C and 3 7 T , there were no significant differences between 20°C and the other storage temperatures, at all confidence levels. However, for most higher chlorinated CBs significant differences between -20°C and 50°C were found after six months storage. It is currently unknown why higher CB concentrations have been found after six months storage at 50°C. Instability normally leads to lower concentrationsofthe analytes. Leakage ofmoisture from the tins was impossible. It was concluded that the CB concentrationsin the mussels are stable at room temperature for a period of at least two years. At 50'C some instability cannot be excluded. Therefore, storage at 20°C or lower temperatures is recommended. However, storage at temperatures below 0°C should be avoided, because moisture present in freezers could cause corrosion ofthe tins,

4.3 Certified Reference Materials for Microanalytical Methods 1.50

CB153

1.40

1.30

1.20

T

1.10

E. 1.00

+I

E I

0.90

0.80

0.70

0.60 0.50

0

6

18

12

monlhs

24

30

Fig, 4.2 Stability of CB 153 in CRM PCBs in mussels (BCR 682); RT ratios of mean values at 20,37 and 50°C versus -20°C (de Boer et al. 2000).

4.3

Certified Reference Materials for Microanalytical Methods

Matthias Rossbach and RolfZeisler

“Microanalytical” or “microchemical” methods, techniques and procedures have been in common use for some time. Chemical dictionaries describe the scale of these methods in various terms. For the purpose of this discussion, the following definition is most appropriate: Microchemistry - a branch of analytical chemistry that involves procedures that require handling ofvery small quantities ofmaterials. Spedically it refers to carrying out various chemical operations (weighing, puriication, quantitative and qualitative analysis) on samples r a n g i n g j o m 0.1 to 10 milligrams. (The Condensed Chemical Dictionary 1971).

To further clarify the scope of our discussion, we present a compilation of existing and developing modern microanalytical techniques in Table 4.3. These techniques routinely use small samples in the aforementioned mass range, with some techniques extending their capabilities to significantly smaller sample sizes.

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4 Particular Deve/oprnents

Tab. 4.3

Techniques used in microanalysis

“Traditional”Solid Sampling Techniques Neutron Activation Analysis X-Ray Fluorescence Particle-InducedX-Ray Emission Particle-InducedNuclear Reaction Analysis Rutherford Backscattering spectrometry Spark Source Mass Spectrometry Glow Discharge Mass Spectrometry Electron Microprobe Analysis Laser Microprobe Analysis Secondary Ion Mass Analysis Micro-PIXE Micro-Synchrotron-InducedXRF

Typical Sample Mass lmg-log 1 mg - 100 mg 0.1 mg - 10 mg 0.1 mg - 10 mg 0.1 mg - 10 mg 0.1 mg - 10 mg

1 Pg - 100 down to 1 pg down to 1 pg down to 1 pg down to 0.1 ng down to 0.1 ng

“Emerging”Solid Sampling Techniques Solid Sampling Atomic Absorption Spectrometry ICP Optical Emission Spectrometry ICP Mass Spectrometry Laser Ablation ICP Optical Emission Spectrometry ICP Mass Spectrometry Transmission Electron Microscopy with Energy-DispersiveX-Ray Analysis

0.1 mg- 1 mg 0.1 mg- 1 mg 0.1mg-lmg 1 ng - 10 ng lng-long down to 1 fg

Organic Analysis Techniques Pressurized Fluid Extraction followed by Gas Chromatography/Mass Spectrometry (PFE-GC/MS) On-line Supercritical Fluid Extraction - Gas Chromatography/Mass Spectrometry (SFE-GC/MS)

0.1 mg - >1g

0.1 mg - 1 mg

With the availability of this wide variety of techniques, microanalysis has becor rapidly developing field. The use of microanalytical techniques falls into two basic gories. In some cases the samples are by nature or necessity smaller and in otl increased analytical sensitivity allows for the use of smaller samples. For trace elen determinations,direct analytical techniques using solid samples are gaining ever-br ening utility in the determination of natural and pollutant constituents in biological environmental materials. These techniques are preferred over the destructive ana techniques because of cost-saving sample preparation and, in many instances, s turn-around times. In addition, in the many uses of destructive analysis techniq inorganic, organic and speciation procedures are miniaturized. They provide inci ingly smaller samples for analytical determination, as a result of more efficient px dures that reduce chemical processes and their waste. These practical and economical aspects compel the analyst to use microanaly techniques. However, when confronted with the task of analyzing a CRM wi

4.3 Cert$ed Refrence Materials for Microanalytical Methods

microanalytical technique, the analyst will realize that suitable CRMs are scarce. The minimum sample sizes currently recommended by CRM producers are generally =IOO mg (IAEA-TECDOC-825/8801gg5/1ggG). These minimum sample sizes were developed for determinations made primarily on dissolved samples. There are, however, some CRMs available that may permit the successful use of much smaller sample aliquots as discussed in the examples below. 4.3.1.

Homogeneity of Components in CRMs

To discuss the development of CRMs for the emerging use of microanalytical techniques, one has to be concerned chiefly with the degree of homogeneity of the components in the material at the designated sample size. Basic indications for the homogeneity properties of a CRM for microanalytical methods and the assessment of these properties can be derived from the general requirements: According to I S 0 Guide 35 (1989). a “material is perfectly homogeneous with respect to a given characteristic if there is no difference between the value of this characteristic from one part (or unit) to another. In practice, a material is accepted to be homogeneous with respect to a given characteristic if the difference between the value of this characteristic from one part (or unit) to another cannot be detected experimentally. The practical concept of homogeneity therefore embodies both a specificity to the characteristic and a parameter of measurement (usually the standard deviation) of the measurement method used, including the defined sample size of the test portion”. The degree of homogeneity of a property in a given material can be determined by repetitive measurements of the property in a number of small units by a method of sufficiently high precision. To assess homogeneity, the distribution of chemical constituents in a matrix is at the core of the investigation. This distribution can range from a random temporal and spatial occurrence at atomic or molecular levels over well defined patterns in crystalline structures to clusters of a chemical of microscopic to macroscopic scale. Although many physical and optical methods as well as analytical chemistry methods are used to visualize and quantify such spatial distributions, the determination of chemical homogeneity in a CRM must be treated as part of the uncertainty budget affecting analytical chemistry measurements. The statistical nature of the homogeneity problem can be treated theoretically and it is clear that it is a matter of large numbers. Either a large number of individual particles in the investigated unit or a large number of individual analyses will produce consistent mean results, whereas the deviations from the mean (fluctuation of individual results) are an indication of the element distribution in the matrix (homogeneity). A “homogeneity index“ or “significancecoefficient” has been proposed to describe area or spatial homogeneity characteristics of solids based on data evaluation using chemometrical tools, such as analysis of variance, regression models, statistics of stochastic processes (time series analysis) and multivariate data analysis (Singer and

I

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4 Particular Developments

Danzer 1984; Liebich et al. 1989; Liebich 1995). A complete strategy for characterizing homogeneity is elaborated to provide sufficient information on the behavior of elements in solid materials. Another frequently used approach, developed for the sampling of geological materials starts with the estimated number of individual grains per sub-sample, and ends up with the calculation of a sampling constant ,(I which is a function of the overall standard deviation and the mean mass of sample used for repetitive analysis (Ingamells and Pitard 1986). This so-called Ingamells’ sampling constant was slightly modified for practical reasons (square root of K,)by Kurfurst et al. (1984) to derive a relative element-specifichomogeneity factor HE (Stoeppler et al. 1985; ICurfurst 1998a).Both approaches have been widely used, particularly for characterizing CRMs and for guiding the production process of such materials (e.g. Pauwels et al. 1990; Luclter et al. 1992; Biangini et al. 1995; Mao Xueying and Chai Chifang 1995; Rossbach et al. 1998). Other authors used a simple -t- z standard deviation criteria or an outlier test ( F test) to check for significant differences between within-bottle and between-bottle results (Martin-Estebanet al. 1997; Quevauviller et al. 1995).The degree ofhomogeneity of elements and compounds in the materials tested in these studies does not seem to be adequately described and, hence, the asigned uncertainties in the mean values may represent only the bias between the analytical methods used in the certification. Particularly for direct microanalytical techniques using
At a first approximation, a gaseous or a liquid sample without suspended particles should have ideal homogeneity characteristics for all the constituents. As we are mainly concerned about solid samples when talking about CRMs for micro-analytical techniques, we should look for materials having expected intrinsic homogeneity for their constituents. Intrinsically homogeneous materials like single cell algae or deep sea sediment showed no need for sample preparation except drying. These materials typically are of such small particle size distribution that, even at very low sample mass, they exhibit good reproducibility of repetitive measurements of many elements (Zeisler 1998). It must be noted that conventional drying procedures were not applicable because they produced a “concrete-like”block which could not returned to the material’s originaI powdered form without milling. This increases the risk of contamination and destroys the original structure (e.g. the cell structure of algae) of these materials. To maintain the fine particle size and structure of naturally grown/

4.3 Cert$ed Reference Materials for Microanalytical Methods

formed material with naturally small particle size distribution, drying with a spray dryer is the preferred method. Other natural materials with expected intrinsic homogeneity properties that could be produced as CRMs include fractionated aerosols, conifer pollen, egg yolk or albumen, plankton of defined origin and size, and certain sediments. However, there arc particular problems related to the collection and preparation, of large enough quantities of such matrices for their preparation as RMs. RM producers might initiate research and development to provide technical solutions to handle some of these problems. For all other samples (biological,geological, environmental etc.), the reduction of sample particle size which is performed in two steps, the initial grinding and fine milling, is the most important concern. Two different milling procedures, jet milling (IAEA-338, IAEA-395) and ball milling (IAEA-jgG), were tested during a Coordinated Research Project (CRP) on “Reference materials for micro-analytical nuclear techniques” organized by the IAEA, Vienna, from 1994 to 1998 (IAEA/AL/ 083 1994, IAEA/AL/o95 1996). Both methods were suitable, but needed to be repeated several times to produce the small particle size that was required. The particle size reduction, e.g. of IAEA-395 from a median size of 30 pm to 3.5 pm, improved the homogeneity of elements. Sampling constants (the minimum mass that can be used to achieve a random error of I % at the 65% confidence level) improved from a factor of 1.2 for Sc, up to a factor of 800 for Au. The average improvement was about a factor of 2-10. (Ni Bangfa et al. 1996). From these initial experiences, it is clear that preparation of reference materials is critical with respect to the final particle size distribution, which should exhibit a low maximum (250 pm) and a narrow range in particle sizes. Milling techniques to meet such criteria are available today, and materials that show intrinsic uniformity are particularly suitable to achieve the desired properties. 4.3.3 Aspects o f Homogeneity Determination

Starting from a qualitative point of view, the particle size distribution of a material can give some indication of homogeneity. The larger the number of individual particles is in a certain aliquot used for a single determination, the higher the probability will be of determining equal concentrations of an analyte in subsequent aliquots. Quantitative estimation of the degree of homogeneity is preferentially carried out by repetitive determination of analytes in the solid by a technique of known intrinsic precision. The total variance of the observations (analyticalresults), Ri is composed of the variance of the analytical method, R: and the sampling variance from the heterogeneity of the study material, @ (Equation4.1) Rf = R;

+ R,’

In order to extract the degree of homogeneity from the variance of repetitive determinations, it is mandatory to determine the variance of the method used for analysis as accurately as possible. On the other hand, it is obvious that the variance of the

I

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132

I applied method can be estimated quite accurately, once the sampling variance (due 4 Particular Developments

to heterogeneity of the analyte) is precisely known. Such investigations lead directly towards a better understanding of uncertainty budgets in analytical chemistry (Rossbach and Grobecker 1999). The sampling variance of the material determined at a certain mass and the number of repetitive analyses can be used for the calculation of a sampling constant, K,, a homogeneity factor, HE,or a statistical tolerance interval (m * A) which will cover at least a 95 % probability at a probability level of I - a = 0.95 to obtain the expected result in the certified range (Pauwels et al. 1994).The value of A is computed as A = P2R, , a multiple of R,, where R, is the standard deviation of the homogeneity determination,. The value of K2 depends on the number of measurements, n, the proportion, P, of the total population to be covered (95%) and the probability level I - M (0.95). These factors for two-sided tolerance limits for normal distribution P2can be found in various statistical textbooks (Owen 1962).The overall standard deviation S; = (s/fi) as determined from a series of replicate samples of approximately equal masses is composed of the analytical error, Ra, and an error due to sample inhomogeneity, R,. As the variances are additive, one can write (Equation4.2):

Ingamells and Pitard (1986) introduced into analytical vocabulary the term “sampling constant”, Ks, defined as (Equation4.3):

K, = Rf . m

(4.3)

where: R,” is sampling variance and m is sample mass. The square root of this expression leads to the so-called relative homogeneity factor HE as introduced by Kurfiirst (Equation4.4): HE = Rs . f i

(4.4)

K, is expressed in the units of mass and is numerically equal to the sample mass necessary to limit the error due to sample heterogeneity (sampling uncertainty) to I % (with 68 % confidence). In order to determine the sampling variance accurately, it is necessary to minimize the individual components of analytical variance as much as possible. Once the sampling constant(s) are determined, one can predict what the magnitude of the sampling variance should be for a given analyte and for various masses of the sample of a given material. 4.3.4 Examples

Not all reference materials producers currently employ the various techniques that would characterize materials for microanalytical use. Such techniques include measurement of particle size distribution, particle composition, and the determination of component homogeneity with microanalyhcal techniques. Nevertheless, some

4.3 Certf7ed Reference Materials for Microanalytical Methods

initial characterization of existing and candidate CRMs by particle sizing techniques (Fajgelj and Zeisler 1998) and analytical determinations of component distributions (as discussed below) have shown that certain groups of materials could easily be developed for use with microanalytical techniques. For possible use of existing CRMs, users of microanalytical techniques can investigate the key properties of a material and apply a microanalytical technique, as outlined below, to establish some materials for their own quality control.

I

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4.3.4.1 Homogeneity Determinations with Solid Sampling Atomic Absorption Spectrometry

A suitable method to determine the degree of homogeneity of an element in a material is by repetitive analysis of a large number of small solid aliquots by direct solid sample analysis. As shown in Figure 4.3, there is a functional relationship between the sample mass used for analysis and the standard deviation of repetitive analysis. The smaller the sample mass is, the higher is the standard deviation of repetitive measurements. The case of Pb in IAEA 393, at masses greater than 2 mg, also shows that the overall standard deviation levels off at an approximate value of the instrument's precision (* 3 %). The constant factor in the fitting curve (4.68) represents the homogeneity factor HE for Pb in this particular material, IAEA 393, single cell algae (Sonntag and Rossbach 1997). Thus the standard deviation, which is

Rsd ph] 25

-

20

-

15

-

10

-

5-

0

Pb 0.0

l

0.2

~

0.4

l

~

0.6

l

0.8

~

1.0

l

sample mass [mg] f ( z ) = 4.68. Fig. 4.3 Relative standard deviation (Rsd) ["/.I over sample mass [mg] for Pb in IAEA 393, Algae. Each data point represents at least 20 individual measurements (Sonntag and Rossbach 1997).

'

1.2

l

1.4

~

l

1.6

'

l

'

i

134

I

4 Particular Developments

now merely related to the material's homogeneity and not influenced by any methodological bias, can be calculated according to Equation (4.4) and the mean values expressed as Equation (4.5)(Rossbach et al. 1998): Concentration= Mean * HE . rn-'I2

(4.5)

As the uncertainty of the mean is now related to the mass used for analysis and it is based on a careful evaluation of the element distribution in the material, this approach could help to evaluate further the total uncertainty budget for various analytical techniques as is required in accreditation and certification campaigns. A statistical tolerance interval for Img sample mass can be calculated according to Equation (4.6).

A minimum sample mass to achieve 5 % precision on a 95% confidence level is derived from Equation (4.7) (Pauwels et al. 1994) using a factor V 2for two-sided tolerance limits of normal distributions as given in standard statistical textbooks. UNC denotes the uncertainty level at which M should be given (in our case + 5 %). In the following example we have calculated the minimum sample mass required to obtain a 5 % relative standard deviation in repetitive measurements for Pb in IAEA-338,lichen: distribution of results: normal uncertainty wanted: 5% 2.2313 V 2(p= 0.95, I-a = 0.95, n= 100): Ro of the homogeneity study: 9.52 % R, for Pb as determined by liquid sample 3.5 % 8.85% R, according to Equation (4.2) 0.211 mg mass used for homogeneity study: According to Equation (4.7)the minimum sample mass is: M = (2.2313 x 8.85%/ 5 %)' x 0.211 mg = 3.3 mg, providing the critical parameter for a potential use of this lichen material with a microanalytical technique. 4.3.4.2 Homogeneity Determinations with Instrumental Neutron Activation Analysis

("4

INAA is well suited to study homogeneity of small samples because of its dynamic range of elemental sensitivity. The technique allows for the use of small solid samples, with the smallest usable sample size in the range of 0.5 mg to I mg as determined by handling and blank considerations. The INAA analytical procedure is well understood and characterized with mathematical relationships, Its analytical uncertainties can be sufficiently controlled and can be well determined for a particular procedure. This allows the calculation of the contribution of material heterogeneity to the uncertainty budget based on experimental data.

4.3 Certified Reference Materials for Microanalytical Methods

4.3.4.3 Uncertainty Budget of INAA

The results of activation analysis are subject to well known and common analytical sources of uncertainty, as well as method specific uncertainties, e.g. summarized by Greenberg (1997). and also in Section 2.2. In order for INAA experiments to measure differences in induced activity, i.e. differences due to heterogeneity in the amount of analyte in a given test portion, the experimental procedure is designed to allow only the following uncertainties to be part of the result: uncertainty due to homogeneity u H O M , uncertainty due to counting statistics u,, uncertainty due to activation uim uncertainty due to the gamma spectrometric measurement u,. Uncertainties relating to the determination of accurate quantitative results are not relevant in these experiments. The observed experimental variance of the INAA results is a summation of the variances of homogeneity and the relevant analytical components as shown in Equation (4.8):

Knowing the listed analytical components of variance allows the determination of the homogeneity component. The determination and control of uncertainty due to counting statistics (u,) is rather straightforward; this uncertainty is largely dependent on the sample composition, the decay characteristics of the indicator nuclides, and the assay parameters. The applied procedure optimizes irradiation, decay and counting parameters to obtain statistical uncertainties in a desirable range of I % to 0.1% relative for the majority of analytes assayed. This requires peak areas of tens to hundreds of thousands of counts. In the case of rapidly decaying activities, this is essentially achieved using the high count rate capabilities of the y spectrometers employed (Zeisler zoooa). The control of the irradiation uncertainties has been discussed by Becker (1989)and verified in this work. Depending on irradiation time and mode, i.e. fixed rabbit position or 180 degree inverted irradiations, uirr ranged at the NIST reactor irradiation facilities from 0.2 % to 0.08 % relative. The uncertainty due to the y spectrometry measurement (u,) is a combination of uncertainties in measurement geometry, spectrometry system data throughput, and y-spectrum evaluation. The measurement geometry for small sample analyses approaches almost ideal conditions since in essence point sources are produced in this INAA procedure. This uncertainty is commonly estimated at several tenths of a percent, but may be negligible in these experiments. The dependency of the y spectrometry measurements from the count rates has been checked by evaluating spectra obtained with the loss free counting (LFC) system in dual counting mode, i.e. accumulating the live spectral data and the loss corrected data simultaneously. No increase beyond corrected counting statistics due to LFC technique was observed in the measurement uncertainty, whereas the application of standard pile-up corrections to the same data was affected by uncertainties in the pile-up correction factor (Zeisler zooob). Uncertainties due to the y-spectrometryevaluationprocess can

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136

I be kept small when the spectrum shape is the same for all counts. Under these condi4 Particular Developments

tions, the relative uncertainties due to the measurement (urn)are estimated in the range of 0.5%to 0.3 %. The observed elemental variances of each measurement experiment and the components of the analytical variances discussed above are used to calculate variances due to heterogeneity for each element which are converted to relative uncertainties (R). These relative uncertainties then provide the input in the two relevant Equations (4.3 and 4.4) that are commonly used to express elemental homogeneity of a sample as a function of sample mass (w). 4.3.4.4

Homogeneity Factors in Test Materials Determinedwith

INAA

The process for determining homogeneity values is exemplified with results from NIST SRM 1648 Air Particulate Matter shown in Table 4.4. The table illustrates that conclusive sampling data can be obtained for all elements where the analytical uncertainty is small compared to the uncertainty from heterogeneity. Elements that are assayed with two y-ray lines give similar results. As shown in other investigated materials, the analysis of the uncertainty budget gives ambiguous results when the overall experimental uncertainty is small. For these cases much smaller sample sizes should be taken. When the observed experimental uncertainty is rather large, e.g. due to large counting statistical uncertainties associated with the INAA assay for some elements as shown in Table 4.4, homogeneity results may be biased. As INAA has low sensitivity for these elements in the investigated materials, their homogeneity must be determined with a different technique. The described process has been used for a number of existing and candidate CRMs. Table 4.5 lists the Kurfurst elemental homogeneity factors for elements Calculation o f homogeneity factors for selected elements in NIST SRM 1648 based on experimental INAA results

Tab. 4.4

Nement(y-energy evaluated}

A1

cu

C1-1642 C1-2178 Mg-1014 Mn-846 Mn-1811 Na-1368

Ti V

[mgl

Observed uncertainty I"/. rel.]

A priori counting uncertainty /% rel.]

Uncertainty due t o hetero(estimate) geneity I/. rel.] @ rel.]

1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2

5.491 2.907 3.084 4.282 0.882 1.480 1.325 1.264 0.654

2.11 0.70 0.44 2.79 0.32 0.30 0.72 0.94 0.34

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

Mass

Other uncertainties

5.061 2.805 3.038 3.233 0.765 1.418 1.069 0.788 0.473

KufUrst

Homogeneity

lngamells Sampling

Factor HE

Constant [mgl

5.54 3.07 3.33 3.54 0.84 1.55 1.17 0.86 0.52

0.01 30.7 9.4 11.1 12.5 0.7 2.4 1.4 0.7 0.3

I

4.3 Certified Reference Materialsfor Microana/yt;ca/Methods Kurfurst elemental homogeneity factors for selected RMs determined from experimental uncertainties with an INAA procedure using short-lived indicator nuclides. The sample masses ranged from 0.5 m g to 2.5 mg; the number o f determinations were 12 for each material

Tab. 4.5

N I S T SRM 1648 Urban particulate matter N I S T SRM 1649 U r b a n dust/ organics N I S T SRM 1547 Peach leaves NISTCandidate SRM Abyssal sediment

IAEA-338

0.10

5.5

3.1

-

3.5

0.84

1.2

0.86

0.52

1.1

<5

5.4

-

2.6

2-0

4.2

<S

1.6

17.7

2.0

6.2

-

2.0

1.5

10.7

2.2

5.1

1.0

-

1.5

-

4.1

1.6

1.6

5-4

1.3

2.1

1.4

1.1

-

5.6

0.30

2.0

4.0

0.7

-

-

3.4

2.9

1.7

8.9

2.5

-

-

Lichen

IAEA-386 Bovine liver

determined with INAA via short-lived nuclides. The results illustrate that existing CRMs cannot be unconditionally used with small sample procedures, but that extensive measurements may show for which elements small sample analysis may produce reliable results. The potential heterogeneity for A1 in SRM 1547 was reported earlier by separating a silicate fraction from the material (Lindstrom et al. 1990).Na and C1 are probably more homogeneous in SRM 1547 than indicated in Table 4.5 due to fluctuations of the blank contribution in INAA. 4.3.5

Conclusion

With the exception of direct microanalytical techniques, analytical methods based on dissolution of materials would likely not be capable of performing analyses on such small individual samples in the mass range described here. Hence, the total analytical uncertainty in an analysis involving dissolution of normally a larger sample is less influenced by the material’s heterogeneity than it is by the uncertainties related to various steps (weighing, digestion, pipetting, diluting and measurement) in the analytical process of these methods. As micro-analytical techniques (performing direct analysis on a 510mg sample mass) have a particularly distinct demand for very homogeneous CRMs, it becomes necessary to provide element-specifichomogeneity information in the CRM certificates. The distribution of elements in a material can be evaluated experimentally by repetitive analysis. The scattering of results from a method with laown intrinsic precision is related to the mass of sample consumed for individual analysis. The

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possibility exists to determine a sample mass related factor to describe an element’s homogeneity characteristics in a reference material. If such factors could be given in the certificate of a CRM, users of the CRM would have a tool for precise evaluation of their methods’ uncertainty, a condition which would clearly enhance the harmonization and traceability of analytical results in analytical chemistry.

4.4

CRMs as calibrants Markus Stoeppler, Ulrich Kufurst and Knut Ohls 4.4.1

Principles

Despite the general recommendation that the preferred use for CRMs is method validation, there are increasing numbers of publications that describe the application of CRMs for direct calibration of elemental contents in technical, environmental and biological materials, generally achieving very good to quite acceptable results. This is particularly the case for methods that do not need decomposition prior to analysis, including solid sampling graphite furnace AAS, to some extent the “socalled slurry technique, for electrothermal vaporization-inductivelycoupled plasmaatomic emission and -mass spectrometry (Kurfurst, Igg8a) and neutron activation analysis (e.g. Rossbach and Stoeppler 1987);see also Section 3.2. How can CRMs be applied in this way with such success? The reasons depend either on the methods used, or the properties of the studied CRMs. For direct calibration often CRM sample masses well below the producers recommended samples masses of at least IOO mg for reliable results (e.g. IAEATECDOC-854) could be used. On the one hand, as an example, with solid sampling graphite furnace AAS using various different technical systems and an appropriate temperature program of the atomizer unit, it was shown that with sample intakes at the low-milligram level or even below (requiring a rather high number of single measurements) a sufficiently small uncertainty and thus acceptable results could be obtained (Kudurst 1984, 199%). This applies also to the other methods mentioned above. On the other hand, homogeneity studies with microanalytical techniques proved that in a significant number of CRMs at sub-sample levels around and even below 20 mg certified values remained valid for calibration purposes (Rosopulo et al. 1983; Pauwels et al. 1991; Pauwels and Vandecasteele 1993; Pauwels et al. 1993). This underlines the fact of a much better homogeneity for many analytes than documented in the certificates of these CRMs; for details see Sections 2.2 and 4.3. Typical examples are materials consisting of very small particles with homogeneous element distribution, e.g. milk powder, single cell algae, fish muscle, lyophilized body fluids and some sediments as well as materials prepared by special procedures such as spray drying and grinding at liquid nitrogen temperature.

4.4 CRMs as calibrants

An important prerequisite for the use of CRMs as calibrants, at least for optical methods and particularly all AAS modes, is that they should match the matrix and level of analyte contents of the materials to be analyzed as closely as possible, so that potential matrix effects will be compensated if calibrant and sample material are affected by the applied method, e.g. the temperature program for furnace techniques, in the same way. Further it is very important for all methods that the CRMs used should not show a “nugget effecY‘, i.e. particles with extremely high analyte content that can lead to a high analyte heterogeneity (Kurfiirst 1991; Kurfurst et al. 1993; Liicker and Thorius-Ehrler 1993).In some cases also for the moisture content a correction factor has to be applied if the actual moisture content differs significantly from that stated in the certificate. For relatively simple matrices, such as pure metallic CRMs synthetic reference materials for direct calibration were prepared and used, for example Bi, Cd, Hg, Pb and Tl in high purity gallium (Hiltenkamp and Jackwerth 1988),Ag in copper (Pauwels et al. 1990) and Au and Pd in silver (Hinds 1993).Direct calibration by solid biological materials with added analyte belongs also to these quite successfully applied techniques (Hofmann et al. 1992). 4.4.2

Calibration Techniques

Apart from relatively rare cases where single point calibrationwith a material very similar in matrix and analyte content is sufficient, for example in industrial product control, it is more common that a calibrationfunction has to be constructed to cover the expected concentration range of the analyte in the unknown material. As an example for solid sampling graphite furnace AAS and with peak area evaluationtwo approaches are common: the use of differentintakes of one suitablematerial or intakes oftwo or more materials with different contents of the analyte. As the certified content in the CRM used may also more or less deviate from the true content (uncertaintyof the certified value), the second approach usually is preferred if suitable materials are at hand. The number of calibration points distributed over the complete range depends mainly on the homogeneity of the CRMs used, often already known from the laboratories’own experience, but must be a sufficiently high number (usually 10-20)to obtain a useful calibration function (Pauwelset al. 1991). Figure 4.4 shows an example for the use of several CRMs from NIST and BCR with certified or indicative values for chromium for the construction of a calibration curve. Increasing intakes of the CRMs were used covering the range 3-23 ng Cr. It is further important to note that if only solid samples are taken, the calibration curve passes through the origin as is obvious in Figure 4.4. An intercept usually caused by blank values from reagents and digestion vessels in wet analysis can be excluded except in the case of matrix modification. The validation of calibration with CRMs is commonly performed by parallel application of independent methods, for details see below.

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mass of Cr I ng

Fig. 4.4 Calibration curve for chromium, constructed from measurements of increasing intakes o f five different CRMs: NIST SRMs 1646 Estuarine sediment, certified content 76 f 3 rng/kg; 1632a Coal, certified content 34.4 + 1.5 mg/kg; BCR CRMs 141 Calcareous loam soil, indicative content 75 mg/kg, 142 Light sandy soil, indicative content 74.9 rng/kg and 143 Over fertilized soil, indicative content 228 mg/kg (Kurfurst 1998b).

4.4.3

Examples 4.4.3.1

Biological Materials

For the determination of As, Cd, Zn, Pb, Mn, Cu and Cr with solid sampling GFAAS in a number of biological samples, calibration curves using one NIES CRM and three NIST SRMs were constructed and successfully used (Atsuyaet al. 1987). Using the slurry-method and GF-AAS in Cu, Cr, Fe and Pb were determined in various pine needle samples, using NIST SRM 1575Pine needles slurry as calibrant. Comparison of the obtained results showed good agreement with wet ashing (Carrion et al. 1988). Cadmium and lead profiles in birds feathers based on samples in the milligram range and below were determined by solid sampling Zeeman-GFAAS, using a feather RM produced by milling at liquid nitrogen temperatures and characterized for its metal contents with different analytical methods (Hahn et al. 1990). Within collaborative work on element concentrations in a number of biological reference materials using solid sampling and other analytical methods, calibration of Cd, Cu, Pb and Zn in BCR CRM 185 Bovine liver with solid CRMs was performed for each element with a reference material of the same matrix, NIST SRM 1577

4.4 CRMs as calibrants

Bovine liver and CRMs of a different matrix and good to acceptable results were obtained. (Schauenburg and Weigert 1991). During production and characterization of various internal animal tissue reference materials for a number of metals, a comparative study was performed for Pb in six bovine teeth and two bovine bone materials using calibration with a solid RM and two versions of wet chemical analysis with GF-AAS and electrochemical (DPASV) detection. There was good agreement in the range of approx. 1.3-3 mg/kg dry weight for all techniques used (Liicker et al. 1992). In a microhomogeneity study of a BCR candidate RM (422 cod muscle) Pb, Cd, Hg, Fe and Zn were determined with solid sampling GF-AAS, calibration was done with four different suitable BCR CRMs (Pauwels et al. 1993). 4.4.3.2

Environmental and Geological Materials

With solid sampling-electrothermal vaporization-inductively coupled atomic emission spectrometry (SS-ETV-ICP-AES),Cu in two environmental CRMs was determined using a third CRM with similar matrix as calibrant. Comparison with a reference solution showed good agreement (Verrept et al. 1993). The same technique together with SS-GF-AAS (solid sampling graphite furnace AAS) was recently systematically investigated for the determination of Cr, Cu, Ni, Pb and Zn in 28 sediments, soils, rocks and advanced ceramics. In order to obtain acceptable sample homogeneity, the sample materials were ground until analyixal results were reproducible and representative for the bulk material. Calibration was done by using a number of suitable CRMs from various producers. Calibration was performed by variation of analyte content using different CRMs for SS-GF-AAS. In this case the matrix composition of the CRMs should have the highest possible similarity to the composition of the sample matrix. Using SS-ETV-ICP-AESthe matrix composition of the different materials was negligible low. Thus calibration was feasible with different intakes of just one CRM with analyte concentration in the required range. Precision and accuracy of SS-GF-AAScould be improved by application of 3-D calibration plots. In this way the two studied methodological approaches complement one another concerning their analytical performance (Schron et al. 2000).

The total content of As, Cd, Cr, Cu, N i and Pb was determined in contaminated soils and sediments using the slurry technique and Zeeman GF-AAS, either by calibration with aqueous solutions of the analytes or slurries of some suitable CRMs. Except for Cr, where only the calibration with a solid CRM was successful, good agreement was found between both calibration approaches (IUemm and Baumbach 1995). Using a newly developed, transversely heated graphite atomizer and D2-background correction (for details see Sections 2.2 and 4.3), Cd, Pb and Cr were determined in cement and river sediment samples. Of the various calibration approaches applied the best results, also in comparison with wet chemical procedures, were achieved with calibration curves constructed by means of different BCR CRMs with different analyte concentrations and usually n = 10individual intakes (Nowlta and Miiller 1997).

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The same instrumentation and the same calibration approach, i.e. using varying weights of two or more different CRMs for the calibration curves, was applied for the quick determination of traces of Cd, Pb, V, Cu, Ni, As and Cr in a number of barytes in comparison to X-ray fluorescence spectrometry and flame AAS after aqua regia extraction. In general good agreement was found, but for some samples additional grinding was necessary, possibly because of inhomogeneous analyte distribution observed for Cu (Nowka et al. 1999). 4.4.3.3

Technical Materials

Normally one can assume that most metallic samples contain elemental traces in a homogeneous distribution. Lead, Bi, Zn, Ag and Sb in steel and nickel-base alloys were determined, first by using the graphite boat technique for routine analysis. Several calibration approaches were studied and it was found that the best results could be obtained by using various amounts of a number of solid alloyed steel or pure iron CRMs and to plot absorbance against concentration of the element sought (Backman and Karlsson 1979). This technique was subsequently modified for the determination of Co, Cu and Mn in pure iron and steel samples (Sommer and Ohls 1979). Solid sampling is, so far, the only method that allows the speciation of Mg contained in metals and other materials. At a temperature of 18oo0C,the metallic Mg is completely vaporized and e.g. MgO appears at 2800°C. So, RMs or synthetic sample mixtures can be analyzed to prepare new RMs (Ohls 1981). For the determination of Pb, Zn and Mn in coal samples with solid sampling GFAAS, calibration curves were prepared either from three appropriate NIST coal SRMs or solution standards. Identical results were obtained for Pb and Zn, while only solid SRMs gave correct results for Mn (Ali et al. 1989). Mercury in various cement products was determined with a special mercury oven for solid samples. Calibration was performed with four BCR CRMs and one NIST SRM with different Hg content as well as with a reference solution and excellent agreement found (Bachmann and Rechenberg 1991). Solid sampling techniques for ICP emission spectrometry need to be calibrated by very carefully characterized RMs or CRMs, if available. For example, the determination of Pb by vaporization of very small sample amounts into an ICP source was shown to be independent from matrix effects by using different CRMs for calibration (Ohls 1989). The composition of steels or other metals is commonly analyzed by emission or X-ray spectrometry during and after the production process. Both methods have to be calibrated by solid samples. These are either exactly analyzed samples taken from the same process or synthetic melted mixtures of the matrix with added accompanying elements (RMs).Available CRMs are then used to control the slope of the calibration function. Today, available RMs and CRMs are increasingly and exclusively used in spectral laboratories as the chemical analysis became much restricted and typical control laboratories were totally closed (Slickers 1993). The increasing application of laser ablation or induced techniques for local and bulk analysis, whereby either the sampled vapor or the excited light becomes trans-

4.5 RMsfor Radioisotopes, Stable Isotopes and Radiopharmaceuticals

ported to an ICP source or to an optical spectrometer directly, makes it necessary to use solid RMs or CRMs, which have to be checked for homogeneity prior to any vaporization of nanogram to microgram amounts (Hoffmann et al. 1997). 4.4.4

Conclusion

Since the availability of high quality CRMs, across a broad range of matrices, is continuously increasing so improved methods for preparation, certification and homogeneity determination can be expected - see Section 4.3, and future trends for reference material activity in Chapter g - the possibility of obtaining suitable materials for a large variety of applications will increase as well. This will certainly be also the case for the use of solid CRMs as calibrants for microanalytical methods. These particular CRMs will be further of paramount importance if ubiquitously occurring elements and elements in difficult to digest geological and industrial materials have to be determined. An example is the growing need for precisely characterized trace elements in semiconductor materials, for which reliable results often cannot be achieved by wet analysis because of external contamination at all analyt~calsteps.

4.5 RMs for Radioisotopes, Stable Isotopes and Radiopharmaceuticals Robert Parr

Isotopic measurements of various kinds are widely practised in a large number of scientific disciplines ranging from radiation safety, to nuclear medicine, studies of climatic processes and the tracing of water supplies. As for the other kinds of measurements discussed in this book, isotopic reference materials are needed for establishing traceability of the measurement results, for quality control, and in some cases also for calibration. This Section gives a brief overview of the main kinds of materials available and the purposes for which they are used. Emphasis is given to materials of natural origin (Acompositionalor Amatrix reference material). 4.5.1

Radioisotopes 4.5.1.1

Requirements and uses

Reference materials for radioisotopes have mainly been used for purposes relating to nuclear and radiation safety. Historically, the development of such materials first arose from the need to assess the risk to human populations caused by worldwide contamination of food and the environment as a consequence of atomic bomb testing - particularly from bombs exploded in the atmosphere. Even now, although atmospheric testing ceased many years ago, the residues from these tests still remain the main source of radionuclides such as 137Csand 90Srin the global environment (though locally, other sources may be more important in some countries).

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The nuclear power plant accident at Chernobyl in April 1986 (IAEA Technical Report 1991)proved to be a much more potent source of environmental contamination in many surrounding countries, over distances up to several thousands of kilometers, and was a cause of worldwide problems in international trade in food products contaminated (or possibly contaminated) with radionuclides. The resulting requirement by many countries to establish systems for monitoring radionuclides in foodstuffs and in the environment led to a large worldwide increase in the demand for suitable reference materials. Fortunately, atomic bomb testing and the Chernobyl accident are slowly becoming mere historical memories that have little current impact on human health. However, most countries still insist on maintaining the ability to monitor radionuclides in food and the environment in order to be prepared for a possible future nuclear accident. To this end, validated measurement techniques are required (IAEA Technical Report 1989) and reference materials play an essential role in establishing this validation. An important relevant international activity in this area is the so-called ALMERA network (Analytical Laboratories for Measuring Environmental Radioactivity) supported by the International Atomic Energy Agency (IAEA AL Report 1997). Similar demands for reference materials also arise in connection with the monitoring of radioactivity in and around nuclear installations (nuclear power plants, nuclear fuel and reprocessing plants, and nuclear waste facilities).These, in fact, are now the main applications of radionuclide reference materials. Some of the intended categories of use of radioisotopic reference material have been reviewed recently by Fajgelj et al. (1999).They include assignment of property values, establishing the traceability of a measurement result, determining the uncertainty of a measurement result, calibration of an apparatus, assessment of a measurement method, use for recovery studies and use for quality control purposes. It should be noted however that, in general, natural matrix reference materials are not recommended for calibration purposes. This should preferably be done with pure chemical forms of the element labelled with the isotope of interest. Calibrated isotopic sources of this kind are available from a number of commercial suppliers and are not the subject of this review. 4.5.1.2

Available Reference Materials

A wide variety of reference materials is now available, covering several different kinds of natural matrix such as food (e.g. milk powder), human tissues (e.g. liver), marine biological materials (e.g. tuna fish) and soils and sediments. The radionuclides of interest cover naturally occurring ones (e.g. 40K,226Ra),fission products (e.g. "Sr, 137Cs)and other nuclides of importance in the processing of nuclear fuels (e.g. 238U,239Pu).The International Atomic Energy Agency (IAEA) is the main, but not the only, producer of such reference materials, as may be seen from Table 4.6 which provides an overview of currently available reference materials for a typical aemitter (241Am),f3-emitter (90Sr),and y-emitter (137Cs).

4.5 RMsfor Radioisotopes, Stable Isotopes and Radiopharmaceuticals Tab. 4.6

Overview of available reference materials for 24'Am, 13'Cs and "St

Marine sediment, NIST

6

'OS~

IAEA

17

l37CS

NIST GBW IAEA

4 2 19

NIST CANMET GBW IRANT

4 2 2 1

Freshwater sediment; soil; human liver & lung Marine sediment, plant, flesh & water; fresh water sediment; soil; terrestrial plants; milk powder; animal bone Freshwater sediment; soil River sediment Marine sediments, plant, flesh 8 water; lake sediment; soil; terrestrial plants; milk powder Freshwater sediment; soil Spruce twigs & needles River sediment Coal fly ash

0.004-1.25 0.2-1310

5-1090 6-20 0.3-12 000

0.3-60 34-110 80-130 1

a) CANMET Canada Centre for Mineral and Energy Technology, Toronto, Canada

GBW National Research Centre for Certified Reference Materials, Beijing, China IAEA International Atomic Energy Agency, Vienna, Austria IRANT Institute of Radioecology and Applied Nuclear Techniques, Kosice, Slovak Republic NIST National Institute of Standards and Technology, Gaithersburg, USA b) Number of reference materials c) Range of activities in the available reference materials (Bq/kg)

Tab. 4.7

IAEA radioisotope reference materials selected for upgrade (Fajgelj et al. 1999) Matrix

Measurands of lnterest

IAEA-152 IAEA-312 IAEA-314 Soil-6 SL-2

Milk Powder Soil Stream Sediment Soil Lake Sediment

IAEA-375

Soil

Radionudides Ra, Th and U Ra, Th and U Sr-90, Cs-137, Ra-226 and Pu-239 I<-40,Sr-90, '2-137, Db-210, Ra-226, Ra-228, Th-228, Th-234, U-238 and h-239/240 K-40, Sr-90, Ru-106, Sb-125, (3-134, Cs-137, Th-232 and U-238

Code

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4.5.1.3

Future Developments

The concept of preparing a reference material with property values traceable to SI units is a relatively recent one which has not yet been applied in practice to natural matrix radioisotopic reference materials. This is one of the main reasons why such materials are generally not recommended for calibration purposes. Traceability, however, is such an important concept that it will undoubtedly also be applied in future to radioisotopic reference materials. The IAEA is giving high priority to such work in its own reference material program. Table 4.7 provides details of some existing materials which have been selected for upgrade. 4.5.2 Stable Isotopes 4.5.2.1

Requirements and uses

Reference materials for stable isotopes have a wide variety of applications, the great majority of them are in areas relating to: isotope hydrology biochemistry, physiology and medicine (3) nuclear material accountability (I)

(2)

In the first two ofthese areas it is mainly isotopes of light elements (e.g.hydrogen, carbon, nitrogen and oxygen) that are of interest; in the third area the isotopes of interest are mainly those of heavy elements (e.g.uranium, thorium and plutonium). The great majority of the reference materials used in such studies are chemically pure compounds, and usually it is the relative deviation of the isotopic ratio from a standard (expressed as &values) that is of interest rather than the absolute isotopic ratio in the given sample. For example, in isotope hydrology (IAEA-TECDOC-825 1995; Groning et al. rggg), the available reference materials are all chemically pure compounds such as carbonates, sulfates, sulfides, nitrates, graphite, or polyethylene; or are nearly pure natural materials such as distilled water, carbonate rock, silicates, refined oil, sugar, cellulose and similar compounds. Some of these materials are primary reference materials which define conventional scales for reporting measurement results. One of the main examples is that of VSMOW (Vienna Standard Mean Ocean Water), which by international agreement has been defined as having zero 6values (Groning et al. 1999). Unfortunately, however, VSMOW is expected to be exhausted within the next seven to ten years, which is the reason why it is now important to develop alternatives. For biochemical and medical applications, the International Atomic Energy Agency (IAEA) has issued a variety of stable isotope reference materials (Parr and Clements 1991) containing elevated levels of 'H, 13C, 15N and l8O. They are all chemically pure compounds and are intended for use in checking the calibration of mass spectrometers used in relevant research and clinical diagnosis (Hein and Roseland Klein 1987). Another major supplier of stable isotope reference materials is the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. These materials

4.5 RMsJor Radioisotopes, Stable Isotopes and Radiopharmaceuticals

may be divided into two major groups: (I) those certified for isotope amount ratios and hence suitable for calibration of measurements of isotope abundance ratios and (2) those certified for isotope amount content and hence suitable for direct use in isotope dilution as "spikes" against which an unknown amount of an isotope or an element can be measured. These reference materials are also suitable for calibration of instruments for isotopic measurements, calibration and evaluation of isotopic measurement procedures, development of isotopic measurement methods, and nuclear material accountability measurements. Many of these IRMM materials are, in fact, concerned with the last of these applications, i.e. in the area of nuclear material accountability. Some of them have been examined by a nuclear experts committee and certified as EC-NRMs (European Community - Nuclear Reference Materials). All values of the IRMM Isotopic Reference Materials are traceable to the SI (the international system of base quantities and base units). Isotopic measurement results corrected by means of these Isotope Ratio Reference Materials have reduced (ISO/BIPM) uncertainties. Isotopic measurements carried out against these Spike Reference Materials are traceable to the SI, if carried out properly. Further details are available from IRMM website; see Chapter 8. 4.5.2.2

Future Developments

An increasing number and variety of stable isotope reference materials will certainly become available in future. At present there are only very few materials for elements of intermediate atomic weight (Mg, Cr, Fe, Zn etc.), mostly from IRMM, and this deficiency will almost certainly have to be rectified. However, there is still also a need for new isotopic standards for some of the lighter elements. For medical studies, the IAEA is considering preparing a new doubly-labelledwater standard for use in e n e r g expenditure studies (Prentice 1990).In isotope hydrology, one of the most important activities will be to create suitable replacements for some of the existing reference materials for which supplies are becoming exhausted, particularly VSMOW (Vienna Standard Mean Ocean Water) (Groning et al. 1999). 4.5.3

Radiopharmaceuticals

Quality assurance of radiopharmaceuticalpreparation and use is obviously a very important topic because of its direct impact on patient diagnosis,treatment and health (see, e.g. Abreu 1996). Reference materials play only a small - but nevertheless important role in this process, mainly in the area of calibration of radioactivity-measuringinstruments. The materials of interest are all pure chemical containing calibrated activities of selected radionuclides used commonly in nuclear medicine (e.g. 57C0,"Ga, '"In, I2'I, 1311, 99Mo/99mTc, 32P, 153Sm,"Sr, 201T1,133Xeand '%). The US National Institute of Standards and Technology (NIST) operates an external quality control program on a varying schedule from one year to the next (Golas1998).Details of the current program are available from the NISTwebsite.

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4.6 References

ABREUSH (1996) The nature of quality assurance in nuclear medicine and the American College of Nuclear Physicians' quality assurance programs. Eur J Nuclear Med 23(1):39-103. ALI AH, SMITHBW, WINEFORDNER JD (1989) Direct analysis of coal by electrothermal atomization atomic-absorptionspectrometry.Talanta 362393-896. ATSUYAI, ITOH K, AKATSUKAK (1987)Development of direct analysis of powder samples by atomic absorption spectrometry using the inner miniature cup technique. Fresenius Z Anal Chem 328338341, BACHMANNG and RECHENBERG W (1991)Aufschluss fur die Atomspektrometrische Quecksilberbestimmung von Stoffen der Zementherstellung. In: WELZ B, ed. 6. Colloquium Atomspektrometrische Superanalytik,pp 699-676. Bodenseewerk Perkin-Elmer GmbH, Uberlingen. BACKMANNS, KARLSSONRW (1979)Determination oflead, bismuth, zinc, silver and antimony in steel and nickel-base alloys by atomic-absorption spectrometry using direct atomization of solid samples in a graphite furnace. Analyst I O ~ : I O I ~ - I O ~ ~ . BECKERDA (1989)NIST Tech. Note 1272: 238. BERMANSS, BOYKOVJ (1992) ICES seventh round intercalibration for trace metals in biological tissue - ICES 7/TM/BT (part 2). ICES Cooperative Research Report No 189. International Council for the Exploration of the Sea, Copenhagen, Denmark. BIAGINIR, DERSCHR, DE FELICE P, JEROME SM, PERKIN EME, PONAC, DE SANOITJ, WOODS JM (1995) Homogeneity testing of spiked reference materials. Sci Total Enviyon 173/174:267-274. BORELLAP, BARGELLINIA, CASELGRANDI E, M E N D I ~A, O PATRIARCA M, TAYLOR A, VIVOLI G (1998) Selenium determination in biological matrices. Microchem J 58325-336. BRAITHWAITERA, GIRLING AJ (1988) Bovine reference materials for accuracy control of blood lead analysis. Fresenius Z Anal Chem 332:704-70g. BROWN SS, NOMOTOS, STOEPPLERM, SUNDERMAN FW JR (1981) IUPAC reference method for analysis of nickel in serum and urine by electrothermal atomic absorption spectrometry. Clin Biochem 14:295-299. BULLOCKDG, SMITHNJ, WHITEHEAD TP (1986) External quality assessment of assays of lead in blood. Clin Chem 32x884-1889. B U R N ED~(1996) Understanding Accreditation.ACB Venture Publications, London. C A R R I ~N, N DEBENZOZA, MORENOB, FERNANDEZ EJ, FLORESD (1988) Determination ofcopper, chromium, iron and lead in pine needles by electrothermal atomisation spectrometry with slurry sample introduction.J Anal At Spectrom 3:479-483. MS, GARDEN LM, HOLDEN AJ, MILESDL (1999) Atomic CAVE MR, BUTLER0, COOKJM, CRESSER spectrometry Update: Environmental Analysis. J Anal Atom Spectrom 14:279-353. CHANCEDH, ADAM BW, SMITHSJ, ALEXANDER JR, HILLMAN SL, HANNON WH (1999) Validation of accuracy-based amino acid reference materials in dried-bloodspots by tandem mass spectrometry for newborn screening assays. Clin Chem 451269-1277. JM (1996) Correcting measurement errors using reference materials in method CHRISTENSEN validation. Mikrochim Acta I Z ~ : Z ~ I - Z ~ O . COBBAERT C, MORALES C, VAN FESSEMM, KEMPERMAN H (1999)Precision and accuracy and linearity ofradiometehEMLTM105 whole blood metabolite biosensors. Ann Clin Biochem 36:730-738. COTLOVEE, HARRIS EK, WILLIAMS G (1970) Biological and analytic components of variation in long-term studies of serum constituents in normal subjects. Physiological and medical implications. Clin Chem 16:1028-1032. DE BOERJ (1997) The preparation of biological reference materials for use in interlaboratory studies on the analysis of chlorobiphenyls, organochlorine pesticides and trace metals. Mar Poll Bull 35:84-92. DE BOERJ and SMEDES F (1997) Effects of storage conditions of biological materials on the contents of organochlorine compounds and mercury. Mar Poll Bull 35:93-108.

4.6 References

BOER J and WELLS DE (1996) The 1994 QUASIMEME laboratory performance studies: chlorobiphenyls and organochlorine pesticides in fish and sediment. Mar Poll Bull 32:654-666. D E BOERJ and WELLS DE (1997) Chlorobiphenyls and organochlorine pesticides in fish and sediDE

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ments -three years of QUASIMEME laboratory performance studies. Mar Poll Bull 35:52-63. BOERJ, TUINSTRA LGMTH,MAIEREA ( 2 0 0 0 ) The certification of the contents (mass fractions) of chlorobiphenyls 28, 52, 118, 138, 149, 153, 170 and 180 in mussels (CRM 682). European Commission DG XII, Brussels, Belgium (in press). D E BOERJ, VAN D E R MEERJ, BRINKMANUATH (1996) Determination of chlorobiphenyls in seal blubber, marine sediment, and fish: interlaboratory study. J Assoc Off Anal Chem 79533-96. DELVES HT (1984) Use reference samples rather than reference methods. Anal Proc 2q91-394 DELVESHT, DIAPERSJ. OPPERT 5, PRESCOTT-CLARKE P, PERIAM J, DONGW, COLHOUN H, GOMPERTZ D (1996) Blood lead concentrations in United Kingdom have fallen substantially since 1984. Brit Med J 313:883-884. Department of the Environment (1983) European Community screening programme for lead United Kingdom results for 1981. Pollution Report No. 18, HMSO, London. European Commission (1993) Guidelines for the production and preparation of BCR reference matenals. Document BCR/48/93. DG XII, Brussels, Belgium. FAIRMANB, HINDSMW, NELMSSM, PENNY DM, GOODALL P (1999) Atomic Spectrometry Update: Industrial analysis: metals, chemicals and advanced materials. J Anal Atom Spectrom 14:2001-2030. FAJGELJ A, RADECKI 2, BURNS KI, MORENOBERMUDEZJ, DE REGGEPP, DANESIPR, BOJANOWSKI R (1999).Intended use of the IAEA reference materials, Part I: Examples on reference materiM, eds. als for the determination of radionuclides or trace elements. In: FAJGELJA, PARKANY The Use of Matrix Reference Materials in Environmental Analytical Processes, pp 65-80. Royal Society of Chemistry, Cambridge. FAJGELJA, ZEISLERR (1998) Particle size determination of some IAEA and NIST environmental and biological reference materials. Fresenius J Anal Chem 360:442-445. Fox MP, REILLYAA, SCHNEIDERE (1999) Effect of the ratio of free to total prostatic-specific antigen on interassay variability in proficiency test samples. Clin Chem 45:1181-1189. FRASERCG, HYLTOFT PETERSON P (1993) Desirable standards for laboratory tests if they are to fulfil medical needs. Clin Chem 39:1447-1455. FRASERCG, HYLTOFT PETERSON P (1999) Analytical performance characteristics should be judged against objective quality specifications. Clin Chem 45:321-322. GERMANY (1979) Technische Regel fur Gefahrenstoffe TRGS 410 - - Statistische QualitatssicherW, THOMAS HP, eds. pp 1-12. C Heymanns, Koln. ung. In Gefahrstoffverordnung, WEINMANN GOLASDB (1998) NIST radiopharmaceutical standard reference materials and the NEI/NIST radiopharmaceutical measurement assurance program. Appl. Radiat Isot 49/4:329-334. GREENBERG RR (1997) in: Trace Elements in Man and Animals - 9, FISCHER PWF, L'ABBEMR, COCKELL KA, GIBSON RS, eds. pp 410-413. NRC Res. Press, Ottawa, Canada. GRONINGM, FROLICHI<, DE REGGEPP, DANESIPR (1999) Intended use of the IAEA reference materials, Part 11: Examples on reference materials for stable isotope composition. In: FAJGELJ A, PARKANY M, eds. The Use of Matrix Reference Materials in Environmental Analytical Processes, pp 81-92. Royal Society of Chemistry, Cambridge. M (1990) Zeeman SS-GFAAS- an ideal method for the evaHAHNE, HAHNK, MOHLC, STOEPPLER luation ofleadand cadmium profilesin birds feathers. Fresenius J Anal Chem 377306-309. HILTENKAMP E, JACKWERTH E (1988) Untersuchungen zur Bestimmung von Bi, Cd, Hg, Pb und Tl in hochreinem Gallium durch Graphitrohr-AASbei Verdampfung fester Proben. Fresenius Z Anal Chem 332:134-139. HINDSMW (1993) Determination of gold, palladium and platinum in high purity silver by different solid sampling graphite furnace atomic absorption spectrometry methods. Spectrochim Acta 48B:435-445. E, LUDKE C, SCHOLZE H (1997) Is laser ablation-ICP-MS an alternative to solution HOFFMANN analysis of solid samples? Fresenius J Anal Chem 359394398, DE

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HOFMANN C, VANDECASTEELE C, PAUWELS J (1992) New calibration method for solid sampling Zeeman atomic absorption spectrometry (SS-ZAAS) for cadmium. Fresenius J Anal Chem 342:936-940. IAEA AL REPORT(1997). First ALMERA Workshop. IAEA/AL/II), IAEA, Vienna. IAEA TECHNICAL REPORT (1991) The International Chernobyl Project. STI/PUB/885, IAEA, Vienna. IAEA TECHNICAL REPORT(1989) Measurement of Radionuclides in Food and the Environment. STI/DOC/IO/Z~S, IAEA, Vienna. IAEA/AL/oSj (1994) Report of the Research Co-ordination meeting on “Reference Materials for Micro-Analytical Nuclear Techniques”13.-16.Dec 1994, Zagreb, Croatia. IAEA/AL/og5 (1996) Report of the 2nd Research Co-ordination Meeting on “Reference Materials for Micro-Analytical Nuclear Techniques”.3 0.05. -5.0 6.I 9 9 6, Mexico, M EX1CO. IAEA-TECDOC-825 (1995) Reference and intercomparison materials for stable isotopes of light elements. IAEA, Vienna. IAEA-TECDOC-854(1995)Survey of reference materials. Vol. I: Biological and environmental reference materials for trace elements, nuclides and microcontaminants. IAEA, Vienna. IAEA-TECDOC-880 (1996) Survey of Reference Materials. Vols. I and 2. International Atomic Energy Agency, Vienna, Austria. INGAMELLS CO, PITARD FF (1986) Applied Geochemical Analysis, pp 1-84.Wiley, New York. International Federation of Clinical Chemistry (IFCC) (1978) Expert Panel on Nomenclature and Principles of Quality Control in Clinical Chemistry. Clin Chim Acta 81: I8gF-zozF. International Organization for Standardization (ISO) (1993) Guide to the expression of uncertainty. Geneva. I S 0 Guide 35 (1989) Certification of reference materials - general and statistical principles. International Organization for Standardization, Geneva, Switzerland. KESSLERA, SIEKMANN L (1999) Measurement of urea in human serum by isotope dilution mass spectrometrj: A reference procedure. Clin Chem 45:15z.-1529. KLEIN PD, ROSELANDKLEIN E (1987) Stable isotope usage in developing countries: safe trace tools to measure human nutritional status. IAEA Bulletin 4141-44. KLEMMW, BAUMBACHG (1995) Trace element determination in contaminated sediments and soils by ultrasonic slurry sampling and Zeeman graphite furnace atomic absorption spectrometry. Fresenius J Anal Chem 353:12-15. KRISTIANSENJ, CHRISTENSEN JM (1998) Traceability and uncertainty in analytical measurements. Ann Clin Biochem 35:371-379. KURFURSTU (1984) Untersuchungen uber die Schwermetallanalyse in Feststoffen mit der direkten Zeeman-Atomabsorptionsspektrometrie.Dissertation Univ. Bremen, FB Physik. KURFURSTU (1991) Statistical treatment of ET-AAS solid sampling data of heterogeneous samples. Pure Appl Chem 63:1205-1211. KURFURST U, ED.(1998a) Solid Sample Analysis - Direct and Slurry Sampling using GF-AAS and ETV-ICP. Springer, Berlin Heidelberg New York. KURFURSTU (1998b) Calibration using certified reference materials. In: KURFURST U, ed. Solid sample Analysis - Direct and Slurry Sampling using GF-AAS and ETV-ICP, pp 37-46, Springer, Berlin Heidelberg New York. KURFURSTU, GROBECKER KH, STOEPPLER M (1984) Homogeneity studies in biological reference and control materials with solid sampling and direct Zeeman-AAS. In: BRXTTER P and SCHRAM E L P, eds. Trace Element Analytical Chemistry in Medicine and Biology, Vol 3, pp 591-601. de Gruyter, Berlin New York. KURFURSTU, PAUWELS J, GROBECKER ICH, STOEPPLERM, MUNTAUM (1993) Micro-heterogeneity of trace elements in reference materials - determination and statistical evaluation. Fresenius J Anal Chem 345x12-120. LAW RJ, DE BOERJ (1995)Quality assurance of analysis of organic compounds in marine matrices: application to analysis of chlorobiphenyls and polycydic aromatic hydrocarbons. In: QUEVAU-

4. G References

PH, Ed. Quality assurance in environmental monitoring - sampling and sample pretreatment. VCH, Weinheim, Germany. LIEBICH V (1995) Characterization of the chemical homogeneity of solid-state materials by chemometric methods: Further multivariate aspects. Fresenius J Anal Chem 352:420-425. LIEBICH V, EHRLICH G, STAHLBERG U, KLUGE W (1989) Characterization of the chemical homogeneity of solid-state standard materials by chemometric methods. Fresenius Z Anal Chem 335945-953. LINDSTROM RM, BYRNEAR, BECICER DA, SMODIS B, G A R R IKM ~ (1990) Characterization of the mineral fraction in botanical reference materials and its influence on homogeneity and analytical results. Fresenius J Anal Chem 338:569-571. LUCICERE, KONIG H, GABRIEL G, ROSOPULOA (1992) Analytical quality control by solid sampling graphite furnace atomic absorption spectrometry in the production of animal tissue reference materials. Fresenius J Anal Chem 342:941-949. LUCKER E, THORIUS-EHRLER S (1993) Solid sampling ZAAS determination of endogenous Pb contamination in muscle tissue causedby calcification. Fresenius J Anal Chem 346:1072-1076. MAOXUEYING, CHAICHIFANG (1995) Evaluation of the homogeneity of the environmental standard reference material - Estuarine Sediment SRM 1646 by INAA and SRXRF. Fresenius J Anal Chem 352x74-178. MARTIN-ESTEBAN A, FERNANDEZ P, CAMARA C, KRAMERGN, MAIEREA (1997) Preparation, homogeneity and stability of polar pesticides in freeze-dried water interlaboratory exercise. Int. J Environ Anal Chem 67:125-141. MOLLERJ, RASMUSSEN K, CHRISTENSEN L (1999) External quality assessment of methylmalonic acid and total homocystein. Clin Chem 45:1536-1542. MORISIG, MENDITTOA, PATRIARCA M, TAYIOR A, eds. (1996) European external quality assessment schemes in occupationaland environmentalmedicine. Ann 1st Sup di Sanita 32(2):191-316. MUSIALCJ, UTHE JF (1983) Interlaboratory calibration results of polychlorinated biphenyl analyses in herring. J Assoc Off Anal Chem 66:22-31. NI BANGFA, WANGPINGSHENG, HE GAOKUI, TIAN WEIZHII(1996) In: Report of the 2nd Research Co-ordination meeting on “Reference Materials for Micro-Analytical Nuclear Techniques”. I AEA/AL/og 5 , 30.05 .-5.0 6.19 9 6, Mexico, pp 55-6 I. MEXICO. NOWKAR, MARRIL, ANSARITM, MULLERH (1999) Direct analysis of solid samples by GFAAS determination of trace heavy metals in barytes. Fresenius J Anal Chem 364:533-540. NOWKAR, MULLERH (1997) Direct analysis of solid samples by graphite furnace atomic absorption spectrometry with a transversely heated graphite atomizer and Dz-background correction system (SS GF-AAS).Fresenius J Anal Chem 3yp32-137. OHLSK (1981) Spurenanalyse metallischer Werkstoffe. Miltrochim Acta, Suppl. g:49-70. OHLSK (1989) Sample introductioninto ICP-OESfor metallic samples. MikrochimActa III:337-346. SA (1991) Intercomparison of enriched stable isotope reference materials PARRRM, CLEMENTS for medical and biological studies. NAHRES-5, IAEA, Vienna. J, DEANGELISL, GROBECKER KH (1991) Solid sampling Zeeman atomic absorption spectroPAUWELS metry in production and use of certified reference materials. Pure Appl Chem 63:1199-1204. J, DE ANGELISL, PEETERMANS F, INGELBRECHT C (1990) Determination of traces of silPAUWELS ver in copper by direct Zeeman graphite furnace atomic absorption spectrometry. Fresenius J Anal Chem 337:2go-293. J, HOFMANN C, VANDECASTEELE C (1994) Calibration of solid sampling Zeeman atomic PAUWELS absorption spectrometry by extrapolation to zero matrix. Fresenius J Anal Chem 348:418-421. J, KRAMERNG, DE ANGELIS L, GROBECKER KH (1990) The preparation ofcodfkh candidate PAUWELS reference material to be certified for Pb, Cd, Hg, Fe, and Zn. Fresenius J Anal Chem 338:515-519. J, KURFURSTU, GROBECKER KH, QUEVAWILLER P (1993)Microhomogeneity study of BCR PAUWELS candidate reference material CRM-422- cod muscle. Fresenius J Anal Chem 345478-481. J , VANDECASTEELE C (1993) Determination of the minimum sample mass of a solid PAUWELS CRM to be used in chemical analysis. Fresenius J Anal Chem 345:121-123. VILLER

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PRENTICE AM, ed. (1990). The Doubly-Labelled Water Method for Measuring Energy Expenditure: A consensus report by the IDECG Working Group. NAHRES-4, IAEA, Vienna. P, RAURETG, URE A, RUBIO R, LOPEZ-SANCHEZJ-F, FIEDLERH, MUNTAUH QUEVAUVILLER (1995) Preparation of candidate certified reference materials for the quality control of EDTAand acetic acid-extractabletrace metal determinations in sewage sludge-amended soil and terra rossa soil. Miltrochim Acta 120:289-300 ROSOPIJLOA, GROBECKER KH, KURFURST U (1983)Untersuchungen uber die Schwermetallanalyse in Feststoffen mit der direkten Zeeman-Atom-Absorptionsspektroskopie.Fresenius Z Anal Chem 319:540-546. ROSSBACHM, GROBECKER KH (1999) Homogeneity studies of reference materials by solid sampling-AAS and INAA. Accred Qua1 Assur 4: 498-503. ROSSBACHM, OSTAPCZUK P, EMONSH (1998) Microhomogeneity of candidate reference materials: Comparison of solid sampling Zeeman-AASwith INAA. Fresenius J Anal Chem 360:380-383. ROSBACH M, STOEPPLER M (1987)Use of CRMs as mutual calibration materials and control of synthetic multielement standards as used in INAA. J Radioanal Nud Chem Articles 113:217-223. M (1995)Development and application of a protocol for quality assurance of trace analSARGENT ysis. Anal Proc 3x71-76. SCHAUENBURG H, WEIGERT P (1991) Determination of element concentrations in biological reference materials by solid sampling and other analytical methods. Fresenius J Anal Chem 342:950-956. G (zooo) Direct solid sample analysis of sediment, soils, SCHRON W, LIEBMANNA, NIMMERFALL rocks and advanced ceramics by ETV-ICP-AES and GF-AAS. Fresenius J Anal Chem 366:79-

88. SHEEHANTM, HALLS DJ (1999) Measurement of selenium in clinical samples. Ann Clin Bio-

chem 36301-315. L (1979) Determination of steroid hormones by the use of isotope dilution mass specSIEKMANN trometry: a definitive method in clinical chemistry. J Steroid Biochem 11:r17-123. L (1985) Determination of creatinine in human semm by isotope dilution mass specSIEICMANN trometry: a definitive method in clinical chemistry. J Clin Chem Clin Biochem z3:137-144. SINGERR, DANZERK (1984) Homogenitatsuntersuchungen von Festkorpem rnit Hilfe h e a r e r Regressionsrnodelle. 2 Chemie 24:339-341. SLICKERSK (1993) The automatic atomic emission spectroscopy. Briihl-Universitatsdruckerei, Giessen, Germany. D, OHLSK (1979) Spurenanalyse mit der flammenlosen AAS unter Verwendung fester SOMMER Proben. Fresenius 2 Anal Chem 298:123-127. SONNTAGT-M, ROSSBACH M (1997) Micro-homogeneity of candidate reference materials characterized by particle size and homogeneity factor determination. Analyst I Z Z : Z ~ - ~ I . STOEPPLERM, KURFURST U, GROBECKER KH (1985) Untersuchungen uber die Schwermetallanalyse in Feststoffen rnit der direkten Zeeman-Atornabsorptions-Spektroskopie. Teil V. Der Homogenitatsfaktor als KenngroBe f i r pulverisierte Feststoffproben. Fresenius 2 Anal Chem 322:6 87-69 I SUTTON AM, HARVIE A, COCKBURN F, FARQUHARSON J, LOGAN RW (1985) Copper deficiency in preterm infants of very low birthweight. Four cases and a reference range for plasma copper. Arch Dis Childh 60:6+-651. A (1988)The use of internal quality control materials for the preparation and maintenance of TAYLOR reliablemethods for measurement oflead in blood. Fresenius 2 Anal Chem 332:73z-735. A (1995) Quality Assurance: Clinical Analysis. Encyclopaedia of Analytical Science, pp TAYLOR 4250-4261. Academic Press, London. A (1996) Reference materials and analytical standards to stimulate improved laboratory TAYLOR performance: Experience from the external quality assessment scheme for trace elements in biological samples. Milcrochim Acta 123:251-260. DJ, OWENL, WHITEM (1999) Atomic Spectrometry Update: Clinical TAYLOR A, BRANCHS, HALLS and biological specimens, foods and beverages. J Anal Atom Spectrom 14:717-781.

4.6 References

The Condensed Chemical Dictionary (1971) 8th Edition revised by G.G.HAwLEY. Van Nostrand Reinhold Co., New York. M, WOODR (1993) International harmonized protocol for proficiency testing of THOMPSON (chemical)analytical laboratories.J of AOAC Intern 76:gz6-g37. G (1982) Report on the sixth ICES trace metal intercomparison exercise for cadmium TOPPING and lead in biological tissue. ICES Cooperative Research Report No III. International Council for the Exploration of the Sea, Copenhagen, Denmark. WA (1985)Capillarygas chromatographic determination TUINSTRA LGMTH,Roos AH, WERDMULLER of some chlorobiphenylsin eel fat: interlaboratory study. J Assoc OffAnal Chem 68:756-759. UTHE JF, MUSIALCJ, MISRARI< (1988)Multi-laboratory study of measurement of chlorobiphenyls and other organochlorinesin fish oil. J Assoc Off Anal Chem 7qGg-372. VAHTERM (1982)Assessment of human exposure to lead and cadmium through biological monitoring. Natl Swed Inst Environ Med, Stockholm. VERREPTP, DAMSR, KURFURSTU (1993) Electrothermal vaporisation inductively coupled plasma atomic emission spectrometry for the analysis of solid samples: contribution to instmmentation and methodology Fresenius 2 Anal Chem 34y1035-1041. WELCHMJ, COHENA, HERTZHS, RUEGG FC, SCHAFFER R, SNIEGOSKILT, WHITEVE (1984) Determination of serum urea by isotope dilution mass spectrometry as a candidate definitive method. Anal Chem 56:713-719. WELLSDE (1988)The need for organic reference materials in marine science. Fresenius Z Anal Chem 332:583-590. DE, DE BOER J, TUINSTRA LGMTH, REUTERGARDH L, GRIEPINXB (1988) Improvement in WELLS the analysis of chlorobiphenylsprior to the certification of seven CBs in two fish oils. Fresenius Z Anal Chem 332:591-597. WESTGARD JO, BARRYPL, HUNTMR (1981) A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem z7:4g3-501. WHITEHEAD TP (1976)Quality Control in Clinical Chemistry. Wile5 Chichester. RR, BUROW M, WISESA, SCHANTZMM, KOSTER BJ, DEMIRALPR, MACKEYEA, GREENBERG OSTAPCZUK P, LILLESTOLETI (1993)Development of frozen whale blubber and liver reference materials for the measurement of organic and inorganic contaminants. Fresenius J Anal Chem 345:27o-z77. R (1998) Reference materials for small-sample analysis. Fresenius J Anal Chem ZEISLER 360:376-379. ZEISLERR (zoooa) Investigationsby INAA for the Development of Natural Matrix Standard Reference Materials (SRMs) Suitable for Small Sample Analysis. J Radioanal Nucl Chem z45:73-

80. ZEISLER R (zooob)Maintaining accuracy in gamma-rayspectrometryat high count rates. J Radioanal Nucl Chem z44:507-510.

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Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

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Reference Materials for “Life” Analysis Edited

by PeterJ Jenks

So far we have examined the reference material and calibration needs of the conventional analytical chemist. But as we move into the zIst Century, new technologies are driving the quality concepts established over the past 30 years by analytical chemists into other analytical disciplines, including microbiology and molecular biology. Indeed, the next major challenge may be the development of reference materials, or the provision of a form of reference material, that gives to the molecular biologist the same ability to control and standardize their determinations and quantification as a chemical reference material gives the analytical chemist. There have been a number of attempts to achieve this objective, but so far the challenge has not been fully met. This Chapter will examine some of the conventional approaches and then go on to consider how recent developments in the use of the Polymerase Chain Reaction (PCR) with DNA for the identification of species and individual organisms by DNA analysis, sometimes known as “DNA Fingerprinting”, have identified a yet unrealized need for a new dimension of certified reference materials. We will also review the development of chemical metrology in the pharmaceutical industry, an industry that is ever more closely related to molecular biology. The pharmacists first developed quality protocols and reference materials more than 40 years ago, quite independently of the activities of I S 0 and the REMCO Technical Committee. Only in the closing years of the 20th Century have these two important groups begun to realize that they share common objectives.

5.1 Standard Reference Materials for Microbiological Assays Jane Tang and Shung-ChangJong

When the American Type Culture Collection (ATCC) was founded in 1925, one of its chief roles was to be a source of standards. In this context we mean standard organisms, rather than standard materials or chemicals known as Reference Materials or Certified Reference Materials and issued in the USA by the National Bureau of Standards (NBS). The National Bureau of Standards evolved into NIST and the abbreviation SRM became a trade mark of NIST.

5.I Standard Reference Materials for Microbiological Assays

ATCC biological standards were known as Type Strains (TS),but as they are used in the same ways as, and fill many of the requirements for, RMs we have described them in this article as biological RMs. In the 1920’s microbiological “type strains” were acquired to support taxonomical studies. Since then many organizations have used ATCC as a repository for other types of biological RMs, especially for use in official assays and standardized test procedures in public health, microbiology and clinical determinations. The range of biological RMs held by ATCC includes bacteria, viruses, fungi, animal and human cell lines, protozoa and, as we move into the 21st Century, also includes genetically modified organisms (GMOs). Unlike chemicals, microorganisms are living materials, so growth conditions, the physiological state of the cells, and their metabolic activities will all influence the results of tests in which they may be used. The reproducibility of a determination is a critical element of a standard method, so they are carefully written to ensure that they can be followed accurately by any qualified laboratory. The designated test biological RMs must not only be reproducible in their reactions to the performance tests, but they must also respond in a predicable manner. In order to guarantee that the same strain of microorganisms could always be available, many of these biological RMs have been deposited with the ATCC and other culture collections around the world. 5.1.1 Standards for Official Assays and Tests

Around the world there are hundreds of official methods from various government and professional sources that specify ATCC microorganisms as biological RMs. The following six examples serve as examples of many organizations around the world that specify ATCC microbial cultures as biological RMs: ASTM, The American Society for Testing and Materials, is a scientific and technical organization that develops standards for a wide variety of materials and products. It is the worlds largest source of voluntary consensus standards. AOAC, The Association of Official Analytical Chemists International, describes methods for analyzing a wide range of materials. These methods are often adopted by other organizations and government regulatory agencies as standards. NCCLS, National Committee for Clinical Laboratory Standards provides a medium for communication among professionals in healthcare, government, and industry in developing guidelines for clinical laboratory practices. USDOD, The United States Department of Defense describes testing procedures for any product or material for use in the military. USFDA, The United States Food and Drug Administration details its standard methods in the Code of Federal Regulations, Title 21.

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USPC, The United States Pharmacopeia is a compendium of information on drug products. The standards set forth in the USP are recognized in both federal and state law. Other organizationswhose standard procedures specify ATCC biological RMs include: American Association of Cereal Chemists (AACC) American Association of Textile Chemists and Colorists (3) American Wood-Preservers’Association (4) British Pharmacopoeia (BP) (5) British Standards Institution (BSI) (6) International Organization for Standardization (I SO) (7) Radio Technical Commission for Aeronautics (8) Technical Association of the Pulp and Paper Industry (9) United States General Services Administration (I)

(2)

For a comprehensive list of quality control and reference strains refer to ATCC QC and Reference Strains, 1st edition, 1997. 5.1.2

Quality Management of Biological RMs

Effective management of microbiology collections requires that the cultures meet current and future international standard requirements of biotechnology industries. The knowledge and competence of the scientists performing cell culture work obviously impact the quality of the cultures. For biological RMs, quality control is the total accumulation of all means and activities by which desirable properties of each microbial culture are precisely and consistently maintained throughout production, storage, shipping, and customer use. To confirm such quality, ATCC have developed a quality system that uses certain principles from International Standard Organization (ISO) standards for quality systems and Good Laboratory Practices (GLPs) and the pharmaceutical industry’s Good Manufacturing Practices (GMP).All of these principles require written standard operating procedure (SOP) manuals, the most important documents for standardization and organization of all observations,tests, and functions in the collections. ATCC issues “Certificates of Analysis” with cultures. Although ATCC Certificate of Analysis do not meet the specific requirements of I S 0 Guide 31, they do show that the biological RM culture has been authenticated and/or specific characteristics have been verified. Each ATCC Certificate of Analysis is lot-specific and includes expiration dates, the specific seeds used for propagation, and selected biochemical and morphological characteristics that are indicative of the culture. ATCC also aims to provide a Product Sheet for each microbial culture, with instructions for propagation, special features of the organism, and any unusual observations or properties. Microbial cultures at ATCC are normally preserved by lyophilization (freeze-drying) and/or storage over liquid nitrogen at -180°C. A seed stock system is used to maintain the distribution stock.

5.1 Standard Reference Materials for Microbiological Assays

The source ofall biological RMs i s “newaccessions”,i.e. a new organism. Once one i s received, it i s grown in fresh medium and a set of seed stock vials are made along with a distribution batch of vials. Quality control is performed on the seed material and the distribution batch. When the first distribution batch is exhausted, another new lot i s made by propagating only from the seed material. The seed stock i s always the closest material to the original deposit available for propagation and verification. The seed stock system allows the minimization of frequent subculturing and passages of microorganisms. With repeated culturing there are risks of contamination and mutation, which will alter the characteristics of the microorganisms. T h i s is especially crucial for maintaining biological RMs, thus every effort i s made to minimize transfers and passages of cultures. Users of biological RMs need to pay particular attention to the risk of change in the identity of the organism caused by subculturing, mutation or contamination. A recent personal communication from Masters and coworkers on human cell line cross-Contamination showed that HeLa cell lines used by a number of workers for work published in the literature were not, when checked by DNA analysis, what the authors thought they were. To ensure microbial strains are viable and pure a suite of morphological, biochemical, and cytochemical tests are used to confirm characteristics specific to their taxons. A number of commercially available rapid identification kits are also employed for some common genera. In addition to these taxon specific tests, many of the cultures are tested for their fatty acid methyl ester (FAME) profiles using the commercial MIDI system. The FAME profiles can be compared to the MIDI database for species identification/confirmation purposes. The Biolog system, which yields a metabolic fingerprint of an organism, i s another alternative for rapid identification. In recent years molecular taxonomic tests have been introduced. There are a number of molecular methods available. However, due to the volume and variety of organisms it i s important that any molecular method meet the following criteria: Be cost effective Be reproducible (3) Provide data that can be converted to a database accessible format (4) Be robust enough to work with a variety of different organisms (5) Be rapid and simple to perfom (I)

(2)

Based on these criteria DNA fingerprinting has started to be used as part of the quality control procedure for Biological RMs. Comparison of DNA profiles generated from the seed material and the distribution batches assure no alteration occurred during the propagation and preservation processes. Due to the diverse nature of the organisms no single system can work well with all strains. Therefore development to investigate other PCR-fingerprinting methods as possible molecular taxonomic characterization of microbial cultures i s ongoing at ATCC. The goal i s to achieve a polyphasic taxonomicapproach for authentication and quality control of microbial strains. Each morphological, physiological, cytochemical, and molecular method has limitations. Combining and comparing the results enable us to obtain a more accurate and complete picture ofthe microorganism’sidentity.

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Certified Microbiological Culture Materials

Although the work of ATCC and others has done much to ensure the reproducibility and even demonstrate some traceability of microbiological reference materials the development of microbiological Certified Reference Materials (CRMs),certified for number of viable life forms is seen as important for control analyses of water and food. Somewhat of a “holy grail” the development of such CRMs has long been hampered by the unstable concentration and insufficient homogeneity of viable organisms in the materials. This Section describes one of the first successful attempts, by a multi national project team funded by the European Commission’s DG XII, to produce and certify a microbiological CRM that is both fit for purpose and meets the requirements of the I S 0 Guides. The first results are two bacterial strains, Enterococcus fueciurn (CRM 506) and Sdmoneha typhimuriurn (CRM 507) (Janning et al. 1995). Both are available as part of the EU “ B C R range of CRMs. The authors demonstrated that the main problems could be overcome by stabilization of the target microorganisms by spray-dryingthem in a preservation medium based on sterile evaporated milk. The resulting highly contaminated milk powder had, depending on the test strain and the contamination level of the milk suspension, a final contamination level of 104-107 colony-forming particles per gram (cfp/ 8).After a period of stabilization while stored at a temperature of +5’C or -2o”C, the contaminated milk powder was mixed with sterile milk powder to achieve the desired contamination level of the final material. Then, gelatin capsules were filled with 0.2-0.3 g of the mixed powder. The capsules stored at -20°C. Fig. 5.1 shows the most important steps of the production process.

Cultivation of bacterial strain

U Suspension in evaporated milk U

Spray drying Highly contaminated milk powder

U

Mixing with sterile milk powder

U

Filling of gelatin capsules

U

Storage at-20°C

Fig. 5.1

Flow diagram: Production of

a microbiological RM.

5.2 Certified Microbiological Culture Materials

The homogeneity determination of the bacteria in the materials is performed by viable count followed by statistical evaluation of the counts of sub-samples from the same capsule solution and of total counts of different capsules of one batch. An example for the homogeneity determination for a batch of capsules containing Enterococcusfaecium is also presented in (Janninget al. 1995). After the performance of collaborative studies organized by the RIVM (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands) and SM&Tcertification studies were performed by a group of eleven or twelve qualified European laboratories. These examined simultaneously the materials using standardized methods following a very strict analytical protocol. Table 5.1 below shows data for CRM 506 (Mooijman et al. 1999). The development of these CRMs has shown that it is possible to produce CRMs for public health microbiology and that whilst such a CRM can largely meet the I S 0 Guides requirements it does not completely satisfy all the requirements of CRMs. It has also demonstrated that their manufacture and certification places considerable challenges before the producers.

Tab.

5.1

Certified number concentration of colony forming particles of Enterococcus faeciurn

(WR63) in CRM 506 from artificially contaminated milk powder (Mooijman et al. 1999)

I S 0 7899/2 (1984) KFAa

76

71-81

9

I

159

160

I

5 Reference Materialsfor “Lif”Ana/ysis 5.3 Reference Materials for DNA Analysis Barbara C. Levin and DennisJ. Reeder

Mention has already been made, in Section 5.1, of the use of DNA-based techniques for the control of microbiological DNA. In parallel to the work of ATCC the USA National Institute of Standards and Technology (NI ST) developed three Standard Reference Materials (SRM 2390, SRM 7,391 and SRM 2392) to provide quality assurance in the analysis of human DNA. Two of these SRMs, the DNA Profiling SRM 2390 and the PCR-based DNA Profiling SRM 2391, are intended for use in forensic and paternity identifications, for instructional law enforcement, or non-clinical research purposes. They are not intended for any human/animal clinical diagnostic use. The third NIST SRM - 2392 - is for standardization and quality control when sequencing the entire or any segment of human mitochondria1 DNA (16569 base pairs) for forensic identification, disease diagnosis or mutation detection. 5.3.1

SRM 2390

The first human DNA SRM developed by NIST was designed in the late 1980’s to standardize Restriction Fragment Length Polymorphism (RFLP) procedures which at that time were very new developments in the application of DNA to forensic analysis. SRM 2390 uses the HaeIII restriction enzyme to examine highly polymorphic human DNA loci containing variable numbers of tandem repeats (VNTR) (Reeder 1991). These sequences resemble mini-satellites in that they are composed of repeats of a core sequence and different individuals have different numbers of repeats. However, unlike mini-satellites, there is usually only one VNTR of a given type in an individual. Therefore, VNTR analysis gives patterns that are simpler and easier to analyze than DNA fingerprints, but several different probes must be used to be convincing. The use of RFLP depends on the presence of a polymorphism which can be as small as a single nucleotide change or as large as the tandem repeat segment in a particular DNA sequence. In this case, the HueIII restriction cleavage sites flank the tandem repeat sequences and will produce different length DNA fragments depending on the number of tandem repeats in an individual. In 1991, the Federal Bureau of Investigation (FBI), the Royal Canadian Mounted Police and many US State and local laboratories were using Hue111 in their RFLP procedures for forensic and paternity testing (Budowle 1988; Eisenberg et al. 1991).The restriction enzyme HueIII was chosen since it is an extremely hardy enzyme, recognizes a four-base sequence (GGCC), cuts approximately every 256 bases, produces smaller DNA fragments, and has better resolution of the fragment size variants than other enzymes, e.g. PstI, HinfI, A h 1 (Budowle 1988). In addition, DNA methylation does not interfere with HaeIII efficiency to recognize or cleave its restriction site.

5.3 Reference Materialsfor DNA Analysis

SRM 2390 provides quality control for the following procedures: (I) (2)

(3)

(4)

(5) (6) (7)

(8)

extracting the DNA from the cell pellet quantifying the extracted DNA determining the restriction enzyme cutting efficiency separating the DNA electrophoretically blotting the separated DNA onto a nylon membrane using an alkaline transfer solution hybridizing the sample DNA to radioactively labelled DNA probes exposing the membrane to X-ray film imaging the developed autoradiogram with a computerized imaging system to determine band sizes

The human DNA in this SRM comes from a female (K562) cell line and a male (TAW) source. The cells and DNA are provided as: a cell pellet containing approximately 3 x 10' cells extracted genomic DNA (3) HaeIII-digested DNA (I)

(2)

In addition, the SRM contains a DNA molecular size standard for sizing the allele fragments; a set of quantitative DNA standards in concentrations of 6-250 ng/6 pL; and a visualization marker set which produces twelve bands ranging from 594 to 35 937 base pairs and which is used to assess the DNA separation on the electrophoretic gel. Twenty-nine laboratories, including NET, participated in an inter-laboratoryevaluation of SRM 2390. All laboratories received the above components plus a set of four bloodstains [DzS44 (YNH24) was the target probe (Nakamura et al. 1987)], instructions, and a questionnaire. All laboratories used the basic FBI protocol (Budowle and Baechtel 1990) or modifications thereof for RFLP-sizing.The participants were asked to provide calculated band sizes for locus DzS44 and any other probes that they were willing to examine. Variations were noted among the participating laboratories in the type of agarose, sources of chemicals and probes, methods for labelling probes and the electrophoretic and imaging equipment. Thirty-four sets of data on DzS44 were sent to NIST. Nineteen laboratories also reported results on D&jg (PHjo), 17 tested D1oS28, 16 laboratories examined D1S7 (MSI), and 12 provided results on D17S7g (VI). The certified values (number of DNA base pairs) for SRM 2390 are shown on the NIST SRM 2390 Certificate. 5.3.2

SRM 2391

The second NET human DNA SRM is a PCR-based DNA Profiling Standard. The PCR was first described by Saiki et al. (1985,1989). Since then it has developed into a highly versatile and widely used detection, identification, manipulation and analysis tool in molecular biology, including DNA profiling. In brief, two short synthetic oligonucleotides, or primers, are used to define an intervening DNA sequence

I

162

I

5 Reference Materials for “L@”Ana/ysis

which is then amplified in vitro using a thermo stable DNA polymerase, DNA precursors, suitable reaction buffer and a thermal cycler. Correct amplification requires a specific interaction of the primers with their target sequence. The specificity of the reaction is generally assessed by gel electrophoresis or hybridization which determines whether the size or internal sequence is consistent with correct amplification. Because of the complexity of the complete DNA profiling procedure, including the PCR stage, NIST developed and certified a specific SRM for use in procedures based on those of the FBI (PCR Typing Protocols, FBI Laboratory, Quantico, VA, 121 12/94). This procedure employs the genetic locus DIS8o (pMCTIr8) (Nakamura et al. 1988) and specific primers (Kasaiet al. 1990).The procedures covered by the use of SRM 2391 are: extraction of DNA from cells spotted on special filter paper amplification of the DNA from the DIS8o locus (Kasai et al. 1990) (3) separation of the amplified alleles by electrophoresis (4) silver staining the gel or fluorescent dye detection plus image analysis (I)

(2)

This SRM contains human cells from two cell culture lines from which DNA can be extracted, genomic DNA from those two cell lines plus eight individuals, PCRamplified DNA from the two cell lines plus four of the eight individuals, a DIS8o allelic ladder for characterization of amplified DNA, and a DNA size marker to assure proper electrophoretic separations. Prior to certification, various components of this SRM were tested by 2 0 laboratories. The 1995 certified values for D1S80 alleles, derived from pooled results from analyses performed at two laboratories plus NIST are shown in the certificate supplied with the SRM. 5.3.2.1

Recertification of SRM 2391

Since 1995, when SRM 2391 was issued, technologies, particularly the use of Short Tandem Repeat (STR) loci for human identity, have developed rapidly. The initial certificate for SRM 2391 had limited STR information on only four STR loci (tetraplex system), HUMTHOI (THoI), HUMFI~AOI(FI~AoI), HUMVWFA~I(vWA)and HUMFESFPS (FES/FPS). The forensic community requested that SRM 2391 include STR information for all the DNA samples and that the STR information include the FBI’s CODIS (Combined DNA Index System) core loci. The CODIS core loci are HUMCSFIPO (CSFIPO), D3S1358, DgS818, D7S820, D8S1179, DqS317, DrGSgjg, DI8Sg1, DZISII, FGA, THoI, HUMTPOX (TPOX), and vWA. In addition, F13Ao1, HUMBFXIII ( F I ~ B ) FES/FPS, , HUMLIPOL (LPL), DrS80, DQAI, and Amelogenin were also analyzed. Therefore, a total of 2 0 loci were analyzed for the recertification of SRM 2391 and seven laboratories contributed data on specific genetic loci. The STR data generated at NIST were based on amplifjmg 1.0 ng of the genomic DNA with fluorescent labelled primers. The PCR amplified products were analyzed by slab gel electrophoresis followed by imaging with a Molecular Dynamics FluorImager 595 or by capillary electrophoresis using a PE-ABI 310 Genetic Analyzer.

5.3 Reference Materials for DNA Analysis

When possible, both methods of analysis were used. The other laboratories used either a PE-ABI 377 sequencer, PE-ABI 310 Genetic Analyzer, or a Hitachi FMBIO imager. Tables in the certificate provided with SRM 2391 provide the data for AmpliType@ HLA DQA1, AmpliType@ PM types including the Amelogenin typing information and information values for the 17 STR loci. 5.3.3 SRM 2392

The third human DNA SRM developed by NIST was designed to meet the need for quality control when amplifying and sequencing human mitochondrial DNA (mtDNA). Human mitochondrial DNA has been completely sequenced and found to be circular double-stranded molecules containing 16 569 base pairs (Anderson et al. 1981).There i s very little wasted space in this genome, since there are no introns, no untranslated leaders or trailers in the encoded mRNA and few if any, bases between structural genes. The human mitochondrial genomes encode two rRNAs, 22 tRNAs and 13 proteins. Each human cell can have a few dozen to several thousand molecules of mtDNA (Bogenhagen and Clayton 1974; King and Attardi 1989). Sequence analysis of mtDNA is being used by the forensic community for human identification especially in those cases where genomic DNA is highly degraded or non-existent (Holland et al. 1993, 1995). Forensic analysis to distinguish between individuals is primarily based on the considerable sequence variation found in the two hypervariable regions (HVI, HV2) located in the non-coding displacement loop (D-loop).The medical community i s also using sequence analysis of mtDNA for diagnoses of diseases associated with specific mutations and deletions (Wallace 1992). A third area of research which is largely unexplored and which needs sequence analysis is the examination of the mutagenic effects of chemical and physical agents on mtDNA (Grossman 1995; Ballinger et al. 1996).

DNA Source Most investigators examining human mtDNA have used the numbering system of Anderson et al. (1981) and have compared their findings to the sequence first described in 1981. However, the DNA sequenced by Anderson is not available for use as a positive control during actual experiments; whereas, NIST SRM 2392 is available. This SRM provides quality control when amplifying and sequencing human mtDNA (Levin et al. 1999). SRM 2392 includes DNA from two lymphoblastoid cell cultures (CHR and 9947A) and cloned DNA from the CHR HVI region which contains a C-stretch and i s difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GMo3798 is provided for comparison. All three DNA templates are from apparently normal individuals. Fifty-eight unique primer sets allow any area or the entire mtDNA (1G569 base pairs) to be amplified and sequenced. By using this number of sets of primers, about 58 overlapping fragments are generated, ensuring complete coverage of the whole genome while 5.3.3.1

I

163

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I

5 Reference Materials for “Life”Analysis

sequencing. While none of the differences in these three templates correspond to published mutations associated with specific diseases (Wallace 1gg2),some of these differences did result in anima acid changes when compared with that published by Anderson et al. (1981). Table 5.2 shows the mtDNA differences compared to the Anderson sequence that were found at NISTwith all three templates. There were 13, g, and 4 differences in the non-coding regions of templates CHR, 9947.4, and GMo3798, respectively and 33, 23, and 19 differences in the coding regions of templates CHR, 9947A and GMo3798, respectively. 5.3.3.2

Inter-Laboratory Evaluation of SRM 2392

Three laboratories in addition to NIST participated in an inter-laboratory evaluation of the CHR template. All of the laboratories essentially followed the NIST protocol. Three of the four labs found essentially the same polymorphisms. Laboratory 4, who had less experience with sequencing mtDNA, did find differences that the other laboratories did not observe. The differences noted by Laboratory 4 confirm and emphasize the need for a standard reference material for sequencing mtDNA. Had Laboratory 4 had run NIST mtDNA SRM 2392 simultaneously with their unknown sample, they would have realized that they were finding an undue number of differences and could have reexamined their procedures to try to determine the reason for these differences. 5.3.4 Summary

These three NIST SRMs have a number of important quality control applications for forensic DNA profiling, medical diagnostics and mutation detection. The main applications are summarized below SRM 2390 can be used in several different ways depending on quality assurance requirements. Components of the SRM are designed to provide assurance that each step of the RFLP protocol is functioning properly, but they can also be used for trouble shooting,for calibrating equipment, and for testing the efficacy ofnew lots ofreagents. SRM 2391 is designed to provide quality assurance to laboratories that perform DNA profiling using PCR methods. This SRM can be used to verify that each step of the analysis system is operating correctly and within the proper limits. SRM 2392 is designed to provide quality control when amplifying and sequencing any region or the entire 16 569 base pairs which comprise human mtDNA. It can also be used as a control when amplifying and sequencing any type of DNA. The two DNA templates (CHR and 9947A) included in the SRM have characteristic polymorphisms throughout the non-coding and coding regions of the DNA and therefore, can serve as positive controls during PCR amplification and sequencing. None of these polymorphisms correspond to any of the published base pair changes that have been correlated with specific diseases. Corroboration ofthe SRM results provides quality assurance that any unknown DNA is also being amplified and sequenced correctly.

5.3 Reference Materialsfor DNA Analysis Tab. 5.2 Primer sets used for PCR amplification o f human m t D N A and differences with the anderson sequence found i n three templates at NlSTfor SRM 2392

Primer set

Amplified region"

Length ofamplijed region

Comparison with Anderson Amino Ander-

1470 73 93 195 204 207 214 263 309.1 309.2 315.1 2

361-921

start 39

start 55

G

-

-

G

-

C C

G A

C -

A

-

-

C(ins) end 436

G G C(ins) C(ins) C(ins) end 473

C (ins) end 454

start 429

start 421

start 415

A G end 891

G

G C(ins)

561 709 750

tart 39

-

end 846

A G end 834

3

756-1425

670

NONE

start 778 start 778 start 818 end 1197 end 1278 end 1146

4

873-1425

553

NONE

start 931 start 928 end 1335 end 1377

5

1234-1769

536

6

7

1587-2216

1657-2216

start 938 end 1323

start 1279 start 1275 start 1295

1438 1719

A G

1719*

G

630

G G G A E end 1738 end 1741 end 1654 start 1632 start 1632 start 1649

560 1719d

G

A end2106

end2106

-

end2031

start 1691 start 1686 start 1715

A end2170

end2173

end2097

8

1993-2216

224

NONE

start 2036 start 2018 start 2069

9

2105-2660

556

NONE

start 2157 start 2150 start 2161

10

2417-3006

590

end 2213 end 2636

end 2217 end 2212 end 2586 end 2560

start 2465 start 2458 start 2483

2706

A

G end 2920

end 2956

-

end 2915

166

I

5 Reference Materialsfor “Li$e”Analysis

1

Tab. 5.2

Primer

11

(Continued) Amplified region‘

2834-3557

length of amplified region

Anderson No.

9947A

724 3010 3106/3107

12

2972-3557

586

13

32343557

324

I

Comparison with Anderson

G

C

Amino acid GMo3798 change

start 2861 start 2869 start 2888 A del del del end3350 end3373 end3243

3106/3107d C 3423 G

start 2999 start 2999 start 3031 del del del E T T Silent end 3422 end 3460 end 3425

G

start 3265 start 3258 start 3292 T T T Silentd end 3548 end 3545 end 3541

3423d 14

3441-3940

500

NONE

start 3487 start 3491 start 3499 end 3916 end 3920 end 3847

15

36354162

528

NONE

start 3667 start 3662 start 3725 end4126 end4061 end4044

16

3931-4728

798 4135

17

41834728

546

18

4392-4982

591

19

20

21

4447-4982

4797-5553

4976-5553

T

start 3964 start 3968 start 3987 Try+ C His end 4399 end 4427 end 4436 start 4208 start 4249 start 4208 end4657 end4657 end4642

NONE

4769

A

start 4449 start 4453 start 4440 G G G Silent end 4860 end 4935 end 4877

4769d

A

start 4492 start 4492 start 4492 G G G Silentd end4958 end4921 end4931

4985 5186

G A

start 4838 start 4845 start 4838 A A A Silent G Silent end5327 end5324 end5215

518Gd

A

start 5000 start 5007 start 5016 G Silentd end5516 end5521 end5400

536

757

578

22

5318-5882

565

NONE

start 5361 start 5360 start 5371 end 5754 end 5758 end 5800

23

5700-6262

563

NONE

start 5741 start 5744 start 5754 end 6149 end 6163 end 6136

5.3 Reference Materialsfor D N A Analysis

7 i Tab. 5.2

Primei

24

25

26

27

(Continued)

Ampli-

Length

region"

plifed region

5999-6526

6242-6526

6426-7030

6744-7255

T C

637Id

C

start 6271 start 6302 start 6293 T Silentd end6520 end6520 end6520

6791 6849"

A A

start 6451 start 6474 start 6487 G Silent G(0.3A)h+: Thr+ end 6916 end 6930 end 6885 Ala"

6849d" 7028

A C

start 6775 start 6782 start 6801 G(0.3A)h+c Thr-t T Ala'k end 7215 end 7221 end 7177 Silent

512

29

7215-7792

578

7645-8215

6221 6371

605

718

30

bp

285

7075-7792

I

NONE

Jemplate CHR

Jemplate 9947A

start 6043 start 6058 start 6047 C Silent Silent T end6442 end6503 end6456

start 7123 start 7123 start 7130 end 7602 end 7601 end 7547

7645

T

start 7263 start 7280 start 7273 C Silent end 7722 end 7769 end 7706

7861

T

start 7671 start 7666 start 7701 C Silent

571

end 8149 end 8155 31

7901-8311

411

32

8164-8669

506

33

34

35

8539-9059

8903-9403

9309-9848

Amino Template acid CMo3798 change

Anderson No.

528

28

I

Comparison with Anderson

NONE

end 8156

start 7960 start 7959 start 7960 end8289 end8288 end8258

8448 8503

T T

start 8211 start 8212 start 8230 C Met+ C Thr end 8646 end 8641 end 8637 Silent

8860

A

start 8581 start 8582 start 8581 G G G Thr+ end 9019 end 8999 end 8991 Ala

9315

T

start 8947 start 8944 start 8951 C Phe-t end 9380 end 9381 end 9370 Leu

9559

G

start 9334 start 9333 start 9333 C C C Argt end 9823 end 9827 end 9800 Pro

521

501

540

I

167

168

I

5 Reference Materiaisfor “Life”Ana1ysis Tab.

5.2

(Continued)

AndersonNo.

36

9449-9995

bp

547 9559d

G

Template CHR

I

Template 9947A

Amino Template acid CMo3798 change

start 9476 start 9485 start 9479 C C C Argi end 9964 end 9940 end 9911 Prod

37

9754 10275

522

NONE

start 9777 start 9781 start 9808 end10225 end10251 end10184

38

1012710556

430

NONE

start 10168 start 10166 start 10180 end 10534 end 10536 end 10524

39

1038611166

781

NONE

start 10410 start 10416 start 10439 end 10899 end 10916 end 10865

40

1070411267

564

NONE

start 10734 start 10742 start 10758 end 11223 end 11197 end 11167

41

1100111600

600

1140311927

525

1176012189

430

44

1190112876

976

45

1235712876

520

46

1260113123

523

47

1279313343

551

48

1318813611

424

42

43

11335

T

start 11026 start 11040 start 11059 C C C Silent end 11461 end 11517 end 11497

11719

G

start 11428 start 11432 start 11456 A Silent end 11795 end 11853 end 11855

11878

T

start 11784 start 11802 start 11802 C Silent end12159 end12164 end12163

NONE

12612 12705

start 11926 start 11926 start 11961 end12404 end12443 end12397 A C

12705~~ C

NONE

13572

start 12404 start 12391 start 12391 G Silent T Silent end12769 end12849 end12775 start 12627 start 12645 start 12643 T Silentd end 13102 end 13045 end 13024 start 12817 start 12807 start 12816 end13295 end13307 end13266

T

start 13238 start 13238 start 13244 C Silent end13587 end13593 end13590

5.3 Reference Materialsfor DNA Analysis Tab.

5.2

(Continued) Comparison with Anderson

region

49

1351813935

I Amino

Anderson No.

418 13572d 13702 13708 13759

T G G G

start 13541 start 13541 start 13571 C C C C A A end 13910 end 13921 end 13900

50

1371514118

404

51

1389914388

490

1418914926

738

52

53

1447014996

527

1490915396

488

1526015774

515

13966

Silentd GlyArg Alai

Thr AlaThr

A

start 13775 start 13760 start 13760 G Thr+ end 14094 end 14110 end 14104 Ala

1396Gd 14199 14272 14365

A G G G

start 13926 start 13927 start 13961 G T T T C C C C C E end 14369 end 14374 end 14342

14272d 1436Sd 14368 14470 14766

G G

14766d

T

G T T

start 14216 start 14216 start 14240 C C C C C C C C c C E C E end 14699 end 14806 end 14698

Thri Alad Pro+ Thr Phei Leu Silent PheLeud Silentd Phe+ Leu Silent Ileh Thr

start 14502 start 14513 start 14527 C C Ileend 14957 end 14972 end 14956 Thrd

54

55

15326

A

1532Gd 15646

A C

start 14941 start 14933 start 14950 G G G Thr-t end 15380 end 15373 end 15359 Ala start 15305 start 15293 start 15287 G G G Thr+ T Alad end 15754 end15950 end 15723 Silent

56

1557416084

511 1564Gd

C

start 15637 start 15599 start 15601 T Silentd end 16056 end 16058 end 16030

I

169

170

I

5 Reference Materials for “L@”Ana/ysis 5.2

(Continued)

Primer set

Amplified region”

Tab.

57 (HV1)

1597116451

Length ofamplified region

Comparison with Anderson

Anderson No.

Template 9947A

481 16183 16189 16311 16357

A

T T T

Amino Template acid GMo3798 change

start 16014 start 16011 start 16004 C C E C -

E

-

C

end 16193 end 16430 end 16403

58

16097-336

809 16183d 1618gd 16311d 16357d 16519

-21 M13‘ cloned DNA

16133-40

A

T T T T

a) Numbers correspond to Anderson sequence (Anderson et al. 1981) B) Base pair change came before the readable sequence. E) Base pair change came after the readable sequence. -) Base pair same as in Anderson sequence.

h“)

PossibIe heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the mtDNA from the second CHR cell culture line. It is DNA from the second CHR cell culture line which is supplied in NIST SRM 2392. c) This primer is used for sequencing the cloned DNA of the HVI region. d) Change also seen in previous primer set. Start)

Start of readable sequence. end) End of readable sequence.

E E

-

C C

end16193

C end59

end103

A

start 16131 C

ND

ND

T

C

477 16183d 16189d 16193.1 16223 16278 1651gd

start 16125 start 16130 start 16151 C C E C -

C(ins) C

T T

T

C end 40

c

5.4 Future Developments in Molecular Reference Materials

5.4

Future Developments in Molecular Reference Materials PeterJJenks and Vanessa Dekou

DNA analysis has become an invaluable tool having very many practical applications that aim to open new frontiers in science. The sequencing of the human genome will provide information that could be applied to the study of genetic disorders as well as complications affecting the behaviour of humans at molecular level. Cloning genes has become an extensively used technique, with applications ranging from production of proteins (e.g. human growth protein) to production of improved new generation vaccines. In the future it may be possible to treat genetic defects by transplanting cloned normal genes into patients whose own genes are damaged. Already the use of gene transfer techniques to produce so-called genetically modified organisms, or “GMOs” in agriculture is extensive and has become rather controversial. Initial efforts by workers at the Institute for Reference Materials and Measurement (IRMM), Geel, Belgium, to produce certified reference materials for GMOs have demonstrated that the provision of suitable reference materials is not easy and that together with the development of suitable analytical methods there are many challenges to be solved ahead. The first two examples produced jointly by the IRMM in Belgium and Flulta Chernie AG in Switzerland were based on “Round-Up ReadyTMSoya and BT 17GTMMaize. The reference materials are needed to validate EU and Swiss regulations which permit non-GMO products to be contaminated by up to 1% GMO material and still be accepted. In each case the grains were supplied by the producers as “100% GMO”. At the IRMM they were milled, heat-treated to inactivate enzymes found in the grain and then blended with non-GMO materials to produce a range of CRMs, from I % GMO to 5 % GMO. Certification was undertaken by 25 labs (soya) and 22 labs (maize) for the PCR qualitative screening method. Whilst the CRM prove completely suitable for the intended purpose later use in quantitative PCR analysis suggests that much more controlled and rigorous procedures may be needed if CRMs suitable for quantitative PCR are to be realized. It is believed that contamination of the exterior of the grains by both non-GMO or other GMO types, the breakdown of DNA in processing, especially if site-specific and the consequences of the polyploidal nature of plant genomes must all be taken into consideration when developing matrix RMs certified for GMO (Pauwelsand Schimmel, personal communication). The detection of modified, or recombinant DNA (rDNA) in processed foods will pose even greater challenges. In the European Union, foods and food ingredients produced by means of, or containing more than I % of GMO material, have to be labelled declaring the fact. Detecting rDNA from GMO ingredients within processed foods will require the development of novel techniques, although some evidence exists to show that rDNA is reasonably stable and can be extracted in sufficient quantity to be amplified (Straub et al. 1999).Certainly Matrix CRMs will be needed, but so far no organization has done more than consider the need.

I

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The efforts by NIST to produce a PCR SRM have already been described. Whilst SRMs 2390 and 2391 both meet the specific requirements of forensic DNA determination by a specific PCR-based method and. SRM 2392 can be used as a general control of PCR-based methods, but it is unrealistic to believe that similar reference materials will be developed for each application of PCR. Even inside the controlled conditions of a research laboratory, analyzing clean and standardized test samples PCR procedures requires careful quality control, taking into consideration differences in sample preparation, variation in pipetting, differences in reaction tube thickness, poor calibration or instability of the thermal cycler, and reagent quality. As nucleic acid amplification techniques are applied to more diverse applications, as has been demonstrated by the experiences of the IRMM, a range of factors including DNA stability, externalcontamination,inhibition from other elements ofthe matrix and the inherent variability of biological matrices all conspiretogether to affect both the ability of the PCR to amplify a target and the specificity of the interaction of primer and target. Together these variables lead to false positives or negatives,over- or under-results, and generally make reliable quality control very difficult. The use of internal standards for monitoring PCR analysis for certain food pathogens has been developed. The internal standard is a DNA fragment flanked by the same primer recognition sites as the target sequence. It can be considered a mimic and can help control false positives or negatives but is no use monitoring the specificity of the PCR reaction. Indeed, as the mimic competes with the intended target it decreases assay sensitivity. As far back as 1989 Wang et al. demonstrated the use of mimics in the quantification of competitive PCR. Unfortunately these and other existing quality control procedures do not answer all problems. There remains a clear need for development of PCR reference materials that will provide information both on quality and quantity levels. For quality the reference materials should be host-specificand PCR primers, for positive control, may correspond to host specific house keeping genes e.g. b-actin. For quantitative analysis, fluorescence dyes in specific primers might be used in order to measure accurately the amount of DNA present. Such practices, and other as yet un-realizedprocedures,w ill be needed to achieve reliable results in the quantification of DNA analysis.

5.5 Reference Substances and Spectra for Pharmaceutical Analysis John

H. McB. Miller, Agnbs Artiges, Ulrich Rose, Vincent Eglofand

Emrnanuelle Charton

The aim of REMCO, The International Standards Organisation Committee on Reference Materials, is to carry out and encourage a broad, international effort for the harmonisation, production, and application of CRMs. Unfortunately, the specifications of pharmaceutical analysis and the use of pharmacopoeia1 reference substances were not taken into account in the preparation of the ISO-REMCO guidelines. Chemical reference substances (CRS) required for the application of monographs for drug substances and pharmaceutical formulations are an integral part of

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

these monographs, which are legally binding quality standards. Pharmacopoeias mostly follow the general philosophy of the ISO-REMCO guidelines. Differences have been recognized by ISO-REMCO with an amendment to the Introduction to I S 0 Guideline 34,znd Edition 2000: ‘‘Pharmacopoeia1standards and substances are established and distributed by pharmacopoeial authorities following the general principles of this Guide. It should be noted, however, that a different approach is used by the pharmacopoeial authorities to give the user the information provided by certificates of analysis and expiration dates. Also, the uncertainty of their assigned values is not stated since it is negligible in relation to the defined limits of the method-specific assays of the pharmacopoeias for which they are used. ’’ 5.5.1 Introduction

Reference substances (and spectra) form an integral part of the majority of monographs of the official compendia and as such their use is mandatory for the testing of pharmacopoeial substances and preparations. Reference substances for pharmaceutical analysis are available from a number of pharmacopoeias world-wide but the most extensively used are those of the European and United States Pharmacopoeias. However, in Europe some national pharmacopoeias may continue to publish additional national monographs; in this case they publish the monographs of the European Pharmacopoeia which require the use of European Pharmacopoeia chemical reference substances (Ph.Eur. CRSs), biological reference preparations (Ph.Eur. BRPs) or reference spectra, while national pharmacopoeial reference materials are required only for the substances or preparations which are not included in the European Pharmacopoeia. The European Pharmacopoeia includes about 1500 monographs which describe the quality specifications for the corresponding substances or preparations. These norms are obligatory standards for these substances/preparations in all signatory countries of the “Convention on the Elaboration of the European Pharmacopoeia” (European Treaty Series 1964) and are further enforced by Directives (EEC Directives 1975ff.) of the European Union. This is also the situation in the United States of America where the USP is the legal instrument for the quality of pharmaceutical material (US Pharmacopoeia 1995).The International Pharmacopoeia chemical reference substances (IPCRSs) have no legal status except in those countries which have adopted the International Pharmacopoeia for the control of pharmaceutical substances, products and excipients within the framework of their national legislation. It is essential to realize that reference materials must only be employed for the purpose(s) for which they are intended. The use of such materials for analytical methods other then those described in the monographs is unacceptable, except when the user carries out the necessary testing to validate the reference substance for a particular test. Chemical and pharmaceutical manufacturers must establish their own internal reference materials, when no pharmacopoeial monograph exists, to satisfy the requirements of Good Manufacturing Practice (European Commission 1997).

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This Section will describe the procurement, characterization, production, packaging, storage, distribution, and repeat testing of pharmacopoeia1 reference materials and will to a large extent reflect the experiences and practices of the European Pharmacopoeia. A number of articles have previously been published describing the characterization and monitoring of chemical reference substances of the European Pharmacopoeia (Miller 1987, 1990, 1992; Rose 1996), chemical reference substances for microbiological assay (Sandrin et al. 1997) and biological reference preparations (The Biological Standardization Programme 1996; Charton et al. 1997). 5.5.2

Definitions and Guidelines

In 1975 the World Health Organization produced a guideline for the establishment, maintenance and distribution of chemical reference substances (WHO 1975). This document was intended to foster collaboration and harmonization of approval for the provision of reference substances by national authorities and organizations responsible for reference substances collections. This guideline was revised in 1982 (WHO 1982) and a further revision was completed more recently (WHO 1999) to take into account progress in pharmaceutical analysis. The latest guideline defines both primary chemical reference substance and secondary chemical reference substance as follows: “A designated primary chemical reference substance is widely acknowledged as having appropriate qualities within a specified context, and whose value is accepted without reliance on comparison to another chemical substance. “A secondary chemical reference substance is a substance whose characteristics are assigned and/or calibrated by comparison with a primary chemical reference substance. The extent of characterization and testing of a secondary chemical reference substance may be less extensive than for a primary chemical reference substance. This definition may apply to some substances termed Working Standards”.

This guideline covers all aspects of chemical reference substance production including procurement of candidate material, the evaluation of the material by various chemical and physico-chemical methods, the assignment of content for those substances to be employed in quantitative analysis, packaging, storage and distribution. However, a series of guidelines have been published by International Standardization Organization (ISO) comprising terms and definitions ( I S 0 Guide 30 1gg2), quality system required ( I S 0 Guide 34 1996) and contents of certificates and labels ( I S 0 Guide 31 1981).A stand-alone guideline for accreditation of producers of reference materials is presently under elaboration ( I S 0 Document N 464 1998) by the ISO-REMCO group. I S 0 defines a reference material as a “material or substance, one or more of whose properties are sufficiently homogeneous and well established to be used for the calibration of an apparatus, the assessment of a measurement method or for assigning values to materials” whilst a certified reference material (CRM) is a “reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

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accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence”. The role of CRMs in chemical analysis is to provide a “measurement benchmarlts” that chemists can use to calculate or assess the accuracy of their analysis. When several laboratories can achieve the same analytical results for a given CRM, they demonstrate comparability of their measurement. The use of reference materials is encouraged whenever possible in the quality system guidelines of I S 0 ( I S 0 1990) and EN 45001 (CEN/CENELEC 1989) where it is stated that reference materials provide essential traceability in chemical measurements and are used to demonstrate the accuracy of results, calibrate equipment and methods, monitor laboratory performance, validate methods, and enable comparison of methods by use of transfer standards. Guidance concerning the extent of testing required is also given in the “Technical Guide for the elaboration of monographs” (1996) of the European Pharmacopoeia. These guidelines are specifically addressed to the establishment of reference substances for pharmacopoeia1use. Thus, it can he seen that IS0 type reference materials or certijied reference materials are intended to he employed for a number of purposes using a variety of diferent analytical procedures, whilst pharmacopoeia1 reference substances/preparations are intended f o r a specijic purpose and are not to he used for methods or procedures which are not described i n the particular monograph. 5.5.3

Uses of Pharmacopoeia1 Reference Substances

Pharmacopoeia1 reference substances/preparations are an integral part of a monograph and employed for a variety of purposes within the monograph (Table 5.3). 5.5.3.1

Reference Substances Used for Identification

Reference substances can be used for confirmation of identity of the substance by, e.g. infrared spectrophotometry where the spectrum of the substance to be examined must be concordant with the spectrum of the CRS, or by thin layer chromatography where the migration and appearance of the spots of both the substance to be examined and the CRS are the same, or by liquid chromatography where the retention time of both the substance to be examined and the CRS are the same. Identification by peptide mapping requires the use of both a CRS and a reference chromatogram. The substance to be examined and the CRS are hydrolyzed with a protease and the resulting digests are chromatographed.The profile of the chromatogram obtained with the substance to be examined must correspond to that of the chromatogram obtained with the CRS and must be similar to the reference chromatogram supplied with the CRS. In the monograph for insulin (Monograph 0276 1999).Figure 5.2, such a test allows the distinction between different insulin species.

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Use of reference products in the European Pharmacopoeia

Code

Uses

Code

Uses

I

Identification by infrared spectrophotometry Identification by thin layer chromatography Identification by melting point

I2

Identification by depolymerization Identificationby liquid chromatography Identification by peptide mapping Identification by electrophoresis Identification by size-exclusion chromatography Identification by gas chromatography Identification by nuclear magnetic resonance spectrometry Identifkation by UV spectrophotometry or colorimetry Test for related substances by UV spectrophotometryor colorimetry Test for related substances by thin-layer chromatography Test for related substances by liquid chromatography Test for related substances by size-exclusionchromatography

IS

Test for related substances by electrophoresis Test for related substances by gas chromatography Assay by UV-spectrophotometiyor colorimetry Assay by liquid chromatography Assay by size-exclusionchromatography Assay by gas chromatography Microbiological assay Bioassay

2

3

3A 4 4‘4

5 5‘4

6 7 8 8A 9 I0 I1

5.5.3.2

13

I4

15‘4

16 I7 I8

I9A

Imrnunoassay Chromogenic substrate assay

20

Assay by volumetric titration

2oA

Assay by hydrolysis rate

21

General methods

22

Other purposes

19

Reference Substances Used for Related Substance Tests

Related substances are defined as known impurities which may be identified or unidentified (Technical Guide for the Elaboration of Monographs 1996).They include intermediates and by-productsfrom a synthetically produced organic substance impurities, co-extracted from a natural product and degradation product ofthe substance. The monographs of the European Pharmacopoeia describe a so-called test for related substances which is intended to control the level of impurities. Reference substances may be used in this test in order to ensure the system suitability and/or to control the content of related substances. (I) System Suitability Tests These chromatographic performance tests are camed out in order to ensure that all impurities to be controlled are well separated from the substance to be examined (HPLC,GC andTLC). For this reason, preferably such reference substances are chosen which elute close to the main compound (HPLC,GC) or which have a similar Rfivalue (TLC) but can still be separated. These may be structurally related compounds which shall be separated with a minimum requirement for the resolution using the chromatographic system described, e.g. such as in the monograph for desmopressin (Monograph0712 1999), Figure 5.3.

5.5 Reference Substances and Spectrafor Pharmaceutical Analysis 1400

1200

I

I11 I1

1000

2 E

800

Porcine Insulin

h

8

> E

600

; : 1 400

I11 I1

200

0

5

10

15

Human Insulin 25 Minutes

20

30

Fig. 5.2 Chromatogram ofthe enzymatic digests of different types o f insulin employed to demonstrate the suitability ofthe digestion procedure and the chromatographic system.

200~

16.76

1

Oxytocin 17.36

150~

100 50~

1

‘L

desmopressin required to confirm the suitability ofthe chromatographic system in the desmopressin monograph.

35

40

45

50

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5 Reference Materialsfor “Life”Ana1ysis

A different approach is the use ofby-productsor degradation products which has the advantage that the impurity itself, which must be controlled, is used to ensure the chromatographicperformance and can simultaneouslyserve as external standard in the purity test. Occasionally, structural or optical isomers are used for these purposes (Rose 1996)such as diltiazem impurity A (“trans-diltiazem”)in the monograph for diltiazem hydrochloride (Monograph1004 1997). Another approach to carry out a system suitability test in liquid chromatographic tests for related substances is the use of “performance test? mixtures or spiked materials. These mixtures are employed to adjust the chromatographic conditions required for the performance of the test by comparison to a representative chromatogram and may serve to identify the impurities on a given test sample. A characteristic example is atenolol for column validation CRS which is supplied together with a typical chromatogram depicted in Figure 5.4 (Monograph 0703 1997). Purity Control The employment of reference substances in the related substances test as external standards for the determination of the content of impurities is often used. The impurities themselves may be described for this purpose but also a

(2)

W 1.4€+04 SPECIMEN CHROMATOGRAM

1.2E+04

1.OE+04

8.OE+03

6.OE+03

AJ 6

4.OE+03

I

2.OE+03

k

O.OE+OO

5.00

10.00

15.00

Minutes

Fig. 5.4 Chromatogram ofatenolol for column validation CRS which is supplied with the CRS. A similar chromatogram must be obtained to assure the suitability ofthe chromatographic system. (Column:4.6x 150mm Nucleosil C-l8[5pm]: Mobile phase: 1.Og sodium octane-sulphonate,0.4 g tetrabutyl ammonium hydroxide, 2.72 g potassium dihydrogen phosphate in 800 ml water [pH 3.01, 20 ml tetrahydrofuran and 180 ml methanol, flow rate: 1.Ornl/min and detection wavelength: 226 nm).

20.00

25.00

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

Fig. 5.5 Chromatogram ofchlorprothixenehydrochloride CRS which contains 2.7% ofthe E-isomer. (Column: 4 x 120 mm hypersil BDS [3 p]. Mobile phase: 6.0 g potassium dihydrogen phosphate, 2.9 g sodium lauryl sulphate, 9.0 g tetrabutylammonium bromide 550 ml water, 50 ml methanol and400 ml acetonitrile, flow rate: 1.5 ml/min and detection wavelength: 254 nm.)

dilution of the test solution can be used provided that the impurity and the monograph substance exhibit a similar detector response. In case of reduced availability of an impurity a possible approach is to prepare a “spiked sample”, i.e. a known amount of impurity is added to the CRS and may serve in a system suitability test as well as for the control of the level of this impurity. An example is given in the monograph for chlorprothixene hydrochloride (Monograph 0815 1999) where the content of the E-isomer is controlled to a level of not more than z per cent, Figure 5.5. 5.5.3.3

Reference Substances Used for Assay

Pharmacopoeia1reference substances are increasingly employed as assay standards since assay of content is determined more and more frequently by separation techniques. In such a case, based on the results of a collaborative study, a content is assigned to the corresponding reference substance which is method specific, i.e. it is only to be used with the method described in the monograph. The establishment of these substances is described in more detail later in the Chapter.

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5.5.3.4

Minimizingthe Use of Reference Substances

It is policy of the Commission of the European Pharmacopoeia to minimize the use of reference substances/preparations since the production, maintenance and distribution of chemical reference substances is a costly and time-consuming undertaking. Therefore the decision to establish a reference substance should not be taken lightly and consideration should be given to other approaches which could be adopted to avoid the use of reference substances. A number of strategies can be employed to reduce the need for reference substances. When the monograph requires the identification of a substance by infrared spectrophotometry,comparison can be made to a reference spectrum. When the test for related substances is a limit test, the peaks of the impurities in the chromatogram of the test solution can be compared to the peak of the test substance in the chromatogram of a dilution of the test solution at the limiting concentration. The approach is valid provided that the response factors of the impurities and the test substance are equivalent using the detector conditions described, otherwise correction factors need to be applied. When there is a system suitability criterion for the resolution of a critical pair it is preferable to describe reagent grade material which is available in commerce rather than to establish a reference substance. Alternatively, it may be possible to describe a degradation procedure of the substance so that a particular impurity profile is obtained which can serve as a chromatographic performance check. This approach has been described (Rose 1998)and applied to a number of monographs of the European Pharmacopoeia, e.g. in the revised monograph for spiramycin (Monograph 0293 1999).This monograph describes a test for related substances by liquid chromatography for the control of impurities and degradation products. Spiramycin is a mixture of three components: spiramycin I, 11 and I11 of which spiramycin I is the most important. One of the impurities to be controlled by the monograph and which may be obtained by acid hydrolysis is neospiramycin I. In the conditions described, neospiramycin I elutes close to spiramycin I and can therefore be used in the system suitability test. Thus, to avoid the use of a chemical reference substance, spiramycin is hydrolyzed as follows: a solution of spiramycin (I mg/ml) in the mobile phase (pH 2.2) is heated for 30 min at G O T . By this procedure peaks of similar heights of spiramycin 1 and neospiramycin I are obtained (Figure 5.6) which can be used for the identification of the impurity and also serves for the calculation of the resolution. Non-specific absolute assay methods, e.g. volumetric titration, can be applied to avoid the establishment of a reference substance. This is only appropriate, however, when the monograph describes a separation test for related substances. This approach is certainly valid for the determination of the content of pharmaceutical raw materials but less acceptable for the assay of content of pharmaceutical preparations where the employment of specific assay methods is recommended (ICH Guideline 1994)to take account of decomposition of the active ingredient during the shelf life of the product and to avoid possible interference from excipients.

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

Minutes Fig. 5.6 Liquid chromatographic separation of spiramycin I from the impurity neospiramycin I produced by acid-hydrolysis.

5.5.4 Procurement o f Candidate Reference Substances

A candidate reference substance is selected from the normal production batches of a manufacturer and is not further usually purified. Candidate reference substances/ preparations should be supplied with: a certificateof analysis including test methods, test results with complete identification by appropriate physico-chemicalmethods, e.g. nuclear magnetic resonance spectroscopy, infrared spectrophotometry,mass spectroscopy etc. stability data of the substance with an indication of the storage conditions to be employed information as to its hygroscopicity and its solid-state properties, e.g. amorphous, crystalline,polymorphic form etc. a material safety data sheet a list of the potential impurities which may be present, if a monograph substance The quantity requested should be sufficient to allow for all the pre-testingrequired and the preparation of a stock ofvials or ampoules which will last for several years. 5.5.5 Requirements for Candidate Reference Substances

Except in rare cases, the quality of the CRS to be established must comply with the requirements of the monograph. The purity of the candidate reference substances

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I depends on the purpose for which it is required in the monograph. In most cases a 5 Reference Materialsfor "L@"Ana/ysis

purity of better than 99.0 % (on an anhydrous and solvent-free basis) is required, but some substances, e.g. antibiotics, and naturally occurring substances may be employed with higher levels of impurity. Where the CRS is intended for a non-specific assay the purity requirement may also be less (ultraviolet and visible spectrophotometric methods). Impurities with physico-chemical characteristics similar to the main component will not impair the use of the chemical reference substance whereas traces of impurities with significantly different properties will render the substances unsuitable for use in a non-specific assay. The response of impurities under the assay conditions should always be assessed. Substances (impurities) which are not the subject of a monograph are usually synthesized or supplied by the manufacturer. In this case a purity in excess of 90.0 % is required provided that the reference substance is employed in a limit test. If the reference substance (impurity) is employed in a quantitative test then the purity requirement is normally better than 99.0 %. When it is less, then an assigned content must be established. 5.5.6 Evaluation

Although pharmacopoeia1 reference substanceslpreparations are employed for specific tests and assays in the monographs of the pharmacopoeia, the candidate substances are tested against a wide variety of analytical methods, including all the tests prescribed in the monograph. However, the extent of testing and the number of laboratories involved depends on the use of the reference substances. The following testing programme is carried out by the European Pharmacopoeia applying a quality assurance system based on EN 45001. 5.5.6.1 Reference Substances Used for Identification The batch must comply to the requirements of the monograph. However, the most important control is the application of the test@)for which the substance is intended. It is usual for at least one laboratory (usually the Ph. Eur. laboratory) to apply all tests of the monograph. The structure of the substance must be clearly identified by comparison of the IR spectrum to spectra published in the literature and interpretation of the NMR and mass spectral data. 5.5.6.2

Reference Substances Used for Related Substance Tests

As previously described three types of reference substances may be used in this test: structurally related compounds by-productsor degradation products (3) performance test mixtures or spiked materials (I)

(2)

The substances described under (I) and (2) are characterized by the following physico-chemical and spectrophotometric techniques:

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

structural identification by 'H-NMR, MS and IR resolution test as described in the monograph purity tests by LC (usually by external standard), TLC (semi-quantitative or densitometric), GC (peak area normalization) or DSC water determination by Karl Fischer or by coulometry, occasionally TGA Although not used for assay purposes, the purity of the CRS should be at least go %. Howevei-, when the substance is used in a quantitative test and the purity is not greater than gg % a content will be assigned. For the mixtures described under (3) it is sufficient to determine the chromatographic profile of the CRS and to demonstrate that all impurities are well separated according to the monograph description. When the spiked sample is also used in the purity control, then the content of the impurity in the CRS material must be determined by appropriate chromatographic methods and a value assigned to the material. 5.5.6.3 Reference Substances Used for Assay Physico-ChemicalAssay Standards

Where the CRS is to be employed as an assay standard, the extent of testing is very much greater. A number of ways for the assignment of content values of reference materials have been described ( I S 0 Guide 34 1996): single definitive method by a single Organization two or more reference methods by one Organization number of methods of known and acceptable accuracy and precision by a network of qualified Organizations method specific approach (inter-laboratorystudy) giving only a method specific assessed property value The laboratory of the European Pharmacopoeia applies the method specific approach (inter-laboratorystudy) as has been previously described (Technical Guide for the Elaboration of Monographs 1996). Several collaborating laboratories (usually five participating laboratories) test the proposed substance using a variety of techniques. The relative reactivity or relative absorbance of the impurities present in a substance must be checked when a nonspecific assay method is employed, e.g. by colorimetry or ultraviolet spectrophotometry. It is particularly important to quantify the impurities when a selective assay is employed. In such a case, it is best to examine the proposed substance by as many methods as practicable, including, where possible, absolute methods. For acidic and basic substances, titration with alkali or acid is simple but other reactions which are known to be stoichiometric may be used. Phase solubility analysis and differential scanning calorimetry may also be employed in certain cases. The European Pharmacopoeia prepares a protocol which must be strictly followed by the participants of the collaborative trial to assign the content. The protocol usually requires:

184

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the determination ofwater (or loss on drying) the estimation of the impurities using the separation technique described in the monograph the estimation of residual solvents by head space gas chromatography when a test for loss on drying is not prescribed the determination of the content of the substance by an absolute method (usually volumetric titration) may be included to ensure that significant levels of inorganic impurities are not present. This is a confirmatory determination and the result is not used in the calculation of the assigned value Summation of the results of the determinations of water, organic solvents, mineral impurities and the organic components amounts to roo %. The results of methods employed to analyze the substance other than these given above are not used for the calibration of the assigned value but are reported to support the results obtained by the defining methods. For most reference substances intended as assay standards the assigned content is normally expressed "as is" so that it is essential (when establishing the CRS) to determine the content ofwater and residual solventsfor a non-specificassay and also,for a selective assay, to determine the content of impurities. The European Pharmacopoeia Commission has adopted the policy that the value assigned to a reference substance as a result of an inter-laboratorytrial should have an uncertainty not greater than a predetermined value. The following formula (Equation 5.1) may be used to calculate the estimated approximate uncertainty

where: n = the number of participating laboratories oi2 = variance for the estimation of impurities ow2 = variance for the determination of water 0s' = variance for the determination of residual solvents t = studentst Assuming an analytical error of +2 % which is based on the analysis of the results of a number of proficiency tests and collaborative trials (Daas and Miller 1998),a m a imum uncertainty of 0.24% would imply a 0.1% probability of rejecting a good result. Given that the uncertainty of a content to be assigned is below a predetermined value, the results of the collaborative trial are acceptable; otherwise it is recommended to repeat the trial in whole or in part. An example for the application of this calculation is given in Table 5.4 which shows the results of a collaborativetrial for the establishment of ciprofloxacinhydrochloride CRS 2. The uncertainty was calculated to be 0.11%. In the specific case of the European Pharmacopoeia reference substances, it is considered that the recommendation in the IS0 guidelines, to give the uncertainty value with the assigned value on the label of the CRS, is not relevant because of the following reasons:

5.5 Reference Substances and Spectrafor Pharmaceutical Analysis Tab. 5.4 Uncertainty ofthe assigned value of ciprofloxacin HCI CRS z fmpurities Ph) Laboratory

Related substances

Wafer

1 2 3 4 5 6 7 Mean

0.08 0.04 0.07 0.05 0.04 0.05 0.03 0.05 (n= 7) 0.018 0.0003

5.88 5.94 6.07 5.77 6.08 6.07 5.96 6.0 (n= 7) 0.116 0.014

SD (0) Variance (0’)

The content of residual solvents is negligible. Approximate Uncertainty=

JT

x 2.447 = 0.11

If the risk of rejecting a good result is considered to be acceptable, there is no justification of giving uncertainty values with the assigned value. The I S 0 guide is particularly concerned with the establishment of reference materials which contain the analyte as a small, or even trace, quantity in a complex matrix. These reference materials serve as measurement benchmarks when applying an appropriate analytical procedure for the determination of an analyte in the sample. The value attributed to the reference material is usually the mean of results obtained from a variety of methods and laboratories. Thus, the value attributed to the substance may have a high degree of uncertainty. A particular reference material is subjected to the same procedure as the test samples so that greater confidence can be given to the results of the test samples provided that the value found for the reference material falls within the given uncertainty. A pharmacopoeia1 reference substance is intended for the determination of the main component of a substance or for the active ingredient of a pharmaceutical formulation which is usually present at a high proportion of the total. The reference substance is to be used as a primary standard in a specific method validated as prescribed in the ICH Guideline “Validationof Analytical Procedure: Methodology” (Technical Guide for the Elaboration of Monographs 1996; ICH Guideline 1gg7), the reproducibility of which is known. This is taken into account when the limits of acceptance (tolerance) for the substance or product are fxed (Daas and Miller 1997,1998). MicrobiologicalAssay Standards

The potencies of some antibiotics described in the European Pharmacopoeia are determined by microbiological assay (Microbiological Assay of Antibiotics 2.7.2

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5 Reference Materials for “1fe”Analysis

The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory,a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in International Units. If an International Standard does not exist, European Pharmacopoeia Units are used. Due to the inherent variability of these assays either by agar-plate diffusion measurement or turbidimetry measurement, the fiducial limits are calculated according 1300

1200

1100

1000

900

800

700

1

2

4

5

Fig. 5.7 Results (IU/mg) of an interlaboratory study to determinate the potency oftylosin CRS 1 by microbiological assay (diffusion method).

6

7

i300r

5.5 Reference Substances and Spectrafor Pharmaceutical Analysis

1200"

"oo-J 1000'.

!ill

goo-.

800'.

700'

All

1

3

4

Fig. 5.8 Results (IU/rng) of an interlaboratory study t o establish the potency of tylosin CRS 1 by microbiological assay (turbidimetric method): individual estimates per assay (empty dots indicate rejected assays).

to thc statistical procedures given in the pharmacopoeia (Statistical analysis of results of biological assays and tests 5.3 1997). The variability obtained for these assays is shown for tylosin CRS (Figures 5.7 and 5.8 for the difision assay and the turbidimetric assay respectively). The decision tree employed for the statistical treatment of the results is shown in Figure 5.9. In this case, the two assay methods were considered equivalent and the fiducial limits were calculated from all valid data. Tylosin was assigned a potency of 1035 IU/mg with confidence limits of roz8 IU/mg to 1044 IU/mg (corresponding to * 0.8 % of the assigned value).

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I

and statistics of validity

1

I

Situation

// //

c No

If I!!7

// arations

smal

Fig. 5.9 Decision tree used to accept or reject results from collaborative trials t o establish the potencies o f antibiotics to be used as chemical reference substances for microbiological assay standards.

Biological Reference Preparations

Biological reference preparations are established for specific assay methods by collaborative trials following a strict protocol. The results are statistically evaluated and potencies are assigned (Statisticalanalysis of results of biological assays and tests 5.3 1997). Reports of such trials are regularly published in special issues of Pharmeuropa (Phamzeuropa Bio, published by the Council of Europe). Since 1996, results of collaborative studies for the establishment of the following BRPs have been published in Pharmeuropa Bio: Low Molecular Mass Heparins, Factor VIII, Human Immunoglobulin Inactivated Poliomyelitis Vaccine, Live Measles Vaccine, Live

5.5 Reference Substances and Spectra for Pharmaceutical Analysis

Rubella Vaccine, Live Mumps Vaccine, rDNA Hepatitis B Vaccine, Oral Poliovirus Vaccine, Erythropoietin,and Factor IX etcetera. 5.5.6.4

CRS as Calibrators

Where the CRS is to be employed as a verification material in general methods published in the European Pharmacopoeia, then like a CRS used as an assay standard, the extent of testing is considerable. It is desirable that several collaborating laboratories test the proposed substances using a variety of techniques to ascertain that its purity as adequate. An appropriate number of collaborating laboratories will also participate, after the substance has been deemed suitable, in the study to establish a value by the measurement of the essential property of the substance using an appropriate instrument. Examples of such substances are calcium oxalate CRS employed for the verification of the electrobalance in thermogravimetry and the solvents, trimethylpentane CRS, toluene CRS and methylnaphthalene CRS, employed for verification of the performance of refractometers. 5.5.7 Monitoring Programme

It is necessary to test the CRSs and the BRPs for stability during their storage, in order to ensure their continued fitness for use. A standardized testing procedure has been introduced which is designed to detect at an early stage any sign of decomposition using appropriate analytical techniques. These screening methods have also been employed during the establishment phase and these initial results served as the “base-line”values. The reference substance collection of the European Pharmacopoeia consists of more than 1300 items. Consequently, for regular screening, the methods must be rapid. They must also be sensitive so that the quantities used are small to avoid depletion of the stock. The monitoring programme includes: determination of water, loss on drying or TGA (not necessary for substances in ampoules) thin-layer chromatography using high loadings of samples and silica as absorbent liquid chromatography usually employing reverse-phase stationary phase as a complement to the thin-layer chromatography. Other stationary phases are also used as considered appropriate when appropriate, differential scanning calorimetry is employed to determine the purity any other specific tests for detecting impurities The frequency of testing of a reference substances depends on the known or suspected stability and of its prescribed use. Reference substances used as assay standards are re-examined on a two-yearly basis whilst the other reference substances are

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tested every three to four years. Substances known to be unstable are monitored more frequently. Any differences observed compared to the last examination will lead to a more extensive examination of the batch and, if considered necessary, a replacement batch is obtained and established. Implementation of this procedure precludes the necessity of giving expiry dates for these substances. However, no studies are undertaken to test the stability ofthe substance in opened vials or ampoules nor the substances in solution. Users of reference substances should not store solutionsof chemical reference substancesor opened vials. 5.5.8

Packaging and Filling

All operations conform to the requirement of Good Manufacturing Practice (European Commission 1997). The reference substances are distributed into suitable containers using appropriate filling conditions. A quantity sufficient to carry out the required tests, generally in duplicate, are filled into the containers. Most substances for which there are no concerns for either toxicity or stability are filled by weighing the appropriate quantity into antibiotic vials in a horizontal laminar flow work station. These operations are carried out in a self-contained cubicle to avoid cross-contamination. The vials are then closed with butyl rubber stoppers and sealed with an aluminium crimp seal using an automatic crimping and labelling machine. A substance which is either hygroscopic or easily oxidized is filled in a pressurized glove box (isolator) fitted with a gas purification system in which the atmospheric air is replaced by argon. Under these conditions the contents of water and oxygen are restricted to less than I ppm. A substance which is highly toxic is filled in a depressurized glove box (isolator). Increasingly, lyophilization is employed for the preparation of chemical and particularly biological reference preparations. Substances which are expensive and available only in small quantities are prepared in solution and distributed by an automatic liquid filling machine into containers which are then placed in a freezedrier. On completion of the lyophilization cycle the vacuum is broken by the introduction of an inert gas. If vials are employed they are automatically stoppered in the freeze-drying chamber. Reference substances prepared in this manner include antibiotic standards for microbiological assay, synthetic peptides, biologicals and expensive chemical substances in short supply. In such cases the content or potency of the substance is expressed as a quantity per vial and the whole quantity has to be dissolved for further use. Instructions on the preparation of the solution used is the assay standard are either given in an information leaflet which accompanies the CRS or in the monograph itself. Thus, CRSs prepared in this manner are standards for which no weighing by the analyst is required.

5.5 Reference Substances and Spectra for Pharmaceutical Analysis 5.5.9 Certificates of Analysis / Expiry Date / Catalogue

Since the CRSs and BRPs are officially certified by the European Pharmacopoeia Commission, which adopts the reports establishing their suitability for the intended use, it should be noted that neither certificates of analysis nor data which are not relevant to the use of the substances as defined by the Ph. Eur. monograph, are provided with the reference products or substances. Information required for the correct use of the chemical reference substance or biological reference preparation is provided. The label on the vials or ampoules gives:

0

the name of the organization the name of the substance both in English and in French (as written in the corresponding monograph) the batch number

If used as an assay standard the following information is also fronted: (I) the assigned content of the chemical entity or (2) the content in mg or ml of the chemical entity or (3) the assigned potency (for microbiological assays or biological tests)

An accompanying explanatory leaflet may also be provided, if necessary, to describe the preparation of the substance for use. In some uses, a chromatogram may also be provided. As mentioned before, no expiry date is indicated because the products and substances comply with the requirements of the corresponding monograph and are monitored regularly. A catalogue is issued three times a year, after each session of the European Pharmacopoeia Commission, where new and replacement batches of chemical reference substances or biological reference preparations are adopted. This catalogue indicates for each substance or preparation: the order code the name of the substance or preparation in English the batch number the assigned value or assigned content or assigned potency, if applicable the validity or status of the previous batch if a replacement batch has been adopted the chemical formula (for impurities) the sales unit the unit quantity the code indicating the presented use(s) of the substance or preparation the corresponding reference number of the monograph(s) where its use is prescribed Both the catalogue and material Safety Data Sheets are available on request or may be obtained from the web site of the European Pharmacopoeia.

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5.5.10 Storage and Distribution

Most of the reference substanceslpreparations are stored in cold rooms controlled at between 2°C and 8°C. However, a number of substances/preparations which are relatively unstable are stored at -20°C or, in a few cases, e.g. vaccines, at -80°C. The reference substanceslpreparations may be stored for years under these conditions and their fitness for use is continually monitored as described above. Thus, the status of each reference substance/preparation is indicated in the catalogue. It is recommended that purchasers only order a sufficient amount for immediate use since the stability of the contents of opened vials or ampoules cannot be guaranteed. Reference substances are distributed world-wide. Special packaging is employed to minimize the risk of damage during transport. Substances which are normally stored at 4’C are dispatched by normal mail. However, products which are stored at low temperatures (-20°C or -80°C) are packed on ice or solid carbon dioxide respectively and dispatched by courier. 5.5.1 1 International Harmonization

Users of reference substances have expressed the wish that reference substances from one pharmacopoeia be employed for a test described in another pharmacopoeia. It is clear from the previous text that this is not always possible since the monograph requirements of the different pharmacopoeias are generally not identical and therefore that the reference substanceslpreparations have not been established for the same purposes. However, the establishment of a new endotoxin standard by the World Health Organization is a recent example of successful international collaboration between the World Health Organization, the United Stated Pharmacopoeia and the European Pharmacopoeia (Poole et al. 1997).Thus this standard is available from any of these organizations to be employed as a reference in the harmonized Livnulus Amoebocyte Lysate test. Exchanges between pharmacopoeias are co-ordinatedby the Pharmacopoeia1 Discussion Group (PDG) (International Harmonisation 1995)and it is frequent that one pharmacopoeia participates in a collaborative study organized by another pharmacopoeia, or that several pharmacopoeias share the same batch of reference substance to be used in their respective monographs: nevertheless, in this case the reference substance can not be considered as harmonized. A new batch of erythromycin was shared between the United States Pharmacopoeia and the European Pharmacopoeia and was established in a common collaborative study both for the microbiological assay (used in the USP for formulations) and the liquid chromatographic assay (used in the Ph. Eur. and USP for bulk material). It is to be hoped that in the future there may be greater harmonization between the pharmacopoeia monographs leading to a greater number of common reference substances or standards.

5.G References 5.6 References

ANDERSONS, BANKIERAT, BARRELLBG, DEBRUJINMHL, COULSON AR, DROUINJ, EPERON IC, NIERLICH DP, ROE BA, SANGERF, SCHREIERPH, SMITHAJH, STADEN R and YOUNG IG (1981)Sequence and organization of the human mitochondria1genome. Nature 290:457-465. Bacteriological analytical manual, 8th edn. (1995).AOAC International, Gaithersburg, MD. BALLINGERSW, BOUDERTG, DAVISGS, JUDICE SA, NICKLASJA and ALBERTINIRJ (1996) Mitochondrial genome damage associated with cigarette smoking. Cancer Res 56:569~-5697. BOGENHAGEND and CLAYTON DA (1974) The number of mitochondrial deoxyribonucleic acid genomes in mouse 1 and human hela cells. Biol Chem z49:7gg1-7995. BUDOWLEB (1988)The RFLP technique. Crime Lab Digest 15:97-98. BUDOWLEB and BAECHTELFS (1990) Modifications to improve the effectiveness of restriction fragment length polymorphism typing; applied theories. Electrophoresis 1:181-187. CEN/CENELEC (1989) General requirements for the competence of testing and calibration laboratories. EN 45001. E (1997) Establishment and use of biological reference substances of the European CHARTON Pharmacopoiea. Pharmeuropa 9:327-330. DAASAGJ and MILLERJHMcB (1997) Content limits in the European Pharmacopoeia (Part I). How to define limits in monographs and how to use them. Pharmeuropa 9:148-156. DAASAGJ and MILLERJHMcB (1998) Content limits in the European Pharmacopoeia (Part 2). Pharmeuropa 10:1j7-146. EDWARDSMJ, ed. (1937) ATCC QC and reference strains, 1st edn. American Type Culture Collection, Rockville, MD. EISENBERGAJ, GIBSON P, NANDIS and WANGL (1991)The development and implementation of a HAE 111-based RFLP system for parentage testing in Texas. Proceedings from the Second International Symposium on Human Identification, Promega Corporation, pp 163-180. European Commission (1997) Good manufacturing practice Vol. 4, DG 111. European Treaty Series (1964) No 50 Convention on the elaboration of the European Pharmacopoeia amended by the protocol to the convention (EuropeanTreaty Series No 134). EEC Directives (1975 ff.) 75/318: 81/85z; 89/342; 89/381; 91/507; 92/18 LI (1995) Mitochondrial mutations and human disease. Env Mol Mutag 25130-37. GROSSMAN WQ (1991) Statistical Intervals: a Guide for Practitioners. John Wiley, NY. HAHNGJ and MEEKER WC, CANIKJJ, MERRILCR and WEEDN VW HOLLAND MM, FISHERD, MITCHELLLG, RODRIQUEZ (1993) Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War. J Forensic Sci 38:542-553. MM, FISHERDL, ROBYRK, RUDERMAN J, BRYSONC and WEEDN VW (1995)MitochonHOLLAND drial DNA sequence analysis of human remains. Crime Lab Digest 2x109-115. HUNTER-CEVERA JC and BELTA (1996) Maintaining cultures for biotechnology and industry. Academic Press, New York. ICH Guideline (1994) Validation of analytical methods (Definition and Terminology). IFPMA, Geneva. ICH Guideline (1397) Validation of analytical procedure: methodology. IFPMA, Geneva. (ref. 31) International Harmonisation (1995) Harmonisation policies of the Pharmacopoeia1 discussion group (Julyr995). Pharmeuropa 7:413-420. I S 0 (1984) I S 0 7899/2 I S 0 (1988)I S 0 6222 I S 0 (1990) ISO/IEC 25 General requirements for the competence of calibration and testing laboratories and ISO/IEC DIS 17025 General requirements for the competence of testing and calibration laboratories. International Standards Organization, Geneva. I S 0 Document N 464 (1998) General requirements for the competence of reference materials producers, revised IS0 Guide 34. International Standards Organization, Geneva.

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5 Reference Materialsfor “L~”Ana1ysis I S 0 Guide 30 (1992)Terms and definitions used in connection with reference materials. Interna-

tional Standards Organization, Geneva. I S 0 Guide 31 (1981) Contents of certificates of reference materials, revised April 1996 as ISO/ REMCO document N 382. International Standards Organization, Geneva. I S 0 Guide 34 (1996) Quality system guidelines for the production of reference materials. International Standards Organization, Geneva. J A N N I N G B, in ’t Veld PH, MOOIJMANI(A, HAVELAAR AH (1995) Development, production and certification of microbiologicalreference materials. Fresenius J Anal Chem 352:240-245. JONG, SC, BIRMINGHAM,J M and CYPESS RH (1998). Internal quality control audits for microbiology laboratories in culture collections. SIM News 4866-69. KASAI I<, NAKAMURA Y and WHITER (1990) Amplification of a variable number of tandem repeats (VNTR)locus (pMCTn8) by the polymerase chain reaction (PCR) and its application to forensic science. J For Sci 35x96-1200. KING MP and ATIARDI G (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246:500-503. LEVIN BC, CHENGH and REEDERDJ (1999) A human mitochondria1 DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 55:135-146. Microbiological Assay of Antibiotics 2.7.2 (1997) European Pharmacopoeia 3rd Edn. Council of Europe, Strasbourg. MILLERJHMcB (1987) Establishment and verification of European pharmacopoeia Reference E, CINGOLANI G and DESIDERID, eds. Proceedings of the 2nd Substances. In: CINGOLANI International Conference on Pharmacopoeias and Quality Control of Drugs. Vol. I, pp 169172. Fondazione Rorer par la Science Mediche, Rome. MILLERJHMcB (1990) Reference substances of the European Pharmacopoeia. Acta Pharm Jugosl 40:63-69. MILLERJHMcB (1992) Substances de rkfkrence de la pharmacopke europkene et de la pharmcopke franpise. STP Pharma Pratiques 2:496-499. Monograph 0276 (1999) Insulin. European Pharmacopoeia 3rd Edn, Suppl. 2000. Council of Europe, Strasbourg. Monograph 0293 (1999) Spiramycin. European Pharmacopoeia Suppl. 2001. Council of Europe, Strasbourg. Monograph 0703 (1997)Atenolol. European Pharmacopoeia3rd Edn. Council of Europe, Strasbourg. Monograph 0712 (1999) Desmopressin. European Pharmacopoeia 3rd Edn, Suppl. zooo. Council of Europe, Strasbourg Monograph 0815 (1999) Chlorprothixene hydrochloride. European Pharmacopoeia 3rd Edn, Suppl. 2000. Council of Europe, Strasbourg. Monograph 1004 (1997) Diltiazem hydrochloride. European Pharmacopoeia 3rd Edn, Council of Europe, Strasbourg . MOOIJMANI(A, HAVELAAR AH, VAN STRIJP-LOCKEFEER NGWN, BRANGERD, SCHIMMEL H (1999) Recertification of the number of colony forming partides of Enterococcusfaecium in I mL suspension ofreconstitutedartificially contaminated milk powder, BCR CRM 506. EUR-report 19281EN. NAKAMURAY, CARLSON M, KRAPCHO V and WHITER (1988) Isolation and mapping of a poly~ )chromosome IP (D1S80).Nucleic Acids Res 16: 9364. morphic DNA sequence ( ~ M C T I Ion NAKAMURA Y, GILLILAN S, O’CONNELL P, LEPPERTM, LATHROP GM, LALOUELJM and WHITER (1987) Isolation and mapping of a polymorphic DNA sequence pYNH24 on chromosome 2 (DzS44). Nucleic Acids Res 15:10073. NCCLS Infobase 99 (1999) NCCLS approved standards and guidelines. National Committee for Clinical Laboratory Standards, Wayne, PA. S, DAWSONP and GAINESRE (1997) Second international standard for endotoxin: calibraPOOLE tion in an international collaborative study. J. Endotoxin Res. 4221-231. REEDER DJ (1991)NIST standards development for RFLP DNA profiling. Proceedings of the Second International Symposium on Human Identification,pp 245-261. Promega Corporation.

5.6 References ROSEU (1996) The establishment and use of reference substances of the European Pharmaco-

poeia. Pharmeuropa 8:455-464. ROSEU (1998) In s i b degradation: a new concept for system suitability tests in monographs of the European Pharmacopoeia. J Pharm Bio Anal 18:1-14. SAIKI RIC, SCHARFS, FALOONAF; MULLISKB, HORNGT, ERLICH HA, ARNHEIMN (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 1985 Dec 20, 210(4712):1350-1354. SAIKIRK, WALSHPS, LEVENSONCH., ERLICH HA (1989) Genetic analysis of amplified DNA with immobilized sequence specificoligonucleotide probes. Proc. Natl Acad Sci USA 86:6230-6234. SANDRINJ, DAASAGJ and CHARTON E (1997) Establishment of reference substances for the microbiological assay of antibiotics. Pharmeuropa 9:327-330. SIMIONEF and BROWNEM (1991)ATCC preservation methods: freezing and freeze-drying. 2nd edn. ATCC, Rockville, MD. Statistical analysis of results of biological assays and tests 5.3 (1997) European Pharmacopoeia 3rd Ed. Council of Europe, Strasbourg. STRAUBJA, HERTEL C, HAMMES WP (1999) The fate of recombinant DNA in thermally treated fermented sausages. Eur Food Res Techno1 210:62-67 Technical Guide for the Elaboration of Monographs (1996) 3rd Edn. Pharmacopoeia Special Issue, January 2000. The Biological Standardization Programme (1996) Pharmeuropa Bio 1:1-5. SPEISERJM, RAUTM A N N G, COUNE C, eds. Council of Europe, Strasbourg. U.S. Pharmacopeia National Formulary. (1995)U S . PharmacopoeialConvention Inc., Ro d d l e, MD. US Pharmacopoeia (1995)XIII-XIV, 23 pp. U. S. Pharmacopoeia1Convention Inc., Rocldle, MD. WALLACE DC (1992) Mitochondria1 genetics: a paradigm for aging and degenerative diseases. Science 256628-632. WANGAM, DOYLEMV, and MARKDF (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 86:9717-9721. WHO (1975) WHO Expert Committee on Specifications for Pharmaceutical Preparations, 25th Report. World Health Organization, Geneva (WHOTechnicalRepoit Series 567) Annex 3, p 98. WHO (1982) WHO Expert Committee on Specifications for Pharmaceutical Preparations, 25th Report, WHO TechnicalReport Series 681, Annex I, p 19.World Health Organization, Geneva. WHO (1999) WHO Expert Committee on Specifications for Pharmaceutical Preparations. 35th Report, WHO Technical Report Series 885. World Health Organization, Geneva.

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Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

6

General Application Fields Edited by PeterJ Jenks

6.1

Workplace Air Monitoring Yngvar Thomassen and Barry Tylee

6.1.1 Introduction

This Chapter provides information on available certified reference and quality control materials relevant for use in the measurement of airborne contaminants in occupational hygiene. The majority of measurements made in this area worldwide are solvents, dust (total, respirable), elements, oil mist, quartz, fiber identification (asbestos,man-made fibers), mists and gases. Many proficiency testing schemes operate in the area of occupational hygiene measurements. Most countries organize their own schemes and there are differences between these schemes at many levels. Some organizers, for example the Workplace Analysis Scheme for Proficiency (WASP), and Proficiency Analytical Testing (PAT), managed by the Health and Safety Laboratory, Sheffield, UIC and National Institute for Occupational Safety and Health, Cincinnati, USA, respectively, prepare their samples simply by analyte spiking of, e.g. air filters with solutions. In several countries specialist facilities are available to produce samples with characteristics that are more realistic representations of the condition and complexities of true samples. These samples are more difficult to analyze than the original standards which proficiency testing schemes send to participants since they contain interferences and complex matrices that do not exist in samples prepared from solutions of pure standards. The demand for such material on a national scale is smaller than the alternative complements because these samples are specialized and designed to mimic those taken from specific atmospheres. Since these samples are also considered much more expensive to produce, the commercialization has been difficult, although the technology does exist for their production. Surprisingly, few certified reference materials or quality control materials for use in the measurement of airborne contaminants are commercially available from world-wide producers. The main reason for the scarcity of such materials is related to great difficulties in producing realistic samples and the lack of interest from

G. 7 Workplace Air Monitoring

potential users. Even though, some materials are available for the most commonly occurring contaminants in workroom atmospheres. 6.1.2 Solvents

Facilities for the preparation of replicate samples from standard atmospheres exist in several countries. Two major institutes; the Vlaamse Installing voor Technologisch Onderzoek in Belgium (Goelen et al. 1992) and the Netherlands Met Institute Van Swindon Laboratory in Netherlands (Hafkenscheid and Mower 1996) both have state of the art facilities that have been used for the production of reference material on behalf of the Standards, Measurements and Testing Programme of the European Commission (SM&T, formerly BCR). These systems can prepare gas phase mixtures at the occupational hygiene and environmental levels and obtain replicate samples with a high degree of accuracy and precision. The volatile organic compounds (VOCs) are injected continuously using methods such as heat/pressure differences etc. through a glass capillary into a manifold fed by a steam of purified air which is then sampled. The amounts of pollutants injected into the system can be calculated from gravimetric measurements and are directly traceable to primary standards (Goelen et al. 1992). In Denmark, a similar system is used by Mi@-Kemi for commercial production of quality control samples for a number of different solvents and some inorganic gases; see Table 6.1.

Tab. 6.1

Reference materials for solvents and gases

Producer .

Carrier

Component(sJ

Milj@-Kemi,

Charcoal tube

Na2C03tube Chromosorb 106 Tenax tube OVS tube 2MP filter HzS04

Benzene; t-Butanol, Heptane; 1-1-Dichloroethylene; Methoxypropanol,Toluene; Toluene Benzo(u)pyrene, Fluoranthene, Naphtalene; Halothane; Phenol Acetic acid Benzene, Vinyl acetate n-Butanol, n-Octane t-Butyl acrylate, Ethylene glycol-dimethacrylate Methylene diphenyl di-isocyanate (MDI) Ammonia

Glass fiber filters Tenax charged tube Charcoal charged tube

Formaldehyde-2,4-dinitrophenylhydrazone Benzene, Toluene, rn-Xylene Benzene, Toluene, rn-Xylene, o-Xylene

XAD2 tube

BCR

* Address: Miljer-Kemi, Smedeskowej $3, 8464 Galten, Denmark; www.miljo-ltemi.dk

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Elements and Inorganic Compounds

The Standards, Measurements and Testing Programme of the European Commission launched a few years ago a project dealing with the development and, ultimately, the production of air filters realistically exposed to welding dust occurring during stainless steel welding (Christensen et al. 1999).This project resulted in the production and certification of a batch of 1100 filters for the Cr (VI) content (40.16 * 0.60 pg/g dust) (CRM 545). In addition, the total leachable Cr content ( 39.37 f 1.30 pg/g dust) was certified as a means to check for total Cr recovery. Realistic loading ofthese filters was carried out using a multi-port sampler developed at NIOH in Oslo (Butlerand Howe 1999).This sampling system basically consists of a thin circular aluminium drum which is evacuated by a large air pump. One face of the drum has up to 120positions for air filter cassettesto be fitted. Each position is equipped with critical orifices to allow air to pass through them at a fmed rate during exposure. This sampling system is capable of producing near identical air fdter samples; at optimal conditionshomogeneities better an I % RSD can be achieved. Filter samples can be prepared to airborne workplace concentrationsby spiking each fdter with aqueous solution containing elements with concentrations gravimetrically traceableto ultrapure metals or stoichiometricallywell defined oxides. The amounts correspond for some of the materials to current threshold limit values of contaminants in workroom atmospheresprovided that the simulated fdter has been exposed to one cubic meter of air. The certified values are based on a gravimetric procedure, i.e. weight per volume composition of the primary reference material dissolved in high purity sub-distilled acids. The National Institute of Occupational Health in Oslo, Norway, has produced several batches of such materials certifiedfor 2 0 elements. Additionally, information values are reported for four other elements; see Table 6.2. From NIST in the USA similar air filters spiked from solutions are also available for a number of elements. Although the element content of these filters is less representative for actual exposure levels in industrial settings, these samples are also of primary interest in the documentation of measurement uncertainties; see also Table 6.2. Tab. 6.2

Elements on filter media, spiked from solution

Producer

Material No.

Elements (levels)order of magnitude: pg or pg/filter

NIOH, Oslo, Norway"

B2 (pg)

Al, As, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Ni, Pb, Sb. Sn, Ti, TI, V, W, Zn, Zr (lower level) Same elements (approx. factor 2 higher level) Cd, Pb, Mn, Zn (three concentration levels) Be, As (three concentration levels) As, Ba, Cd, Cr, Fe, Mg, Mn, Ni, Pb, Se, V, Zn

NIST

*

A2 (M)

SRM 2676d (pglfilter) SRM 2677a (p,g/filter) SRM 3087a (pg/filter)

National Institute for Occup. Health (NIOH), P.O. Box 8149 DEP 0022 Oslo, Norway http://www.stami.no

6.2 Clinical Application Fields

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Quartz on filter media in a clay matrix is also available from NIST. The SRM 2679a is certified for quartz at three levels; 30.8, 80.2 and 202.7 pglfilter respectively. Respirable silica in powder form is also issued by NIST; SRMs 1878a and 1879a are crystalline silica materials with particles in the respirable range and they are intended for use in X-ray diffraction and infrared spectroscopy. 6.1.4 Asbestos

Optical microscope asbestos reference standards for use in identifying and quantifying asbestos types are available both from NIST and the Institute of Occupational Medicine (IOM) in Edinburgh, Scotland. The IOM materials consists of various asbestos materials; actinolite, amosite, anthophyllite, chrysotile (both from Cassiar, Canada and Zimbabwe), crocidolite and tremolite. The NIST material SRM 1866a consists of a set of three common bulk minegrade asbestos materials; chrysotile, amosite and crocidolite, and one glass filter sample. SRM 1867 consists of a set of three uncommon mine-grade asbestos materials; antophyllite, tremolite and actinolite. The optical properties of SRMs 1866a and 1867 have been characterized so that they may serve as primary calibration standards for the identification of asbestos types in building materials. SRM 1868 consists of a set of two common bulk mine-grade asbestos materials; chrysotile and amosite, contained in matrices simulating building materials (calcium carbonate and glass fiber), in quantities at just below the U.S. EPA regulatory limit of I %. This material is certified by weight for the quantity of each asbestos material present. SRM 1876b is intended for use in evaluating transmission electron microscopy (TEM) techniques used to identify and count chrysotile fibers. This SRM consists of sections of mixed-cellulose-ester filters containing chrysotile fibers deposited by an aerosol generator. RM 8411 consists of a section of collapsed mixed-cellulose-esterfilters with a high concentration (138 fiberslo.01 mm) of chrysotile and a medium concentration (43 fiberslo.01 mm) of amosite. It is intended for use in evaluating the technique used to identify and count asbestos fibers by TEM.

6.2

Clinical Application Fields Robert FM Herber andJan P Straub

6.2.1

Introduction

Reference materials have been long used in clinical chemistry; the first biological reference material was developed by Paul Ehrlich in 1897 (Buttner 1995). The routine use of RMs in clinical chemistry started in the early 1970’s and was driven by

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to give both reliable and comparable results. Physicians use data from the laboratory to help decide whether the patient has a certain disease, or not, and patients move from hospital to hospital. The direct and immediate use of laboratory data in medical diagnostic decision making is unique, so the proper use of reference materials in conjunction proficiency testing clinical chemistry is vital if false and mis diagnosis is to be avoided. A second reason for using reference materials in clinical chemistry is to ensure values obtained are traceable to those in a recognized, authoritative reference material (Johnson et al. 1996). As a result, the assignment of values of secondary and tertiary reference materials, calibrants, controls, and proficiency samples should be performed as precisely as possible (Johnsonet al. 1996). Surprisingly there is still debate on this topic, and on the need for clinical chemistry to incorporate the principals of analytical quality assurance (Dybkaeret al. 1999). A number of international organizations are active in the field of clinical chemistry reference materials and proficiency testing, these include:

the International Federation of Clinical Chemistry (IFCC) the International Union of Pure and Applied Chemistry (IUPAC) the World Health Organization (WHO) Various External Quality Assessment Working Groups These and other organizations have published a number of papers on this subject, (Dybkaer and Stornng 1995; Heuck and Magrath 1995; McQueen et al. 1995; Wagner 1997).In the United States, the National Committee for Clinical Chemistry Laboratory Standardization (NCCLS) edits in the National Reference System for the Clinical Laboratory series of standards and guidelines on reference for assigning the best reference quality analytical and characteristic values. In one of the guidelines (NCCLS 1995). it has been stated, that CRMs must be produced under conditions specified by the Food and Drug Administration’s current Good Manufacturing Practice Guidelines. In addition, the council of the National Reference System for the Clinical Laboratory (NRSCL) may accept the CRM produced on base of information of the institutions to serve as a source of the CRM, and the description of the CRM. This last issue must imply the CRM name, batch number, labelling and units; source of the material; homogeneity of the CRM samples; and certified materials. Recent developments in Europe mean that reference materials included in diagnostic kits are covered by the recent directive controlling the production and use of in vitro diagnostic devices, and must be licensed before use. As in other reference materials, a hierarchy exists with three levels (Johnson et al.

1996). Primary reference materials, i.e. international or national certified reference materials, CRMs) Secondary reference materials, i.e. manufacturers’ in-house calibrants and controls (including commercial reference materials) Tertiary reference materials, i.e. controls and working calibrants produced by the user

G.2 Clinical Application Fields

At the end of 1996, a collaboration agreement was signed between the European Commission and the International Federation of Clinical Chemistry (IFCC) for the joint IRMM/IFCC production and certification of clinical reference materials. The certification of a cortisol reference serum panel came to an end with the introduction of IRMM/IFCC-4yat the end of 1999. It consists of 34 sera (80-750 nmol/L) and has been certified using ID-GC/MS as a reference method. Ongoing projects include the certification of enzyme CRMs for GGT, LD, ALAT, CIC-MB, ASAT, ALP and a-amylase at 37T, according to adapted IFCC methods. The first four materials (IRMM/IFCC-452,453, 454, 455) are expected to be released during 2000. Projects on the certification of reference materials for cardiac marker (myoglobin)and total protein concentration in serum are under discussion. Even so the number of available CRMs for clinical chemistry and occupational toxicology is still limited. This has to do with the complexity of physiological compounds (e.g. proteins), the instability (e.g. enzymes), or the volatility (e.g. solvents). In clinical chemistry, a great number of components are to be determined. These components may be classified according to their physiological function. In occupational toxicology, a division into functional chemical components may be a better classification. In Tables (3.3 and 6.4 RMs of three major producers are mentioned, i.e. the World Health Organization (WHO, International Standards),BCR (EuropeanUnion, CRMs) and the National Institute of Standards and Technology (NIST, USA, SRMs). Some important national producers of clinical reference materials are: the Chemicals and Inspection Testing Institute (CITI, Japan), National Institute for Biological Standards and Control (NIBSC, UIC), and Deutsche Gesellschaft fiir Klinische Chemie (DGIK). There are numerous commercial producers of secondaryreference materials. 6.2.2

Elements

Elements are mostly classified to their abundance in the earth crust. The most abundant elements are known as bulk elements (H, C, N, 0, F, Na, Mg, Al, Si, P, S, C1, K, Ca), the others are considered as trace elements, with the exception of Fe. (Geldmacher-von Mallinckrodt and Meissner 1994). In case of clinical chemistry/occupational toxicology subdivisions are followed according to function: Essential electrolytes Essential trace elements Elements therapeutically used Non-essential elements Because the physiological function of the elements vary widely, and for a number of elements different compounds with different effects physiologically exist, this group of compounds is described more comprehensively in the next sections. In a recent review the current problems with e.g. the determination of some trace metals are illustrated (Herber 1999).

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6.2.2.1

Essential Electrolytes

In case of essential elements, the most important issue is deficiency. Deficiency may be diagnosed by determining the element or compound containing the element. Sometimes it will be necessary therapeutically to administer the element in ion form. In this case higher concentrations than the normal levels can be expected. Sodium, Na(1) has a normal concentration in human serum of 136-145 mmol/L (Tohda 1994) and makes up about 90 % of the cations present. (Many extracellular body fluids possess ranges from 7 mmol/L [mature milk] via 33 [saliva]to 145 mmol/ L [bile]).The reference method for determination is potentiometry with ion-selective electrodes (PISE). Potassium is abundant in animal and plant cells (Birch and Pradgeham 1994). Hypoltalemia (deficiency)and hyperlalemia (accumulation of K[I]) may both occur. As the normal range of K[I] in plasma is small, and the consequences of hyperltalemia fatal, the method of determination must be precise and accurate to detect lower and higher than normal levels (hypokalemia and hyperltalemia, respectively). The preferred method of determination is PISE. Calcium exists in the human body as Ca(I1) protein-bound and free Ca (11) ions (Dilana et al. 1994). For total extracellular Ca in plasma, serum and urine a definitive isotope dilution-massspectrometry (ID-MS) method exist. Free Ca(I1)in plasmalserum can be determined with PISE, but no definitive and reference methods exist. For Ca in faeces, tissue and blood flame atomic absorption (FAAS) is used widely. Magnesium deficiency has been long recognized, but hypermagnesia also occurs (Anderson and Talcott 1994). Magnesium can be determined in fluids by FAAS, inductively coupled plasma atomic emission spectrometry (ICP-AES)and ICP-MS. In tissue Mg can be determined directly by solid sampling atomic absorption spectrometry (SS-AAS) (Herber 1994a). Both Ca and Mg in plasma/serum are routinely determined by photometry in automated analyzers. Chloride can be determined by photometry, by a coulometric titration method or PISE. There are no special problems with the reconstitution or handling of these kinds of materials for electrolytes. The concentration of electrolytes is high, and no contamination problems are to be expected. Many commercial suppliers deliver reference materials for electrolytes in serum and urine. 6.2.2.2

Essential Trace Elements

Iron is, as part of several proteins, such as hemoglobin, essential for vertebrates. The element is not available as ion but mostly as the protein ligands transferrin (transport), lactoferrin (milk), and ferritin (storage), and cytochromes (electron transport) (Alexander 1994). Toxicity due to excessive iron absorption caused by genetic abnormalities exists. For the determination of serum Fe a spectrophotometric reference procedure exists. Urine Fe can be determined by graphite furnace (GF)-AAS, and tissue iron by GF-AAS and SS-AAS (Alexander 1994; Herber 1994a). Total Iron Binding Capacity is determined by fully saturated transferrin with Fe(III),but is nowadays mostly replaced by immunochemical determination of transferrin and ferritin.

6.2 Clinical Application Fields

Copper appears as the a2-globulin ceruloplasmin in the human body (Sarkar 1994). Deficiency of this protein in serum is characteristic of both Menkes’ and Wilson’s diseases. Wilson’s disease is an abnormal storage of Cu(I1) in body tissues. Cu(I1) in biological material can be determined by spectrophotometry or by FAAS, ceruloplasmin in seium by a spectrophotometric method. Selenium is required, but levels must fall into a narrow window. Both deficiency and toxicity symptoms occur. The element is also used therapeutically in cancer treatment. It is the co-factor of the enzyme glutathione peroxidase which is thought to play an important role in oxygen toxicity. The determination of Se in blood or serum is not easy, as many incorrect, inaccurate and imprecise methods have been published (Magee and James 1994).A suggested procedure for Se in body fluids is based on GF-AAS (Thomassen et al. 1994). For tissues SS-AAS may be used (Herber 1994a). Recent developments by Turner et al. (1999) show that LC-ICP-MS is sensitive and reproducible at low levels. Cobalt is present in animals in vitamin B12 (cyanocobalamine)and thus is essential for humans (Thunus and Lejeune 1994).The determination of Co has little significance for the diagnosis of deficiency of cyanocobalamine. Instead, cyanocobalamine itself must be determined in serum. The determination of methyl malonic acid in urine seems more reliable (McCann et al. 1996). Chromium deficiency may be related to the glucose tolerance factor (Herold and Fitzgerald 1994). The determination of this deficiency, however, is questioned, because the lack of accuracy of the Cr determination in the earlier publications. Deficiency of manganese may lead to vitamin K deficiency (Chiswell and Johnson 1994)and to problems in prenatal and neonatal development of the brain. Deficiency of molybdenum cofactor can lead to sulphite oxidase deficiency (Anke and Glei 1994). Although vanadium seems to be an essential element, no deficiencies have been reported (Blotcky et al. 1994). Tin is essential for animals, but the essentiality for humans is not clear (Anger and Curtes 1994). Iodine is incorporated in thyroid proteins to form thyroxin and 3-I-thyroxine,both hormones essential for life. They are determined by immunochemical methods. Deficiency of I may lead to crop disease. Fluorine as the anion F(1) is found in bone and tooth. Enhanced levels are toxic. F(1) can be determined by PISE. There are a number of CRMs available for this group of elements in serum and urine (see Table 6.3). The most severe problems with the determination of these trace elements are contamination and loss. Therefore, strict protocols are necessary to prevent these problems. Contamination can be prevented by cleaning thoroughly all used utensils, and the use of highly purified chemicals. Loss is mostly due to exchange between the container walls and can be prevented by working at a pH<2. 6.2.2.3

Elements Therapeutically Used

Lithium is therapeutically used in the prevention of major changes in mood which are characteristic of the affective disorders (Birch et al. 1994). The therapy can be

I

203

204

I monitored by the determination of G General Application Fields

Li in serum, usually by F-AAS or flame emission photometry. As normal concentrations of Li in serum are very low, monitoring of the therapeutic high dose ( Li2C03, 30 mmol/day) is easy to perform. Concentrations of Li in serum must be in the range of 0.4-0.8 mmol/L. Higher levels of Li in serum may be toxic. Gold is used therapeuticallyin chronic inflammations as rheumatic arthritis (Ishida and Orimo 1994).The dose is given in the form ofa gold complex, such as gold sodium thiomalate and Auranofin;toxic effects due to overdoses may appear.The most common method to monitor the therapeutic dose in serum or urine is GF-AAS. Platinum is used therapeutically as cis-platin, carboplatin and iproplatin against tumors (Konig and Schuster 1994). Determination of Pt in serum must be carried out after enrichment procedures followed by GF-AAS. 6.2.2.4

Non-Essential Elements

These elements are sometimes called “toxic” elements. Although many elements belong to this group, a few only are encountered in the clinical or occupational laboratory. This is also reflected in the literature. For this reason only the most common elements are mentioned. Lead has been known to be toxic since the Roman Empire. There are a number of acute effects known, e.g. anorexia, dyspepsia, constipation, colics, and toxic encephalopathy (Christensen and Kristiansen 1994). Most common effects to chronic exposure to lead are inhibition of heme synthesis, leading in severe cases to anemia, effects on the peripheral nervous system, and effects on the renal tubules. Exposure to Pb can take place both in the occupational and the general environment. Since the early 1980’s there have been many programs designed to reduce lead from the environment, including the removal of lead from gasoline, the banning of lead as a paint pigment, and replacement of lead water pipes. The most common method used to monitor inorganic Pb is the determination of Pb in whole blood by GF-AAS. Exposure to organic lead (i.e. tetraethyl lead) can be monitored by the determination of Pb in urine by GF-AAS (Christensen and 10-istiansen 1994). Early effects of exposure to Pb on the heme synthesis can be monitored by determination of the inhibition of the enzyme 6-aminolevulinicacid dehydratase in whole blood or 6-aminolevulinicacid in urine by spectrophotometry. Acute exposure to cadmium may lead to chemical pneumonitis and edema, but is rare nowadays (Herber 19g4b). Chronic exposure to Cd affects mostly the renal tubules and the lung. Exposure to Cd can take place both in the occupational and environmental area. Since the 1980’s occupational and environmental exposure to Cd has been reduced due to banning of Cd in pigments, plastic softeners, fertilizers, and batteries. In some European countries (e.g. Sweden) Cd has been banned completely. Monitoring of Cd exposure can take place by the determination of Cd in whole blood (reflects recent exposure) or urine (reflects body burden) by GF-AAS. Early effects can be monitored by the determination of a tubular protein (e.g. µglobulin, retinol binding protein, a2-microglobulin) or the activity of an enzyme (e.g. N-acetyl @-D-glucosaminidase) in urine.

G.2 C/inical Application Fields

Mercury exists as inorganic Hg (I) and Hg(I1) and as organic mercury. The toxic effects depend crucially on the binding form. In the past a number of local effects were reported when using sublimate (HgC12),e.g. effects on the oral cavity and the gastrointestinal tract. Exposure to high concentrations of mercury vapor may lead to a number of acute effects to the lungs. The central nervous system is the critical organ for the long-term exposure to mercury vapor or organomercury vapor compounds (Drasch 1994). Other effects are reported on the kidney (inorganic mercury) and on skin and mucous membranes (thiomersal). Incidence of amalgam contact allergy is rare. Biomonitoring of inorganic mercury can be performed by cold vapor atomic absorption spectrometry (CV-AAS) of mercury in urine. Organic mercury can be monitored by determination of mercury in blood by CV-AAS. Aluminium is the most abundant element of the lithosphere. Although a large number of persons are exposed world-wide to Al, the incidence of pulmonary effects is low (Schaller et al. 1994).In the 1970’s the effect of A1 appearing in dialysis solutions on the central nervous system has become well known. increased Al could also be detected in several brain regions of patients with Alzheimer’s disease. For the determination in biological materials the most widely used method is GF-AAS. From arsenic a number of different compounds exist, however, by far the most toxic forms are the inorganic As compounds (Stoeppler and Vahter 1994). In human environment exposure to As(V) is generally found. Acute exposure may lead to gastrointestinal effects, muscular cramps, and cardiac abnormalities but are rare nowadays. Long-term exposure through inhalation or ingestion may lead to a number of adverse health effects, including lung cancer, effects on the liver, the cardiovascular system, the heme system, and the nervous system. Exposure to inorganic As can be monitored by the determination of As in urine, but as all As species are excreted in urine, the determination of total As in urine gives an overestimation of the exposure. Instead a hydride AAS technique can be used to monitor the sum of

Tab. 6.3

RMs for essential electrolytes ( I ) , essential and therapeutically used trace elements (2) and nonessential elements (3)

Category

Producer

Type of RM

1

NIST

Na, K, Ca, Mg and CI in serum, K, Ca, Mg and CI in serum, Ca in urine, C1 in serum Ca and Mg in serum

BCR 2

NIST BCR WHO

3

NIST

BCR

Cu and Se in urine, Fe in serum, F in urine, Li in serum, Au and Pt in urine Cu as ceruloplasmin and Fe as transferrine in serum, Li in serum Fe as ferritin in serum, Co as vitamin BIZ in serum Pb in blood, As, Be, Cd, Cr, Cu, Mn. Ni and V in urine, Hg in urine Pb and Cd in lyophilized bovine blood

I

205

206

I As(III), As(V),monomethyl arsonic acid, and dimethylarsenic acid, which gives G General Application Fields

a

quick and reliable estimation of exposure to inorganic arsenic. There are a number of other elements appearing from time to time in the laboratory. From these, chromium and nickel are most common. Both appear in enhanced concentrations in workers exposed to welding fumes, in galvanization processes, and in processing of ores. Prolonged exposure to Cr and/or N i causes cancer and affects the kidney. Preferred methods of determination of Ni and Cr in urine are GF-AAS. Because of the risk of contamination of the very low concentrations in urine, extreme precautions in sample handling and analysis must be carried out. Table 6.3 lists reference materials for the elements mentioned in Sections 6.2.2.1-6.2.2.4. 6.2.3 Organic Compounds

More than 70000 chemicals are recorded in the European Inventory of Known Chemical Substances and can be used in industry. As the majority are organic compounds, the importance of these compounds will be clear. Moreover, many of these organic compounds are solvents and volatile and may thus expose humans through the lungs or the skin, or are persistent and lipophilic and remain within the human body for many years as deposits in the fatty tissues. Solvents Many organic solvents, including hydrocarbons, chlorinated hydrocarbons, alcohols, esters, and ketones have the potential upon acute high level vapor exposure to cause narcosis and death (Andrews and Snyder 1996). Effects may be disorientation, euphoria, giddiness, confusion, paralysis, unconsciousness, convulsions and ultimately death. Distinct from the central nervous system (CNS) depression actions of solvents are specific toxicities associated with them (Andrews and Snyder 1996). Examples of specific toxicities are: hemopoietic toxicity of benzene, (CNS) depressant effects of alkylbenzenes, hepatoxicity of certain chlorinated hydrocarbons, ocular toxicity of methanol, hepatoxicity and CNS depressant effects of ethanol, neurotoxicity of n-hexane and certain ketones, reproductive toxicity of ethylene glycol ethers, and carcinogenicity of dioxane. Because of the usage of solvents in many industries (i.e. paint, metal cleaning, glues, organic synthesis) and their volatile character, there is considerable exposure of workers to these solutions. In the general environment there may be sometimes exposure due to soil pollution. Solvents or their metabolites are commonly determined by GC (Toltunaga et al. 1974) or GC-MS. In spite of the high importance of exposure to solvents, and the great number of determinations performed worldwide, reference materials for solvents in serum or urine are virtually nonexistent. There are a number of reference materials used in occupational hygiene, for example the ethanol in water standard from NIST (SRM 1828a)is commonly used in the clinical laboratory. 6.2.3.1

G.2 Clinical Application Fields

6.2.3.2

Polyaromatic Hydrocarbons (PAH)

PAHs, more correctly known as polyaromatic compounds (PACs) are common in the human environment, e.g. the exhaust of diesel engines, bitumen and asphalt production. Some of the PAHs are genotoxic and carcinogenic (e.g benz(a)pyrene). Even though PAHs are commonly determined by GC or HPLC, there are no matrix reference materials for PAHs in urine or serum. A number of reference materials certified for PAHs in animal tissue are available, but they are intended for environmental applications, see Section 3.4. 6.2.3.3

Pesticides

Pesticides are used to control pests in agriculture and animal breeding. It has been known since the 1950’sthat pesticide contamination occurs worldwide (e.g. DDT). Many pesticides are neurotoxicants poisoning the nervous system. A number of pesticides are acetyl cholinesterase inhibitors (Serat and Mengle 1973).Generally, pesticides determination has been performed by GC since the IgGo’s (Morrison and Durham 1971; Fournier et al. 1978).There are no reference materials for pesticides in urine or serum, although as with PAHs there are a number biological matrices certified for the content of various pesticides available for environmental food and agriculture analysis and which may have some application in clinical chemistry. 6.2.3.4

Persistent Compounds

Polychloro-benzenes, polybromo-benzenes, and dioxins (TCDD) are among these compounds. They were discovered when the analysis techniques improved. Especially the development of GC-MS has contributed to the knowledge of the distribution of these compounds. Effects on humans are the development of chloracne, suppression of the immune system, and some compounds are probably carcinogens (Shaw 1993). As a consequence of the ubiquitous nature of PCBs, humans are exposed via many sources. Other than NIST SRM 1589, PCBs in human serum, there are no reference materials for these compounds in urine or serum. A number of reference materials are available for environmental samples, food and agriculture. 6.2.4 Proteins and Enzymes

Proteins are essential to all living systems. Proteins are macromolecules and, like all biological macromolecules, polymers (Alberts et al. 1994). The structural units of proteins (monomers) are about 2 0 amino acids. Although no clear line exists, proteins are generally considered to have minimal chain lengths of about 50 amino acids, corresponding to molecular masses near 5000 daltons. The most complicated proteins contain several thousand amino acids and have molecular masses of several million daltons. The functional diversity ranges from: Enzymes, which have catalytic properties Antibodies, which serve as a weapon in the defense arsenal of the organism

I

207

208

I

6 General Application Fields

Structural elements, e.g. to define and maintain the architectural construction Transport devices, e.g. for ions, oxygen, and lipids Metabolic regulators, including hormones. Animal proteins are classified as follows: Albumins, characterized by their solubility in water, and in diluted aqueous salt solutions Globulins, soluble in diluted aqueous salt solutions, but insoluble or slightly soluble in distilled water Protamines, among the smallest proteins with a molecular mass of 5000 daltons. They have a high basicity and are found in sperm Histones, which also have a high basicity and are found in combination with nudeic acid Scleroproteins, insoluble in most solvents. Localized in connective tissue, bone, hair, and skin. The two principal classes are the collagens and keratins Nucleoproteins, nucleic acid Lipoproteins. The lipid moiety of the lipoproteins is quite variable, both qualitatively and quantitatively Mucoproteins are carbohydrate in nature Chromoproteins are pigments There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997).Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectrophotometric measurement of the ninhydrin-amino acid complex. Currently, a number of methods are available , including spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specificity (cross-overreactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-artof protein determination was mentioned by Walker (1996). Enzymes are mostly determined by some spectrophotometric methods in clinical chemistry laboratories, but immunochemical and molecular biological techniques are finding their way into routine laboratory procedures. Protein macromolecules present in biological fluids are almost invariably heterogeneous in their characteristics.They may be products of more than one gene in the population (allotypesin the case of proteins; isoenzymes in the case of enzymes), or a single individual (isotypes of proteins, allelozymes of enzymes), or be subject to post-translational modification. The result of this inherent molecular heterogeneity is that different forms of the same protein may behave differently with respect to

6.2 Clinical Application Fields

binding to anti-sera, while certain enzyme isoforms may also display different catalytic properties. The heterogeneity spectrum of reference and control materials may differ from that of biological fluids, and this can lead to differing behavior in the determination. This again, may lead to the problem of method-dependent results (Moss and Whicher 1995). The solution may be a rigorous standardization. This has been done for enzyme determination, but other than BCR CRM 470 hardly at all for protein determination. 6.2.5

Lipids

Lipids in living systems are by solvents extractable compounds. Among the lipids are the fatty acids, glycerides, steroids, terpenes, and complex lipids as lipoproteins. Fatty acids can be compared with detergents and have the capacity, i.e. in the form of micelles to solubilize organic material. Fatty acids play an important role as a risk factor for cardiovascular diseases, that is by forming plaques within the arteria. Low density lipoproteins (LDL) are seen as the most important risk factor. In the clinical chemistry laboratory, both LDLs and HDLs (high density lipids, considered as an anti-atherogenic factor) are determined. Most used methods are spectrophotometry and immunochemistry, in dedicated laboratories ultracentrifugation. A handbook on this material has been written by Rifai et al. (1997). Certified reference materials for the determination of lipids in serum are provided by NIST. 6.2.6 Other Compounds

There are numerous other compounds which can be determined in human body fluids. For some classes of compounds, e.g. nitrogen compounds, hormones, and sugars, a few reference materials are available. Nitrogen compounds commonly determined are creatinine, urea, and uric acid. Creatinine is an end product of the energy process occurring within the muscles, and is thus related to muscle mass. Creatinine in urine is commonly used as an indicator and correction factor of dilution in urine. Creatinine in serum is an indicator of the filtration capacity of the kidney. Urea is the end product of the nitrogen urea cycle, starting with carbon dioxide and ammonia, and is the bulk compound of urine. The production of uric acid is associated with the disease gout. In some cases, it appears that the excess of uric acid is a consequence of impaired renal excretion of this substance. Hormones are regulatory substances from small molecules (e.g. nucleotides and steroids) to large polypeptides (e.g. insulin). Carbohydrates (sugars) catabolism provides the major share of the energy requirement for maintenance of life and performance of work. Table 6.4 lists examples of reference materials mentioned in Sections 6.2.46.2.6.

I

209

210

I

G General Application Fields RMs for proteins in serum a n d purified materials (4)

Tab. 6.4

(I),

enzymes in serum

( 2 ) , lipids

a n d other in serum (3), pure

Category

Producer

Type of RM

1

WHO

Alphafoetoprotein,Ancrod, Anti-D Immunoglobulin, Antithrombin 111 total, Apolipoprotein B, Haemoglobin A2 and F Lysate, Heparin, Protamine; Protein C, Fibrinogen, Plasmin; a-Thrombin, Antithrombins, 6-Thrornboglobulin,Haemiglobincyanide cxl-acid Glycoprotein, Albumin. Alphafoetoprotein, a,-anti-Chymotrypsin, a,-anti-Trypsin, a2-Macroglobulin,Apolipoproteine A1 and A2, Complements C3 and C4, C-reactiveprotein, Haemiglobincyanide, Haptoglobulin, Immunoglobuline A,G,M, Prostate specific antigen (PSA),Transthyretrin, Thromboplastine, Thyroglobulin Albumin Alanine transferase, Alkaline phosphatase, a-Amylase, Creatine kinase, Creatine kinase; y-Glutamyltransferase,Lactate Dehy drogenase isoenzyme 1,Prostatic Acid Phosphatase Plasminogen Activator Inhibitor-1, Prekallikrein Activator, Streptokinase, Streptokinase and Streptodornase, Tissue Plasminogen Activator, Urokinase Cholesterol, Cholesterol HDL and LDL, Glycerides, Total glycerides, Glucose, Fat soluble vitamins Cortisol, Progesterone, 17 6-Estradiol,Creatinine, Creatinine interfering substances Chorionic gonadotropin, Follicle stimulating hormone Urea, Uric add, Bilirubin, Cortisol,o-Mannitol.o-Glucose, Sodium p y v a t e , 4-hydroxy-3-methoxymandelic acid, 4-Nitrophenol, 17Amino adds in HCl, Angiotensin-I,Tripalmitin,Bone meal (8elements), Bone ash (8elements), Lithium carbonate Luteinizing hormone, Thyroid stimulating hormone

BCR

2

NIST BCR

WHO

3

NIST BCR

4

WHO NIST

WHO

6.3 Food/Biological Milan lhnat and Wayne R Wolf

6.3.1 introduction

Generation of data on the nutrient content of agricultural products and foods forms the basis for estimating nutrient intakes of populations via dietary surveys, nutritional labelling for consumer protection, nutrition education for consumer food choice, home and institution menu planning and food purchase, and for research in nutrient requirements and metabolism. toxicant chemical composition is used to assess effects of farm management practices, crop culture, and food processing on chemical content and implications for human health.

6.3 Food/Biological

Accurate food composition data can only be obtained by utilization of sound analytical methodologies and quality assurance systems. The use of well defined RMs is a vital part of this quality assurance. A summary of procedures for RM selection and utilization is presented here as a guide for monitoring and maintaining analytical data quality in the determination of inorganic and organic measurands in food and biological materials. The aggregate of all steps such as sampling, sample manipulation and measurement, subsequent to the point at which the RM is introduced into the scheme of analysis, will be monitored. 6.3.2

Food Matrix Triangle

Much of the analytical data on the nutrient content of foods is generated using official methods of analysis (e.g.AOAC International). An evaluation of AOAC Methods of Analysis for Nutritional Labelling is available (Sullivan and Carpenter 1993). While these methods have often been studied for a variety of food matrices, applicability over the entire range of food matrices has not been formally studied in most cases. In addition, RMs are not available over the entire range of food matrices (Wolf

1993). In order to define this variety of food matrices, chemical composition differences that primarily influence chemical analytical measurements have to be considered. Major food components determining basic chemical make-up are the proximate composition of fat, protein, carbohydrate, ash, and moisture. Variations in ash content in general have a minor influence on analytical methods for other constituents and impact of moisture content can be controlled. Thus the major components influencing analytical performance are the relative levels offat, protein, and carbohydrate. Southgate (1987) discusses the range of available RMs in terms of their fat, protein, and carbohydrate content. These constituents are presented graphically via a triangle wherein the relative position of each of these three proximate components is represented as IOO % at a separate apex and o % at the opposite side of an equilateral triangle as shown in Figure (5.1. Drawing on this representation, an approach has been described to systematically describe selection of food products to evaluate the applicability of collaboratively studied methods over a range of food matrices (Wolf and Andrews 1995).A food matrix is described by its location in one of the nine sectors in the triangle. Foods falling within the same sector are chemically similar and thus should behave in a similar analytical manner. This same scheme can be used to select food matrices representing each sector for development of a series of RMs representing all foods. In order to begin this selectionprocess, the entire range offood matrices can be examined. The proximate components of foods are plotted to determine into which of the nine sectors the foods fall. To develop an x/y plot of these sectors, values for x and yare assigned using the following equations based upon the% fat (a),% protein (b) and% carbohydrate (c) relative to the sum of these three components,normalizing these components to a dry weight, ash-freebasis. Thus the s u m of a + b + cis IOO %.

I

*11

212

I

G General Application Fields

Fat fOWo

Fig. 6.1 Schematic layout o f f o o d matrices suggested for a col. laborative study based on protein (Prot), fat and carbohydrate (Cho) content, excldding moisture and ash

y = a;

x = ( a + ab)/tan

a = ( % fat x

IOO)/

(60")

(% fat + % protein + % carbohydrate)

b= (% protein x IOO)/(% fat + % protein +% carbohydrate) c = (% carbohydrate x IOO)/(% fat +% protein + % carbohydrate)= [IOO - (a + b)] In some sectors, several materials may be necessary to account for differences in the type of protein, fat or carbohydrate. For example, some high-carbohydrate foods are high in sugar whereas others are high in starch or dietary fibre. As a preliminary approach, data contained in USDA databases on nutrient content of foods (USDA 1993) have been sorted and plotted using this food matrix organizational system (Wolf and Andrews 1995). These databases contain single ingredient items as well as multi-ingredient foods whose proximate content was determined by recipe. The normalized fat, protein and carbohydrate-by-difference data for 6675 foods are plotted in a scatter plot (Figure 6.2) on a dry weight basis. The data in each of the nine sectors were further analyzed to determine both the mean and the distance each food lies from the mean. Mean x and y values and the number of items for each sector are plotted in Figure 6.3, showing many more foods at the high end of the carbohydrate axis (Sector 5). The foods in each sector were then ranked by distance from the mean and cross checked with frequency-of-usedata (USDA 1991).

6 3 Food/Biological 100

. -.. ...\ .. *. .

.

.

.. . .

. . ..:: > .. ,. . .:. . . . . ’’...... :i.

z 20 20

0

40

60

Normalized Percent

80

100

Fig. 6.2 Plot of6675 foods from the USDA Databasefor Food Consumption Survey (USDA 1989-91)

1

F

20

40

60

Fig. 6.3

Number offoods and mean point per sector from Figure 6-2

The resulting tables (i.e. Table 6.5) list commonly consumed foods that are near the mean x and y for each sector. Commonly consumed foods are a real advantage when preparing RMs, because they tend to be readily available and inexpensive. In Table 6.5 (sector 4), eggs, cheese, and chicken are commonly consumed foods and are possible candidates for RMs for foods falling into the proximate ranges for this sector (fat content 34-66 %, protein content 34-66 %, and carbohydrate content 0-33 %). Multi-ingredient items like soups, sandwiches and low-fat frozen dinners are the commonly consumed foods whose proximate contents are close to the mean for sector 7. These mixed dishes are possible candidates for RMs for foods with fat

I

213

214

I

G General Application Fields Tab. 6.5

List of commonly consumed foods closest to the sector 4 mean (Wolf and Andrews 1995)

(57)

Description

Fat

Egg, whole boiled Cheese, swiss Egg, whole poached Egg, omelet/scrambled egg, no fat added Chicken wing with skin, baked

43.5 46.3 42.2 40.2 43.7

Protein

51.8 47.9 52.6 48.8 51.5

(55)

Carbohydrate (57)

4.6 5.7 5.1 10.9 4.8

content 0-33 %, protein content 34-66 %, and carbohydrate content 34-66 %. A list of key foods that supply 75 % of a given nutrient to the population has been developed (Haytowitz et al. 1996). It would be of interest to examine this list using this food matrix triangle approach to develop further recommendations for candidate RMs. The Standard Reference Materials Program of NIST has utilized this approach in setting priorities for development of several new food-based RMs. One outcome was the additional characterization of proximate contents for a number of RMs presently available from NIST. 6.3.3 Available Reference Materials

Table 6.6 lists most of the available RMs, a listing of major producers and suppliers of all kinds of RMs is given in Chapter 8. Many materials have been characterized for elemental, isotopic and radionuclide content, but increasingly, materials are becoming available for a wide variety of organic measurands; see Section 2.1. An example of concentrations of nitrogen in biologicallfood RMs is given in Table 6.7. Tabulations as in Tables 6.6 and 6.7 (e.g. Ihnat 1988,1998b; International Atomic Energy Agency 1995; National Oceanic and Atmospheric Administration 1995) enable the analyst to locate materials of appropriate natural matrix composition and measurand concentration.Individual catalogues and reports (e.g. Bowman 1994; International Atomic Energy Agency 1998; Trahey 1998; Institute for Reference Materials and Measurements 1999) and appropriatewebsites should also be consulted. 6.3.4 Mode o f Application and Application Examples

A good presentation of general principles relating to RM and data quality concepts and use is given by Taylor (1993).Specific guidelines for the selection and utilization of RMs for monitoring and maintaining analpcal data quality in the measurement of inorganic measurands in plants and soils have been published (Ihnat 1993, 1998a, b). In order to properly use RMs, it is imperative that compliance with several preliminary requirements be established (I) An appropriate analytical method must be applied to the task on hand by appropriately qualified and trained personnel.

6 3 Food/Bio/ogica/ Tab. 6.6

Examples of biological, food, agricultural and related RMs for chemical composition available from, principally, government agency suppliers (Ihnat 1988, 1992,1998a; International Atomic Energy Agency 1998; Institute for Reference Materials and Measurements 1999; National Oceanic and Atmospheric Administration 1995; Trahey 1998)4 RM group

SupplieF:

Material

Animal tissues

BCR

Bovine liver, pig kidney, mussel tissue (also for butyltin compounds),tuna fish (methylmernuy),tuna fish tissue (As speciation) Non-defatted lobster hepatopancreas, lobster hepatopancreas, dogfish liver, dogfish muscle Fish flesh Fish tissue Bovine liver, oyster tissue Animal muscle (pork),carrot powder, total diet, wheat flour Skim milk powder (elements),whole meal flour, bovine muscle, wholemeal flour, brown bread, cod liver oil (PCBs),rye flour, haricots verts (beans),pork muscle, mixed vegetables, carrot, bran breakfast cereal, unspiked milk powder (PCDDs, PCDFs), spiked milk powder (PCDDs, PCDFs), milk powder Rye flour, milk powder, whey powder Pork meat Tea leaves, rice flour, oyster tissue, wheat flour, rice flour Nonfat milk powder, spinach leaves, corn kernel, bovine muscle powder, whole egg powder, microcrystalline cellulose, wheat gluten, whole milk powder, durum wheat flour Aquatic phnts (Lugurosiphonmajor, Platihypnidium riparioides), olive leaves, beech leaves, hay powder, lichen, single cell protein, sea lettuce, rye grass, white glover, plankton Spruce twigs and needles Cotton cellulose, hay powder, grass Oriental tobacco leaves, Virginia tobacco leaves Pepperbush, chlorella, sargasso, Apple leaves, citrus leaves, pine needles, corn stalk Sewage sludge (PAHs),sewage sludge (domestic origin), sewage sludge (industrial origin), waste mineral oil (low level PCBs)

NRCC

Foodstuffs

IAEA EPA NIST ARC BCR

IAEA LIVSVER NIES NIST

Plants

Biological waste materials 4

?<*

BCR

CANMET IAEA INCT NIES NIST BCR

The majority of these RMs are available from the issuing organizations; several older materials may not be available from primary sources but may still be available in the secondary market (e.g. from existing stock in laboratories). For the suppliers EPA, ARC, LIVSVER, CANMET and INCT listed above that are not already mentioned in other chapters sources for further information are given in Chapter 8.

Suitable quality control and quality assurance procedures should be in place and the analytical system must be in a state of statistical control. (3) It must be ascertained that the method is measuring all of the measurand and the correct moiety. (2)

216

I

6 General Application fields Example of nitrogen concentrations in biological,food, agricultural and related reference materials listed in increasing order of concentrationa)

Tab. 6.7

Material

Microcrystalline Cellulose Corn Starch Corn Stalk Corn Bran Haricot beans Bush branches and leaves Pine Needles Rye flour Corn Kernel Rye bread flour Bush branches and leaves Pork meat Soft Winter Wheat Flour Spruce needles Olive Leaves Wheat bread flour Sea Lettuce Wheat flour Apple leaves Wheat flour Poplar leaves Beech leaves Hard Red Spring Wheat Flour Durum Wheat Flour Cabbage Citrus Leaves Peach leaves Tomato leaves Tea Aquatic Plant (I?. riparioides) Total diet Hay powder Aquatic Plant (L. major) Whole Milk Powder Kale Whole milk powder Tea Non fat milk powder Skim Milk Powder Spinach leaves Slum milk powder Whole Egg Powder Oyster tissue Bovine Liver Pork liver Single Cell Protein

Codeb)

Concentrationc)

NIST-RM-8416 NIST-RM-8432 NIST-RM-8412 NIST-RM-8433 BCR-CRM-383 GBW-07602 NIST-SRM-1575 BCR-CRM-381 NIST-RM-8413 CSRM-12-2-05 GBW-07603 LIVSVER-SMRI-941 NIST-RM-8438 BCR-CRM-101 BCR-CRM-062 CSRM-12-2-04 BCR-CRM-279 BCR-CRM-382 NIST-SRM-1515 GBW-08503 GBW-07604 BCR-CRM-100 NI ST-RM-8437 NIST-RM-8436 GBW-08504 NIST-SRM-1572 NIST-SRM-1547 NIST-SRM-1573a GBW-07605 BCR-CRM-061 NIST-SRM-1548 BCR-CRM-129 BCR-CRM-060 NIST-RM-8435 BOWEN’S KALE BCR-CRM-380 GBW-08505 GBW-08509 BCR-CRM-063 NIST-SRM-1570a BCR-CRM-O63R NIST-RM-8415 NIST-SRM-1566a NIST-SRM-1577a GBW-08551 BCR-CRM-273

200 670 6970 8840 10500 12000 12000 12500 13750 14000 15000 16800 17560 18890 19500 20000 20800 21200 22500 23900 25600 26290 26900 27070 28000 28600 29400 30300 33200 33500 34400 37200 41200 41820 42790 45000 48800 55100 58800 59000 62300 63000 68100 107000 108600 121000

6.3 Food/Bio/ogica/ Tab. 6.7

Continued

Material

Code b,

Concentrationc)

Pork muscle Bovine Muscle Powder Prawn Wheat Gluten Human hair

BCR-CRM-384 NIST-RM-8414 GBW-08572 NIST-RM-8418 GBW-07601

137000 137500 143000 146800 149000

a) Information has been adapted from International Atomic Energy Agency (1995)and Ihnat (1988)as well as personal information and includes certified and informational concentration values. b) Code is a combination of supplier code and identity assigned to the product. c) Concentrations are reported as mg/kg on a dry basis.

This table is for information and discussion only; original certificates or information sheets provided with the Reference Materials must be consulted for actual concentrations and uncertainties.

6.3.4.1

Procedures for Reference Material Selection and Use

The RM and the commodity undergoing analysis must be very similar with respect to matrix and measurand concentration and form (e.g. native form, speciation). By consulting information as in Table 6.6, giving a descriptive name of the product, select the RM(s) approximating the laboratory sample to be controlled with respect to matrix (i.e. by RM name). By consulting a measurand concentration table, as exemplified in Table 6.7 for nitrogen, independently select RMs based on concordance of the measurand concentration in the RM and the level anticipated in the sample. Use these two selection criteria to arrive at appropriate RMs. Several RMs, spanning the concentration range of interest is preferred, but this is not always possible due to inadequacy of the world RM repertoire. 6.3.4.2 Procedures for Reference Material Utilization

Following RM certificate instructions for material usage and handling, incorporate the RM into the scheme of analysis at the earliest stage possible, i.e. prior to the beginning of sample decomposition. Take it through the entire analytical procedure at the same time and under the identical conditions as the actual analytical samples in order to correctly monitor all the sample manipulation and measurement steps. 6.3.4.3 Performance Interpretation and Corrective Action

Results from the analysis of the RM and the certified value and their uncertainties are compared using simple statisticaltests (Ihnat 1993,1998a).If the measured concentration value agrees with the certified value, the analyst can deduce with some confidence that the method is applicableto the analysis of materials of similar composition. Ifthere is disagreement, the method as applied exhibits a bias; and underlying causes of error should be sought and corrected,or their effects minimized.

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G General Appiication Fie/ds 6.3.4.4 Examples ofApplication o f R M s to Certification ofother R M s

Incorporation of RMs into schemes of analysis is still evolving. Official and recommended methods of analysis often still do not stipulate RMs as an integral part of the analytical method. An important major use of existing RMs is quality control of analysis and certification activities for the developmentofnew RMs. In earlier years of RM development, and surprisingly even more recently, very little allusion is made to the use of existing RMs for quality control. Examples of recent usage are the certificationactivities by BCR (IRMM) (Quevauder et al. 1993; Pendlington et al. 1996; Lamberty and Kramer 1gg8),NIST (Donais et al. 1g97), IAEA (Horvat et al. 1997). and Agriculture and Agri-Food Canada (AAFC) (Ihnat 1990.1994). In some BCR work (Quevauvilleret al. 1gg3),the report simply indicates that the performance of the method was verified by analyzing available RMs or other materials. In the BCR certification of several foods for dietary fibre (Pendlington et al. 1996), Rye Flour BCR-CRM-381was used for quality control of one of the fibre methods, while in the certificationof the trace elemental conR and Kramer 1gg8), the pretent of a replacement bovine liver, C R M - I ~ ~(Lamberty vious similar material, CRM-185, was used. In the NISTcharacterizationofthree mussel SRMs (1g74a,2974 and 2976) for Hg and MeHg (Donais et al. 1gg7),previously analyzed SRM-2976was used as a control for measurements on the other two materials. Additionally, Oyster Tissue SRM-1566a, Montana I Soil sRM-2710, and Lobster Hepatopancreas NRCC-TORT-Iwere used as controls for all three tissues. The IAEA certification oftotal Hg and MeHg in mussel IAEA-142 (Horvat et al. 1gg7), involved Dogfish Muscle NRCC-DORM-I and DORM-2, Lobster Hepatopancreas NRCC-TORT-I and TORT-2, Non-defatted Lobster Hepatopancreas NRCC-LUTS-I, Marine Sediment NRCC-MESS-I, Tuna Fish Homogenate IAEA-350, Copepod Homogenate IAEA-MA-AI, Oyster Tissue NIST-SRM-1566and 1566a,Orchard Leaves NIST-SRM-1571,Albacore Tuna NIST-RM-50, and Human Hair BCR-CRM-397. The authors judged that only NIST-SRMs 1566 and 1566a fulfilled the matrix/concentration matching criteria exactly. RM development at Agriculture and Agri-Food Canada of 12 food RMs for NIST, incorporated existing RMs for quality control (Ihnat 1990,1994).Homogeneity assessment by solid sampling graphite furnace atomic absorption spectrometry of Pb and Cu in Bovine Muscle Powder 136 (initial assigned numbering), Wheat Gluten 184, Corn Bran 186, Durum Wheat Flour 187, and Whole Egg Powder 188 (Ihnat 1990) used Spiked Skim Milk Powder BCR-CRM-150, Wheat Flour N I S T - S R M - I ~and ~~~ Bovine , Muscle BCR-CRM-184for checking instrument performance and comparative behavior. In the subsequent full-scaleinter-laboratorycharacterization campaign RMs included: Non Fat Milk Powder NIST-SRM-1549,Rice Flour NIST-SRM-1568,Orchard Leaves NIST-SRM-1571,Citrus Leaves NIST-SRM-1572,Tomato Leaves NIST-SRM-1573, Pine Needles NET-SRM-1575, Bovine Liver NIST-SRM-1577, Corn Stak NET-RM-8412, Corn Kernel NIST-RM-8413, Pepperbush NIES-CRM-I, Chlorella NIES-CRM-3, Tea Leaves NIES-CRM-7,Rice Flour NIES-CRM-10,Wheat Flour IAEA-V-Z/I,Animal Muscle IAEA-H-4,Rye Flour IAEA-V-8,Cotton Cellulose IAEA-V-9,and Bowen’s Kale. 6.3.4.5

Examples of Applying RMs in Analyses

Several recent examples of the incorporation of food RMs in analytxal work are presented in Table 6.8. These applications do not necessarily adhere to the foregoing

ICP-AES Q-ICP-MS HR-ICP-MS

As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Pt, Sn, V, Z n in Italian honey

*

Caroli et al. 1999

Fingerova and Roplik 1999

Method abbreviations: D-AT-FAAS (derivative flame AAS with atom trapping), ETAAS (electrothermal AAS), GC (gas chromatography), HGAAS (hydride generation AAS), HR-ICP-MS (high resolution inductively coupled plasma mass spectrometry),ICP-AES (inductively coupled plasma atomic emission spectrometry), ICP-MS (inductively coupled plasma mass spectrometry), TXRF (total reflection X-ray fluorescence spectrometry), Q-ICP-MS (quadrupole inductively coupled plasma mass spectrometry)

Na, Mg, P, I<, Ca, Mn, Fe, Co, Ni Cu, Z n Se, Mo i n soybean flour

ICP-AES ICP-MS FAAS ICP-AES ICP-MS

K, Mg, Ca, Mn, Zn, Fe, Cu, Al, Na, Ni, As, Mo, Cd in Vietnamese rice

NIST SRM 1568a, Rice flour NIST SRM 1515, Apple leaves BCR CRM 281, Rye grass BCR CRM 191. Brown bread

Larsen et al. 1999 Mnnoz et d.1999

BCR CRM 063R, Skim milk powder NRCC DORM-1 and DORM-2, Dogfish muscle NRCC TORT-2, Lobster hepatopancreas, BCR CRM 278, Mussel NIST SRM 156Ga, Oyster NIES CRM 10, Rice flour

ICP-MS HGAAS

Butyric acid in edible fats P, S, K Ca, Ti, Mn, Fe, Ni, Cu, Zn, Se, Rb, Ba, Pb in tea 55 major and trace elements in Danish crops PCBs in cod, mussel and shrimp from Belgian continental shelf I in Danish dairy products As(II1) and As(V) in seafood

Phuong et al. 1999

Van Cauwenbergh et al. 1997 Muller et al. 1997 Zhang et al. 1998 Ulberth 1998 Xie et al. 1998 Bibak et al. 1998 Roose et al. 1998

NIST SRM 1567, Wheat flour IAEA H-9, Mixed human diet GBW 08571, Mussel BCR CRM 164, Anhydrous milk fat GBW 08505, Tea NIST SRM 1567a, Wheat flour BCR CRM 349, PCBs in Cod liver oil

ETAAS ETAAS D-AT-FA A S GC TXRF HR-ICP-MS GC

A1 in tea and coffee Cd in vegetables

Fe in duplicate diet samples

Reference

Metho&<

RM used

Examples of recent applications offood RMs for quality control in the analysis of foods and food products

Matrix, measurand

Tab. 6.8

P

0s.

0

m

2

F

lu

P

220

I

G General Application Fields

suggested mode of application principles, but exemplify current usage. Increasing interest in control of analytical data quality and awareness of the usefulness of RMs is leading to more routine use of RMs in daily analytical activities.

6.4 Applications o f Reference Materials in the Geological Sciences Jean Kane

6.4.1

Introduction

The geological sciences are involved in studying the naturally occurring materials of the earth and solar system: (I) to understand the fundamental processes of crustal formation on earth and solar system evolution, and ( 2 ) to evaluate the crustal materials of potential economic value to man. Prior to the I ~ ~ o ’analyses s, were carried out exclusively using classical analytical techniques, with detection limits on the order of 0.01-0.1 % (mass fraction). The number of elements contained in any sample could be as extensive as the periodic table, but very few of these could be determined. The development of instrumental techniques revolutionized the analysis of geochemical samples, beginning in the 1930’s. Natural matrix reference materials of geological samples to support geochemicalanalyses were among the earliest to have been produced by NBS or NIST. For example, an argillaceouslimestone was certified in 1906, while zinc, manganese, and iron ores date to 1910. All of the geological reference samples developed prior to G-I and W-I (Ahrens 1951) support the economic valuation of ores for commerce; none support basic geochemical studies of crustal processes. Regardless, classic work of both Clarke and Washington (1924) and Goldschmidt (1929) in deriving the average abundances of igneous rocks in the earth’s crust was completedwithout reference sample support;that work has stood the test of time extremely well. The same can be said for the work of Goldschmidt and Thomassen (1924)in developing data for rare earth geochemistry. A significant change in geochemical analysis, and in the demands for natural matrix reference samples to support the analysis of geochemical materials, came with the advent of instrumental methods of analysis. The historical roots of these instrumental methods date from the mid-1880’s to the early goo's, but only later were the methods used in routine rock analysis. This use of instrumental methods of analysis generated a demand for rock reference samples, that could be used for calibration purposes. The earliest of them were analyzed by classical methods to determine major and minor oxides The development of G-I and W-I (Ahrens 1951; Fairbairn et al.1951; Stevens et al. 1gG0) was the response to this demand with respect to dc arc emission spectrography. As similar samples are used routinely in calibration for XRF and INAA analyses, many geological samples have been developed as reference materials since that time to support geoanalysis (Potts et al.Iggz). Just as the change from classical to instrumental methods of analysis changed the nature of demands for reference

6.4 Applications of Reference Materials in the Geological Sciences

samples, so the new methods changed the nature of geochemical research (Potts et al. 1993). As it became possible to determine many elements simultaneously, and at lower detection limits than were achieved with classical methods, it became possible to study previously hidden geological processes. That in turn generated new demands on analytical capabilities, to probe even further into unknown geological processes. The synergistic process continually redefines what is needed in the way of reference samples to support method validation and to provide calibration materials. Table 6.9 summarizes the history of the issuance of geochemical reference samples that was inspired by, and resulted in, production of these materials. Tab. 6.9 Timeline of key geological reference materials"

Year issued

Reference sample description

Designation

1906 1919 1910 pre-1918 1910 1950s

Argillaceous limestone Zinc ore Manganese ore Sibley iron ore Norrie iron ore Granite Diabase Columbia River basalt Essey-la-Cote basalt Kitamatsuura basalt Tholeiitic basalt Sphalerite concentrate Nickel ore concentrate Molybdenum ore Platinum ore Jasperoid hot springs deposit Copper mill head soils Hawaiian basalt Rhyolite Basalt Icelandic basalt Devonian Ohio shale Zinnwaldite Estuarine sediment Buffalo River sediment Contaminated soils Trace elements in glass Mean oceanic water Belemite

NBS 1 NBS 2 NBS 25 NBS 27 NBS 28 USGS G-1 USGS W-1 USGS BCR-1 CRPG BR and BE-N JGS JB-1, JB-la JGS JB-2 JGS 19 JGS 20 CANMET PR-1 MINTEK SARM-7 USGS GXR-1, -3 USGS GXR-4 USGS GXR-2, -5, -6 USGS BHVO-1 NIST SRM 278 NIST SRM 688 USGS BIR-1 USGS SDO-1 IWG ZW-C NIST SRM 1646 NIST SRM 2704 NIST SRMs 2710,2711 NIST SRMs 610-617 IAEA VSMOW IAEA VPDB

1967 1964,1980 1968,1984 1982 1970 1970 1971 1975 1975 1975 1975 1976 1981 1981 1984 1990 1994 1982 1988 1992 1982 1987 1987

*

Over 300 geological RMs have been developed over the years. Those listed above have been mentioned in the text, and may be considered representative of the full number available. A complete listing may be found in Potts et al. (1992) or in the July 1994 special issue of Geostandards Newsletter

I**'

222

I

G General Application Fields

The types of natural matrix materials available for geochemistq the means of production, and the applications of these materials in analytical problem-solving are highly varied. It is the purpose of this Section to briefly review these materials and their uses, citing a number of specific applications. Because reference samples for environmental analyses form the subject of another book (Zschunke ~ O O O ) ,this particular application in using geological reference materials will receive only brief mention here, despite a very close link between geological and environmental RMs in terms of both exploration and global mapping applications, as discussed below. With apologies to the reader, space limitations dictate that only a few key references of the hundreds that might support this review can be included. Each cited reference contains many others that present the broader perspective. 6.4.2 Producers of Geochemical Reference Materials

Historically, national geological surveys around the world, working in collaboration with university laboratories, and not national metrology laboratories, have been the major producers of reference materials supporting studies in baseline geochemistry. This is seen in the history of the development of G-I and W-I by the United States Geological Survey (USGS).The initial cooperative investigation of analyses involved the Department of Geology at Massachusetts Institute of Technology, the Office of Naval Research in Washington, DC, Carnegie Institute of Washington, and the Geochemistry and Petrology Branch of USGS (Fairbairn et al. 1951; Stevens et al. 1960). The aim was to provide calibration samples for the developing technique of dc arc atomic emission spectrography. In the end, these samples were recognized as the first geochemical reference samples supporting the analysis of typical crustal rocks, rather than ores and concentrates. Since then, numerous other natural rod< reference materials were developed by USGS, by the Centre de Recherches Petrographique et Geochimiques and Association National de la Recherche and by others (Pottset al. 1993) to assure the closest possible matrix match between calibration and analytical samples. In 1981two rocks were issued by NBS as certified reference materials (Uriano 1981).Today the Japanese Geological Survey and the Chinese Institute of Geophysical and Geochemical Exploration are perhaps foremost among producers in terms of numbers of reference samples supporting geological studies that have been produced in the past decade or two. Reference samples supporting mineral exploration came both from metrology laboratories in countries having extensive mineral potential (e.g. Canada, South Africa) and from geological surveys (e.g. USGS, British Geological Survey). More recently, the geological materials that metrology laboratories like NIST have certified as reference materials have focused on elements important to studies of environmental pollution. It should be noted that many of these environmentally toxic metals are also ore “pathfinder” elements, of great importance in geochemical exploration for untapped mineral resources. For research analysis, the demand is generally in front of the availability of reference materials. For routine production analysis, this is less often the case, but in

6.4 Appfications ofRe&rence Materids in the Geobgical Sciences

some instances is still a problem. For example, rare earth elements and high field strength elements are crucial in understanding the evolution of crustal rocks from the mantle. While close to 300 reference samples have been developed since 1951 (Potts et al. 1993).few reference materials are yet available with well established reference values for these important elements, or for the platinum group elements so important in exploration programs today (Kane 1991;Potts and Kane 1992).Further, over IOO research and routine laboratories are performing microanalysis on geologic samples using the electron probe, the ion probe, laser ablation-mass spectrometry, etc. to determine these elements, but not a single reference material has been certified either in bulk rock or at microsample masses for these applications (Pearce et al. 1997;Potts 1997). 6.4.3

General Application: Calibration o f Instrumental Measurements

The use of reference samples for method calibration and development/validation occurred hand-in-hand with the development of all modern instrumental methods of analysis. In fact, the two developments are intimately linked with one another. As already noted, G-I and W-I (Fairbairn et al. 1951; Stevens 1960) illustrate first instance of reference samples specifically developed for calibration purposes. Following that, the use of BCR-I as a reference sample throughout the lunar program (Science 1970)is a prime illustration of the quality assurance and method validation applications in large-scale inter-laboratorymeasurement programs. The Fairbairn et al. (1951)report on the analysis of G-I and W-I presents a comparison of data obtained by 34 chemists using classical methods of analysis and participating in “the first step in what is probably the most comprehensive study ever taken in rock analysis.” The report goes on to state that “The disparity in results is too great at this preliminary stage to justify the assignment of “correct”values for the composition of the samples.” Thus began the first comprehensive attempt to certify natural matrix geological materials for many commonly determined elements in each such sample. The process “to locate and correct discrepancies” and thus produce an “improvement of analytical procedures and a more accurate estimate of the actual composition” follows the process currently recommended in I S 0 Guide 35 (1989) for certifications of reference materials. The process continues today throughout the geoanalyhcal community, with varying degrees of success. It is important in this regard to recognize two things. First, I S 0 Guide 32 (1993) recommends using at least ten reference samples to establish a calibration line which is the least squares fit of their signals and concentrations. Few method protocols specify the use of so many standards in defining the calibration line. Typically only one standard is used in instrumental neutron activation analysis; the software used with some commercial inductively coupled plasma spectrometric instruments is based on two-point calibration. However, the calibration error must be incorporated into the overall uncertainty of the measurement; and that calibration error will be relatively high unless uncertainties for reference material values are very small and/or many such samples are used in defining the calibration line. Second, there

I

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are not enough reference samples characterized for some elements to meet the I S 0 Guide requirements for calibration with reference samples in many cases. And yet that i s a key application of these materials in geoanalysis. Examples of using reference samples for calibration can be found in several chapters of the USGS Methods for Geochemical Analysis (Baedecker 1987). Solid reference sample powders are used in calibrating the dc arc emission, energy-dispersive X-ray and instrumental neutron activation analyses described, while acid-dissolved rock reference samples are used for ICP emission analyses and fused reference samples are used for wavelength-dispersiveX-ray analyses. 6.4.4 General Application: Method Development and Validation

Identification of sources of analpcal bias in method development and method validation i s another very important application of reference materials in geochemicallaboratories. USGS applied simplex optimization in establishing the best measurement conditions when the ICP-AES method was introduced as a substitute for AAS in the rapid rock procedure for major oxide determinations (Learyet al. 1982).The optimized measurement parameters were then validated by analyzing a number of USGS rock reference samples for which reference values had been established first by classical analyses. Similar optimization of an ICP-AES procedure for a number of trace elements was validatedby the analysis of USGS manganese nodule P-I (Montaseret al. 1984). Another excellent example of the use for method development and validation appears in Morrison and Richardson (1996).Their laboratorywas analyzing many samples of Li ore and related samples for Ba, among other elements, using a routine XRF procedure. The reference sample chosen as a control sample for the run was the zinnwaldite ZWC (Govindarajuet al. 1gg4),for which analyses produced a value approximatelytwice the reference value. Investigationofthat result identified a Rb overlap in the X-ray spectrum that had not previously been observed in use of the method. Similarly, some INAA data contributed to the derivation of a reference value for Ba in SDO-I were biased high by an interference from Io3Ru (Wandless 1993). The lo3Ruis a fission product of U, whose concentration of 40 pg/g i s relatively high in SDO-1. In this case, no appropriate reference sample was available for analysis to control the SDO-1 results; the interference was identified through the disagreement between INAA data and data produced using XRF and ICP-AES methods on the same sample. A bias-free method again resulted when analysis of an atypical type led to detection of a rarely encountered but sizeable spectral overlap. Once identified, correction was straightforward. 6.4.5 General Application: Quality Control in Multilaboratory, or Long-Term Within Laboratory, Studies

Frequently, many laboratories around the world are joint participants in major geochemical studies. One example already mentioned i s the lunar program of almost

G.4 Applications of Reference Materials in the Geological Sciences

thirty years ago (Science 1970). In such studies, it is important that all laboratories involved analyze a common sample. This allows true variability between samples for the program to be distinguished from analytical variability due to method discrepancies between laboratories. BCR-I, a Columbia River basalt developed by USGS, was the prime sample used for this purpose in the lunar program. While many participating laboratories reported data for BCR-I, there was little overlap between laboratories in the elements determined. As a result the use of the sample contributed more to extending the characterization of BCR-I than to providing control oflunar program analytical data (Flanagan1976).BCRI and other basalts, (e.g. BHVO-I, BIR-I, BR, BE-N, JBI, JBIa, JB2, and JB-3) continue to be used for this purpose: they are particularly appropriate control samples in studies of large igneous provinces, including continental flood basalts. These studies will be discussed for the specific application of petrogenic modelling below. In global mapping programs, extensive analytical data from many laboratories, that may or may not be in harmony, is used (Xie 1995). These mapping programs are direct outgrowths of geochemical exploration programs. They aim to uni@, in a single international database, all exploration data developed throughout the world, in order to obtain a consistent global overview of mineral occurrences. In this regard, reference samples provide a means of normalizing all data to a common basis, to provide a coherent world-wide map. Once the procedure is established, the mapping could cover environmental pollution as well as mineral source identification. The difficulty in such data normalization is that the same reference sample is not necessarily used in, or even available to, all laboratories throughout the long life of the mapping program. Similarly, within laboratory, use of a common reference sample throughout the life of an analytical program spanning months or years, assures coherent data over the life of the program. General quality assurance and control using reference materials, as carried out at the Ontario Geological Survey, is described by Richardson et al. (1996), Likewise, the use of an in-house reference basalt BB-I is described in Taggart et al. (1993). Specific applications of this use of reference materials supporting a variety of geochemical studies are discussed below. 6.4.6

Specific Application: Geochemical Exploration

Applications of geological reference samples to mineral prospecting and economic evaluation of ore potential is the only application with a history dating back before the issuance of G-I and W-I in 1951. It is an area in which data quality or lack thereof has serious economic impacts, hence the very early development of certified reference materials mentioned previously. An extensive study of the state of ore analysis was undertaken by the Institute of Geological Sciences (now the British Geological Survey). Nineteen ores and concentrates, of varied matrix, were distributed to 38 laboratories; more than 1532 results were received (Lister and Galagher 1970). The data showed many analytical discrepancies that highlighted the need for ore reference samples of different matrices than those already available and/or certified

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I for additional elements. An outgrowth of that survey was another conducted almost G General Application Fields

immediately by IGS for the purpose of developing reference ores (Lister 1g77), and concurrently, the development of reference ores by both South Africa (Steele et al. 1975) and Canada (Faye 1971). Mineral exploration, the search for economic ore deposits, requires somewhat different reference samples than those used in ore valuation. Soil or sediment and water samples are frequently used in the search when mineralized areas of abundant outcrop or those covered only by thin locally derived overburden are being evaluated. In such cases, it is virtually impossible not to detect the mineralization from an analysis of ore elements in these types of samples. Later, as the mineral deposits closest to the surface were exploited and then played out, new deposits occurred at progressively greater depths, and these sample types were less and less effective as markers in the search (Hoffman 1989). New exploration techniques, and new reference materials in support of them, were needed. One major change was in the use of ore pathfinder elements, rather than the ore elements themselves, for exploration purposes. For example, instead of analyzing samples for the primary Au ore element, samples were analyzed for As, Hg, and W pathfinders that pointed to “hidden” gold deposits. The pathfinder elements occur in association with ore veins, but have a much broader spread than the mineralized area itself. However, measurement of the pathfinder elements requires methods with better detection limits than were needed in earlier exploration programs, as the pathfinders typically are not as enriched as the ore elements, in comparison to baseline crustal levels. Another major change was the shift from extensive use of field laboratory exploration techniques to the laboratory techniques like ICP-AES and INAA. These produce a higher quality data than had resulted from the dc arc and other field techniques, with respect to both repeatability of measurement and improved detection limits. The metrology laboratory certifications for As and Hg in soils and sediments as key environmental toxins provided strong support to mineral exploration programs. The GXR samples illustrateboth sound use and serious misuse of reference samples developed specifically for mineral exploration.The series was developed in an expanded USGS program to identify exploration targets as likely sources of strategic minerals, and thus intended primarily for calibration and/or control of dc arc emission “six-step” field analyses (Alcott and Laltin 1975).These field analyses provided data with a ‘33 % relative standard deviation. Reference values with such wide uncertainty intervals were adequate in establishing a yes/no conclusion regarding the presence of an ore element at an economically recoverable concentration, so long as the concentration was well below or well above the cutoff value. The GXR samples were also analyzed extensively by AAS following an aqua regia leach procedure that efficiently attacked sulfide minerals, but not silicates, thereby highlighting primary mineralization patterns (Ward et al. 1969). These data contributed extensively to establishing the reference values for the ore elements. Used in control of similar analyses,these samples played a very important role in supporting explorationprograms. However, laboratories later began to use the GXR samples for very different purposes. Control of INAA and XRF determinations of the ore elements was sometimes

6.4 Applications of Reference Materials in the Geological Sciences

attempted, but was inappropriate as these techniques measure total, not sulfide-specific, concentrations of the ore elements. Additionally, uncertainties of the reference values exceeded those of the XRF and INAA measurements in-laboratory, further confounding the use of the materials for calibration and/or control. Serious problems resulted from this misapplication of the GXR reference samples in analytical laboratories. A further misapplication occurred when the materials were used for controlling the analysis of additional elements that could be measured by XRF, in particular for the major oxides. Homogeneity of the materials had been established as adequate for analysis of the ore elements. However, that conclusion could not be extended to all elements that later were measured in the samples (Kane et al. 1992). Also consider the use of NIST sediments 1646, 2704, and soils 2709-2711 in exploration geochemistry. These samples were certified largely in view of the demand for samples to support monitoring of toxic elements in environmental samples. However, many of the elements certified overlap either the list of primary ore elements or the list of “pathfinder” elements. Thus, these samples may legitimately be used in a very different application than the one that prompted certification. The sample matrix is ideal for the alternative application, and so is the suite of certified elements. Assume that without the proper use of reference samples in an exploration program, a site is purchased that is in fact barren. Hill (1974).for example, cites a 20 % added analytical cost for quality control and quality assurance. He further cites a possible cost of $220 million for purchasing and developing a mine site. The analytical expense for QA\QC based on use of reference samples is trivial in comparison to the potential loss, if the analyses of exploration survey samples are faulty and the mine worthless as a result. 6.4.7 Specific Application: Petrogenic Modelling Based on Bulk Rock Analysis

Among the quality control uses of reference samples, petrogenic studies to understand the genesis of large igneous provinces and zones of mineralization during earths long history were cited. This application is one which illustrates the synergy of developing analytical methodology and geochemical models for earth processes particularly well. Clarke and Washington (1924) published the first attempt at calculating average crustal abundances of major and minor oxides in crustal rocks. Shortly thereafter, Goldschmidt (1929) published results of a similar calculation, based on the analysis of different suites of rock. In both cases, the data used was obtained entirely using classical methods of analysis, and was limited to the 12 most abundant oxides that form the rock matrix. At about the same time, Goldschmidt and Thomassen (1924) used X-ray diffraction to make the first extensive geochemical study of the rare earth elements. Modern geochemical studies use data for a much larger suite of elements, determined at much lower concentrations, to model the tectonic movements of continental plates, and to understand the sources of magma generated in that process (e.g. Lightfoot 1993; Sutcliffe 1993).The key elemental suites include the “incompatible”

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patterns of these elements in igneous rocks as compared to average crustal abundances "fingerprint" the source areas for different volcanic events. Major oxide patterns are also informative in distinguishing magma types. The USGS basalts BCR-I, BHVO-I and BIR-I are among the most frequently used reference samples for analyses performed by XRF, INAA, and ICP-MS. The reference values available in many cases do not meet the Uriano and Gravatt (1977) criterion of being known with three to ten times greater certainty than the data produced in routine analysis. Until metrology laboratories develop a certified reference basalt, with values for these critically important elements, the situation is unlikely to change. Reference values developed through inter-laboratory programs, and based on the agreement in results between two or more different methods of analysis, have not produced values with uncertainties that can be achieved in metrology laboratories based on definitive methods (Kane 1992; Kane and Potts 1997). Isotopic compositions are also critical in these petrogenic modelling studies, to provide ages of the different magmatic episodes. Among the most important ones are 87Sr/86Sr,206Pb/204Pb,207Pb/204Pb,143Nd/144Nd,and 147Sm/144Nd.NBS-certified isotopic reference materials are available for control of the Sr and Pb ratio measurements. USGS Columbia River basalt BCR-1 is used extensively in controlling Nd and Sm isotopics. While the material was not issued for this purpose, in 1990, approximately 50 % of all Nd isotopic ratio measurements reported in the geological literature were for BCR-1, despite the fact that, at the time, it had not been distributed by USGS for about ten years. Similarly, the La Jolla sample is used for this purpose (Wasserburget al. 1981). 6.4.8 Specific Application: Petrogenic Modelling Based on Microanalysis

Microanalytical techniques are currently being used almost as extensively in petrogenic modelling studies as bulk rock methods. The lack of suitable reference materials for this application is particularly acute. Again the lack of participation by national and international metrology laboratories in resolving the problem must be noted. At this time, geochemical laboratories rely on the NIST glasses SRMs 610-617 for the purpose (Pearce et al. 1997; Kane 1998). Although the reference values available are not entirely adequate, in the absence of any alternative they provide needed support to an emerging analytical approach that is of growing importance in geochemistry (Potts 1997). 6.4.9 Application: Studies of Paleoclimates

There are a number of light stable isotope measurements that provide very important data in the study of paleoclimates to better understand and interpret anthropogenic contributions to present-day climate change (Fritz and Fontes 1980). These measurements involve the determination of carbon and oxygen isotopes in fresh-

G.5

References

water shells; the ratios give an indication of water temperature at the time the shells formed. Similarly deuterium in organic matter i s an indicator of paleoclimate factors. The principle collection of light stable isotope ratio reference materials was first developed at NBS (Mohler 1960). These were augmented by the work of The International Atomic Energy Agency’s Consultancy Group on Stable Isotope Reference Samples for Geochemical and Hydrological Investigations (Hut 1987). Important contributions include the absolute reference values of isotopic ratio standards VSMOW for hydrogen and oxygen isotopics, and Vienna PeeDee Belemite for carbon isotopics. The group has further determined isotopic values expressed as parts per thousand difference from these standards for a number of other materials. All are of value to geochemistry laboratories engaged in studies of changes in the earth’s environment over geological time. 6.4.1 0 Summary

The above discussion touches rather briefly on some key applications for the use of reference samples in geology and geochemistry. Many others could be cited, but space restrictions prohibit doing so. Regardless, it has been seen that every change in measurement capability over the past fifty years has led to new and unmet demands for reference values in natural matrix samples. These values might be for elements previously not considered measurable, or for elements at progressively lower and lower concentration ranges. As the needed reference values have become available, even when not fully up to I S 0 standards for reference value quality, geochemical interpretation of the earth’s crustal processes has moved forward, sometimes quite dramatically. Further progress i s essential, but great pride can be taken in accomplishments to date regarding reference sample production and continual data quality improvement in the geosciences.

6.5 References

AHRENS LH (1951)A story of two rocks. Geostds News1 1:157-161. ALBERTS B, BRAYD, LEWIS J, RAFFM, ROBERTSK, WATSON JD (1994) Molecular biology of the cell. Garland, New York, Chapter 3, p 89 ff. ALCOTTGH, LAKIN HW (1975) The homogeneity of six geochemical exploration reference samWK, eds. Geochemical Exploration 1974. Proc 5th International ples. In: ELLIOTTIL, FLETCHER Geochemical Exploration Symposium, pp 659-681. ALEXANDER NM (1994) Iron. In: SEILER HG, SICELA, SICELH, eds. Handbook on metals in dinical and analytical chemistry. Dekker, New York. ANDERSON KA, TALCOTIPA (1994) Magnesium. In: SEILERHG, SIGELA, SIGELH, eds. Handbook on metals in clinical and analytical chemistry. Dekker, New York. ANDREWS LS, SNYDERR (1996) Solvents. In: KLAASSENCD, ed. C A S A Rand E ~ Doul’s Toxicology. McGraw-Hill,New York. ANGERJP, CURTES JP (1994) Tin. In: SEILER HG, SIGEL A, SICELH, eds. Handbook on metals in clinical and analytical chemistry. Dekker, New York.

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ANKEM, GLEI M (1994) Molybdenum. In: SEILERHG, SIGELA, SIGELH, eds. Handbook on metals in clinical and analytical chemistry, Dekker, New York. BAEDECKER PA, ed. (1987)US Geological Survey Bulletin 1770 Methods of Geochemical Analysis, Department of the Interior, Washington DC. BIBAKA, BEHRENSA, STURUP S , KNUDSENL, GUNDERSEN V (1998) Concentrations of 55 major and trace elements in Danish Agricultural crops measured by inductively coupled plasma mass spectrometry. 2 . Pea (Pisurn sativum Ping Pong). J Agric Food Chem 463146-3149. BIRCH NJ, PRADGEHAM C (1994) Potassium. In: SEILER HG, SIGELA, SIGELH, eds. Handbook on metals in clinical and analytical chemistry. Deldter, New York. BIRCH NJ, PRADGEHAM C, HUGHES MS (1994) Lithium. In: SEILERHG, SIGEL A, SICELH, eds. Handbook on metals in clinical and analytical chemistry, Dekker, New York. BLOTCKYAJ, DUCKWORTH WC, HAMEL FG (1994) Vanadium. In: SEILER HG, SIGELA, SIGELH, eds. Handbook on metals in clinical and analytical chemisby. Deltker, New York. BOWMANWS (1994).Catalogue of Certified Reference Materials, CCRMP 94-1E. Canadian Certified Reference Materials Project, Natural Resources Canada, Ottawa. BUTLEROT, HOWE AM (1999) Development of an international standard for the determination of metals and metalloids in workplace air using ICP-AES: evaluation of sample dissolution procedures through an interlaboratorytrial. J Environ Monit 123-32. B U ~ N E JR (1995) The need for accuracy in laboratory medicine. Europ J Clin Chem Clin Biochem 33:981-988. S, FORTEG, IAMICELI AL, GALOPPIB (1999) Determination of essential and potentially toxic CAROLI trace elements in honey by inductively coupled plasma-basedtechniques. Talanta 50327-336. B, J O H N S O N D (1994) Manganese. In: SEILER HG, SIGELA, SIGELH, eds. Handbook CHISWELL on metals in clinical and analytical chemistry. Dekker, New York. VERCOUTERE , K, CORNELIS R, QUEVAUVILLER PH (1999) CertificaCHRISTENSEN JM, BYRIALSENI< tion of Cr(VI) and total leachable Cr contents in welding dust loaded on a filter (CRM 545). Fresenius J Anal Chem: 363:28-32. JM, KRISTIANSENJ (1994) Lead. In: SEILER HG, SIGELA, SIGELH, eds. Handbook CHRISTENSEN on metals in clinical and analytical chemistry, Dekker, New York. HS (1924) The composition of the earth's crust. US Geological Survey CLARKE FW, WASHINGTON Professional Paper 127, 117 pp. S (1994) Calcium. In: SEILER HG, SIGEL A, SIGELH, eds. HandDILANA BA, LARSSONL, OHMAN book on metals in clinical and analytical chemistry. Dekker, New York. DONAISMI<, SARASWATI R, MACKEY E, DEMIRALP R, PORTER B, VANGELM, LEVENSONM, MANDIC V, AZEMARD S, HORVAT M, MAYK, EMONSH, WISES (1997) Certification of three mussel tissue standard reference materials (SRM) for methylmercury and total mercury content. Fresenius J Anal Chem 39424-430. DOUMASBT, PETERS T Jr. (1997) Serum and urine albumin: a progress report on their measurement and clinical significance.Clin Chim Acta 258:3-20. DRASCHG (1994) Mercury. In: SEILERHG, SIGEL A, SIGELH, eds. Handbook on metals in clinical and analytical chemistry. Dekker, New York. U, ULDALL A, RICHTERW (1999) Metrology in laboratory medicine - A DYBKAER R, ORNEMARK necessity. Accredit Qua1 Assur 4:349-351. D Y B I ~ ER, R STORRING PL (1995) Application of IUPAC-IFCC recommendations on quantities and units to WHO biological reference materials for diagnostic use. Europ J Clin Chem Clin Biochem 33:623-625. FAIRBAIRN HWet al. (1951) U.S. Geological Survey Bulletin 980. A cooperative investigationofprecision and accuracy in chemical, spedrochemicaland modal analysis of silicate rocks. 71 pp. FAYEGH (1971) Canadian Mines Branch Technical Bulletin TB139 Molybdenum Ore PR-I: its characterization and preparation for use as a standard reference material. FINGEROVA H, KOPLIXR (1999) Study of minerals and trace element species in soybean flour. Fresenius J Anal Chem 363:545-549.

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IHNATM (1992) Selection and preparation of relevant reference materials for agricultural purJD, OSTAPCZUK P, eds. Specimen Banking - Environmenposes. In: ROSSBACHM, SCHLADOT tal Monitoring and Modem A n a l y t d Approaches, pp 57-73. Springer, Berlin. IHNAT M (1993) Reference materials for data quality. In: CARTER MR, ed. Soil Sampling and Methods of Analysis, pp 247-262. Lewis Publishers, Boca Raton, FL. IHNAT M (1994) Development of a new series of agricultural/food reference materials for analytical quality control of elemental determinations. J AOAC International 77:1605-1627. IHNAT M (1998a) Reference materials for data quality control. In: KALRA YP, ed. Handbook of Reference Methods for Plant Analysis, pp zog-zzo. CRC Press, Boca Raton. IHNAT M (199%) Plant and related reference materials for data quality control of elemental content. In: KALRA YP (ed.) Handbook of Reference Methods for Plant Analysis pp 235-284. CRC Press, Boca Raton. Institute for Reference Materials and Measurements (1999) BCR Reference Materials 1999. Reference Materials Unit, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium. International Atomic Energy Agency (1995) Survey of Reference Materials Volume I and Volume 2. IAEA-TECDOC-854,International Atomic Energy Agency, Vienna. International Atomic Energy Agency (1998) AQCS Intercomparison Runs. International Atomic Energy Agency, Vienna. K, ORIMOH (1994) Gold. In: SEILER HG, SIGEL A, SIGELH, eds. Handbook on metals in ISHIDA clinical and analytical chemistry. Dekker, New York. IS0 Guide 32 (1993) Linear calibration using reference materials. Internatlonal Organization for Standardization, Geneva, Switzerland,31 pp. IS0 Guide 35 (1989) Certification of reference materials- general and statistical principles. International Organization for Standardization, Geneva, Switzerland,32 pp. J O H N S O N AM, SAMPSON EJ, BLIRUP-JENSENS, SVENDSEN PJ (1996) Recommendations for the selection and use of protocols for assignment of values to reference materials. Europ J Clin Chem Clin Biochem 34;279-285. KANE JS (1991) Review of geochemical reference sample programs since G-I and W-I: progress to date and remaining challenges. Spectrochim Acta 46B:1623-1638. KANE JS (1992) Reference samples for use in analytical geochemistry: their availability, preparation, and appropriate use. Journ Geochem Explor 44:37-63. KANE JS (1998) An assessment of the suitability of NISTglass SRM literabre data for the derivation of reference values. Geostds Newslett: The Journal of Geostandards and Geoanalysis 22:15-31. KANE JS, P o n s PJ (1997) I S 0 Guidelines for reference material certification and use, Geostds Newslett: The Journal for Geostandards and Geoanalysis ~1:51-58. KANE JS, SIEMSDF, ARBOGAST BF (1992) Geochemical exploration reference samples GXR-I to GXR-4 and GXR-6: evaluation of homogeneity based on high precision analyses. Geostds Newslett 16:45-54. KONIG KH, SCHUSTER M (1994) Platinum group metals. In: SEILER HG, SIGEL A, SIGELH, eds. Handbook on metals in clinical and analytical chemistry. Dekker, New York. LAMBERTY A, KRAMERGN (1998)The Certification of the Mass Fraction ofAs, Cd, Cu, Mn, Pb, Se and Zn in Bovine Liver CRM 185R. European Commission, Report EUR 18841EN, Luxembourg. LARSEN EH, KNUTHSENP, HANSEN M (1999) Seasonal and regional variations of iodine in Danish dairy products determined by inductively coupled plasma mass spectrometry. J Anal At Spectrom 14:41-44. LEARYJJ, BROOKESAE, DORRZAPF AF 1%GOLIGHTLY DW (1982)An objective function for optimization techniques in simultaneous multiple-element analysis by inductively coupled plasma spectrometry. Applied Spectroscopy36:37-40. LIGHTFOOT PC (1991)The Interpretation of Geoanalyhcaldata in analysis of geological materials. In: RIDDLEC, ed. Analysis of Geological Materials, pp 377-455. Marcel Dekker, Inc. New York.

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General Application Fields

Pons PJ, HAWICESWORTH CJ,VAN CASTEREN P, WRIGHT IP (1993)In: PRITCHARD H M, ALABASTER T, HARRIS NBW, NEARYCD, eds. Magmatic Processes and Plate Tectonics, pp 501-520. Geological Society Special Publication No 76. POTTS PJ, TINDLE AG, WEBBPC (1992) Geochemical Reference Material Compositions. CRC Press Boca Raton FL. PH, VAN RENTERGHEM D, GRIEPINICB, SPARKESST, KRAMERGN (1991)The CertiQUEVAUVILLER fication of the Contents (Mass Fractions) of Ca, Cu, C1, I, Fe, I(, Mg, P, Pb, N, Na and Zn in Skim Milk Powder (CRM o63R) Commission of the European Communities, Community Bureau of Reference Report EUR 15021 EN, Luxembourg. RICHARDSONJM, LIGHTFOOT PC, DESOUZA H (1996) Current geosciences laboratories geoanalytical programs and their quality assurance underpinnings. Geostds Newslett 20:141-156. RIFAI N, WARNICIC GR, DOMINICZAIC MH, eds. (1997) Handbook of protein testing. AACC Press, Washington. ROOSEP, COOREMAN I(, VYNCKEW (1998) PCBs in cod (Gadus rnorhua), flounder (Platichthysflesus), blue mussel (Mytilus edulis) and brown shrimp (Crangon crangon) from the Belgian continental shelf:relation tobiologicalparameters and trend analysis. Chemosphere37:2199-2210. SARKARB (1994) Copper. In: SEILER HG, SIGELA, SIGEL H, eds. Handbook on metals in clinical and analytical chemistry. Dekker, New York. SCHALLER KH, LETZEL S, ANGERER J (1994) Aluminium. In: SEILER HG, SIGELA, SIGELH, eds. Handbook on metals in clinical and analytical chemistry. Defier, New York. Science (1970) “The Moon 1ssue”Apollo11 Lunar Conference Volume. Vol167 No 918. SERAT WF, MENGLEDC (1973) Quality control in the measurement of blood cholinesterase activities among persons exposed to pesticides. Bull Environ Contam Toxicol9:24-27. SHAWG (1993) Polychlorinated biphenyls (PCB’s)effects on humans and environment. In: CORN M, ed. Handbook of hazardous materials. Academic Press, San Diego. SOUTHGATEDAT (1987).Reference material for improving quality of nutritional Composition data for foods. Fresenius J Anal Chem 326:660-664. TW et al. (1975) The preparation and certification of a reference sample of a precious STEELE metal ore. National Institute for Metallurgy, Report 1696. RE et al. (1960) Second report on a cooperative investigation of the composition of two STEVENS silicate rocks. U.S. Government Printing Office, Washington DC, 126 pp. M, VAHTERM (1994) Arsenic. In: HERBER RFM, STOEPPLERM, eds. Trace element STOEPPLER analysis in biological specimens. Elsevier, Amsterdam. SULLIVAND, CARPENTER D, eds. (1991) Methods of Analysis for Nutrition Labelling. AOAC International, Gaithersburg MD, USA. SUTCLIFFE RH (1993) An Overview of Geochemical Research Implications for the Geoanalyst in Analysis of Geological Materials. In: RIDDLEC, ed. Analysis of Geological Materials, pp 1-35. Dekker, New York. JE JR, LINDSAY JR, SCOTT BA, VIVIT DV, BARTELAJ, STEWARTK C (1993) Analysis of TAGGART geological materials by wavelength-dispersive X-ray fluorescence spectrometry. In: BADECKER PA, ed. U.S. Geological Survey Bulletin 1770. Methods for Geochemical Analysis, pp E I - E I ~ . TAYLOR,J.K. (1993) Standard Reference Materials: Handbook for SRM Users. NIST Special Publication 260-100, National Institute of Standards and Technology, Gaithersburg, MD. THOMASSEN Y, LEWIS SA, VEILLONC (1994) Selenium. In: HERBER RFM, STOEPPLER M, eds. Trace element analysis in biological specimens. Elsevier, Amsterdam. L, LEJEUNER (1994) Cobalt. In: SEILER HG, SIGEL A, SIGELH, eds. Handbook on metals THUNUS in clinical and analybcal chemistry. Deldten; New York. K (1994) Sodium. In: SEILER HG, SIGELA, SIGELH, eds. Handbook on metals in clinical TOHDA and analytical chemistry, Dekker, New York. R, TAKAHATA S , ONODA M, ISHI-I T, SATO K (1974) Evaluation of the exposure to TOKUNAGA organic solvent mixture. Comparative studies on detection tube and gas-liquid chromatographic methods, personal and stationary sampling, and urinary metabolite determination. Internationales Archiv Arbeitsmedizin 33:257-267.

6 5 References I 2 3 5 TRAHEY NM, ed. (1998) NIST Standard Reference Materials Catalog 1998-99, NIST Special Publication 260. National Institute of Standards and Technology, Gaithersburg, MD. J, HILLSJ, EVANSEH, FAIRMANB (1999) The use of ETV-ICP-MSfor the determination TURNER of selenium in serum. J Anal Atomic Spectrom (JAAS) 14:121-126. ULBERTHF (1998) A rapid headspace gas chromatographic method for the determination of the butyric acid content in edible fats. 2 Lebensm Unters Forsch 2oGA305-307. GA (1981) Certificates of Analysis for SRM 278 and SRM 688. U.S. Department of ComURIANO merce, Washington DC, 2 pp each. URIANOGA, GRAVATICA (1977)The role of reference materials and reference methods in analytical chemistry. CRC Crit Rev Anal Chem 6361-411. USDA (1989-91) Nutrient Data Base for Food Consumption Surveys, Agricultural Research Service, Beltsville Human Nutrition Research Center, Survey Systems/Food Consumption Laboratory, Beltsville MD, USA. USDA (1991) Continuing Survey of Food Intakes by Individuals (CSFII).Agricultural Research Service, Beltsville Human Nutrition Research Center, Survey Systems Laboratory, Beltsville, MD, USA. USDA (1993)Nutrient Data Base for Standard Reference, Release. No. 10.Agrinrltural Research Service, Beltsdle Human Nutrition Research Center, Nutrient Data Laboratory, Beltsville MD, USA. VAN CAUWENBERGH, HENDRIX P, ROBBERECHT H, DEELSTRA H (1997) Daily dietary iron intake in Belgium using duplicate portion sampling. 2 Lebensm Unters Forsch zo5A401-406. LR (1997) The college of American pathologist, 1946-1996: laboratory standards. Arch WAGNER Path Labor Med 121:536-541. JM, ed. (1996) The protein protocols handbook. AACC Press, Washington. WALKER KV, WARDI<M, MAHANJD, WISMATT DI< (1989) Current concepts in proteinuria. Clin WALLER Chem 35:755-765. GA (1993) Instrumental neutron activation analysis of Devonian Ohio Shale SDO-I. WANDLESS In: IQNE JS (ed.) U.S. Geological Survey Bulletin 2046. The USGS Reference Sample Devonian Ohio Shale SDO-Ipp DI-D~. FN, NAKAGAWA HM, HARMS TF, VANSICKLE GH (1969)Atomic-absorptionmethods ofanalysis WARD useful in geochemicalexploration.U.S. Government Printing Oflice, Washington DC, 45 pp. DJ. MCCULLOCK MT, WENT (1981) Precise determinaWASSERBURG, GJ, JACOBSEN SB, DEPAOLO tion of Sm/Nd ratios, Sm and Nd isotopic abundances in standard solutions. Geochim Cosmochim Acta 45:2311-2323. WIEDEMANN G, UMBREITA (1999) Determination of urinary protein fractions by different electrophoretic methods in comparison to quantitatively determined protein concentrations. Clinica Labratorio 45257-262. D, CARPENTER D, eds. Methods of Analysis WOLFWR (1993) Reference materials, In: SULLIVAN for Nutrition Labelling, pp 112-115. AOAC INTERNATIONAL, Gaithersburg MD. WOLFWR, ANDREWS I<W (1995)A system for defining reference materials applicable to a11 food matrices. Fresenius J Anal Chem 35x73-76. XIE M, VON BOHLENA, K L O C K E N ~ M P R, E RJIAN X, GUNTHERI< (1998) Multielement analysis of Chinese tea (Camellia sinensis) by total reflection X-ray fluorescence. ZLebensm Unters Forsch 207A31-38. XIEX (1995)Analytical requirements in internationalgeochemical mapping. Analyst 120:1497-1504. ZHANGD-Q, LI C-U, YANG L-L, SUNH-W (1998) Determination of cadmium in vegetables by derivativeflame atomic absorption spectrometry with atom trapping technique. J Anal At Spectrom 13:1155-1158. A, ed. ( 2 0 0 0 ) Reference Materials in Analytical Chemistry, Springer Berlin HeidelZSCHUNKE berg New York.

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

7

Proper Usage o f Reference Materials PeterJ Jenks and RolfZeisler

7.1

Selection, Use, and Abuse of RMs

Since the early 1970’sthere has been a growing belief that chemical measurements must not only be done correctly, but that data, the product of the measurement process, must be seen to be accurate, precise, and reliable. Analpcal data have become another manufactured product and like all manufactured products, the customers demand that Quality Assurance (QA)must be built in. There is an abundance of references defining and describing the role played by QA, Quality Control (QC) and Total Quality Management (TQM) in a modem commercial analytical laboratory. The role played by reference materials (RMs)and certified reference materials (CRMs) in the pursuit of analytical measurement accuracy is also well documented. It has become an accepted wisdom that the use of RMs or CRMs will help to improve the accuracy and precision of an analybcal process. This belief has led to a rapid growth in the use of RMs and CRMs in commercial laboratories. The authors and many analysts the world over support this view, but also recognize that in far too many cases inexperience and carelessness conspire together with the result that error accumulates and often unreliable data are produced. In this Chapter we highlight the practical considerations that must be understood by all users of RMs and CRMs; we look at some of the issues of traceability and make the CRM user aware of the uncertainty budgets that need to be considered with the use of CRMs. No attempt will be made to advise CRM users on the proper use of statistics in the analytical measurement process and no statistical approaches on the establishment of measurement uncertainty will be given. There are a number of good texts on the subject which should be consulted. These are listed in the “Further Reading” section of the references at the end of this Chapter and include Miller and Miller (1993) and Taylor’s work for NIST (Taylor 1985).

7.1 Selection, Use, and Abuse of RMs

7.1.1 Conventional “Proper” Uses o f RMs

There are many applications in which RMs and CRMs are used, but those that are relevant to analytical chemistry, including environmental, industrial, bio-medical, and forensic applications and that directly influence Total Quality Management (TQM)can briefly be grouped into the main categories listed below. 7.1.1.1

Method Development and Evaluation

Evaluation and verification of the precision and accuracy of test methods Development of reference test methods Evaluation of field methods Validation of methods for specific uses, and developing new or improved techniques and methods. 7.1.1.2

Assurance o f Measurement Compatibility

Direct calibration of methods and instrumentation; i.e. ensuring that an analytical device is giving a correct reading. For some types of direct solid sample analysis, sample results can be calibrated using several CRMs with suitable matrices (Kurfiirst 1998);see also Section 4.4. Internal (intra-laboratory)quality assurance External (inter-laboratory)quality assurance Demonstration of the integrity and performance of a complete analytical system, from initial sampling to data manipulation 7.1.1.3

Establishment o f Measurement Traceability

Development and implementation of traceability protocols Development of secondary (in-house) standards Direct laboratory use Verification of laboratory competence to satisfy organizational or customer needs There are a number of prerequisites for properly using CRMs in these tasks, including established quality control of the laboratory’s analytical measurement operations and proven statistical control of the analytical measurement process. Publications describing the use of RMs and CRMs are not as plentiful as those on how CRMs are made but, in addition to the ISO/REMCO Guides 30-35, the ISO/REMCO publication “The role of reference materials in achieving quality in analytical chemistry”(IS09000 1987).the NIST Handbook for SRM users (Taylor 1995),and the various LGC-VAM publications listed under “Further Reading” should all be consulted.

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7.1.2

Mis-Use and Other Causes o f Errors

Despite all that has been written about the requirements of carefully performed analytical quality control including the usage of CRMs, there are still many laboratories that either have neither proper internal QC nor CRMs, or perhaps do not want to admit to using them! Many more common problems start because the new users do not really understand their analytical systems, a problem described as the "NintendoTM scientist" syndrome by Jenks (1995).Inexperienced scientists are often not sufficiently discriminating in their selection and use of CRMs. But incorrect choice can also be due to the unavailability of suitable matched matrix CRMs, or surprisingly often because the laboratory believes it cannot afford the ideal product. We have described a selection of uses of CRMs in Chapters 4, 5, and 6. There are copious additional references in the literature, as we explain in Chapter 8. But most of these references do not explain how the CRM was used, only why. The authors assume that the reader knows how to use a CRM. We have discovered from a number of years experience in advising on the use of CRMs that the same mistakes and mis-understandings occur time and time again. The main causes of error can be conveniently grouped together, as follows: 7.1.2.1

Documentation Errors

Such errors include mis-reading certificate or report data supplied by the producers, using secondary information from catalogs and/or literature listings, and reporting incorrect data. For the correct use of a RM/CRM, it is essential to read the information that accompanies the product once an appropriate RM/CRM has been obtained. The only reliable source of information is the Certificate of Analysis or Report of Assigned Values issued with the RM/CRM, and it must be the most up to date version available. Failure to follow a producer's recommendationwill invariably result in error. Unfortunately, these rather basic errors are distressingly common, yet cause much unnecessary dissatisfaction. No printer is perfect, and relying on catalog data can result in the publication of incorrect data in a paper. This occurred, e.g. in 1994 when data was taken from an out-of-dateNIST catalog, rather than the appropriate certificate. Published in the Journal of Analytical Atomic Spectroscopy,the paper by Soares et al. (1994) cited a certified value for Cr in NIST SRM 1548, when consultation of the Certificate would have shown that for several technical reasons the element value reported could not be certified. Supplementary information on many RMs/CRMs may also exist in the form of publications in the open literature, describing their production and recommended uses. That information should also be consulted by the users. But users must be aware that some values reported in these publications are indicative only and may lack the complete evaluation of data that eventually forms the certified value. Even more caution is advisable when data compilations are used in lieu of certified values. For many years a cobalt value in SRM 1577, Bovine Liver, was propagated in this way at about 120 % of the true value.

7.1 Selection, Use, and Abuse of RMs

Selection Errors Choosing an inappropriate RM/CRM, or failing to understand the matrix effect is another common source of error. In many types of environmental and biological reference materials the effect of the matrix can be profound, especially when using routine sample preparation and analysis methods. Nevertheless, it should not be forgotten that some direct analysis procedures, e.g. instrumental neutron activation analysis (INAA), are much less sensitive to matrix mis-match because of their matrix-independence and dynamic range. The use of such direct procedures in the certification of a RM has to be taken into consideration when matching matrix and RM. The issue of matrix-matching is considered in more detail later in this Chapter. The literature includes a number of mis-matches, the following standing as examples for the many! The use of bovine liver and other animal tissues for QC in the analysis of human body fluids should not be considered by analysts. The matrix and the levels of trace elements do not match the levels to be analyzed, which may lead to serious errors. An even more severe mis-use was recently reported by Schuhmacher et al. (1996) for NIST SRMm1577aBovine Liver, which was used for QC in the analysis of trace elements in plant materials and soil samples in the vicinity of a municipal waste incinerator. Also recently, Cheung and Wong (1997) described how the quality control for the analysis of trace elements in clams (shellfish) and sediments was performed with the same material NIST SRM 1646, Estuarine sediment. Whilst the selected SRM was appropriate for sediments, its usefulness as a QC tool for clams is difficult to prove; see also Chapter 8. This inappropriate use is the more mystifying because a broad selection of suitable shellfish RMs from various producers is available. How critically interdependent matrix and analybcal methods can be is illustrated in the example of the analysis of a soil sample. Table 7.1 shows the method dependent certified values for some common trace elements. The soil had been subjected to a multi-national, multi-laboratory comparison on a number of occasions (Houba et al. 1995) which provided extensive data. The data was subjected to a rigorous statistical program, developed for the USEPA by Kadafar (1982).This process allowed the calculation of certified values for a wide range of inorganic analytes. Uniquely, for the soil there are certified values for four very different sample preparation methods, as follows: 7.1.2.2

Acid Extraction - Aqua regia extraction is comparable with DIN 38 414part 7, NEN 6465 and many other European routine procedures. Calcium Chloride Extraction - A 0.01M calcium chloride solution extraction. This method has been shown to be a step forward in the development of a universal extractant for nutrients and metals by Erp et al. (1998). Nitric Extraction - A 2 M hot nitric acid. This method is comparable with the EPA 3050A extraction procedure. Total - The complete dissolution of the matrix by methods such as hydrofluoric acid, or measurement by nondestructive methods such as INAA.

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The Influence of analytical technique on analytical values (Data extracted from Certificate for CRM RTH 912, Loess Soil from Switzerland, produced by Promochem CmbH, Germany, with permission). All units mg/kg

Tab. 7.1

Cadmium Chromium Cobalt

Copper

Iron

Lead

13.4

127.0

35 600

71.6

103-120

12.4-14.3

123-132

34 200-37 000 63.8-79.4

62.3-160

8.61-18.1

98.2-156

26 800-44 400 27.9-115

1.20

67.5

12.2

120.0

32 500

1.16-1.24

65.7-69.3

12.0-12.5

118-121

31 900-33 000 63.2-65.3

0.67-1.73

40.2-94.8

9.48-15.0

100-139

25 300-39 600 47.5-81.0

1.22

36.6

9.53

117.0

No Data

1.19-1.24

35.3-37.9

9.28-9.78

115-118

63.3-66.0

0.90-1.53

22.9-50.3

6.90-12.2

99.7-133

49.6-79.7

(0.124)

(0.122)

No Data

0.492

1.13

0.413-0.571

0.74-1.53

0.0062-0.949

0.00-3.23

Confidence Interval Prediction Interval Acid Extraction Confidence Interval Prediction Interval Nitric Acid Extraction Confidence Interval Prediction Interval Chloride Extraction Confidence Interval Prediction Interval

64.2

64.6

(3.03)

The enormous difference in certified values between methods and between analytes illustrates well how much care is needed in matrixlmethod matching. Further evidence of the importance of matrix matching is provided by an interlaboratory study on trace elements in soil reported by Maier et al. (1983)and the certification of a sewage sludge described by Maaskant et al. (1998). In trace organic analysis there is usually an extraction or clean-up process, rather than a sample dissolution. Here not only must the matrix effect be considered, but also the recovery yield of the extraction. Frequently an external spike standard is added, but there is often no way of knowing if the recovery of the spike standard matches the analyte in question. There is considerable evidence that the US EPA method for VOA analysis (Minnich 1993)is subject to such error, as reported by Schumacher and Ward (1997).The analyst must always consider the possibility of such an error, especially when using CRMs to control methods that are applied in routine mode.

7.7 Selection, Use, and Abuse of RMs

7.1 2 . 3

Handling and Use Errors

As mentioned before, RM producers go to great lengths, as are required by the I S 0 Guide 31 (1996)to prove homogeneity and stability, and to establish the best sample size and storage conditions for an optimal shelf life. This information is normally provided in the RM certificate. Nevertheless, users must pay particular attention to a number of procedures in the use of RMs to avoid invalid results. 7.1 2 . 4

Storage

The stability of individual analytes within a matrix material is often quite variable. A good example is shown by NIST SRM 96% fat soluble vitamins and cholesterol in serum. The material must be shipped and stored at -80°C. The SRM is certified for a range of vitamins, most of which are quite stable at -2o"C, or even +4"C, but the beta-carotene and other components are not. It is therefore essential to ensure the material, if the carotene components are of interest, is shipped and stored correctly. Manufacturers of CRMs go to considerable lengths to ensure that the CRMs are stable, therefore attention must be paid to specified storage conditions and shelf life. It must not be forgotten that in most cases the shelf life stated by the producer refers to an unopened unit, and that once opened shelf life is often not guaranteed. This applies especially to CRMs packed under a protective atmosphere and stored at reduced temperatures; see Sections 2.1, 3.1, and 7.1.2.5for further consideration of these issues. Unfortunately not all producers provide specific storage instructions, and even for those that do it is unlikely that they will have updated certificates produced 5-10 years ago. Therefore, in the absence of clear guidelines for storage after opening, one approach is to divide the material into a number of aliquots, taking into consideration the recommended minimum sample size, and to re-seal each aliquot before storage at appropriate temperatures (for sensitive materials in deep freezers at e.g. -2o"C, for less sensitive, e.g. gamma-radiation sterilized biological matrices, storage at + 4 T may suffice) under controlled humidity and protected from light. Reports on evaluations of long-term storage of biological and environmental materials under several conditions can be found in the literature (Zeisler et al. 1988; Mackey et al. 1999). 7.1 2.5

Shelf Life and Expiration Dates

Most matrix reference materials are regarded stable for their application within a certain time frame; see also Section 2.2. They are usually produced in large batches designed to ensure that the same material is available for a number of years as well as to spread the high cost of production over as many units as is possible. The stability is closely monitored from initial production by the producer; lot numbers or even individual unit numbers are allocated and the producers closely monitor, by regular analysis, the condition and quality of their reference materials over time. Because of such careful control, and to minimize waste, the tendency has been for producers to give a "usable life from receipt" to the customer, commonly 12-24 months. HOWever, the producers can give this shelf life expectancy only for unopened units,

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stored as recommended by the producer. Accordingly, users are advised to buy fresh CRMs when required and not to store them for a prolonged period. The most recent revision of I S 0 Guide 31(1ggG)includes a recommendation that each certificate include a clear expiration date. This change reflects increasing demands by laboratory quality management procedures for all consumable materials to have a clear use by date. Industries have welcomed this move; and a large user sector has reacted by claiming that when there is a change to an I S 0 Guide, the RM producers should quickly up-date all their certificates. Funding does not always exist to do so, therefore certificates are sometimes up-dated when RMs are re-certified,or new information is added to the certificate. The authors have found that industries are unwilling to understand this approach and also contradict such requirements with the demand for long shelf lives and usage periods. Other industries, especially the highly regulated pharmaceutical industry, believe that CRMs are designed to be taken by the scientific community as of the highest quality on a metrological basis and they should therefore be in perfect compliance with all appropriate rules at all times. Compliance with such demands may force producers to increase production and certification capacities beyond reasonable limits and contribute to dwindling variety in available RMs. 7.1.2.6

Sampling and Preparation o f RMs for Analysis

An important point for achieving results that can be compared to the certified values is the use of the appropriate sample size. The required amount is usually specified by the producer. However, some users take either a smaller amount (sometimes much smaller as required by the analytical technique; see Sections 4.3 and 4.4). and many may even take larger amounts. These users must be aware of two problems: (I) The specified amount is, in many instances, just a piece of guesswork since expensive studies of sampling behavior were not incorporated in the certification process. However, the certification process has established that the recommended amount provides sufficient analyte for a reproducible value with many of the analytical techniques operating at an optimal level. Deviation in sample size may change the optimum and reproducible response of some analytical techniques. ( 2 ) If a different amount is taken, other than which is specified in the certificate, then this has a significant impact on the confidence interval for the certified value in that particular sample. Extrapolation of uncertainty to different sample sizes, in particular uncertainties due to inhomogeneity at smaller sample size, is not possible without extensive sampling studies. Even so, RM producers should support analysis procedures that require different sample sizes by supplying sampling information such as sampling constants; see also Section 4.3.

Correct determination of the sample mass is critical. Usually drying conditions are specified by the producer. The user has to be aware that there is an amazing variety of recommended drying conditions presented in the various certificates. Since the optimum conditions are very dependent on matrix and composition, it is of utmost

7.1 Selection, Use, and Abuse of RMs

importance that the user follows the instructions, even if they are not a standard procedure in the user’s laboratory. The influence of different drying conditions on the uncertainty of the mass determination, as well as on the content of certain analytes is generally unknown; so serious errors can result from a non-validatedsample preparation. Another common error is caused by contaminating the RM during removal of samples. Contamination can be caused by other samples in the laboratory, but also by bacteria, fungi and dust. Once removed for later use, RM aliquots should never, ever, be returned to the original RM container after sampling, but must be discarded. Contamination may be the source of many apparently subtle sources of error, especially in the use of matrix CRMs. Users may wish to exceed the normal precautions taken against contamination of their analytical samples, and only open and weigh out CRMs in a laminar flow hood, or other “clean bench” environment, to assure long-term validity of the RM unit. 7.1.2.7

Sample Characteristics

Discontinuity between the physical form of the sample and reference material used can lead to error. This is another manifestation of the matrix effect, but one which has to be considered when analyzing biological and environmental samples. There is no easy answer to the relationship between particle size and homogeneity. It is a popular assumption that the smaller the particle size the less the degree of heterogeneity. In some cases this may be true but there are a number of considerations. Reducing particle size does nothing to make the individual particles the same, it only makes more of them and increases total surface area, with the probability of higher exchange factors for constituents leaving or entering the sample. (2) The resultant material in many cases is not “flowable”,which means it tends to cake or agglomerate. It is then difficult to get good particle distribution which increases the tendency toward heterogeneity and means mixing of the material before sampling is critical. ( 3 ) The importance of particle size is directly proportional to the sub-sample size recommended by the analytical method. The larger the sub-sample size the larger the acceptable particle size. For sub-sample sizes of Ig or greater a soil sieved through a Imm screen is generally acceptable. Therefore if the sample is relatively coarse, e.g up to zmm particles and the matrix CRM is an uniform sub-micron powder, it may be necessary to use a much larger sample from the material under test than for the CRM. (4) The more a source material is processed the less it behaves and reacts like a typical field sample, and if a real-world contaminated soil is ground to reduce the particle size the heat of frictionlshearing may alter the composition and constituents may volatilize. (I)

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7.1.3 Continuity

Lack of continuity in the supply of a matrix CRM can often be a cause of error: this can be either discontinuity caused by the re-certification of a CRM or advances in analytical technology rendering certified values unreliable. Once a particular batch of a given matrix CRM is sold out, there may be a considerable delay before a replacement is available. Replacements often differ in analyte profile and value, although the producers generally attempt to hold these differences to a minimum. Analysts must be aware of changes in the composition which sometimes may be subtle; at other times, however, they may be significant. For example, a change in the milling technique such as using a zirconia mill will introduce significant levels of Zr and Hf to the sample, possibly interfering with the analytical procedure. Standard solution RMs are normally replaced with an identical product and without a significant break in supply. However, the supplier may have changed the solution matrix in pH or composition to assure better stability. This may cause a significantly different response in the analytical technique used. Evolution of analytical techniques can cause data, once considered to be "state of the art" to be shown to be unreliable. A good example is provided by the work of Houba et al. (1995).who demonstrated that a number of older methods for the determination of trace levels of boron in plant materials were subject to the interference by high levels of copper. This and other evidence suggest that older data, even when presented on a certificate, have to be viewed critically; see also Section 3.2. The analyst must stay aware of developments and be ready to disregard certified values if the date of certificationof the CRM predates the release of new developments and the certification authority concerned cannot confirm that the certified value is good in the light of the new knowledge. 7.1.4 Artifacts

In certain areas, particularly the rapidly developing area of organo-metallicspeciation, concern has been expressed that artifacts may lead to false results. One example are the doubts about the accuracy and suspicion of possible artifact formation of methylmercury (MeHg)during analytical procedures, mainly distillation and alkaline dissolution, which were expressed for the first time at the Conference "Mercury as a Global Pollutant" in 1996(Hintelmann and Evans 1997; Hintelmann et al.1997). Stable isotope dilution ICP-MS was used to study the accuracy of mercury speciation analysis. It was found that, on spiking sediments with relatively high quantities of inorganic mercury (Hg2+in acidic aqueous solution) enriched with 202Hgisotope, an increase of "'MeHg was observed after water vapor distillation, suggesting in situ formation of MeHg from inorganic Hg. This was also proved by conventional analytical techniques, described by Bloom et al. (1997). Although the amount of MeHg formed was very small, varying from about 0.005 % to a maximum of 0.1 % of the spiked Hg2+,it could represent an important proportion of MeHg in samples that normally contain low concentrations of MeHg (a factor up to 1.5 was reported).

7. I Selection, Use, and Abuse of RMs

The controversy was serious enough for the European Commission to finance a Worltshop which was held in Wiesbaden, Germany on 28-29 May 1998. Reports of this work led many laboratories to doubt the value of the CRMs they use for their quality control, but at this stage the findings on artifact formation of MeHg are not considered to give sufficient ground to claim that all MeHg data produced worldwide were overestimated (Quevauviller and Horvat 1999). Even so the experience serves as a salutary warning that assumptions based on experience can so easily be overturned with the arrival of new, more revealing, methodologies. 7.1.5 Data Interpretation Errors

Users expect “certifiedvalues” to be correct - with a probability of 95 % - within the stated uncertainty intervals. They assume, perhaps naively, that all statements of uncertainty are the same. In practice the stated uncertainties may have quite different meanings because they have been based on quite different principles. This issue is discussed in more detail below. But for most users neither the differences nor the consequences of the differences are always evident, or understood. I S 0 Guide 33 (1989)recommends that “CRMs are used on a regular basis to ensure reliable measurements”. In reality, the expression “to ensure reliable measurements” can have a wide range of interpretations, including: “to transfer information on property values” “to assure traceability to unit scales or to standards” “to assess precision and/or trueness of measurement processes” Although the user will require differing types of information from the producer to properly use the CRM for each applications, there is a tendency to provide only a certified value and an uncertainty value, which is generally said to be a 95 % confidence interval, or something similar. The relevance of this was made clear by Jorhem (1998),but it is not always evident from the supplied documentation. One of the most common complaints from the inexperienced user is that the result obtained in the routine laboratory does not fall in the confidence interval. Pauwels (1999) makes considerable reference to this problem, which he calls the ‘‘Jorhem Paradox”. Even though Pauwels goes on to explain this paradox, in doing so he highlights the problem when he states “two results (the certified value and the subsequent laboratory determination) which both claim to contain the most probable mean value of the material with a probability of 95 % do, effectively, not overlap”. How can this situation arise? It is because most certification bodies are not in a position to consider other uncertainty components than those associated with the certification process. The proper application of the I S 0 Guide to the expression of uncertainty in measurement requires that all sources of uncertainty are included. In practical terms this means an “uncertainty budget” has to be developed also by the user. The development of an uncertainty budget, and the consequences for both analysts and producers is described later in Section 7.2.

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Considering the different uses of RMs in the estimation of uncertainty would mean that more routine results overlap the certified value, but it would also make the certified values given by some agencies less useful for the broad user community. So there needs to be a better way of expressing uncertainty, possibly by showing the existing uncertainty and a wider total uncertainty. A form of this approach has long been followed by RT Corporation in the USA. In their certification of soils, sediments and waste materials they give a certified value, a normal confidence interval and a “prediction interval”. A rigorous statistical process is employed, based on that first described by Kadafar (1982),to produce the two intervals: the prediction interval (PI) and the confidence interval (CI). The prediction interval is a wider range than the confidence interval. The analyst should expect results to fall “19 times out of 20” into the prediction interval. In real-world QC procedures, the PI value is of value where Shewhart (1931)charts are used and batch, daily, or weekly QC values are recorded; see Section 4.1. Provided the recorded value falls inside the PI gs % of the time, the method can be considered to be in control. So occasional abnormal results, where the accumulated uncertainty of the analytical procedure cause an outlier value, need no longer cause concern. Pauwels (1999) argues that the certified values of CRMs should be presented in the form of an expanded combined uncertainty according to the I S 0 Guide on the expression of uncertainty in measurement, so that coverage factor should always be clearly mentioned in order to allow an easy recalculation of the combined standard uncertainty. This is needed for uncertainty propagation when the CRM is used for calibration and the I S 0 Guide should be revised accordingly. The use of the expanded uncertainty has been policy in certification by NIST since 1993 (Taylor and Kuyatt 1994). There are a number of other problems relating to the manipulation and interpretation of data that cause difficulty. The most common are: (I) uncertainty about the number of replicate results required for proper comparison of the certified reference value, and (2)the actual analytical result and how gross outlier results should be handled. These issues and how to deal with data that falls outside the confidence limit are reviewed in detail by Walker and Lumley ( ~ g g g who ) , conclude that whilst customer requirements may provide answers the judgement of the analyst must always be the final arbiter in any decision! 7.1.6

Reporting Errors

In his survey of the use of CRMs in food related publications on the subject of trace elements for the years 1990-1996, Jorhem (1998)checked 82 papers published in five international journals. He found that in 42 papers there was no mention of CRM results and assumed that no CRMs were used. He wrote: “Since the importance of incorporating CRMs in the AQA-activities today is well recognized, it is surprising that firstly so many laboratories still do not use CRMs and secondly that scientific journals accept papers describing analytical results without the use of reference materials, as part of the verification of the analytical results”.

7.2 Statistical Consequences in the Assurance of Measurement Compatibility

Of these 4 2 papers, 13 came from countries within the EU, eight from European countries outside the EU, and 12 from North America. He mentioned that the papers dealing with the use of CRMs did not make reference to any user guide such as I S 0 Guide 33 (1989) (see Section 1.2) and further that “the comparison between the found results and the certified means and intervals are often presented in rather vague, or non statistical terms”. The reasons for such vagueness may lie in a combination of factors, the lamentable level of proper understanding of statistics amongst many analysts and, as mentioned above, the inconsistent and complex manner in which many certification bodies use statistics to produce their certified values and the willingness of journals to accept papers that lack proper validation of results and do not describe the proper use of CRMs.

7.2 Statistical Consequences in the Assurance of Measurement Compatibility 7.2.1

Uncertainty

In this Section we aim to make the CRM user aware of the uncertainty budgets that need to be considered with the use of CRMs. Certified values in CRMs are the property values (mass fraction, concentration, or amount of substance) and their uncertainty, the uncertainty being in many instances a specified confidence interval for the certified property. As we discussed before, this uncertainty value is not always a complete uncertainty budget for an analytical process from sampling to production of data. But even when disregarding the subtle differences in the certificates, the way a CRM is used has serious consequences on the uncertainty budget that has to be applied to a user’s result. This is summarized in Table 7.2. These uses may affect accuracy claims as well as traceability claims. It is the user’s obligationto establish comTab. 7.2 uses

Overview on uncertainties (variances for more convenient formalism) with different CRM

Investigated Material

CRM Use

Matrix Match Matrix Related Matrix Relation Inferred Matrix Not Related

Calibration

Matrix Match etc.

Uncertainty of User Result

u,Z

=

urn2+ ( u C R M ) ~

~ , =2urn2+ urn,: + ( u ~ R M ) ~ u,Z = urn2+ n urn,: + ( u ~ R , ) ~

Only useable with truly matrix- U,” = Urnz+ ( UCRM)’ independent procedures Control Measurement u,Z = 2( urn)’+ k(U c R M ) 2 Rapidly Increasing Uncertainties

U,: Combined Uncertainty Urn: Measurement Uncertainty Urn,,: Uncertainty in Materials Properties (n= potential multiplier for differences) k Coverage factor to be considered for normally fewer measurements of CRM

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plete uncertainty budgets and statistical control for each user component of the analytical process and be certain that the recipient of the data understands the consequences of the stated uncertainty.The authors experienceis that this is often not the case. From the formulae presented it is clearly evident that the uncertainty of the CRM may become a strong component in the user’s combined uncertainty, but it is not the only component. To this effect, the user’s result will always have a larger uncertainty than the uncertainty stated for the CRM. Only a direct matrix match of sample and CRM, and the CRM’s use as a direct calibrant will allow the user to demonstrate accuracy and subsequently traceability close to the uncertainties established during the CRM certification ( note: matrixmatching may not be necessary with matrix-independent techniques). This reality places a significant burden on the CRM producers, since large uncertainties in the certified values may degrade the perceived value of the CRM. On most occasions CRMs are used as Quality Control materials, rather than as “calibrations”. As outlined above, this common application adds significantly to the user’s uncertainty budget, since at a minimum it is necessary to consider at least two independent measurement events (Urn), so increasing the combined uncertainty of the results. Again this process rapidly increases the combined uncertainty with increasing complexity of the analytical system; and so the usefulness of a control analysis may be downgraded when a correct uncertainty budget is formulated. 7.2.2

Indicative Approach to Quantifying Uncertainty

Mention has already been made of the EPA recommended use of both confidence interval and prediction interval. However, many users and their customers may be satisfied by some simplistic comparisons. Two methods for comparing experimental results with certified values are presented here. The users are referred to statistical handbooks for comparing results of sets of determinations such as usingftest, etc. Users may conclude that the analyhcal procedure gives correct results if the difference between the analyst’s experimental mean(s) (xe)and the certified value(s) (xc) is less than the combined uncertainty (IS standard deviation) of the experimental and certified means (Equation. 7-I), with s, and s, representing the estimates of the respective standard deviations.

In practice this evaluation is difficult to apply because the standard deviation of the certified value is usually neither stated in the certificate nor can it be derived from the quoted confidence interval. Another assessment is based on the presumption that there is no significant difference between the certified values and the experimental results when the confidence intervals of the two values overlap. Using this method, the analyst needs a

7.3 Traceability

data set of n independent measurements from which a confidence interval must be calculated (Equation 7-2) with t,05being the student’s t value at the 95 % probability level with (n-I) degrees of freedom.

If this range overlaps the stated confidence interval of the certified value, then the analytical procedure may be assumed to be under satisfactory statistical control. Discussion about the accepted degree of overlap may inevitably occur; so when reporting results it is good practice that the certified value of the CRM should be within the experimentallydetermined confidence interval. The disadvantage of both tests is that the user obtains only a small set of test data compared to the certification measurements that lead to the certified value and its uncertainty. But this is a fact of life in the world of the routine, rather than research, laboratory . Therefore the user of the data is always forced to compare differently obtained values and uncertainties.

7.3

Traceability 7.3.1

Definition

To the users of CRMs, the concept of “traceability”is very closely related to the statistical considerations in the measurement process and the quality of the measurements in the users’ laboratory. Traceability is defined in the international vocabulary on metrology (VIM) as: “The property of a result of a measurement or standard whereby it can be related to stated reference, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties” (VIM 1993). Traceability is at the core of the answers to one of the key questions for analytical determinations: How reliable is the data? The answers can be rather complex and range from qualitative statements, such as: “the specificity of a certain chemical measurement for the chemical of interest” to quantitative statements, such as: “the accuracy and uncertainty associated with measured value of the mass fraction or amount of substance determined”.

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It is in the declaration of the latter where a certain quality of the measured value can be described with traceability. This relation to measurement quality has been expressed in the literature for some time: “Traceability to designated standards (national, international, or well-characterized reference standards based upon fundamental constants of nature) is an attribute of some measurements. Measurements have traceability to the designated standards if and only if scientifically rigorous evidence is produced on a continuing basis to show that the measurement process i s producing measurement results (data) for which the total measurement uncertainty relative to a national or otherwise designated standard is quantified (Belanger 1980). Although there are many similar definitions for traceability, the essence of traceability is an unbroken pathway to the definition of the accepted units used to express the measurement result and a measurement process in which quality assurance is an integral component. Considering the proper use of a CRM in a measurement process, the user may wish for a recipe to establish traceability, so that the data produced can be claimed to be of the highest quality, or to satisfy regulatory requirements, contractual agreements, and to comply with the conditions of written standards. In view of these issues, it should be noted that there i s a component of “legal traceability” that expands the obligations of a measurement laboratory beyond the demonstration of “good laboratory practice” that commonly support a laboratory’s claims for accuracy in their measurements. Regardless of these extensions, in this section we review some uses of CRMs that can support claims on the traceability of an analytical chemist’s result. But in every instance, the user of a CRM must understand, as we have explained above, that the uncertainty of the CRM used in the assessment will expand the uncertainty of the result. 7.3.2 Practical Aspects

It should be recognized that, in some cases, it is not difficult to set up a traceable measurement system. The best examples of this are in physical metrology where traceability i s often based on “direct” measurements of the SI units. There i s also general agreement that a similar SI link is highly desirable in the case of chemical measurements, but, for a variety of reasons, direct “chemical”traceability i s difficult to achieve in most of the analytical chemistry applications. Only a very few analytical chemistry procedures exhibit a direct measurement capability that allows the set-up of a traceable measurement pathway such as in physical metrology measurements; most of these procedures have been accepted as primary methods if carried out under certain constraints (CCQM 1998). The majority of analytical chemistry procedures, especially those in commercial laboratories, depend on a matrix of controllable variables that include sampling,

7.3 Traceability

calibration, chemical yields and instrumental efficiencies, specificity of chemical and/or instrument responses, and the material and/or matrix in which the analysis is conducted. Therefore, demonstration of traceability for analytical chemistry procedures requires a complex approach in the determination and validation of the aforementioned matrix of variables. Whilst CRMs can be used in many of these assessments and the degree to which traceability will be demonstrated may correspond with the degree of demonstrated accuracy for a certain measurement, or may be only inferred through comparisons and established knowledge of the processes involved. Nevertheless, there are a few elementary pathways through which the use of a CRM may establish traceability to the declared reference, e.g. the certified mass fraction of a chemical in the CRM. The possible pathways include the use of a CRM as a calibrant in the analytical process, the comparison of measurement results with results obtained in measuring a CRM under essentially equal conditions, or the quality of performance of the analytical procedure on a number of CRMs over time. In discussing these pathways and considering the broad spectrum of analytical chemistry applications in contrast to the limited supply of CRMs, one has to keep in mind that the unbroken chain of comparisons may be literally demonstrated in only a few instances and that for a long time to come data are accepted by inferred quality, including traceability. The use of a CRM as calibrant in an analytical procedure is probably the simplest way to comply with the VIM definition. CRM producers may give guidance to the users for the proper use of the CRM, e.g. the certificate for SRM@ 2031a, Metal on Fused Silica Filters for Spectrophotometry, states: “To demonstrate that a user’s measurements are traceable within acceptable limits to the accuracy transferred by SRM@ 2031a, the user must first determine the required tolerances or acceptable uncertainty for the application in question. It is recommended that a number of replicate measurements be made for each filter and wavelength, with removal and replacement of the filter between replicate measurements. The user should then compare each mean value and the user-defined tolerance with the NIST certified value and expanded uncertainty (given in the certificate). An acceptable level of agreement between a user’s measurement and the certified value is demonstrated if any part of the range defined by the NIST certified value and its expanded uncertainty overlaps any part of the user’s tolerance band defined by the measured mean and the user-defined level of acceptable uncertainty” (NIST 1994; SRMP 1997). In a different example, traceability in the amount-of-substanceanalysis of natural potassium, thorium, and uranium by the method of passive gamma-ray spectrometry was demonstrated by Nir-El (1997). For an absolute quantitative determination, accurate values of two parameters were required: (I) the emission probability of a gamma-ray in the decay of the respective indicator radionuclides, and (2)the detection efficiency of that gamma-ray. This work employed a number of CRMs in the critical calibration of the detection efficiency of the gamma-ray spectrometer and the establishment of precise emission probabilities. The latter results compared well with literature values and provided smaller uncertainties for several gamma-rays that were critical for the traceability claim. The amount-of-substance analytical 235U,and 238U results of the long lived naturally occurring radionuclides 40K,232Th,

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I were shown to be traceable to radioactivity 7 Proper Usage of Reference Materials

CRMs. It must be noted, however, that in many instances the known nuclear and atomic parameters may have substantial uncertainties and, in most cases, a CRM will provide only a single point calibration, whose extension to a broader bandwidth of mass fractions or concentration levels may be erroneous. In their broadest application, CRMs are used as “controls” to verify in a direct comparison the accuracy of the results of a particular measurement; parallel with this verification, traceability may be demonstrated. Under conditions demonstrated to be equal for sample and CRM, agreement of results, e.g. as defined above, is proof. Since such possibilities for a direct comparison between samples and a CRM are rare, the user’s claims for accuracy and traceability have to be made by inference. Naturally, the use of several CRMs of similar matrix but different analyte content will strengthen the user’s inference. Even so, the user still has to assess and account for all uncertainties in this comparison of results. These uncertainty calculations must include beyond the common analytical uncertainty budget: (I) a component that reflects material matrix effects, (2) a component that reflects differences in the amount of substance determined, ( 3 ) the uncertainty of the certified or reference value(s) used, and 4) the uncertainty of the comparison itself. All this information certainly supports the assertion of accuracy in relation to the CRM. However, the requirement of the “unbroken chain of comparisons”will not be formally fulfilled.

7.4

Conclusions

We have shown that CRMs can have a positive influence and help identify sources of error when used as the producer intended and when the user understands the limitations of the CRM in the particular application. If users are to benefit from the implementation and/or verification of traceability in analytical chemistry the unbroken pathway of references must be kept short. The uncertainty of the references (CRMs) used may significantly widen the uncertainty a user must attach to the result of his measurement when addressing accuracy and traceability through comparison with a CRM. These comparisons should be only considered in a first or second level step as to keep the uncertainties of the results within limits fit for the purpose. The producers of CRMs must keep their uncertainties sufficiently small to allow introduction of the CRM at different points in the analytical pathway, without limiting the usefulness of results through unduly expanded uncertainties. The producers need to establish, in general terms or for specific applications of CRMs, clear instructions for the user on how to establish traceability to the stated reference and (implied in the use of CRMs of national metrology organizations) the SI. But the authors’ experience is that, as the workload in commercial analytical laboratories increases (as analytical laboratories seek to demonstrate their competence and quality to an increasingly discriminating

7.5 References I 2 5 3

marketplace) the pressures on analysts to produce product increases. One result is that, in an attempt to meet pressure from both management and customers, corners are cut. CRMs are used inappropriately and without fully understanding the various traceability and uncertainty issues, leading to: Accumulation of unstated error = incorrect data = useless product = unhappy customers!

7.5

References BELANCERBC (1980)Traceabilty an evolving concept. Standardization News 822-28. BLOOMNS, COLMAN JA and BARBERL (1997)Artifact formation of methyl mercury during aqueous distillation and alternative techniques for the extraction of methylmercury from environmental samples. Fresenius J Anal Chem 358371377, CCQM (1998) Consultative Committee on the Quantity of Material, Bureau International des Poids et Mesures (BIPM),Minutes of Fifth Meeting, S k e s , France. CHEUNG YH and WONGMH (1997) Depuration and bioaccumulation of heavy metals by clams from To10 harbour, Hongkong. Toxic01 Environ Chem 58:103-116. ERP P VAN, HOUBAV and BEUSICHEMM VAN (1998) One Hundredth Molar Calcium Chloride Extraction Procedure Part I. Commun Soil Sci Plant Anal 29:1603-16~3. HINTELMANN H and EVANSRD (1997)Application of stable isotopes in environmental tracer studies - measurement of monomethylmercury (CH,Hg+) by isotope dilution ICP-MS and detection of species transformation. Fresenius J Anal Chem 358:378-385. HINTELMANN H, FALTER R, ILGEN G and EVANSRD (1997) Determination of artifactual formation of monomethylmercury (CH,Hgf) in environmental samples using stable Hg2+isotopes with ICP-MS detection: calculation of contents applying species specific isotope addition. Fresenius J Anal Chem 358:363-370. HOUBA V, UITTENBOGAARD J and PELLEN P (1995)Wageningen Evaluating Programmes for Analytical Laboratories (WEPAL)Organisation and Purpose. Commun Soil Sci Plant Anal 27:421431. HOUBA V, NOVOZAMSKY I and LEE J VAN D E R (1995) Influence of Storage of Plant Samples on the Chemical Composition. Sci Total Environ 176:73-79. I S 0 9000 (1987)Quality management and quality assurance standards - Guidelines for selection and use. I S 0 Publications, Casa Postale $5, CH 1211 Geneva 20, Switzerland. I S 0 Guide 31 (1996) Contents of certificates of reference materials, and Document N 382, revision of I S 0 GUIDE 31. I S 0 Publications, Casa Postale 56, CH 1211 Geneva 20, Switzerland. I S 0 Guide 33 (1989) Uses of certified reference materials (under revision). I S 0 Publications, Casa Postale 56, CH 1211 Geneva 20, Switzerland. J E N K S PJ (1995)Editorial in Fresenius J Anal Chem 352:3-4. JORHEM L (1998) Non-use and misinterpretation of CRMs. Can the situation be improved? Fresenius J Anal Chem 360:370-373. KADAFARK (1982) A bi-weight approach to the one sample problem. J Am Sta Assn 77 No 378416- 424. KURFURSTU, ed. (1998) Solid sample analysis. direct and slurry sampling using GF-AAS and ETV-ICP. Springer Berlin Heidelberg New York. MAASKANT J, BOEKHOLT A, J E N K S P and RUCINSKIR (1998)An international interlaboratory study for the production of a sewage sludge certified reference material for routine use in inorganic quality control. Fresenius J Anal Chem 360:406-409.

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MACICEY EA, DEMIRALPR, FITZPATRICK IW, PORTER BJ, WISESA, BECICERPR and GREENBERG RR

(1999) Quality assurance in analysis of cryogenically stored liver tissue specimens from the NJST National Biomonitoring Specimen Bank (NBSB).Sci Total Environ 226:165-176. MAIER E, GRIEPINK B, MUNTAUH and VERCOUTERE J< (1993) Report EUR 15283 EN. European Commission, Luxembourg. MINNICHM (1993) Behavior and determination of volatile organic compounds in soil: A literature review. USEPA, Las Vegas, NV, EPA/6oo/R-93/140 U.S. Government Printing Office, Washington, DC NIR-EL Y (1997) Traceability in the amount-ofisubstance analysis of natural potassium, thorium and uranium by the method of passive gamma-ray spectrometry. Accred Qua1 Assur 2:193198. NJST (1994) Guidelines for the expression of uncertainties o f NJST measurement results. NJST Tech. Note 1297, Gaithersburg, MD, USA. J (1999) How to use matrix certified reference materials? Examples of materials proPAUWELS M, eds. The use of duced by JRMM’s reference materials unit. In: FAJGELJA and PARKANY matrix reference materials in environmental analytical processes, pp 31-45. Royal Society o f Chemistry, Cambridge. M (1999) Artifact formation of methylmercury in sediments. LetQUEVAUVILLER PH and HORVAT ter to the Editor. Anal Chem ~ I : I ~ ~ A - I ~ G A . M, GRANERO S, BELL&M, LLOBETJ M and DOMINGOJ L (1996) Levels of metals SCHUHMACHER in soils and vegetation in the vicinity of a municipal solid waste incinerator. Toxic01 Environ Chem 56:119-132. SCHUMACHER BA and WARDSE (1997) Quantitation reference compounds and VOC recoveries from soils by purge-and-trap GC/MS. Environ Sci Techno1 31:2287-2291. SHEWHART W (1931) Economic Control of Quality of Manufactured Products. Van Nostrand, New York. M, BASTOSM and FERRERIA M (1994) Determination of Total Chromium and Chromium SOARES (IV) in Animal Feeds by Electrothermal AAS. J Anal Atom Spectrom 9:1zGg-1272. SRMP (1997) Certificate Standard Reference Material@ 2031a, SRM@ Program, Gaithersburg, MD, USA. BN and KUYATI CE (1994) Guidelines for evaluating and expressing the uncertainty of TAYLOR NIST measurement results. NIST Technical Note 1297, U.S. Government Printing Ofice, Washington, DC. JK (1995) Handbook for SRM Users. NBS Special Publication 260-100, US. Department TAYLOR of Commerce. VIM (1993) International vocabulary of basic and general terms in metrology 2nd edition. ISO, Geneva, Switzerland R, LUMLEYJ (1999) Pitfalls in terminology and use of reference materials. Trends Anal WALKER Chem 18:594-616. RR, STONE SF, SULLIVANTM (1988) Long-term stability of the elemental ZEISLER R, GREENBERG composition of biological materials. Fresenius 2 Anal Chem jp:G1~-615.

7.6

Further Reading BRM and BERM special issues of Fresenius J Anal Chem (availableissues: BRM-2, 1987; BRM-3,

1988; BERM-4, 1990; BERM-5, 1993; BERM-6, 199s and BERM-7, 1998; for an overview of these symposia see Chapter 8. CSUROS M (1997) Environmental Sampling and Analysis, Laboratory Manual. CRC Press. DOERFFEL I< (1994) Assuring trueness of analytical results. Fresenius J Anal Chem 348x83-184

7. G Further Reading

GUNZLER H (1995) Accreditation and Quality Assurance in Analytical Chemistry. Springer, Germany. ISO/REMCO (1997)The role of reference materials in achieving quality in analytical chemistry. I S 0 Publications, Casa Postale 56, CH 1211 Geneva 2 0 , Switzerland KOHL H (1994)Qualitatsmanagement im Labor. Springer, Germany; LGC - VAM Publications: (I) The Fitness for Purpose of Analytical Methods, A Laboratory Guide to Method Validation and Related Topics, ( 2 ) Practical Statistics for the Analytical Scientist: A Bench Guide By TJ Farrant, ( 3 ) Trace Analysis: A structured Approach to Obtaining Reliable Results By E Pritchard, (4) Quantifying Uncertainty in Analytical Measurement, and (5) Quality in the Analytical Chemistry Laboratory. LGC/RSC Publications, London, England. MESLEYRJ et al. (1991)Analytical Quality Assurance - A Review. Analyst 116:975-990. MILLERJC and MILLER J N (1993) Statistics for Analytical Chemistry, 3rd edition. Ellis Honvood Prentice Hall Series in Analytical Chemistry, Prentice Hall. TAYLOR J K (1985)Principles of Quality Assurance of Chemical Measurements. NBS (now NIST) U.S. Department of Commerce. THOMPSON M (1997) Comparability and Traceability in Analytical Measurements and Reference Materials. Analyst I ~ Z : I Z O I - I ~ O ~ . YOUDENWJ (1991)Experimentation and Measurement. NIST Special Publication 672, Reprint of 1961,U.S. Department of Commerce.

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Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

8

Availability and Sources o f Information Peter] Jenks and Harry Klich Introduction

In the first Chapter we noted the increasing demand for reliable chemical analyses; and this theme is discussed in terms of the future need for RMs in Chapter 9. The need for accurate results, essential for reliable decisions in many fields, has been well argued by several of the contributors to this book. It is accepted that reliable analysis plays a decisive role in many important areas, including worker protection, medical and forensic investigations. Furthermore, reliable measurements are essential for the enforcement of many Governmental regulations. The structure of the 5th Framework ofthe EU SMT Program (whichwill fund many metrology related activities, including the production of CRMs between 2000 and 2005) reflects this belief. Reported data should achieve a clear, purpose-oriented level of accuracy and precision, especially when data produced by several laboratories often needs to be compared as part of a decision process. It has become accepted by more and more analysts that to overcome differences between national standards and specifications and measurement procedures or to make Total Quality Management (TQM) an achievable reality, freely available and internationally agreed points of reference are needed. Therefore RMs and CRMs must be easily available: indeed the availability of reference materials has been described as an issue of “strategic importance” to the EU (Maier et al. 1997). Simply producing reference materials is not enough: they have to be used properly. Chapter 7 reviewed the proper use of RMs, but unless RMs and CRMs can be easily obtained and moved from one country to another without delays, duty or unnecessary controls, their use will be inhibited. Likewise, information about the materials that are available, their use, and their application should be freely available if analysts are to make informed decisions about the selection and use of a RM or CRM. It has already been made clear in previous Chapters that the correct choice of reference material for a particular application requires not only information about the RM, which is relatively easily available, but also information about the uses and applications of the reference material. Unfortunately,neither the availability of information about the reference materials and their uses nor their free movement across national borders are at all satisfactory. In this Chapter we review the main sources of information about reference materials and their use, and we direct the user to reliable sources of information about

8.7 Printed Publications

not only the RMs and the producers, but also the reported uses. Increasingly the Internet is becoming a vital source of information, and access to a web browser equipped PC is now as important as access to a library, for most analysts. Knowing where to obtain a CRM is not enough, knowing how to obtain it is also important. We describe some of the difficulties associated with obtaining RMs and CRMs from overseas, and argue the case for using specialist suppliers with local offices in many countries, such as LGC and Promochem, to facilitate supply.

8.1 Printed Publications 8.1.1 Catalogs, Lists, and Directories

For many years, the main source of information about reference materials was limited to catalogues issued by the various national metrology institutes that produced and certified RMs. Indications that new materials existed and could be used was gleaned from posters and papers given at meetings, or printed in the literature. In the mid-1970’s, the International Union of Pure and Applied Chemistry (IUPAC) produced the first “Catalogue of Reference Materials” (IUPAC 1976). As interest in RMs grew, various organizations started to consider how information from the many diverse sources could be brought together in a structured manner. The first, and so far the only regularly revised source, was the COMAR Database, which is described in more detail later in this Chapter. From the mid-~@o’s,a number of initiatives was made to produce reference publications describing the current state of RM availability, the activities of the producers, and their future plans. Whilst the documents themselves were useful, they were generally out of date by the time they were published, were not regularly updated and, with one or two exceptions, have not endured. None was completely comprehensive, with the IAEA and Cantillo’s work focused on environmental analysis. (IAEA 1985, 1990, 1996; Delve and Keune 1992; Rasberry 1994, 1996; Cantillo 1995). Rasberry’s surveys, produced for ISO/REMCO, identified more than zoo organizations that have, at some time, been involved with the production and certification of reference materials. Later research suggested that there may be more than 300 “producers”worldwide. The most active of the organizations are listed in Table 8.1, together with mailing addresses. For up to date web addresses, use a search engine or visit the BAM (Bundesanstalt fur Materialforschung und priifung, Berlin, Germany) database, which is regularly updated; for website see Table 8.4 below. The diversity of suppliers and the difficulty of maintaining up to date lists made the maintenance of print directories difficult and expensive. Most are now either out of print or have migrated to database format. Nevertheless a review of the older directories is interesting, and they should be retained as they provide valuable background reading.

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Major producers a n d suppliers o f reference materials for inorganic and organic chemical composition quality control. The table lists in alphabetical order the popular names, or abbreviations, which may not be the full official title

Tab. 8.1

Name or Abbreviation

Address

Agriculture Canada

Dr. M. Ihnat, Pacific Agri-Food Research Centre, Agriculture and AgnFood Canada, Summerland, BC VOH 120,Canada (see NIST)

A1 Micltiewicza Metals Faculty of Physics and Nuclear Techniques, University of Mining and Metallurgy, A1 Micltiewicza30, 30.059 Krakow, Poland ARCF

Food Research Institute, Laboratory of Food Chemistry, Agricultural Research Centre of Finland, SF-31600 Jokioinen, Finland

BCR

Management of Reference Materials Unit (MRM),Institute for Reference Materials (IRMM), Retieseweg, B-2440 Geel, Belgium

CALNRI

Central Analyhcal Laboratory, Nuclear Research Institute, CZ-25068 Rez near Prague, Czech Republic

CANMET

Canadian Certified Reference Materials Project, Canada Centre for Mineral and Energy Technology,Natural Resources Canada, 555 Both Street, Ottawa, ON KIA OGI, Canada

CSRM

Pb-Anal, Garbiarslta 2, 040 01Kosice, Slovakia

CZIM

Chemical Measurement Department, Slovak Institute of Metrology, Karloveslta 63,842 55 Bratislava, Slovakia

EPA

Environmental Monitoring Systems Laboratory,Quality Assurance Research Division, US Environmental Protection Agency, Cincinnati, OH 45268, USA

GBW

Ofice of CRMs, National Research Centre for Certified Reference Materials, No. 18 Bei San Huan Dong Lu, Hepingjie, 100013 Beijing, China

IAEA

Analytical Quality Control Services, International Atomic Energy Agency, P.O. Box 100,A-1400 Vienna, Austria

INCT

Department of Analytical Chemistq Institute of Nuclear Chemistry and Technology,d. Dorodna 16, 03-195 Warszawa, Poland

LIVSVER

Chemistry Division 2 , Swedish National Food Administration, P.O. Box 622, S-75126 Uppsala, Sweden

LGC

Reference Materials, LGC (Teddington) Ltd., Queens Road, Teddington, Middlesex TWII OLY, United Kingdom

NIES

Division of Environmental Chemistry, National Institute for Environmental Studies, 16-2 Onagowa, Tsnkuba, Ibaraki 305, Japan

NIST

Standard Reference Materials Program, National Institute of Standards and Technology, Room 204, Building 202, Gaithersburg, MD 20899, USA

NRCC

Institute for National Measurements Standards, National Research Council Canada, Ottawa, ON KIA oRG, Canada and Marine Analytical Chemistry Standards Program, Institute for Marine Biosciences, National Research Council, Canada, 1411 Oxford Street, Halifax, NS BjH 321, Canada

8.1 Printed Publications Tab. 8.1

(Continued)

Name or Abbreviation

Address

Promochem

Promochem GmbH, P.O. Box 10 09 55,46469 Wesel, Germany

RTC

R.T. Corporation, P.O. Box 1346, Soldier Springs Road, Laramie, WY 82070, USA

SABS

South African Bureau of Standards; I Dr. Lategan Road, Groenldoof, Pretoria; Postal Address: Private Bag X191, Pretoria 0001,RSA

8.1.2 Journals

A number of scientific journals regularly publish papers reviewing the state of the

art in the RM business as well as original contributions on certification,inter-laboratory comparisons, and on RM/CRM applications. Table 8.2 lists the most popular and widely cited. The most prolific journals are Fresenius Journal, JAAS, Science of the Total Environment and Water, Air, Soil Pollution, all with around or even more than 50 papers mentioning reference materials from 1998 and 1999. Of these, Fresenius Journal led in 1998, with more than 80 papers. This was partly because it traditionally publishes, so far in six special issues, a series of papers presented at the International Biological and Environmental Reference Materials Symposia (BERM) series. The role and contribution of the BERM series of meetings is reviewed below. Reviewing the published literature can be most fmstrating. Jorhem (1998) reviewed 82 papers in five journals and found that in 42 of them, where a RM or CRM might have been expected to be used, there was no mention. Since then the editors of this book have conducted a review of all the papers published in most of the journals shown in Table 8.2. The findings, which were reported at the BERM meeting held in Bethesda in September 2000, showed little improvement. Some journal editors and to a much greater extent many, possibly most, authors of analyhcal and research papers still neglect the importance of correct reporting and use of reference materials. Only in about 50 % of the abstracts of the surveyed papers is the use of the CRMs described in these papers mentioned. Even in the abstracts of some highly respected journals the editors (certainlyalso the reviewers) and authors appear not to consider it worthwhile mentioning that RMs were used. This is of considerable importance for interested persons, as mainly just the abstracts of papers are easily available in electronic media. It may be concluded that many authors are convinced that it is sufficient to report the use of CRMs in an incomplete form and without giving any details of results, and not reporting the type and producer of CRMs used. Not infrequently it could be noted that CRMs from one producer were attributed to another, showing that the author(s) and reviewer really do not know from which source the RMs applied originated. Others use materials that do not match the samples analyzed or, even worse, do not see the need to use any CRM despite the availability of suitable and not too expensive materials to check the reliability of their work. From all this it is clear that,

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Main journals for papers concerning RMs and CRMs and with actual internet data, if

available Journal

Accreditation and Quality Assurance

Pub/isher/lnternet Access

Springer, Heidelberg, Germany http://link.springer.de Analusis EDP Sciences,Wiley-VCH; http://www.edpsciences.com Analyst (includes from 2000 Analytical Royal SOC.Chem.; http://www.rsc.org/analyst Communications) Elsevier Science; Analytica Chimica Acta http://www.elsevier.nl/locate/ContentsDirect Analytical Chemistry Am. Chem. SOC.;http://pubs.acs.org/ac Analytical Letters" Marcel Dekker Inc., New York, Basel; www.de1dter.com Applied Organometallic Chemistry;? John Wiley & Sons Limited www.interscience.wiley.com The Perkin Elmer Corporation, Norwalk CT, USA Atomic Spectroscopy Elsevier Science B.V.; Chemosphere http://www.elsevier.nl/locate/Contents/Direct Environmental Science and Technology Am. Chem. SOC.;http://pubs.acs.org/EST European Food Research and Springer, Heidelberg, Germany Technology" (formerly 2 http://link.springer.de LebensmUntersForsch A) Fresenius Journal of Analytical Springer, Heidelberg, Germany; http://link.springer.de Chemistry Geostandards Newsletter Geostandards, CPRG, B.P. 20,54501Vandoeuvre-16sNancy, Cedex, France; http:/ /www.crpg.cnrs-nancy.fr/Geostandards at http://www.crpg.cnrs-nancy.fr Gordon and Breach; http://www.gbhap.com Intern Journal of Environmental Analytical Chemistry$< Royal SOC.Chem.; http://www.rsc.org/jaas Journal of Analytical. Atomic Spectrometry (JAAS) AOAC International, Gaithersburg; Journal of AOAC International http://www.aoac.org Elsevier Science B.V.; Journal of Chromatography A http://www.elsevier.nl/locate/ContentsDirect (Chemistry) Journal of Radioanalytical and Nuclear Chemistry Springer, Vienna, Austria; http://www.springer.at Mikrochimica Acta Elsevier Science B.V. Table of contents in ESTOCThe Science of the Total Environment Elsevier Science Tables of Contents Service, URL addresses: http://www.elsevier.nl/locate/scitotenvor http://www.elsevier.com/locate/scitotenv Elsevier Science B.V.; http://www.elsevier.nl/locate/ SpectrochimicaActa B estoc or http://www.elsevier.com/locate/estoc Talanta Elsevier Science B.V.; www.elsevier.nl/locate/ContentsDirect

8.7 Printed Publications Tab. 8.2

Journal

(Continued) Pub/isher/lnternet Access

Toxicological Environmental Chemistry" Gordon and Breach; http://www.gbhap.com Trends in Analytical Chemistry (trac) Elsevier Science B.V.; http://www.elsevier.nl/locate/tac Water Air and Soil Pollution Kluwer Academic Publishers;

http://www.wl
5~

Journals marked with an asterisk usually contain around or less than five papers annually dealing with RMs

even in cases where correct data were given for type and producer of the applied materials, the proper use and statistical evaluation might be questionable. As examples for the many the following examples are offered: Error 1 CRMs completely or partly not identical with the matrix to be analyzed: Bermejo-Barrera et al. (1999)studied enzymatic hydrolysis procedures using pronase E as sample pretreatment for multi-element - Cu, Fe, Mg, Zn, Ag, As, Cd, and Pb determination in biological materials, mussel samples and human hair.

Comment: CRMs NRCC CRMs DOLT-I (dogfish liver) and DORM-I (dogfish muscle) used, but no use of mussel or hair CRMs, despite availability of both. Acar et al. (1999) determined Bi, In and Pb in geological and seawater samples by ET-AAS Comment :Wismuterz I1 (AGV-I) and Blei JG-Iawere used as certified geological samples, but no CRM used for the (more difficult) water analysis Error 2 The use of CRMs is mentioned, but the type of CRMs not mentioned, nor the results of their analysis. Hellou et al. (1999) determined PCBs, DDTs, chlordanes, dibenzo-p-dioxins(ds), dibenzofurans (Fs), PAHs and sulphur heterocycles in marine harbor sediments.

Comment: Use of spiked sediment and RMs but no details of the used RMs given. Marlholm and Bennett (1998)determined mercury accumulated in transplanted lichens downwind of a C1-alkali plant. Comment: A certified marine sediment reference material mentioned (NIST, NRCC?) But no other details given on the CRM used.

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8 Availability and Sources oflnformation 8.2 Electronic Sources and the Internet

There can be no doubt that the adoption of the “world wide web” as a means of distributing information about their organization, services and products by virtually all the agencies and organizations involved in the production and distribution of reference materials (and by the journals and publishers associated with analytical chemistry) has revolutionized the ease with which information can be obtained. Since mid-Igg8 anyone with access to a reasonably fast Internet connection can obtain, in a few hours, a mass of data about reference materials - if they know where to look. As with all “e-activity”there is quantity, but finding quality can be hard work. Using a popular search engine, “google.com”and entering “certified reference materials” produced GI 800 hits, which would take some sorting. Even more specialized topics produced many hits, with “Dioxin” finding 14 zoo and even TBTO finding 120. The posting of product and certificate data on a producers web site is but a first step. Through the work of REMCO and others, it has been realized that there is a need to organize this mass of data into a usable form, so that analysts can quickly find the information they need about both a RM and its applications. It is anticipated that by about zooz there will be available searchable databases, either on line or as a CD-ROM, that will open the door to the needed information, and act as a gateway to the mass of data provided by the producers and in the literature. When selecting a RM it is important that the user understands the mission of the producer, the way in which the matrix material has been produced and the analytical data collected. Information about the main CRM and RM producers together with a brief overview of their activities and lists their “url” or e-mail address can be also found at the BAM website; see Table 8.4 below. 8.2.1 The “COMAR” Database

The first reference material data base was established in 1984. Using what was then “state of the art” software [dBASE 3 and Clipper!] the “Code of Reference Materials” (COMAR),was developed. By the release of the last edition at the beginning of 1999 it contained information on more than 10 ooo RMs from over 200 producers in over 20 countries. The COMAR database is distributed only as a PC DOS-based program that provides the name and general description of the RM, address of the producer, form of the material, the properties certified, their values and the field of application. Although very useful for the experienced researcher COMAR is not completely suitable for general use. Efficient use of the search engine needs considerable experience and background knowledge about the producers and the method of coding. This is because of the way data is compiled, through a network of national coding centers and the limited amount of information that can be retrieved for each entry. Also the COMAR database is beginning to show its age, especially as DOS-based

CRM Producer

CRM Producer

CRM Producer

CRM Producer

4 , COMAR National Coding Center

COMAR National Coding Center

International Database

Other sources

CRM Producer

Producer Database

COMAR Core Data

Homepage

Certificates

COMAR Hyperlinks

Fig. 8.2 Diagram showing how additional sources of informa. tion can be connected by hyperlinks to the intended COMAR structure.

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opments have seen responsibility for the development of the database move to BAM (Bundesanstalt fur Materialforschungund priifung) in Berlin, Germany. Substantial further development is planned; for COMAR website see Table 8.4. The next generation of COMAR will be an Internet-based Information center for CRMs which uses the well established structure: see Figure 8.1. Additional sources of information, e.g. literature can be connected by hyperlinks; see Figure 8.2. It is intended that the database will include details of literature citations, making the database a truly useful source of information about the use and availability of reference materials. 8.2.2 The IAEA Database

The International Atomic Energy Agency (IAEA) maintains a database on internationally available certified reference materials. The information presented refers to reference materials for trace elements, organic constituents, radionuclides, stable isotopes, inorganic compounds, ionic compounds, extractable compounds and organometallic compounds of natural origin. Originally issued in print volumes, as described above, since 1998 the database has been comprehensively revised and expanded, now containing eight matrix classifications and 11 measurand groups in anticipation of the release of a searchable database that can be accessed through the Internet. The revised database holds over 23 ooo analyte values for GGo measurands and 1670 reference materials produced by $5 different producers. from 22 countries. The database is restricted to natural matrix materials (i.e. made from naturally occurring materials, excluding calibration standards manufactured from pure chemicals). Information has been extracted from the relevant certificates of analysis, information sheets, and other reports provided by the reference material producers. As a general rule, the authors have only included in the compilation reference materials for which a certificate of analysis or similar documentation is on file. Information included in the survey is on values for measurands determined in reference materials, producers, suppliers, the cost of the materials, the unit size supplied, and the recommended minimum weight of material for analysis, if available. The new searchable database has been designed to help analysts to select reference materials for quality assurance purposes that match as closely as possible, with respect to matrix type and concentrations of the measurands of interest and their samples to be analyzed; see Table 8.3. The measurands of the matrices listed in Table 8.3 are divided into 11 groups: Trace and macro elements Inorganic compounds (e.g. oxides, chlorides) Extractable constituents (e.g. aqua regia, EDTA, acid) Radionuclides

8.2 Electronic Sources and the internet

Stable isotopes Organo-metallicconstituents Agrochemical contaminants Aliphatic and aromatic hydrocarbons Chlorinated hydrocarbons Organic compounds other than hydrocarbons and chlorinated substances Veterinary drugs and other toxins

Tab. 8.3

Matrix classification of the IAEA database

Main ClassificationExplanation

Body Fluids; human and animal Fuel; e.g. coal, coal ash Food Products and Animal Feedstuffs; including meat, milk and flour Rocks and Geological Materials AnthropogenicPollution Materials; e.g. sludges, dust, fly ash Soil and Sediments Aquatic and Terrestrial Biological Products including vegetables Water

Number of CRMs (approximately)

475

95 135 300 170 270 120

70

The user can find suitable materials in a number of different ways. For instance any of the above measurands can be chosen and a search made within a specific matrix type. A list of the measurand values in all materials of the selected matrix classification sorted by decreasing concentration will be produced, including the uncertainties in percent, the certification status and the material identification code. Other search methods are possible, selection by material gives a table with values of all measurands in the chosen material in alphabetical order and additional information about the price, the unit size, the issuing date, the suppliers and the exact material name. A further option is to list all materials from a producer. The website can be found through the IAEA homepage and directly via www.iaea.org/programmes/nahunet/e~/nmrm/index.htm 8.2.3 WinRefPro Database of Elements in Metals

A searchable, relational database was first developed by Mats Alfredsson and Sandberg Analytical Data AB in Sweden to enable rapid identification of suitable reference materials for element analysis in metallurgical samples. Since then the database has been expanded; and it is now supplied with information about more than 11 ooo RMs and CRMs. The database also allows continuous addition of RMs of interest to the user. Searches can be made for samples matching given criteria, by entering one or more element concentration. Sample type or range of sample types can also be speafied. If

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I internal or supplier product codes are used in the database, a search mask can also be 8 Availability and Sources oflnforvnation

specified for this field. The search mask may also contain so called “wild card characters, where “-‘P means “anycharacter(s)”and “?” means one character. An searchable “element-concentration-criteria”may consist of a minimum concentration, a maximum concentration or a concentration range. After searching is complete, a hit list is displayed, from which more information about any listed CRM can be selected. For further information contact: [email protected] 8.2.4 Website Addresses

Table 8.4 below lists all website addresses mentioned in this Chapter for international and national institutions. The web addresses shown in it were correct at the time of going to print, but as the nature of the www is for websites to be regularly reviewed, changed or updated, web addresses do change without warning. If any of the addresses shown below cannot be found, it may be necessary to use a search engine to find the latest site address.

Tab. 8.4

Websites for national and international organizations mentioned in this chapter

Institution

Abbrev.

WWM Address

Bundesanstalt fur Materialforschung und -Priifung, Berlin, Germany

BAM

http://www.bam.de/a-i/crm/

COMAR database

COMAR

http://www.bam.de/ai/comar/scr/titel.htm

International Atomic Energy Agency

IAEA

http://www.iaea.org

International Standards Organization, Reference materials Committee

ISOREMCO

http://www.iso.ch/REMCO

AOAC International, Technical Division of Reference Materials (TDRM)

AOACTDRM

http://www.aoac.org

http://www.vtt.fi/ket/eurachem/contacts.htm

EURACHEM UI< Valid Analytical Measurement Programme

VAM

http://www.lgc.co.uk

United States pharmacopoeia

USP

http://www.usp.org/index

The European Pharmacopoeia

EP

http://www.pheur.org/

The British Pharmacopoeia

BP

http://www.phannacopoeia.org.ulc/

8.3 Organizations and Symposia

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8.3 Organizations and Symposia

A number of international organizations and scientific meetings play a pivotal role in the RM and CRM world. Without their involvement there would be few reference materials and little cooperation. Users of reference materials should ensure that they monitor the activities of these organizations and understand the consequences of any changes in recommended procedures or guidelines.

Information about forthcoming meetings and symposia can be found on the www sites of the organizations described above (see Table 8.4). Thejournal Accreditation and Quality Assurance published by Springer (see Table 8.2) includes a comprehensive review of “RM related” meetings. 8.3.1 Organizations 8.3.1 .I

The International Standards Organization, I S 0

The field of general and specific information and guidance on all aspects of RMs is covered comprehensively by the International Organization for Standardization (ISO), Geneva, Switzerland, a worldwide federation of national standards bodies from some 120 countries. The mission of I S 0 is to promote the development of standardization and related activities in the world with a view to facilitating the international exchange of goods and services and to developing cooperation in the spheres of intellectual, scientific, technological and economic activity. I So’s work results in international agreements which are published as International Standards. The Reference Materials Committee (REMCO) of I S 0 was established in 1976 and was initially known as REMPA. The aim of this committee is to carry out and encourage a broad international effort for the harmonization and promotion of CRMs and their production and applications. Through the I S 0 website (Table 8.4), it is easy to access vast amounts of information about the activities of I S 0 and its various technical committees. 8.3.1.2

AOAC International

Established in 1894,AOAC International is an independent association of scientists and organizations in the public and private sectors devoted to promoting methods validation and quality measurements in the analytical sciences. AOAC has a mission to ensure the development, testing, validation, and publication of reliable chemical and biological methods of analysis for foods, drugs, feed-stuffs, fertilizers, pesticides, water, forensic materials and other substances affecting public health and safety and the environment. Interest in promoting the use of reference materials in method validation studies led in March 1993 to the formation of a Technical Division on Reference Materials, TDRM (Heavner 1995).The stated purpose of the TDRM is to improve the quality of

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information

analytical measurements through the use of reference materials in validation and use of AOAC methods through goals which include: Promoting policy changes within AOAC to incorporate reference materials in AOAC methods. To identify and facilitate availability of reference materials needed for use with AOAC methods. To plan and conduct educational activities on use of reference materials in AOAC. Providing a pool/network/resource of experts in RM areas. The issue of data reproducibility had been the previous focus of the AOAC Official Methods Program. The TDRM will assist AOAC in responding to an expanded scope of it mission to include statements of accuracy with AOAC methods. This extra step requires independent verification of the accuracy of measurements generated by the method. Accuracy of an analytical method must be verified at three separate points: When the method is first developed and collaboratively validated. When the method is implemented in a different laboratory. (3) When the method is used routinely. Proper use of reference materials in each of these three areas is essential. (I)

(2)

TDRM has conducted activities in specific areas such as: matching available reference materials to specific AOAC method; determining reference material needs of the future, and establishing acceptance criteria for the production of reference materials from multi-laboratory studies. In addition to organizing workshops on RM at AOAC meetings, the TDRM is the sponsoring organization for the international symposia series on Biological and Environmental Reference Materials (BERM). Additional information on activities and membership in AOAC and the TDRM can be found on the AOAC website (Table 8.4). 8.3.1.3

EURACHEM

Established in 1989, EURACHEM provides a focus for analytical chemistry and quality related issues in Europe. EURACHEM is a network of organizations in Europe, having the objective of establishing a system for the international traceability of chemical measurements and the promotion of good quality practices. It provides a forum for the discussion of common problems and for developing an informed and considered approach to both technical and policy issues. Within Europe there are thousands of organizations concerned with analytical measurement. However, within the broad field of testing, chemical measurement has generally been poorly represented. EURACHEM was established to address this concern by enabling analytical laboratories to work together, across international boundaries, on analytical measurement issues. EURACHEM’s uniqueness as an organization comes from its primary concern, which is the analytical quality of chemical measurement.

8.3 Organizations and Symposia

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Membership of EURACHEM is open to all EU/EFTA countries and the Commission of the European Communities. Other European countries may participate in EURACHEM as Associate Members and representatives of organizations. For example, AOAC International and the Federation of European Chemical Societies (FECS) can attend EURACHEM meetings as observers. All delegates to the EURACHEM Committee are eligible to voice their opinions and participate in projects of interest. The only requirement is that members actively participate and share the costs of activities by contributing support for selected tasks. More information can be found at the Eurachem web site: see Table 8.4, where details of activities and publication, most downloadable, are provided. 8.3.1.4

VAM

The UK Government has, for more than six years, funded the Valid AnalFcal Measurement (VAM) Programme, which is aimed at improving the quality and comparability of analyhcal measurements. The work undertaken within VAM is key to the underpinning of a modern physico-chemical and biochemical National Measurement System. By disseminating the activities of VAM across international boundaries and linking with other national measurement system VAM also aims to ensure the comparability of data worldwide. Thus VAM provides an infrastructure under which reliable measurements can be made for trade, regulation and health and safety provision. The practical aims of the VAM Programme is to help analysts move towards the goal of delivering reliable measurements first time, every time. The VAM concept describes six principles of good analflcal practice which, if adopted by any laboratory, will improve your overall business performance through increased efficiency, reduction of costs, and avoidance of the risks associated with passing on incorrect results to customers. The implementation of VAM in any laboratory can be aided by selecting from a growing range of VAM products. More details can be found at in the VAM section of the LGC website (Table 8.4). Given the key role that reference materials play in the quality assurance of analytical measurements, VAM supports the production of a wide range of reference materials covering many analytical disciplines. Other products include books (described in the "Further Reading" section of Chapter 7) and videos, the VAM Bulletin magazine, seminars and training courses and an advisory and consultancy service. All of these materials and services are available world-wide from LGC and its authorised distribution network. 8.3.2 Conferences and Meetings 8.3.2.1

BERM

The First International Symposium on the Production and Use of Reference Materials, held in 1979in Berlin, Germany was one of the starting points for special RM meetings; see Section 1.1. Two other formative meetings took place around that time. A symposium held by the American Association for Clinical Chemistry in

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8 Availability and Sources oflnformatron

US Clinical Measurement infrastructure. It is also where the classic figure of the hierarchy of methods and RMs appeared: from SI units through Definitive methods, SRMs, Reference Methods, Secondary RMs to Field Methods (Boutwell 1978). A research symposium held by NIST, then NBS, in 1976 on methods and standards for enmronmental measurements set out the foundations for environmental measurement. (IGrchhoff1977). But it was the first BRM symposium in 1983 which started a series of symposia that focused specifically on technical and scientific aspects of this topical area. That first BRM symposium took place in September 1983, in conjunction with the FACSS meeting held in Philadelphia PA. The idea for this meeting arose during an extensive tour by Wayne Wolf in the autumn of 1982 to ten European institutions involved with RM production. During this tour, the strong mutual interests of the visited institutions became clear, as did the strong interest in programs at other institutions on the itinerary. The genesis of the idea for the BEM Symposium was the statement “I do not know those people personally” made by many people to Wayne Wolf. In a meeting with Herbert Muntau, Ispra Italy, the idea was conceived that it was time to get these people together in a workshop/symposium. So upon a subsequent wonderful train ride through the Austrian Alps, from Zurich to Vienna, the organizational structure for the (BRM-I) symposium was born. BRM-I consisted of about 25 attendees with 16 presentations over a two-day session focusing on biological reference materials (BRM), especially in the area of food and nutrient analysis. Wiley Interscience published a book of the papers presented at this meeting. The session was very well received and encouraged to continue. So with the assistance of Markus Stoeppler, Julich, Germany, the next BRM meeting was organized in Neuherberg (Munich) Germany in the spring of 1986. It attracted 111 participants from zg countries and extended for 4 I/Z days. As a result of the experiences of publishing a camera-ready copy book for the first BRM meeting, a generous offer was gladly accepted to publish some 35 papers as a special volume of Fresenius Zeitschrift fur Analytische Chemie. Thus began a series of alternating BERM Symposia between Europe and the United States, co-organized by Markus Stoeppler and Wayne Wolf. The format of a 4 I/Z day meeting and publication as a special issue of Fresenius has remained basically the same. BRM-3 was held in Bayreuth, Germany, May 1988, with 115 registrants, from 17 countries presenting 63 papers. At this meeting the name was changed to BERM to include the strong interest in Environmental RMs. BERM-4 was held in Orlando, FL, USA, in February 1990, with over IOO participants and 70 presentations. BERM-5 was convened in Aachen, Germany in May 1992 with 141 registrants representing some 30 countries and more than 130 oral and poster presentations. BERM-6 was held in Kona, Hawaii in April 1994, with 105 registrants from zg countries and IIO oral and poster presentations. Following that meeting, with the general global paradigm shift to analyhcal quality, a number of national and regional conferences in the area of RMs took place. As a result of this increased interest, it was decided at BERM-6 in 1994 to hold the International BERM symposia at three year intervals. Therefore BERM-7 was convened in Antwerp Belgium in April 1997, with ZII participant from 31 countries and 182 oral

8.3 Organizations and Symposia

and poster presentations. BERM-8 was held in Bethesda, MD, USA, in September 2000.

Proceedings of the BEM and BERM symposia have been used to assess emerging trends in the development of RMs to meet Analytical Quality Control requirements for clinical, food, nutrition, and environmental health areas. (Iyengar and Wolf 1998): BRM-1 reflected a strong need to initiate and expand KM activities for certifying organic nutrients in foods. BRM-z highlighted the distinction between primary (certified)and secondary (e.g. check samples for proficiency testing) RMs. BRM3 identified the need for producing different levels of an analyte in a given matrix (spiked standards) to address matrix related measurement problems in foods. BERM-4 highlighted the need for a global vision in dealing with standards, illustrated by the activities of GESREM. Also, the logistics required for setting up intercomparison programs related to food safety monitoring programs were outlined. BERM-5 presented the changing outlook of the AOAC International in recognizing the usefulness of incorporating RMs for use in conjunction with their methods validation protocol. BERM-6 brought to the forefront the concern for traceability of chemical measurements to internationally recognized standards. B ERM-7 recognized the need for interdisciplinary approaches for preparation of certain types of CRMs, partly in response to the measurement needs arising from governmental regulations dealing with food safety and environmental health criteria.

Finally, BERM symposia have promoted a meaningful dialogue on the RM needs of African, Asian and South American countries and provided the developing countries opportunitiesto discuss their problems with the international analytical community. The BERM symposia have served further to clarify understanding of the basic attributes of a good reference standard that is indispensable for establishing new frontiers in the science of measurements, and to highlight the invaluable infrastructure and proven record of metrological excellence available at the National Measurement Institutes to accomplish such tasks. These symposia, in bringing together experts from international metrology and standards institutions, managers from governmental regulatory agencies, and the private producers and users of RMs, have documented an impressive list of accomplishments. Questions raised during, and largely answered by, various sessions of the symposia include: The lack of RMs certified for organic nutrients to satisft regulatory measures; The need for new clinical and speciated RMs to meet the requirements of health and food safety,

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The need for development of matrix specific standards for a variety of pollutants to answer questions in marine environmental health; A realization that concerted efforts needed in identifylng standards that were lacking (e.g. microbiology and biotechnology, among others); Concerns about aspects of refinement in material processing and stability of RMs. The BERM platform has also helped to highlight the need for systematic use of secondary RMs to retain consistency in the quality of field investigations. In doing so, it has succeeded in demonstrating the importance of using a well-characterized RM for method validation, besides proving to be a successful conduit in linking the use of RMs to official methods such as those recommended by the AOAC International. Finally, the global version shared by the BERM symposia concept is illustrated in its interactions with entities such as the GESREM, UNEP, and IAEA among others in recognizing the need for providing a global platform for the developing countries to discuss their measurement problems and, with the assistance of their international colleagues, seek effective solutions. 8.3.2.2 Other Meetings

Several individual countries’ efforts in the European region consider hture needs, including during the biannual German Analytica expositions and conferences, the Eurachem series of symposia such as that held in Nordwijkerhout in The Netherlands in 1995 on Interlaboratory Studies, and the April 1999working party on Harmonization of QA schemes for analytical laboratories organized by EURACHEM, IUPAC, ISO/REMCO and BAM in Berlin; papers from this event were published subsequently by CRC (Fajgelj and Parkany 1999).The next meeting of similar kind was announced by EURACHEM for 22-23 May 2000 “RMs for technologies in the new millennium”. In India, following participation at BERM-6 in 1994,a national reference materials program was begun, organized by the Central Pollution Control Board (CPCB) in Delhi. Resulting from a national conference on reference materials (DUREM-I, organized in 1996 and based upon the BERM model), a National Task Force on Reference Materials in India (REMTAF) was formed. REMTAF, consisting of a number of nodal agencies (with coordination by CPCB, Delhi), has begun a number of programs to identify and prepare priority reference materials (Mahwar et al. 1998). In North America at the annual PittCon exposition there are papers and workshops concerning the future need for RMs, including the CITAC workshops. In Canada, in June 1993in Quebec as part of the Annual Conference of the Canadian Society for Chemistry, a RM symposium was held; lectures from it were the basis for a CRC RM book (Clement et al. 1997).Further examples include reports of regional activities in China (Chai 1993)and Australia (Millar et al. 1995).

8.4 The Pharmacopoeia 8.4

The Pharmacopoeia

The role of the European, U. S. and other pharmacopoeia in the production, certification and use of reference substances has already been described. Section 5.5 reviewed the processes and techniques used by the European pharmacopoeia in their production of reference substances. The main pharmacopoeias maintain comprehensive web sites and issue publications describing their activities, but as they are not yet a main part of the I S 0 REMCO community, it is necessary to visit their websites for further information. 8.4.1 United States Pharmacopoeia

The 2000 U.S. Pharmacopoeia and National Formulary, USP 24-NF 19,was published in October 1999.It became official on 01.01.2000. USP-NF is one of, if not the most, widely recognized of the worlds pharmacopoeia1 compendia of standards for drug strength, quality, purity, packaging, labelling and storage. USP-NF also provides standards for devices and diagnostics, as well as nutritional supplements. The reputation of USP arises from its authoritative position in the USA, where its drug standards and monographs are supported in law, designated as “official compendia” in the US Federal Food, Drug and Cosmetic Act. In addition USP and NF standards of strength, quality, purity, packaging and labelling are enforced by the U.S. Federal Drug Administration (FDA). USP-NF Supplements are published twice a year. They contain all the approved changes to USP-NF Monographs and General Chapters as well as newly adopted monographs. The Pharmacopoeial Forum fulfills a vital role in promoting industry-wide communication between those involved with quality assurance, the development of standards and analytical methods. It contains an up to date list of official USP Reference Substances, with current and recently changed lot numbers. Reference Substances not yet available and those under development are also described. The publication acts as an international open forum in which scientists are invited to express their views, suggestions, ideas and comments regarding new drug standard development and revisions to existing monographs. Pharmacopoeial Forum is published, on a subscription basis, six times a year, back issues are available;for USP website see Table 8.4. 8.4.2 The European Pharmacopoeia

The EP is published under the direction of the Council of Europe and is available in English and French. It is the result of obligations undertaken in a convention set up under the auspices of the Council of Europe and signed by 19countries. The EP has two important aspects, the unification of national pharmacopoeia and the unification of methods of quality control of medicines.

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Pharmeuropa is available by annual subscription in English and Frendi from the Council of Europe and is published four times per year. Like the USP’s Pharmacopoeia1 Forum it contains draft monographs for comment, official announcements, scientific notes and the contents to the fascicles issued by the EP; for EP website see Table 8.4. 8.4.3

The British Pharmacopoeia

The 1999 edition of the British Pharmacopoeia and British Veterinary Pharmacopoeia, which became official on December I, 1999, is published by the Stationary Office. With this new edition, the BP have combined the three-volume book form and the complete work on CD-ROM; for website see Table 8.4.

8.5 The Movement of Reference Materials

As the use of RMs increases and international trade in these materials becomes established, it has become clear that they can be subject to high levels of import duty and delay when clearing customs. In 1998 ISO/REMCO established Task Group G to investigate the problems involved in shipping reference materials (and materials that are in the process of being made into reference materials, such as proficiency testing materials) across national boundaries. Most of the difficulties occur when reference materials cross national borders and, as a result of specific national or international regulations, are in some way impeded. Generally the impediments include questions involving tariff numbers and customs duty coupled with environmental and health and shipping regulations. Many times, these questions arise because reference materials are shipped in small quantities and not used directly in commerce. For example: trace elements in milk powder are not consumed as milk, and moisture in transformer oil is not used in transformers, yet matrix reference materials based on milk are imported as food and are subject to health certificationrequirements and sometimes import quotas. Likewise a matrixbased on oil is identified as fuel or lubricating oil and is both classified as a hazardous material and subject to mineral oil tax. These problems arise because RMs are frequently incorrectly classified by specific title of their matrix (as Reference Material of Trace Elements in Rice is classified as rice) and not as reference material which is the intended use. The movement of all types of goods from one country to another is restricted by a wide variety of regulations and restrictions. For substances concerned with chemical and physical analysis the main areas are: Labelling All reference materials have to be labelled. Presently they are considered to be laboratory materials, so the label should include:

Identification - including UN Number Health, risk and safety phrases

8.5 The Movement of Reference Materials

Country of origin Name, address and phone number of producer There are standard formats for labels, but the standards issued by the EU, USA, Canada and Australia are all largely incompatible, so it is impossible for a producer to produce a single label that meets all requirements. Customs Tariff Numbers The movement of any traded material across borders cannot take place without recording the transaction for both statistical and fiscal purposes. An internationally agreed means identification of the goods exists to ensure correct recording and taxation by the selling and buying nations. To achieve the above, a so-called “Harmonized Customs Tariff Code Number” is used. These numbers are:

Based on a common system of numbering managed by the international customs body in the Netherlands. Allocated on the basis of contentlmalte up and use. Described in a handbook issued by the customs authorities of every trading nation and regularly updated. Used as the basis for the collection of import duty: this ranges from o % to 150% or more. Tariffs specific to a certain product or service are allocated, but only when there is an unambiguous description and the value of trade is significant. There are many products and services for which no specific code is allocated, but it is easy to allocate them to a code. In some cases, such as reference materials, it is not so easy to allocate a specific code as a number of possible headings can be used. There is no tariff code that is specific for reference materials. This is because: There is no all encompassing definition issued by either I S 0 or any other competent international body. The value of world trade in RMs is small - even though the value of trade consequent on the availability of reference materials is high. When a specific tariff code is not available, the seller and buyer have to use their judgement as to the most suitable tariff code. If a specific use is not listed in the handbook of numbers, the content or makeup is used to find a number. This decision is subject to review by the customs officer at point of entry. This leads to anomalies and unnecessary expense. Health and Safety Data Sheet This document is required to provide anyone who comes into contact with the material with sufficient information needed to react to a spillage or breakage. The labelling and health and safety sheet regulations are not yet consistent through out the world and at the time of writing there is no way to label a product SO that it conforms to US, Canadian, European and Australian requirements! The Safety data sheet issue is less confused, the US OSHA and EU forms ofthe so called “material safety data sheet”are moving to conform with an I S 0 Standard.

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8 Availabilityand Sources oflnformotion Health, Environmental, Quarantine and Other Regulations: Many countries have strict regulations designed to protect the ecosystem and agrochemical business. For example any matrix material derived from pork, beef, sheep or horse tissue has to be accompanied with a Veterinary Certificate confirming that the matrix material is free of certain specified diseases before it can be imported into the EU. The Australian import restrictions are even tougher and require the importer to obtain prior permission to import plant and animal materials and products derived from biological materials. To get an import license it is necessary to complete an application, which includes information from the producer about the actual production process used to prepare the matrix! Other regulations that have an impact on the movement of reference materials include:

CITES: International regulations designed to prevent the trafficking of narcotics and other controlled substances and endangered species Weapons Precursors: International restrictions designed to prevent the production of chemical and biological weapons Montreal Protocol: The U N convention on the trade of materials damaging the ozone layer Hazardous Goods: International regulations that specify how and in what way all types of hazardous materials can be transported

To comply with all of the above, copious documentation is required. The trigger is the Customs Tariff Number: as soon as it is entered into the customs computer, all the other requirements are highlighted. Any errors in documentation and the goods will, at best, be held up for a week or so, whilst the correct data is supplied. At worst the goods will be seized or destroyed. There is perhaps only one way that the free movement of reference materials can be made more certain: the establishment of a single, specific Customs Tariff code. This might be achieved by revising the existing REMCO Guide definitions for RM, CRM, etc into a single definition, couched in terms that can be identified by customs organizations. This would best be issued by I S 0 as an I S 0 Standard that can be built into the Standards of all participating nations. With a specific tariff number, free movement of reference materials will be more certain as: Shippers will have to make only one statistical declaration per shipment Receivers will have to make only one statistical declaration per shipment Shipments will not be held in customs whilst importer and customs haggle over identity and duty rates Governments can be encouraged to apply o % duty, as to do so will not have consequences on other imports But for now it is often easier to purchase through a specialist organization, such as LGC or Promochem, who have the resources and experience to make the movement of goods as easy as possible - but there is a cost to be paid!

8.G References

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References ACAR0, K I I I C 2, ' ~ ' U R K E RAR (1999)Determination of bismuth, indium and lcad in geological and sea-water samples by electrothermal atomic absorption spcctrometry with nickcl containing chcmical modifiers. Anal Chim Acta j8z:pg-)38. RFKMEJO-BAKKEKA P, I;ERNANI)FZ-NOCELO S, MOREDA-PIREIRO A and BEKMEJO-BARR~KA A (1999) Uscfiilness of enzymatic hydrolysis procedures bascd on the use of pronase E as sample prc-treatment for multi-element determination in biological materials. J Anal At Spcctrom 14:1893-1900. BOUTWI;I.I.J H , ed. (1978) A Kational Understanding for the Developmcnt of Reference Materials and Mcthods for Clinical Chemistry. Proceedings of a Conference. Am Assoc for Clinical Chemistry, Washington D.C. ISBN 0-915274-06-X. CANTILLO AY (1995)Standard and Keference Materials for Marine Science 2nd Edition. NOAA, US Department of Commercr, Kockville, MD, USA. CIIAIC (1993) Present status and future trends in biological and environmental reference materials in China. Fresenius J Anal Chem 34593-98. CI.EMENT RE, KEITH LII, SIU KWM, cds. (1997)Kcference materials for environmental analysis. CRC Press Inc. Boca Raton New York I.ondon Tokyo. DEIVFM, KEUNE H. (1992)Survey of Institutions, Organizations and Laboratories Manufacturing Supplying or Using KMs. UNEP-HEM, Published by GSF Ncuherberg, Germany. FAJGELJ A and PARKANY M, eds. (1999)The use of matrix refcrence materials in environmental analytical processes. RSC, Cambridge. HI:AVNER GR (1995)An update on AOAC INTERNATIONAL new program activities. Fresenius J Anal Chem 352:19-22. J , MACKAYD and BANOUBJ (1999)Lcvels, persistence and bioavailability of organic conHELLOU taminants present in marine harbor sedimcnts impacted by raw sewage. Chemosphere 381457-473. IAEA (1985) [MURAMAI'SU Y, PARRRMI Survey of currently availabk reference materials for use in connection with the detcrmination of trace elements in biological and environmental materials. Report IAEA/KL/r28, International Atomic Energy Agency, Vienna, Austria. TOHOE, PARRRM, CLEMENIS SA] Biological and environmcntd reference IAEA (1990)[CORTES materials for trace elements, nuclides and organic microcontaminants. Report IAEA/RI./ 128(Rcv.1),International Atomic Energy Agency, Vienna, Austria. IAEA (1996)Database on lntcrnationally Available Certified Reference Matcrials. IAEA-TECDOC-854 and IAEA-TECDOC-880, International Atomic Energy Agency, Vienna, Austria. IUPAC (1976) Physico-Chemical Measurements: A Catalogue of Reference Materials from National Laboratories. Pure Appl Chem 48:joj-515. IYENCAK V, WOLFW (1998)Global activities in the world of reference matcrials including the needs for developing countries. Frcscnius J Anal Chem 360:282-286. J O R H E M L (1998)Non-use and misinterpretation of CRMs. Can the situation be improved? Fresenius J Anal Chem 360:370-373. KI KCHHOFI' WH, cd. (1977) Methods and Standards for Environmental Measurements. Proceedings of the 8th Materials Rescarch Symposium, NUS, Gaithersburg, MD 20234. Sep 1976. NRS Special Pub 4 6 4 U.S. Govt Printing Office, Washington D.C. SK, BISWASDK (1998)Development and use of reference MAHWAR RS, VERMANK, CHAKRABARI.~ materials i n India-status and plans. Frescnius J Anal Chem jGo:z91-~95. MAII:RFA, BOI:NKEA and M ~ K I C U Pc E T(1997) Importance of the Certified Reference Materials Programme for the Furopean Union. Trends Anal Chcm 16:496-503. MARKiioLM MM and BENNETI J P (1998)Mercury accumulation in transplanted Ilypogyrnnk physodes lichens downwind of Wisconsin chlor-alkali plant. Water, Air Soil Poll 102:427-436. MILLAR RC, ARMISHAWP, WILSONMC, MAJEWSKI1.M (1995).Recent developments in Australiareference materials and proficiency testing. Frescnius J Anal Chem 3 52128-32.

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RASBERRYSD (1994) World Wide Production of CRMs. ISO-REMCO Status Report. I S 0 Publica tions, Casa Postale 56, CH 1211 Geneva 20, Switzerland. RASBERRY SD (1996)World Wide Production of CRMs. Accred Qua1 Assur r:130-134.

Reference Materials for Chemical Analysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

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Future Trends for Reference Material Activity Wayne R Wolf

Introduction

As so eloquently stated in the Foreword to this book, measurement science has undergone a significant period of development and growth over the past 20 years into a mature discipline. This growth has resulted in a strong focus on Quality Assurance (QA) and the role of standards in the foundation of demonstrating that measurement data are reliable and sufficiently accurate for their intended purpose. As this is being written, at the dawn of a new century, it is safe to predict that these concepts in measurement science will continue to develop and to be applied in improving the quality of analytical chemical measurements. An important aspect in assuring this quality of chemical measurements is the availability and proper use of appropriate Reference Materials (RMs). As detailed in Chapter 7, these RMs fulfil various purposes including: calibration of equipment, verification of result accuracy, aids in improvement and performance of methods, daily quality control and achievement of traceability to basic measurement units (Griepink 1990).Present and past activities to meet these purposes have been well documented in this book. There are a number of trends that will continue to influence future RM activity and a number of areas which are not yet very significant that will become more so. One important source of information on developments in the area of RMs is the series of International Symposia on Biological and Environmental Reference Materials (BERM), organization of which was described in Chapter 8. The Proceedings of these successive BERM Symposia have given a series of detailed snapshots of activity on a 2-3 year cycle over nearly two decades (BRM-I 1985 - BERM-7 1998).Most of the information and projections in this Chapter are taken from various papers published in these BERM proceedings. Good projections about future use are especially important to RM producers (Rasberry 1993, 1998).The producers need to know what kinds of RMs will be required in the future, by whom, and for what purposes. They need to laow what batch sizes to prepare of different kinds and how to best allocate limited resources. In order to understand the trends in needs for RMs, it is important to understand the more general trends in legislation, health, environment, manufacturing, and commerce. An overview of the issues underlying these trends, as they bear on and result in a significant growth of interest in reliably accurate measurements, is summarized in the following Sections,as outlined in a stepwise fashion by Rasberry (1998).

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9.1

Overview o f General Issues 9.1.1

Review o f Trends

Prediction without reasoned examination of key trends, driving forces, or potential technological breakthrough is risky at best (Rasberry 1998).One well known inaccurate prediction was that of Thomas J. Watson, founder of the International Business Machines Company. On first considering the new technology of computers, in 194.3, Watson forecast: “I think there is a world market for maybe five computers” (Adams 1937). In 1981, Bill Gates, founder of Microsoft, characterized the appropriate size of personal computer random access memories by saying: “640 K ought to be enough for anybody” (Adams 1997). The most outstanding trend within analytical chemistry over the past century has been the inexorable change from classical to instrumental analysis, which now covers nearly every analytical need. From the emergence of spectrophotometers in the I ~ ~ o ’tos ,the development of mass spectrometers and infrared, visible, and x-ray spectrometers in the 1940’s and I ~ ~ o ’instrument-operating s, technicians replaced chemists in field laboratories. In normal use, these instruments needed calibration and validation of results through the use of RMs. Thus the need for RMs, as a way of linking accuracy to a higher authority grew dramatically (Rasberry 1993). Today, virtually all applied analytical chemistry relies on computer-controlled instruments linked to laboratory information systems. Thus, the future growth of RMs will be based on both the growth in the number of instruments and the kinds of methods employed. Over the past decade, a major trend has been the development of the use of proficiency testing (PT) or evaluation materials (Fox 2000). PT materials are a type of reference material, which aid in assessment of analytical laboratory measurement quality. There will be an increased use of such materials as part of laboratory accreditation programs and other new quality assurance efforts, including internal audits. At the same time, a number of providers have used PT schemes to produce a form of RM intended to meet the ever-growingneed for RMs required for routine QC use (Jenl~s1995,1997). Thus, even a cautious view of recent trends would allow for considerable growth in the application of RMs into the early years of this new century. Indeed, in volume terms the usage of RMs within Western Europe has grown by up to 20 % per year over the last decade (Quevauviller 1999). More speculative considerations are required to extend our view for perhaps another 50 years. 9.1.2 Projection of Challenges

The foremost longer-term global challenge lies in the projection that by 2050 world population will increase from the present 6 billion to close to 10 billion (Census

9.1 Overview ofCeneral Issues

1996). This population growth has a role in almost every challenge that we can foresee in the next fifty years, including air, water and soil pollution, and insufficiencies and maldistribution in safe food production. Statistics gathered to express these tends clearly show that increased development and use of technology will continue to greatly contribute to help secure clean air and water, grow sufficient food, and produce energy adequate to meet needs and comfort of future generations. For example, the portion of gross domestic product applied to the health field has more than doubled in the quarter century from 1965 to 1990 (Health Care 1992). U.S. employment in the environment industry is growing at a rate of 5 %. which is outpacing U.S. population growth five-fold! (Statistical Abstract 1994).While these statistics cover only the US.,they are representative ofconcerns throughout the world in the fields served by biological and environmental reference materials (Rasberry1998). 9.1.3

Analysis of Driving Forces and Construction of Scenarios

Twelve driving forces that seem likely to shape the production and use of reference materials over the next quarter century are listed in Table 9.1(Rasberry 1998). Rasbeny (1998) gives a detailed description of the potential interaction of these pairs of driving forces on reference material usage. For example for the first set, his view is that method diversity will increase, increasing the demand for RMs. However, that demand will be offset by increased equipment and method robustness, giving a neutral impact on future need because of these forces. The scenario selected for the third set is that increases in both extent of regulation and number of field laboratories will produce an increased demand for RMs. The other sets or forces are similarly explored. Although there is certain arbitrariness in this selection of driving forces, this approach does give a qualitative view of future possibilities, and is an excellent guide to factors that bear careful observation into the future needs and usage of reference materials.

Tab. 9.1 Selected driving forces that affect extent of reference material usage (arranged by pairs, in

order of increasing external forces) Equipment and method robustness

Method diversity

RM producer co-operation

Challenges to be met

Number of field laboratories

Extent of regulation

Lack of trust in commerce

Extent of trade

International programs

Per capita wealth

Spread of war

Population density

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9.1.4 Thinking “Outside the Box”

Thinking “outside the box” or “what if’ is a way to bring fresh perspectives to a given issue, question, or problem. Even if the “what if’ is not true today, it may be true tomorrow (Rasberry 1998).Consider the following six questions: What if proficiency testing (PT) materials were more integrated into the theory and use of reference materials? World-wide, the number of active PT programs is steadily grow-

ing. There are far more than can be mentioned here. Examples include new programs of AOAC International (Fox zooo), the series of PT Programs run by LGC in the U.K. and the programs run by RT Corporation and others in the USA to meet the needs of laboratories accredited to undertake environmental testing required by U.S. EPA regulations. In a database prepared in 1997 by LGC, under the U.K. VAM Programme, a total of 296 PT schemes were identified in the areas of food, agriculture, industrial production, water and environmental analysis, biomedicine and forensic and occupational hygiene analysis. Clearly one of the foreseeable economies of such programs is preparing amounts of materials that are large enough for subsequent application as RMs. Costs of material collection, preparation, and initial characterization could be more widely spread in the future by expanded use of such materials (Jenlis et al. 1998; Rasberry 1998). What if instrument manufacturers would “adopt” specific RMs? Until now, there has

been a general tendency for instrument manufacturers to avoid admitting the need for RMs. If instrument manufacturers could be convinced to make a serious appraisal of needs in the field, they might provide some resources to help meet those needs (Rasberry 1998).After all, the extra cost of providing suitable RMs, as a part of an annual service contract costing US$ 20 ooo on an instrument that is worth US$ 2 million is quite insignificant. What if instruments could be designed which were remotely selfcalibrating with respect to calibration services of a National Metrology Institute (NMI)? While this may sound very

“far-fetched,it is not difficult to speculate that future application of telemetry, transducers, intrinsic standards, detectors and computers will link some instruments directly to a NMI to obviate their need for RMs (Rasberry 1998). What if radically different ways to use existing instruments are developed in the future? Al-

most trivial examples, from today’s perspective, are “screening tests” that have been developed to displace the need for further testing in many cases. An example of this is the determination of chlorine in environmental samples. If chlorine is found to be lower than a critical value, one can conclude that polychlorinated biphenyls (PCB) are not present in the sample at or above an associated level and a more expensive determination of PCBs is not required. This simple example is an inadequate indication of what may he possible in the future. It may be possible to arrange millions of sensors, including sorting and detecting systems, upon chips and connect them with massive

9. I Overview ofCeneral h u e s

computing power. Such devices may be able to answer our real questions about real specimens with little or no recourse to external reference materials. (Rasberry 1998). Which leads to the next question. What if the use of chemometric approaches significantly increases? Breakthroughs in

sensor technology have augmented the chemist’s measurement repertoire by introducing new kinds of detectors with improved selectivity and the capacity to perform simultaneous multi-species measurements (Meglen 1990).Thus, the electronic revolution has qualitatively and quantitatively changed the data matrices to which the analyst (problem-solver)has access. The new chemical sub-discipline of chemometrics is developing powerful mathematical and statistical data analysis tools to exploit the electronic windfall and enhance data interpretation. Principal component analysis and graphical procedures have been used to examine the mult-variate suitability of current reference materials in matching the concentration ranges and matrices for quality control of various food analyses. Use of the food triangle concept (discussed in more detail in Chapter 6) with this approach may enable identification of a finite number of primary RMs to define the entire range of food matrices (Wolf and Andrews 1995). What if clinical testing is replaced with total DNA analysis? The worlds first DNA array

“biochip”(Stipp 1997) holds considerable promise for revolutionizing clinical laboratory testing. The aim of this new technology is to produce arrays and scanners which can be used on a few cells from a patient not only to analyze genetic risk for hundreds of diseases, but even diagnose the current status of the patient. Such a development would have a profound effect on current practices of clinical chemical testing and could dramatically reduce requirements for clinical reference materials, with U.S. sales exceeding a million units per year (Rasberry 1998). 9.1.5

Future Projections

There is no doubt that the rate of sales growth of RMs will continue. While perhaps there is a valid argument for limiting the availability of primary CRM, the issue of availability of appropriate secondary standards needs to be addressed. There is a need for good materials, produced in bulk and subjected to stringent analysis with data that are traceable to a primary CRM. It is easy to say what is needed, but the funding issue must not be overlooked. Whilst demand for RMs rises, Government funding for the production of RMs has been cut back. A consequence is the wish of some National Metrology Institutes to distance themselves from “secondary” RM production, due to capacity and cost constraints. One approach to providing secondary RMs is the NIST Traceable Reference Material (NTRM) program for gas standards (Jenks et. al. 1998; NIST 1997). The NTRM program is to be extended to metals, trace elements and pure substances. It may be possible to extend this concept to other types of RM, but it is difficult to see how it can easily be applied to the production of complex matrix CRMs.

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Another route to providing secondary RMs is the multi-laboratory collaborative study through performance testing (PT). It seems sensible to utilize all the work that goes on every month as part of PT programs in countless laboratories around the world testing blind standards. These programs could produce a stock of material that could have statistically valid values assigned and then be subsequently used as within batch control material. If NIST, IRMM, or similar organizations could set standards for such materials, then they would be much the better. The College of American Pathologists already does this in many respects for the clinical chemistry community (Jenks 1995). Having RMs available is only one aspect, obtaining them is another. There are real difficulties in distribution of RMs through different Customs and Quarantine systems. These obstructions in movement of RMs are not only in the developing world, but also in all global areas. Materials that cross international borders must be accompanied by a (or maybe 2 0 ! ) customs document. One of the key pieces of information on that document is correct allocation of the tariff number to the product. The customs tariff number directly impacts on the resolution of such matters as, most importantly, the rate of import tax and the speed through customs. Unfortunately at present there is no specific tariff number for RMs. For most producers, tariff number 382200000 seems to fit the bill. As discussed in Chapter 7, this issue will need to be resolved at International levels before easy movement and distribution of RMs can occur. Admitting an uncertainty of at least 5 0 %, Rasberry (1998)has reached his own “semi-quantitative guesses” and conclusions about the future trends for reference materials. The author together with the Editors has modified Raspberry’s views slightly - upwards.

Ten-year horizon. Based on recent trends in the production of analytical instmments, requirements for RMs should grow by more than 5% per annum. Because PT material usage is growing from a smaller and less structured base, its growth should be about 15 % per annum. Twenty-yearhorizon. Looking at the potential challenges faced by the world, RM usage should continue to grow by at least 5 % per annum. The annual growth rate for PT materials should peak during the period at about 20 % per annum. Such a large rate will not be maintained due to the growth of base usage and consolidation of providers into more structured and commercial organizations. Thirtyyear horizon. Increased instrument sophistication and breakthroughs in technology will temporarily halt the growth of RM usage at about fourfold above the present level. There will be a continued replacement ofobsolete types with materials to address new challenges. The base of PT usage will have grown substantially,and the rate of increased growthwill slow to 5 % per annurn.

9.7 Overview ofCeneral Issues 9.1.6 Selecting Strategies

The real work of setting strategies to meet future needs rests with RM producers. Fundamental to their success will be early recognition and acknowledgement of change. Several strategies can be provided as examples. RM producers could centre on developing systematic collection and monitoring of trends that affect the field. Surveys from the I S 0 Reference Materials Committee (REMCO) and by the EU have made significant contributions in tabulating current and future activities to certify and issue new reference materials (Quevauviller 1999; Rasberry 1996). Greater co-operation among producers could be developed. The World Wide Web has provided a model of sharing information based on very clear technical principles and formats, but with no formal bureaucracy or external control. Researchers using the Web can move easily among computers of many research institutions that are linked to the Internet. Perhaps the National Measurement Institutions that produce RM could make their instruments and laboratories just as open and available to researchers who are characterizing a new material (Rasberry 1998). Increased attention could be focussed on accreditation of RM producers at every level in the metrological hierarchy. In Australia the first National Metrology Institute (NMI) has been accredited against the requirements of I S 0 Guide 34. Others will follow. The growth rate of RM production at the secondary and lower levels is increasing. There is little formal accreditation or control of producers at these levels. Some producers cite the I S 0 Guides 30 to 35 in their I S 0 9000 accreditation, but this is by no means universal. The accreditation of NIST Traceable Reference Material (NTRM) producers by NIST, described in Section 9.4 below, is a move in the right direction. No doubt the arrival of I S 0 Standard 17025, the formalized form of what was I S 0 Guide 25, will help further. An emphasis of increasing quality and confidence at this level would make that growth more productive in meeting RM needs, while at the same time saving scarce resources of the NMIs for producing the primary standards (Rasberry 1998). RM producers could obtain greater support for research, production and certification of RMs. Increasingly, laboratories must convince policy level managers that these RMs serve major objectives of human welfare. Instead of presentations which detail the minutiae of a somewhat arcane technical field, presentations (directed at those who control resources) are needed which discuss the external driving forces and expand on the contributions of RMs to the most fundamental and serious problems facing the world (Rasberry 1998).

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9 Future Trendsfor Reference Material Activity 9.2

Needs for Specific Reference Materials

The need for a number of specific analyte and matrix RMs can be identified. Ihnat (1995)gives a very extensive overview of these needs. Quevauviller (1999) has reported on more recent work, undertaken in Europe to study future demand for RMs. Both authors identify a number of common themes, which may be summarized as follows. There is a dearth of elemental concentration data for a wide range of nutritionally, toxicologically, clinically, and environmentally pertinent elements. Some of the elements for which total concentration information is still required, usually at the low end of concentration range but occasionally at the high end, are: Al, Ba, B, Be, Br, Cs, F, I, Li, Mo, N, Pt, S, Sb, Si, Sn, Th, Ti, TI, U, V, W, rare earth elements, and radionuclides. Thus, it would seem advisable to certify each new RM for as many elements as possible so that certified values would be available for a larger number of elements in addition to the small number of core elements typical of many current RMs.

Total Elements

Little information is available for RMs with respect to the chemical forms or species in which elements occur. In the first approximation, bioavailable, extractable, or leachable levels of elements are of interest. Secondly, at a higher degree of sophistication, data on the levels of the actual species or inorganic moieties such as nitrate, ammonium, phosphate, bromide, bromate, iodide, iodate, and molecular species of which the elements are constituents would be of relevance to those conducting mechanistic and speciation research. Reference materials that are certified for extractable elemental concentrations are not available to monitor the usual procedures in soil science based on extraction. As usually viewed by the reference material producers, a fundamental philosophy of certification rests on the concept of independent methodology,which is the application of theoretically and experimentally different measurement techniques and procedures to generate concordant results leading to one reliable assigned value for the property. Such assigned values are thus method-independent.Extractable concentrations are generated by specific procedures and are thus method-dependent,an idea that has to be rationalized with the fundamental method-independent concept in reference material certification work. Speciated Components

Organic Constituents Increasingly, interest is being directed to the reliable characterization of materials for organic constituents. Although reliable elemental determinations are sufficiently challenging, the determination of organics has an additional dimension of quantitativelyconsistent extraction from the matrix without alteration or destruction of the organic analyte. Organic analytes of interest requiring additional RMs include those such as vitamins, fat, lipids and carbohydrates that are of nutritional significance in foods, and are required for nutritional labelling. Other organic constituents in foods, feedstuffs and environmental materials of toxicological concern requiring additional RM which include chlorinated pesticides, PCBs, PAHs, drug resi-

9.3 Reference Material Needsfor Regulatory Nutrient Analysis

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dues, sterols, aflatoxins, and toxins in shellfish. In the organic/biologicaldomain, RM for DNA measurement and characterization, as described in Chapter 7, constitute a new and important field in biotechnology and forensic science that has begun to be addressed. The development of control materials for microbiological measurements, is in its infancy, and presents a challenging endeavor to be pursued. The thrust towards the development of required, but poorly represented analytes is, however, tempered by not only limited availability of funds, but also the need for and possible shortage of highly competent analysts required for certification work. The many difficulties of good work at trace and ultra-tracelevels, and correction of methodological deficiencies for specific analytes will require highly skilled research expertise. Matrix Components The term matrix component refers to the constituents in the material aside from those being determined, which are denoted as analyte. Clearly, what is a matrix component to one analyst may be an analyte to another. Thus, in one hand for the case of analyses for elemental content, components such as dietary fibre, ash, protein, fat, and carbohydrate are classified as matrix components and are used to define the nature of the material. On the other hand, reference values are required to monitor the quality of determinations of these nutritionally significant "matrix" components. Hence, there is a challenging immediate need for certified values for dietary fibre, ash, protein, fat, and carbohydrate. Concomitantly, these values must be accompanied by scientifically sound definitions (e.g. total soluble dietary fibre, total sulphated ash, total unsaturated fat, polyunsaturated fat, individual lipids, simple sugars, and complex carbohydrates). Pure standards encompass solid elements, inorganic and organic compounds, as well as certified solutions of such products used to prepare calibration standards or solutions. Although basic products are available from many commercial sources, it is worthwhile to have certified materials available from RM agencies for use either as a starting point for preparation of all calibration standards or at least for monitoring the quality of starting materials. Increasingly such RMs are becoming available from a variety of sources (see Chapter 8). Emphasis should be placed on products in short supply such as pure organics, toxins, etc. Naturally-occurring matrices to be considered in continuing and future developments include: animal tissues, sewage sludge, wastes of various kinds, soils representative of various important agricultural areas, marine tissues and oils, fresh full-fat food and marine products, waters (drinking, fresh surface, ground, sea, and waste), human tissues, and plastics.

9.3 Reference Material Needs for Regulatory Nutrient Analysis

For regulatory purposes, food-based RMs play an important role in validating accuracy of analytical data from use of routine methodology. For example, the quality of data obtained by analytical measurements serves an important function with regard to ensuring nutritional label claims. Unfortunately, and historically in some cases, assay data for the same analyte can vary greatly from laboratory to laboratory. Evalua-

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tions of these variations are truly dependent upon whether one is developing a method, comparing a new method to an existing one, or testing whether a method is performing as expected (Chase and Long 1997).

First, to address and monitor the inherent variability in methods development, a systematic statistical approach and collaborative evaluation utilizing appropriate controls is essential. Known spiked amounts and a statistically based set of data points will clearly show the limitations of the method. Second, the newly developed and collaboratively evaluated method may need to be compared to another validated procedure if available, which requires an additional and different data set. fiivd, in order to ensure that a collaborated method is being performed properly, and that resultant data obtained is sound, an appropriate set of controls or reference materials is needed to test the method as it is being used (Chase and Long 1997). However, there is a difference in how one views the aforementioned concepts. For example, analytical method development is a process-oriented approach in which each step of the process is continually tested whereas, the use of a RM, standard control or comparison with another method is a result-oriented view (Tanner et al. 1995). Currently, nutrient analytical methods development often utilizes the method of standard additions as an intrinsic aspect of the development process. Essentially, the analyte to be measured exists in the matrix to which an identical known pure standard is added. The spiked and non-spiked matrix is extracted and analysed for the nutrient of interest. By spiking at increasing levels the researcher can establish, to some degree of certainty, the recovery and linearity of the standard additions. One can also evaluate data to determine reproducibility, precision, and accuracy. Unfortunately, the method of standard additions does not allow the evaluation of the method at nutrient concentrations less than IOO % of the endogenous level. From a regulatory perspective, the method must be able to evaluate a nutrient claim at not only greater than, but also less than the label declaration. For example, some nutrients have an upper as well as a lower legal value. If the method has been developed using the method of standard additions, nutrient claims at greater than IOO % can be faithfully evaluated. As stated previously, methods developed using the method of standard additions cannot answer questions relative to method performance at less than IOO % of label claim. This complicates regulatory decisions relative to lower level determinations (Chase and Long 1997). Chase and Long (1997) propose that this conundrum can be eliminated by the use of Zero Reference Materials (ZRMs)in analytical methods development to fully evaluate the method. A ZRM is a product matrix that lacks those nutrient components that are to be assayed, i.e. a “blank” matrix. The use of a ZRM in method development can and will give a true indication as to how the method will perform as the spiked nutrient levels approach zero. For example, two products, Corn Starch (NIST RM 8432) and Microcrystalline Cellulose (NIST RM 8416),contain very low elemental concentrations and could conceivably serve as real sample blanks or ZRMs in some analytical procedures.

9.4 Perspectivesfrom Distributorz of Certified Reference Materials

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289

ZRMs for fabricated foods could be easily manufactured following recommendations for development of in-house RM (Craft and Boyer 1993).Batch records, published information, and so on allow the analysts to quickly determine the compositional makeup of fabricated foods and thus to formulate and make a ZRM devoid of a specific micronutrient. Since the complexity and nature of sample matrices for foods is vast, in effect, so is the difficulty and economics of ZRM production for all foods. In addition, most testing laboratories do not possess the food processing equipment or capital necessary to manufacture ZRMs. Since the average laboratory usually analyzes more than one type of product, the feasibility of having every applicable ZRM becomes unattainable. Applying the food triangle concept as described in Chapter 6 (Wolf and Andrews 1995) to provide ZRMs for a host of food products may help to provide a solution to this problem. The ZRM approach applied to nutrient analytical methods development research will add a significant and valuable level of assurance that the developed method will meet regulatory needs at both the lower and upper legal regulatory level (Chase and Long 1997).

9.4

Perspectives from Distributors of Certified Reference Materials

The Promochem Group was the first international specialist supplier of certified reference materials (CRMs) and pharmaceutical reference substances used in environmental, medical and trace element analysis. Their experience provides a viewpoint that echoes, reinforces and expands on many of the trends discussed above (Jenks 1997). From the middle of the r980’s, sales of CRM by Promochem increased between 10% and 20 % annually, depending on the market sector and application. Since then National and International Metrology Institutes, such as the now privatized U.K. Laboratory of the Government Chemist (LGC), the European and U.S. Pharmacopoeias, the E.U. IRMM and others have recognized that efficient distribution of RMs, backed by available technical support, is as important as production and certification. Thus, they have moved to spread their influence outside their national origins. The Web and e-commerce will continue to grow as major facilitators of better information dissemination and supply of CRMs. In addition to real growth caused by the intelligent use of CRM by informed and aware scientists, there has also been growth by what might be described as negative sales influences (Jenks1995,1997).These influences include:

Laboratory Accreditation and “Quality Systems”:Together these make laboratory managers look for added security in assuring data quality and thus invoke a desire in them to fall back on the best available standard. The result is that a CRM is often used instead of a working standard. This is a tendency that is supported and encouraged by some RM providers but discouraged by others. Inappropriate Pricing by Producers: There are still some certification organizations which issue CRM without a rational commercial regard for the cost of

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9 Future Trendsfor Reference Material Activity

production or the perceived worth of the material. This is normally because the funding for the development of the CRM has been supported by government grant or some other means. Producers then find customers using the CRM because it is “better and cheaper” than the commercial secondary standard. This is fine, until the material has to be replaced. The results are situations such as happened in 1gg1/g2 in The Netherlands, when a new requirement for within-batch control for use by Dutch environmental laboratories resulted in the rapid depletion of BCR CRM 142, 143,and 146. It is worth noting that the replacements of these CRM were priced 2.5 times higher than the originals (Jenks 1gg8)! Nothing Else Available: When there is no commercial working standard, and there is a CRM available, many scientists will not bother to spend the time and effort required to characterize an in-house standard.

9.5

RM Needs in Developing Countries

With few exceptions, many laboratories in developing countries continue to face limitations in their efforts to introduce a scientifically sustainable level of quality assurance as an integral part of their analybcal programs. These limitations include: inadequacy of laboratory facilities, lack of opportunities to participate in recognized proficiency testing or inter-comparison programs, technical restrictions in accessing relevant QA sources of information, and finally, a paucity of required RM to validate methods and enhance the QC credibility of a given laboratory. The RM problem is multi-fold lack of resources to buy RMs, suitability of the RM matrix for the problems at hand, and insufficient expertise to prepare secondary RM in the users’ own laboratories. These problems have no simple solutions. They encompass broad issues such as national economies, education, training, and regulatory statutes requiring AQC as a component for compliance. Notwithstanding the above-mentioned difficulties, there are efforts in progress in numerous global regions to address the issue of reliability in chemical measurements through development and utilization of RM (lyengar and Wolf 1998). Examples include: Development of basic pharmaceutical reference substances now in use by the ASEAN group of countries, including Indonesia, Malaysia, Philippines, Singapore, and Thailand, and initiated by the WHO, South American projects led by Chile, Brazil, Argentina, Uruguay, and other countries to produce environmental and food based RMs, IAEA efforts related to both CRMs and others, such as hair for total mercury and methyl mercury (prepared in India), and lichen for multi-element certification (prepared in Portugal) to assist biomonitoring programs in Brazil, Chile, China, Czech Republic, India, Italy, Malaysia, Slovenia, and Vietnam, F A 0 efforts through its network of laboratories,

9.G References

A similar effort by Australia to activate AQC efforts and promote proficiency testing in South East Asian and Pacific Rim Regions providing RMs for organo-chlorinesand phosphates in animal fat (Millar et al. 1gg5), In South Asia the “DUREM” series of meetings held in India, based on the style of the BERM meetings and organized by the Indian Central Pollution Control Board, which are designed to present RM and metrology matters in the context of analytical chemistry in India (Mahwar et al. 1998), Programs to provide RMs in China (Chai 1993). For many years the International Atomic Energy Agency (IAEA) has been promoting analyhcal quality assurance and quality control in its Member States with emphasis on measurands that are amenable to analysis by nuclear and related techniques, i.e. radionuclides, trace elements, and stable isotopes. A description of these activities, (particularly in relation to the needs of participants in developing countries, arising out of co-ordinated research programs, technical co-operation projects and global and regional networks is given by Parr (1998). All of these efforts serve to develop an ever-longer “wish list” of RMs. Those reflecting the specified needs of many African and Asian countries are still awaiting action. Among others, this list includes animal feeds, fertilizers, drinking water standards, and typical plant and soil RMs for tropical conditions. Also needed are foodstuffs representing typical cereals, pulses, and edible oils (processed and unprocessed) required to estimate the dietary intake of toxic substances via ingestion. Importantly, high purity water and common chemicals in sufficient purity are not easily available in some laboratories in these regions. Training, the most essential component of all, is still very scarce in many countries. Therefore, the concept of establishing regional laboratories to help developing countries deserves serious consideration (WHO 1996).

9.6

References ADAMSD (1997)Science and technology quotations. http:// falcon.cc.ukans.edu/-dadams. BRM-I (1985) Proceedings. In: WOLFWR, ed. Biological Reference Materials: Availability, Uses, and Need for Validation of Nutrient Measurement.Wdey, New York. BRM-z (1987) Proceedings: Fresenius J Anal Chem 326:597-745. BRM-3 (1988)Proceedings: Fresenius J Anal Chem 332:517-743. BERM-4 (1990) Proceedings: Fresenius J Anal Chem 338:359-581. BERM-5 (1993) Proceedings: Fresenius J Anal Chem 345:79-350. BERM-6 (1995) Proceedings: Fresenius J Anal Chem 352:~-270. BERM7 (1998),Proceedings: Fresenius J Anal Chem 360(3-4):275-504. CENSUS(1996) U.S. Bureau of Census, The international database-up-dated 15 May 1996. Government Printing Office, Washington USA. CHAI C (1993)Present status and future trends in biological and environmental reference materials in China. Fresenius J Anal Chem 345:93-98. CHASEGW and LONG AR (1997) Nutritional Metrology, PART 4, The Role of a “Zero Control” Reference Material. Food Testing and Analysis, Jun/Jul I997,3:30-33.

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CRAFT N and BOYERI< (1993) Guidelines for preparation of in-house quality assurance control materials. The Referee, AOAC International, Gaithersburg, MD, 17(5):6-8. Fox A ( 2 0 0 0 ) AOAC International Offers Leadership in Proficiency Testing. Food Testing and Analysis, 5 (6):7-9. B (1990) The role of CRM’s in measurement systems. Fresenius J. Anal Chem. GRIEPINK

338:360-362.

Health care financing review. Health Care Financing Administration. U.S. Health Care (1992) Government Printing Office, Washington, USA. IHNAT M (1995)Key analytes and matrices lacking in the CRM-system and future needs for CRMs. Fresenius J Anal Chem 352:5-G. IYENGARV and WOLFWR (1998) Global activities in the world of reference materials including the needs of developing countries. Fresenius J Anal Chem 360282-286. J E N K S PJ (1995) Use, abuse, and availability of certified reference materials. Fresenius J Anal Chem 3yx-4. J E N K S PJ (1997)Reference Materials, Their Use, Abuse and Development. Anal Eur 4:22-28. J E N K S PJ, BOEKHOLTAH, MAASKANT JFN, and RUCINSKIRD (1998) Are certified reference materials a victim of quality systems? The need for working matrix-certified reference materials. Fresenius J Anal Chem 360:366-369. MAHWARRS, VERMANK, CHAKRABARTI SK, and BISWASDK (1998) Development and use of reference materials in India-status and plans. Fresenius J Anal Chem 360:291-295. MEGLENRR (1990) A n a l y t d problem solving, reference materials, and multivariate quality control: A chemometrics approach. Fresenius J Anal Chem 338:363-367. MILLARRC, ARMISHAW P, WILSON MC, and MAJEWSKILM (1995)Recent developments in Australia-reference materials and proficiency testing. Fresenius J Anal Chem 352:28-32. NIST (1997)Special Publication zG0-126. PARRRM, FAJGELJ A, DEKNER R, VERARUIZ H, CARVALHO FP, and POVINEC PP (1998)IAEA analytical quality assurance programs to meet the present and future needs of developing countries. Fresenius J Anal Chem 360:287-290. QUEUVAUVILLER P (1999)Reference Materials, an enquiry into their use and prospects in Europe. Trends in Anal Chem 18:76-85. RASBERRY SD (1993) Measurements: Developments in the USA. Fresenius J Anal Chem, 45237-

89.

RASBERRYSD (1996)Accred Qua1 Assur 1:1jo-134,186-89, z . z . ~ - z ~ z . RASBERRY SD (1998) Reference materials in the world of tomorrow. Fresenius J Anal Chem 360:277-281. (1994)Tables 373. Government Printing Office. Washington, DC, USA. STATISTICAL ABSTRACT STIPP D (1997) Fortune 135:54-73. TANNER JT, WOLFWR, and HORWITZ W (1995)Nutritional Metrolom. The role of reference materials in improving quality of analytical measurements and data on food components. In: GREENFIELD H, ed. Quality and Accessibility of Food-Related Data, pp 99-104. AOAC International, Gaithersburg MD. WHO (1996),Trace Elements in Human Nutrition and Health. Geneva, Switzerland. V (1996) Nutritional metrology: Food-based reference materials. Food WOLFWR and IYENGAR Testing &Analysis 2(6):27-31. A system for defining reference materials applicable to all WOLPWR and ANDREWS KW (1995) food matrices. Fresenius J Anal Chem 35x73-76.

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

I About the genesis ofthis book

It is usual for the editors to set the scene for the writing of such a book as this: unusually the asked me to contribute, from the Publishers Chair. This book is all about using reference materials. The editors have been closely involved with the users of reference materials for many years: they Dr. Markus Stoeppler, Dr. Wayne Wolf and Peter Jenks. In the discussions that lead up to commissioning this book I talked to them about the world of reference materials and asked them what drove them to undertake this work. Markus told me that Wayne was perhaps the first person to see the need to plan a special RM meeting, and that he and Markus had already met, in the I ~ ~ o before ’s, this meeting was organized. Markus said of those early days: “We both felt from the beginning of our co-operation that we should spend a significant part of our scientific life to the improvement of analytical science, particulary the reliability of methods”. Wayne‘s work was in basic analytical methodology and food science. He worked at the US Department of Agriculture Human Nutrition Laboratories in Beltsville, just outside Washington DC. Markus work was devoted to the development and improvement of methods for the analysis of elements. They both recognized in the early 1970’sthe “must” of using reference and so-called “control”materials. In 1971 Markus became responsible for a new analytical section in his institute at the Research Center Juelich, Germany, better known as the “KFA”. This section participated, from that time, in many international analytical inter comparisons, including those within IUPAC and subsequently in numerous BCR certification campaigns. At that time Markus first met, and then worked for decades with Ben Griepink, BCR, who has written a thoughtful foreword to this volume, and this scientific partnership remained valid until Markus’ retirement. Another analytical impetus came from the joint efforts with colleagues from NISTwith the 1979 start of the joint USGerman Environmental Specimen Banking program. In the course of the first BERM meeting, wich later became the BERM series of RM symposia both Marlms and Wayne had the impression that in addition to the special Symposia Issues that gave just impressions on the “state of the RM art” there was a real need for a well organized book to guide the interested reader systematically through the numerous steps and pitfalls of RM planning, production,

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I use. Wayne and Markus started, together with a number of willing co-authors, in About the genesis of this book

the late 1980, to prepare a handbook on the use of reference materials, but for a number of reasons it did not reach fruition. A new stimulus came when Wayne and Markus met Peter Jenks at the 6” BERM Symposium in Hawaii in 1994. Peter was working for Promochem GmbH in Germany, a small company that had developed a unique position as a supplier of reference materials produced by a wide range of governmental agencies around the world. Wayne said of that meeting “We were impressed by Peter’s experience in the RM business and clear formulated ideas how the users of RMs should be educated to avoid the nasty errors that often change the expected benefit of using reference materials into just its opposite”. Peter had started work as a Clinical Chemist, studying under Prof. Tom Whithead at the Wolfson Institute in Birmingham, EngIand. He moved into the commercial world, spending 13 years with BDH Chemicals Ltd in England, before moving to Promochem. Peter told me: “Working with Promochem I began to realize that the application of Quality Systems, reference materials, proficieny testing, laboratory accreditation, and so on, wich had been pioneered by Tom Whithead in the UK Clinical Chemistry laboratoris of the early 1970’s had not yet migrated fully to mainstream analytical chemistry. The problems I had faced as a young scientist in the mid 1g7o’swere still challenging analysts in the mid ~ggo’s”. Many discussions, continued after this symposium, showed that the three were thinking along very similar, almost identical lines. They wanted to include the wisdom and ideas of the many scientists they had worked with, and to write a cohesive book, not a collection of review articles, including just the necessary basic information and avoiding too much theory and lengthy chapters. So, based on former ideas but with regard to the rapid development in electronic media, especially the Internet and e-mail which would allow regular exchange of text and ideas between authors and editors, they designed this book as a guide for practitioners. At the Berm 7 Meeting, held in Antwerp during 1997, they agreed with VCH, now Wiley-VCH, to publish the book. A favorable situation was that Markus, now a retired person, had time enough to assist his co-editors in completing the many urgent organizational and technical tasks needed to bring the manuscripts to completion. Needless to say that this project had not to be completed without the help of many of the editors colleagues (the reader may be suprised by the relatively large number of contriburors) from around the world, all of whom are highly respected experts. This book is published to coincide with the 8thBERM meeting, held in Bethesda, Maryland, in 2000. We do expect that this work wilI be useful for newcomers as well as for more experienced readers. Since there is another book an reference materials due to be released at the same time, edited by Adolf Zschunlte, BAM, Germany, we have cooperated with Prof. Zschunlte to try reduce any overlap with hope that both books will be complementary rather than competitive. Steffen Pauly, Wiley-VCH

Reference Materials for ChemicalAnalysis Certification, Availability, and Proper Usage

Edited by Markus Stoeppler,Wayne R. WOK PeterJjenks 0 Wiley-VCH Verlag GmbH, 2001

Subject Index a Airborne contaminants 197,

198

Analytical methods, general Elemental content 62, 63 Organic constituents 91-

96,286,287 Organometallic species 76ff. 286 Preconcentration,isolation, separation 61 Sample pretreatment 61 AOAC 155 ASTM,USA 155 ATTC, USA 2 , 154-157,160

b BAM,Germany 3 BAS Ltd., UK 3 BCR, 5 (organizationnow SM&T) BCR CRMs Clinical analysis 86, 205, 210

Elements and Elemental Species 65 67,72,79,80,

87,197,205,210,215-219 Microbiology 156 Organic constituentsin matrix CRMs 86, 88,

98,100,126,127,197, 7-19

Pure organic substances and calibrationsolutions 84,

85

Vitamins, food 86,87 BERM/BRM Symposia 270-

272.279 BGS, UK 222,223

BNM, France 3 Bowens Kale 4,216 Preparation 26,59 C

Calibration with CRMs 133 ff., 223,224,251 Certification examples, elements BCR CRM 482, Lichen 65 NIST SRM 1573a, Tomato Leaves 70.71 NIST SRM 1633a, Coal Fly Ash 69 NIST SRM 2704, River Sediment 65 Certification examples, organic constituents Benzo[b]fluoranthene:BCR CRM 088, Sewage Sludge 98 PAHs: NIST SRM 1649a, Urban Dust 93.96 Certification, BCR approach

97-101 Certification,NIST approach Definitions 89-92 Certified, Reference and Information values 89 CITI, Japan 201 CRM 3 Definitions 8,49 CRMs for Elemental species Separationand detection methods 77- 82 CRS 172-192

d DGKC, Germany 201 DNA analysis 160ff., 283 DNA fingerprinting 157,160 DUREM Conferences, India

272,291 e

Error detection/NAA 73,74 EURACHEM 268

f

FB1,USA 160 Frozen RMs 122

g

GBW CRMs of NRCCRM, China 6 Elements 216,217 Radioisotopes 145 Geological CRMs, table 221

h Homogeneitydeterminabon, definition Homogeneity factors 136 Ingamells sampling constant 130,132 Kurfbrst relative homogeneityfactor 132,

I37

Homogeneity determination, methods INAA 33-34,62,66-75 PIXE 35 SS-AAS, SS-GFAAS 31.33,

35,218

SS-ETV-ICP-AES/MS 36,

141

296

I

Subject fndex SS-ZAAS 3536 Various for organic compounds 36

i IAEA 4,144-146,291 IAEA CRMs Metals and organometallic species 73,74,80,137, 221

Radioisotopes, stable isotopes 145 IFCC 200 Information sources Catalogues 4,191, 257 Comar database 7,262265 IAEA database 7, 257 Internet 257 Journals 258-2 GI Search engines 262 Website addresses table 266 WinRefPro database 265 IRMM 5,23,37,41,146,147, 171,172 IRMM CRMs DNA materials 171 Stable isotopes 146,147 IS0 7,156,267 I S 0 Guides Guide 30 8 , q . 50,174,237 Guide 31 8,174,237.242 Guide 32 8,223,237 Guide 33 9,1o, 237 Guide 34 10~40,174,237 Guide 35 I I , I ~14,40,223, , 237 ISO-REMCO 6,172,285 ISOtypeRMs 175 IUPAC 75,200

j

JGS (Japanese)geological CRMs 221

I

LGC, UK 5,282,289 Life analysis DNA fingerprinting 154, I57 Molecular taxonomic tests I57

Polymerase Chain Reaction ( P W I54

m Microbiological assays Assay standards 183 Bacteria etc. 153 GMOs 153,171 Microbiological Culture Materials Enterococcusfaen’um 158, I59 Salmonella typhirnurium 158 Microbiological RMs 158 Production 159 Microchemistry,definition 127 Mil@-KEMI,Denmark CRMs I97 Movement of RMs Custom tariff numbers 274 Health and safety data sheet 191,275 Labelling 274 REMCO Guide 276 n NBS 2,jff. NCCLS, USA 155,200 NIBSC,UK 201 NIES 6 NIES CRMs 30,215 NIOH CRMs, Norway 198 NIST 2,160-172, see below NIST SRMs Clinical analytes 86,205, 209,210

Elements/various matrices. 64,67969,70-731797 136,137,198,199, 205, 210,215-219,221 Human DNA analysis 160-170,172 Organic constituents 86, 87?96 Pure organic substances and calibration solutions 85 Radioisotopes and radiopharmaceuticals I459 147 Vitamins, food 86

NIST traceable RMs 283,285 Nitrogen concentrations in RMs 216,217 NRCC, Canada 5 NRCC CRMs, Elements/elemental species 29,67,80,215,218,219 Organic constituents 5 NRCCRM, China 6 NWRI, Canada 5

P

PAHs 91ff. PCBs 91ff. Pharmaceutical analyses 172, I73 Pharmacopoeiae BP (British Pharmacopoeia) 274 EP (European Pharmacopoeia) 173192,2737 274 USP (United States Pharmacopoeia) I, 155, 273 Pharmacopoeia1Reference Substances (PRS) Candidate RS 181 Certificates 191 Evaluation 182 Expirydate 191 Identification 175,182 International harmonization 192 Purity control 179 Related substance test 176, 183 Storage and distribution 192 System suitability tests 176 Used for assay 179,183 Uses, overview 176 Promochem 240,25g,289 Proper usage of RMs Consensus mean 117 Consensus values 119 External Quality Assessment 2,112,118 External Quality Assurance 117-119 Internal QC 113 Laboratory accreditation 115

Subject Index I 2 9 7

Levis-Jenningschart 115, Measurement bias 9,1o Measurement traceability

237

Performance score 117 Proficiency testing (PT)

112,280,282 Quality Assurance (QA)

236,279

Quality Control (QC)

Clinical tissues and fluids

28

116

112,

236

Reporting errors 246,247 Shewhart chart 246 Total Quality Management (TQM) 236,256 PTB, Germany 3

4

Quality management Good analybcal practice I20

Good laboratory practice

156

Good manufacturing practice 156

Foods and agricultural products 27 Plant tissues 26 Sediments 30 Soils 29 Wastes and sludges 29 Waters 29 RM producers and distributors 258,259 RM usage, driving forces 281 RMs, definition 8 RMs, general Analytes (measurands) 22,

5

Secondary RMs 201,284 SM&T (formerly BCR) 59.84 Solid sampling techniques Applications 138-143 Calibration curve 140 Methods 128 Specimen Bank 7 SRM, Trade Mark of NIST 3 Statistical definitions Confidence interval 115,

245,246,248,249

Expanded uncertainty 95 Prediction interval 246,

248

Standard deviation of certified value 248,249 Uncertainty budget 135,

62,63

Characterization, certification z5,26,49 ff. Classes 21,22, 27 Collection, preparation 15, 22 ff. Contamination 243 Documentation 26 Drying, drying conditions

136,245,247,252

Uncertainty intervals 245 Uncertainty value 95,245,

247

Uncertainty, uncertainty calculations 10,13, 95, 135,136,252

24,242,243

Expiry date 31,41,43,191,

241

r REMTAF, India 272 RIVM,NL 159 RM artifacts Methyl mercury 80, 244 RM certification, general Bar charts 65, IOO Definitive methods 52 Independent reference and validated methods 54, 55 Independent reference methods 53, 55 Methods: elemental contents 60-64 Methods: organic constituents 88-101 Specific examples 58-59 Specific methods 57 Statistical evaluation 59,

Grain (particle)size 12,13, 28, 31732,243 Homogeneity/heterogeneity 14,31,33-377 957 98,

95.101 Volunteer analysts - various methods 56,57 RM preparation/examples Animal tissues 26 Ashes and dusts 29

14,3L40-43,959 99,122, 126,127 Sterilization 4,28 Storage 189,241 Water activity 38-40

123-126, 129-134

Homogenization, milling

25,28, 131, 244

Humidity, Karl Fischer titration 37-40 Isochronous measurements 41,42 Material safety data sheets

1917 275

Material selection 24 Methods: properties 31 Packaging, filling 28,122-

t TBT 76 TDRM 267 TPhT 76 Traceability definition 8,249,250 examples 59, 111, 251 legal traceability 250 Measurement traceability

237

SIunits

250

U

USDOD 155 USFDA 155 USGS 3,222ff. USGS materials

221

124,190

Sample size 242 Shelflife, stability 7,11, 12,

V

VAM Programme, UK 269 W

WHO 200 WHO RMs Z

205,210

ZERO RMs 288,289