Manual of Pesticide Residue Analysis, Volume 2

DFG

Manual of Pesticide Residue Analysis Volume II

VCH

© VCH Verlagsgesellschaft mbH, D-6940 Weinheim (Federal Republic of Germany), 1992 Distribution: VCH, P.O. Box 101161, D-6940 Weinheim (Federal Republic of Germany) Switzerland: VCH, P.O. Box, CH-4020 Basel (Switzerland) United Kingdom and Ireland: VCH (UK) Ltd., 8 Wellington Court, Cambridge CB1 1HZ (England) USA and Canada: VCH, 220 East 23rd Street, New York NY 10010-4606 (USA) ISBN 3-527-27017-5 (VCH, Weinheim)

ISBN 0-89573-957-7 (VCH, New York)

DFG Deutsche Forschungsgemeinschaft

Manual of Pesticide Residue Analysis Volume II Edited by Hans-Peter Thier and Jochen Kirchhoff Working Group "Analysis"

Pesticides Commission

VCH

Deutsche Forschungsgemeinschaft Kennedyallee 40 D-5300 Bonn 2 Telefon: (0228) 885-1 Telefax: (0228) 8852221

Published jointly by VCH Verlagsgesellschaft mbH, Weinheim (Federal Republic of Germany) VCH Publishers Inc., New York, NY (USA)

Translators: J. Edwards t and Carole Ann Traedgold

Library of Congress Card No. applied for.

A catalogue record for this book is available from the British Library.

Deutsche Bibliothek Cataloguing-in-Publication Data: Manual of pesticide residue analysis / DFG, Deutsche Forschungsgemeinschaft, Pesticides Commission. Ed. by Hans-Peter Thier and Jochen Kirchhoff. [Transl.: J. Edwards and Carole Ann Traedgold]. Weinheim; Basel (Swizerland); Cambridge; New York, NY: VCH. NE: Thier, Hans-Peter [Hrsg.]; Deutsche Forschungsgemeinschaft / Kommission fur Pflanzenschutz-, Pflanzenbehandlungs- und Vorratsschutzmittel Vol. 2 (1992) ISBN 3-527-27017-5 (Weinheim ...) ISBN 0-89573 957-7 (New York)

© VCH Verlagsgesellschaft mbH, D-6940 Weinheim (Federal Republic of Germany), 1992 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation into 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 a 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. Composition: Filmsatz Unger & Sommer GmbH, D-6940 Weinheim. Printing: betz-druck Gmbh, D-6100 Darmstadt 12 Printed in the Federal Republic of Germany

Preface During more than two decades, the Working Group on Pesticide Residue Analysis of the "Senatskommission fur Pflanzenschutz-, Pflanzenbehandlungs- und Vorratsschutzmittel" (Pesticides Commission), Deutsche Forschungsgemeinschaft (DFG), has edited a loose-leaf Manual of residue analytical methods. All the methods contained in this Manual were validated prior to their publication, by at least one independent laboratory. Therefore, the Manual has met with acceptance far beyond the frontiers of the Federal Republic of Germany, particularly since many of the methods are included in the List of Recommended Methods of Analysis issued by the Codex Committee on Pesticide Residues (CCPR) of the FAO/WHO Codex Alimentarius Commission. Many residue analysts are, however, not well versed in German. Therefore, to overcome this language barrier and to render the methods accessible to a far wider international circle of analysts, the Working Group decided to translate the most important sections of the Manual into English. This mission was sponsored by the Deutsche Forschungsgemeinschaft. Volume 1 of the English edition was published in 1987. It contained 23 compound-specific ("single") analytical methods selected from the 6th and 7th instalments (issued in 1982 and 1984, respectively) of the German edition, 17 multiresidue analytical methods and 6 cleanup methods (both 1984 status) as well as all pertinent general sections, e. g. on the collection and preparation of samples, on the limits of detection and determination, and on micro methods and equipment for sample processing. The present Volume 2 of the English edition is a direct continuation and completion of the first volume. It contains 32 single methods, many of them designed for the determination of recently developed compounds. These methods were adopted, in most cases, from the 8th to 11th instalments of the German edition issued between 1985 and 1991. Furthermore, Volume 2 contains five new multiresidue analytical methods (coded S) published in German since the first volume went to press, and some tables providing supplementary data on the broad applicability of Methods S 8 and S 19 and Cleanup Method 6, both described in Volume 1. Special features of Volume 2 are Part 5, presenting six multiple methods for analysis of residues in water (coded W), and Part 6 on analytical methods for determining residues in water using the Automated Multiple Development (AMD) technique. Moreover, two new cleanup methods for the solid-phase extraction of water samples on alkyl-modified silica gel are included. An additional chapter introduces a new concept for deriving the limits of detection and determination by the calibration curve technique, thus providing a commendable alternative to the procedure proposed in Volume 1. Finally, a comprehensive table gives massspectrometric El data for confirmation of gas-chromatographic results. In some cases, the Editorial Committee has also partly changed or updated the original German version in order to better adjust it to the needs of today's methodology. A cumulative index for Volumes 1 and 2 provides easy access to all pertinent compounds and information. The Working Group on Pesticide Residue Analysis had hoped that it would render a major contribution to pesticide residue analytical methodology by carrying on the German and English editions. However, the Working Group had to terminate its activities in 1989, after so many years of engagement in matters of pesticide residue analysis, because the Senate of

VI

Preface

the DFG modified the basic structures of its advisory commissions with the consequence that the mandate of the Pesticides Commission and its Working Groups expired. Nevertheless, the Editorial Committee (J. Kirchhoff (chairman), H. Frehse, H.-G. Nolting, H.-P. Thier) was charged, by the DFG, with the commitment to finalize any going publication activities. Thus, the Committee first edited the last, 11th Instalment (issued 1991) of the German Manual on the basis of the validated methods that had become available yet by 1989. Next, the Editorial Committee did its very best to compile Volume 2 of the English edition. Parts of it are based on the very competent contributions of the late James Edwards (died 1987) who had translated the text of Volume 1. The remaining text was basically translated by Carole Ann Traedgold and edited by the Committee. The Editorial Committee hopes that this two-volume compilation of procedures and methods will prove useful to all concerned with the analysis of pesticide residues.

Contents Contents of Volume 1 Senate Commission for Pesticides, Deutsche Forschungsgemeinschaft Working Group on Residue Analysis, Senate Commission for Pesticides Part 1: Introduction and Instructions (contd.) Derivation of the Limits of Detection and Determination Applying the Calibration Curve Concept Mass-Spectrometric El Data for Confirmation of Results Part 2: Cleanup Methods (contd.) Cleanup Method 6. Cleanup of crude extracts from plant and animal material by gel permeation chromatography on a polystyrene gel in an automated apparatus (updated) Cleanup Method 7. Solid phase extraction of water samples on alkyl-modified silica gel using disposable columns Cleanup Method 8. Solid phase extraction of water samples on alkyl-modified silica gel Part 3: Individual Pesticide Residue Analytical Methods (contd.) Amitrole, 4-A*) Anilazine, 186 Benomyl, Carbendazim, Thiophanate-methyl, 261-378-370 Bitertanol, 613-A Bitertanol, Triadimefon, Triadimenol, 613-425-605 Bromoxynil, Ioxynil, 264-212 Carbendazim, 378 Carbosulfan, Carbofuran, 658-344 Chlorflurenol, Flurenol, 275-215 Chloridazon, 89-A Chlorsulfuron, Metsulfuron, 664-672 Copper Oxychloride, 147-A Cymoxanil, 513 2,4-D, Dichlorprop, 27-A-38-A Dichlobenil, 225-A Dichlofluanid, Tolylfluanid, 203-371 Dichlofluanid, Tolylfluanid, 203-A-371-A Dinobuton, Binapacryl, 255-8 Fonofos, 288 Fosetyl, 522

IX XI XV

3 25

31 37 41 49 59 69 77 87 99 107 113 127 135 145 153 157 163 169 177 191 197 205 211

*) Code numbers according to which the analytical methods are identified in the German issue of the Manual. The number without affixed letter corresponds to the BBA registration number of the individual compound.

VIII

Contents

Glufosinate, 651 Glyphosate, 405 Metaldehyde, 151-A Metribuzin, 337 Nitrothal-isopropyl, 416 Oxamyl, 441 Phenmedipham, 233-B Propachlor, 310 Propiconazole, 624 Sulphur, 184-B Thiabendazole, 256-A Thiabendazole, 256-B

217 229 239 245 253 261 269 275 281 287 291 295

Part 4: Multiple Pesticide Residue Analytical Methods (contd.) Pesticides, Chemically Related Compounds and Metabolites Determinable by the Multiresidue Methods in Parts 4 to 6: Supplement to the Table of Compounds, pp. 221 ff, Vol. 1 S 8 Organohalogen, Organophosphorus and Triazine Compounds (updated) S 19 Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides (updated) S 22 Natural Pyrethrins, Piperonyl Butoxide S 23 Pyrethroids S 24 Organotin Compounds S 25 Methyl Carbamate Insecticides S 26 Phthalimides

317 323 333 343 349 359

Part 5: Multiple Pesticide Residue Analytical Methods for Water W 4 Phenoxyalkanoic Acid Herbicides W 5 Fungicides W 6 Organochlorine Insecticides W 7 Phenoxyalkanoic Acid Herbicides W 8 Triazine Herbicides W 13 Desalkyl Metabolites of Chlorotriazine Herbicides

369 377 387 393 403 413

Part 6: Pesticide Residue Analytical Methods for Water Using the AMD Technique Thin-Layer Chromatographic Analysis of Pesticides and Metabolites Using the Automated Multiple Development (AMD) Technique Examples for Applying the AMD Technique to the Determination of Pesticide Residues in Ground and Drinking Waters Cumulative Indexes for Volumes 1 and 2 Index of Determinable Pesticides, Metabolites and Related Compounds (Index of Compounds) Index of Analytical Materials List of Suppliers Referenced in the Text-Matter of the Manual Author Index

301 313

423 435

449 459 479 483

Contents of Volume 1 Senate Commission for Pesticides, Deutsche Forschungsgemeinschaft Members and Guests of the Working Group on Residue Analysis, Senate Commission for Pesticides Part 1: Introduction and Instructions

Explanations Notes on Types and Uses of Methods Important Notes on the Use of Reagents Abbreviations Preparation of Samples Collection and Preparation of Soil Samples Collection and Preparation of Water Samples Use of the Term "Water" Micro Methods and Equipment for Sample Processing Limits of Detection and Determination Reporting of Analytical Results Use of Forms in the Reporting of Analytical Results Part 2: Cleanup Methods

Cleanup Method 1. Separation of organochlorine insecticides from hexachlorobenzene and polychlorinated biphenyls Cleanup Method 2. Cleanup of crude extracts from plant and animal material by sweep codistillation Cleanup Method 3. Cleanup of crude extracts from plant material by gel permeation chromatography on Sephadex LH-20 Cleanup Method 4. Cleanup of crude extracts from plant material by gel permeation chromatography on polystyrene gels Cleanup Method 5. Cleanup of large quantities of fats for analysis of residues of organochlorine and organophosphorus compounds Cleanup Method 6. Cleanup of crude extracts from plant and animal material by gel permeation chromatography on a polystyrene gel in an automated apparatus Part 3: Individual Pesticide Residue Analytical Methods

Acephate, Methamidophos, 358-365 Aldicarb, 250 Captafol, 266 Captafol, 266-A Captan, 12-A Chlorthiophos, 465 Dalapon, 28 Dichlobenil, 225

X

Contents of Volume 1

Diclofop-methyl, 424 Ethylene Thiourea, 389 Folpet, 91-A Heptenophos, 427 Metalaxyl, 517 Methomyl, 299 1-Naphthylacetic Acid, 434 Nitrofen, 340 Paraquat, 134-A Pirimicarb, 309 Pirimiphos-methyl, 476 Pyrazophos, 328 Tetrachlorvinphos, 317 Triazophos, 401 Vinclozolin, 412 Part 4: Multiple Pesticide Residue Analytical Methods Pesticides, Chemically Related Compounds and Metabolites Determinable by the Multiresidue Methods (Table of Compounds) S6 Substituted Phenyl Urea Herbicides S 6-A Substituted Phenyl Urea Herbicides S 7 Triazine Herbicides S 8 Organohalogen, Organophosphorus and Triazine Compounds S9 Organochlorine and Organophosphorus Pesticides S 10 Organochlorine and Organophosphorus Pesticides S 11 Potato Sprout Suppressants Propham and Chlorpropham S 12 Organochlorine Pesticides S 13 Organophosphorus Insecticides S 14 Triazine Herbicides and Desalkyl Metabolites S 15 Dithiocarbamate and Thiuram Disulphide Fungicides S 16 Organophosphorus Pesticides with Thioether Groups S 17 Organophosphorus Insecticides S 18 Bromine-Containing Fumigants S 19 Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides S 20 Phthalimide Fungicides (Captafol, Captan, Folpet) S 21 Ethylene and Propylene Bisdithiocarbamate Fungicides Indexes Index of Determinable Pesticides, Metabolites and Related Compounds (Index of Compounds) Index of Analytical Materials List of Suppliers Referenced in the Text-Matter of the Manual Author Index

Senate Commission for Pesticides, Deutsche Forschungsgemeinschaft Members

Prof. Dr. Rudolf HeitefuB (Chairman from 1987 to 1989)

Institut ftir Pflanzenpathologie und Pflanzenschutz der Universitat GrisebachstraBe 6, D-3400 Gottingen-Weende

Prof. Dr. Horst Borner

Institut fur Phytopathologie der Universitat OlshausenstraBe 40/60, D-2300 Kiel

Dr. Dietrich Eichler

Shell Forschung GmbH D-6501 Schwabenheim

Dr. Helmut Frehse

Bayer AG, PF-A/CE-RA, Pflanzenschutzzentrum Monheim D-5090 Leverkusen-Bayerwerk

Dr.-Ing. Siegbert Gorbach

Hoechst AG, Analytisches Laboratorium, Pflanzenschutz-Analyse, G 864 Postfach 800320, D-6230 Frankfurt 80

Prof. Dr. Friedrich GroBmann

Institut ftir Phytomedizin der Universitat Hohenheim Otto-Sander-Stral3e 5, D-7000 Stuttgart 70

Prof. Dr. Hans-Jurgen Hapke

Institut ftir Pharmakologie der Tierarztlichen Hochschule Bischofsholer Darnm 15, D-3000 Hannover 1

Dr. Manfred Herbst

Asta Pharma AG WeismtillerstraBe 45, D-6230 Frankfurt 1

Dr. Giinther Hermann

Bayer AG, PF-A/CE-Okobiologie, Pflanzenschutzzentrum Monheim D-5090 Leverkusen-Bayerwerk

Dr. Wolf-Dieter Hormann

Division Agrochemie der CIBA-GEIGY AG CH-4002 Basel/Schweiz

Dr. Hans Th. Hofmann

Lorscher StraBe 10, D-6700 Ludwigshafen

Dr. Horst Hollander

Hoechst AG, Toxikologie-Gewerbetoxikologie Postfach 800320, D-6230 Frankfurt 80

Prof. Dr. Georg Kimmerle

Bayer AG, Institut ftir Toxikologie Friedrich-Ebert-StraBe 217, D-5600 Wuppertal 1

Dr. Jochen Kirchhoff

Institut ftir Phytomedizin der Universitat Hohenheim Otto-Sander-StraBe 5, D-7000 Stuttgart 70

XII

Senate Commission for Pesticides

Prof. Dr. Fred Klingauf

Biologische Bundesanstalt fur Land- und Forstwirtschaft Messeweg 11-12, D-33OO Braunschweig

Dr. Claus Klotzsche

Bruelweg 36, CH-4147 Aesch/Schweiz

Prof. Dr. Werner Koch

Institut fur Pflanzenproduktion in den Tropen und Subtropen der Universitat Hohenheim Kirchnerstrafle 5, D-7000 Stuttgart 70

Prof. Dr. Ulrich Mohr

Abteilung fur experimentelle Pathologie der Med. Hochschule Konstanty-Gutschow-Strafle 8, D-3000 Hannover 61

Prof. Dr. Friedrich-Karl Ohnesorge

Institut fur Toxikologie der Universitat Moorenstrafk 5, D-4000 Dusseldorf 1

Prof. Dr. Christian Schlatter

Institut fur Toxikologie der ETH und Universitat Zurich Schorenstrafle 16, CH-8603 Schwerzenbach/Schweiz

Prof. Dr. Heinz Schmutterer

Institut fiir Phytopathologie und angewandte Entomologie der Universitat Ludwigstrafk 23, D-6300 Gieften

Prof. Dr. Fritz Schonbeck

Institut fiir Pflanzenkrankheiten und Pflanzenschutz der Universitat Herrenhauser Strafie 2, D-3000 Hannover 21

Prof. Dr. Fidelis Selenka

Institut fiir Hygiene der Ruhr-Universitat Postfach 102148, D-4630 Bochum

Prof. Dr. Hans-Peter Thier

Institut fiir Lebensmittelchemie der Universitat Piusallee 7, D-4400 Miinster

Dr. Ludwig Weil

Institut fiir Wasserchemie und Chemische Balneologie der Technischen Universitat Marchioninistrafie 17, D-8000 Miinchen 70

Prof. Dr. Heinrich Carl Weltzien Institut fiir Pflanzenkrankheiten der Universitat Nuflallee 9, D-5300 Bonn 1

Permanent Guests Prof. Dr. Fritz Herzel

Bundesgesundheitsamt Postfach 330013, D-1000 Berlin 33

Prof. Dr. Alfred-G. Hildebrandt

Institut fiir Arzneimittel des Bundesgesundheitsamtes Postfach 330013, D-1000 Berlin 33

Senate Commission for Pesticides

XIII

Secretaries of the Senate Commission for Pesticides Frau Dr. Dagmar Weil until 1986

Institut fur Wasserchemie und Chemische Balneologie der Technischen Universitat Marchioninistrafie 17, D-8000 Munchen 70

Dr. Friedhelm Dopke from 1987 to 1989

Institut fur Pflanzenpathologie und Pflanzenschutz der Universitat Grisebachstr. 6, D-3400 Gottingen-Weende

Assessor Wolfgang Bretschneider t 1990

Deutsche Forschungsgemeinschaft Kennedyallee 40, D-5300 Bonn 2

Working Group on Residue Analysis, Senate Commission for Pesticides Members and Guests

Dr. Hans-Gerd Nolting Biologische Bundesanstalt fur Land- und Forstwirtschaft (Chairman from 1988 to 1989) Messeweg 11-12, D-3300 Braunschweig Prof. Dr. Hans-Peter Thier Institut fur Lebensmittelchemie der Universitat (Chairman from 1976 to 1988) Piusallee 7, D-4400 Munster Prof. Dr. Hans Zeumer t 1988 (Chairman from 1961 to 1976) Dr. Gtinther Becker

Chemisches Untersuchungsamt Charlottenstrafie 8, D-6600 Saarbrucken

Prof. Dr. Winfried Ebing

Biologische Bundesanstalt fur Land- und Forstwirtschaft Konigin-Luise-Straite 19, D-1000 Berlin 33

Dr. Siegmund Ehrenstorfer

Landesuntersuchungsamt fur das Gesundheitswesen, Fachabteilung Chemie Fritz-Hintermayr-Strafle 3, D-8900 Augsburg

Dr. Dietrich Eichler

Shell Forschung GmbH D-6501 Schwabenheim

Dr. Helmut Frehse

Bayer AG, PF-A/CE-RA, Pflanzenschutzzentrum Monheim D-5090 Leverkusen-Bayerwerk

Dr. Ing. Siegbert Gorbach

Hoechst AG, Analytisches Laboratorium, Pflanzenschutz-Analyse, G 864 Postfach 800320, D-6230 Frankfurt 80

Prof. Dr. Fritz Herzel

Bundesgesundheitsamt Postfach 330013, D-1000 Berlin 33

Dr. Wolf-Dieter Hormann

Division Agrochemie der CIBA-GEIGY AG CH-4002 Basel/Schweiz

Prof. Dr. Antonius Kettrup

Fachbereich Chemie u. Chemietechnik der Universitat Postfach 1621, D-4790 Paderborn

Dr. Jochen Kirchhoff

Institut fur Phytomedizin der Universitat Hohenheim Otto-Sander-StraBe 5, D-7000 Stuttgart 70

Prof. Dr. Hans Maier-Bode

Tannenweg 7, D-7884 Rickenbach b. Sackingen

XVI

Working Group on Residue Analysis

Dr. Egon Mollhoff

Bayer AG, PF-A/CE-RA, Pflanzenschutzzentrum Monheim D-5090 Leverkusen-Bayerwerk

Dr. Ludwig Weil

Institut fur Wasserchemie und Chemische Balneologie der Technischen Universitat Marchioninistrafie 17, D-8000 Miinchen 70

Editorial Committee

Prof. Dr. Hans Zeumer t (Former Chairman, until 1986) Dr. Jochen Kirchhoff (Present Chairman, appointed in 1986) Dr. Helmut Frehse Dr. Hans-Gerd Nolting Prof. Dr. Hans-Peter Thier

Parti Introduction and Instructions

Derivation of the Limits of Detection and Determination Applying the Calibration Curve Concept (German version published 1991)

1 Introduction It is a familiar experience in trace analysis that analytical results can become uncertain or even entirely unreliable if the substance to be analyzed (the analyte) is present in very low concentrations. This can be due to various causes which can also occur simultaneously, e. g.: — Co-extractives from the matrix simulate the analyte, thus leading to blank values. — The analyte is lost during the cleanup in varying proportions, so that the results from parallel analyses vary to an unacceptable extent. — The minute amounts of the analyte are not, or are only inadequately substantiated by the measuring system. Consequently there are three categories in which an analytical result can fall: A. The presence of the analyte is shown; a quantitative determination is possible. B. The presence of the analyte can indeed still be shown, but a reliable quantitative determination is no longer possible. C. The presence of the analyte can no longer be established with sufficient probability; the analyte must, therefore, be considered as "not detectable". Categories A and B are separated by the limit of determination (LDM), categories B and C by the limit of detection (LDC). For these reasons, a convention needs to be established on how to define LDM and LDC. *) Both can be used in different respects: 1. By specifying LDM and/or LDC, the author of an analytical method can give other analysts using the method an indication as to its performance. 2. The analyst can more accurately characterize his findings with the aid of LDM and/or LDC, e.g. by presenting a result (in case B only!) as "Content of compound X in the sample < [LDM]", or in case C, as "Compound X not detectable in the sample, LDC = [LDC]". The letters in brackets denote the numerical value for either LDM or LDC; see also p. 45, Vol. 1.

*) In the absence of a defined limit of determination, it may be expedient for the analyst to use the routine limit of determination (RLDM; see p. 43, Vol. 1) as the reporting level, if the analytical problem permits such an approach. In this case, however, the analyst must clearly state that the RLDM was used as the threshold when reporting the result of an analysis as " < ...", thus indicating that quantitation below this level was not attempted and there is no evidence whether or not the analyte is determinable when present in concentrations smaller than the RLDM.

4

Limits of Detection and Determination

Numerical values for LDM and LDC are valid only for each special case of the analyst's instrumental and operating conditions. "Generally valid" statements such as "The method has a LDM (LDC) of ..." are, therefore, not appropriate. The pertinent literature contains numerous recommendations for a mathematical definition of the LDC and/or LDM. Nevertheless, often even the nomenclature is not uniform. Frequently LDC and LDM are used as synonyms; additionally there are other, and sometimes even incorrect terms in use, e.g. "sensitivity". Older recommendations, in part still used today, relate the LDC or LDM to the blank value or instrument noise and their random scatter (standard deviation). The measured signal may then be considered to be significant if its mean value differs from the mean of the blank or noise by a given multiple of the standard deviation. This kind of evaluation, however, is only justified if the errors inherent in the measurement procedure are caused exclusively by the instrumental conditions, e.g. with photometric measurements after a wet ashing, or with establishing a calibration curve from standard solutions in gas chromatography. It is, therefore, not comprehensive enough for application in residue analysis. The results of residue analyses are decisively affected by primary factors from the preceding extraction and cleanup steps, such as variable "recoveries". For use in this Manual, therefore, the derivation of the LDC and, from it, of the LDM, is based upon results obtained from complete analytical procedures. Moreover, the additional Requirements II and III were introduced for the definition of the LDM. The LDM is defined as the smallest value for the content of an analyte in an analytical sample that satisfies the three following requirements: I The LDM is greater than, and significantly different from the LDC. II The recovery (sensitivity) at the LDM is equal to, or greater than 70%. Ill The coefficient of variation at the LDM, from replicate determinations, is equal to, or smaller than 0.2 (equivalent to 20%). The recommendation given in the Section on Limits of Detection and Determination on pp. 37 ff, Vol. 1, still required the existence of blank values for estimating LDC and LDM. However, it also demanded the requirement of the recoveries exceeding 70% to be checked (II) by stepwise fortification and calculation of the regression line. In addition, the smallest fortification level had to meet Requirement III. With the progress of analytical techniques, however, blank values often do not show any more, or are not significant for the interpretation of an analytical result. In order to enable a convention on the definition of the LDC and/or LDM in these cases, the calibration curve concept (also familiar from many publications) is proposed here for application in residue analysis. A special advantage of this concept is that the LDC can be determined with actually measured values, and that neither authentic control samples nor blank values are required. This concept will be presented and mathematically sustained. For its routine application from a series of measurements, the use of a suitably programmed computer is recommended. In individual cases, both limits can be derived graphically, with relatively little calculation effort and with sufficient accuracy, from a plot of the calibration curve and its prediction interval. For an example, see 9.3.

Limits of Detection and Determination

5

2 Calibration curve concept 2.1 Basic considerations The aim of the concept is to define the limit of detection and, resulting from it, the limit of determination for the results obtained when a given analyte is determined with a particular analytical method in an individual laboratory. The definition proceeds from the calibration curve obtained with the analytical method and employs the upper and lower limits of the prediction interval of the curve for deriving LDC and LDM. The prediction interval is used here as the confidence interval. The operation described for determining LDM permits an appropriate consideration of Requirement I. Requirement III is integrated into the formulae used to calculate LDM. Determining the slope of the calibration curve will check Requirement II. 2.2 Establishing the calibration curve To obtain the calibration curve, a series of fortification experiments is run with k given levels

for which the corresponding signal values

are measured, with m, replicate experiments per level Xt. The number of replicate exit periments may be different on each fortification level X{. In total, n = £ m^ value pairs [ l for X and Y will be obtained. = The given levels (X) of the analyte in the samples and the corresponding signal values (Y) are connected by the method-specific calibration function, which will be linear — at least locally — in good approximation. In this case, only few value pairs for X and Y from fortification experiments are required (see 3.3.1). From the total of the n value pairs obtained, the calibration curve is established. It is represented by the regression line which is calculated according to the least squares method. The function equation of the regression line is Y=A+BX where Y = measured signal value for the content X X = content of the analyte in the sample A = intercept on the signal axis at the point X = 0 B = slope of the regression line (sensitivity of the method) The prerequisite to deriving LDC and LDM is a certain minimum value for the slope B of the calibration curve (see 4.3). Fortification experiments which do not meet this requirement are useless and must be repeated under improved and appropriate experimental conditions.

6

Limits of Detection and Determination

Next, the prediction interval is calculated which symmetrically envelopes the linear calibration curve (see Figure 1). The curves for the upper (Y + ) and lower (Y_) limits of the prediction interval define that interval in which future ("predicted") signal values for any content X are to be expected at a selected level of statistical significance.

x=0

X = Concentration

Fig. 1. Calibration curve with upper and lower limits of the prediction interval. Intersection of the line Y = Yo with the prediction interval and the calibration curve; intersection points = Xl5 X3, X2. A = theoretical blank value. The points (Y UP , YLO), where both curves Y + and Y_ intersect with the signal axis, indicate the confidence interval for signal values yielded by samples with a "nil" content. Note that this range is extrapolated from the results of the fortification experiments and is not derived from the measurement of blank values. Each signal value Yo > YUP yields three possible points of intersection with the calibration curve and the limits of its prediction interval. They correspond, respectively, to the values Xj, X 3 and X 2 (Figure 1) and form the basis for further derivations. When the curvature of both the upper and lower limits of the prediction interval is negligible, X 3 can be considered the arithmetic mean of X{ and X 2 with good approximation. X 2 is corresponding to LDC* (Figure 4) and X£ v (Figure 5). Xj and X 2 can be calculated from the formulae IIa and l i b (see 6.6, see also 3.2).

Limits of Detection and Determination

3 Limit of detection (LDC) 3.1 Definition The limit of detection is defined by the smallest content of the analyte in an analytical sample, for which the particular analytical method yields signal values which differ, with a selected level of significance a, from signal values obtained from samples with a "nil" content (blank signal values). The level of significance, a, can be arbitrarily chosen. In most cases, values of a between 5 and 1% will allow a sufficient margin of safety. In residue analyses, usually a level of a = 5%, i.e. a confidence level of S = 1 - a = 95%, is chosen. Based on the n results (X^Yj) from the fortification experiments and the given significance level, a, the limit of detection for an analyte is derived from the calibration curve. It is represented by the smallest value X L D C for which the confidence intervals of the corresponding signal value Y LDC and of the signal value for a "nil" content do not overlap (Figure 2).

X=0

LDC

X = Concentration

Fig. 2. Definition of the limit of detection (LDC).

3.2 Decision rules The limit of detection corresponds, on the linear calibration curve, to a signal value Y LDC . For the interpretation of further measurements, this implies: - When the measured signal value Y is smaller than Y LDC , the analyte is considered not detectable.

Limits of Detection and Determination

8

— When the measured signal value Y is greater than YLDC, the analytical sample is assigned a content of X = (Y - A)/B (see X3 in Figure 1). The confidence interval belonging to X is equivalent to the range from X{ to X2 in Figure 1. At the limit of detection, the probability for the false proof of detecting the analyte which in reality is not present in the sample (error of the first kind) is just equivalent to the selected a of 5%. This is illustrated in Figure 2: The distribution curves for the signal values A and YLDC each overlap by 2.5 area percent, corresponding to a test with a = 5% (two-sided). The error of the second kind (false negative result in spite of a real content of the analyte being present in the sample) depends on the actual content present. For X > LDC (corresponding to Y > YLDC), it is smaller than 50%, for X = LDC, it is exactly 50% (Figure 3), i. e. a signal value Y > YLDC will be caused, with a probability greater than 50%, by an analyte content >0.

C/3 II

^ s ^

"LDC

V A

_.

X=0

-

ki

LDC

^ -

Y=A+BX

X = Concentration

Fig. 3. Confidence interval of a result X at the point LDC with distribution curve (schematic illustration).

3.3 Determining the limit of detection 3.3.1 Establishing the calibration curve

For the fortification experiments, it is advantageous to use authentic control samples, if available, but other comparable material can also be used provided it does not contain any substances that would interfere with the analysis. The fortification levels extend from the anticipated LDC into the expected working range. Note that the prediction interval which envelopes the calibration curve is narrowest at the point X. Therefore, by suitable experimental planning and by making use of experience available, a higher degree of precision can be reached through choosing fortification levels in the neighbourhood of the anticipated LDC.

Limits of Detection and Determination

9

For best reliability of LDC, more than 4 evenly spaced fortification levels should be used (k > 4), each with several replicate derminations (up to m = 4). However, for economical reasons it will often not be possible for this statistically required number of measurements to be carried out. A substantial reason for this is certainly the fact that for a given analyte, depending on the sample material, different values for LDC can result, so that an accordingly great number of fortification experiments would be required. In general, it will be sufficient to choose 4-5 different fortification levels if the experiments are repeated at least once on each level. Note that it is beneficial to use fewer replicates on a greater number of levels, rather than to carry out more repetitions on fewer levels. The number of replicates may, however, be different on the individual levels. Moreover, increasing the number of fortification levels can, if need be, render it feasible to check the adequacy of the model assumption, e.g. linearity. Although to the disadvantage of statistical precision, in practice it may often be unavoidable to derive the LDC from only one single measurement per fortification level. In this case, however, a minimum of 6-8 measured values is required. Measured values are only valid for the calculations if they represent the results of complete analyses. It would be malpractice, for example, to split an extract obtained from a sample into halves and to analyze these two portions separately in order to get "two" measured values. The performance of the measurement set-up must be thoroughly checked before the fortification experiments are undertaken. Only such instrumentation which is in good condition, complies to the standards, and produces sensitive and reproducible signal values, will be suitable for establishing the calibration curve. Note that the instruments often produce quite different signal values for the same amount of the analyte if the analyses are not carried out consecutively. The signals must not be evaluated if they exhibit a drift, or if their quality declines due to other reasons. Moreover, the signal values must not be adversely affected by co-extractives from the sample material. Using a suitable programmed computer, the parameters of the linear calibration curve and the two limits of the prediction interval can easily be calculated from the individual value pairs X{,Y{. The curves are best drawn by a plotter. For illustration, Figure 6 shows a graph and print-out generated by computer, using Example 1 (cf. 9.1). In Section 9.3, a description is given on how to proceed without the aid of a computer. 3.3.2 Graphical derivation of LDC

In the graph obtained (Figure 2; cf. Figure 7), draw a straight line, parallel with the abscissa, from the point of intersection, YUP, to the point where it intersects the lower limit of the prediction interval. This point corresponds, on the abscissa, to the value of LDC, the limit of detection (cf. 9.3). For computer calculation of LDC, see 6.6.

4 Limit of determination (LDM) 4.1 Definition Residue analyses are frequently performed to monitor foodstuff for compliance with established maximum residue limits. For this reason, both risks, namely erroneously to state the content of a sample either as conforming to, or exceeding the maximum residue limit, must

Limits of Detection and Determination

10

be kept to a minimum. This can only be achieved when the coefficients of variation from replicate determinations are small and systematic errors can be excluded. It is also for these reasons that the limit of determination must fulfill particular requirements, especially when the maximum residue limits to be enforced were set at or about this limit. For the purpose of residue analyses, therefore, the LDM is defined as the smallest content of the analyte satisfying the Requirements I, II and III given in the Introduction. 4.2 Consequences of Requirement I According to Requirement I, the LDM should be greater than, and significantly different from the LDC. This condition is met when the confidence interval (a = 5%) of a concentration determined at X = LDM does not extend into the range below the LDC. If Xj denotes that concentration whose confidence interval just borders the LDC, the LDM cannot be smaller than Xj. Xj can be determined graphically in a simple manner from the plot of the calibration curve (Figure 4; cf. Figure 8): First, the point of intersection, Y c , of the vertical line X = LDC with the upper limit of the prediction interval is determined by drawing a parallel to the Y (signal) axis through the point X = LDC; cf. 9.3 (for formulae to calculate Yc, see 6.7). Next, Xj is obtained as the X value of the intersection of the horizontal line Y = Yc with the calibration line: X! = (Yc - A)/B; see 6.8.

I CO

^ ^

UP A

x=o

Y=A+BX Y_

r LDC

LDC

X = Concentration

Fig. 4. Limit of determination (LDM). Condition: LDM > Xx, with XT = (Yc - A)/B. The value of Xx can be taken as LDM if it can be shown that the Requirements II and III are fulfilled at this concentration as well. However, a value for LDM being greater than X! may be exacted through Requirement III in many cases (see 4.4).

Limits of Detection and Determination

11

4.3 Consequences of Requirement II The usual way to obtain a calibration curve is to plot each signal value (Y) versus the corresponding given content (X) of the analyte in the sample material. For checking Requirement II, however, a different ordinate scaling is used for plotting the line. For this purpose, standard solutions are used in order to determine which amount of analyte results in which signal value (calibration curve for the standard solutions). Next, the individual signal values obtained from the fortification experiments are converted into the corresponding concentrations of the analyte. For the given fortification levels (X) and the corresponding measured concentrations thus obtained (Y), the parameters of the regression line are calculated according to the regression equation given in 2.2. If the slope B of the regression line is B = 1, the recovery is 100%. Requirement II asks for a recovery of > 70% which means that the slope must be B > 0.7. 4.4 Consequences of Requirement III According to Requirement III, the coefficient of variation from replicate determinations at the limit of determination should be equal to, or less than 0.2 (equivalent to 20%). One way of checking whether this requirement is met could be to calculate the coefficient of variation for each fortification level individually. This can, of course, only be done if several repeat measurements (at least m = 4) for each level were made. Then, the LDM is given by the smallest fortification level at which Requirement III is satisfied, provided that Requirements I and II are met as well. This approach corresponds to the procedure recommended on p. 40, Vol. 1. In practice, however, there may be a need to derive the LDC and/or LDM from only one measurement (anyway from m < 4) each per fortification level (see 3.3.1). In such cases, fortunately, the calibration curve concept offers an elegant possibility to obtain the required information in a different manner. Each future determination of a signal value at a definite concentration can be assigned a coefficient of variation V which is given by

Y(X) where SY = standard deviation of a future determination of the signal value Y (X) at the point X Y (X) = signal value on the calibration curve at the point X. According to Requirement III, therefore, a position X must be found from which onward V becomes smaller than the required value Vo = 0.2, presuming that V decreases with increasing values of X. This position X is given by the intersection of the line Y = (A + B • X) • (1 + t • Vo) with the upper limit of the prediction interval (for explanation, see 6.10). X c v is the X coordinate of the intersection point (see Figure 5; cf. Figure 9).

Limits of Detection and Determination

12

-H C/3

^ Y(i+tv0)

x=o

^

^Y=A+BX

—*y

*CV

X

CV

X = Concentration

Fig. 5. Limit of determination (LDM). Condition: LDM > X m , with X in = (Ycv - A)/B.

To determine this line, calculate for two points X' and X" the corresponding values Y' and Y" on the calibration curve, using the equation Y = A + B • X. Both Y' and Y" are each multiplied by the factor (1 + 0.2 • t), where t is the factor t from the formula for the prediction interval, see 6.3 and Table 2. The two points obtained are connected by a straight line. In analogy to 4.2, Y cv is the Y value corresponding to the intersection point X c v . Next, X m is defined as the X value of the intersection point of the line Y = Y cv (parallel to the abscissa) with the calibration curve (cf. 9.3): Xin = ( Y c v - A ) / B . X m is the second lower bound for LDM. 4.5 Determining the limit of determination For the final determination of the LDM, the values obtained for Xj (see 4.2) and X m (see 4.4) are compared. The larger one of the two numbers is the limit of determination, LDM. For formulae to calculate X c v , Y cv and X m , see 6.11-6.13.

5 Comments The concept of deriving the LDC and LDM requires that the calibration curve is linear in the range of the fortification levels used, or that it can be converted to a linear form by a simple coordinate transformation. It is assumed that the variation of the signal values for each for-

Limits of Detection and Determination

13

tification level X follows a Gaussian distribution around the mean value. Moreover, the variation of the signal values must be homogeneous for all fortification levels. For this reason it is advantageous to choose such levels which are close to the anticipated LDC. If these conditions are not satisfied, the calibration curve must be fitted with the aid of a non-linear regression. The Gaussian distribution may possibly be obtained by suitable transformation of the signal values. If the variation of the signal values is dependent on concentration, it must be measured and taken into account in the calculation of the regression, so that the calibration curve concept can be maintained as described, even under aggravated conditions, such as non-linear functions. The fortification levels chosen may include some which one later discovers to lie below the LDC. Although these levels are not significantly distinguishable from blank values, they can nevertheless be used for calculating and constructing the calibration curve. The calibration curve can, alternatively, be obtained by converting the signal values into their corresponding concentrations (see 4.3) and plotting these values on the Y axis instead of the signal values. This form of the calibration curve was used in the Examples (see 9).

6 Mathematical formulation of the concept 6.1 General Here, and in many other aspects of calibration statistics, the low-cost computer programs available for most personal computers (or even pocket calculators) are very helpful. For calculating the regression line (calibration curve) by hand, as well as for further calculations required, first compute the following sums: Formula

Auxiliary Term

E m,X,

E

i= 1 k (mjAj )

r

i= 1

v i yY• i = i Vj = i

iv) i=i\j=i k / mi

/

H

14

Limits of Detection and Determination

6.2 Derivation of the calibration straight line The means of the X and Y values are each determined using the sums obtained from 6.1. — 1 k E X = — £ m; X; = — = mean of the fortification levels X; n i= 1 n — 1 k /mi \ G Y = — E l E Y n ) = — = mean of the corresponding signal values Y; n i = i\j = i '7

n

In addition, the following square and product sums are needed: Formula

Auxiliary '.Term

k

= K == F i= 1 k

(Xi-X)E(Yifj-Y)

T

=J-

=M=H

E2 n EG n G2 n

I? N= ~K

The auxiliary term will also be required.

The terms for the slope, B, and the axis intercept, A, of the calibration straight line are obtained through: L B=—

Slope

A = Y- B • X

Theoretical Blank Value

6.3 Prediction interval (I) Y± = Y + B ( X - X ) ± t s , r S R - | / l + ^ +

(X

X)2 K

t s f = critical value of the t distribution (two-sided) for a confidence level S, and f = n — 2 degrees of freedom (in the following text designated as "t" only) sR = y

—- E (Yj - A - BXj)2 = standard error of estimate: "mean" deviation of the signal values Y{ from the calibration line

Limits of Detection and Determination

15

With £ ( Y - A - B X ) 2 = M - L2/K, the standard error can also be expressed as

6.4 Confidence limits of X = (Y - A ) / B for given signal value Y

K 2

C

= B2 -

(t • sR ; ) = auxiliary term K

In Figure 1, the confidence interval for X 3 is shown, with limits X{ and X 2 . 6.5 Intersection point Y UP (Figure 1) YUP is obtained by setting X = 0 in the formula for the prediction limits, using the positive portion of equation I: (III)

YUP = Y - B X + t - s R -

6.6 Derivation of LDC LDC is obtained by inserting the value for Y UP (6.5) in equation IV:

Equation IV is directly deduced from equation II. This, for describing Figure 1, is taking the form: C

v

C

V \

n

K

Substituting YUP for Yo results in X 2 being identical with LDC, and one obtains (IV).

16

Limits of Detection and Determination

6.7 Calculation of Y c The expression for Yc is immediately obtained from (I) as: (V)

1 (LDC — X) 2 Yc = Y + B (LDC - X) + t - sR • |/ 1 + — + — n

Jv

6.8 Calculation of X, The value obtained for Yc from 6.7, when inserted in equation VI, gives Xji (VI) XI = (Y C -A)/B 6.9 Determination of SY SY is identical with the product of sR and the square root expression in equation I (cf. 6.11). The product t • SY describes half the width of the prediction interval of the calibration line (see Figure 1 and 6.10). 6.10 Definition of X c v Inserting the expression for V, as given in 4.4, in the formulation of Requirement III (4.4), one obtains: -|-:gV0

or

S Y gV 0 -Y(X)

Multiplying both sides with the factor t, and by adding Y(X) on both sides, one obtains: Y(X) + t • SY ^ Y(X) + t • Vo • Y(X) =| Y(X) • (1 + t • Vo) The expression on the left side of the less-or-equal sign is now just identical with the expression for the upper limits of the prediction interval (cf. 6.9). On the right hand side, the values on the calibration straight line are multiplied by the factor (1 +1 • Vo). In the limit of the equal sign, and in graphical interpretation, the formula describes the intersection of the line Y • (1 + t • Yo) with the curve of the upper prediction limits (Figure 5). The X-coordinate of the point of intersection is X c v . 6.11 Derivation of X c v Solving the equation Vo • Y = SY for X gives the value for X c v , whereby 1 -

Limits of Detection and Determination

17

and

Squaring both sides gives

(V0)2(Y + BZ)2 = (l + i + ^pj (sR)2 with Z = X c v - X . This is a quadratic equation for Z which can be solved for Z in a straightforward way, leading to the following solution:

(VI.)

K D

(ST.)2

= (B V o ) 2 - ^ L J ^- = auxiliary term; K.

(Vo)2 = 0.04

6.12 Calculation of Y c v The value for Y, corresponding to the point of intersection, is calculated by inserting Xcv in (VIII): (VIII) Y c v = (A + B • X c v ) • (1 + t • Vo)

6.13 Calculation of X m (IX) X m = (Y c v -A)/B

7 References C. /. Bailey, E.A. Cox and J.A. Springer, High pressure liquid chromatographic determination of the intermediates/side reaction products in FD & C Red No. 2 and FD & C Yellow No. 5: Statistical analysis of instrument response, J. Assoc. Off. Anal. Chem. 61, 1404-1414 (1978). L. Oppenheimer, T.P. Capizzi, R.M. Weppelman and H. Mehta, Determining the lowest limit of reliable assay measurement, Anal. Chem. 55, 638-643 (1983). H. Frehse and H.-P. Thier, Die Ermittlung der Nachweisgrenze und Bestimmungsgrenze bei Ruckstandsanalysen nach dem neuen DFG-Konzept, GIT Fachzeitschrift fur das Laboratorium 35, 285-291 (1991).

18

Limits of Detection and Determination

8 Authors German version prepared for publication by: Bayer AG, Research Department TPP 4, Leverkusen, Bayerwerk, H.-E Walter Hoechst AG, Department of Informatics and Communication - Software, Frankfurt/Main, K.-H. Holtz; in collaboration with H. Frehse, S. Gorbach and H-R Thier English version prepared for this Manual by H.-P. Thier and H. Frehse

9 Examples In this chapter, examples will be given on how to derive the LDC and LDM from a series of measurements. The measured values listed in Table 1 will be used for this purpose. Table 1. Measured values from recovery experiments. Example 1 Concentration [|ig/kg] Added (X) Found (Y) 0 20 40 80 100 120 170 200

12 26 45 63 105 115 153 180

Example 2 Concentration [mg/kg] Added (X) Found (Y) 0.03 0.03 0.03 0.03 0.05 0.05 0.05 0.05 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2

0.031 0.027 0.029 0.025 0.037 0.042 0.045 0.047 0.088 0.080 0.093 0.080 0.159 0.177 0.159 0.186

For the calculations to be made, all individual value pairs (measured values vs. respective fortification level) must be used. The degrees of freedom increase with the number of calibration points, n, whereby LDC or LDM may shift to lower values. Therefore, do not form arithmetic means from the individual Y values obtained on a given level X! The formulae needed for deriving the terms and quantities at the different steps of the calculation are given in the text. They will be quoted here either as numbers of equations or in the form of the auxiliary terms as given under 6. For better legibility, the dimension terms (mg/kg or M-g/kg, respectively) are omitted. 9.1 Example 1: Calculation of LDC and LDM Note that in this case only one measurement per fortification level was made. Number of measurements: n = 8.

Limits of Detection and Determination

19

When proceeding according to 6.1 and 6.2, the following results will be obtained: E F G H J

= 730 = 101700 = 699 = 86873 = 93670

X=

91.25,

K = L = M= N =

35087.50 29886.25 25797.88 25456.01

Y = 87.38

The terms L and K, as well as X and Y, yield the parameters of the calibration straight line: B = 0.8518,

A = 9.6516.

The equation of the straight line, therefore, is (with parameters rounded): Y = 9.65 + 0.85 • X. The Intersection Point, Y UP , is obtained from eq. Ill in 6.5, where A can be inserted for Y - BX; t = 2.45 for f = n - 2 = 6, see Table 2: YUP = 31.21. Accordingly, YLO is obtained through YLQ = A — t • sR • square root term in eq. Ill = —11.91 (for quick calculation, use YLO = 2 • A — Y UP ). Note that the calculations outlined thus far must also be made when the procedure is continued graphically "by hand" (see 9.3). The following steps are, however, given for illustration, so that users carrying out the estimation of LDC or LDM either graphically or by computer (cf. Figure 6) can check their results versus the figures given here. Using eqs. II (for C) and IV, one obtains LDC = 48.83. Eq. V yields Y c = 71.28. Y c is the Y value, on the calibration line, for X = Xl.

20

Limits of Detection and Determination

20

40 i

80

100

120

140

160

180

200

Cone, added X (ug/kg) LDC=48.830

LDM=72.3486

Fig. 6. Evaluation of Example 1 by computer. Calibration curve with prediction interval; S = 95%. Therefore, Xr

= 72.35 (cf. eq. VI).

X c v and Y c v are obtained from eqs. VII and VIII, yielding X c v = 37.36, and Y c v = 61.78. Y c v is the Y value, on the calibration line, for X = X m . Therefore, X m = 61.20 (cf. eq. IX). As the result, Xz > X m , and Xj can be regarded as LDM, since Requirement II (see Sections 1 and 4.3) is satisfied by B = 0.85, corresponding to a recovery of 85%. Requirement I is satisfied by LDM > LDC.

9.2 Example 2 For comparison, and in order to demonstrate the importance of choosing suitable fortification levels and measured values, the LDC and LDM are calculated by two different ways in this

Limits of Detection and Determination

21

example. In the first case, all values given in Table 1 are used (n = 16), while in the second case the calibration points obtained for the fortification level 0.2 were disregarded (n = 12). Calculations in analogy to the one outlined in Example 1 will yield the following results:

A B sR YUP YLO

LDC x

i

Xcv YCV x m LDM (rounded)

n = 16 (t = 2.145)

n = 12 (t = 2.228)

0.0016 0.8415 0.0074 0.0188 -0.0156 0.0402 0.0599 0.0439 0.0551 0.0635 0.06

0.0026 0.8240 0.0045 0.0146 -0.0094 0.0280 0.0414 0.0269 0.0358 0.0403 0.04

In both cases, recoveries were_fully sufficient (84 and 82%). In the second case (n = 12), X (= 0.06) is closer to LDC and LDM, and the prediction interval is a little narrower (cf. the difference in the values for sR) than in the first case (n = 16), where X = 0.095.

9.3 Example 1: Graphical derivation of LDC and LDM The mathematical expressions (Chapters 6.6-6.13) were given in order to describe the model, at the same time serving as a basis for establishing suitable computer programs. Without computer support, it is rather laborious to construct the upper and lower prediction limits. In general, however, these limits are only needed in a region between X = 0 and X = X 2 (see Figure 1). For a graphical derivation of the LDC and LDM it is, therefore, permissible to substitute the curves for these limits by straight lines, which are drawn, parallel to the calibration line, through the points YUP and Y ^ . (A mathematical check of the quality of the approximation can be made by calculating some values for Y+ and Y_ using equation I.) This simplification permits the derivation of the LDC and LDM "by hand" on graph paper. The graphical procedure is described here, using Example 1: - Calculate the terms E through N, as well as X, Y, A, B, YUP and Y ^ as described in 9.1 — Draw the calibration line by connecting two points, e. g. X = 0, Y = 9.65 and X = 100, Y= 94.8, using the linear equation Y = 9.65 + 0.85 • X to obtain the Y values - Draw two lines, parallel to the calibration line, through YUP and Y ^ - Derive LDC as illustrated in Figure 7, obtaining a value of approx. 50 on the X-axis — Derive Xj as shown in Figure 8, obtaining a value of approx. 75 on the X-axis — For deriving X m , draw a straight line through two points, e.g. X', Y* and X", Y**, so that it intersects the upper parallel as illustrated in Figure 9. Y* and Y** are obtained from the equation of the calibration line by multiplying the resulting Y values (Y' and Y") by 1 + 0.2 • t (for explanation, see 4.4); in this case, t = 2.45 (see Table 2).

22

Limits of Detection and Determination

Example: X' = 20, Y' = 26.7,

Y* = 39.8

X" = 60, Y" = 60.8, Y** = 90.5 The point of intersection corresponds to Y c v and yields X m being approx. 62. The outcome of the graphical derivation is in good agreement with the results obtained from calculation (9.1). The approximation achieved by such a procedure will be sufficient for residue analyses in most cases.

LDC

Fig. 7. Graphical derivation of LDC.

UX

X

Fig. 8. Graphical derivation of

Fig. 9. Graphical derivation of Xu

Limits of Detection and Determination

23

Appendix Table 2. Critical values of the t distribution at a 95% level of statistical significance in relation to the degree of freedom f = n — 2.

two-sided 1 2 3 4 5 6 7 8 9

12.71 4.303 3.182 2.776 2.571 2.447 2.365 2.306 2.262

f

t two-sided

10 11 12 13 14 15 20 30 40

2.228 2.201 2.179 2.160 2.145 2.131 2.086 2.042 2.021

Mass-Spectrometric El Data for Confirmation of Results GC/MS is an excellent tool for the confirmation of results in pesticide residue analysis. For this reason, the six most abundant fragments and their relative intensities for approx. 150 pesticides and derivatives are listed in the following Table. The data relate to electron impact ionization at 70 eV and can be helpful for identifying suitable fragments when multiple ion detection (MID) is used. The data given for the relative intensities, however, may vary to some extent according to the type of the mass-spectrometer or the mass-selective detector used. Therefore, any confirmation of identity is best based on comparison of mass spectra which were obtained under identical instrumental conditions.

Table. Main fragments and their relative intensities for pesticides and some derivatives. P

,

Acephate Alachlor Aldicarb Aldrin Allethrin Atrazine Azinphos-methyl Barban Benazolin methyl ester Bendiocarb Bromacil Bromacil N-methyl derivative Bromophos Bromophos-ethyl Bromoxynil methyl ether Captafol Captan Carbaryl Carbendazim Carbetamide Carbofuran Chlorbromuron Chlorbufam cis-Chlordane trans-Chlordane Chlorfenprop-methyl Chlorfenvinphos Chloridazon Chloroneb

Molar mass*) 183 269 190 362 302 215 317 257 257 223 260 274 364 392 289 347 299 201 191 236 221 292 223 406 406 232 358 221 206

1 43 (100) 45 (100) 41 (100) 66 (100) 123 (100) 43 (100) 77 (100) 51 (100) 170 (100) 151 (100) 205 (100) 219 (100) 331 (100) 97 (100) 291 (100) 79 (100) 79 (100) 144 (100) 159 (100) 119 (100) 164 (100) 61 (100) 53 (100) 373 (100) 373 (100) 125 (100) 81 (100) 77 (100) 191 (100)

2

Main fragments m/z (intensities) 3 4 5 44 (88) 188 (23) 86 (89) 91 (50) 79 (40) 58 (84) 160 (77) 153 (76) 134 (75) 126 (58) 207 (75) 221 (68) 125 (91) 65 (35) 88 (77) 80 (42) 80 (61) 115 (82) 191 (57) 72 (54) 149 (70) 46 (24) 127 (20) 375 (84) 375 (93) 165 (64) 267 (73) 221 (60) 193 (61)

136 160 58 79 43 44 132 87 198 166 42 41 329 303 276 77 77 116 103 91 41 62 51 377 377 75 109 88 206

(80) (18) (85) (47) (32) (75) (67) (66) (74) (48) (25) (45) (80) (32) (67) (28) (56) (48) (38) (44) (27) (11) (13) (46) (53) (46) (55) (37) (60)

94 (58) 77 (7) 85 (61) 263 (42) 81 (31) 200 (69) 44 (30) 222 (44) 257 (73) 51 (19) 70 (16) 188 (41) 79 (57) 125 (28) 289 (55) 78 (19) 44 (44) 57 (31) 104 (37) 45 (38) 58 (25) 63 (10) 164 (13) 371 (39) 371 (47) 196 (43) 269 (47) 220 (35) 53 (57)

47 146 87 65 91 68 105 52 172 58 206 190 109 359 293 151 78 58 52 64 131 60 223 44 272 51 323 51 208

(56) (6) (50) (35) (29) (43) (29) (43) (40) (18) (16) (40) (53) (27) (53) (17) (37) (20) (32) (37) (25) (9) (13) (36) (36) (43) (26) (26) (39)

95 224 44 101 136 215 104 63 200 43 162 56 93 109 248 51 149 63 51 74 122 124 70 109 237 101 91 105 141

(32) (6) (50) (34) (27) (40) (27) (43) (31) (17) (12) (37) (45) (27) (50) (13) (34) (20) (29) (29) (25) (8) (10) (36) (30) (37) (23) (24) (35)

26

List of Mass-Spectrometric Data

Table, (contd.) Compound Chlorotoluron 3-Chloro-4-methylaniline (GLC degradation product of chlorotoluron) Chloroxuron Chlorpropham Chlorpyrifos Chlorthal-dimethyl Chlorthiamid Cinerin I Cinerin II Cyanazine Cypermethrin 2,4-DB methyl ester Dalapon Dazomet Demeton-S-methyl Desmetryn Dialifos Di-allate Diazinon Dicamba methyl ester Dichlobenil Dichlofenthion Dichlofluanid 2,4-D isooctyl ester 2,4-D methyl ester Dichlorprop isooctyl ester Dichlorprop methyl ester Dichlorvos Dicofol o,p'-DDT p,p'-DDT Dieldrin Dimethirimol methyl ether Dimethoate DNOC methyl ether Dinoterb methyl ether Dioxacarb Diphenamid Disulfoton Diuron Dodine Endosulfan Endrin Ethiofencarb Ethirimol Ethirimol methyl ether

Molar mass*)

1

212

72 (100)

141 290 213 349 330 205 316 360 240 415 262 142 162 230 213 393 269 304 234 171 314 332 332 234 346 248 220 368 352 352 378 223 229 212 254 223 239 274 232 227 404 378 225 209 223

141 (100) 72 (100) 43 (100) 97 (100) 301 (100) 170 (100) 123 (100) 107 (100) 44 (100) 163 (100) 101 (100) 43 (100) 162 (100) 88 (100) 213 (100) 208 (100) 43 (100) 137 (100) 203 (100) 171 (100) 97 (100) 123 (100) 43 (100) 199 (100) 43 (100) 162 (100) 109 (100) 139 (100) 235 (100) 235 (100) 79 (100) 180 (100) 87 (100) 182 (100) 239 (100) 121 (100) 72 (100) 88 (100) 72 (100) 43 (100) 195 (100) 67 (100) 107 (100) 166 (100) 180 (100)

Main fragments m/z (intensities) 4 5 2 3 44 (29)

140 245 127 195 299 60 43 93 43 181 59 61 42 60 57 210 86 179 205 173 279 92 57 45 57 164 185 111 237 237 82 223 93 165 209 122 167 89 44 73 36 81 69 209 223

(37) (37) (49) (59) (81) (61) (35) (57) (60) (79) (95) (81) (87) (50) (67) (31) (62) (74) (60) (62) (92) (33) (98) (97) (83) (80) (18) (39) (59) (58) (32) (23) (76) (74) (41) (62) (86) (43) (34) (80) (95) (67) (48) (17) (23)

6

167 (28)

132 (25)

45 (20)

77(11)

106 44 41 199 303 171 93 121 68 165 41 62 89 109 58 76 41 152 234 100 223 224 41 175 41 59 79 141 165 165 81 181 125 89 43 166 165 61 73 59 237 263 77 167 85

142 (36) 75 (21) 45 (20) 65 (27) 332 (29) 172 (49) 121 (27) 91 (50) 212 (48) 91 (41) 162 (36) 97 (59) 44 (73) 142 (17) 198 (58) 173 (17) 44 (25) 93 (47) 188 (26) 136 (24) 109 (67) 167 (27) 55 (54) 145 (70) 71 (48) 189 (56) 187 (6) 75 (18) 236 (16) 236 (16) 263 (17) 224 (3) 58 (40) 90 (57) 91 (35) 165 (42) 239 (21) 60 (39) 42 (20) 55 (47) 41 (89) 36 (58) 41 (26) 96 (12) 181 (12)

143 (28) 45 (19) 44 (18) 47 (23) 142 (26) 205 (35) 81 (27) 149 (35) 41 (47) 77 (33) 69 (28) 45 (59) 76 (59) 79 (14) 82 (44) 209 (12) 42 (24) 153 (42) 97 (21) 75 (24) 162 (53) 63 (23) 71 (41) 111 (69) 55 (47) 63 (39) 145 (6) 83 (17) 199 (12) 75 (12) 77 (17) 42 (2) 47 (39) 212 (48) 77 (33) 73 (35) 152 (17) 97 (36) 232 (19) 72 (46) 24 (79) 79 (47) 81 (21) 194 (4) 55 (10)

77 (25) 63 (16) 129 (16) 314 (21) 221 (24) 173 (29) 150 (27) 105 (33) 42 (34) 51 (29) 63 (25) 44 (47) 43 (53) 47 (11) 171 (39) 357 (10) 70 (19) 199 (39) 201 (20) 50 (19) 251 (46) 77 (22) 69 (27) 109 (68) 162 (41) 191 (35) 47 (5) 251 (16) 75 (12) 239 (11) 108 (14) 109 (2) 63 (33) 51 (47) 254 (33) 45 (31) 168 (14) 65 (23) 187 (13) 100 (46) 75 (78) 82 (41) 45 (17) 55 (2) 96 (9)

(68) (31) (35) (53) (47) (50) (33) (53) (60) (68) (39) (67) (79) (24) (66) (20) (38) (65) (27) (31) (90) (29) (76) (94) (61) (62) (17) (33) (33) (37) (30) (10) (56) (69) (36) (46) (42) (40) (25) (52) (91) (59) (29) (14) (14)

List of Mass-Spectrometric Data

27

Table, (contd.) Compound

Molar mass *)

Etnmfos Fenarimol Fenitrothion Fenoprop isooctyl ester Fenoprop methyl ester Fenuron Flamprop-isopropyl Flamprop-methyl Formothion Heptachlor Iodofenphos Ioxynil isooctyl ether Ioxynil methyl ether Isoproturon Jasmolin I Jasmolin II Lenacil Lenacil N-methyl derivative Lindane Linuron MCPB isooctyl ester MCPB methyl ester Malathion Mecoprop isooctyl ester Mecoprop methyl ester Metamitron Methabenzthiazuron Methazole Methidathion Methiocarb Methomyl Metobromuron Metoxuron Metribuzin Mevinphos Monocrotophos Monolinuron Napropamide Nicotine Nitrofen Nuarimol Omethoate Oxadiazon Parathion Parathion-methyl Pendimethalin Permethrin Phenmedipham

292 330 277 380 282 164 363 335 257 370 412 483 385 206 330 374 234 248 288 248 340 242 330 326 228 202 221 260 302 225 162 258 228 214 224 223 214 271 162 283 314 213 344 291 263 281 390 300

Main fragments m/z (intensities) 2 3 4 5 125 (100) 139 (100) 125 (100) 57 (100) 196 (100) 72 (100) 105 (100) 105 (100) 93 (100) 100 (100) 125 (100) 127 (100) 385 (100) 146 (100) 123 (100) 107 (100) 153 (100) 167 (100) 181 (100) 61 (100) 87 (100) 101 (100) 125 (100) 43 (100) 169 (100) 104 (100) 164 (100) 44 (100) 85 (100) 168 (100) 44 (100) 61 (100) 72 (100) 198 (100) 127 (100) 127 (100) 61 (100) 72 (100) 84 (100) 283 (100) 107 (100) 110 (100) 43 (100) 97 (100) 109 (100) 252 (100) 183 (100) 133 (100)

292 (91) 107 (95) 109 (92) 43 (94) 198 (89) 164 (27) 77 (44) 77 (46) 125 (89) 272 (81) 377 (78) 57 (96) 243 (56) 72 (54) 43 (52) 91 (69) 154 (20) 166 (45) 183 (97) 187 (43) 57 (81) 59 (70) 93 (96) 57 (94) 143 (79) 202 (66) 136 (73) 161 (44) 145 (90) 153 (84) 58 (81) 46 (43) 44 (27) 41 (78) 192 (30) 67 (25) 126 (63) 100 (81) 133 (21) 285 (67) 235 (91) 156 (83) 175 (92) 109 (90) 125 (80) 43 (53) 163 (100) 104 (52)

181 (90) 47 (84) 111 (40) 219 (39) 47 (57) 79 (62) 41 (85) 196 (63) 59 (82) 55 (36) 119 (24) 91 (22) 276 (21) 106 (18) 276 (20) 106 (14) 42 (49) 126 (68) 274 (42) 237 (33) 47 (64) 79 (59) 41 (34) 43 (33) 370 (41) 127 (13) 44 (35) 128 (29) 55 (34) 93 (25) 135 (69) 93 (67) 110 (15) 109 (15) 168 (12) 165 (12) 109 (89) 219 (86) 189 (29) 124 (28) 71 (45) 43 (62) 77 (40) 107 (25) 127 (75) 173 (55) 41 (70) 169 (77) 59 (58) 141 (57) 42 (42) 174 (35) 135 (69) 163 (42) 124 (36) 187 (31) 93 (32) 125 (22) 45 (40) 109 (37) 45 (59) 105 (69) 91 (13) 60 (15) 183 (23) 228 (22) 57 (54) 43 (39) 67 (20) 109 (27) 97 (23) 109 (14) 153 (42) 214 (34) 44 (55) 128 (62) 42 (18) 162 (17) 50 (55) 202 (55) 203 (85) 139 (60) 79 (39) 109 (32) 57 (84) 177 (60) 291 (57) 139 (47) 79 (26) 263 (56) 41 (41) 57 (43) 44 (15) 165 (25) 132 (34) 91 (34)

153 (84) 56 (73) 141 (33) 251 (31) 93 (40) 63 (44) 71 (60) 198 (59) 87 (34) 223 (31) 44 (11) 42 (14) 51 (5) 278 (7) 230 (12) 44 (11) 87 (40) 47 (48) 102 (33) 93 (54) 109 (49) 37 (16) 55 (26) 386 (10) 88 (9) 45 (28) 161 (25) 91 (24) 81 (23) 55 (66) 121 (58) 152 (13) 136 (10) 124 (9) 123 (6) 111 (75) 217 (68) 44 (23) 46 (28) 41 (42) 69 (29) 41 (22) 142 (20) 99 (35) 158 (37) 142 (69) 55 (52) 228 (54) 107 (50) 77 (24) 103 (19) 69 (30) 58 (25) 159 (24) 163 (23) 47 (21) 58 (20) 91 (31) 58 (21) 42 (55) 47 (52) 258 (13) 170 (12) 45 (21) 73 (15) 47 (38) 74 (36) 43 (8) 193 (7) 58 (14) 192 (13) 46 (29) 125 (25) 115 (41) 127 (36) 161 (15) 105 (9) 63 (37) 139 (37) 123 (46) 95 (35) 47 (21) 58 (30) 42 (35) 258 (22) 125 (41) 137 (39) 93 (18) 63 (18) 281 (37) 253 (34) 184 (15) 91 (13) 44 (27) 165 (3D

28

List of Mass-Spectrometric Data

Table, (contd.) „

, P

Phosalone Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Propachlor Propanil Propham Propoxur Pyrethrin I Pyrethrin II Quintozene Resmethrin Simazine Tecnazene Terbacil Terbacil N-methyl derivative Tetrachlorvinphos Tetrasul Thiabendazole Thiofanox Thiometon Thiophanate-methyl Thiram Tri-allate Trichlorfon Tridemorph Trietazine Trifluralin Vamidothion Vinclozolin

Molar mass*) 367 238 333 305 211 217 179 209 328 372 293 338 201 259 216 230 364 322 201 218 246 342 240 303 256 297 229 335 287 285

1 182 72 168 290 120 161 43 110 123 91 142 123 201 203 160 56 109 252 201 57 88 44 88 43 109 128 200 43 87 54

(100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100)

2

Main fragments m/z (intensities) 3 4 5 121 166 318 276 77 163 93 152 43 133 237 171 44 201 161 174 329 254 174 42 60 73 42 86 79 43 43 264 58 53

(48) (85) (94) (93) (66) (70) (88) (47) (62) (70) (96) (67) (96) (69) (99) (79) (48) (67) (72) (75) (63) (97) (25) (73) (34) (26) (81) (33) (47) (93)

97 42 152 125 93 57 41 43 91 161 44 128 186 108 117 175 331 324 63 68 125 159 44 41 47 42 186 306 44 43

(36) (63) (88) (69) (36) (64) (42) (28) (58) (55) (75) (52) (72) (69) (69) (31) (42) (51) (12) (39) (56) (89) (20) (43) (26) (18) (52) (32) (40) (82)

184 44 304 305 43 217 120 58 81 117 214 143 68 215 42 57 79 108 202 61 61 191 208 42 44 44 229 57 61 124

*) Molecular ions; chloro and bromo compounds with Cl = 35 and Br = 79.

(32) (44) (79) (53) (35) (16) (24) (27) (47) (48) (67) (49) (63) (60) (45) (24) (20) (49) (11) (38) (52) (80) (18) (31) (20) (13) (52) (7) (29) (65)

154 43 180 233 51 165 65 41 105 107 107 81 173 44 41 176 333 75 64 55 47 86 73 70 185 129 214 42 59 212

(24) (24) (73) (44) (30) (11) (24) (21) (45) (47) (62) (38) (57) (57) (41) (23) (14) (40) (11) (34) (49) (72) (15) (23) (17) (11) (50) (6) (26) (63)

111 238 42 42 41 219 137 111 55 160 212 91 96 213 162 41 93 322 65 47 93 150 45 44 80 55 42 290 60 187

(24) (23) (71) (41) (27) (9) (23) (20) (43) (43) (61) (28) (40) (51) (37) (20) (9) (40) (9) (33) (47) (71) (10) (21) (8) (5) (48) (5) (25) (61)

Part 2 Cleanup Methods (contd.)

Cleanup Method 6 (updated) Cleanup of crude extracts from plant and animal material by gel permeation chromatography on a polystyrene gel in an automated apparatus Since the publication of Volume 1 of this Manual, analytical experience has shown that many more compounds can be analyzed by using Cleanup Method 6 than those listed in the Table on pp. 77 f, Vol. 1. The following Tables 1 and 2 show the elution volumes for approx. 350 pesticides and related compounds and approx. 60 non-pesticidal compounds, respectively, as they were published in the 9th Instalment (1987) of the German edition of the Manual. In addition, 15 pesticides are listed in Table 3 that cannot be gel-chromatographed under the conditions set out in step 5.3 (p. 76, Vol. 1). Table 1. Elution volumes of pesticides and related compounds under the conditions of gel permeation chromatography set out in step 5.3 (p. 76, Vol. 1). Compound

Elution volume range

Compound

ml

Acephate Alachlor Aldicarb Aldicarb sulphone Aldrin Allidochlor Ametryn Amidithion Amitraz Anilazine Anthraquinone Atraton Atrazine Azinphos-ethyl Azinphos-methyl Aziprotryne Barban Bendiocarb Benfluralin Benodanil Bensulide Benzoylprop-ethyl Bifenox Binapacryl Biphenyl Bis(4-chlorophenyl)methanol (DBH)

115-145 125-150 115-140 110-135 120-150 125-160 115-190 115-145 125-155 105-135 145-185 115-140 110-135 130-160 145-180 120-150 105-140 130-160 100-130 135-160 115-135 125-150 115-150 100-130 155-185 125-155

Elution volume range ml

Bitertanol Bromacil Bromophos Bromopho s-ethyl Bromopropylate Bromoxynild) Bromoxynil octanoate Brompyrazon Camphechlor (Toxaphene) Captafol Captan Carbaryl Carbofuran Carbophenothion Carbophenothion-methyl Carbophenothion oxon Chinalphos see Quinalphos Chinomethionat (Quinomethionate) Chloranil Chlorazine Chlorbenside Chlorbenside sulphone Chlorbromuron Chlorbufam a-Chlordane y-Chlordane

100-130 105-140 120-150 110-140 95-135 120-150 120-150 140-170 110-150 120-150 120-150 125-170 125-155 120-140 120-160 130-155 170-200 140-160 115-140 120-155 130-160 125-150 115-145 110-140 100-130

32

Cleanup Method 6 (updated)

Table 1. (contd.) Compound

Elution volume range

Compound

ml

ml

110-140 Chlordecone Chlorfenethol 115-145 Chlorfenprop-methyl 125-150 Chlorfenson 120-150 Chlorfenvinphos 110-140 Chloridazon (Pyrazon) 130-155 Chlormephos 115-145 Chlorobenzilate 100-135 2-Chloro-4-nitroaniline 105-140 Chloroneb 145-170 4-Chlorophenoxyacetic acidb) 100-130 Chloropropylate 100-135 Chlorothalonil 125-165 Chlorotoluron 115-150 Chloroxuron 130-155 Chlorpropham 110-135 Chlorpyrifos 110-140 Chlorpyrifos-methyl 120-150 Chlorthal-dimethyl 135-160 Chlorthiophos 115-155 Coumaphos 135-165 Coumithoate 105-135 Crotoxyphos 105-145 Crufomate 100-140 Cyanazine 110-135 Cyanofenphos 115-145 Cyanophos 115-150 Cycluron 140-160 Cymoxanil 110-130 Cypermethrin 100-135 2,4-Db) 100-130 135-165 2,4-D methyl ester b 100-130 2,4-DB > p,p'-DDA 120-160 o,p'-DDD 110-140 p,p'-DDD 100-140 o,p'-DDE 120-150 p,p'-DDE 120-150 o,p'-DDT 120-150 p,p'-DDT 110-140 Decachlorobiphenyl 130-165 Deltamethrin 100-135 Demephion (mixture of demephion-O and demephion-S) 125-165 Demeton (mixture of demeton-O and demeton-S) 130-155 Demeton-S-methyl 125-155

Elution volume range

Demeton-S-methyl sulphone Demeton-S sulphone Demeton-S sulphoxide N-Desethyl-pirimiphos-methyl Desmethyl-norflurazon Desmetryn Dialifos Di-allate Diazinon Dichlobenil Dichlofenthion Dichlofluanid Dichlone 2,6-Dichlorobenzamide p, p'-Dichlorobenzophenone 2,4-Dichlor ophenoxy-phenoxypropionic acidb) Dichlorprop (2,4-DP)b) Dichlorvos Diclofop-methyl Dicloran Dicofol Dicrotophos Dieldrin Dienochlor Dimefox Dimethachlor Dimethoate Dimethoxy-anilazine Dimethylaminosulphanilide (DMSA) Dimethylaminosulphotoluidide (DMST) Dinitraminea) Dinobuton Dinocap Dinoseb acetate Dioxacarb Dioxathion Diphenamid Diphenylamine Dipropetryn Dipropyl isocinchomeronate Disulfoton Disulfoton sulphone Disulfoton sulphoxide Ditalimfos

120-160 115-140 140-170 120-155 105-130 120-175 110-140 120-150 105-135 125-155 110-140 100-140 155-180 110-150 125-155 100-130 95-130 115-140 135-165 105-145 100-150 130-160 120-150 130-160 120-155 135-165 120-150 140-170 125-150 120-145 105-130 110-140 100-120 100-140 140-170 110-140 145-175 130-160 105-130 130-160 115-150 110-140 120-150 120-150

Cleanup Method 6 (updated)

33

Table 1. (contd.)

Compound Dithianon Edifenphos a-Endosulfan 3-Endosulfan Endosulfan sulphate Endrin EPN Ethidimuron Ethion Ethoprophos Ethoxyquin Etridiazole Etrimfos Famophos Fenamiphos Fenarimol Fenazaflor Fenchlorphos Fenitrothion Fenoprop (2,4,5-TP)b> Fenson Fensulfothion Fensulfothion sulphone Fensulfothion sulphone, oxygen analogue Fensulfothion sulphoxide, oxygen analogue Fenthion Fenthion sulphone Fenthion sulphone, oxygen analogue Fenthion sulphoxide Fenthion sulphoxide, oxygen analogue Fenvalerate Fluazifop-butyl Flubenzimine Fluchloralin Fluotrimazole Fluroxypyrc) Fluroxypyr n-butyl ester Fluroxypyr-( 1 -methylheptyl) Fluvalinate Folpet Fonofos Fonofos oxon Formothion

Elution volume range ml 140-175 130-160 110-150 110-150 100-140 130-160 135-160 120-150 100-140 120-155 125-150 140-160 105-140 125-155 105-140 125-150 115-140 120-150 120-150 95-130 130-160 120-150 125-155 125-155 135-165 130-160 145-175 140-170 155-185 150-180 105-135 105-130 85-120 100-120 100-140 95-125 110-130 90-120 95-120 140-180 120-150 145-175 120-150

Compound

Elution volume range ml

Fuberidazolea) 120-160 Genite 135-165 a-HCH 120-150 (3-HCH 100-130 6-HCH 100-130 8-HCH 105-135 Heptachlor 110-140 cis-Heptachlor epoxide 125-155 trans-Heptachlor epoxide 125-155 120-150 Heptenophos Hexachlorobenzene 140-165 Hexazinone 155-190 Imazalila> 120-150 120-150 Iodofenphos Ioxynild> 120-150 Ioxynil octanoate 125-155 Ipazine 105-135 Iprodione 115-145 Isobenzan 105-140 Isocarbamid 130-165 Isodrin 120-150 Isofenphos 95-125 Isomethiozin 125-150 Isopropalin 110-135 8-Keto-endrin 135-165 Lenacil 130-160 120-150 Leptophos Leptophos, desbromo derivative 120-150 Lindane 110-140 Linuron 120-140 110-140 Malaoxon 110-140 Malathion 100-130 MCPAb> MCPA-(2-butoxyethyl) 115-145 MCPA pentafluorobenzyl ester 100-125 MCPBb> 100-130 Mecarbam 105-145 95-130 Mecopropb) 140-170 Mephosfolan 125-145 Merphos 115-150 Metalaxyl Metamitron 140-170 150-180 Methabenzthiazuron 125-165 Methacrifos 120-150 Methamidophos 130-165 Methidathion

34

Cleanup Method 6 (updated)

Table 1. (contd.) Compound

Elution volume range

Compound

ml

Methoprotryne Methoxychlor Metobromuron Metolachlor Metribuzin Mevinphos Mirex Molinate Monocrotophos Monolinuron Morphothion Naled 2-Naphthoxyacetic acidb) Napropamide Neburon Nicotine Nitralin Nitrapyrin Nitrofen 4-Nitrophenold) Nitrothal-isopropyl Norazin Norflurazon Octachlorodipropyl ether (S 421) Omethoate Oxadiazon Oxamyl Oxychlordane (Octachlor epoxide) Oxydemeton-methyl Paraoxon Paraoxon-methyl Parathion Parathion-methyl Pencycuron methyl derivative Pendimethalin Pentachloroaniline Pentachloroanisole Pentachlorobenzene Pentachlorophenold) Permethrin Perthane Phenkapton Phenmedipham Phenthoate Phorate

115-140 125-155 125-150 130-160 125-150 120-150 130-160 150-175 115-140 125-150 130-170 115-155 110-140 135-165 110-140 145-195 115-145 135-160 135-165 115-140 105-135 110-140 125-150 110-130 140-160 115-145 140-165 100-160 135-165 110-140 140-170 110-140 120-150 130-160 125-155 110-140 125-160 125-165 105-140 115-145 110-140 115-145 105-130 115-150 115-145

Elution volume range ml

Phorate oxon Phorate sulphoxide Phosalone Phosfolan Phosmet Phosphamidon Phoxim Piperonyl butoxide Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Plifenate Procymidone Profenofos Profluralin Prometon Prometryn Propachlor Propanil Propargite Propazine Propetamphos Propoxur Propyzamide Prothiofos Pyrazophos Pyrethrinsa) Quinalphos (Chinalphos) Quintozene Rabenzazolea) Resmethrin Salithion Secbumeton Simazine Simeton Simetryn Strobane T Sulfallate Sulfotep Sulphur Sulprofos 2,4,5-Tb> 2,4,5-T amyl ester 2,4,5-T-butyl 2,4,5-T hexyl ester 2,4,5-T-(iso-octyl) Tecnazene

130-165 125-155 110-140 150-180 145-180 110-145 120-150 100-130 130-170 100-135 105-145 125-155 120-150 130-155 100-125 105-200 110-140 125-150 105-130 105-135 95-125 110-135 110-130 95-125 105-145 110-140 100-130 115-155 135-165 120-160 100-130 125-165 115-140 95-135 125-190 120-150 125-160 145-175 100-130 215-245 115-155 100-130 110-140 100-130 115-145 105-135 130-160

Cleanup Method 6 (updated)

35

Table 1. (contd.) Compound

Elution volume range

Compound

ml

Terbacil Terbufos Terbuthylazine Terbutryn 2,3,4,6-Tetrachloroanisole 2,3,4,5-Tetrachloronitrobenzene Tetrachlorvinphos Tetradifon Tetramethrin O,O,O',O'-Tetrapropyl dithiopyrophosphate Tetrasul Thiabendazole Thiofanox sulphone Thiometon

120-145 125-155 105-130 115-175 130-160 130-160 120-140 120-150 120-150 105-135 125-155 130-160 120-150 145-185

Elution volume range ml

Thionazin Tolylfluanid Triadimefon Triadimenol Tri-allate Triamiphos Triazophos Trichlorfon Trichloronat Trietazine Trifluralin Vamidothion Vamidothion sulphone Vamidothion sulphoxide Vinclozolin

120-150 105-135 100-130 100-130 120-150 125-160 120-140 100-140 110-140 115-150 100-130 135-165 125-155 150-180 100-130

a)

with complete exclusion of light; b) gel-chromatographed as acid, determined as methyl ester; c) gelchromatographed as acid, determined as n-butyl ester; d) gel-chromatographed as phenol, determined as acetyl derivative.

Table 2. Elution volumes of selected non-pesticidal compounds under the conditions of gel permeation chromatography set out in step 5.3 (p. 76, Vol. 1). Compound

Elution volume range

Compound

ml

ml

Ethylvanillin Aroclor 1016 Aroclor 1221 Aroclor 1232 Aroclor 1242 Aroclor 1248 Aroclor 1254 Aroclor 1260 Aroclor 1268 3,4-Benzpyrene (Benzo[a]pyrene) Butylhydroxyanisole Butylhydroxytoluene Chloroparaffins (C{2 — C{8) 2-Chlorophenold) 3-Chlorophenold) 4-Chlorophenold) Clophen A 30 Clophen A 40

115-150 140-165 140-170 120-170 120-170 120-170 130-165 120-165 120-165 195-240 110-140 105-135 105-130 120-140 115-145 120-140 120-165 120-165

Elution volume range

Clophen A 50 Clophen A 60 Clophen T 64 Coumarin Dibutyl phthalate 1,2-Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene 2,3-Dichlorophenold) 2,4-Dichlorophenold) 2,5 -Dichlorophenold) 2,6-Dichlorophenold) 3,4-Dichlorophenold) 3,5-Dichlorophenold) Dihydrocoumarin Halowax 1000 Halowax 1013 Halowax 1051 Hexabromobiphenyl

120-165 120-160 135-160 135-170 120-150 140-165 135-165 135-165 120-145 120-145 120-140 120-145 120-140 120-145 135-170 140-180 140-180 140-200 160-190

36

Cleanup Method 6 (updated)

Table 2. (contd.) Compound

Elution volume range

Elution volume range

Compound

ml

Hostatox (chlorinated indene) 6-Methylcoumarin Octachlorostyrene Polychlorinated terphenyl (5O°7o Cl)

1,2,3,4-Tetrachlorodibenzodioxin (1,2,3,4-TCDD) 1,2,3,4-Tetrachlorobenzene 1,2,3,5 -Tetrachlor obenzene 1,2,4,5-Tetrachlorobenzene 2,3,4,5 -Tetr achlorophenold) d)

110-150 140-160 140-160 110-140 145-175 125-165 140-165 125-165 105-140

ml d)

2,3,4,6-Tetrachlorophenol 2,3,5,6-Tetrachlorophenold) 1,2,3-Trichlor obenzene 1,2,4-Trichlorobenzene 1,3,5-Trichlorobenzene 2,3,4-Trichlorophenold) 2,3,5-Trichlorophenold) 2,3,6-Trichlorophenold) 2,4,5-Trichlorophenold) 2,4,6-Trichlor ophenold) 3,4,5-Trichlorophenold)

gel-chromatographed as phenol, determinated as acetyl derivative.

Table 3. Pesticides not determinable using gel permeation chromatography. Chlormequat Diquat Dodine Ethirimol Mancozeb Maneb Metham-sodium Methylmetiram

Metiram Morfamquat Nabam Paraquat Propineb Zineb Ziram

120-145 105-140 125-155 125-150 125-150 120-140 115-145 115-145 100-130 115-145 115-135

Cleanup Method 7 Solid phase extraction of water samples on alkyl-modified silica gel using disposable columns (German version published 1991)

1 Introduction The solid phase extraction (SPE) of water samples on alkyl-modified silica gel (reversed-phase material, RP-18) is a labour and solvent saving alternative to the usual extraction procedures involving liquid-liquid partitioning. Various manufacturers (e. g. Analytichem, Baker, Merck, Supelco) offer suitable extraction systems which simplify the cleanup and enhance the consistency of sample recovery. The systems are composed of disposable columns, a vacuum unit with manometer and vacuum control valve, and a rack to carry the collection vessels. The disposable columns are polypropylene cartridges filled with 0.1 to 2.5 g reversed-phase material, of which the 0.5 and 1-g versions are most customary. The great advantage of the solid phase extraction systems is that many samples can be worked up simultaneously, thus facilitating its application in routine work. Most of the procedures described up to now differ only in type and amount of the column filling and eluting solution, the flow rate of the water samples through the column, and the time required to dry the column after passage of the sample.

2 Outline of method A disposable column is conditioned with methanol and water. A 1-1 water sample is passed through this column with suction, whereby the compound residues are retained in the column. The column is dried by passing an air stream through it, and then eluted with a dichloromethane-methanol mixture. The eluate is evaporated to a definite small volume and used directly for the final chromatographic determination. Heavily contaminated water samples require an additional cleanup of the eluates.

3 Apparatus Solid phase extraction system, e.g. Supelco Visiprep SPE Vacuum Manifold (Supelco No. 5-7030) Activated charcoal cartridge, e. g. disposable filter column, volume 3 ml (Baker No. 7121-03), filled with granular activated charcoal (DeguSorb AS IV, Degussa) Adapter for disposable columns (Baker No. 7122-00)

38

Cleanup Method 7

Funnel column (reservoir for disposable columns), 75-ml, with adapter (Baker No. 7120-03) Vacuum tube, 8 mm i.d. T-piece, for 8 mm i.d. vacuum tube Woulfe bottle Water jet pump Needle valve Graduated cylinder, 1-1 Beaker, 1-1 Pear-shaped flask, 50-ml Rotary evaporator, 30-40 °C bath temperature

4 Reagents Dichloromethane, for UV spectroscopy (Fluka No. 66745) Methanol, HPLC quality (Fluka No. 65541) Water, HPLC quality (Baker No. 4218) Eluting mixture: dichloromethane + methanol 7:3 v/v Sodium chloride, p. a. Disposable column, volume 3 ml, filled with 500 mg Octadecyl (RP-18 material) (Baker No. 7020-03)

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Column conditioning Mount the reservoir, using the adapter, onto the disposable column, and mount these onto the vacuum manifold. Condition the column by passing, with suction, successively 10 ml methanol and 10 ml water through the column within 1 to 2 min, not allowing the column to run dry. Remove the reservoir. 6.2 Preparation of the analytical sample Measure the volume (approx. 1 1) of the analytical sample in a graduated cylinder. Transfer the sample to a 1-1 beaker and add 0.002 vol.% methanol or approx. 50 g/1 sodium chloride in order to optimize the extraction. For extraction of phenoxyalkanoic acid residues, adjust the pH of the sample to 2.

Cleanup Method 7

39

6.3 Solid phase extraction Remove the disposable column from the vacuum manifold and immerse it into the prepared analytical sample derived from 6.2. Using water jet pump suction, allow the sample to pass through the disposable column at a flow rate of 15 to 20 ml/min (see Figure). Rinse the graduated cylinder and the beaker with 10 ml water and suck the rinsings also through the column. Mount the activated charcoal cartridge onto the disposable column using an adapter, and dry the column filling by passing a stream of air through it for 5 min. Remove the activated charcoal cartridge.

Figure. Extracting a water sample. 1, Beaker; 2, disposable column; 3, adapter; 4, vacuum tube; 5, Woulfe bottle; 6, needle valve; 7, water jet pump.

6.4 Elution Mount the reservoir, using the adapter, onto the disposable column, and mount these onto the vacuum manifold. With suction, pass 30 ml eluting mixture through the column. Collect the eluate in a 50-ml pear-shaped flask and evaporate it to a small volume or just to dryness. Use the residue directly for the chromatographic determination. 6.5 Additional cleanup If required, clean up the residue derived from 6.4, e. g. proceeding as described in the respective chapters of the Multiresidue Methods S 8 or S 19 (see pp. 283 ff and pp. 383 ff, respectively, Vol. 1). If the water sample contains humic acids, use a silica gel cartridge for cleanup, see Section 6.5 in Cleanup Method 8, p. 44, this Volume.

7 Important points Polar compounds which are poorly retained on the RP-18 material may yield poor recoveries.

40

Cleanup Method 7

8 References W. Weber, J. Hahn and V. Lang, Bestimmung von u.a. carboxyl- und hydroxylgruppenhaltigen Pflanzenbehandlungsmitteln und ahnlichen Stoffen in Wasser, Lebensmittelchem. Gerichtl. Chem. 41, 15-16 (1987). U. Oehmichen, K. Karrenbrock and K. Haberer, Erfahrungen zur Analytik von Pflanzenschutzmitteln aus stark belasteten Oberflachengewassern, in: B. Bohnke (ed.), Gewasserschutz — Wasser — Abwasser, Nr. 106, pp. 110-135, Gesellschaft zur Forderung der Siedlungswasserwirtschaft an der RWTH Aachen e.V., Aachen 1989.

9 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, D. Gottschild, H. Kohle and H.-G. Nolting

Cleanup Method 8 Solid phase extraction of water samples on alkyl-modified silica gel (German version published 1991)

1 Introduction The solid phase extraction (SPE) of water samples on alkyl-modified silica gel (reversed-phase material, RP-18) is a labour and solvent saving alternative to the usual extraction procedures involving liquid-liquid partitioning. This method uses glass concentration devices, several of which can be arranged in rows for routine operation. The procedure is especially suitable when the final determination of the analyte will be performed by the automated multiple development (AMD) of thin-layer chromatograms. The final determination will also be possible by gas chromatography or highperformance liquid chromatography.

2 Outline of method A chromatographic tube filled with 3 g RP-18 material is successively conditioned with acetonitrile and sodium chloride solution. A 1-1 water sample, with internal standard added, is passed through this column with suction, whereby the compound residues are retained in the column. The column is dried by passing a nitrogen stream through it, and then eluted with an acetonitrile-methanol mixture. The eluate is evaporated to a small volume and used directly for the final chromatographic determination. Heavily contaminated water samples require an additional cleanup of the eluates.

3 Apparatus Glass reservoir, 25 mm i.d., 25 cm long, with male ground joint NS 12.5 at the lower end Chromatographic tube, 9 mm i. d., 14 cm long, with female ground joint NS 12.5 at the upper end, lower end drawn out to a capillary, 1 mm i.d., 1 cm long Filter paper discs, 9 mm dia. (Schleicher & Schull No. 2294) Erlenmeyer flask, 1-1, with ground joint NS 29 Erlenmeyer flask adapter (home-made, see Figure), with male ground joint NS 29 and a side tube for connecting the activated charcoal cartridge. A glass tube, i. d. 6 mm, extending to the base of the Erlenmeyer flask, passes through the adapter and, outside the flask, is bent twice at right angles so that its end points vertically downwards (dimensions, 22 cm x 13 cm x 5 cm). This end is fitted with a spherical ground joint KS 13.9 (ball) Capillary tube, 1 mm i.d., 150 cm long, with a spherical ground joint KS 13.9 (cup) at the upper end and a male ground joint NS 12.5 at the lower end

42

Cleanup Method 8

Activated charcoal cartridge, e. g. disposable filter column, volume 3 ml (Baker No. 7121-03), filled with granular activated charcoal (DeguSorb AS IV, Degussa) Concentrating device (see Figure): Place the Erlenmeyer flask adapter, with the activated charcoal cartridge being attached, onto the Erlenmeyer flask, and connect the shorter end of the glass tube via the capillary tube to the chromatographic column. Clean the device with water (HPLC quality) before use

Figure. Concentrating device assembly. 1, Erlenmeyer flask; 2, Erlenmeyer flask adapter; 3, activated charcoal cartridge; 4, glass tube; 5, spherical joint connection; 6, capillary tube; 7, chromatographic column. pH meter Glass syringe, 50-ml PTFE tube, 1 cm i.d., 3.5 cm long Peristaltic pump, capacity 1 ml/min Sample vials, 5-ml, with cone-shaped inside, e.g. conic ampoules N 18-5 (Macherey-Nagel No. 702240) Device for evaporating solvents in a nitrogen stream, suitable to take the 5-ml sample vials, e. g. Silli-Therm heating module with Silli-Vap evaporator and Reacti-Bar heating block (Pierce No. 19793, 19792 and 19785, respectively) Separatory funnel, 20-ml

4 Reagents Acetonitrile, HPLC quality (Promochem No. 2856) Dichloromethane, for chromatography (Riedel-de Haen No. 32222) Methanol, p. a. (Merck No. 6009) Water, HPLC quality (Baker No. 4218) Eluting mixture: acetonitrile + methanol 8:2 v/v Internal standard solution: 10 mg/ml diphenyl sulphone in methanol Hydrochloric acid, p. a., cone. (Riedel-de Haen No. 30721) and 2 mol/1 HC1 ortho-Phosphoric acid, p.a., 85% (Merck No. 573) and 2 g/100 ml H 3 PO 4

Cleanup Method 8

43

Buffer solution: 1.38 g/1 trisodium phosphate, adjusted to pH 10 with phosphoric acid (2 g/100 ml) Sodium chloride solution: 50 g/1 sodium chloride, adjusted to pH 2 with hydrochloric acid (2 mol/1) Sodium chloride, p. a. (Riedel-de Haen No. 31434), heated for 12 h to 530 °C Trisodium phosphate, pure (Riedel-de Haen No. 04278) Column filling material RP-18, 40 |j,m, e.g. Bakerbond reversed phase Octadecyl (Baker No. 7025-00) Disposable column, volume 3 ml, filled with 500 mg silica gel (Baker No. 7086-03); only for samples containing humic acids Nitrogen, passed through an activated charcoal cartridge

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Extraction column preparation and conditioning Place a filter paper disc in the bottom of the chromatographic tube, add 3 g column filling material and top it with another filter paper disc. Mount the reservoir, and condition the column first with 10 ml acetonitrile followed by 100 ml sodium chloride solution. Remove the reservoir and make sure that the column is filled with sodium chloride solution up to the joint. 6.2 Preparation of the analytical sample Transfer 1000 ml of the analytical sample and 50 g sodium chloride into the Erlenmeyer flask and adjust the pH to 2 with hydrochloric acid (2 mol/1). Add 6 \i\ internal standard solution. 6.3 Solid phase extraction Place the Erlenmeyer flask, filled according to 6.2 and with the adapter attached, on a shelf about 2 m above the working bench. Attach the capillary tube to the doubly bent glass tube and connect the glass syringe to the conical joint of the capillary tube, using the PTFE tube. Suck the water from the Erlenmeyer flask, using the syringe, until the tube system is completely filled with liquid. Pull off the tube, attaching the syringe, and connect the prepared extraction column to the capillary tube (see Figure). Connect the peristaltic pump to the column outlet and set the pump to give a flow rate through the column of 1 ml/min. When the analytical sample has been entirely sucked through the column, dry the filling by passing a stream of purified nitrogen through the column for at least 12 h.

44

Cleanup Method 8

6.4 Elution Successively add two 3-ml portions of eluting mixture to the extraction column. Collect the first 3 ml of the eluate in a sample vial, place the vial into the heating block (35 °C) of the evaporation device and evaporate to dryness under a stream of nitrogen. Use the evaporated residue directly for the chromatographic determination. 6.5 Additional cleanup If required, clean up the residue derived from 6.4, e. g. proceeding as described in the respective chapters of the Multiresidue Methods S 8 or S 19 (see pp. 283 ff and pp. 383 ff, respectively, Vol. 1). 6.5.1 Water with low humic acid content Condition the disposable silica gel column with 10 ml eluting mixture. Before performing the elution as described in 6.4, place this column under the outlet of the extraction column, so that the first 3 ml of the eluate will pass through the silica gel column. Following the elution of the extraction column, elute the silica gel column further with 1 ml methanol. Collect the total eluate in a sample vial, place the vial into the heating block (35 °C) of the evaporation device and evaporate to dryness unter a stream of nitrogen. 6.5.2 Water with unusually high humic acid content (only for basic and neutral analytes) Transfer the evaporated residue derived from 6.4 into a 20-ml separatory funnel, using a total of 5 ml dichloromethane to complete the transfer, and extract three times with 5-ml portions of buffer solution. Save the dichloromethane phase. Re-extract the combined aqueous phases with 3 ml dichloromethane. Combine the dichloromethane phases and evaporate them successively to dryness in a sample vial under a stream of nitrogen, using the heating block (35 °C) of the evaporation device.

7 Important points The amount of 3 g RP-18 column filling and the slow flow rate used for the extraction of the analytical sample shall ensure that as many differently polar compounds as possible will be retained. This will be supported by the addition of sodium chloride to the analytical sample. The long drying period following the extraction is necessary in order to completely remove the water from the column filling.

8 Reference K. Burger, Multiple method for ultratrace determination: Pesticide active ingredients in ground and drinking water analyzed by TLC/AMD (Automated Multiple Development), Pflanzenschutz-Nachr., Engl. edition, 41, 175-228 (1988).

Cleanup Method 8

9 Author Bayer AG, Analytical Laboratories, Dormagen, K. Burger

45

Part 3 Individual Pesticide Residue Analytical Methods (contd.)

Amitrole

4-A

Apples, cherries, grapes, must, pears, wine Soil, water

Gas-chromatographic determination

(German version published 1991)

1 Introduction Chemical name Structural formula

3-Amino-lH-l,2,4-triazole (IUPAC) N II

C—NH2 II H

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100ml at 20°C)

Other properties

C2H4N4 84.1 157-159°C No data 3.3 • 10"7 mbar at 20 °C Readily soluble in water (30 g); slightly soluble in isopropanol (2.7 g), very sparingly soluble in dichloromethane (11 mg), virtually insoluble in toluene (2 mg) and n-hexane (<1 mg) Stable in acid, neutral and alkaline media

2 Outline of method Amitrole residues are extracted from plant material with aqueous ethanol. The ethanol is evaporated from the extract and the aqueous residue, after the addition of acetic acid, is shaken with dichloromethane. From soil samples, amitrole residues are also extracted with aqueous ethanol. Pre-cleaned extracts from plant material, extracts from soil, and water samples are concentrated. Subsequent addition of ethanol and acetic anhydride results in the conversion of amitrole to its monoacetyl derivative. The derivative is partitioned into dichloromethane and cleaned up by both gel permeation chromatography and column chromatography on silica gel. Monoacetyl-amitrole is determined by gas chromatography using a thermionic detector.

3 Apparatus High-speed blendor, e.g. Waring Blendor fitted with 2-1 metal jar Glass funnels, 16 cm and 8 cm dia. Fluted filter paper

50

Amitrole

Centrifuge Graduated cylinder, 500-ml Round-bottomed flasks, 1-1, 500-ml, 250-ml, 100-ml and 50-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Separatory funnels, 500-ml and 250-ml Polyethylene bottle, 500-ml, with screw cap Laboratory mechanical shaker, 150 r.p.m. Buchner porcelain funnel, with round filter paper Graduated test tubes, 10-ml, with ground stoppers Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Glass syringe, 10-ml, with Luer-lock fitting Chromatographic tube, 18 mm i.d., 35 cm long, with extended outlet and PTFE stopcock Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-ul

4 Reagents Cyclohexane, for residue analysis Dichloromethane, for residue analysis Ethanol 96%, denatured with ethyl methyl ketone Ethyl acetate, for residue analysis Eluting mixture: cyclohexane + ethyl acetate 1:1 v/v Extraction mixture 1: ethanol + water 2:1 v/v Extraction mixture 2: ethanol + water 3:2 v/v Amitrole standard solution: 10 jag/ml ethanol Derivative standard solution: 1.5 ng/ml monoacetyl-amitrole in ethanol, prepared as follows: Derivatize 5 \xg amitrole as described in 6.2.2, clean up the acetylated product as described in 6.3 and 6.4, and dissolve it in 5 ml ethanol Glacial acetic acid, p.a. Acetic anhydride, p.a. Sodium sulphate, p.a., anhydrous Filter aid, e.g. Celite 545 Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Silica gel, deactivated with 4% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734), for 5 h at 130 °C, allow to cool in a desiccator, and store in a tightly stoppered container in the desiccator. To 100 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 4 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container Cottonwool, chemically pure Air, synthetic, re-purified Helium 4.6 (> 99.996 vol. %) Hydrogen 5.0 (> 99.999 vol. °/o) Nitrogen 4.6 (> 99.996 vol. %)

Amitrole

51

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material (apples, cherries, grapes, pears)

Homogenize 200 g of the analytical sample (G) with 300 ml extraction mixture 1 for 1 min in the blendor, then add 10 g filter aid and mix for a further 10 s. Measure the total volume of the homogenate (VEx). Filter the homogenate through a fluted filter paper, or centrifuge, whichever is best. Take a volume of the filtrate or of the supernatant, respectively, corresponding to one half of volume VEx (approx. 250 ml, VR1), and rotary-evaporate the ethanol. Add 1 ml glacial acetic acid to the aqueous residue; then transfer the mixture into a 500-ml separatory funnel and shake with 150 ml dichloromethane for approx. 15 s. Discard the dichloromethane phase, and drain the aqueous phase into a 1-1 round-bottomed flask. Rinse the funnel with approx. 10 ml ethanol and add the rinsing to the aqueous phase in the roundbottomed flask. Rotary-evaporate the combined solutions to approx. 10 ml, not allowing the water bath temperature to rise above 40 °C. 6.1.2 Soil To 100 g soil (G), add sufficient water to yield 40% of its maximum water capacity (see 8. Important points). Transfer the prepared soil into a polyethylene bottle, add 250 ml extraction mixture 2, cap the bottle, and shake on a mechanical shaker for 2 h. Suction-filter the extract through a filter paper in a Buchner porcelain funnel. Return the filter cake to the polyethylene bottle, add a further 250 ml of extraction mixture 2, and shake again on the mechanical shaker for 1 h. Repeat the second extraction. Combine the filtrates in a 1-1 roundbottomed flask and rotary-evaporate to approx. 10 ml. 6.1.3 Must, wine, water

Take 100 g of the analytical sample (G), add 1 ml glacial acetic acid to must and wine samples, and rotary-evaporate to approx. 10 ml. 6.2 Acetylation 6.2.1 Plant material, must, wine, soil and water To the concentrated extract derived from 6.1, add 20 ml ethanol and 1 ml acetic anhydride, swirl, and allow to stand for 10 min at room temperature. Add 10 ml water, and immediately transfer the solution into a 250-ml separatory funnel. Rinse the flask with approx. 80 ml dichloromethane, add the rinsing also to the separatory funnel, and shake vigorously for 15 s. Save the dichloromethane phase, and shake the aqueous phase with a further 80 ml of

52

Amitrole

dichloromethane for 15 s. Discard the aqueous phase. Filter the combined dichloromethane phases through a fluted filter paper containing 5 g sodium sulphate into a 500-ml roundbottomed flask, ignoring a slight turbidity. Rinse the filter with approx. 40 ml dichloromethane. Rotary-evaporate the combined filtrates to approx. 2 ml, not allowing the water bath temperature to rise above 40 °C, and concentrate further using a gentle stream of nitrogen, warming the flask in the hand, to obtain an oily residue of approx. 0.2 ml.

6.2.2 Amitrole standard solution

Transfer 0.5 ml amitrole standard solution (equivalent to 5 \xg amitrole) into a 250-ml separatory funnel. Add 20 ml ethanol, 5 ml water and 1 ml acetic anhydride, swirl, and allow to stand for 10 min at room temperature. Add 10 ml water to the reaction mixture, and immediately extract it twice with 80-ml portions of dichloromethane. Discard the aqueous phase, and continue to process the combined dichloromethane phases as described in 6.2.1.

6.3 Gel permeation chromatography Take up the residue derived from 6.2 in exactly 5.0 ml ethyl acetate and swirl; then add exactly 5.0 ml cyclohexane (VR2 = 10 ml). Filter the solution through a cottonwool plugged filter funnel, containing a 1 cm deep layer of sodium sulphate, into a test tube. Using a 10-ml syringe, load the 5-ml sample loop (VR3) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with a standard solution of monoacetyl-amitrole; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. — Elution volumes ranging from 130 to 180 ml were determined for monoacetylamitrole on Bio-Beads S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 130 to 180-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to approx. 2 ml with 40 °C bath temperature. Then proceed to step 6.4. Check the elution range every 500 samples, and determine anew whenever a new gel column is used.

6.4 Column chromatography Insert a cottonwool plug into the bottom of the chromatographic tube, and introduce a slurry of 10 g silica gel suspended in ethyl acetate. Remove air bubbles with the aid of a glass rod, allow the packing to settle, and drain the solvent to the top of the packing. Dissolve the residue derived from 6.3 in 5 ml ethyl acetate. Add the solution to the column and allow to percolate. Rinse the flask twice with 5-ml portions of ethyl acetate, and add these rinsings successively to the column, each time draining the supernatant solution, at a rate of 40 drops per min, to the top of the silica gel. Next elute monoacetyl-amitrole, at the same dropping rate, with 150 ml ethyl acetate, allowing the column to run dry. Rotary-evaporate the eluate to approx. 2 ml with 40 °C bath temperature, then remove the residual solvent using a gentle stream of nitrogen, warming the flask in the hand.

Amitrole

53

6.5 Gas-chromatographic determination Dissolve the residue derived from 6.4 in 1 or 2 ml ethanol (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column 1 Column temperature 1 Injection port temperature Detector Gas flow rates

Linearity range Injection volume Retention time for monoacetyl-amitrole Alternative conditions Gas chromatograph Column 2 Column temperature 2 Column 3 Column temperature 3 Injection port temperature Split ratio Detector Gas flow rates

Linearity range Injection volume Retention times for monoacetyl-amitrole

Varian 3700 Fused silica capillary, 0.53 mm i.d., 25 m long; coated with SE-54, CB 2.0 (Macherey-Nagel) Programmed to rise at 10 °C/min from 80 to 280 °C, then isothermal at 280 °C for 10 min 280 °C, splitless Thermionic nitrogen-specific detector Temperature 300 °C Helium carrier, 8 ml/min Nitrogen purge gas, 30 ml/min Hydrogen, 4 ml/min Air, 170 ml/min 1-20 ng 2 nl 4 min 35 s Hewlett-Packard 5880 A Capillary column, 0.5 mm i.d., 20 m long; coated with SE-30, film thickness 2.5 u.m 90 °C Capillary column, 0.5 mm i.d., 50 m long; coated with SE-54, film thickness 2.5 \im 140 °C 150 °C 1:10 Thermionic nitrogen-specific detector Temperature 225 °C Helium carrier, 7.5 ml/min Helium purge gas, 12 ml/min Hydrogen, 3 ml/min Air, 45 ml/min 1-20 ng 1-2 u.1 Column 2

Column 3

ca. 4 min

ca. 6 min

0.00

2.00

4 .00

6.00

8.00

Fig. 1. Amitrole in grapes (2 ul, column 1). Chromatogram 1: Untreated control sample. Chromatogram 2: Derivative standard solution; peak (arrow) representing 1 |ug/ml amitrole.

Amitrole

55

8.02 2.00 4.01 6.01 0.00 mm Fig. 2. Amitrole in grapes (2 \i\, column 1). Chromatogram 3: Derivative standard solution added to cleaned-up extract of untreated control sample; peak (arrow) representing 1 ng/ml amitrole. Chromatogram 4: Untreated control sample fortified with 0.01 mg/kg amitrole.

56

Amitrole

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas or peak heights of the sample solutions and comparing them with the peak areas or peak heights obtained for dilutions of the derivative standard solution. Equal volumes of the sample solutions and the derivative standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas or heights (see also 8. Important points). 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with amitrole at levels of 0.01 mg/kg to plant material, must, wine and soil, and of 0.001 to 0.1 mg/1 to tap water, ranged from 76 to 103% (see Table). Table. Percent recoveries from plant material, must, wine, soil and water, fortified with amitrole. . , A , . , Analytical material

Added mg/kg

Recovery %

Apples Cherries Grapes Must Pears Wine Soil Standard soil 2.1 Standard soil 2.2 Standard soil 2.3 Tap water

0,01 0,01 0,01 0,01 0,01 0,01

82 (5) 98 (5) 83 (5) 77 (2) 79 (3) 76 (2)

0,01 0,01 0,01 0,001 *) 0,01 *) 0,1*)

82 (3) 85 (4) 91 (3) 78 (2) 103 (4) 79(1)

Gas-chromatographic measurement with column 1. Numbers in brackets: number of recovery experiments. *) mg/1. The soils used for the recovery experiments had the following characteristics:

Soil type Standard soil 2.1*) Standard soil 2.2*) Standard soil 2.3*)

Organic carbon

Particles <0.02 mm

pH

0.31 2.64 1.06

8.5 14.5 24.7

6.0 6.0 7.0

*) Standard soils as specified by Biologische Bundesanstalt fiir Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

Amitrole

57

Blank values for plant material and soils did not occur or, if so, they were less than 0.001 mg/kg. The routine limit of determination was 0.01 mg/kg for plant material, must, wine and soil, and 0.001 mg/1 for water.

7.3 Calculation of residues The residue R, expressed in mg/kg amitrole, is calculated from the following equation:

FsfVRrVR3-VrG where G

= sample weight (in g) or volume (in ml)

VEx

= total volume of plant material homogenate from 6.1.1 (in ml)

VR1

= portion of filtrate from 6.1.1 used for further clean-up (in ml)

VR2

= volume of solution prepared for gel permeation chromatography in 6.3 (before filtration) (in ml) = portion of volume VR2 injected for gel permeation chromatography (volume of sample loop) (in ml)

VR3

V E n d = terminal volume of sample solution from 6.4 (in ml) Vj

= portion of volume V E n d injected into gas chromatograph (in ul)

W St

= a m o u n t of amitrole equivalent injected with derivative standard solution (in ng)

FA

= peak area or height obtained from Vj (in m m 2 or integrator counts)

F St

= peak area or height obtained from W St (in m m 2 or integrator counts)

8 Important points The water content and the maximum water capacity of soil samples must be determined before the soil extraction is performed as described in 6.1.2. To determine the water content, dry 10 to 20 g of the soil to constant weight in an oven at 105 °C (approx. 15 h). To determine the maximum water capacity, weigh 20 g of the soil into a plastic cylinder (26 mm i. d.) perforated with 12 holes at the junction of the side wall with the bottom (bored so that no water can collect in the bottom of the cylinder). Allow the cylinder to stand in a Petri dish containing just as much water as can be absorbed through the holes. After approx. 1 h remove the cylinder, wipe it with filter paper, and let it stand on a pad of filter paper for 10 min to allow excess water to flow out. Wipe the cylinder again and re-weigh it. Calculate the maximum water capacity from 5 such determinations, considering the actual water content. The analysis can only be interrupted after the acetylation of the extracts or the standards. The final determination should be performed without delay because acetylated amitrole in solution is stable only for a short time even in a refrigerator.

58

Amltrole

The aqueous phases derived from 6.1.1, 6.1.2 or 6.1.3 must not be evaporated to dryness, and the water bath temperature must not exceed 40 °C. Gas chromatography with columns 2 or 3 under the conditions given in 6.5 requires at least 30 min between injections to allow time for interfering peaks from the previous chromatogram to elute. Gas chromatography with column 1 requires only 20 min between injections because of the temperature programme. The peak areas of monoacetyl-amitrole on the capillary columns used are dependent on the sample matrix (compare Chromatograms 2 and 3). Therefore, the derivative standard solution should be added to the cleaned-up extract from an untreated control sample to yield a reference standard solution suitable for quantitation according to 7.1. For preparing monoacetyl-amitrole in larger amounts, 1 g amitrole is acetylated as described in 6.2, but using 5 times the volumes of the solvents and of acetic anhydride. The derivative can also be synthesized according to Staab and Seel who give its formula as 3-acetylamino-1,2,4-triazole. When preparing standard solutions from monoacetyl-amitrole analytical standard material, consider that its molar mass (126.1) is 1.5 times greater than that of amitrole; accordingly, 1.5 |iig monoacetyl-amitrole is equivalent to 1 |ug amitrole.

9 References W. Jaggi, Die Bestimmung der CO2-Bildung als Mass der bodenbiologischen Aktivitat, Schweizerische landwirtschaftliche Forschung 75, 371-380 (1976). H. J. Jarczyk, Methode zur gaschromatographischen Bestimmung von 3-Amino-l,2,4-triazolRiickstanden in Apfeln, Birnen, Kirschen, Weintrauben und Wasser mit N-spezifischem Detektor, Bayer AG, Report No. RA-988, 16.10.1985 (unpublished). H. 1 Jarczyk and E. Mollhoff, Methode zur gaschromatographischen Bestimmung von 3Amino-l,2,4-triazol-Ruckstanden in Pflanzen, Boden und Wasser mit Stickstoff-spezifischem Detektor, Bayer AG, Report No. RA-785, 7.7.1988 (unpublished). J. M. van der Poll, M. Vink and J. K. Quirijns, Capillary gas chromatographic determination of amitrole in water with alkali flame ionization detection, Chromatographia 25, 511-514 (1988). H.A. Staab and G. Seel, Uber N-Acyl-Derivate des 3-Amino-l,2,4-triazols, Chem. Ber. 92, 1302-1306 (1959).

10 Authors Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, H. J. Jarczyk and E. Mollhoff

Anilazine

186

Artichokes, aubergines, barley (green matter, grains and straw), beans (green), clover, coffee (raw), garlic, hop cones, onions, potatoes, radicchio, rape (green matter), spinach, sweet peppers, tomatoes, turnips (foliage and edible root), wheat (green matter, grains and straw) Soil, water

Gas-chromatographic determination

(German version published 1989)

1 Introduction 2,4-Dichloro-6-(2-chloroanilino)-l,3,5-triazine (IUPAC) Chemical name H

Structural formula Cl

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C9H5C13N4 275.53 159 °C No data 9 • 10-8 mbar at 20 °C (extrapolated) Virtually insoluble in water; soluble in dichloromethane (5-10 g), slightly soluble in toluene (2-5 g), sparingly soluble in n-hexane (0.1-0.2 g) and isopropanol (0.5-1 g) Colourless crystals; stability to hydrolysis (half lives at 22 °C): 730 h at pH 4, 790 h at pH 7, 22 h at pH 9

2 Outline of method Anilazine residues are extracted with methanolic sodium hydroxide solution, whereby they are converted to dimethoxy anilazine. The extract is filtered and the methanol evaporated. After the addition of sodium chloride solution, the aqueous residue is shaken with a dichloromethane-acetone mixture. The organic phase is dried on sodium sulphate and evaporated to dryness.

60

Anilazine

Anilazine

Dimethoxy anilazine

With plant material, the subsequent cleanup includes chromatography on silica gel, followed by gel permeation chromatography on a polystyrene gel. The silica gel column chromatography can be omitted for soil and water samples. In the concentrated eluates, dimethoxy anilazine is determined by gas chromatography using a thermionic detector.

3 Apparatus Homogenizer Wide neck glass bottle, 1-1 or 500-ml, with ground joint Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 1-1 Round-bottomed flasks, 1-1, 500-ml, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C and 55 °C bath temperature Separatory funnels, 1-1 and 500-ml, with ground stoppers Polyethylene bottle, 500-ml, with cap Laboratory mechanical shaker Chromatographic tube, 17.5 mm i.d., 30 cm long, with PTFE-stopcock Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Glass syringe, 10-ml, with Luer-lock fitting Test tubes, 10-ml, with ground stoppers Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-jul

4 Reagents Acetone, for residue analysis Cyclohexane, p. a. Dichloromethane, for residue analysis Ethyl acetate, for residue analysis Methanol, for residue analysis Toluene, p. a. Dichloromethane + acetone mixture 4:1 v/v Eluting mixture 1: cyclohexane + ethyl acetate 85 :15 v/v Eluting mixture 2: cyclohexane + ethyl acetate 7:3 v/v Eluting mixture 3: cyclohexane + ethyl acetate 1:1 v/v

Anilazine

61

Anilazine standard solutions: 1.0-1000 ng/ml ethyl acetate Dimethoxy anilazine standard solutions: 0.1-100 ng/ml ethyl acetate Preparation of dimethoxy anilazine [2-(2-chloroanilino)-4,6-dimethoxy-l,3,5-triazine]: Dissolve 1 g anilazine in 50 ml methanolic sodium hydroxide solution and stir for 1 h at room temperature, then rotary-evaporate the solution to dryness and dissolve the residue in dichloromethane-acetone mixture. Shake this solution three times with water, separate the organic phase, dry on sodium sulphate, and rotary-evaporate to dryness with 40 °C bath temperature. Recrystallize the residue twice from cyclohexane to yield colourless crystals, m.p. 102 °C Methanolic sodium hydroxide solution: 0.5 mol/1 NaOH p.a. in methanol Sulphuric acid, 3 g/100 ml H2SO4 p. a. Sodium chloride solution, 15 g/100 ml NaCl p. a. Sodium sulphate, p.a., anhydrous Filter aid, e. g. Celite 545 Silica gel 60, 0.2-0.5 mm (Merck No. 7733) Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Cottonwool, chemically pure Air, synthetic, re-purified Hydrogen 5.0 (> 99.999 vol. Vo) Nitrogen 4.6 (> 99.996 vol. %)

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. When it is not possible to analyze the samples straight away, store them in a freezer at — 20 °C. For taking water samples, observe the guidelines given on pp. 23 ff, Vol. 1. Water samples must be analyzed without delay.

6 Procedure 6.1 Extraction 6.1.1 Plant material

According to the sample material, the following amounts (G) are used as analytical samples: Aubergines, potatoes, sweet peppers, tomatoes 100 g; Artichokes, beans, cereals (green matter and grains), clover, coffee (ground), garlic, onions, radicchio, rape (green matter), spinach, turnips (foliage and edible root) 50 g; Cereal straw 25 g; Hop cones 10 g. Transfer the comminuted analytical sample into a wide neck glass bottle. Add 250 ml methanolic sodium hydroxide solution, shake the mixture well and allow it to stand for 1 h at room temperature. Next homogenize the mixture for 3 min. Add approx. 15 g filter aid to the homogenate, in the case of cereal samples also add approx. 10 g filter aid to the filter paper in a Buchner porcelain funnel, and filter the homogenate with gentle suction. Wash the bottle and filter cake twice with 100-ml portions of methanol. Allow the filter cake to pull dry, then

62

Anilazine

discard it. Rotary-evaporate the filtrate with 55 °C bath temperature to an aqueous residue and, without delay, add 200 ml sodium chloride solution (for coffee samples, add 500 ml sodium chloride solution to prevent the formation of emulsions). Extract the mixture twice, first with 150 ml, followed by 100 ml of the dichloromethane-acetone mixture (for coffee samples, use 200 ml and 150 ml of the mixture). Shake the combined organic phases with 150 ml sulphuric acid. Dry the organic phase on sodium sulphate, and rotary-evaporate to dryness with 40 °C bath temperature. Proceed as described in 6.2. 6.1.2 Soil Add 200 ml methanolic sodium hydroxide solution to 100 g soil (G) in a 500-ml polyethylene bottle, cap the bottle, and shake on a mechanical shaker for 1 h. Filter the suspension, with gentle suction, through a filter paper in a Buchner porcelain funnel containing approx. 10 g filter aid. Shake the filter cake for 1 h with a further 200 ml of methanolic sodium hydroxide solution on the mechanical shaker. Filter as before and wash the bottle and filter cake twice with 50-ml portions of methanol. Allow the filter cake to pull dry, then discard it. Rotaryevaporate the combined filtrates to an aqueous residue (approx. 10 ml) with 55 °C bath temperature. Add 200 ml sodium chloride solution and extract twice, first with 150 ml, followed by 100 ml of the dichloromethane-acetone mixture. Dry the combined organic phases on sodium sulphate, and rotary-evaporate them to dryness with 40 °C bath temperature. Proceed as described in 6.3. 6.1.3 Water

To 400 ml water (G), add 200 ml methanolic sodium hydroxide solution, shake vigorously, and allow to stand for 1 h at room temperature. Next rotary-evaporate the methanol with 55 °C bath temperature. Shake the remaining aqueous solution twice, first with 150 ml, followed by 100 ml of the dichloromethane-acetone mixture. Dry the combined organic phases on sodium sulphate, and rotary-evaporate them to dryness with 40 °C bath temperature. Proceed as described in 6.3. 6.2 Column chromatography Fill the chromatographic tube, in this order, with 10 ml toluene, a cottonwool plug, 15 g silica gel (mixed to a slurry with toluene; filling height approx. 13 cm), approx. 5 g sodium sulphate, and a loose cottonwool plug. Then drain the toluene down to the top of the sodium sulphate layer. Dissolve the residue derived from 6.1.1 in 10 ml toluene. Transfer the solution onto the column, using a pipet, and allow to percolate to the top of the sodium sulphate. Rinse the flask twice with 10-ml portions of cyclohexane. Pre-wash the column first with the rinsings and then by a further 30 ml of cyclohexane, followed by 50 ml eluting mixture 1. Next elute dimethoxy anilazine with 100 ml eluting mixture 2, collecting the eluate in a 250-ml round-bottomed flask. Rotary-evaporate the eluate to dryness with 40°C bath temperature; then proceed as described in 6.3. 6.3 Gel permeation chromatography Transfer the residue derived from 6.1.2, 6.1.3 or 6.2 into a test tube, using a total of 10 ml eluting mixture 3 (VR1) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample

Anilazine

63

loop (VR2) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with a standard solution of dimethoxy anilazine; cf. Cleanup Method 6, pp. 75ff, Vol. 1. — Elution volumes ranging from 150 to 180 ml were determined for dimethoxy anilazine on Bio-Beads S-X3 polystyrene gel, using eluting mixture 3 as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 150 to 180-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to dryness with 40 °C bath temperature. Then proceed to step 6.4. Check the elution range every 500 samples, and determine anew whenever a new gel column is used. 6.4 Gas-chromatographic determination Dissolve the residue derived from 6.3 in a definite volume (e. g. 5 ml) of ethyl acetate (VEnd) and transfer the solution to a test tube. Inject 5 \i\ of this solution (Vj) into the gas chromatograph. Next inject 5 L| L1 of a dimethoxy anilazine standard solution. Repeat each injection. When using a capillary column, inject 1 \i\. Operating conditions Gas chromatograph Column 1 Column 2 Detector Gas flow rates Attenuation Recorder Linearity range Injection volume Column temperatures Injection port temperatures Detector temperatures Retention times for dimethoxy anilazine Alternative conditions Gas chromatograph Column 3 Column temperature

Varian 3700 or Varian 6000 Glass, 3 mm i.d., 1.8 m long; packed with 1.5% SP2250 + 1.95% SP-2401 on Supelcoport, 100-120 mesh Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE30 on Chromosorb W-HP, 80-100 mesh Thermionic nitrogen-specific detector Nitrogen carrier, approx. 40 ml/min Hydrogen, 4.5 ml/min Air, 175 ml/min 1 • 10"11, plot attenuation 1.0 1 mV; chart speed 5 mm/min 0.5-50 ng 5 Hi Column 1 220 °C 250 °C 250 °C

Column 2 210 °C 260 °C 35O°C

3 min 24 s

2 min 42 s

Varian 3700 Fused silica capillary, 0.53 mm i.d., 15 m long; coated with OV-17 RTX 50, crossbond, film thickness 0.5 M-m (Restek No. 10537) Isothermal at 100 °C for 2 min, programmed to rise at 20°C/min from 100 to 230 °C, then isothermal at 230 °C for 10 min

64

Anilazine

Injection port temperature Detector Gas flow rates Attenuation Recorder Linearity range Injection volume Retention time for dimethoxy anilazine

300 °C Thermionic nitrogen-specific detector Temperature 280 °C Nitrogen carrier, 14.6 ml/min Hydrogen, 4.5 ml/min Air, 175 ml/min 1 • 10~n 1 mV; chart speed 5 mm/min 0.5-50 ng 1 ul 8 min 9 s

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the dimethoxy anilazine standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. 7.2 Recoveries and limit of determination Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with known amounts of anilazine dissolved in 1-2 ml ethyl acetate. The results are given in the Table. Table. Percent recoveries from plant material, soil and water, fortified with anilazine; duplicate experiments. Analytical material Artichokes Aubergines

Barley Green matter Grains Straw Beans

Added (mg/kg)

Range (%)

0.02 0.2 0.02 0.2 2.0 20

86-94 81-85 89 84-97 107-108 94-99

0.04 1.0 0.02 0.2 0.04 0.4 0.02 0.2

84-93 87-95 96-97 83-91 92-95 86-87 109-111 87-95

Anilazine Table, (contd.) Analytical material Clover Coffee Garlic Hop cones Onions Potatoes

Radicchio

Rape green matter Spinach

Sweet peppers

Tomatoes

Turnips Foliage Edible root Wheat Green matter

Grains Straw Soil Standard soil 2.1 Standard soil 2.2 "Laacherhof West'

Added (mg/kg)

Range (%)

0.04 0.04 1.04 0.04 0.4 4.0 0.04 0.02 0.1 0.2 1.0 0.02 0.2 2.0 0.04 0.02 0.2 2.0 20 0.02 0.2 2.0 20 0.02 0.2 2.0

79-81 92-96 72-80 84-85 82-91 82-87 90-91 85-98 72-82 73-81 75-79 91-103 93-96 114 79-86 97 94-99 98-103 94-99 86 85-91 100-104 98-100 75-76 81 76-79

0.04 0.04

64-70 90-91

0.04 0.4 1.0 10 0.02 0.2 0.04 0.4

86-95 84-90 76-88c> 74-88 a> 85-91 78-90 77-86a> 81-87a>

0.02 1.0 0.02 1.0 0.02 1.0

92-101 91-92 95-105 88-91 87-90 71-72

65

66

Anilazine

Table, (contd.) Range (%)

Added (mg/kg)

Analytical material

82-93 b> 88-94a> 87-94 90-94

0.005 0.01 0.05 0.5

Water

Different number of recovery experiments:

a)

4,

b)

5,

c)

6.

The soils used for the recovery experiments had the following characteristics: Soil type

Organic carbon %

Particles <0.02 mm %

PH

0.31 2.64 1.78

8.5 14.5 20.5

6.0 6.0 6.1

Standard soil 2.1 * } Standard soil 2.2*> "Laacherhof West"

*) Standard soils as specified by Biologische Bundesanstalt filr Land- und Forstwirtschaft (BRA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

The data for water relate to tap water, spring water, drainage water, leaching water, water from lysimeter trials, and water from trials for the determination of fish toxicity. The limit of determination was 0.02 mg/kg for artichokes, aubergines, beans, cereal grains, potatoes, radicchio, spinach, sweet peppers, tomatoes and soil, 0.4 mg/kg for hop cones, 0.04 mg/kg for other plant material, and 0.005 mg/1 for water. 7.3 Calculation of residues The residue R, expressed in mg/kg anilazine, is calculated from the following equation: R R

Fst.VR2.Vi.G

L033

where G

= sample weight (in g) or volume (in ml)

V R1 VR2

= volume of solution prepared for gel permeation chromatography in 6.3 (in ml) = portion of volume V R1 injected for gel permeation chromatography (volume of sample loop) (in ml)

VEnd

= terminal volume of sample solution from 6.4 (in ml)

Vj

= portion of volume V E n d injected into gas chromatograph (in ul)

W St

= a m o u n t of dimethoxy anilazine injected with standard solution (in ng)

FA

= peak area obtained from Vj (in m m 2 )

FSt

= peak area obtained from W St (in m m 2 )

1.033

= factor for conversion of dimethoxy anilazine to anilazine

Anilazine

67

8 Important points To avoid any loss of residues, deep frozen plant material and soil samples should not be thawed prior to starting the analysis. When processing extracts from plant material as described in 6.1.1, the aqueous residue resulting from concentrating the filtrate, on prolonged standing, tends to solidify to a jelly-like mass, which dissolves in the sodium chloride solution only after warming to approx. 50 °C. Therefore, sodium chloride solution should be added to the aqueous residue without delay.

9 References R. Brennecke, Methode zur gaschromatographischen Bestimmung von ®Dyrene-Ruckstanden in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 38, 11-32 (1985). C. E. Mendoza, P. J. Wales and G. V. Hatina, Alkoxy derivatives of Dyrene: Identification and carboxylesterase inhibition, J. Agric. Food Chem. 19, 41-45 (1971). P. J. Wales and C. E. Mendoza, Investigation on determination and confirmation of Dyrene added to plant extracts: GLC and TLC of Dyrene and products of its reaction in methanolic sodium hydroxide, J. Assoc. Off. Anal. Chem. 53, 509-513 (1970).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke

Benomyl, Carbendazim, Thiophanate-methyl

261-378-370 High-performance liquid chromatographic determination

Lettuce, wheat (grains and straw)

(German version published 1987)

1 Introduction Benomyl Chemical name

Methyl l-(butylcarbamoyl)benzimidazol-2-ylcarbamate (IUPAC) CONH(CH2)3CH3

Structural formula

a>

Empirical formula Molar mass Melting point Boiling point Solubility Other properties

C14H18N4O3 290.32 Undergoes decomposition on heating Not distillable Virtually insoluble in water Decomposed in acid and alkaline media

Carbendazim Chemical name

Methyl benzimidazol-2-ylcarbamate (IUPAC)

N

Structural formula

Empirical formula Molar mass Melting point Boiling point Solubility (in 100 ml at 20 °C)

/V-NHCOOCH3

C9H9N3O2 191.19 307-312°C (with decomposition) Not distillable Virtually insoluble in water at pH 7; readily soluble in acetic acid and dimethylformamide; sparingly soluble in ethanol (30 mg);

70

Benomyl, Carbendazim, Thiophanate-methyl

Other properties

virtually insoluble in benzene (3.6 mg) and dichloromethane (6.8 mg) Stable to light and acids, decomposed in alkaline media

Thiophanate-methyl Chemical name

Dimethyl 4,4'-(o-phenylene)bis(3-thioallophanate) (IUPAC) NHCSNHCOOCH,

Structural formula NHCSNHCOOCH3

Empirical formula Molar mass Melting point Boiling point Solubility (in 100 ml at 20 °C)

Other properties

C12H14N4O4S2 342.40 178 °C (with decomposition) Not distillable Virtually insoluble in water; soluble in acetone (5.8 mg); slightly soluble in acetonitrile (2.4 g), chloroform (2.6 g), ethyl acetate (1.2 g) and methanol (2.9 g) Stable to light, decomposed in acid and alkaline media

2 Outline of method In plant tissues, benomyl and thiophanate-methyl are partially converted to carbendazim. Therefore, in this method the sum of the three compounds is determined and expressed as carbendazim. The residues are extracted from the plant material with methanol or aqueous methanol, whereby any existing benomyl is converted to carbendazim. An aliquot of the extract is concentrated in the presence of dimethylformamide, and any residual thiophanate-methyl is converted to carbendazim with ammonia. The reaction mixture is diluted with buffer solution (pH 7) and extracted with dichloromethane. The dichloromethane phase is cleaned up by column chromatography on acidic aluminium oxide, and carbendazim is determined by highperformance liquid chromatography using a UV detector.

3 Apparatus Beater-cross mill Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) Knife mill Wide neck bottle, 500-ml, with screw cap Laboratory mechanical shaker

Benomyl, Carbendazim, Thiophanate-methyl

71

Buchner porcelain funnel, 6 cm dia. Filter paper, 6 cm dia., e.g. MN 713 (Macherey-Nagel) Filtration vessel after Witt Volumetric flask, 250-ml Round-bottomed flasks, 500-ml and 250-ml, with ground joints Rotary vacuum evaporator, 40-45°C, 58-65 °C and 80-85°C bath temperature Water bath, 80 °C temperature Separatory funnel, 500-ml Chromatographic tube with sintered glass disk, 16 mm i.d., 30 cm long, with PTFE stopcock Test tubes, 10-ml, graduated High-performance liquid chromatograph equipped with UV detector Microsyringe, 10-|il

4 Reagents Dichloromethane, distilled in glass N,N-Dimethylformamide, p. a. (Fluka No. 40250) Ethanol, absolute, p. a. (Merck No. 983) Ethyl acetate, p. a. (Merck No. 9623) n-Hexane, for residue analysis (Merck No. 4371) Methanol, distilled in glass Eluting mixture 1: n-hexane + ethyl acetate 8:2 v/v Eluting mixture 2: n-hexane + ethyl acetate 7:3 v/v Mobile phase: ethanol + n-hexane + phosphoric acid 700:300:0.2 v/v/v Carbendazim standard solutions: dilute a solution of 10 mg/ml carbendazim in ethanol to 0.03, 0.09, 0.24 and 0.6 ng/ml with mobile phase ortho-Phosphoric acid 85%, p. a. (Merck No. 573) Ammonium hydroxide solution 25%, p. a. (Merck No. 5432) Sodium chloride solution, saturated Phosphate buffer solution (pH 7): 0.041 mol/1 Na2HPO4 + 0.028 mol/1 KH2PO4 Sodium sulphate, p.a., anhydrous Aluminium oxide, activity grade V: To 100 g Alumina Woelm A Super I (ICN Biomedicals) in a 300-ml Erlenmeyer flask (with ground joint), add 19 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h Dry ice Cottonwool

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Lettuce is pre-homogenized, wheat grains are finely ground in the beater-cross mill in the presence of dry ice. Wheat straw is coarsely cut in the knife mill, and also finely ground in the beatercross mill in the presence of dry ice.

72

Benomyl, Carbendazim, Thiophanate-methyl

6 Procedure 6.1 Extraction 6.1.1 Wheat grains Weigh 25 g of the analytical sample (G) into the wide neck bottle and add 35 ml water. Mix for about 1-2 min with a glass rod, add 150 ml methanol, and shake the mixture for 30 min on the mechanical shaker. Filter the mixture with gentle suction through a filter paper in the Buchner porcelain funnel and collect the filtrate in the 250-ml volumetric flask which stands in the filtration vessel. Rinse the bottle and the filter cake with 60 ml methanol, suction-filter the rinsings also into the volumetric flask, and make up to the mark with methanol (VEx). 6.1.2 Wheat straw Weigh 5 g of the analytical sample (G) into the wide neck bottle and add 25 ml water. Mix for about 1-2 min with a glass rod, rinse the glas rod with a little methanol which is given into the bottle, and leave to stand for about 10 min. Add 150 ml methanol and shake the mixture for 30 min on the mechanical shaker. Then proceed as described in 6.1.1. 6.1.3 Lettuce Transfer 25 g of the analytical sample (G) into the wide neck bottle with 150 ml methanol and homogenize at high speed. Rinse the mixer blade with 25 ml methanol which is given into the bottle, and shake the mixture for 30 min on the mechanical shaker. Then proceed as described in 6.1.1. 6.2 Conversion of thiophanate-methyl to carbendazim Use the following portions (VR1) of the extracts obtained in 6.1: 30 ml (6.1.1), 75 ml (6.1.2), or 60 ml (6.1.3). Place the portion into a tared 500-ml round-bottomed flask, add 10 ml (approx. 9.4 g) dimethylformamide, and rotary-evaporate the solution to less than 9 g, with 58-65 °C bath temperature. Add dimethylformamide until the net weight is 9.4 g, and add 1 ml ammonium hydroxide solution. Seal the flask with a metal-clamped ground stopper, shake the solution for a short time, and allow the flask to stand in a water bath at 80 °C for 20-25 min with occasional shaking. Rotary-evaporate the solution to less than 1 g with 80-85 °C bath temperature. 6.3 Liquid-liquid partition Add 50 ml phosphate buffer solution and 50 ml dichloromethane to the residue obtained in 6.2, shake, and transfer the mixture into the separatory funnel. Rinse the flask with a further 50 ml of phosphate buffer solution and 50 ml of dichloromethane, and add the rinsings to the separatory funnel. Next add 40 ml sodium chloride solution, shake, and allow the phases to separate. Filter the dichloromethane phase through 25 g sodium sulphate on a cottonwool plug into a tared 500-ml round-bottomed flask. Shake the aqueous phase twice more with 70-ml portions of dichloromethane, filter the dichloromethane phases through the sodium

Benomyl, Carbendazim, Thiophanate-methyl

73

sulphate into the round-bottomed flask, and rotary-evaporate the solution to less than 0.6 g, with 40-45 °C bath temperature. The residue consists mainly of dimethylformamide, which holds the carbendazim residue in solution. In these minor quantities, dimethylformamide does not have a detrimental effect on the following aluminium oxide cleanup. 6.4 Column chromatography Introduce 30 ml eluting mixture 1 and then 30 g aluminium oxide into the chromatographic tube. Drain the supernatant liquid to the top of the column packing. Next dissolve the residue derived from 6.3 in 20 ml eluting mixture 1, add the solution to the column, and drain the supernatant liquid to the top of the packing again. Rinse the flask twice with 25-ml portions of eluting mixture 1, add the rinsings to the column, and allow to percolate in the same manner as before. At this point the elution rate as a rule decreases. Therefore, thoroughly stir the top 4-5 mm of the column packing with a Pasteur pipet for some seconds, and then elute carbendazim with 85 ml eluting mixture 2. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to dryness with 40-45 °C bath temperature. 6.5 High-performance liquid chromatographic determination Dissolve the residue derived from 6.4 in 4.0 ml (VEnd) of the mobile phase. Inject an aliquot of this solution (Vi) into the sample loop of the high-performance liquid chromatograph. Operating conditions Pump Injector Column Mobile phase Flow rate Detector Attenuation Recorder Injection volume Retention time for carbendazim

Constant volume pump, model LC 250/1 (Kratos) Injection valve 70-10 fitted with sample loop 70-11 (Rheodyne) Stainless steel, 4 mm i.d., 12 cm long (Knauer); packed with LiChrospher Si 100, medium particle size 10 urn (Merck No. 9312) Ethanol + n-hexane + phosphoric acid 700:300:0.2 v/v/v 1.5 ml/min UV detector Spectroflow 773 (Kratos) Wavelength 285 nm Detector range 0.009 AUFS 5 mV; chart speed 5 mm/min 50 ul 3 min 50 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject 50 ul of the carbendazim standard solutions (equivalent to 1.5, 4.5, 12 and 30 ng carbendazim) into the high-performance liquid chromatograph. Plot the heights of the peaks

74

Benomyl, Carbendazim, Thiophanate-methyl

obtained vs. ng carbendazim. Also inject 50-^1 aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of carbendazim from the calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with benomyl, carbendazim and thiophanate-methyl at levels of 0.04 to 1.0 mg/kg, ranged from 73 to 94% and averaged 84% (see Table). Blank values did not occur. The routine limit of determination was 0.02 mg/kg for lettuce, 0.04 mg/kg for wheat grains, and 0.08 mg/kg for wheat straw. Table. Percent recoveries from lettuce, wheat grains, and wheat straw, fortified with benomyl, carbendazim and thiophanate-methyl, expressed as carbendazim equivalents. Compound added (mg/kg)

Analytical material Carbendazim Lettuce

Thiophanatemethyl

Benomyl

0.04 0.4

Wheat straw

0.02 0.2 0.1 0.5 0.05 0.2 0.2 1.0 0.1 0.5

Recovery

78

0.06 0.6

Wheat grains

Carbendazim equiv. *)

0.02 0.2

0.2 1.0 0.05 0.2 0.4 1.0 0.1 0.5

0.06 0.6 0.02 0.2

0.034 0.34 0.04 0.40 0.044 0.44

0.05 0.2

0.12 0.56 0.11 0.44

0.1 0.5

0.22 0.56 0.22 1.11

78 73 81 85 77/85/88 85/88/88 82 86 79/82 81/82/89/91 84 83 93 84 79 73 78/90/94 79/83/86

*) The factors for conversion of thiophanate-methyl and benomyl to carbendazim are 0.558 and 0.659, respectively.

7.3 Calculation of residues The residue R of benomyl, carbendazim and thiophanate-methyl, expressed in mg/kg carbendazim, is calculated from the following equation:

w A -v E x -v E n d VR1.VrG

Benomyl, Carbendazim, Thiophanate-methyl

75

where G V Ex VR1 V End Vj WA

= = = = =

sample weight (in g) volume of extract solution from 6.1 (in ml) portion of volume V Ex used for further processing (in ml) terminal volume of sample solution from 6.5 (in ml) portion of volume V End injected into high-performance liquid chromatograph (volume of sample loop) (in \i\) = amount of carbendazim for Vj read from calibration curve (in ng)

8 Important points No data

9 Reference H. Suzuki et al., Rapid and systematic determination of thiophanate methyl (TPM), benomyl and MBC (methyl benzimidazolecarbamate) by a combined method of alumina column cleanup and UV spectrophotometry, Agric. Biol. Chem. 46, 549-552 (1982).

10 Authors Ciba-Geigy AG, Agricultural Division, Basle, Switzerland, G. Formica, C. Giannone and W. D. Hormann

Bitertanol Apples, barley (green matter, grains and straw), cherries (fruit, conserves, juice and press pulp), cucumbers, pears, plums (fruit, jam and puree), sugar beet (foliage and edible root), tea (dry leaf, liquor and infused leaf), tomatoes Soil, water

613-A High-performance liquid chromatographic determination

(German version published 1991)

1 Introduction For data on physico-chemical properties of bitertanol, see Method for Bitertanol, Triadimefon, Triadimenol on p. 87, this Volume.

2 Outline of method Bitertanol residues are extracted from cereal samples (green matter, grains and straw) and tea leaves with an acetone-water mixture, and from other plant material with acetone. Soil samples are refluxed in aqueous methanol to extract the residues. The extract is concentrated to an aqueous residue, which is made up to a definite volume. An aliquot of this solution is transferred onto a disposable extraction column. Water and liquid samples are transferred directly onto the disposable extraction column. The column is eluted with a cyclohexane-ethyl acetate mixture, and the eluate is evaporated to dryness. Bitertanol is determined by highperformance liquid chromatography using a fluorescence detector.

3 Apparatus Homogenizer Wide neck glass bottles, 1-1 and 500-ml, with ground joints Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia., fast flow rate Filtration flask, 1-1 Round-bottomed flasks, 1-1, 500-ml and 250 ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Glass funnel, 10 cm dia. Reflux condenser Heating mantle for 1-1 round-bottomed flask Solvent dispensers, 50-ml and 10-ml Graduated cylinders, 500-ml and 250-ml

78

Bitertanol

Volumetric flasks, 100-ml, 50-ml and 25-ml, with ground joints Volumetric pipets, 100-ml, 50-ml, 10-ml, 5-ml, 2-ml and 1-ml Centrifuge, e. g. Variofuge (Heraeus-Christ), with 10-ml glass tubes Ultrasonic bath Test tubes, 10-ml, graduated, with ground stoppers High-performance liquid chromatograph equipped with fluorescence detector Microsyringe, 100- JLXI

4 Reagents Acetone, for residue analysis Acetonitrile, for chromatography Cyclohexane, for residue analysis Ethyl acetate, for residue analysis Methanol, for residue analysis Water, ultrapure Acetone + water mixture 2:1 v/v Acetonitrile + water mixture 1:1 v/v Methanol + water mixture 7:3 v/v Eluting mixture: cyclohexane + ethyl acetate 85:15 v/v Mobile phase: acetonitrile + water mixture 55:45 v/v Bitertanol stock solution: 1000 M-g/ml ethyl acetate Bitertanol standard solutions: 0.05-100 M-g/ml acetonitrile-water mixture 1:1 v/v Filter aid, e. g. Celite 545 Disposable extraction columns, 100-ml and 50-ml (Chem Elut CE 20100 and CE 2050; Analytichem) Air, synthetic, re-purified Helium 4.6 (> 99.996 vol. %)

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material with high water content

Transfer 100 g of the analytical sample (G) into the 1-1 glass bottle with 200 ml acetone and homogenize for approx. 3 min. For tomatoes and other material from which it is difficult to take a representative 100-g sample, homogenize 200 g with 300 ml acetone.

Bitertanol

79

Add approx. 15 g filter aid, and filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, using gentle suction. Rinse the filter cake and the bottle several times with a total of 150 ml acetone-water mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 round-bottomed flask and rotary-evaporate to an aqueous residue (approx. 150 ml for a 100-g sample, approx. 250 ml for a 200-g sample). Make up the aqueous residue with water to 200 ml (100-g sample) or 400 ml (200-g sample) in a graduated cylinder (VEx). Pipet 50 ml (VR1) of this solution onto a dry disposable 50-ml extraction column and allow the solution to soak in. Elute the column three times with 50-ml portions of the eluting mixture. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.1.2 Cereals Transfer 50 g of cereal green matter or grains, or 25 g of straw (G) into the 1-1 glass bottle with 450 ml (for grains, 300 ml) acetone-water mixture, allow to stand for 10 min, and then homogenize for approx. 3 min. Add approx. 15 g filter aid, and filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, using gentle suction. Rinse the filter cake and the bottle several times with a total of 150 ml acetone-water mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 round-bottomed flask and rotary-evaporate to an aqueous residue. Make up the aqueous residue with water to 200 ml in a graduated cylinder (VEx). Pipet 100 ml (VR1) of this solution onto a dry disposable 100-ml extraction column and allow the solution to soak in. Elute the column three times with 100-ml portions of the eluting mixture. Collect the eluate in a 500-ml round-bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.1.3 Tea leaves Transfer 25 g of dry tea leaves or 10 g of infused tea leaves (G) into the 500-ml glass bottle with 100 ml acetone-water mixture and homogenize for approx. 3 min. Add approx. 15 g filter aid, and filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, using gentle suction. Rinse the filter cake and the bottle several times with a total of 150 ml acetone-water mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 round-bottomed flask and rotary-evaporate to an aqueous residue. Make up the aqueous residue with water to 150 ml in a graduated cylinder (VEx). Pipet 50 ml (VR1) of this solution onto a dry disposable 50-ml extraction column and allow the solution to soak in. Elute the column three times with 50-ml portions of the eluting mixture. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.1.4 Beverages (e. g. cherry juice, tea liquor)

Pipet 50 ml of the analytical sample (G) directly onto a dry disposable 50-ml extraction column and allow the liquid to soak in. Elute the column three times with 50-ml portions of the eluting mixture. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2.

80

Bltertanol

6.1.5 Soil Weigh 50 g soil (G) into a 1-1 round-bottomed flask, add 300 ml methanol-water mixture, and heat under reflux for 4 h. Allow to cool, and filter the suspension with gentle suction through a fast flow-rate filter paper, covered with approx. 15 g filter aid, in a Buchner porcelain funnel. Rinse the flask and filter cake twice with 50-ml portions of methanol-water mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 round-bottomed flask and rotary-evaporate to an aqueous residue (approx. 130 ml). Make up the aqueous residue with water to 200 ml in a graduated cylinder (VEx). Pipet 100 ml (VR1) of this solution onto a dry disposable 100-ml extraction column and allow the solution to soak in. Elute the column three times with 100-ml portions of the eluting mixture. Collect the eluate in a 500-ml roundbottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.1.6 Water Pipet 100 ml of the water sample (G) directly onto a dry disposable 100-ml extraction column and allow the water to soak in. Elute the column three times with 100-ml portions of the eluting mixture. Collect the eluate in a 500-ml round-bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.2 High-performance liquid chromatographic determination Dissolve the residue derived from 6.1 in a definite volume (VEnd, e.g. 5 ml; for water, 2 ml) of acetonitrile-water mixture, immersing the flask in an ultrasonic bath. Transfer the solution to a test tube, and stopper. Remove any undissolved material by centrifugation or allow it to settle by standing overnight in a refrigerator. Inject an aliquot of the clear supernatant (V{) into the high-performance liquid chromatograph. Operating conditions Chromatograph Column Column temperature Mobile phase Column inlet pressure Flow rate Detector Attenuation Integrator Injection volume Retention time for bitertanol

Spectra-Physics 8100 fitted with autosampler Stainless steel, 4 mm i.d., 12.5 cm long; packed with LiChrospher RP 18, particle size 5 |nm (Merck) 40 °C Acetonitrile + water 55:45 v/v Approx. 60 bar 1 ml/min Fluorescence detector RF 530 (Shimadzu) Wavelengths: excitation 254 nm, emission 322 nm Detector range 2 (for water, range 1) Sensitivity high Integrator Spectra-Physics SP 4270 or Laboratory Data System LAS on a Hewlett-Packard HP 1000 A 10 ul (for water, 25 \i\) 4 min 36 s

Bitertanol

0 00

i.25

2.50

3.75

5.00

6.25

7.50

81

8.75

Fig. 1. Bitertanol in apples (VEnd = 5 ml each). Chromatogram A: Extract from untreated control sample, fortified with standard solution; peak representing 1 ng bitertanol. Chromatogram B: Untreated control sample fortified with 0.02 mg/kg bitertanol. Chromatogram C: Untreated control sample.

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.75

10.00 mm

82

0 00

Bitertanol

1.25

2.50

3.75

5.00

6.25

7.50

8.75

10.00

mm

Fig. 2. Bitertanol in sugar beet foliage (VEnd = 5 ml each). Chromatogram A: Standard solution representing 1 ng bitertanol. Chromatogram B: Untreated control sample fortified with 0.02 mg/kg bitertanol. Chromatogram C: Untreated control sample.

^ yv

0.00

1.23

2.50

3.75

3.00

6.26

7.51

8.76

10.01

Bitertanol

83

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the bitertanol standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. The linearity range examined for bitertanol extended from 0.5 to 500 ng; the recommended measuring range is from 0.5 to 10 ng. 7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material, beverages, soil and water, fortified with known amounts of bitertanol dissolved in 1-2 ml ethyl acetate. The results are given in the Table. Table. Percent recoveries from plant material, beverages, soil and water, fortified with bitertanol; duplicate experiments. . . . . . . Analytical material Apples

Barley Green matter

Grains

Straw

Cherries Fruit

Conserves

Juice

Press pulp

Added mg/kg e

0.02 > 0.2 2.0 0.02 0.2 2.0 0.02 0.2 2.0 0.02 0.2 2.0 0.02 0.2 2.0 20 0.02 0.2 1.0 0.002b> 0.02b> 0.2b> 1.0b> 0.02 0.2 1.0

Range %

80-110a> 96-98 96-99 105 96-98 93-97 104-105 97-101 86-98 90a> 81-93 87-91 95-99 95-97 93-97 98-102 98-100 a) 93-103 94-96 100 100 95-101 101-103 81-87a> 92-99 89-101

84

Bitertanol

Table, (contd.) , . , . . Analytical material Cucumbers

Pears

Plums Fruit

Jam Puree Tea Liquor Dry leaf

Infused leaf Tomatoes

Sugar beet Foliage

Edible root

Soil Standard soil 2.1

Standard soil 2.2

Pisserfeld soil

Laacherhof soil

Added

Range

mg/kg

%

0.02 0.2 2.0 0.02 0.2 2.0

93-94 89-95 93-95 95-97 97 97-98

0.02 0.2 2.0 0.02d> 0.2d> 0.02d> 0.2d>

85-88 81-93 82-84 70-77 75-80 66-71 65-78

0.002b> 0.02 b> 0.02 0.2 2.0 10 2.0 0.02 0.2 2.0

100 100-105 75-80a> 83-86 84 79-86 99-100 101-104 98 100-102

0.02 0.2 2.0 0.02 0.2 2.0

96 93-97 99-100 97-99 92-98 96-101

0.01 0.1 1.0 0.01 0.1 1.0 0.01 0.1 1.0 0.01 0.1 1.0

91-95 86-88 90-95 100-107 94-98 93-94 91-94 92-96 81-90 89-90 83-89 94-98

Bitertanol

85

Table, (contd.) . . . . ^ . . Analytical material

Added

Range

mg/kg

Water Tap water Process water

%

0.1 c> 0.1 c> 1.0c> 10 c> 0.1c'e>

Leaching water

91-99 91-97 85-88 99-100 75-98

a) Because of blanks, the evaluation of the recovery experiments was performed with the aid of the "Standard Addition Method" (for details, see Method for Glufosinate, p. 217, this Volume), b) mg/1, c) |ig/l, d) 4 recovery experiments, e) 6 recovery experiments.

The soils used for the recovery experiments had the following characteristics:

_ ., Soil type

Organic carbon %

Standard soil 2.1*> Standard soil 2.2* > "Pisserfeld" "Laacherhof, Miete A "

0.79 2.06 1.65 1.45

Particles < 0.02 mm %

7.4

11.0 55.0 20.2

pH 5.7 6.0 6.7 5.8

*) Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

The data for water relate to tap water, process water and leaching water. Blank values of approx. 0.002 to 0.004 mg/kg were observed with apples, barley, cherries, pears, and tea leaves. The routine limit of determination was 0.02 mg/kg for plant material, 0.002 mg/1 for beverages, 0.01 mg/kg for soil and 0.1 ng/1 for water.

7.3 Calculation of residues The residue R, expressed in mg/kg bitertanol, is calculated from the following equation: R __

F A -V E x -V E n d -W s t Fst-V^-Vi-G

where G VEx VRI VEnd

= = = =

sample weight (in g) or volume (in ml) volume of concentrated extract after dilution with water (in ml) portion of volume VEx used for further cleanup (in ml) terminal volume of sample solution from 6.2 (in ml)

86

Bitertanol

Vj

= portion of volume V End in injected into high-performance liquid chromatograph (in ul)

W St

= amount of bitertanol injected with standard solution (in ng)

FA

= peak area obtained from Vj (in mm 2 or integrator counts)

F St

= peak area obtained from W St (in mm 2 or integrator counts)

8 Important points No data

9 References R. Brennecke, Methode zur gaschromatographischen Bestimmung von Riickstanden des Fungizids ®Baycor in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 38, 33-54 (1985). R. Brennecke, Methode zur hochdruckfltissigchromatographischen Bestimmung von Riickstanden des Fungizids ®Baycor in Pflanzenmaterial und Getranken durch FluoreszenzDetektion, Pflanzenschutz-Nachr. 41, 113-135 (1988). M. C. S. Mendes, A gas chromatographic method for the determination of residues of bitertanol, J. Agric. Food Chem. 33, 557-560 (1985). W. Specht and M. TilIkes, Gaschromatographische Bestimmung von Riickstanden an Pflanzenbehandlungsmitteln nach Clean-up iiber Gelchromatographie und Mini-KieselgelSaulenchromatographie. 2. Mitt.: Bestimmung der Fungizide Bitertanol, Fluotrimazol, Fuberidazol, Imazalil, Rabenzazole, Triadimefon und Triadimenol in Pflanzen und Boden, Pflanzenschutz-Nachr. 33, 61-85 (1980).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke

Bitertanol, Triadimefon, Triadimenol 613-425-605 Apples, bananas, barley (green matter, grains and straw), Gas-chromatographic determination cucumbers, fruit juices, melons, peaches, pears, sugar beet (foliage and edible root) Soil, water Bitertanol (additionally): Apricots, artichokes, beans (green), cherries, peanuts (kernels and shells), plums Triadimefon and triadimenol (additionally): Grapes, hop cones, must, rye (green matter, grains and straw), sweet peppers, tomatoes, wheat (green matter, grains and straw), wine (German versions published 1987)

1 Introduction Chemical name

Structural formula

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Bitertanol a//-rac-l-(Biphenyl-4-yloxy)-3,3-dimethyl-l-(lH-l,2,4triazol-l-yl)butan-2-ol (IUPAC) OH CH33 I I - O - C H - C H - C — C H 33 I I CH A 3 (l N N U

C2oH23N302 337.42 139.8 °C (diastereoisomer A) 146.3 °C (diastereoisomer B) 118.0°C (eutectic) No data <10~ 5 mbar at 20 °C (extrapolated) Virtually insoluble in water; readily soluble in dichloromethane (20-50 g [A] and [B]); soluble to slightly soluble in 2-propanol (2-5 g [A] and [B]); slightly to sparingly soluble in toluene (1-2 g [A], 0.1-0.2 g[B]); sparingly soluble in n-hexane (0.2 g [A] and [B])

88

Bltertanol, Triadimefon, Triadimenol

Other properties

Chemical name

Colourless crystals. Stability to hydrolysis: No breakdown after 12 months in acid (pH 3), neutral, and alkaline (pH 10) media Triadimefon

Triadimenol

l-(4-Chlorophenoxy)-3,3dimethyl-l-(lH-l,2,4-triazoll-yl)butanone (IUPAC)

l-(4-Chlorophenoxy)-3,3dimethyl-l-(lH-l,2,4-triazoll-yl)butan-2-ol (IUPAC) OH I

Structural formula

Empirical formula Molar mass Melting point Vapour pressure Solubility

Other properties

CH3 I

O - C H - C H - C - C H 33 I I N CH3 (I

N

N

U

C14H16C1N3O2 293.76 82.3 °C

C14H18C1N3O2 295.77 110°C (eutectic mixture of the two diastereoisomers) < 1 0 " 5 mbar at 20 °C < 1 0 " 5 mbar at 20 °C Virtually insoluble in water; Very sparingly soluble in water; readily soluble in readily soluble in dichlorodichloromethane, 2-propanol methane and 2-propanol; and toluene; soluble in toluene; soluble in n-hexane very sparingly soluble in n-hexane Colourless crystals, resistant Colourless crystals, resistant to to hydrolysis hydrolysis

2 Outline of method Bitertanol, triadimefon and triadimenol residues are extracted from plant material of low water content with an acetone-water mixture, and from other plant material with acetone. The extracts are saturated with sodium chloride and shaken with dichloromethane. Soil samples are refluxed in aqueous methanol to extract the residues. The methanol is evaporated, and the aqueous residue is shaken with dichloromethane. Dichloromethane is used for direct extraction from water samples. The dichloromethane phases are evaporated to dryness. With plant material and soil, the extracts are cleaned up on a silica gel column, followed by gel permeation chromatography on a Bio-Beads S-X3 column. For water samples, and in the case of triadimefon and triadimenol residues also for soil samples, the silica gel cleanup step can be omitted. The compounds are determined by gas chromatography using a thermionic detector.

Bitertanol, Triadimefon, Triadimenol

89

3 Apparatus Homogenizer Wide neck glass bottle, 1-1, with ground joint Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia., fast flow rate Filtration flask, 1-1 Separatory funnels, 1-1, 500-ml and 250-ml, with ground stoppers Round-bottomed flasks, 1-1, 500-ml, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Glass funnel, 10 cm dia. Reflux condenser Heating mantle for 1-1 round-bottomed flask Chromatographic tube, 17.5 mm i.d., 30 cm long, extended outlet fitted with PTFE-stopcock Glass syringe, 10-ml, with Luer-lock fitting Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Test tubes, 10-ml, with ground stoppers Gas chromatograph equipped with thermionic nitrogen-specific detector Micro syringe, 10-ul

4 Reagents Acetone, for residue analysis Cyclohexane, for residue analysis Dichloromethane, for residue analysis Ethyl acetate, for residue analysis Methanol, for residue analysis Toluene, for residue analysis Acetone + water mixture 2:1 v/v Methanol + water mixture 7:3 v/v Eluting mixture 1: cyclohexane + ethyl acetate 85:15 v/v Eluting mixture 2: cyclohexane + ethyl acetate 2:8 v/v Eluting mixture 3: cyclohexane + ethyl acetate 1:1 v/v Compound standard solutions: 0.2-250 ^ig/ml bitertanol, 0.1-100 fig/ml triadimefon and 0.2-200 M-g/nil triadimenol in ethyl acetate Sodium chloride, p. a. Sodium sulphate, p.a., anhydrous Filter aid, e.g. Celite 545 Silica gel 60, 0.063-0.200 mm (Merck No. 7734) Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Cottonwool, chemically pure Glass wool Air, synthetic, re-purified Hydrogen 5.0 (> 99.999 vol. %) Nitrogen 4.6 (> 99.996 vol. %)

90

Bitertanol, Triadimefon, Triadimenol

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material with high water content (e.g. apples, apricots, bananas, beans, cherries, cucumbers, grapes, melons, peaches, pears, plums, sugar beet, sweet peppers, tomatoes)

Transfer 100 g of the analytical sample (G) to the 1-1 glass bottle with 200 ml acetone and homogenize for approx. 3 min. Add approx. 15 g filter aid, swirl the bottle several times, and filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, using gentle suction. Rinse the filter cake and the bottle with two 100-ml portions of the acetonewater mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 separatory funnel, saturate with approx. 40 g sodium chloride, and shake with 100 ml dichloromethane. Let the phases separate and discard the lower aqueous phase. Rotaryevaporate the remaining organic phase in a 1-1 round-bottomed flask to a volume of 40-50 ml. Add 50 ml dichloromethane and approx. 50 g sodium sulphate (for triadimefon and triadimenol, add 25 ml dichloromethane and approx. 30 g sodium sulphate), and filter through a cottonwool plug overlaid with an approx. 3-cm layer of sodium sulphate in a glass funnel. Collect the filtrate in a 500-ml round-bottomed flask. Rinse the 1-1 round-bottomed flask and the funnel three times with 50-ml portions of dichloromethane. Rotary-evaporate the combined filtrates to dryness, and proceed as described in 6.2. 6.1.2 Artichokes, cereals, peanuts (for bitertanol)

Transfer 50 g of cereal green matter, cereal grains, peanut kernels or artichokes, or 25 g of cereal straw or peanut shells (G) to the 1-1 glass bottle, add 150 ml water, and leave to stand for 20 min. Add 300 ml acetone, and homogenize for about 3 min. For homogenizing cereal grains and peanut kernels, use 100 ml water and 200 ml acetone. Proceed as described in 6.1.1. 6.1.3 Cereals, hop cones (for triadimefon and triadimenol) Transfer 50 g of cereal green matter or grains, 25 g cereal straw, or 10 g hop cones (G), to the 1-1 glass bottle with 450 ml acetone-water mixture and homogenize for approx. 3 min. Then proceed as described in 6.1.1. 6.1.4 Fruit juice, must, wine Transfer 100 g of the analytical sample (G) to a 1-1 separatory funnel. Add 200 ml acetone and 40 g sodium chloride, and shake the mixture with 100 ml dichloromethane. After phase separation, discard the lower aqueous phase and continue to process the organic phase as described in 6.1.1.

Bitertanol, Triadimefon, Triadimenol

91

6.1.5 Soil Weigh 50 g soil (G) into a 1-1 round-bottomed flask, add 300 ml methanol-water mixture, and heat under reflux for 4 h. Allow to cool, and filter the suspension with gentle suction through a fast flow-rate filter paper covered with approx. 15 g filter aid in a Buchner porcelain funnel. Rinse the flask and filter cake twice with 50-ml portions of methanol-water mixture. Allow the filter cake to pull dry, and discard it. Rotary-evaporate the filtrate to its aqueous residue (approx. 100 ml), and transfer to a 250-ml separatory funnel. Rinse the flask with dichloromethane and shake the aqueous residue three times with dichloromethane (100, 100, 50 ml). Filter the organic phase successively through a cottonwool plug overlaid with an approx. 3-cm layer of sodium sulphate in a glass funnel. Collect the filtrate in a 500-ml round-bottomed flask. Wash the sodium sulphate three times with 25-ml portions of dichloromethane, and rotary-evaporate the combined filtrates to dryness. For bitertanol, proceed as described in 6.2; for triadimefon and triadimenol proceed to 6.3. 6.1.6 Water

Extract 400 ml water (G) three times with 200-ml portions of dichloromethane. In the case of only small amounts of water being available (e. g. 100 ml), extract the sample with correspondingly smaller portions of dichloromethane. Filter the organic phases successively through a cottonwool plug overlaid with an approx. 3-cm layer of sodium sulphate in a glass funnel. Collect the filtrate in a 1-1 round-bottomed flask. Wash the sodium sulphate three times with 25-ml portions of dichloromethane, and rotary-evaporate the combined filtrates to dryness. Then proceed as described in 6.3. 6.2 Column chromatography Fill the chromatographic tube, in this order, with 10 ml toluene, a cottonwool plug, 15 g silica gel (mixed to a slurry with toluene, filling height approx. 13 cm), approx. 1 cm sodium sulphate, and a loose glass wool plug. Then drain the toluene down to the top of the sodium sulphate layer. Dissolve the residue derived from 6.1.1, 6.1.2, 6.1.3, 6.1.4, and (for bitertanol residues) 6.1.5 in 10 ml toluene, transfer the solution onto the column, and allow to percolate to the top of the sodium sulphate. Rinse the flask twice with 10-ml portions of eluting mixture 1. Pre-wash the column with the rinsings followed by a further 80 ml of eluting mixture 1. Next elute the compounds from the column with 100 ml eluting mixture 2 into a 250-ml round-bottomed flask. Rotary-evaporate the eluate to dryness, then proceed as described in 6.3. 6.3 Gel permeation chromatography Transfer the residue derived from 6.1.5 (for triadimefon and triadimenol), 6.1.6 or 6.2 into a test tube, using a total of 10 ml eluting mixture 3 (VR1) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR2) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with standard solutions containing approx. 40 [ig/ml of each compound; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. — Elution volumes ranging from 100 to 130 ml were

92

Bitertanol, Triadimefon, Triadimenol

determined for triadimefon and triadimenol, and from 120 to 140 ml for bitertanol, on BioBeads S-X3 polystyrene gel, using eluting mixture 3 as eluant, pumped at a flow rate of 5.0 ml/min. Collect the appropriate fraction, according to the elution volumes of the compounds, in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to 6.4. Check the elution ranges from time to time, and determine anew whenever a new gel column is used. 6.4 Gas-chromatographic determination Dissolve the residue derived from 6.3 in 5 ml ethyl acetate (VEnd) and transfer the solution to a glass stoppered test tube. Inject 5 ul of this solution (Vj) (if necessary, after dilution with ethyl acetate to an appropriate volume) into the gas chromatograph. Operating conditions Gas chromatograph Column 1

Varian 3700 Glass, 3 mm i.d., 1.8 m long; packed with 1.5% SP2250 + 1.95% SP-2401 on Supelcoport, 100-120 mesh Column 2 Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE30 on Chromosorb W-HP, 80-100 mesh Injection port temperature 280 °C Detector Thermionic nitrogen-specific detector Temperature 35O°C Gas flow rates Hydrogen, 4.5 ml/min Air, 175 ml/min Attenuation 10- 11 Recorder 1 mV; chart speed 5 mm/min Linearity range Bitertanol 1-50 ng Triadimefon 0.5-50 ng Triadimenol 1-100 ng Injection volume 5ul Column 2 Column 1 Conditions for bitertanol: Column temperature 245 °C 255 °C Carrier gas flow rates Nitrogen, 55 ml/min Nitrogen, 30 ml/min Retention times for 2 min 54 s bitertanol 3 min 54 s Conditions for triadimefon and triadimenol: Column temperature 215 °C 195 °C Carrier gas flow rates Nitrogen, 40 ml/min Nitrogen, 35 ml/min Retention times for triadimefon 3 min 3 min 18 s triadimenol 3 min 54 s 4 min 6 s

Bitertanol, Triadimefon, Triadimenol

93

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the compound standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas.

7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material, soil, and water, fortified with known amounts of the compounds dissolved in 1 -2 ml ethyl acetate. The results are given in the Table. Table. Percent recoveries from plant material, soil, and water, fortified with bitertanol, triadimefon and triadimenol; duplicate experiments. Analytical material Apples Fruit

Juice Pulp Apricots Artichokes Bananas Fruit

Peel

,,

Bitertanol

0.02-0.05 0.4-0.5 1.0-5.0 0.02-0.05 0.02-0.05 0.05 0.5 0.05 0.5

89-110e> 80-100c> 102-107 87-105c> 96-108b> 89-92 87-92 95 80-86

0.01-0.05 0.01-0.05 0.4-0.5 1.0 0.01-0.05 0.01-0.05 0.4-0.5 1.0

83-89b) 82-90 83-89b)

0.04-0.08 0.2-0.4 0.04-0.08 1.0-4.0 0.04-0.08 0.4-0.8 0.04-0.08 1.0-2.0 0.04-0.08 0.4-0.8 0.04-0.08 1.0-4.0 0.05 0.5

82-89 82-89 92-93 87-91 86-91 87-91

88-95b> 88-95b> 85-92

Triadimefon

84-91 92-99 84-87 80-81

87-91 85-103 95-99 82-85

Triadimenol

94-99 85-86 99-100 92-93

91-93 89-108 92 92-96

Barley Green matter

Grains

Straw

Beans

95-104 86-97 95-104 85-92 83-86

90-105b> 87-97 86-87 84-91b> 92-93 77-83 89-99b> 90-102 8 5_ 89 b)

90-104b> 87-89 87-91 90-93 b> 95 85-89 92-104b> 87-95 93-95b>

94

Bitertanol, Triadimefon, Triadimenol

Table, (contd.) Analytical material Cherries Fruit Juice Cucumbers

Grapes

Hop cones Melons

Must Peaches

Peanuts Kernels Shells Pears Fruit

Juice Plums Rye Green matter

Grains Straw Sugar beet Foliage

Edible root

Added mg/kg

Bitertanol

Triadimefon

0.05 0.5 0.05 0.5 0.02-0.05 0.1-0.5 1.0 0.02-0.05 0.4 1.0 0.3-0.5 3.0-5.0 0.02-0.05 0.4 1.0 0.02-0.05 0.02-0.05 0.25-0.5 1.0

82-90b> 89-102b> 87-91 93-97 89-105b> 85-98b>

0.02-0.05 0.5 0.05 0.5

83-92b> 88-89 91-92 86-90

0.02-0.05 0.1-0.5 1.0 0.02-0.05 0.05 0.5

90-104^ 87-110«)

81-91 100-101

86-100b> 93-105 82-83

80-81

95 86-88

93-99

84-88 81-86 88-97 90-92

86-96 93-100 85-94b>

86-93 86 83

84-87 93-95 107-110 92-100 101-104 85-93 93-102 97-98 85-86 100-103 96-97

0.04-0.08 0.4-0.8 1.0-2.0 0.04-0.08 1.0-2.0 0.04-0.08 1.0-2.0 0.02-0.05 0.4-0.5 1.0 0.02-0.05 0.4-0.5 1.0

90-94 88-92

Triadimenol

98-104b> 84-94 86-88 91-104b> 86-88 83-93

87-95 85 89-90

75_98b) 83-89 86-90 82-103 92-96 90-103b> 78-79

81-97b> 91-96 94-102 92-106 94-98 91-101b>

80 85-86

90-93

88-91 89-100

78-86

96-98 82-87 94-105

Bitertanol, Triadimefon, Triadimenol

95

Table, (contd.) Analytical material Sweet peppers

Tomatoes

Wheat Green matter

Grains Straw Wine

Soil Water

Added mg/kg

Bitertanol

0.02-0.05 0.4 1.0 0.02-0.05 0.4 1.0 0.04-0.08 0.4-0.8 1.0-2.0 0.04-0.08 0.4-0.8 0.04-0.08 0.4-0.8 0.02-0.05 0.4 1.0 0.04-0.08 0.4-0.8 0.005*)

o.oi*) 0.5*>

Triadimefon 88-91 82-87

Triadimenol 97-105 95-100 90-96

90-97 89-94

100-106 90-110b) 85-91 80-87 90-94 89 84-104a) 96-105 102-109a) 87-94a) 83-107d) 79-101d> 91-114e> 96-105 104

96-103°) 94-108°) 88-108e) 97-112

83-98b) 90-99 83-101b> 93-99 98-99 93-1093) 103-112 92-1033) 87-1033) 104-108c) 94_1 nc) 93-103°) 94

*) Equivalent to 5, 10, and 500 ng/1, respectively. Different number of * recovery experiments: a) 3, b) 4, c) 6, d) 7, e) 8, ° 10, «> 12.

The soils used for the recovery experiments had the following characteristics: Soil type Standard soil 2.1*) Standard soil 2.2* > Standard soil 2.3* ) Alluvial soil

Organic carbon °/o

Particles < 0.02 mm %

p H

0.31 2.64 1.06 1.47

8.5 14.5 24.7 26.6

6.0 6.0 7.0 7.0

*) Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

The data for water relate to tap, spring, well, lysimeter, ground, and drainage waters as well as to water used for fish toxicity studies. The routine limit of determination for bitertanol was 0.01 mg/kg in bananas, 0.02 mg/kg in apples, pears and peanut kernels, 0.05 mg/kg in other plant material and soil, and 0.005 mg/1 in water. The routine limit of determination for triadimefon was 0.02 to 0.04 mg/kg in plant material, 0.04 mg/kg in soil, and 0.005 mg/1 in water. For triadimenol, it was 0.05 to 0.08 mg/kg in plant material, 0.08 mg/kg in soil, and 0.005 mg/1 in water.

96

Bitertanol, Triadimefon, Triadimenol

7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified compound is calculated from the following equation: R

_

F A -V R1 -V End -W St Fs,-VR2-VrG

where G

= sample weight (in g) or volume (in ml)

VR1 = volume of solution prepared for gel permeation chromatography in 6.3 (in ml) VR2 = portion of volume VR1 injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.4 (in ml) (if necessary, take account of a dilution) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

W st

= amount of bitertanol, triadimefon or triadimenol, respectively, injected with standard solution (in ng)

FA

= peak area obtained from Vj (in mm 2 or integrator counts)

FSt

= peak area obtained from WSt (in mm 2 or integrator counts)

8 Important points Triadimefon is reduced to triadimenol in plants, soil and water. Therefore, both compounds can appear as residues after the application of triadimefon. Use both gas chromatographic columns to analyze sample solutions with a high content of plant co-extractives (e. g. from cereal samples). As an additional gas-chromatographic column the following can be used: Glass column, 3 mm i.d., 1.8 m long; packed with 4% SE-30 + 6% OV-210 on Chromosorb W-HP, 80-100 mesh. On the gas-chromatographic columns described here, neither the two diastereoisomers of bitertanol nor those of triadimenol will be separated; one peak will be obtained with a shoulder appearing to a greater or lesser degree.

9 References R. Brennecke, Methode zur gaschromatographischen Bestimmung von Riickstanden der Fungizide ®Bayleton und ®Bayfidan in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 37, 66-91 (1984). R. Brennecke, Methode zur gaschromatographischen Bestimmung des Fungizids ®Baycor in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 38, 33-54 (1985). R. Brennecke and K. Vogeler, Methode zur gaschromatographischen Bestimmung von Riickstanden verschiedener Fungizide in Wasser, Pflanzenschutz-Nachr. 37, 44-65 (1984).

Bitertanol, Triadimefon, Triadimenol

97

W. Specht and M. Tillkes, Gaschromatographische Bestimmung von Riickstanden an Pflanzenbehandlungsmitteln nach Clean-up iiber Gelchromatographie und Mini-KieselgelSaulenchromatographie. 2. Mitt.: Bestimmung der Fungizide Bitertanol, Fluotrimazol, Fuberidazol, Imazalil, Rabenzazole, Triadimefon und Triadimenol in Pflanzen und Boden, Pflanzenschutz-Nachr. 55, 61-85 (1980).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke

Bromoxynil, Ioxynil

264-212

Barley (grains and straw), wheat (grains and green matter) Gas-chromatographic Soil, water determination (German version published 1989)

1 Introduction

Chemical name

Ioxynil Bromoxynil 3,5-Diiodo-4-hydroxybenzonitrile 3,5-Dibromo-4hydroxybenzonitrile (IUPAC) (IUPAC)

Structural formula Br

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C7H3I2NO C7H3Br2NO 370.92 276.93 212-213 °C, 194-195°C, octanoate 59-60 °C octanoate 45-46°C Not distillable Not distillable <10" 5 mbar at 20 °C <10" 5 mbar at 20°C Virtually insoluble in water; Very sparingly soluble in soluble in acetone (7 g) and water; methanol (2 g) readily soluble in acetone (17 g), soluble in methanol (9 g) Commercial products contain bromoxynil and ioxynil mostly as octanoates or alkali metal salts

2 Outline of method After refluxing the analytical sample with methanolic potassium hydroxide solution, bromoxynil and ioxynil residues are partitioned into dichloromethane. The extract is cleaned up by acid-base partitioning, whereupon the compounds are methylated with diazomethane. The methyl ethers are chromatographed on a Florisil column and are determined by electron capture gas chromatography.

3 Apparatus High-speed blendor, e.g. Waring Blendor Round-bottomed flasks, 1-1, 500-ml, 250-ml and 100-ml, with ground joints

100

Bromoxynil, loxynil

Heating mantles, 1-1 and 500-ml Reflux condenser (Dimroth type) Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate Rotary vacuum evaporator, 40 °C bath temperature Glass funnel Fluted filter paper, 15 cm dia. (Schleicher & Schull) Separatory funnels, 1-1 and 500-ml Membrane filter, e.g. SM 11605-025 N, 0.65-u.m (Sartorius), and Millex-HV4 filter unit (Millipore) Glass syringe, 10-ml, with Luer-lock fitting Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Chromatographic tube, 15 mm i.d., 40 cm long Methylation apparatus, see Fig. 1, p. 130, Vol. 1 Graduated cylinders, 1-1, 500-ml, 250-ml, 100-ml and 50-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Acetone, high purity Cyclohexane, for residue analysis Dichloromethane, techn. pure, dist. Diethyl ether, high purity, dried over calcium chloride Ethyl acetate, for residue analysis n-Hexane, high purity Methanol, high purity Toluene, high purity 2,2,4-Trimethyl pentane (isooctane), p. a. Eluting mixture 1: cyclohexane + ethyl acetate 1:1 v/v Eluting mixture 2: n-hexane + toluene 8:2 v/v Eluting mixture 3: n-hexane + toluene 2:8 v/v Compound standard solutions: 1.46, 14.6, 146 and 1460 u.g/ml bromoxynil octanoate or 1.34, 13.4, 134 and 1340 |iig/ml ioxynil octanoate (equivalent to 1, 10, 100 and 1000 ng/ml bromoxynil or ioxynil) in methanol Derivative standard solutions: bromoxynil methyl ether or ioxynil methyl ether (equivalent to 0.01 to 0.1 ng/ml bromoxynil or ioxynil) in isooctane. Reflux 728 [ig bromoxynil octanoate or 670 |ug ioxynil octanoate (equivalent to 500 fig bromoxynil or ioxynil) for 1 h in 200 ml methanolic potassium hydroxide solution. Allow to cool and rinse the condenser with 20 ml methanol. Filter through a fluted filter paper and wash with 20 ml methanol. Add 20 ml water and rotary-evaporate to an aqueous residue. Transfer the residue into a 250-ml separatory funnel, using 100 ml water to complete the transfer, acidify with 3 ml sulphuric acid, and extract the compound with dichloromethane (see 6.2.1). Perform the methylation as described in 6.2.3. Remove the solvent, dissolve the residue in isooctane, and dilute this solution to the concentrations given above

Bromoxynil, loxynil

101

Glacial acetic acid, p. a. Sulphuric acid, p.a., cone. Ethanolic potassium hydroxide solution: Dissolve 7 g KOH p.a. in 10 ml water and make up to 100 ml with ethanol Methanolic potassium hydroxide solution: 3 g/100 ml KOH p.a. Sodium sulphate, p.a., anhydrous Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Florisil, 60-100 mesh, deactivated with 5% water: Heat a weighed sample of Florisil for at least 8 h at 130 °C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared; next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Filter aid, e.g. Celite 545 Diazomethane solution in diethyl ether (for apparatus see Fig. 1, p. 130, Vol. 1): Dissolve 1.2 g N-methyl-N-nitroso-p-toluenesulphonamide in 10 ml diethyl ether and transfer to the dropping funnel. Slowly add this solution dropwise to 5 ml ethanolic potassium hydroxide solution contained in the reaction vessel, and sweep the generated diazomethane into 20 ml diethyl ether, using a gentle stream of nitrogen, while the receiver containing the ether is cooled in an ice + sodium chloride freezing mixture Cottonwool, exhaustively extracted with dichloromethane Helium Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material (except grains)

Reflux 50 g of finely cut plant green matter or 25 g of chopped straw (G) with 400 ml methanolic potassium hydroxide solution for 1 h. Allow to cool and rinse the condenser with 20 ml methanol. Filter the mixture through a fast flow-rate filter paper covered with filter aid in a Buchner porcelain funnel and wash the filter cake with 80 ml methanol. Add 20 ml water to the filtrate and rotary-evaporate to approx. 100 ml. 6.1.2 Cereal grains, soil

Reflux 50 g of finely ground grains or 50 g soil (G) for 1 h with 200 ml methanolic potassium hydroxide solution. Then proceed as described in 6.1.1.

102

Bromoxynil, loxynil

6.1.3 Water

Reflux 500 ml of the water sample (G) with 200 ml methanolic potassium hydroxide solution for 1 h. Allow to cool and rinse the condenser with 20 ml methanol. Filter the solution through a fluted filter paper, wash the filter with 30 ml methanol, and rotary-evaporate the filtrate to an aqueous residue. Then proceed as described in 6.2.1. 6.2 Cleanup 6.2.1 Acid-base partition

Quantitatively transfer the residue derived from 6.1.1 into a 500-ml separatory funnel, using 100 ml water to complete the transfer. Quantitatively transfer the residue derived from 6.1.3 into a 1-1 separatory funnel. Acidify to pH 1-2 with 3-7 ml of sulphuric acid. Allow to cool to room temperature, and shake three times with dichloromethane (50, 25, 25 ml) for 2 min each time. Dry the combined dichloromethane phases on sodium sulphate and filter through a fluted filter paper covered with sodium sulphate. Wash the filter with 30 ml dichloromethane, and rotary-evaporate the filtrate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. 6.2.2 Gel permeation chromatography (see 8. Important points) Dissolve the residue derived from 6.2.1 in 10 ml eluting mixture 1 (VEx) and filter the solution through a membrane filter. Using a 10-ml syringe, load the 5-ml sample loop (VR1) of the gel permeation chromatograph with 8 to 9 ml of the filtrate. Set the gel permeation chromatograph at the eluting conditions determined beforehand with standard solutions of bromoxynil and ioxynil; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. — Elution volumes ranging from 80 to 140 ml were determined for bromoxynil and ioxynil on Bio-Beads S-X3 polystyrene gel, using eluting mixture 1 as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 80 to 140-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. Check the elution range from time to time, and determine anew whenever a new gel column is used. 6.2.3 Methylation Dissolve the residue derived from 6.2.1 or 6.2.2 in 1 ml methanol, add 5 ml of the diazomethane solution, and allow to stand for 15 min with occasional swirling. Remove excess diazomethane and solvent with a gentle stream of nitrogen. 6.2.4 Column chromatography Insert a cottonwool plug into the bottom of the chromatographic tube and add 5 ml hexane. Fill the chromatographic tube with a slurry of 15 g Florisil in 35 ml hexane, while gently tapping the tube walls. As soon as the Florisil has settled, trickle in 5 g sodium sulphate. Drain the supernatant to the top of the sodium sulphate layer. Dissolve the residue derived from 6.2.3 in 1 ml eluting mixture 2 and transfer the solution quantitatively onto the column. Rinse the flask portionwise with a total of 20 ml eluting mixture 2, add the rinsings to the column, and

Bromoxynil, loxynil

103

allow to percolate. Wash the column, the stopcock being opened a little, with a further 80 ml eluting mixture 2, and discard this eluate. Next elute the derivatives with 100 ml eluting mixture 3. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to near dryness. 6.3 Gas-chromatographic determination Dissolve the residue derived from 6.2.4 in isooctane and dilute to an appropriate volume (VEnd), e.g. 10 ml. Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions 6.3.1 Packed column

Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Recorder Injection volume Retention times for bromoxynil methyl ether ioxynil methyl ether

Carlo Erba Fractovap 230 Glass, 2 mm i.d., 1.5 m long; packed with 7.5% DC200 + 7.5% QF-1 on Gas Chrom Q, 80-100 mesh 200 °C 250 °C 63 Ni electron capture detector ECD HT-25, ECD Control Module 250 Temperature 275 °C Nitrogen carrier, 25 ml/min Nitrogen purge gas, 10 ml/min 1 mV; chart speed 5 mm/min 1-3 ul 1 min 24 s 3 min 36 s

6.3.2 Capillary column

Gas chromatograph Column Column temperature Injection technique Detector Gas flow rates Recorder Injection volume Retention times for bromoxynil methyl ether ioxynil methyl ether

Carlo Erba Fractovap 4160 with on-column injector Fused silica capillary, 0.32 mm i.d., 15 m long; coated with OV-1, crossbond, film thickness 0.10-0.15 |im (Carlo Erba Mega) 90 to 170 °C at maximum heating rate, programmed to rise at 2°C/min from 170 to 200 °C Cold on-column at 90 °C oven temperature with secondary cooling 63 Ni electron capture detector ECD HT-25, ECD Control Module 251 Temperature 300 °C Helium carrier, 2 ml/min Nitrogen purge gas, 30 ml/min 10 mV; chart speed 5 mm/min 1 nl 2 min 20 s 4 min

104

Bromoxynil, loxynil

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject equal volumes of each derivative standard solution (equivalent to 0.01 to 0.1 ng bromoxynil or ioxynil, respectively) into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng bromoxynil or ioxynil. Also inject equal volumes of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of bromoxynil or ioxynil from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples, fortified with bromoxynil and ioxynil (as octanoates) at levels of 0.05 to 10 mg/kg, ranged from 68 to 99% and averaged 85%. The limit of detection was 0.01 mg/kg, and the limit of determination was 0.05 mg/kg. The recoveries from tap and pond waters ranged from 71 to 93% and averaged 83%; the limit of detection was 0.05 jxg/1, and the limit of determination was 1 fxg/1. 7.3 Calculation of residues The residue R, expressed in mg/kg bromoxynil or ioxynil, is calculated from the following equation: r

w A -v E x -v E n d VR,-V,-G

where G

= sample weight (in g) or volume (in ml)

VEX VR1

= volume of solvent used to dissolve the residue from 6.2.1 (in ml) = portion of volume VEx injected for gel permeation chromatography (volume of sample loop) (in ml)

VEnd = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WA

= amount of bromoxynil or ioxynil, respectively, for Vj read from calibration curve (in ng)

8 Important points The gel permeation chromatographic cleanup (6.2.2) is only required when interfering peaks are observed. In some cases, e.g. for ground and tap waters, the column chromatographic cleanup (6.2.4) can be omitted.

BromoxynJI, loxynil

105

With straw samples, sometimes more dichloromethane will be required to wash the precipitate formed during the acidification in 6.2.1. Centrifugation is sometimes required to obtain separation of the dichloromethane phase during the partitioning steps.

9 Reference G. Zweig, Analytical methods for pesticides, plant growth regulators and food additives, Bromoxynil, Vol. V, 347-362, and Vol. VI, 605-610, Academic Press, New York and London, 1967 and 1972.

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, J. Siebers and M. Blacha-Puller

Carbendazim

378

Cereals (green matter, grains and straw) Soil

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name Structural formula Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

Methyl benzimidazol-2-ylcarbamate (IUPAC)

o

C9H9N3O2 191.19 307-312 °C (decomp.) 6.5-10- 10 mbar at 20°C Virtually insoluble in water; readily soluble in dimethylformamide and glacial acetic acid; very sparingly soluble in ethanol (30 mg) and ethyl acetate (13.5 mg); virtually insoluble in benzene (3.6 mg), dichloromethane (6.8 mg) and n-hexane (0.05 mg) Stable in weakly acid media; slowly decomposed under alkaline conditions; soluble in diluted acids, with salt formation

2 Outline of method Carbendazim residues are extracted from the sample material by adding sodium hydrogen carbonate to the substrate and homogenizing it with ethyl acetate. Neutral and acidic coextractives are separated by acid-base partition. An aliquot of the extract is derivatized with pentafluorobenzyl bromide. The pentafluorobenzyl derivative of carbendazim is cleaned up by chromatography on a silica gel minicolumn and determined by electron capture gas chromatography.

3 Apparatus High-speed blendor fitted with leak-proof glass or stainless steel jar and explosion-proof motor Buchner porcelain funnel, 13.5 cm dia.

108

Carbendazim

Filter paper, 12.5 cm dia., fast flow rate (Schleicher & Schtill), extracted with dichloromethane Filtration flask, 1-1 Separatory funnels, 1-1 and 250-ml, with ground stoppers and FIFE stopcocks Volumetric flasks, 100-ml, with ground joints Round-bottomed flask, 100-ml, with ground joint and 9-cm long neck Drying cabinet, 50 °C temperature Rotary vacuum evaporator, 40 °C bath temperature Chromatographic tube, 7.5 mm i. d., 23 cm long, with extended outlet Test tubes, 12 to 15 ml, with ground stoppers and graduation mark at 10.0 ml Gas chromatograph equipped with electron capture detector Microsyringes, 10-ul and 100-ul

4 Reagents Prepare all aqueous solutions with distilled water, not with deionized water Acetone, for residue analysis Dichloromethane, for residue analysis Ethyl acetate, for residue analysis n-Hexane, for residue analysis Toluene, for residue analysis Water, distilled, not deionized Eluting mixture: toluene + acetone 8:2 v/v Derivative standard solution, equivalent to 25 ng/ml carbendazim, in eluting mixture: Pipet 0.5 ml of a solution containing 10 ng/ml carbendazim in ethyl acetate into a 100-ml long neck flask, rotary-evaporate to dryness, and process as described in steps 6.2 and 6.3. Make up the column eluate to a volume of 10.0 ml, and dilute 1.0 ml of the solution wih 19.0 ml of eluting mixture Sulphuric acid, 0.5 mol/1 H2SO4 p.a. Potassium carbonate solution, 30 g/100 ml K2CO3 p. a. Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p. a. Pentafluorobenzyl bromide, 99 + % (Aldrich No. 10,105-2) Sodium hydrogen carbonate, p.a. Sodium sulphate, p.a., heated at 550°C for at least 2 h Filter aid, e.g. Celite 545 Glass wool, extracted exhaustively with dichloromethane Silica gel, deactivated with 1.5% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734), for at least 5 h at 130 °C, allow to cool in a desiccator, and store in a tightly stoppered container in the desiccator. To 98.5 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 1.5 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container. Before use, the deactivated silica gel must be checked for its separating efficiency by testing it with the derivative standard solution as described in 6.3 Universal indicator paper (pH 2-10) Cottonwool, extracted with dichloromethane Argon + methane mixture 9:1 v/v

Carbendazim

109

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1.

6 Procedure 6.1 Extraction and acid-base partition For samples of cereal plant green matter, cereal grains and soil, weigh 50 g (G) into the blendor jar and add 30 ml water. For samples of cereal straw, weigh 25 g (G) into the jar and add 60 ml water. Then add 10 g sodium hydrogen carbonate (20 g for soil samples), and homogenize with 200 ml ethyl acetate (VEx) for 3 min. For cereal samples (green matter, grains and straw), add 10 g filter aid, homogenize again for 10 s, and check the pH of the mixture with indicator paper. If the mixture still does not show an alkaline reaction, add more sodium hydrogen carbonate, and homogenize again. Filter the homogenate, with gentle water jet pump suction, through a fast flow-rate filter paper in a Buchner porcelain funnel, and transfer the filtrate to a 1-1 separatory funnel. Allow the phases to separate, and discard the aqueous phase. Transfer 100 ml of the organic phase (VR1) to a 250-ml separatory funnel, and extract successively with three 25-ml portions of sulphuric acid for 1 min each time. Combine the sulphuric acid phases, transfer to a 1-1 separatory funnel, and wash with 25 ml dichloromethane for 30 s. Discard the dichloromethane phase. Neutralize the acidic solution by adding a total of 200 ml sodium hydrogen carbonate solution in small portions. Add more sodium hydrogen carbonate solution as required to adjust the pH to 7-8. Extract the solution successively with three 25-ml portions of dichloromethane by shaking for 2 min each time. Filter the combined dichloromethane phases through a cottonwool plug layered with about 1 cm sodium sulphate in a funnel, collect the filtrate in a 100-ml volumetric flask, wash with dichloromethane, and make up with dichloromethane to the 100-ml mark (VR2). 6.2 Derivatization Transfer 2.0 ml (VR3) of the dichloromethane solution derived from 6.1 (4.0 ml for straw samples) into a round-bottomed flask with long neck, and rotary-evaporate to dryness. Then add 2.5 ml acetone, 50 ul potassium carbonate solution and 10 \i\ pentafluorobenzyl bromide, loosely stopper the flask with a glass stopper, place in a drying cabinet, and heat the mixture at 50 °C for 4 h. Remove the flask from the cabinet, let the mixture cool, add 5 ml toluene, and rotary-evaporate to a volume of approx. 1 ml. 6.3 Column chromatography Pack the chromatographic column successively with a glass wool plug, 1.0 g deactivated silica gel, a 5 to 10-mm layer of sodium sulphate, and another glass wool plug. Immediately before use, rinse the column with 5 ml hexane, and discard the eluate. As soon as the hexane has drained to the top of the silica gel, add the solution derived from 6.2 using a pipet, and allow

110

CarbendazJm

it to trickle in. Pour 2.0 ml toluene into the flask, swirl, and transfer the solution in the same way to the column. Allow the toluene to drain to the top of the silica gel, rinse the column with 6.0 ml toluene, and discard the eluate. Then place a graduated test tube under the column, pour 2.0 ml eluting mixture into the flask, swirl, and transfer the solution quantitatively to the column using a pipet. As soon as the solution has drained to the top of the silica gel, elute the column with 6.0 ml eluting mixture, and make up the eluate with the mixture to a volume of 10.0 ml (VEnd). 6.4 Gas-chromatographic determination Inject an aliquot (Vj) of the solution derived from 6.3 into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Carrier gas flow rate Attenuation Recorder Injection volume Retention time for carbendazim pentafluorobenzyl derivative

Hewlett-Packard 5710 G Glass, 4 mm i.d., 1.8 m long; packed with 3% OV-61 + 7.5 % QF-1 + 3% XE-60 on Chromosorb W-HP, 100-120 mesh 245 °C 250 °C 63 Ni electron capture detector Temperature 300 °C Argon-methane, 46 ml/min 64 1 mV; chart speed 15 inch/h (38.1 cm/h) 5 nl 8 min 15 s

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas or peak heights of the sample solutions and comparing them with the peak areas or peak heights obtained for the derivative standard solution or dilutions thereof. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas or heights. 7.2 Recoveries and limit of determination The recoveries from untreated control samples, fortified with carbendazim at levels of 0.05 to 0.5 mg/kg, ranged from 65 to 90% and averaged 75% for cereals (green matter, grains and straw), and ranged from 56 to 64% for soil. The limit of determination was 0.05 mg/kg.

Carbendazlm

111

7.3 Calculation of residues The residue R, expressed in m g / k g carbendazim, is calculated from the following equation:

R

_

FA-VEx-VR2-VEnd-WSt FSfVR1-VR3-VrG

where G

= sample weight (in g)

VEX

=

VRI

= portion of volume V E x used for acid-base partition (in ml)

V R2

= volume of extract after acid-base partition in step 6.1 (in ml)

V R3

= portion of volume V R 2 used for derivatization in step 6.2 (in ml)

VEnd

= terminal volume of sample solution from 6.3 (in ml)

Vj

= portion of volume V E n d injected into gas chromatograph (in ul)

W St

= a m o u n t of carbendazim injected with standard solution (in ng)

FA

= peak area or height obtained from V{ (in m m 2 or m m )

Fst

= peak area or height obtained from W S t (in m m 2 or m m )

volume of solvent used for extraction of analytical sample (in ml)

8 Important points Any residues of benomyl that may be present are completely converted to carbendazim during the extraction and cleanup steps, and are co-determined. Thiophanate-methyl, however, is converted to carbendazim to a much lesser degree (less than 10% at a level of 5 mg/kg). The method is applicable also to the analysis of tap water. Extraction is performed by adding 10 g sodium hydrogen carbonate to 100 g of the analytical sample, and shaking the mixture with 200 ml ethyl acetate for 3 min. Then a 100-ml aliquot of the organic phase is processed as described in 6.1.1. However, considerable variations (60 to 95%) in recovery rates were observed.

9 Reference G. H. Tjan and J. T. A. Jansen, Gas-liquid chromatographic determination of thiabendazole and methyl 2-benzimidazole carbamate in fruits and crops, J. Assoc. Off. Anal. Chem. 62, 769-773 (1979).

112

Carbendazim

10 Author Institute for Residue Analysis Dr. Specht & Partner, Hamburg, W. Specht

Carbosulfan, Carbofuran Grapes, head cabbage, lettuce, maize (kernels), rape (green matter and seeds), sugar beet (edible root), tomatoes Soil

658-344 Gas-chromatographic determination

(German version published 1991)

1 Introduction Carbosulfan Chemical name

2,3-Dihydro-2,2dimethylbenzofuran-7-yl (dibutylaminothio)methylcarbamate (IUPAC) CH 2 -CH 2 -CH 2 -CH 3

Carbofuran 2,3-Dihydro-2,2dimethylbenzofuran-7-yl methylcarbamate (IUPAC)

O=C-NH-CH 3

Structural formula CH,

Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

Chemical name

C20H32N2O3S 380.5 96°C (flashpoint) 4.1 • 10"7 mbar (no temp. data) Virtually insoluble in water; soluble in most organic solvents, e.g. acetone, dichloromethane, hexane, methanol and xylene

Hydrolyzed in aqueous media depending on pH, more stable at higher pH values. Half lives in water at 25 °C: pH 4: lh; pH 6: 22 h; pH 7: 7.6 d; pH 9: 58.3 d

C12H15NO3 221.3 153-154°C 2.6-10"5 mbar at 33 °C Virtually insoluble in water; readily soluble in acetone (15 g), acetonitrile (14 g) and dichloromethane (12 g); soluble in benzene (4 g) and ethanol (4 g); sparingly soluble in petroleum ether and xylene Unstable in alkaline media

3-Hydroxy-carbofuran (main metabolite) 2,3-Dihydro-3-hydroxy-2,2-dimethylbenzofuran-7-yl methylcarbamate

114

Carbosulfan, Carbofuran O=C-NH-CH 3 O

Structural formula OH

Empirical formula Molar mass Melting point Vapour pressure Solubility Other properties

C 12 H 15 NO 4 237.1 No data No data No data No data

2 Outline of method Carbosulfan and carbofuran residues are extracted from plant material and soil with acetone, and from rape with a hexane-isopropanol mixture. Water and sodium chloride are added to an aliquot of the extract, followed by shaking with hexane. Interfering co-extractives are removed by chromatography on activated charcoal-silica gel and Florisil columns. Carbosulfan and carbofuran are determined by gas chromatography using a thermionic detector. Residues of the main metabolite, 3-hydroxy-carbofuran, are extracted from plant material with hydrochloric acid after conjugates have been hydrolyzed. The extract is shaken with ethyl acetate. Interfering co-extractives are removed by chromatography on a silica gel column. 3-Hydroxy-carbofuran is determined by gas chromatography using a mass-selective detector operated in the SIM mode.

3 Apparatus Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) High-speed blendor fitted with glass jar Beaker, 1-1 Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 500-ml Graduated cylinder, 500-ml, with ground joint Separatory funnel, 250-ml Round-bottomed flasks, 500-ml, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Erlenmeyer flask, 500-ml Erlenmeyer flask, 100-ml, with ground joint Laboratory mechanical shaker Centrifuge, 2800 r. p. m., with 200-ml glass tubes Reflux condenser, with ground joint Heating mantle, for 500-ml round-bottomed flask

Carbosulfan, Carbofuran

115

Chromotographic tube 1: 25 mm i.d., 40 cm long, with sintered glass disk and stopcock Chromatographic tube 2: 10 mm i.d., 40 cm long Test tubes, graduated, with ground stoppers Gas chromatograph equipped with thermionic nitrogen-specific detector Gas chromatograph equipped with mass-selective detector Micro syringe, 10- \i\

4 Reagents Acetone, p. a. Dichloromethane, p. a. Diisopropyl ether, HPLC quality Ethanol, p. a. Ethyl acetate, p. a. n-Hexane, fractionally distilled 2-Propanol (isopropanol), p. a. Toluene, p. a. 2,2,4-Trimethyl pentane (isooctane), fractionally distilled Solvent mixture 1: n-hexane + isopropanol 2:1 v/v Solvent mixture 2: dichloromethane + acetone + toluene 10:2:2 v/v/v Solvent mixture 3: n-hexane + ethyl acetate 97:3 v/v Solvent mixture 4: n-hexane + ethyl acetate 85:15 v/v Solvent mixture 5: n-hexane + ethyl acetate 7:3 v/v Solvent mixture 6: n-hexane + ethanol 7:3 v/v Standard solutions: 0.3-3.0 ng/ml each of carbosulfan, carbofuran and 3-hydroxy-carbofuran in isooctane Internal standard solutions: 0.3-3.0 ng/ml carbofuran in diisopropyl ether Hydrochloric acid, 0.25 mol/1 HC1 p. a. Sodium chloride, p.a., exhaustively extracted with dichloromethane and dried for 8 h at 120 °C Sodium sulphate, p. a., anhydrous, exhaustively extracted with dichloromethane and dried for 8 h at 120 °C Filter aid, e.g. Celite 545 Silica gel 60, 0.063-0.200 mm (Merck No. 7734) Florisil, 60-100 mesh, deactivated with 3% water: Heat a weighed sample of Florisil for at least 16 h at 120 °C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 3 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Activated charcoal, p. a. (Merck No. 2186) Glass wool (Merck No. 4086) Sea sand, p. a. (Merck No. 7712) Air, synthetic Helium Hydrogen, re-purified

116

Carbosulfan, Carbofuran

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Rape seeds are ground in a high-speed blendor.

6 Procedure 6.1 Extraction (carbosulfan, carbofuran) 6.1.1 Plant material (except rape green matter)

Weigh 100 g of the analytical sample (G) into a beaker, add 150 ml acetone, and homogenize for 3 min. Rinse the homogenizer with 50 ml acetone. Suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel. Rinse the beaker and wash the filter cake with 60 ml acetone. Transfer the filtrate to a graduated cylinder and make up the volume to 300 ml (VEx) with acetone. Mix thoroughly, and pipet off one fifth (60 ml, VR1) of the filtrate into a separatory funnel. Add 10 g sodium chloride, 50 ml water and 100 ml hexane, and shake for 2 min. Separate the layers, and extract the aqueous phase twice more with 100-ml portions of hexane. Dry the combined organic extracts for 30 min in an Erlenmeyer flask on sodium sulphate; then filter the solution through a fluted filter paper into a 500-ml round-bottomed flask. Wash the Erlenmeyer flask and the filter with 50 ml hexane. Rotaryevaporate the combined filtrates to 1-2 ml and remove the last traces of solvent with a gentle stream of nitrogen. 6.1.2 Rape (green matter) Weigh 20 g of the analytical sample (G) into a centrifuge tube, add 100 ml solvent mixture 1 and homogenize for 3 min. Rinse the homogenizer with 40 ml solvent mixture 1. Centrifuge the suspension for 15 min at 2800 r.p.m. Decant the supernatant liquid through a glass wool plug into a graduated cylinder. Repeat the extraction. Combine the extracts and make up the organic phase to a total volume of 240 ml (VEx) with solvent mixture 1. After carefully mixing, pipet off a quarter (60 ml, VR1) of the organic phase into a separatory funnel, and proceed as described in 6.1.1. 6.1.3 Soil Weigh 50 g of the analytical sample (G) into a 100-ml Erlenmeyer flask, add 25 ml water, stopper, and shake the mixture thoroughly for 5 min to distribute the water evenly. Add 50 ml acetone, and shake for 30 min on the mechanical shaker. Decant the supernatant liquid through a fast flow-rate filter paper covered with a layer of filter aid in a Buchner porcelain funnel. Repeat the extraction with a further 50 ml of acetone; then suction-filter the homogenate and wash the flask and the filter cake with a total of 70 ml acetone. Transfer the combined filtrates to a graduated cylinder and make up the volume to 200 ml (VEx) with acetone. Mix thoroughly, and pipet off a quarter (50 ml, VR1) of the filtrate into a separatory funnel. Proceed as described in 6.1.1.

Carbosulfan, Carbofuran

117

6.2 Extraction (3-hydroxy-carbofuran) 6.2.1 Rape (green matter), tomatoes

Weigh 50 g of the analytical sample (G) into a 500-ml round-bottomed flask, add 200 ml hydrochloric acid, and heat to reflux for 1 h. Allow the mixture to cool, and suction-filter through a Buchner porcelain funnel containing a layer of glass wool covered with sea sand. Rinse the flask and the filter cake with 50 ml hydrochloric acid. Transfer the combined filtrates to a graduated cylinder and make up the volume to 300 ml (VEx) with water. Mix well, then transfer a quarter of the solution (75 ml, VR1) into a separatory funnel. Extract the aqueous phase three times with 90-ml portions of ethyl acetate. Dry the combined organic phases for 30 min on sodium sulphate in an Erlenmeyer flask. Filter the solution through a fluted filter paper into a 500-ml round-bottomed flask and rinse the Erlenmeyer flask and the filter with 50 ml ethyl acetate. Rotary-evaporate the combined filtrates to 1-2 ml and remove the last traces of solvent with a gentle stream of nitrogen. 6.2.2 Rape (seeds) Weigh 20 g of the ground analytical sample (G) into a 250-ml round-bottomed flask, add 150 ml hydrochloric acid, and heat to reflux for 1 h. Allow the mixture to cool, and suctionfilter through a Buchner porcelain funnel containing a layer of glass wool covered with sea sand. Rinse the flask and the filter cake with 50 ml hydrochloric acid. Transfer the combined filtrates to a graduated cylinder and make up the volume to 200 ml (VEx) with hydrochloric acid. Mix well, then transfer a quarter of the solution (50 ml, VR1) into a separatory funnel, and continue as described in 6.2.1. 6.3 Cleanup (carbosulfan, carbofuran) 6.3.1 Activated charcoal-silica gel column (see Method S 8, pp. 283 ff, Vol. 1) Fill a chromatographic tube (type 1) with dichloromethane to a level of 1 cm. Slurry 5 g silica gel in 15 ml solvent mixture 2, and pour the slurry into the tube. Drain off the supernatant. Next, thoroughly mix 15 g silica gel and 1 g activated charcoal in a 50-ml beaker, and slowly add, with stirring, 35 ml solvent mixture 2 (Caution! Generation of heat!). Do not add more than 35 ml solvent mixture otherwise the suspension will become separated into phases, resulting in poor passage of material through the column. Add the activated charcoal-silica gel mixture onto the silica gel in the chromatographic column, by pouring it through a funnel, at first slowly and then in a gush, at the same time stirring constantly and with the column stopcock open. Use any eluate that has already passed through column for rinsing the flask. Drain the solvent mixture to a level of 2 cm above the packing, and top the column with a total of 5 g sodium sulphate added in small portions. Next pre-wash the column with 50 ml solvent mixture 2. Quantitatively transfer the residue derived from 6.1 to the column, using a total of 15 ml dichloromethane to complete the transfer. Collect liquid already flowing through the column and subsequent eluate in a 250-ml round-bottomed flask. Elute carbosulfan and carbofuran with 140 ml solvent mixture 2. Rotary-evaporate the eluate to approx. 2 ml, and remove the last traces of solvent with a gentle stream of nitrogen. Immediately dissolve the residue in 5 ml hexane.

118

Carbosulfan, Carbofuran

6.3.2 Florisil column

Slurry 10 g Florisil in 30 ml hexane, and pour the slurry into a chromatographic tube (type 2). Allow to settle, then top the Florisil with 5 g sodium sulphate. Pre-wash the column packing with 50 ml hexane. Quantitatively transfer the residue derived from 6.3.1 onto the column, using a total of 30 ml hexane to complete the transfer. Wash the column with 55 ml solvent mixture 3, and discard all eluates obtained up to this point. Next, elute carbosulfan and carbofuran with 210 ml solvent mixture 4, collecting the eluate in a 250-ml round-bottomed flask. Rotary-evaporate the eluate to near dryness, add 5 ml acetone to the residue, and rotary-evaporate to near dryness again. Transfer the residue to a test tube with a total of 5 ml acetone, and evaporate the solution to dryness in a nitrogen stream. If required, carbosulfan and carbofuran can be separated by collecting carbosulfan in the first 90 ml of the eluate in a separate flask. Carbofuran will appear in the following 120 ml eluate. 6.4 Cleanup (3-hydroxy-carbofuran) Slurry 10 g silica gel in 30 ml hexane, and pour the slurry into a chromatographic tube (type 2). Allow to settle, then top the silica gel with 5 g sodium sulphate. Pre-wash the column packing with 50 ml hexane. Quantitatively transfer the residue derived from 6.2 onto the column, using a total of 10 ml solvent mixture 5 to complete the transfer. Wash the column with 80 ml solvent mixture 5, followed by 25 ml hexane. Discard all eluates obtained up to this point. Next, elute 3-hydroxy-carbofuran with 55 ml solvent mixture 6, collecting the eluate in a 100-ml round-bottomed flask. Rotary-evaporate the eluate to near dryness. Transfer the residue to a test tube with a total of 5 ml diisopropyl ether, and evaporate the solution to near dryness using a gentle stream of nitrogen. 6.5 Gas-chromatographic determination Dissolve the residue derived from 6.3.2 in isooctane and make up to a definite volume (VEnd), e.g. 1 ml. Inject an aliquot of this solution (Vj) into the gas chromatograph equipped with a thermionic detector. To the residue derived from 6.4, add a definite volume of the internal standard solution, and make up with diisopropyl ether to a definite volume (VEnd), e.g. 1 ml. Inject an aliquot of this solution (Vj) into the gas chromatograph equipped with a mass-selective detector. Operating conditions 6.5.1 Thermionic detector

Gas chromatograph Column Column temperature

Hewlett-Packard 5890, equipped with cold injection system KAS (Gerstel) Fused silica capillary HP-1 (methyl silicone), 0.32 mm i.d., 25 m long; film thickness 0.17 urn (HewlettPackard) Programmed to rise at 20°C/min from 100 to 160 °C, and at 9°C/min from 160 to 240 °C, then isothermal at 240 °C for 3 min

Carbosulfan, Carbofuran

Injection technique

Detector Gas flow rates

Attenuation Integrator Injection volume Retention times for carbofuran 3 -hydroxy-carbofuran carbosulfan

119

Splitless for 48 s Temperature of KAS injection system: Programmed to rise at 10°C/s from 70 to 250 °C, then isothermal at 250 °C for 1 min Thermionic nitrogen-specific detector Temperature 300 °C Helium carrier, 2.5 ml/min Helium purge gas, 25 ml/min Hydrogen, 5 ml/min Air, 85 ml/min 21 Hewlett-Packard 3396 A, chart speed 5 mm/min I nl 5 min 8 s 6 min 27 s II min 53 s

6.5.2 Mass-selective detector

Gas chromatograph Column Column temperature Injection technique

Detector Carrier gas flow rate Printer Injection volume Retention times for carbofuran 3-hydroxy-carbofuran carbosulfan SIM parameters for 3-hydroxycarbofuran

Hewlett-Packard 5890/5970 B, equipped with cold injection system KAS (Gerstel) Fused silica capillary HP-1 (methyl silicone), 0.2 mm i.d., 12 m long; film thickness 0.33 |nm (HewlettPackard) Programmed to rise at 20°C/min from 70 to 250 °C, then isothermal at 250 °C for 3 min Splitless for 48 s Temperature of KAS injection system: Programmed to rise at 10°C/s from 70 to 250 °C, then isothermal at 250 °C for 1 min Mass selective detector HP 5970 El, 70 eV, interface 280 °C Helium, < 1 ml/min Paint Jet (Hewlett-Packard) 8 min 13 s 9 min 4 s 11 min 59 s

Group 1 (internal standard carbofuran): Start 7 min, stop 8 min 40 s Dwell time for m/z = 164 and 221:100 ms each Group 2 (3-hydroxy-carbofuran): Start 8 min 40 s, stop 9 min 40 s Dwell time for m/z = 137 and 180:100 ms each When using carbofuran as an internal standard, make sure that residues of carbofuran present have been quantitatively removed by the column cleanup in 6.4.

120

Carbosulfan, Carbofuran Carbofuran

3-Hydroxy-carbofuran

Carbosulfan

10 15 min Fig. 1. Gas chromatogram of a standard mixture representing 1 ng each of carbofuran, 3-hydroxycarbofuran, and carbosulfan. Conditions as described in 6.5.1.

Abundance 6000000 5000000 -

Carbofuran J 3-Hydroxy-carbofuran ,

Carbosulfan

I

4000000 3000000 2000000 1000000 n.

A

JL__J 9.0

12.0 min

Fig. 2. Total ion chromatogram of a standard mixture representing 50 ng each of carbofuran, 3-hydroxycarbofuran, and carbosulfan. Conditions as described in 6.5.2.

Carbosulfan, Carbofuran a)

121

Scan 55 (8.216 min)

Abundance

Coxbofuran

164

1000000 800000 149 600000 400000 122

2J0000-

15

1

0

58

33

,!.

ll.

c

V

J...J ,h.

I.

40

I,

... LI. III.

80

b)

103

191

til I

, I,

120 Mass/Charge

160

221 [

200

Scan 92 (9.007 min)

Abundance 280000 -

137

3-Hydroxy-carbofuran

240000200000 -

180

160000120000 151 60000 40000 :

28 | 1,

ill,..

4-

Z

58 /

91

1,1.1. ,1,1 hi. ..1. 40

u1 80

c]

III

237

123 i..i.. ...i,U

1.

1,

120 Mass/Charge

i.

160

i

i 200

240

Scan 231 (11.979 min)

Abundance J60

Carbosulfan 1000000118 800000 600000 400000200000 0

323

li

76 221

252

200 Mass /Charge

Fig. 3. Mass spectra of a) carbofuran, b) 3-hydroxy-carbofuran, c) carbosulfan. Conditions as described in 6.5.2.

122

Carbosulfan, Carbofuran

7 Evaluation 7.1 Method 7.1.1 Determination with the thermionic detector Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of the standard solutions (equivalent to 0.3 to 3.0 ng of carbosulfan, carbofuran and 3-hydroxy-carbofuran, respectively) into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng of compound. Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the standard solutions should be injected. For the areas or heights of the peaks obtained for the sample solutions, read the appropriate amounts of the identified compound from the corresponding calibration curve. 7.1.2 Determination with the mass-selective detector Quantitation is performed using an internal standard. For this purpose, prepare a sample solution from an untreated control sample. Fortify this solution, in the same order of magnitude as the anticipated residue, with equal amounts of 3-hydroxy-carbofuran and internal standard carbofuran. The volume of this measuring solution should be equal to that of the analytical sample solution. Inject an aliquot of the measuring solution into the gas chromatograph and determine the ratio, f, of the peak areas (or heights) obtained for the metabolite and the internal standard, f must be determined separately for each sample material. Add internal standard to the analytical sample solution in the same order of magnitude as the anticipated residue. Inject an aliquot of this solution into the gas chromatograph and perform the evaluation using the ratio of the peak areas (or heights) obtained for the metabolite and the internal standard. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples of plant material, fortified with carbosulfan and carbofuran at levels of 0.05 to 5 mg/kg, ranged from 65 to 110% and averaged 88%. The limit of detection was 0.02 to 0.03 mg/kg, depending on the sample material, and the limit of determination was 0.05 mg/kg. Carbosulfan and carbofuran in rape seeds were not examined. The recoveries from soil samples, fortified with carbosulfan and carbofuran at levels of 0.05 to 1 mg/kg, ranged from 86 to 113% and averaged 99%. The limit of detection was 0.02 mg/kg, and the limit of determination was 0.05 mg/kg. The recoveries from untreated control samples of rape (green matter and seeds) and tomatoes, fortified with 3-hydroxy-carbofuran at levels of 0.1 to 1 mg/kg, ranged from 68 to 115% and averaged 87%. The limit of detection was 0.01 to 0.02 mg/kg, depending on the sample material, and the limit of determination was 0.05 mg/kg. The average recoveries are given in the Table.

Carbosulfan, Carbofuran

123

Table. Percent recoveries from plant material and soil, fortified with carbosulfan, carbofuran and 3-hydroxy-carbofuran; means from 2 to 5 experiments. Analytical material Grapes Head cabbage Lettuce Maize Rape Green matter Seeds Tomatoes Sugar beet Soil

Added mg/kg

Carbosulfan

3-Hydroxycarbofuran

Carbofuran

0.05-5 0.05-0.5 0.05-0.5 0.05-5

65 95 80 103

88 65 86 83

— —

0.05-1 0.1-1 0.05-1 0.05-5 0.05-1

91 — 103 86 92

103 — 88 80 106

79 77 111 — -

7.3 Calculation of residues The residue R, expressed in mg/kg carbosulfan, carbofuran or 3-hydroxy-carbofuran, is calculated from the following equations: for carbosulfan and carbofuran: R =

WA • VF V

v

End

R1 • Y - G

where G

= sample weight (in g)

VEx

= total volume of organic phase after addition of solvent or solvent mixture, respectively, to filtered extract (in ml)

VR1

= portion of volume VEx used for further cleanup (in ml)

VEnd

= terminal volume of sample solution from 6.3.2 (in ml)

Y

= portion of volume VEnd injected into gas chromatograph (in ul)

WA

= amount of carbosulfan or carbofuran, respectively, for Y curve (in ng)

for 3-hydroxy-carbofuran: R =

where

f =

Ik

S t - E v - YEx * VEnd

F st .f-v Rr v r G

reac

^ from calibration

124

Carbosulfan, Carbofuran

and G VEx

= sample weight (in g) = total volume of filtered extract from 6.2 after addition of hydrochloric acid or water, respectively (in ml)

V R1

= p o r t i o n of volume V E x used for further cleanup (in ml)

VEnd

= terminal volume of sample solution from 6.4 (in ml)

Vj

= portion of volume V End injected into gas chromatograph (in ul)

St

= amount of internal standard injected with sample solution (in ng)

FA

= peak area or height for 3-hydroxy-carbofuran obtained from Vj (in integrator counts)

F St

= peak area or height for internal standard carbofuran obtained from Vj (in integrator counts)

¥'A Fs t

= peak area or height for 3-hydroxy-carbofuran (in integrator counts), and = peak area or height for internal standard carbofuran (in integrator counts), both from injection of the measuring solution (see 7.1.2)

8 Important points Carbosulfan, carbofuran and 3-hydroxy-carbofuran have different stabilities in solution. The stability of the standard solutions must therefore be checked at least once a week. Extracts from sugar beet obtained in 6.1.1 should be suction-filtered through a layer of filter aid on the fast flow-rate filter paper in order to prevent the filter from being clogged. The elution range in 6.3.2 should be checked from time to time, as the activity of the Florisil can change on storage. Blank value problems can occur with 3-hydroxy-carbofuran in rape using a thermionic detector; it is, therefore, preferably determined with a mass-selective detector in the SIM mode using carbofuran as internal standard. For carbosulfan and carbofuran, mass-selective detection is used only to confirm results obtained. The respective mass spectra are given in Fig. 3.

9 References E. Mollhoff, Uber die Riickstandsanalyse von N-Methylcarbamat-Insektiziden, Pflanzenschutz-Nachr. 28, 388-395 (1975). E. Mollhoff, Methode zur gaschromatographischen Bestimmung der Riickstande von Curaterr in Pflanzen- und Bodenproben unter Beriicksichtigung von Metaboliten, PflanzenschutzNachr. 28, 370-381 (1975). R. F. Cook, R. P. Stanovick and C. C. Cassil, Determination of carbofuran and its carbamate metabolite residues in corn using a nitrogen-specific gas chromatographic detector, J. Agric. Food Chem. 17, 277- 282 (1969).

Carbosulfan, Carbofuran

125

FMC Europe SA Agriculture, Chemical Group, Determination of 3-hydroxy-carbofuran crop residues by nitrogen selective gas chromatography, Internal Report INT 35001-2, Brussels 6/79. FMC Europe SA Agriculture, Chemical Group, Determination of FMC 35001 and carbofuran crop residues by nitrogen selective gas chromatography, Internal Report INT 35001-4, Brussels 6/79.

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, J. Siebers, H. Kohle and H.-G. Nolting

Chlorflurenol, Flurenol

275-215

Barley (grains and straw), cucumbers, wheat (grains and straw) Soil, water

Gas-chromatographic determination

(German version published 1987)

1 Introduction

Chemical name

Chlorflurenol

Flurenol

2-Chloro-9-hydroxyfluorene9-carboxylic acid (IUPAC)

9-Hydroxyfluorene-9-carboxylic acid (IUPAC)

Structural formula COOH

Empirical formula Molar mass

C14H9C1O3 260.68

C14H10O3 226.23

Data for

Chlorflurenol methyl ester

Flurenol n-butyl ester

71 °C 10" 6 mbar at 25 °C Virtually insoluble in water; very readily soluble to readily soluble in most organic solvents, e.g. very readily soluble in acetone (145 g) and methanol (150 g); readily soluble in benzene (95 g) and ethanol (70 g); sparingly soluble in petroleum ether (0.7 g) Cream coloured, crystalline, Beige, crystalline, practically Other properties odourless odourless Commercial products contain chlorflurenol as methyl ester and flurenol as n-butyl ester, sometimes as amine salt. As the acids are relatively unstable and the esters are of greater importance, the physical data for the esters have been given.

Melting point Vapour pressure Solubility (in 100 ml at 20 °C)

152 °C 6.7-10" 5 mbar at 25 °C Virtually insoluble in water; readily soluble to soluble in most organic solvents, e.g. readily soluble in acetone (26 g) and methanol (15 g); soluble in benzene (7 g) and ethanol (8 g); sparingly soluble in petroleum ether (0.16 g)

2 Outline of method Residues, which can be present as esters (I), or in the case of flurenol as salts, are extracted under acid conditions with acetone or acetonitrile. If present, the conversion products II and

128

Chlorflurenol, Flurenol

IV, as well as the relatively unstable product III, are also extracted. Next, the extract is washed with toluene or petroleum ether. I is hydrolyzed with potassium hydroxide in the concentrated extract. II is decarboxylated with sulphuric acid to yield compound III which is subsequently oxidized to 2-chlorofluorenone or fluorenone (IV) with chromium trioxide. After column chromatographic cleanup, the oxidation products IV are determined by electron capture gas chromatography.

Chlorflurenol: R2 = Cl; R2 = CH 3 Flurenol: R2 = H; R2 = C 4 H 9

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) Sintered glass filter funnels, porosity 2, 13 cm and 6 cm dia. Filtration flask, 500-ml Volumetric flasks, 500-ml, 250-ml, 10-ml and 5-ml Separatory funnels, 2.5-1, 500-ml and 250-ml Laboratory mechanical shaker, suitable for holding separatory funnels Round-bottomed flasks, 1-1, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Centrifuge, with 250-ml glass tubes (may be required) Chromatographic tube, 10 mm i.d., 40 cm long Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Acetone, p.a., fractionally distilled Acetronitrile, p.a., fractionally distilled Petroleum ether, saturated with acetonitrile Petroleum ether, technical grade, fractionally distilled, boiling range 40-70 °C Toluene, p.a., fractionally distilled Acetone + water mixture 7:3 v/v Eluting mixture: petroleum ether + toluene 1:1 v/v 2-Chlorofluorenone standard solutions: 0.01, 0.02, 0.04, 0.08 and 0.1 [ig/ml petroleum ether Fluorenone standard solutions: 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 ng/ml petroleum ether Sulphuric acid, 1 mol/1 H2SO4 p. a.

Chlorflurenol, Flurenol

129

Potassium hydroxide solution, 10 g/100 ml KOH p. a. Chromium trioxide solution, 60 g CrO3 p. a. in 100 ml sulphuric acid (1 mol/1) Sodium chloride solution, saturated, washed twice with toluene Sodium sulphate, p.a., anhydrous, exhaustively extracted with petroleum ether Aluminium oxide: To 100 g Alumina Woelm A, activity grade I (ICN Biomedicals) in a 300-ml Erlenmeyer flask (with ground joint), add 2 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h Florisil, 60-100 mesh, 7% water content: Heat a weighed sample of Florisil to constant weight at 200 °C and allow to cool in a desiccator. To 93 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 7 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Quartz wool Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Cereal grains, straw

Homogenize 50 g of the comminuted grains or 20 g straw (G) with 5 ml sulphuric acid and 150 ml acetonitrile in the blendor for 3 min. Suction-filter the homogenate through the 13-cm glass filter funnel and wash the filter cake with approx. 50 ml acetonitrile. Repeat the extraction with a further 150 ml acetonitrile. Wash the filter with 50 ml acetonitrile. Combine the filtrates in a 500-ml volumetric flask and make up to the mark with acetonitrile (VEx). Shake a 100-ml aliquot (VR1) twice in a 250-ml separatory funnel, each time for 5 min, with 50-ml portions of petroleum ether saturated with acetonitrile. Collect the lower acetonitrile phase in a 250-ml round-bottomed flask and rotary-evaporate to 1 -2 ml. Proceed as described in 6.2. 6.1.2 Cucumbers Homogenize 50 g of the comminuted material (G) with 5 ml sulphuric acid and 100 ml acetone for 3 min with the homogenizer. Suction-filter the homogenate through the 6-cm glass filter funnel. Wash the filter cake three times with 30-ml portions of acetone. Combine the filtrates in a 250-ml volumetric flask and make up to the mark with acetone (VEx). Transfer a 100-ml aliquot (VR1) into a 500-ml separatory funnel, dilute with 150 ml water and 75 ml sodium chloride solution and shake twice, each time for 5 min, with 50-ml portions of toluene. Filter

130

Chlorflurenol, Flurenol

the upper toluene layers through sodium sulphate and wash the filter with 10 ml toluene. Combine the filtrates in a 250-ml round-bottomed flask and rotary-evaporate to 1-2 ml. Proceed as described in 6.2. 6.1.3 Soil Acidify 100 g soil with 50 ml sulphuric acid and add 100 ml acetone. Allow to stand for 1 h, and homogenize for 3 min in the blendor. Suction-filter through the 13-cm glass filter funnel and repeat the extraction twice, each time with 100 ml acetone-water mixture. Collect the filtrates in a 500-ml volumetric flask and make up to the mark with acetone (VEx). Transfer a 100-ml aliquot (VR1) into a 500-ml separatory funnel, dilute with 150 ml water and 75 ml sodium chloride solution and shake twice, each time for 3 min, with 50-ml portions of toluene. Filter the upper toluene layers through sodium sulphate and wash the filter with 10 ml toluene. Collect the filtrates in a 250-ml round-bottomed flask and rotary-evaporate to 1-2 ml. Proceed as described in 6.2. 6.1.4 Water

Acidify a 2-1 water sample (G) in a 2.5-1 separatory funnel with 5 ml sulphuric acid and extract twice, each time for 5 min, with 250-ml portions of toluene on the mechanical shaker. Filter the separated toluene layers through sodium sulphate into a 1-1 round-bottomed flask and rotary-evaporate to about 50 ml. Transfer into a 250-ml round-bottomed flask and concentrate further to 1-2 ml. Proceed as described in 6.2. 6.2 Hydrolysis of the esters Treat the residual solution derived from 6.1 with 20 ml potassium hydroxide solution. Allow the flask to rotate without suction on a rotary evaporator for 20 min in a boiling water bath. 6.3 Decarboxylation and oxidation Transfer the cooled solution derived from 6.2 with 40 ml sulphuric acid into a 250-ml separatory funnel. Add 5 ml chromium trioxide solution and 30 ml petroleum ether. Shake the mixture for 1 h on the mechanical shaker. Separate the petroleum ether layer, using a centrifuge if required. Shake the aqueous phase for 5 min with a further 30 ml petroleum ether. Filter the organic layers through sodium sulphate, wash the filter with 10 ml petroleum ether, and rotary-evaporate to 1-2 ml. 6.4 Column chromatography Place a quartz wool plug in the bottom end of a chromatographic tube half-filled with the eluting mixture. Add 1.5 g aluminium oxide and then, after settling, 4 g Florisil whilst gently tapping the tube walls. Wash the prepared column with 30 ml eluting mixture. Drain the solvent to the top of the adsorbent. Transfer the petroleum ether solution derived from 6.3 quantitatively onto the column with 5 ml eluting mixture. Allow to percolate, and elute with 180 ml eluting mixture. Discard the first 40 ml, collect the subsequent eluate in a 250-ml roundbottomed flask and rotary-evaporate to approx. 2 ml.

Chlorflurenol, Flurenol

131

6.5 Gas-chromatographic determination Transfer the solution derived from 6.4 into a 5 or 10-ml volumetric flask with petroleum ether and make up to the mark (VEnd). Inject 1 JLXI of this solution (Vj) (if necessary, after dilution with petroleum ether) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Carrier gas flow rate Attenuation Recorder Linearity range Injection volume Retention times for fluorenone 2-chlorofluorenone

Varian 3700 Glass, 2 mm i.d., 1.3 m long; packed with 1.5% OV17 + 2% OV-210 on Gas Chrom Q, 100-120 mesh 200 °C 210 °C 63 Ni electron capture detector Temperature 320 °C Nitrogen, 15 ml/min 16-10"11 1 mV; chart speed 5 mm/min 2-Chlorofluorenone 0.01-0.1 ng Fluorenone 0.005-0.1 ng 1 ul 2 min 24 s 4 min 6 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 1 JLLI of each 2-chlorofluorenone or fluorenone standard solution, respectively, (equivalent to 0.01 to 0.1 ng 2-chlorofluorenone or 0.005 to 0.05 ng fluorenone) into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng of compound. Also inject 1-ul aliquots of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of compound from the corresponding calibration curve. 7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples fortified with chlorflurenol methyl ester, flurenol n-butyl ester, or the conversion products II and IV. The recoveries and the routine limits of determination were as follows:

132

Chlorflurenol, Flurenol

Analytical material

Added mg/kg

Cereal grains Cereal straw Cucumbers Soil

0.02-0.1 0.2-1.0 0.02-0.2 0.05-0.5

Recovery °7o

Routine limit of determination mg/kg

70-85 70-90 75-95 70-85

0.02 0.1 0.02 0.02

Blanks usually did not occur or, if so, they were less than 0.01 mg/kg (grains and soil) or 0.05 mg/kg (straw). From water, the recoveries were 70 to 90% when fortified with 0.05 to 20 ng/1. The routine limit of determination was 0.05 fxg/1.

7.3 Calculation of residues The residue R, expressed in mg/kg chlorflurenol or flurenol, is calculated from the following equations:

for chlorflurenol

R=

W

^'

V

?'^

n d

• 1.214

V

R1 V

for flurenol

Ex ' V End

1.255

R1 • V r G

V

where G

= sample weight (in g)

V Ex

= total volume of extract (in ml)

VRI

= portion of volume V Ex used for t h e analysis (in ml)

VEnd

= terminal volume of sample solution from 6.5 (in ml)

Vj

= portion of volume V E n d injected into gas chromatograph (in ul)

WA

= a m o u n t of 2-chlorofluorenone or fluorenone, respectively, for Vj read from calibration curve

1.214

= factor for conversion of 2-chlorofluorenone t o chlorflurenol

1.255

= factor for conversion of fluorenone to flurenol

For conversion of 2-chlorofluorenone to chlorflurenol methyl ester, t h e conversion factor is 1.280; for conversion of fluorenone t o flurenol n-butyl ester, t h e factor is 1.567.

Chlorflurenol, Flurenol

133

8 Important points 2,7-Dichloro-9-hydroxyfluorenecarboxylic acid methyl ester, which is present as an impurity in technical chlorflurenol methyl ester, and the corresponding 2,7-dichlorofluorenone can also be determined by this method. The retention time for 2,7-dichlorofluorenone was 7 min 12 s.

9 References H. Sieper, Residue analysis and degradation of morphactins, Proc. 2nd Intern. IUPAC Congr. Pestic. Chem., Vol. 6, pp. 157-174. Gordon and Breach, New York 1972. E. Amadori and W. Heupt, Chlorflurecol-methyl, in: G. Zweig, Analytical methods for pesticides and plant growth regulators, Vol. X, Academic Press, New York-San FranciscoLondon 1978. E. Amadori and W. Heupt, Flurecol, in: G. Zweig, Analytical methods for pesticides and plant growth regulators, Vol. XI, Academic Press, New York-San Francisco-London 1980. D. Eichler, W. Heupty IRE. Anderson, K. H. Domsch and G. Jagnow, Chlorflurecol-methyl in soil: Degradation, leaching and effects on microbiological processes, Arch. Environm. Toxicol. 11 185-193 (1982).

10 Authors Shell Forschung GmbH, Schwabenheim, D. Eichler and W. Heupt

Chloridazon

89-A Gas-chromatographic and high-performance liquid chromatographic determination

Mangold, red beet (foliage and edible root), sugar beet (foliage and edible root) Soil, water

(German version published 1987)

1 Introduction 1.1 Chloridazon Chemical name

5-Amino-4-chloro-2-phenylpyridazin-3(2H)-one (IUPAC)

Structural formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

•NH9

C 10 H 8 ClN 3 O 221.65 205-206°C No data 10 ~ 7 mbar at 20 °C Very sparingly soluble in water; slightly soluble in acetone (2.8 g) and methanol (3.4 g); sparingly soluble in dichloromethane (0.33 g) and ethyl acetate (0.6 g); very sparingly soluble in benzene (0.07 g) Rapidly photolyzed in sunlight

1.2 Metabolite A Chemical name

5-(N-Glucosyl)amino-4-chloro-2-phenylpyridazin3(2H)-one Cl

o Structural formula Empirical formula Molar mass

\ ^

-J

X

N=

1P\ H ^»AOH

y— —^\

o6

C l 6 H18C1N 3

383 .79

CH 2 0H 0•N —. / H \ H H

r—

OH

H

136

Chloridazon

Melting point Boiling point Vapour pressure Solubility Other properties

Approx. 220 °C with decomposition No data No data Readily soluble in water at 20 °C No data

1.3 Metabolite B Chemical name formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility Other properties

5-Amino-4-chloropyridazin-3(2H)-one

H-N

NH2

C4H4CIN3O

145.55 Approx. 315 °C with sublimation No data No data Very sparingly soluble in water at 20 °C Readily soluble in dilute alkali

2 Outline of method Residues of chloridazon, metabolite A and metabolite B are extracted from plant material and soil with methanol. The methanol extract is divided in two. One half, or a water sample, serves for the combined determination of chloridazon and metabolite A (as chloridazon), the other half, or a second water sample, for the determination of metabolite B. The half of the plant extract used for the chloridazon analysis is purified from plant pigments and sugars by precipitation; then metabolite A is converted to chloridazon by acid hydrolysis. The extract so prepared, half of a soil extract, or a water sample is evaporated in acid medium, and cleaned up by partition between water and dichloromethane on an Extrelut column, and by column chromatography on aluminium oxide. Chloridazon is determined by electron capture gas chromatography using a fused silica capillary column. The second half of the extract, or a second water sample, is used to determine metabolite B. After a partition step on an Extrelut column, followed by gel permeation chromatography on Sephadex LH-20 and column chromatography on silica gel, determination in performed by high-performance liquid chromatography using a UV detector at 286 nm.

3 Apparatus Homogenizer, e.g. Ultra-Turrax T 45 N (Janke & Kunkel) Ice bath

Chloridazon

137

Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schiill) Filtration flask, 1-1 Volumetric flask, 500-ml Erlenmeyer flask, 500-ml, with ground joint Allihn reflux condenser Hotplate with magnetic stirrer, e. g. Ika-Combimag RTC (Janke & Kunkel), with PTFE stirring rod Rotary vacuum evaporator, 40-60 °C bath temperature Glass funnel, 12 cm dia. Fluted filter paper, 24 cm dia. Round-bottomed flasks, 1-1, 500-ml and 250-ml Erlenmeyer flask, 1-1 Chromatographic tube, 18 mm i.d., 30 cm long Pear-shaped flask, 10-ml Ultrasonic bath Glass syringe, 10-ml, with Luer-lock fitting Membrane filter holder, i.d. 25 mm, with filter membranes, 25 mm dia. (e.g. SM 13400, Sartorius) Automated instrument for gel permeation chromatography, e. g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories), equipped with 5-ml sample loops and chromatographic tube, 25 mm i.d., 45 cm long; column filling 75 g Sephadex LH-20 pre-swelled in methanol Gas chromatograph equipped with electron capture detector Microsyringes, 100-ul and 10-ul High-performance liquid chromatograph equipped with autosampler, pump, and variable wavelength detector

4 Reagents Acetone, dist. Chloroform, dist. Dichloromethane, dist. Ethanol, dist. Methanol, dist. 2-Propanol (isopropanol), dist. 2,2,4-Trimethyl pentane (isooctane), dist. Dissolving mixture: dichloromethane + methanol + triethylamine 91:8:1 v/v/v Eluting mixture 1: chloroform + ethanol 98:2 v/v Eluting mixture 2: dichloromethane + isopropanol 85:15 v/v Eluting mixture 3: chloroform + ethanol 95:5 v/v Eluting mixture 4: chloroform + methanol 8:2 v/v Injection mixture: isooctane + isopropanol 9:1 v/v Mobile phase: dichloromethane + methanol + triethylamine 940:60:1 v/v/v Chloridazon standard solutions: 0.5, 1, and 2 ng/ml injection mixture Metabolite B standard solutions: 0.5, 1, and 2 ng/ml dissolving mixture

138

Chloridazon

Hydrochloric acid, 1 mol/1 HC1 p. a. Sodium hydroxide solution, 10 mol/1 NaOH p. a. Coagulating solution: 20 g/1 ammonium chloride p.a. in hydrochloric acid (1 mol/1) Sodium chloride solution, saturated Triethylamine, for synthesis grade (Merck-Schuchardt No. 808353) Sodium chloride, p. a. (Merck No. 6404) Aluminium oxide 90, Brockmann standardized, activity grade II-III, 0.063-0.200 mm (Merck No. 1097) Extrelut pre-packed column (Merck No. 11737) Extrelut refill pack (Merck No. 11738) Sephadex LH-20 (Pharmacia) Silica gel 60, 0.1-0.2 mm (Macherey-Nagel No. 81534) Sea sand, p. a., washed with hydrochloric acid and ignited Cottonwool Helium re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material Homogenize 50 g of the analytical sample (G) with 300 ml methanol for 10 min, immersing the flask in an ice bath. Suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake twice with 50-ml portions of methanol. Transfer the combined filtrates to a 500-ml volumetric flask, make up to the mark with methanol (VEx), and shake well. 6.1.2 Soil Reflux 50 g of the soil sample (G) with 250 ml methanol in an Erlenmeyer flask, fitted with an Allihn condenser, on the stirrer-hotplate with magnetic stirring for 60 min. Allow to cool, and rinse the condenser with 20 ml methanol. Filter the mixture through a fast flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake twice with 50-ml portions of methanol. Transfer the combined filtrates to a 500-ml volumetric flask, make up to the mark with methanol (VEx), and shake well.

Chloridazon

139

6.1.3 Water Treat each 500 g of water sample (G) as described in 6.2.1.4 or 6.3.1.3. 6.2 Cleanup for chloridazon and metabolite A 6.2.1 Preparation 6.2.1.1 Mangold and beet foliage Transfer 250 ml of the methanolic extract derived from 6.1.1 (VR1) into a 1-1 round-bottomed flask, add 100 ml coagulating solution, and rotary-evaporate to approx. 100 ml, with 50 °C bath temperature. Filter the solution through a fluted filter paper into a tared 500-ml roundbottomed flask. Wash the filter with 10 ml water, and discard the filter residue. Reflux the aqueous filtrate for 15 min on the stirrer-hotplate, whereby metabolite A is quantitatively converted to chloridazon. Allow to cool, rinse the condenser with 10 ml water, and rotaryevaporate to exactly 27 g of aqueous residue (equivalent to 25 ml, VR2), with 60 °C bath temperature. 6.2.1.2 Red beet and sugar beet (edible roots) Rotary-evaporate 250 ml of the methanolic extract derived from 6.1.1 (VR1) in a 1-1 roundbottomed flask to about 10 ml, with 50 °C bath temperature. Add 20 ml sodium chloride solution and give the solution a good swirl to dissolve the residue. Add 400 ml acetone and shake well for 1 min. Leave to stand for a short time, and then decant the acetone solution. Extract the semi-crystalline aqueous residue again with 150 ml acetone. Combine both acetone solutions in a 1-1 Erlenmeyer flask and leave to stand for at least 60 min to allow complete precipitation of sugars and sodium chloride to take place. Filter the suspension through a fluted filter paper into a tared 1-1 round-bottomed flask. Wash the filter with about 20 ml acetone. Rotary-evaporate the filtrate to 5-10 ml with 40 °C bath temperature. Add 50 ml hydrochloric acid and reflux for 15 min on the stirrer-hotplate, whereby metabolite A is quantitatively converted to chloridazon. Allow to cool, rinse the condenser with about 10 ml water, and rotary-evaporate to exactly 25 g of aqueous residue (equivalent to 25 ml, VR2), with 60 °C bath temperature. 6.2.1.3 Soil Transfer 250 ml of the methanolic extract derived from 6.1.2 (VR1) into a tared 500-ml roundbottomed flask, add 25 ml hydrochloric acid, and rotary-evaporate to exactly 25 g (equivalent to 25 ml, VR2), with 50 °C bath temperature. 6.2.1.4 Water Transfer 500 g of the analytical sample (G) into a tared 1-1 round-bottomed flask, add 25 ml hydrochloric acid, and rotary-evaporate to exactly 25 g (equivalent to 25 ml, VR2), with 60 °C bath temperature.

140

Chloridazon

6.2.2 Partition on an Extrelut column Fit an empty Extrelut tube on its lower end with a 10-mm dia. filter disc. Pour 3 g Extrelut into the tube and cover the Extrelut with 3 g sea sand to give an approx. 2 mm deep layer. Add 4 ml sodium hydroxide solution and allow it to soak in. Next add the contents of an Extrelut refill pack (about 12 g). Hold the filling in place with the basket insert and a 24-mm dia. filter disc. Transfer 20.0 ml of the aqueous extract from 6.2.1 (VR3) onto the Extrelut column, using a pipet, and allow the liquid to soak into the column. After 15 min, elute with four 25-ml portions of dichloromethane. Combine the eluates in a 250-ml round-bottomed flask and rotary-evaporate to dryness with 40 °C bath temperature. 6.2.3 Column chromatography on aluminium oxide Insert a cottonwool plug into the lower end of the chromatographic tube, half-fill the tube with chloroform, and trickle in 10 g aluminium oxide. Drain the chloroform until the adsorbent is just covered with liquid. Dissolve the residue derived from 6.2.2 in 5 ml chloroform and transfer the solution onto the column. Allow to percolate, and rinse the flask three times with 5-ml portions of chloroform. Successively add the rinsings to the column, allowing each portion to drain to the top of the aluminium oxide before the next one is added. Next elute chloridazon with 100 ml eluting mixture 1. Collect the total eluate in a 250-ml round-bottomed flask and rotaryevaporate to 1-2 ml with 40 °C bath temperature. Transfer the solution quantitatively with a few ml of dichloromethane into the pear-shaped flask and rotary-evaporate to dryness. 6.3 Cleanup for metabolite B 6.3.1 Preparation 6.3.1.1 Plant material Transfer 250 ml of the methanolic extract derived from 6.1.1 (VR1) into a tared 1-1 roundbottomed flask, add 6.5 g sodium chloride, and rotary-evaporate to an aqueous residue with 50 °C bath temperature. Make up to 24.5 g with water. 6.3.1.2 Soil Transfer 250 ml of the methanolic extract derived from 6.1.2 (VR1) into a tared 1-1 roundbottomed flask, add 6.5 g sodium chloride and 25 ml hydrochloric acid, and rotary-evaporate to a residue of 24.5 g with 50 °C bath temperature. 6.3.1.3 Water Transfer 500 g of the analytical sample (G) into a tared 1-1 round-bottomed flask, add 6.5 g sodium chloride and 25 ml hydrochloric acid, and rotary-evaporate to a residue of 24.5 g with 60 °C bath temperature. 6.3.2 Partition on an Extrelut column Transfer the aqueous solution derived from 6.3.1 onto an Extrelut column and allow the solution to soak in. After 15 min, pre-elute the column four times with 25-ml portions of

Chloridazon

141

dichloromethane, which have been used beforehand to wash the flask in order to transfer residues of water from the flask to the column. Discard this eluate. Next, elute metabolite B with six 25-ml portions of eluting mixture 2. Combine the eluates in a 250-ml round-bottomed flask and rotary-evaporate to dryness with 40 °C bath temperature. Dissolve the residue in 10.0 ml methanol (VR4), immersing the flask in an ultrasonic bath.

6.3.3 Gel permeation chromatography Filter the methanolic solution derived from 6.3.2, using the 10-ml syringe fitted with a membrane filter. Load the 5.0-ml sample loop (VR5) of the gel permeation chromatograph with 7 to 8 ml of the filtrate. Elute the column with methanol using an elution rate of 5.0 ml/min (column inlet pressure 0.8 bar) and set the following parameters at the instrument: Dump 41 min, equivalent to 205 ml Collect 20 min, equivalent to 100 ml Collect the eluate in a 250-ml round-bottomed flask, and rotary-evaporate to dryness with 50 °C bath temperature. Check the instrument parameters from time to time by injecting 250 |ng of metabolite B in methanol solution and follow the elution, with a UV detector attached, at 280 nm wavelength.

6.3.4 Column chromatography on silica gel Insert a cottonwool plug into the lower end of the chromatographic tube and trickle in 10 g silica gel. Dissolve the residue derived from 6.3.3 in 2 ml methanol and dilute with 60 ml chloroform. Add the solution to the column and allow to percolate. Rinse the flask with 25 ml of eluting mixture 3, add the rinsing to the column, allow the column to run dry, and discard the eluate. Next, elute metabolite B with 150 ml eluting mixture 4, collect this eluate in a 250-ml round-bottomed flask, and rotary-evaporate to 1-2 ml, with 50 °C bath temperature. Transfer the solution with a minimum of methanol into the pear-shaped flask and rotaryevaporate to dryness.

6.4 Gas-chromatographic determination of chloridazon Dissolve the residue derived from 6.2.3 in 2.0 ml (VEnd) injection mixture. Inject an aliquot of this solution (Y{) — if necessary, after dilution with injection mixture — into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector

Varian 3700 Fused silica capillary, i.d. 0.28 mm, 25 m long; coated with SE-54, film thickness 0.5 |xm 270 °C 300 °C 63 Ni electron capture detector Temperature 300 °C

142

Chloridazon

Gas flow rates

Attenuation Recorder Injection volume Retention time for chloridazon

Helium carrier, inlet pressure 1 bar Nitrogen purge gas, 30 ml/min Helium septum purge gas, 170 ml/min Helium split gas, 25 ml/min 32-10" 12 1 mV; chart speed 5 mm/min 1 nl 7 min 30 s

6.5 High-performance liquid chromatographic determination of metabolite B Dissolve the residue derived from 6.3.4 in 2.0 ml (VEnd) dissolving mixture, immersing the flask in an ultrasonic bath (no layer of solid material should remain on the walls). Inject an aliquot of this solution (Vj) into the high-performance liquid chromatograph. Operating conditions Chromatograph Columns Mobile phase Column inlet pressure Flow rate Detector Attenuation Recorder Injection volume Retention time for 5-amino-4chloropyridazin-3(2H)-one (metabolite B)

HPLC instrument fitted with pump LC-410 and autosampler MSI 660 (Kontron) Two stainless steel columns, each 3.2 mm i. d. and 10 cm long; packed with Silica Spheri-5 (Brownlee Labs); connected in series Dichloromethane + methanol + triethylamine 940:60:1 v/v/v Approx. 80 bar 1.8 ml/min UV detector Uvikon LC-720 (Kontron) Wavelength 286 nm 0.01 E 10 mV; chart speed 5 mm/min 100 nl 3 min 13 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare two calibration curves prior to each series of measurements as follows. Dilute aliquots of the chloridazon standard solutions with appropriate amounts of the injection mixture to yield a set of chloridazon measuring standards. Inject 1 ul each of these solutions (equivalent to 0.025 to 0.2 ng chloridazon) into the gas chromatograph. Likewise inject 100 ul of each metabolite B standard solution (equivalent to 50 to 200 ng metabolite B) into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng chloridazon or ng metabolite B, respectively. Also inject 1-ul or 100-ul aliquots, respectively, of the sample solutions. For the peaks obtained for

Chloridazon

143

these solutions, read the appropriate amounts of chloridazon or metabolite B from the respective calibration curve.

7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with chloridazon and metabolite B at levels of 0.05 to 0.5 mg/kg (water 0.5 to 5 |ig/l). The recoveries are given in the Table. The routine limit of determination was 0.05 mg/kg for plant material and soil, and 0.5 |ig/l for water. Table. Percent recoveries from plant material, soil and water, fortified with chloridazon and metabolite B. . . . , .. Analytical material

Chloridazon

Mangold Red beet Foliage Edible root Sugar beet Foliage Edible root Soil Water

Metabolite B

Range

Mean

Range

Mean

63-106

85

81-91

86

71- 76 81-109

74 96

62- 85 70- 78

74 73

86-100 86-104 96-103 83- 87

92 95 98 85

77- 83 67-104 73- 91 80- 92

79 86 81 84

7.3 Calculation of residues The residue R, expressed in mg/kg chloridazon or metabolite B, is calculated from the following equations: for chloridazon

R=

W

*/ \ '

VR 2

for metabolite B

R= ^ % '

VR4

,/ > "

"VEnd

V R 5 • Vi • G

where G

= s a m p l e weight (in g)

VEx

= total v o l u m e o f extract o b t a i n e d in 6.1.1 o r 6.1.2 (in m l )

VR1

= p o r t i o n of v o l u m e V E x used for c l e a n u p (in m l )

VR2

= v o l u m e o f a q u e o u s residue o b t a i n e d from evaporation in 6.2.1 (in m l )

VR3

= p o r t i o n o f v o l u m e V R 2 used for c l e a n u p o n t h e Extrelut c o l u m n in step 6.2.2 (in m l )

VR4

= v o l u m e of m e t h a n o l used t o dissolve t h e d r y residue in 6.3.2 (in m l )

144

Chloridazon

VR5 = portion of volume VR4 injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.4 or 6.5 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph or high-performance liquid chromatograph (in \i\)

WA = amount of chloridazon or metabolite B, respectively, for V{ read from calibration curve (in ng)

8 Important points Broad peaks with strong tailing and decreased sensitivity can occur during the gas chromatography of metabolite A (as chloridazon). Shortening the inlet end of the column by approx. 2 cm can greatly restore the sensitivity (see 9. Reference). Due to the presence of hydrochloric acid in the aqueous solutions from 6.3.1, ferric chloride can be released from the Extrelut column during the cleanup in step 6.3.2. In order to prevent any precipitation in the HPLC column, which would adversely affect the separation efficiency, the ferric chloride is removed by the preceding gel permeation chromatography as described in 6.3.3. If a gas chromatograph fitted with capillary columns is not available, chloridazon determination can also be performed on packed columns, under the following conditions: Glass column, i.d. 2.5 mm, 90 cm long; packed with 5% Silar-5 CP on Gas Chrom Q, 100-120 mesh; column temperature 240°C; injection port temperature 260°C; 63Ni electron capture detector, temperature 260°C; carrier gas argon + methane 9:1 v/v, 120 ml/min; retention time for chloridazon about 11 min. In most cases, chloridazon residues can also be determined by HPLC. Operating conditions then differ from those given in 6.5 in the following points: Mobile phase isooctane + isopropanol + methanol 88:10:2 v/v/v; flow rate 2 ml/min, column inlet pressure 20 bar; attenuation 0.03 E; retention time for chloridazon 6 min 36 s.

9 Reference F. Kuhlmann, Ruckstandsbestimmung von Pyrazon und seinen Metaboliten in Zuckerriiben, Z. Lebensm. Unters. Forsch. 775, 35-39 (1981).

10 Authors BASF, Agricultural Research Station, Limburgerhof, W. Keller and S. Otto

Chlorsulfuron, Metsulfuron

664-672 High-performance liquid chromatographic determination

Cereals (green matter, grains and straw) Soil, water (German version published 1989)

1 Introduction

Chemical name

C1

Structural formula

Empirical formula Molar mass Melting point Vapour pressure Solubility

Other properties

Chlorsulfuron

Metsulfuron (as metsulfuron-methyl)

l-(2-Chlorophenylsulphonyl)3-(4-methoxy-6-methyl-l,3,5triazin-2-yl)urea (IUPAC)

Methyl 2-[3-(4-methoxy-6methyl-l,3,5-triazin-2yl)ureidosulphonyl]benzoate (IUPAC)

CH,

ff

N-

SO2-NH-C-NH—(/

CO2CH3 O SO2-NH-C-NH—(/

N

CH, ^ N

OCH3

OCH,

C12H12C1N5O4S 357.78 174-178°C 6.1-10 " 6 mbar at 25 °C Very sparingly soluble in water at 25 °C; slightly soluble in dichloromethane (1-2 g), sparingly soluble in acetone (0.5-1 g) and methanol (0.1-0.2 g), very sparingly soluble in toluene (0.01-0.1 g), virtually insoluble in n-hexane (<0.01 g), in 100 ml each at 22 °C Hydrolyzed slowly in acid media, heat sensitive, little sensitivity to sunlight and UV radiation

C14H15N5O6S 381.37 163-166°C 7.7-10" 5 mbar at 25 °C Virtually insoluble in water at 25 °C; readily soluble in dichloromethane (10-20 g), soluble in acetone (2-5 g), sparingly soluble in ethanol and methanol (0.2-0.5 g), very sparingly soluble in xylene (0.01-0.1 g), virtually insoluble in n-hexane (<0.01 g), in 100 ml each at 20 °C Stable in acid and alkaline media, little sensitivity to sunlight and UV radiation. Metsulfuron is present as the methyl ester in commercial preparations

146

Chlorsulfuron, Metsulfuron

2 Outline of method Chlorsulfuron and metsulfuron residues are extracted from cereal and soil samples with a slightly alkaline buffer solution. The extract is washed with dichloromethane; the aqueous phase is acidified, and the compounds are partitioned into toluene. Water samples are acidified and extracted with dichloromethane. The extracts are cleaned up using a disposable silica gel cartridge. Chlorsulfuron and metsulfuron are determined by high-performance liquid chromatography using a photoconductivity detector.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 500-ml Laboratory mechanical shaker Laboratory centrifuge, 3800 r.p.m. Centrifuge tubes, 500-ml and 250-ml Volumetric flask, 100-ml Graduated cylinders, 500-ml, 250-ml, 100-ml, 50-ml and 10-ml Separatory funnels, 2-1 and 500-ml Magnetic stirrer, with stirring rod Beakers, 400-ml and 250-ml pH Meter Round-bottomed flasks, 500-ml, 250-ml and 50-ml, with ground joints Volumetric pipets, 50-ml, 25-ml, 2-ml and 1-ml Rotary vacuum evaporator, 45 °C bath temperature Glass funnel, 9 cm dia. Glass syringe, 10-ml, with Luer-lock fitting Test tubes, 10-ml, with ground stoppers Vacuum filtration unit, with 0.45 fim membrane filter Ultrasonic bath High-performance liquid chromatograph equipped with photoconductivity detector Microsyringe, 100-ul

4 Reagents Acetone, p. a. Cyclohexane, HPLC quality Dichloromethane, p. a. Diethyl ether, p. a. Ethyl acetate, p. a. Glacial acetic acid, HPLC quality

Chlorsulfuron, Metsulfuron

147

Methanol, HPLC quality 2-Propanol (isopropanol), HPLC quality Toluene, p. a. Water, bi-distilled Extraction solution: acetone + buffer solution B 4:1 v/v Mobile phase: Prepare a mixture consisting of 690 ml cyclohexane + 195 ml isopropanol + 115 ml methanol + 2 ml glacial acetic acid + 1 ml of a glacial acetic acid-water mixture (9:1 v/v). Mix well, and de-gas before use in a vacuum filtration unit using water jet pump suction Conditioning solution: Prepare a mixture consisting of 200 ml isopropanol + 200 ml methanol + 200 ml glacial acetic acid + 20 ml water. Mix well, and de-gas before use in a vacuum filtration unit using water jet pump suction Stock solutions: 10 mg/100 ml each of chlorsulfuron and metsulfuron-methyl in ethyl acetate. Pipet 1 ml of each stock solution into a 100-ml volumetric flask. Remove the solvent with a gentle stream of nitrogen, dissolve the residue and make up to the mark with mobile phase Chlorsulfuron and metsulfuron-methyl standard solutions: 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 M-g/ml of each in mobile phase, de-gassed in an ultrasonic bath. The solutions are stable for approx. two weeks when stored in a refrigerator Hydrochloric acid, cone., 10 g/100 g and 1 mol/1 HC1 p. a. Sodium sulphate, p.a., anhydrous Buffer solution A: 10.6 g/1 sodium carbonate anhydrous p. a. and 8.4 g/1 sodium hydrogen carbonate anhydrous p. a. Buffer solution B: 0.82 g/1 sodium acetate anhydrous p. a. Silica gel disposable cartridge: Sep-Pak Cartridge Silica (Millipore No. 51900) Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Cereals

Homogenize 10 g of the analytical sample (G) (25 g of grains) with 150 ml extraction solution in the mixer for 2 min. Suction-filter the supernatant liquid through a fast-flow rate filter paper in a Buchner porcelain funnel. Homogenize the residue in the glass jar and suction-filter twice more as above, each time using 120 ml extraction solution (for cereal grains, use 100 ml each time). Finally, rinse the glass jar and the filter with a further 80 ml of extraction solution. Transfer the filtrate to a volumetric flask and make up to a definite volume, e.g. 500 ml (VEx). Next transfer a tenth of this solution (VR1) to a 500-ml separatory funnel, add 100 ml buffer solution A, and proceed to step 6.2.1.

148

Chlorsulfuron, Metsulfuron

6.1.2 Soil Weigh 50 g of the analytical sample (G) into a 500-ml centrifuge tube, add 100 ml buffer solution A, and vigorously shake on a mechanical shaker for 1 h. Centrifuge the suspension at 3800 r.p.m. for 15 min, and transfer the supernatant liquid into a 500-ml separatory funnel. Repeat the extraction once more, combine the extracts, and proceed to step 6.2.1. 6.1.3 Water Transfer 2 1 water (G) into a separatory funnel, adjust the pH to 3-4 with hydrochloric acid (1 mol/1), and extract the water three times with 100-ml portions of dichloromethane. Filter the combined dichloromethane phases through a layer of sodium sulphate, contained in a funnel, into a 500-ml round-bottomed flask. Rinse the funnel with 50 ml dichloromethane. Add 1 ml glacial acetic acid and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml round-bottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and rotary-evaporate to 1-2 ml. Evaporate the solution to dryness, using a gentle stream of nitrogen, and proceed to step 6.2.2. 6.2 Cleanup 6.2.1 Liquid-liquid partition (only cereals and soil)

Shake the extracts derived from 6.1.1 or 6.1.2 twice with 50-ml portions of dichloromethane (for soil extracts, use two 50-ml portions of diethyl ether) for 3 min. Discard the organic phases. Break emulsions, if required, by centrifugation. Transfer the aqueous phase to a beaker and acidify, with vigorous stirring, to pH 5-6 (pH meter) with concentrated hydrochloric acid. Further, adjust the pH to 3.5 using hydrochloric acid (10% w/w) (see 8. Important points). Transfer the solution back to the separatory funnel, rinse the beaker with 5 ml water and 50 ml toluene, also add the rinsings to the separatory funnel, and shake for 3 min. Separate the aqueous layer, drain the organic phase into a 250-ml centrifuge tube, rinse the separatory funnel with 5 ml water, and add the rinsings to the centrifuge tube. Repeat the extraction twice, each time using 50 ml toluene and adding the organic phases and water rinsings to the centrifuge tube. Add a further 10 ml of water to the centrifuge tube if the phase boundary is difficult to see after the third extraction. Centrifuge for 15 min at 3800 r.p.m., then transfer the upper toluene phase into a 250-ml round-bottomed flask, using a 50-ml volumetric pipet. Add 30 ml toluene to the aqueous phase remaining in the centrifuge tube, mix, centrifuge for 5 min, and likewise pipet off the top layer into the 250-ml round-bottomed flask. Add 1 ml glacial acetic acid to the combined toluene phases, and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml roundbottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and rotaryevaporate to 1 -2 ml. Evaporate the solution to dryness, using a gentle stream of nitrogen, and proceed to step 6.2.2. 6.2.2 Silica gel cartridge Draw 10 ml of the mobile phase into the glass syringe, attach a silica gel cartridge to the syringe, and force the mobile phase through to condition the cartridge packing. Repeat the condi-

Chlorsulfuron, Metsulfuron

149

tioning with a further 10-ml portion of mobile phase. Next detach the cartridge, pull the plunger out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1.3 or 6.2.1 in 1 ml mobile phase and transfer the solution quantitatively into the syringe with the aid of a Pasteur pipet. Rinse the 50-ml flask with 1 ml mobile phase and also add the rinsings to the syringe. Re-insert the plunger into the syringe and force the liquid through the cartridge, collecting the eluate in a 10-ml test tube. Detach the cartridge, remove the plunger from the syringe, and re-attach the cartridge. Force a further 5 ml mobile phase through the cartridge, proceeding in a similar manner as described above, and collect the eluate in the same test tube. Evaporate the solution to dryness, using a gentle stream of nitrogen. 6.3 High-performance liquid chromatographic determination Dissolve the residue derived from 6.2.2 in mobile phase and dilute to an appropriate volume, e.g. 10 ml (V End ). Inject an aliquot of this solution (Vj) into the high-performance liquid chromatograph. Operating

conditions

Chromatograph Injector Column

Recorder

Spectra-Physics SP 8700 Injection valve 7125 with sample loop (Rheodyne) Zorbax Sil, 4.6 mm i. d., 25 cm long (Du Pont No. 880952701) 25 °C Cyclohexane-isopropanol-methanol-acetic acid-water 0.5 ml/min Isopropanol-methanol-acetic acid-water Photoconductivity detector (Tracor 965), operated with a mercury lamp at 254 nm, ATT = 5 5 mV; chart speed 5 mm/min

Injection volume

20 JLAI

Retention times for chlorsulfuron metsulfuron-methyl

13 min 15 min

Column temperature Mobile phase Flow rate Conditioning solution Detector

The Zorbax Sil column must be conditioned before use. For this end, pump conditioning solution through the column for 4 h at a flow rate of 0.7 ml/min. Next equilibrate the column for 3 h with mobile phase at the same flow rate. Moreover, pump conditioning solution at a flow rate of 0.15 ml/min through the system over night, changing to mobile phase at a flow rate of 0.5 ml/min for 1 h before beginning a new series of measurements the next morning.

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of each chlorsulfuron and metsulfuron-methyl standard solution into the high-performance liquid chromatograph. Plot the areas or heights of the peaks obtained vs.

150

Chlorsulfuron, Metsulfuron

ng chlorsulfuron or metsulfuron-methyl, respectively. Also inject equal volumes of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of compound from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated cereal control samples, fortified with chlorsulfuron and metsulfuron-methyl at levels of 0.05 to 10 mg/kg, ranged from 68 to 103% and averaged 80%. The limit of detection was 0.02 mg/kg, and the limit of determination was 0.05 mg/kg or, for cereal green matter, 0.1 mg/kg. Blanks usually did not occur or, if so, they were less than 0.02 mg/kg. The recoveries from soils, fortified at levels of 0.01 to 1 mg/kg, ranged from 70 to 112% and averaged 91%. The limit of detection was 0.005 mg/kg, and the limit of determination was 0.01 mg/kg. Blanks were less than 0.0025 mg/kg. The recoveries from tap water fortified at 1 |ig/l ranged from 70 to 100%, and the limit of determination was 1 u.g/1. 7.3 Calculation of residues The residue R, expressed in mg/kg chlorsulfuron or metsulfuron-methyl, is calculated from the following equations:

for soil and water R =

WA

' VEnd VrG

where G

= sample weight (in g) or volume (in ml)

VEx = total volume of solution after addition of extraction solution to filtered cereal extract from 6.1.1 (in ml) VR1 = portion of volume VEx used for further cleanup (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume VEnd injected into high-performance liquid chromatograph (volume of sample loop) (in \i\)

WA = amount of chlorsulfuron or metsulfuron-methyl for Vj read from calibration curve (in ng)

8 Important points The pH adjustment in 6.2.1 takes longer than one would expect and must be carried out very carefully. Only at pH 3-4, chlorsulfuron and metsulfuron-methyl are existing in the unionized form which can be transferred into the organic phase. If the pH is too low, decomposition

Chlorsulfuron, Metsulfuron

151

is likely to occur; if it is too high, both compounds are partially ionized and will not be extracted quantitatively. A LiChrosorb column can also be used for the HPLC measurement: 4 mm i. d., 25 cm long; packed with LiChrosorb Si 60, particle size 7 urn; other conditions as described in 6.3. When evaporated to dryness, the cleaned-up extract can be stored in a freezer for a maximum of four days if immediate measurement is not possible. In order to make allowance for baseline drifting, the balance is best set to approx. 30% above the zero point of the recorder prior to each measurement. The balance must be readjusted after each measurement, if necessary. If possible, leave the mercury lamp switched on over night.

9 References E.W. Zahnow, Analysis of the herbicide chlorsulfuron in soil by liquid chromatography, J. Agric. Food Chem. 30, 854-857 (1982). R. V. Slates, Determination of chlorsulfuron residues in grain, straw, and green plants of cereals by high-performance liquid chromatography, J. Agric. Food Chem. 31, 113-117 (1983).

10 Authors DuPont de Nemours & Co., Biochemicals Department, Research Division, Experimental Station, Wilmington, DE, U.S.A., L. W. Hershberger, R. V. Slates and E. W. Zahnow Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, M. BlachaPuller, H. Kohle, H.-G. Nolting and J. Siebers

Copper Oxychloride (as copper)

147-A Atomic absorption spectrophotometric determination

Grapes

(German version published 1985)

1 Introduction Chemical name Structural formula Empirical formula Molar mass Melting point Solubility

Other properties

Dicopper chloride trihydroxide (IUPAC) Cu2(OH)3Cl or CuCl2 • 3Cu(OH)2 Cu2H3ClO3 or Cu4H6Cl2O6 213.56 or 427.12 Above 220 °C, decomposition with elimination of hydrochloric acid to oxides of copper Virtually insoluble in water and organic solvents; soluble in mineral acids yielding the corresponding copper salts; soluble in ammonia, amine and EDTA solutions, under complex formation Largely stable in neutral media, decomposed by warming in alkaline media, yielding oxides

2 Outline of method Copper containing residues are stripped from the grapes with a lead-doped aqueous solution of ethylenedinitrilo tetraacetic acid (EDTA, disodium salt). The copper content of this solution is determined by flame atomic absorption spectrophotometry (FAAS) at 324.7 nm.

3 Apparatus Volumetric flasks, 1-1 and 100-ml Wide neck bottle, 500-ml, with ground stopper Water bath, 40 °C temperature Glass funnel, 7 cm dia. Fluted filter paper, 15 cm dia. (Schleicher & Schull) Flame atomic absorption spectrophotometer

154

Copper Oxychloride (as copper)

4 Reagents Cu standard solution: A solution of 2.116 g cupric chloride (CuCl2) in water (Merck No. 9987) is made up to 1 1 in a volumetric flask. The solution contains 1 mg/ml Cu Pb standard solution: A solution of 1.598 g lead nitrate [Pb(NO3)2] in water (Merck No. 9969) is made up to 1 1 in a volumetric flask. The solution contains 1 mg/ml Pb EDTA solution: 1 g/100 ml of ethylenedinitrilo tetraacetic acid disodium salt dihydrate p. a. (Merck No. 8418) Stripping solution: 10 g ethylenedinitrilo tetraacetic acid disodium salt dihydrate p.a. and 20 ml Pb standard solution are made up to 1 1 in a volumetric flask. The solution contains 20 mg/1 Pb Acetylene, 99.6 vol. °/o Air, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

6 Procedure 6.1 Extraction Transfer 100 g of the unchopped analytical sample (G) into the wide neck bottle, add 100 ml stripping solution (VEx), and allow to stand for 1 h with occasional swirling. With deepfrozen grapes, warm the solution in a water bath at 40 °C. Filter the solution through a fluted filter paper.

6.2 Atomic absorption measurement Measure the absorbance at 324.7 nm (Cu) and at 217.0 nm (Pb), using the filtrate derived from 6.1. Operating conditions Atomic absorption photometer Light source Wavelength Slit width Burner Burner gases

Perkin-Elmer 420 Hollow cathode lamp (Perkin-Elmer), operating current Cu 15 mA, Pb 10 mA Cu 324.7 nm, Pb 217.0 nm 0.7 nm Standard burner head Acetylene, input pressure 0.6 bar Air, input pressure 3.2 bar

Copper Oxychloride (as copper)

Deuterium background compensation Measurement value reading Measurement time

155

Only with Pb measurement Digital 3s

7 Evaluation 7.1 Method The evaluation is based on instrument internal standardization or on a calibration curve. For either procedure, take 0.5, 1.5 and 3.0 ml of the Cu standard solution, respectively, and make up to 100 ml with stripping solution (equivalent to 5, 15 and 30 mg/1 Cu). Measure the absorbances of these solutions at 324.7 nm. In order to take into account an eventual dilution of the analytical solution during the extraction step by some grape juice and/or condensate, measure the absorbance of the 20 mg/1 Pb containing stripping solution at 217.0 nm, and the blank of the EDTA solution without the addition of lead ions. Next, determine the Cu content (in mg/1) of the analytical solution from the instrument internal standardization or from the calibration curve, and ascertain the percent deviation of the Pb concentration from the expected value (20 mg/1 = 100%). Assume the blank of the EDTA solution without the addition of lead ions as 0%. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with copper oxychloride at levels of 2 to 20 mg/kg Cu, ranged from 97 to 100%. The routine limit of determination was 0.2 mg/kg. Blanks were approx. 0.1 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg Cu, is calculated from the following equation: R = 100 •

wAvEx G F

where G

= sample weight (in g)

VEx = volume of stripping solution used to extract copper containing residues from the surface of the grapes (in ml) WA = copper concentration of analytical solution, derived from internally generated standard, or read from calibration curve (in mg/1) F

= recovery rate of lead ions (in °/o)

156

Copper Oxychloride (as copper)

8 Important points No data

9 Reference R. Ipach, B. Altmayer and K. W. Eichhorn, Neue Methode zur AAS-Bestimmung von Kupferriickstanden auf Weintrauben, Fresenius Z. Anal. Chem. 314, 157-158 (1983).

10 Author Landes-Lehr- und Forschungsanstalt fiir Landwirtschaft, Weinbau und Gartenbau, Abteilung Phytomedizin, Neustadt/W., R. Ipach

Cymoxanil

513

Grapes (berries and must), potatoes, tomatoes Soil, water

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

l-(2-Cyano-2-methoxyiminoacetyl)-3-ethylurea (IUPAC)

Structural formula NOCH3

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 25 °C)

Other properties

C7H10N4O3 198.18 160-161 °C Not distillable 8 • 10 ~7 mbar at 25 °C (extrapolated) Sparingly soluble in water (approx. 0.1 g); readily soluble in dichloromethane (approx. 15 g); soluble in acetone (approx. 10 g) and methanol (approx. 4 g); very sparingly soluble in n-hexane (<0.1 g) White crystals, odourless; stable under normal storage conditions

2 Outline of method Cymoxanil residues are extracted from crop or soil samples with ethyl acetate. The aqueous extract is initially washed with hexane. The aqueous solution is then extracted with dichloromethane. Water samples are directly extracted with dichloromethane. In both cases, the dichloromethane phase is rotary-evaporated, the residue is dissolved in ethyl acetate, and the solution is cleaned up on a silica gel column. Cymoxanil is determined by gas chromatography using a thermionic detector.

3 Apparatus Homogenizer, e. g. Ultra-Turrax (Janke & Kunkel) Laboratory centrifuge, Type UJ 3 (Heraeus-Christ), with 340-ml glass tubes Round-bottomed flasks, 500-ml and 250-ml, with ground joints

158

Cymoxanil

Rotary vacuum evaporator, 40-50 °C bath temperature Separatory funnel, 250-ml Laboratory mechanical shaker Chromatographic tube, 15 mm i.d., 30 cm long Volumetric flasks, 10-ml Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-ul

4 Reagents Acetone, p. a. Dichloromethane, p. a. Ethyl acetate, p. a. n-Hexane, p. a. Eluting mixture 1: ethyl acetate + n-hexane 1:9 v/v Eluting mixture 2: ethyl acetate + n-hexane 4:6 v/v Cymoxanil standard solutions: 0.5-5 ng/ml acetone Filter aid, e.g. Celite 545 Silica gel, deactivated with 10% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734) for 12 h at 130 °C, allow to cool in a desiccator, and store in a tightly stoppered container in the desiccator. To 100 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 10 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container Sodium sulphate, p.a., anhydrous, washed with dichloromethane Glass wool Compressed air, re-purified Helium, re-purified Hydrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

Cymoxanil

159

6 Procedure 6.1 Extraction 6.1.1 Plant material, soil Weigh 50 g of the analytical sample (G) into a 340-ml centrifuge tube, and add 100 ml ethyl acetate and 5 g filter aid. Homogenize the mixture for 5 min, and then centrifuge. Decant the supernatant liquid into a 500-ml round-bottomed flask. Extract the residue with 100 ml ethyl acetate for 5 min on a mechanical shaker. Centrifuge and decant the liquid phase. Combine the extracts, add 50 ml water, and rotary-evaporate until the water begins to condense. Quantitatively transfer the aqueous phase into a 250-ml separatory funnel, and wash successively with three 30-ml portions of hexane, with gentle swirling. Discard the organic phases. Extract the aqueous solution successively with three 50-ml portions of dichloromethane for 5 min on a mechanical shaker. Separate the lower organic phases (centrifuge, if necessary), and filter through sodium sulphate into a 250-ml round-bottomed flask. Wash the sodium sulphate with dichloromethane. Rotary-evaporate the solution almost to dryness. 6.1.2 Water Extract 50 to 200 ml water (G) successively with three 50-ml portions of dichloromethane for 5 min on a mechanical shaker. Separate the organic phases (centrifuge, if necessary), and filter through sodium sulphate into a 250-ml round-bottomed flask. Wash the sodium sulphate with dichloromethane. Rotary-evaporate the solution almost to dryness. 6.2 Column chromatography Plug the bottom end of the chromatographic tube with glass wool and add about 5 ml hexane. Slurry 10 g silica gel in 20 ml hexane, and slowly pour the slurry into the column, gently tapping the glass walls. Allow to settle, and add 3 g sodium sulphate. Drain the hexane to the top of the sodium sulphate. Dissolve the residue derived from 6.1 in 1 ml ethyl acetate, and transfer quantitatively to the prepared column, using three 3-ml portions of eluting mixture 1 to complete the transfer. Let the solution percolate into the column packing (flow rate of 1-2 drops per s). Elute the column firstly with 200 ml of eluting mixture 1. Discard this fraction. Then elute cymoxanil with 200 ml of eluting mixture 2. Collect the eluate in a 250-ml flask, and rotaryevaporate almost to dryness. 6.3 Gas-chromatographic determination Quantitatively transfer the residue derived from 6.2 into a volumetric flask, using acetone to complete the transfer, and dilute the solution with acetone to a given volume, e. g. 10 ml (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column

Hewlett Packard 5880 A Glass, 2 mm i.d., 50 cm long; packed with 2% OV-101 on Chromosorb W-HP, 100-120 mesh

160

Cymoxanil

Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder Injection volume Linearity range Retention time for cymoxanil

Programmed to rise at 30°C/min from 100 to 190 °C, then isothermal at 190 °C for 4 min 190 °C Thermionic nitrogen-specific detector Temperature 320 °C Helium carrier, 25 ml/min Hydrogen, 4.5 ml/min Air, 70 ml/min 4 1 mV; chart speed 5 mm/min 1 \x\ 0.5-10 ng 2 min 24 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject 1 ul of each cymoxanil standard solution (equivalent to 0.5 to 5.0 ng cymoxanil) into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng cymoxanil. Also inject 1-ul aliquots of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of cymoxanil from the calibration curve. For control, repeat each injection. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with cymoxanil at levels of 0.05 to 0.5 mg/kg, ranged from 85 to 95%. Blanks usually did not occur or, if so, they were less than 0.03 mg/kg (however, blank values were higher for samples of unripe tomatoes). The routine limit of determination was 0.05 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg cymoxanil, is calculated from the following equation: p

WA-VEnd

where G VEnd Vj WA

= = = =

sample weight (in g) or volume (in ml) terminal volume of sample solution from 6.3 (in ml) portion of volume VEnd injected into gas chromatograph (in jul) amount of cymoxanil for V{ read from calibration curve (in ng)

Cymoxanil

161

8 Important points No data

9 Reference R. R Holt, Determination of residues of l-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea (DPX-3217) by gas-liquid chromatography, Pestic. Sci. 70, 455-459 (1979).

10 Authors Shell Forschung GmbH, Schwabenheim, D. Eichler and W. Heupt

2,4-D, Dichlorprop (2,4-DP)

27-A-38-A

Cereals (green matter, grains and straw), grapes, grass, potatoes

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

2,4-D (2,4-Dichlorophenoxy)acetic acid (IUPAC)

Dichlorprop 2-(2,4-Dichlorphenoxy)propionic acid (IUPAC)

Cl

P

CH3

Structural formula

Cl—/ V - O-CH2-COOH

C l — ^ V - O - C H — COOH

Empirical formula Molar mass Melting point Vapour pressure

C8H6C12O3 221.04 139.5-140.5 °C 0.53 mbar at 160 °C; 93 mbar at 251 °C Very sparingly soluble in water; readily soluble in acetone, chloroform, diethyl ether, ethanol and ethyl acetate No data

C9H8C12O3 235.07 117-118°C No data

Solubility

Other properties

Very sparingly soluble in water readily soluble in acetone, chloroform, diethyl ether, ethanol and ethyl acetate No data

2 Outline of method Residues of 2,4-D and dichlorprop, which may be present as free acids, salts or esters, are extracted from the plant material with aqueous methanol. The methanol is evaporated from the extract in alkaline medium, resulting in hydrolysis of the esters. The extracts are partitioned between dichloromethane and water, and then the compounds are esterified with a mixture of methanol and sulphuric acid. The resultant methyl esters of 2,4-D and dichlorprop are determined by electron capture gas chromatography on a fused silica capillary column.

3 Apparatus Wide neck bottle, 500-ml Homogenizer, e. g. Ultra-Turrax T 45 N (Janke & Kunkel) Buchner porcelain funnel, 9 cm dia.

164

2,4-D, Dichlorprop (2,4-DP)

Filter paper, 9 cm dia., fast flow rate (Schleicher & Schiill) Filtration flask, 500-ml Round-bottomed flasks, 1-1 and 100-ml Rotary vacuum evaporator, 30 to 40 °C or 60 °C bath temperature Graduated cylinder, 50-ml Separatory funnels, 250-ml and 50-ml Centrifuge, with 250-ml glass tubes Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Dichloromethane, dist. n-Hexane, dist. Methanol, dist. Methanol + water mixture 8:2 v/v 2,4-D and dichlorprop solutions for recovery experiments: 5 |ug/ml 2,4-D or dichlorprop in methanol Derivative standard solutions: 0.05, 0.1 and 0.2 |ng/ml 2,4-D methyl ester or dichlorprop methyl ester in n-hexane Sulphuric acid, cone, and 3 mol/1 H2SO4 p. a. Methylating mixture: Methanol + sulphuric acid, cone. 9:1 v/v. Prepare fresh daily Sodium hydroxide solution, 10 mol/1 NaOH p. a. Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p. a. Sodium sulphate, p.a., anhydrous Argon + methane mixture 9:1 v/v Helium, 99.996%

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1. Straw must be finely chopped.

6 Procedure 6.1 Extraction Place 50 g of the analytical sample (20 g for straw) (G) in a wide neck bottle. Add 300 ml methanol-water mixture and homogenize for 10 min, with ice water cooling. Suction-filter the homogenate through a filter paper in a Buchner porcelain funnel. Wash the filter cake successively with three 30-ml portions of methanol-water mixture. Combine the extracts in a 1-1 round-bottomed flask, alkalize with 10 ml sodium hydroxide solution, and rotary-evaporate to an aqueous residue at 60 °C bath temperature.

2,4-D, Dichlorprop (2,4-DP)

165

6.2 Liquid-liquid partition Transfer the aqueous residue derived from 6.1 to a 250-ml separatory funnel, using 50 ml water to complete the transfer. Extract with two 25-ml portions of dichloromethane by shaking. Allow the phases to separate (centrifuge, if necessary), and discard the dichloromethane phase. Acidify the water phase with 25 ml sulphuric acid (3 mol/1) (test with pH indicator paper), and extract successively with three 25-ml portions of dichloromethane by shaking (centrifuge if phases separate poorly). Filter the combined dichloromethane phases through sodium sulphate. Wash the sodium sulphate with 10 ml dichloromethane. Collect the whole filtrate in a 100-ml round-bottomed flask, and rotary-evaporate to dryness at 30 °C bath temperature. 6.3 Methylation To the residue derived from 6.2, add 5 ml methylating mixture, and allow to stand for 10 min at room temperature with occasional swirling. Then add 15 ml water, and extract with 10.0 ml hexane (VEnd). Allow the phases to separate (centrifuge, if necessary), and discard the aqueous phase. Wash the hexane phase with 15 ml sodium hydrogen carbonate solution, and dry with a little sodium sulphate. 6.4 Gas-chromatographic determination Inject an aliquot of the hexane solution derived from 6.3, e.g. 1 ul (Vj), into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Carrier gas Detector purge gas Septum purge gas Split ratio Splitter exit Attenuation Recorder Injection volume Retention times for dichlorprop methyl ester 2,4-D methyl ester

Perkin-Elmer F 22 Fused silica capillary, 0.28 mm i.d., 25 m long; coated with SE-54, film thickness 0.5 \xm 195 °C 250 °C 63 Ni electron capture detector Temperature 300 °C Helium, regulator pressure 1 bar Argon-methane, 30 ml/min Helium, 0.5 ml/min 1:10 Helium, 15 ml/min 32-10" 4 1 mV; chart speed 10 mm/min 1 L| L1 5 min 18 s 5 min 48 s

166

2,4-D, Dichlorprop (2,4-DP)

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prior to each set of measurements, prepare a calibration curve as follows. Inject 1 ul of each derivative standard solution (equivalent to 0.05 to 0.2 ng 2,4-D methyl ester or dichlorprop methyl ester) into the gas chromatograph. Plot the heights of the peaks obtained vs. ng derivative. Also inject 1-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of 2,4-D methyl ester or dichlorprop methyl ester from the calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with 2,4-D and dichlorprop at levels of 0.05 to 5 mg/kg, ranged from 70 to 100%. The routine limit of determination was 0.05 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg 2,4-D or dichlorprop, is calculated from the following equations: for 2,4-D

R=

WA VEnd

for dichlorprop

R=

WA

'

• 0.940

' VEnd • 0.944 VrG

where G

= sample weight (in g)

VEnd

= volume of hexane used for extraction in step 6.3 (in ml)

Vj

= portion of volume V E n d injected into gas chromatograph (in ul)

WA

= a m o u n t of derivative for Vj read from calibration curve (in ng)

0.940 = factor for conversion of 2,4-D methyl ester to 2,4-D 0.944 = factor for conversion of dichlorprop methyl ester to dichlorprop

8 Important points Laboratories equipped with a combined GC-MS system can confirm the results by mass spectrometry, with the aid of the multiple ion detection technique, as follows:

2,4-D, Dichlorprop (2,4-DP)

Operating conditions Gas chromatograph Mass spectrometer Column Column temperature Injection port temperature Separator temperature Ionization mode Electron voltage Carrier gas Injection volume Fragment ions usable for quantitation of 2,4-D methyl ester dichlorprop methyl ester

167

Finnigan 9500 Finnigan 3300 equipped with Finnigan Glass Jet Separator System and 6000 Computer Data System Glass, 2.5 mm i.d., 1.5 m long; packed with 3% OV17 on Gas Chrom Q, 100-120 mesh 180 °C 260 °C 250 °C El (MID) 70 eV Helium, 25 ml/min 5 nl

m/e 175, 177, 234, 236 m/e 162, 164, 248, 250

9 References No data

10 Authors BASF, Agricultural Research Station, Limburgerhof, W. Keller and S. Otto

Dichlobenil

225-A High-performance liquid chromatographic determination

Apples, grapes, grass, pears, red currants Soil, water (German version published 1987)

1 Introduction Chemical name

2,6-Dichlorobenzonitrile (IUPAC) CN Cl

Cl

Structural formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C7H3C12N 172.02 145-146°C 270 °C 7.33 • 10 " 4 mbar at 20 °C 2.0-10" 2 mbar at 50 °C Virtually insoluble in water; readily soluble in acetone (11.1 g) and dimethylformamide (15.8 g); soluble in dichloromethane (9.2 g), dimethyl sulphoxide (7.7 g) and xylene (4.3 g); slightly soluble in methanol (2.2 g) Hydrolyzed in strong acid and alkaline media, thermally very stable, steam volatile

2 Outline of method Dichlobenil residues are separated by steam distillation from plant material, soil, and water samples containing high amounts of organic and inorganic matter. The distillate is filtered through a membrane filter. Dichlobenil in the filtrate is concentrated on a short HPLC column that is connected to the analytical column with a 10-port valve. After elution from the column, dichlobenil is determined by HPLC using a UV detector at 210 nm. Water samples with a dichlobenil content greater than 0.1 mg/1 are filtered through a membrane filter, and the compound is determined directly by high-performance liquid chromatography. If the concentration is less than 0.1 mg/1, dichlobenil must be concentrated using the short HPLC column.

170

Dichlobenil

3 Apparatus High-speed blendor, e.g. Waring Blendor Beater-cross mill Round-bottomed flasks, 1-1 and 500-ml, with ground joints Glass tube, 15 cm long, with standard ground joints, used as fractionating column Distillation head, with ground joints, fitted with Liebig condenser (jacket length 20 cm) with vertical outlet Gas inlet tube, glass or FIFE, with sintered bottom, for steam inlet Aluminium foil Heating mantles, for 1-1 and 500-ml round-bottomed flasks Steam generator Volumetric flask, 250-ml Water bath, 35 °C temperature Glass syringes, 20-ml and 5-ml, with Luer-lock fittings (Waters) Swinny filter holder, stainless steel (Millipore) Filter membranes, for filtration of aqueous solutions, pore size 0.45 \im (Millipore) Sample vials, 5-ml, with septum caps Amber glass bottle, 50-ml, with glass stopper High-performance liquid chromatograph equipped with variable wavelength UV detector for measurement at 210 nm Injection set-up for sample volumes of 0-2000 \i\ Recording integrator, HP 3385 A (Hewlett-Packard) Sample loop, 0.5-ml 10-port valve (Valco Instruments) 2 HPLC pumps, M 6000 A (Waters)

4 Reagents Acetonitrile, LiChrosolv (Merck No. 30) Methanol, LiChrosolv (Merck No. 6007) Mobile phase 1: methanol + water 7:3 v/v Mobile phase 2: acetonitrile + water 54:46 v/v Water, bi-distilled Dichlobenil standard solutions, 0.002, 0.004, 0.008, 0.02, 0.08, 0.1, 0.2, 0.4, 0.8 and 1.0 fig/ml water Calcium chloride solution, saturated

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

Dichlobenil

171

6 Procedure 6.1 Extraction by steam distillation 6.1.1 Apples, grapes, pears, soil

Transfer 100 g of comminuted plant material or air dried soil (free from large particles and finely ground) (G) into a 500-ml round-bottomed flask and add 150 ml bi-distilled water. Place the flask into a heating mantle, attach the 15-cm glass tube to the flask, and connect the distillation head incl. Liebig condenser to the glass tube. Next slip the gas inlet tube through the distillation head and the glass tube into the flask, so that the gas distribution frit is positioned near to the bottom of the flask. Wrap aluminium foil around the vertical glass tube, then steam distil, using bi-distilled water in the steam generator. Maintain a constant water level in the flask during steam distillation by adjusting the heat on the heating mantle control. Collect the distillate in an ice-cooled 250-ml volumetric flask. Terminate the steam distillation when a little less than 250 ml of distillate have been collected, and make up to the mark (VEx) with bi-distilled water at 20 °C. Proceed as described in 6.2.3. 6.1.2 Red currants Transfer 100 g of finely comminuted red currants (G), together with 15 ml calcium chloride solution as a foam suppressant, and 150 ml bi-distilled water into a 500-ml round-bottomed flask. Steam distil as described in 6.1.1. 6.1.3 Grass Transfer 100 g of finely comminuted grass (G) into a 1-1 round-bottomed flask, add 400 ml bi-distilled water, and steam distil as described in 6.1.1. 6.1.4 Water (clean water samples, dichlobenil content > 0.1 mg/1) Subject the sample directly to the cleanup as described in 6.2.1. 6.1.5 Water (clean water samples, dichlobenil content < 0.1 mg/1) Subject the sample directly to the cleanup as described in 6.2.2. 6.1.6 Water (highly contaminated water samples, e. g. river water)

Transfer 250 ml of the water sample (G) into a 500-ml round-bottomed flask, and steam distil as described in 6.1.1. 6.2 Cleanup by filtration 6.2.1 Water (dichlobenil content > 0.1 mg/1) Filter approx. 13 ml of the water sample, using the 20-ml Luer-lock syringe fitted with the Swinny filter holder, through the filter membrane. Discard the first 10 ml, then filter a few

172

Dichlobenil

ml into a sample vial with an air-tight septum cap. HPLC determination should be performed immediately afterwards. If this is not possible, the filtrate can be stored in a refrigerator for 1-2 d. 6.2.2 Water (dichlobenil content <0.1 mg/1) Filter approx. 30 ml of the water sample as described in 6.2.1 and collect in an amber glass bottle. HPLC determination should be performed immediately afterwards. If this is not possible, the filtrate can be stored in a refrigerator for 1-2 d. 6.2.3 Plant material, soil, and highly contaminated water Filter approx. 30 ml of the steam distillate derived from 6.1.1, 6.1.2, 6.1.3, or 6.1.6 as described in 6.2.2. 6.3 High-performance liquid chromatographic determination 6.3.1 Determination by direct injection Inject an aliquot of the filtrate derived from 6.2.1 (Vj) into the high-performance liquid chromatograph. Operating conditions Chromatograph Column Column temperature Mobile phase 1 Flow rate Detector Recorder Injection volume Retention time for dichlobenil

Hewlett-Packard HP 1084 A Stainless steel, 4 mm i.d., 12.5 cm long; packed with LiChrosorb RP-18, 5 p (Merck No. 9333) 25 °C Methanol + water 7:3 v/v 1.0 ml/min Variable wavelength UV detector for measurement at 210 nm 10 mV; chart speed 5 mm/min 20-40 JLXI 3 min 40 s

6.3.2 Determination after enrichment See Diagrams 1 and 2 for the required apparatus set-up and the column switching. Use 1 to 1.5 ml of the filtrate derived from 6.2.2 or 6.2.3 to fill the sample loop (volume 0.5 ml, V{), see Diagram 1. Switch the 10-port valve (see Diagram 2). Flush the sample loop with 1.5 to 2 ml of bidistilled water, delivered by pump 2, thereby transferring the sample solution onto the short enrichment column. Switch the valve to the starting position again (Diagram 1), and pump mobile phase 2, delivered by pump 1, in the opposite direction through the enrichment column to the analytical column. Dichlobenil is thereby eluted from the enrichment column and chromatographed on the analytical column. The enrichment step for the next determination can be started 2 min after the last valve switching, i.e. before the chromatographic separation is terminated.

Dichlobenil

173

Glass syringe Sample loop Waste

Pump 2 (bi-dist. water)

Waste

Pump 1 (mobile phase) Diagram 1

Jl Diagram 2 Diagrams 1 and 2. Set-up of apparatus for HPLC determination of dichlobenil using enrichment column with column switching (for explanation, see step 6.3.2).

174

Dichlobenil

Operating conditions Pumps Injector Enrichment column Column temperature Enrichment flow rate Analytical column Column temperature Mobile phase 2 Flow rate Detector Recorder Retention time for dichlobenil

Two pumps M 6000 A (Waters) 10-port valve (Valco No. C. 10UN60), fitted with 0.5ml sample loop Stainless steel, 4 mm i.d., 17 mm long; packed with Spherisorb ODS (RP-18) (Phase Separations) 25 °C Bi-distilled water, 1.5 ml/min Stainless steel, 4 mm i.d., 12.5 cm long; packed with LiChrosorb RP-18, 5 \xm (Merck No. 9333) 25 °C Acetonitrile + water 54:46 v/v 1.0 ml/min Variable wavelength UV detector for measurement at 210 nm 10 mV; chart speed 5 mm/min 4 min 30 s

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the dichlobenil standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas.

7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material and soil, fortified with dichlobenil at levels of 0.01 to 0.1 mg/kg. The percent recoveries were as follows: Analytical material Apples Grapes Grass Pears Red currants Soil

0.01

83 88 84 91 73 95

Dichlobenil added (mg/kg) 0.05 91 98 90 93 88 97

0.1

93 99 88 95 92 94

Dichlobenil

175

The soils used for the recovery experiments had the following characteristics: _ ..

Organic carbon

Particles <0.02 mm

or

07

p H

0.48 2.19

8.3 15.2

6.5 5.6

Soil type Standard soil 2.1 *) Standard soil 2.2*)

*) Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig

Recoveries ranging from 98 to 100% were obtained for dichlobenil from clean water, fortified at levels of 0.01 to 0.1 mg/1, and from river water, fortified at levels of 0.05 to 1.0 mg/1. The routine limit of determination was 0.01 mg/kg for plant material and soil, 0.001 mg/1 for clean water, and 0.002 mg/1 for river water.

7.3 Calculation of residues The residue R, expressed in mg/kg dichlobenil, is calculated from the following equation: R

_

F A -V E x -W s t

where G

= sample weight (in g) or volume (in ml)

VEx = volume of steam distillate (in ml) Vj

= portion of volume VEx injected into the high-performance liquid chromatograph or, when enriched as described in 6.3.2, volume of sample loop (in ul)

WSt = amount of dichlobenil injected with standard solution (in ng) FA

= peak area obtained from Vj (in mm 2 )

F st

= peak area obtained from WSt (in mm 2 )

8 Important points The routine limit of determination for dichlobenil in water can be reduced by using a larger sample loop, e.g. 2.0 ml, for enrichment.

176

Dichlobenil

9 References F. Herzel, Zur Ruckstandsbestimmung von 2.6-Dichlorbenzonitril aus Boden und Wasser, J. Chromatogr. 193, 320-321 (1980). M. Schmidt, A. Hiltawki, W. Maasfeld and A. Kettrup, Chromatographic investigations of the behaviour of dichlobenil and 2,4,5-T in water during slow sand filtration, Int. J. Environ. Anal. Chem. 13, 289-307 (1983).

10 Authors Department of Analytical Chemistry, University of Paderborn, A. Kettrup, M. Schmidt and R. Hamann

Dichlofluanid, Tolylfluanid

203-371

Apples, grapes, lettuce, must, raspberries, strawberries, tomatoes, wine

Gas-chromatographic determination

(German version published 1991)

1 Introduction Chemical name

Structural formula

Dichlofluanid N-DichlorofluoromethylthioN\N'-dimethyl-N-phenylsulphamide (IUPAC) (CH3)2N— S O 2 — N - / S-CC12F

Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C9HUC12FN2O2S2 333.23 105-106°C <10" 6 mbar at 20 °C, 3-1O"5 mbar at 50 °C Virtually insoluble in water; readily soluble in dichloromethane (>20 g) and toluene (10-20 g); soluble in xylene (7 g); slightly soluble in isopropanol (1-2 g) and methanol (1.5 g); sparingly soluble in n-hexane (0.1-0.2 g) Hydrolyzed in aqueous media depending on pH; more stable at lower pH values. Stability to hydrolysis (half lives at 22 °C): approx. 15 d at pH 4, approx. 19 h at pH 7, ^10 min at pH 9

Metabolite (DMSA) Dimethylaminosulphanilide

(CH3)2N— SO2—P I H

C8H12N2O2S 200.2 87-89°C 2.2-10- 6 mbar at 20°C Sparingly soluble in water (0.13 g); readily soluble in acetone, acetonitrile, dichloromethane (each > 20 g) and isopropanol (13 g); soluble in toluene (4.3 g) and octanol (3.7 g); very sparingly soluble in n-hexane (24 mg) Stable between pH 4 and pH 9; half life in water at 22 °C > lyear

178

Dichlofluanid, Tolylfluanid

Chemical name

Structural formula

Tolylfluanid N-DichlorofluoromethylthioN',N'-dimethyl-N-p-tolylsulphamide (IUPAC) (CH3)2N— SO 2 —N-

-CH 3

S-CC1 2 F

Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C 10 H 13 Cl 2 FN 2 O 2 S 2 347.26 95-97°C 1.6-lO" 6 mbar at 20°C, 9-10" 5 mbar at 50 °C Virtually insoluble in water; readily soluble in benzene (54 g), dichloromethane, toluene (each > 20 g) and xylene (23 g); soluble in isopropanol (2-5 g) and methanol (4.6 g); sparingly soluble in n-hexane (0.5-1 g) Hydrolyzed in aqueous media depending on pH; more stable at lower pH values. Stability to hydrolysis (half lives at 22 °C): approx. 12 d at pH 4, approx. 29 h at pH 7, < 10 min at pH 9

Metabolite

(DMST)

Dimethylaminosulphotoluidide

(CH 3 ) 2 N—S0 2 —N-

•CH-,

H

C 9 H 14 N 2 O 2 S 214.2 88-90°C 8.8-10" 7 mbar at 20 °C Very sparingly soluble in water; readily soluble in acetone, acetonitrile, dichloromethane and isopropanol (each >20 g); soluble in toluene (9.8 g) and octanol (5.4 g); very sparingly soluble in n-hexane (53 mg) Stable between pH 4 and pH 9; half life in water at 22 °C > 1 year

2 Outline of method Dichlofluanid, DMSA, tolylfluanid and DMST residues are extracted from plant material with acetone. The extract is concentrated to an aqueous residue, which is made up to a definite volume. An aliquot of this solution is transferred onto a disposable extraction column. Beverages such as must and wine are transferred directly onto the disposable extraction column. The column is eluted with a cyclohexane-ethyl acetate mixture, and the eluate is cleaned up on a small silica gel-activated charcoal column. The parent compounds and the metabolites are determined by gas chromatography using a thermionic or a sulphur-specific flame photometric detector.

3 Apparatus Homogenizer Wide neck glass bottle, 1-1, with ground joint

Dichlofluanid, Tolylfluanid

179

Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 1-1 Round-bottomed flasks, 1-1 and 250-ml, with ground joints Pear-shaped flask, 50-ml Rotary vacuum evaporator, 40 °C bath temperature Glass funnel, 10 cm dia. Solvent dispensers, 50-ml, 10-ml and 5-ml Graduated cylinders, 1-1, 500-ml, 250-ml and 100-ml Volumetric flasks, 100-ml, 50-ml and 25-ml, with ground joints Volumetric pipets, 50-ml, 10-ml, 5-ml, 2-ml and 1-ml Glass syringe, 10-ml, with Luer-lock fitting Test tubes, 10-ml, with ground stoppers Gas chromatograph equipped with thermionic nitrogen-specific detector or sulphur-specific flame photometric detector Microsyringe, 10-ul

4 Reagents Acetone, for residue analysis Cyclohexane, for residue analysis Dichloromethane, for residue analysis Ethyl acetate, for residue analysis Acetone + water mixture 2:1 v/v Eluting mixture 1: cyclohexane + ethyl acetate 85 :15 v/v Eluting mixture 2: cyclohexane + ethyl acetate 97:3 v/v Compound stock solution: 1000 pig/ml of each dichlofluanid, DMSA, tolylfluanid and DMST in ethyl acetate. The solution can be stored in a refrigerator for approx. 6 months Compound standard solutions: 0.2-100 M-g/ml of each dichlofluanid, DMSA, tolylfluanid and DMST in ethyl acetate. The solutions can be stored in a refrigerator for approx. 6 months Filter aid, e. g. Celite 545 Disposable extraction column, 50-ml (Chem Elut CE 2050; Analytichem) Mini silica gel column: Disposable column, volume 6 ml, filled with 1 g silica gel (Baker No. 7086-07), with adapter and funnel column (reservoir) 75-ml (Baker No. 7122-00 and 7120-03) Activated charcoal, p. a. (Riedel-de Haen No. 31616) Glass wool Air, synthetic, re-purified Helium 4.6 (> 99.996 vol. %) Hydrogen 5.0 (> 99.999 vol. %) Nitrogen 4.6 (> 99.996 vol. %)

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

180

Dichlofluanid, Tolylfluanid

6 Procedure 6.1 Extraction 6.1.1 Plant material with high water content

Transfer 100 g of the analytical sample (G) to the 1-1 glass bottle with 200 ml acetone, and homogenize for approx. 3 min. For tomatoes, grapes, and other material from which it is difficult to take a representative 100-g sample, homogenize 200 g with 200 ml acetone as well. Add approx. 15 g filter aid, and filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, using gentle suction. Rinse the filter cake and the bottle several times with a total of 200 ml acetone-water mixture. Allow the filter cake to pull dry, and discard it. Transfer the filtrate to a 1-1 round-bottomed flask and rotary-evaporate to an aqueous residue (approx. 170 ml for a 100-g sample, approx. 270 ml for a 200-g sample), without evaporating any water, which would result in compound losses. When a precipitation occurs as a result of the evaporation, re-dissolve it using a maximum of 10% acetone. Make up the aqueous residue with water to 250 ml (100-g sample) or 500 ml (200-g sample) in a graduated cylinder (VEx). Pipet 50 ml (VR1) of this solution onto a dry disposable extraction column and allow the solution to soak in. Elute the column three times with 50-ml portions of eluting mixture 1 (see 8. Important points). Collect the eluate in a 250-ml roundbottomed flask and rotary-evaporate to dryness. Proceed to step 6.2. 6.1.2 Beverages (must, wine)

Pipet 50 ml of the analytical sample (G) directly onto a dry disposable extraction column and allow it to soak in. Elute the column three times with 50-ml portions of eluting mixture 1 (see 8. Important points). Collect the eluate in a 250-ml round-bottomed flask and rotaryevaporate to dryness. Proceed to step 6.2. 6.2 Cleanup 6.2.1 Column preparation Using a 10-ml syringe with an adapter, or employing pneumatic pressure, force eluting mixture 2 down through the mini silica gel column until the packing is free from air bubbles (approx. 10-20 ml). The packing should appear jelly-like translucent and should strongly contrast with the white frit. As long as this is not the case, continue conditioning, since air bubbles present will drastically reduce the flow rate. Clamp the column to a stand, trickle 100-150 mg activated charcoal into the supernatant eluting mixture (see 8. Important points) and top with a loose glass wool plug. Allow the supernatant to soak into the column; there is no danger that the column will run dry. 6.2.2 Column chromatography

Dissolve the residue derived from 6.1 in 3 ml dichloromethane. Using a Pasteur pipet, transfer the solution onto the prepared column and allow it to soak into the charcoal layer. Rinse the flask twice with 3-ml portions of dichloromethane and also add the rinsings successively to the column. Fasten the reservoir with adapter to the column, and continue to elute with a

Dichlofluanid, Tolylfluanid

181

further 10 ml of dichloromethane (see 8. Important points). Collect the entire eluate (approx. 19 ml) in a pear-shaped flask and rotary-evaporate to dryness. Dissolve the residue in 5-10 ml ethyl acetate and rotary-evaporate to dryness again to remove the last traces of dichloromethane.

6.3 Gas-chromatographic determination Dissolve the residue derived from 6.2 in a definite volume of ethyl acetate, e. g. 5 ml (VEnd), and transfer the solution to a test tube. Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph 1 Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Integrator Injection volume Retention times for DMSA DMST dichlofluanid tolylfluanid Gas chromatograph 2 Column Column temperature Injection port temperature Detector Gas flow rates

Varian 3700 Glass, 3 mm i.d., 1.8 m long; packed with 3% OV-17 on Supelcoport, 80-100 mesh Isothermal at 180 °C for 4 min, programmed to rise at 10°C/min from 180 to 225 °C, then isothermal at 225 °C for 18 min 280 °C Thermionic nitrogen-specific detector Temperature 320 °C Nitrogen carrier, 90 ml/min Hydrogen, 4.5 ml/min Air, 175 ml/min 10"11 Integrator Spectra-Physics SP 4270 or Laboratory Data System LAS on a Hewlett-Packard HP 1000 A 5 nl

2 min 3 min 5 min 6 min

24 s 30 s 24 s 36 s

Varian 6000 Fused silica capillary, 0.53 mm i.d., 10 m long; coated with OV-1 CB, film thickness 2.0 \xm (Macherey-Nagel No. 723520) Isothermal at 60 °C for 2 min, programmed to rise at 10°C/min from 60 to 220 °C, then isothermal at 220 °C for 15 min 220 °C Thermionic nitrogen-specific detector Temperature 300 °C Nitrogen carrier, 12 ml/min Hydrogen, 4.5 ml/min Air, 175 ml/min

182

0.00

Dichlofluanid, Tolylfluanid

1.25

2.50

3.75

5.00

6.25

7.50

8.75

10.00

IO.OO

nun

Fig. 1. Dichlofluanid (DCFA), DMSA, Tolylfluanid (TYFA) and DMST in strawberries (VEnd = 5 ml each, packed column, TSD). Chromatogram A: Standard solution, representing 1 ng of each compound. Chromatogram B: Untreated control sample fortified with 0.05 mg/kg of each compound. Chromatogram C: Untreated control sample.

o.oo

min

Dlchlofluanid, Tolylfluanid

0.00

1.25

2.50

3.75

183

5.00

Fig. 2. Dichlofluanid (DCFA), DMSA, Tolylfluanid (TYFA) and DMST in strawberries (VEnd = 5 ml each, capillary column, FPD). Chromatogram A: Extract from untreated control sample, fortified with standard solution; peaks representing 0.4 ng of each compound. Chromatogram B: Untreated control sample fortified with 0.05 mg/kg of each compound. Chromatogram C: Untreated control sample.

3.73

3.00

7.50

6.75

IO.OO

min

184

Dichlofluanid, Tolylfluanid

7.00

8.25

7.00

8.25

9.50

10.75

12.00

13.25

14.50

15.75

17.00

min Fig. 3. Dichlofluanid (DCFA), DMSA, Tolylfluanid (TYFA) and DMST in grapes (VEnd = 5 ml each, capillary column, TSD). Chromatogram A: Extract from untreated control sample, fortified with standard solution; peaks representing 0.2 ng of each compound. Chromatogram B: Untreated control sample fortified with 0.05 mg/kg of each compound. Chromatogram C: Untreated control sample.

7.00

8.25

17.00

min

Dichlofluanid, Tolylfluanid

0,00

1.25

2.50

3.75

5.00

6.25

7.50

8.75

185

10.00

IO.OO

min

Fig. 4. Dichlofluanid (DCFA), DMSA, Tolylfluanid (TYFA) and DMST in grapes (VEnd = 5 ml each, capillary column, FPD). Chromatogram A: Extract from untreated control sample, fortified with standard solution; peaks representing 0.4 ng of each compound. Chromatogram B: Untreated control sample fortified with 0.05 mg/kg of each compound. Chromatogram C: Untreated control sample.

io.oo

min

186

Dichlofluanid, Tolylfluanid

Attenuation Integrator Injection volume Retention times for DMSA DMST dichlofluanid tolylfluanid Gas chromatograph 3 Column Column temperature Injection port temperature Detector Gas flow rates

Attenuation Integrator Injection volume Retention times for DMSA DMST dichlofluanid tolylfluanid

10-12 Integrator Spectra-Physics SP 4270 or Laboratory Data System LAS on a Hewlett-Packard HP 1000 A 1 1*1 10 min 11 min 13 min 14 min

18 s 24 s 30 s 30 s

Carlo Erba Mega 5300 Fused silica capillary, 0.53 mm i.d., 10 m long; coated with CP-SIL 5 CB, film thickness 5.0 \xm (Chrompack No. 7645) Isothermal at 150 °C for 1 min, programmed to rise at 10°C/min from 150 to 220 °C, then isothermal at 220 °C for 12 min 270 °C Flame photometric detector (Melpar), equipped with 394-nm sulphur filter Temperature 250 °C Helium carrier, 48 ml/min Helium purge gas, 30 ml/min Hydrogen, 150 ml/min Air, 200 ml/min 1-16; excitation 600 Integrator Spectra-Physics SP 4270 or Laboratory Data System LAS on a Hewlett-Packard HP 1000 A 2 nl 2 min 3 min 5 min 6 min

30 s 30 s 30 s 30 s

7 Evaluation 7.1 Method Quantitation is performed within the recommended measuring ranges (see below) by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the compound standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. When using capillary columns, perform the evaluation with the aid of sample solutions derived from untreated control material which, after the column chromatographic cleanup (6.2.2), have been fortified with the compound standard solutions to yield "fortified calibration solutions".

Dichlofluanid, Tolylfluanid

187

Using the thermionic nitrogen detector, a linear relationship was observed between peak area and amount of compound injected. The flame photometric detector, however, was linear only over a very small range (from 0.4 to 2 ng for the model used). The recommended ranges for quantitative evaluation of the compounds are 1 to 10 ng for gas-chromatographic system 1, 0.2 to 2 ng for gas-chromatographic system 2, and 0.4 to 2 ng for gas-chromatographic system 3. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with dichlofluanid, tolylfluanid, DMSA and DMST at levels of 0.05, 0.5 and 5 mg/kg, are given in the Table. Table. Percent recoveries from plant material, must, and wine, fortified with dichlofluanid, DMSA, tolylfluanid and DMST. Dichlofluanid

DMSA s

Analytical material

Added mg/kg

n

X

s

X

Apples

0.05 0.5 5.0 0.05 0.5 5.0 0.05 0.5 5.0 0.05 *> 0.5 •> 5.0*) 0.05 0.5 5.0 0.05 0.5 5.0 0.05 0.5 5.0 0.05*) 0.5 *> 5.0*)

8 8 8 8 8 8 8 8 6 6 6 4 8 8 4 8 6 6 6 6 6 6 6 6

95 96 98 94 89 85 101 97 101 96 99 99 99 95 92 93 93 84 89 85 88 97 97 100

4.5 6.4 4.8 8.1 1.4 3.8 9.3 4.7 1.9 5.1 3.1 1.7 8.6 8.5 9.3 8.5 6.5 2.5 4.4 7.8 8.9 4.1 1.3 4.5

92 93 94 92 87 88 101 97 99 101 100 100 95 89 83 98 95 88 92 94 86 91 97 95

Grapes

Lettuce

Must

Raspberries

Strawberries

Tomatoes

Wine

5.6 7.0 4.4 9.6 8.6 7.1 4.3 9.0 2.6 5.0 3.8 1.7 5.0 11.1 6.2 9.8 4.2 6.0 6.9 7.9 5.5 3.6 1.8 3.7

Tolylfluanid

DMST s

X

s

X

96 97 95 91 88 87 99 97 102 99 101 103 98 95 95 92 96 87 92 87 90 97 98 98

5.6 5.5 2.2 6.0 3.6 4.1 7.6 5.1 2.5 4.7 2.7 1.5 8.4 9.1 14.2 7.8 5.4 1.9 4.9 6.9 6.1 3.6 1.6 3.9

92 97 96 94 89 88 97 99 99 99 100 101 98 93 86 96 94 88 102 96 90 87 94 96

7.9 8.2 4.8 4.9 3.8 6.0 4.3 6.7 2.6 7.0 6.8 0.8 7.1 8.9 5.6 6.0 8.6 7.9 6.3 7.7 2.9 2.3 2.5 3.3

*) mg/1

The routine limit of determination for the compounds was 0.05 mg/kg. Using the flame photometric detector, blanks usually did not occur or, if so, they were less than 0.01 mg/kg. Using the thermionic detector, blanks were also clearly less than 0.01 mg/kg, except for DMSA (maximum 0.01 mg/kg) and DMST (0.01 to 0.1 mg/kg).

188

Dichlofluanid, Tolylfluanid

7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified compound is calculated from the following equation: p

F A -V E x -V E n d -W s t FsfVR1-VrG

where G

= sample weight (in g) or volume (in ml)

VEx

= volume of concentrated extract after dilution with water (in ml)

VR1

= portion of volume VEx used for further cleanup (in ml)

VEnd = terminal volume of sample solution from 6.3 (in ml) Vi

= portion of volume VEnd injected into gas chromatograph (in ul)

W st

= amount of compound injected with standard solution or "fortified calibration solution" (in ng)

FA

= peak area obtained from Y{ (in mm 2 or integrator counts)

FSt

= peak area obtained from WSt (in mm 2 or integrator counts)

8 Important points The pH of the homogenate in step 6.1.1 should be measured using a pH meter. If it is higher than pH 5, adjust it to pH 4-5 with hydrochloric acid (10 g/100 g HC1 p.a.) in order to prevent hydrolysis of dichlofluanid and tolylfluanid. In a few cases, the complete elution of the compounds from the disposable extraction column required four 50-ml portions of eluting mixture 1 instead of three portions. Therefore, the conditions given in 6.1.1 and 6.1.2 should be checked before a new series of analyses is started or whenever a new batch of the disposable extraction columns is used (observe batch number); if required, increase the number of elutions to four. To obtain complete elution of DMSA and DMST, do not use more than 150 mg activated charcoal for preparing the silica gel-charcoal column as described in 6.2.1. The eluting conditions given in 6.2.2 should be checked before starting a new series of analyses, or whenever a new batch of the mini silica gel columns is used (observe batch number); if required, re-define them. Using the flame photometric detector, the signal-to-noise ratio can be improved by dissolving the residue derived from 6.2 in n-hexane instead of ethyl acetate. However, not all sample material residue will dissolve completely in hexane; additionally, DMSA and DMST are only very sparingly soluble in hexane. The analyst must therefore balance the chance of a better signal-to-noise ratio against the risk of not completely determining the residues present.

Dichlofluanid, Tolylfluanid

189

9 References T. Aizawa, Improved analytical method for the determination of ®Euparen [N-(dichlorofluoromethylthio)-N',N'-dimethyl-N-phenylsulfamide] in vegetables and fruits, Nihon Tokushu Noyaku Seizo K.K. (Nitokuno), Tokyo, Report No. 1092, 7.2.1979 (unpublished). R. Brennecke, Methode zur gaschromatographischen Bestimmung von Riickstanden der Fungizide ®Euparen, ®Euparen M und ®Folicur in Pflanzenmaterial und Getranken, Pflanzenschutz-Nachr. 42, 237-298 (1989). K. Vogeler, Methode zur gaschromatographischen Bestimmung von Riickstanden von Dichlofluanid, Tolylfluanid sowie deren Abbauprodukten DMSA und DMST und von Chinomethionat in Pflanzenmaterial, Bayer AG, Report No. RA-524, 25.6.1984 (unpublished). K. Vogeler and W. Piorr, Gaschromatographische Methode zur Bestimmung von Dichlofluanid- bzw. Tolylfluanid-Ruckstanden sowie deren Abbauprodukten Dimethylaminosulfanilid bzw. Dimethylaminosulftoluidid in Pflanzenmaterial und Boden, Bayer AG, Report No. RA-956, 19.12.1977 (unpublished).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke

Dichlofluanid, Tolylfluanid Strawberries Dichlofluanid (additionally): Grapes, must, wine Tolylfluanid (additionally): Apples, apple juice, apple pulp, pears, pear conserves, pear juice

203-A-371-A Gas-chromatographic determination

(German versions published 1989)

1 Introduction For data on physico-chemical properties of dichlofluanid and tolylfluanid, see the Method on p. 177, this Volume.

2 Outline of method Dichlofluanid and tolylfluanid residues are extracted from plant material with acetone. The extract is concentrated to an aqueous residue. The compounds are partitioned into dichloromethane, and the dichloromethane phase is evaporated to dryness. From fruit juice, must and wine, dichlofluanid and tolylfluanid are directly extracted with dichloromethane; the dichloromethane phase is evaporated to dryness. The residue is dissolved in a cyclohexane-ethyl acetate mixture and cleaned up by gel permeation chromatography on a polystyrene gel. The compounds are determined by gas chromatography using a sulphur-specific flame photometric detector. If interfering peaks occur, a supplemental cleanup on a silica gel column is necessary.

3 Apparatus Homogenizer Wide neck glass bottle, 1-1, with ground joint Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia., fast flow rate Filtration flask, 500-ml Graduated cylinders, 500-ml and 100-ml Round-bottomed flasks, 500-ml, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Separatory funnel, 250-ml, with ground stopper Glass funnel, 10 cm dia. Fluted filter paper Glass syringe, 10-ml, with Luer-lock fitting

192

Dichlofluanid, Tolylfluanid

Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Test tubes, 10-ml, with ground stoppers Chromatographic tube, 17.5 mm i.d., 30 cm long, with PTFE stopcock Gas chromatograph equipped with sulphur-specific flame photometric detector Microsyringe, 5-ul

4 Reagents Acetone, for residue analysis Cyclohexane, for residue analysis Dichloromethane, for residue analysis Ethyl acetate, for residue analysis n-Hexane, for residue analysis Toluene, for residue analysis Eluting mixture: cyclohexane + ethyl acetate 1:1 v/v Compound standard solutions: 1-100 fig/ml of each dichlofluanid and tolylfluanid in n-hexane Silver nitrate solution, 0.1 mol/1 AgNO3 p. a. Sodium chloride solution, saturated Sodium chloride, p. a. Sodium sulphate, p.a., anhydrous Filter aid, e.g. Celite 545 Silica gel 60, 0.2-0.5 mm (Merck No. 7733) Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Cottonwool, chemically pure Air, synthetic, re-purified Hydrogen 5.0 (> 99.999 vol.%) Nitrogen 4.6 (> 99.996 vol.%)

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Apples, apple pulp, grapes, pears, pear conserves, strawberries

To 100 g of the analytical material (G) add 200 ml acetone; for pear conserves, also add 50 ml silver nitrate solution (see 8. Important points). Homogenize the mixture for 3 min. Add approx. 20 g filter aid, mix well, and suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel. Wash the filter cake with approx. 100 ml acetone. Determine the total volume of the filtrate (VEx), and rotary-evaporate an aliquot (e. g. half this

Dlchlofluanid, Tolylfluanid

193

volume) (VR1) in a 500-ml round-bottomed flask to an aqueous residue. Transfer the residue to a separatory funnel, add 20 ml saturated sodium chloride solution, and extract the aqueous phase with 100 ml dichloromethane used earlier for rinsing the flask. Repeat rinsing and extraction twice more, using 100-ml portions of dichloromethane each time. Dry the combined dichloromethane phases on sodium sulphate and rotary-evaporate the solution to dryness. 6.1.2 Fruit juice, must, wine Filter 50 g of the analytical sample (G) through a fluted filter paper into a separatory funnel, and extract the filtrate three times with 100-ml portions of dichloromethane. Dry the combined dichloromethane phases on sodium sulphate and rotary-evaporate the solution to dryness. 6.2 Gel permeation chromatography Transfer the residue derived from 6.1.1 or 6.1.2 into a test tube, using a total of 10 ml eluting mixture (VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR3) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with standard solutions of dichlofluanid or tolylfluanid; cf. Cleanup Method 6, pp. 75ff, Vol. 1. — Elution volumes ranging from 110 to 150 ml were determined for both dichlofluanid and tolylfluanid on Bio-Beads S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 110 to 150-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to step 6.3 or 6.4. Check the elution ranges every 500 samples, and determine anew whenever a new gel column is used. 6.3 Column chromatography Insert a cottonwool plug into the bottom of the chromatographic tube, and introduce a slurry of 15 g silica gel suspended in toluene. Insert another cottonwool plug onto the top of the silica gel, trickle in approx. 3 g sodium sulphate and drain the toluene to the top of the sodium sulphate layer. Dissolve the residue derived from 6.2 in 5 ml toluene. Add the solution to the column and allow to percolate. Rinse the flask twice with 5-ml portions of toluene, add these rinsings successively to the column and also allow to percolate. Next elute dichlofluanid or tolylfluanid with 130 ml toluene. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to dryness. 6.4 Gas-chromatographic determination Dissolve the residue derived from 6.2 or 6.3 in 2 ml hexane (VEnd). Inject 5 |j,l of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column

Tracor 560 Glass, 2 mm i.d., 1.2 m long; packed with 5% DC200 on Gas Chrom Q, 80-100 mesh

194

Dichlofluanid, Tolylfluanid

Column temperature Injection port temperature Detector Gas flow rates Recorder Injection volume Retention times for dichlofluanid tolylfluanid

180°C 250°C Flame photometric detector equipped with 394-nm sulphur filter Temperature 200 °C Nitrogen carrier, 30 ml/min Hydrogen, 120 ml/min Air, 170 ml/min 10 mV; chart speed 5 mm/min 5 \i\ 3 min 24 s 6 min 24 s

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the compound standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. 7.2 Recoveries and limit of determination The recoveries from untreated control samples, fortified with dichlofluanid and tolylfluanid at levels of 0.1 to 1.0 mg/kg, ranged from 77 to 118% for dichlofluanid, and from 75 to 105% for tolylfluanid (see Table). The limit of determination was 0.1 mg/kg for each compound. 7.3 Calculation of residues The residue R, expressed in mg/kg dichlofluanid or tolylfluanid, is calculated from the following equation: R

_ FA-VEx-VR2-VEnd-WSt F s ,-V R1 -V R 3.V r G

where G

= sample weight (in g)

VEx = total volume of filtrate from 6.1.1 (in ml) VRI = portion of volume VEx used for further processing (in ml) VR2 = volume of solution prepared for gel permeation chromatography in 6.2 (in ml) VR3 = portion of volume VR2 injected for gel permeation chromatography (volume of sample loop) (in ml)

Dichlofluanid, Tolylfluanid

195

Table. Percent recoveries from fruits, fruit conserves, fruit juice, and wine, fortified with dichlofluanid, tolylfluanid, and DMST; duplicate experiments. Analytical material Apples Fruit Juice Pulp Grapes Fruit Must Wine Pears Fruit Conserves Juice Strawberries

Added (mg/kg)

Dichlofluanid

0.1 1.0 0.1 0.1 0.1 1.0 0.1 1.0 0.1 0.1 1.0 0.1 0.1 0.1

Tolylfluanid

DMST

97- 98 98-105 85- 89 84- 94

92-104 101-106 88

98-109

100-105 93- 97 107-108 88- 90 116-118

77-104

8780768575-

88 81 78 86 95

98-108 99-103 92- 93 96-103

VEnd = terminal volume of sample solution from 6.4 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

Wst = amount of dichlofluanid or tolylfluanid, respectively, injected with standard solution (in ng) FA

= peak area obtained from Vj (in mm2)

FSt

= peak area obtained from WSt (in mm2)

8 Important points Lower recoveries were obtained from processed fruits, especially pear conserves, as compared to fresh fruits. This probably results from thiol containing plant constituents. Therefore, silver nitrate is added to the pear conserves, as recommended by Aizawa (1979) for dichlofluanid analysis. The method permits the simultaneous determination of the metabolites dimethylaminosulphanilide (DMSA) and dimethylaminosulphotoluidide (DMST). For that purpose, it is necessary to carry out the column chromatography on silica gel as described in 6.3. After dichlofluanid (or tolylfluanid) has been eluted with 130 ml toluene, change the receiver and elute the respective metabolite with 100 ml of a mixture of acetone + toluene, 1:1 v/v. Rotaryevaporate both fractions to dryness, and proceed with each residue as described in 6.4. The retention time for DMSA was about 2 min under the operating conditions given in 6.4; for DMST it was 2 min 24 s at a column temperature of 185 °C. The recoveries from untreated control samples, fortified with DMST at levels of 0.1 to 1.0 mg/kg, ranged from 88 to 109% (see Table). The limit of determination for each metabolite was approx. 0.1 mg/kg.

196

Dichlofluanid, Tolylfluanid

A thermionic nitrogen-specific detector can be used instead of the flame photometric detector. For the detection of dichlofluanid or tolylfluanid alone, an electron capture detector can also be used.

9 References T. Aizawa, Improved analytical method for the determination of ®Euparen [N-(dichlorofluoromethylthio)-N',N'-dimethyl-N-phenylsulfamide] in vegetables and fruits, Nihon Tokushu Noyaku Seizo K.K. (Nitokuno), Tokyo, Report No. 1092, 7.2.1979 (unpublished). K. Vogeler, Methode zur gaschromatographischen Bestimmung von Ruckstanden von Dichlofluanid, Tolylfluanid sowie deren Abbauprodukten DMSA und DMST und von Chinomethionat in Pflanzenmaterial, Bayer AG, Report No. RA-524, 4.6.1984 (unpublished).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, K. Vogeler

Dinobuton, Binapacryl

255-8 High-performance liquid chromatographic determination

Apples, cucumbers Soil, water (German version published 1989)

1 Introduction Binapacryl

Dinobuton Chemical name

2-sec.Butyl-4,6-dinitrophenyl 2-sec.Butyl-4,6-dinitrophenyl isopropyl carbonate (IUPAC) 3-methylcrotonate (IUPAC) CH 3

Structural formula

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

O-CH CH-C 2 H 5

CH-C 2 H 5

CH 3

CH 3

C 14 H 18 N 2 O 7 326.30 61-62°C Not distillable <10" 5 mbar at 20 °C Virtually insoluble in water; very readily soluble in acetone (120 g); readily soluble in xylene (89 g); soluble in ethanol (8.3 g); slightly soluble in n-hexane (1.9 g) Hydrolyzed in alkaline media

C 15 H 18 N 2 O 6 322.32 68-69°C Not distillable 4.2-10- 7 mbar at 20 °C Very sparingly soluble in water; readily soluble in acetone (78 g), ethanol (11 g), dichloromethane (75 g) and xylene (70 g)

Hydrolyzed in concentrated acids and dilute bases; long exposure to UV light causes decomposition

2 Outline of method Dinobuton and binapacryl residues are extracted from plant material with a hexane-methanol mixture. An aliquot of the organic extract is shaken with water to remove interfering polar co-extractives, and cleaned up further by both gel permeation chromatography and adsorption chromatography on a Florisil cartridge. From soil samples, the residues are extracted with methanol. An aliquot of the extract is diluted with water, and the compounds are partitioned

198

Dinobuton, Binapacryl

into dichloromethane. Further cleanup is as with plant material extracts, by gel permeation and adsorption chromatography. From water samples, the residues are extracted with dichloromethane; the extract is cleaned up on a Florisil cartridge. Dinobuton and binapacryl are determined by high-performance liquid chromatography using a UV detector at 254 nm.

3 Apparatus High-speed blendor fitted with leak-proof stainless steel jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., medium flow rate Filtration flask, 500-ml Graduated cylinder, 500-ml Separatory funnels, 2-1 and 250-ml Round-bottomed flasks, 500-ml, 250-ml, 100-ml and 25-ml, with ground joints Glass funnel, 8 cm dia. Rotary vacuum evaporator, 30-35 °C bath temperature Erlenmeyer flask, 500-ml, with ground stopper Laboratory mechanical shaker Test tubes, 10-ml and 3-ml, graduated Glass syringes, 10-ml and 2-ml, with Luer-lock fitting Membrane filter, e.g. SM 11605-025 N, 0.65 nm (Sartorius), and Millex HV4 filter unit (Millipore) Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) High-performance liquid chromatograph equipped with UV detector Microsyringe, 100-ul

4 Reagents Acetone, p. a. Cyclohexane, for residue analysis Dichloromethane, dist. Ethyl acetate, for residue analysis n-Hexane, for residue analysis Methanol, p. a. Methanol, HPLC quality (only for mobile phase) Water, HPLC quality (only for mobile phase) Extraction mixture: n-hexane + methanol 95:5 v/v Eluting mixture 1: cyclohexane + ethyl acetate 1:1 v/v Eluting mixture 2: n-hexane + acetone 98 :2 v/v Mobile phase: methanol + water 8:2 v/v Standard solutions for recovery experiments: 1, 10, 100 and 1000 M
Dinobuton, Binapacryl

199

Dinobuton and binapacryl standard solutions for HPLC measurement: 0.1-2 ng/ml of each in mobile phase Sodium sulphate, p.a., anhydrous, washed with dichloromethane and heated for 4 h at 500°C Filter aid, e.g. Celite 545 Florisil disposable cartridge: Sep-Pak Cartridge Florisil (Millipore No. 51960) Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Cottonwool, exhaustively extracted with dichloromethane

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material Homogenize 50 g of the analytical sample (G) with 5 g filter aid and 100 ml extraction mixture for 3 min. Suction-filter the homogenate through a medium flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake with a further 80 ml extraction mixture. Transfer the filtrate to a graduated cylinder, and make up the organic phase to a definite volume (VEx) (max. 200 ml), ignoring the lower aqueous phase. Transfer an aliquot (VR1) of the organic phase, equivalent to 20 g of the analytical sample, into a 250-ml separatory funnel and wash twice with 20-ml portions of water. Discard the aqueous phases. Dry the organic phase on sodium sulphate, filter through cottonwool, and wash the filter residue with 10 ml hexane. Rotary-evaporate the filtrate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. 6.1.2 Soil In a separate 10 to 20-g aliquot of the laboratory sample, determine the water content by drying in an open weighing glass at 105 °C to constant weight (approx. 15 h). Discard this aliquot. Adjust the water content of 100 g soil (G) to 30% in a 500-ml Erlenmeyer flask (see 8. Important points), add 150 ml methanol, and shake for 30 min on the mechanical shaker. Suction-filter the extract through a medium flow-rate filter paper in a Buchner porcelain funnel. Re-extract the filter cake with a further 150 ml methanol and filter as described above. Wash the filter cake with 50 ml methanol and make up the combined filtrates to 350 ml (VEx) with methanol in a graduated cylinder. Transfer a 70-ml aliquot (VR1), equivalent to 20 g of the analytical sample, into a 250-ml separatory funnel, add 30 ml water, and extract three times with 50-ml portions of dichloromethane. Wash the combined dichloromethane solutions twice with 40-ml portions of water. Dry the organic phase on sodium sulphate, filter through cottonwool, and wash the filter residue with 10 ml hexane. Rotary-evaporate the filtrate to near dryness, and remove the last traces of solvent by swirling the flask in the hand.

200

Dinobuton, Binapacryl

6.1.3 Water

Extract 1 1 of the water sample (G) three times with 100-ml portions of dichloromethane. Dry the combined organic phases on sodium sulphate, filter through cottonwool, and wash the filter residue with 10 ml hexane. Rotary-evaporate the filtrate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. Proceed to step 6.2.2. 6.2 Cleanup 6.2.1 Gel permeation chromatography

Transfer the residue derived from 6.1.1 or 6.1.2 into a test tube, using a total of 10 ml eluting mixture 1 (VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR3) of the gel permeation chromatograph with 8 to 9 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with standard solutions of dinobuton and binapacryl; cf. Cleanup Method 6, pp. 75ff, Vol. 1. — Elution volumes ranging from 80 to 100 ml were determined for both compounds on Bio-Beads S-X3 polystyrene gel, using eluting mixture 1 as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 80 to 100-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. Check the elution range from time to time, and determine anew whenever a new gel column is used. 6.2.2 Florisil cartridge Draw 10 ml hexane into the glass syringe, attach a Florisil cartridge to the syringe, and force the hexane through to condition the cartridge packing. Repeat the conditioning with a further 10-ml portion of hexane. Next detach the cartridge, pull the plunger out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1.3 or 6.2.1 in 1 ml hexane and transfer the solution quantitatively into the syringe with the aid of a Pasteur pipet. Re-insert the plunger into the syringe and force the liquid through the cartridge. Detach the cartridge, remove the plunger from the syringe, and re-attach the cartridge. Force 10 ml hexane through the cartridge, proceeding in a similar manner as described above, and discard the eluates. Next elute the compounds with 10 ml eluting mixture 2 from the Florisil and collect the eluate in a 25-ml round-bottomed flask. Rotary-evaporate to near dryness and remove the last traces of solvent by swirling the flask in the hand.

6.3 High-performance liquid chromatographic determination Dissolve the residue derived from 6.2.2 in mobile phase and dilute to an appropriate volume (VEnd). Filter the solution through a membrane filter. Inject an aliquot of this solution (Vj) into the high-performance liquid chromatograph. Operating conditions Chromatograph Injector

Spectra-Physics SP 8700 Injection valve 7125 with sample loop (Rheodyne)

Dinobuton, Binapacryl Column 1

201

Attenuation Recorder Injection volume

Cartridge column, 4 mm i. d., 25 cm long, and precolumn, 4 mm i. d., 3 cm long (Knauer) Hyperchrome NC column, 4.6 mm i. d., 25 cm long (Bischoff) LiChrosorb RP-18, 5-um (Merck) Methanol + water 8 :2 v/v 1.0 ml/min UV/VIS detector Uvicon 720 LC (Kontron) Wavelength 254 nm Detector range 0.02 AUFS 20 mV; chart speed 5 mm/min 50 \i\

Retention times for dinobuton binapacryl

Column 1 8 min 48 s 10 min 42 s

Column 2 Column packing Mobile phase Flow rate Detector

Column 2 8 min 48 s 9 min 48 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of each dinobuton and binapacryl standard solution into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng dinobuton or binapacryl, respectively. Also inject equal volumes of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of compound from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples, fortified with dinobuton and binapacryl at levels of 0.05 to 5.0 mg/kg, ranged from 70 to 116% (average 82%) for plant material, and from 78 to 98% (average 90%) for soil. The recoveries for tap water, fortified at levels of 0.5 u.g/1 to 0.5 mg/1, ranged from 73 to 112% and averaged 92%. The limit of detection was 0.03 mg/kg for apples and cucumbers, and 0.01 mg/kg for soil. Due to reagent blanks, the limit of detection for tap water was 0.2 ng/1; a lower limit of detection of 0.05 |ig/l can possibly be achieved by using reagents of higher purity. The limit of determination was 0.05 mg/kg for plant material and soil, and 0.5 |ig/l for tap water. 7.3 Calculation of residues The residue R, expressed in mg/kg dinobuton or binapacryl, is calculated from the following equations: for plant material and soil

R=

WA • VEFx • VR2 • VEnd * _x_—R_2_ J; nd V

R1 * VR3 ' V i *

U

202

Dinobuton, Binapacryl

for water

R=

where G

= sample weight (in g) or volume (in ml)

VEx = total volume of organic phase after addition of extraction mixture to filtered extract from plant sample in 6.1.1 (in ml), or total volume of filtered soil extract from 6.1.2 after addition of methanol (in ml) VR1 = portion of volume VEx used for further cleanup (in ml) VR2 = volume of eluting mixture 1 used to take up the residue from 6.1.1 or 6.1.2 (in ml) VR3 = portion of volume VR2 injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume VEnd injected into high-performance liquid chromatograph (in ul)

WA = amount of dinobuton or binapacryl for Vj read from calibration curve (in ng)

8 Important points Before beginning a series of analyses, check the reagents for purity, particularly n-hexane and sodium sulphate. When the filtration of extracts from plant material causes difficulties, slurry the filter aid onto the medium flow-rate filter paper with water, instead of adding it to the sample. When soil samples containing less than 30 percent water are taken for analysis, the phases will generally not separate after the soil extract has been shaken with dichloromethane. In this case, phase separation will occur when water is added. The HPLC measurement can also be performed on a n-Bondapak Column C-18 (Millipore), 4 mm i. d., 30 cm long, substituting the mobile phase with a methanol + water mixture 75:25 v/v. For the quantitative determination, a wavelength of 254 nm is used; fewer interfering peaks occur at this wavelength than at the absorption maxima of dinobuton and binapacryl at 242 nm.

9 Reference H. Roseboom and H. A. Herbold, Determination and confirmation of binapacryl and dinobuton residues on apples and cucumbers by high-performance liquid chromatography, J. Chromatogr. 208, 137-140 (1981).

Dinobuton, Binapacryl

203

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, M. Blacha-Puller and J. Siebers

Fonofos

288

Brussels sprouts, carrots, cauliflower, head cabbage, kohlrabi, maize (kernels), onions, radishes (large and small types), red cabbage Soil, water

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

O-Ethyl S-phenyl (RS)-ethylphosphonodithioate (IUPAC)

Structural formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility Other properties

C10H15OPS2

246.34 No data 130°C at 0.133 mbar 2.8-10"4 mbar at 25 °C Virtually insoluble in water; miscible with organic solvents such as acetone, diethyl ether, n-hexane, methanol and xylene Light yellow liquid; hydrolyzed in acid and alkaline media

2 Outline of method Fonofos residues are extracted from plant material or soil with acetone. The extract is diluted with water, and fonofos is partitioned into petroleum ether. Petroleum ether is used for direct extraction from water samples. The organic phase is rotary-evaporated and cleaned up on a Florisil column. Fonofos is determined by gas chromatography using a thermionic detector.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Ball grinding mill Glass filter funnel, porosity G2, 13 cm dia. Filtration flask, 1-1

206

Fonofos

Separatory funnels, 1-1 and 250-ml Round-bottomed flasks, 500-ml, with ground joints Laboratory mechanical shaker Rotary vacuum evaporator, 30-40 °C bath temperature Chromatographic tube, 15 mm i.d., 40 cm long Volumetric flasks, 10-ml Gas chromatograph equipped with thermionic phosphorus-specific detector Microsyringe, 10-ul

4 Reagents Acetone, dist. Acetonitrile, dist., saturated with petroleum ether Diethyl ether, dist. n-Hexane, p. a. Petroleum ether, dist., boiling range 40-80°C Eluting mixture: petroleum ether + diethyl ether 98:2 v/v Fonofos standard solutions: 0.5-5 M-g/ml n-hexane Fenchlorphos solution (internal standard): 5 mg/ml n-hexane Florisil, 60-100 mesh, 6% water content: Determine the water content of commercial Florisil by heating a given amount for 1 h in a muffle furnace at 400 °C, followed by re-weighing. To 100 g commercial Florisil in a 300-ml Erlenmeyer flask (with ground joint), add an appropriate amount of water dropwise from a burette to adjust the water content to 6%. Shake vigorously for 5 min until all lumps have disappeared, next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Sodium chloride solution, saturated Sodium sulphate, p.a., anhydrous Glass wool Compressed air, re-purified Hydrogen, re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

Fonofos

207

6 Procedure 6.1 Extraction 6.1.1 Plant material and soil

Weigh 100 g of the analytical sample (G) into the blendor jar, and homogenize with 200 ml acetone for 3 min. Suction-filter the mixture, and thoroughly wash the blendor jar and filter cake with a total of 75 ml acetone. Transfer the extract into a 1-1 separatory funnel, and add 150 ml sodium chloride solution and 250 ml water. Extract the mixture twice with 100-ml portions of petroleum ether by shaking on a mechanical shaker for 5 min each time. Combine the organic phases, and filter through sodium sulphate into a 500-ml round-bottomed flask. Wash the sodium sulphate with petroleum ether. Rotary-evaporate the solution to a volume of 5 ml; during the analysis of maize kernels, concentrate the solution to a residual volume of approx. 50 ml. Then proceed to 6.2.2; for maize kernel samples, however, first clean up the solution as described in 6.2.1. 6.1.2 Water Extract a water sample (G) twice with equal volumes of petroleum ether (at least 100 ml) by shaking for 5 min each time on a mechanical shaker. Combine the organic phases, and filter through sodium sulphate into a 500-ml round-bottomed flask. Wash the sodium sulphate with petroleum ether. Rotary-evaporate the solution to a volume of approx. 5 ml. With extracts from water samples containing only small amounts of co-extractives, proceed directly to gaschromatographic analysis in 6.3, otherwise proceed firstly to 6.2.2. 6.2 Cleanup 6.2.1 Liquid-liquid partition (maize kernels) Transfer the solution derived from 6.1.1 to a 250-ml separatory funnel, and extract successively with four 50-ml portions of acetonitrile. Combine the acetonitrile phases in a 1-1 separatory funnel, dilute with 125 ml sodium chloride solution and 350 ml water, and extract twice with 100-ml portions of petroleum ether by shaking for 5 min each time. Combine the organic phases, filter through sodium sulphate into a 500-ml round-bottomed flask, and rotaryevaporate to a volume of approx. 5 ml. 6.2.2 Column chromatography Place a glass wool plug in the bottom end of a chromatographic tube, and half-fill the tube with petroleum ether. Slowly add 20 g Florisil to the column, gently tapping the tube walls. Allow the adsorbent to settle, and drain the petroleum ether until the top of the Florisil is just covered with liquid. Then quantitatively rinse the concentrated solution derived from 6.1.1, 6.1.2 or 6.2.1 onto the prepared column with a little petroleum ether. Allow the solution to percolate into the column at a rate of 1-2 drops per s, and then elute the column with 200 ml eluting mixture. Rotary-evaporate the eluate almost to dryness.

208

Fonofos

6.3 Gas-chromatographic determination Quantitatively rinse the residue derived from 6.2.2 into a 10-ml volumetric flask with hexane, and dilute to the mark with hexane. Carry out a preliminary measurement to estimate approximately the concentration of fonofos. If its concentration is higher than 5 ng/ul, dilute with hexane. On the other hand, if the solution has a very low content of fonofos, it can be concentrated to a lower volume. Using a microsyringe, add to the thus prepared sample solution a pl-amount of the internal standard fenchlorphos solution (shake!) equivalent to the mlvolume (VEnd) of the sample solution. Inject an aliquot of this solution into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder Injection volume Linearity range Retention times for fonofos fenchlorphos

Varian Aerograph 2740 Glass, 2 mm i.d., 1.5 m long; packed with 5% SE-30 on Chromosorb W-AW-DMCS, 80-100 mesh 200 °C 220 °C Thermionic phosphorus-specific detector Temperature 240 °C Nitrogen carrier, 40 ml/min Hydrogen, 15 ml/min Air, 170 ml/min 64 • 10~u 1 mV; chart speed 10 mm/min 1 ul 0.25-5 ng 2 min 42 s 4 min 24 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. To 10 ml of each fonofos standard solution (equivalent to 5 to 50 |ug fonofos), add 10 JLLI fenchlorphos solution (equivalent to 50 \ig fenchlorphos) as internal standard (shake!). Inject 1-ul aliquots of these solutions into the gas chromatograph. For graphic representation of the calibration curve, divide the peak heights of fonofos by the peak heights of the internal standard, and plot each quotient vs. the concentration of fonofos in the standard solutions (ng/^il). From the gas chromatogram of each injected sample solution, measure the peak heights, calculate the quotients as described above, and read the concentration of fonofos in ng/|nl (WA) from the calibration curve.

Fonofos

209

7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with fonofos at levels of 0.02 to 0.5 mg/kg, ranged from 85 to 95%. The routine limit of determination was 0.02 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg fonofos, is calculated from the following equation: R=

w A -v E n d

where G = sample weight (in g) or volume (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) WA = concentration of fonofos read from calibration curve (in ng/ul)

8 Important points No data

9 References No data

10 Author Shell Forschung GmbH, Schwabenheim, D. Eichler

Fosetyl

522 Gas-chromatographic determination

Grapes, hop cones, lettuce, strawberries, wine Water (German version published 1989)

1 Introduction

Chemical name

Fosetyl (as fosetylaluminium)

Main metabolite

Aluminium tris(O-ethyl phosphonate) (IUPAC)

Phosphorous acid

o Structural formula

C 2 H 5 O-P-O-

OH HO-P-OH

Other properties

C6H18A1O9P3 354.10 200 °C with decomposition Not distillable <10" 5 mbar at 20 °C Readily soluble in water (12 g/100 ml at 20 °C); virtually insoluble in organic solvents, e.g. acetonitrile (8 mg), methyl glycol (8 mg), in 100 ml each at 20 °C Stability to hydrolysis: Half life in aqueous solution (1 g/1) > 100 d; hydrolyzed in strong acid and alkaline media

I

H

H

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility

o HO-P-OH

H3PO3

81.99 73.6°C 200 °C with decomposition No data Very readily soluble in water (309 g/100 ml at 0°C, 694 g/100 ml at 40 °C); soluble in ethanol

Forms a tautomeric equilibrium predominantly in favour of phosphonic acid

2 Outline of method Fosetyl-aluminium and its main metabolite, phosphorous acid, are extracted from plant material with dilute sulphuric acid. Wine and water samples are acidified with concentrated sulphuric acid. An aliquot of the plant extract, or the acidified water or wine samples, are diluted with isopropanol. After methylation with a diazomethane solution in diethyl ether, the resulting O-ethyl O-methyl and O,O-dimethyl phosphonates are determined by gas chromatography using a phosphorus-specific flame photometric detector.

212

Fosetyl

3 Apparatus Homogenizer, e. g. Ultra-Turrax (Janke & Kunkel) Laboratory centrifuge with 250-ml glass tubes Glass funnel, 9 cm dia. Volumetric flasks, 100-ml, 50-ml and 5-ml Round-bottomed flask, 50-ml, with ground joint Methylation apparatus, see Fig. 1, p. 130, Vol. 1 Gas chromatograph equipped with phosphorus-specific flame photometric detector Microsyringe, 10-nl

4 Reagents Diethyl ether, high purity, dried over calcium chloride 2-Propanol (isopropanol), p. a. Isopropanol + water mixture 9:1 v/v Fosetyl standard solutions for recovery experiments: 1, 10, 100 and 1000 ng/ml fosetylaluminium or phosphorous acid in water Derivative standard solutions: Prepare solutions of 100 pig/ml of fosetyl-aluminium and phosphorous acid, respectively, in isopropanol-water mixture (dissolve fosetyl-aluminium in water and dilute with isopropanol to yield a solution containing isopropanol and water in the proportion 9:1 v/v). Transfer 10 ml each of these solutions into volumetric flasks, add 25 \i\ of concentrated sulphuric acid, make up to 100 ml with isopropanol-water mixture and shake. Derivatize 5 ml of the solutions as described in 6.2. Concentrate the reaction mixtures to 3 ml, transfer to 5-ml volumetric flasks and make up to 5 ml with isopropanol. Dilute these solutions progressively to obtain solutions containing O-ethyl O-methyl phosphonate or O,Odimethyl phosphonate equivalent to 0.01, 0.02, 0.05, 0.08, 0.1, 0.15, and 0.2 ng/ml of fosetylaluminium or phosphorous acid Sulphuric acid, p. a.; cone, and 1 g/100 ml Ethanolic potassium hydroxide solution: Dissolve 7 g KOH p.a. in 10 ml water and make up to 100 ml with ethanol Diazomethane solution in diethyl ether (for apparatus see Fig. 1, p. 130, Vol. 1): Dissolve 1.2 g N-methyl-N-nitroso-p-toluenesulphonamide in 10 ml diethyl ether and transfer to the dropping funnel. Slowly add this solution dropwise to 5 ml ethanolic potassium hydroxide solution contained in the reaction vessel, and sweep the generated diazomethane into 20 ml diethyl ether, using a gentle stream of nitrogen, while the receiver containing the ether is cooled in an ice + sodium chloride freezing mixture Glass wool Cottonwool, exhaustively extracted with acetone Air, synthetic Hydrogen, re-purified Nitrogen, re-purified

Fosetyl

213

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material (except hop cones) Weigh 50 g of the analytical sample (G) with a water content of x g (see 8. Important points) into a centrifuge tube, add 50 ml dilute sulphuric acid (VEx), and homogenize for 1 -2 min. Centrifuge for 15 min at 2500 r.p.m. Filter the supernatant through glass wool, transfer 5 ml of the filtrate (VR1) into a volumetric flask, and make up to 50 ml (VR2) with isopropanol. Shake the solution and filter through cottonwool to remove precipitated material. 6.1.2 Hop cones Weigh 5 g of the analytical sample (G) into a centrifuge tube, add 100 ml dilute sulphuric acid, and homogenize for 1-2 min. Then proceed as described in 6.1.1. 6.1.3 Wine, water Weigh 10 g of the analytical sample (G) into a volumetric flask, add 25 jil concentrated sulphuric acid, and make up to 100 ml (VR2) with isopropanol. 6.2 Methylation Transfer 5 ml (VR3) of the solution derived from 6.1 into a 50-ml round-bottomed flask, and add diazomethane solution (5-10 ml) until the yellow colour produced remains. Stopper the flask and allow to stand for 15 min with occasional swirling. Remove excess diazomethane and concentrate to approx. 3 ml with a gentle stream of nitrogen. 6.3 Gas-chromatographic determination Quantitatively transfer the solution derived from 6.2 into a volumetric flask and make up to an appropriate volume (VEnd), e.g. 5 ml, with isopropanol. Inject an aliquot of this solution (VA) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature

Carlo Erba Fractovap 2101 AC Glass, 2 mm i.d., 2 m long; packed with 15% Carbowax 20M on Chromosorb 750, 100-120 mesh 130 °C 200°C

214

Fosetyl

Detector

Flame photometric detector, Model SSD 250, equipped with 526-nm phosphorus filter Temperature 175 °C Nitrogen carrier, 90 ml/min Hydrogen, 60 ml/min Air, 200 ml/min 1 • 32 10 mV; chart speed 5 mm/min 5 ul

Gas flow rates Attenuation Recorder Injection volume Retention times for dimethyl phosphonate ethyl methyl phosphonate

3 min 3 min 36 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 5 ul of each derivative standard solution (equivalent to 0.05 to 1.0 ng fosetyl-aluminium or phosphorous acid, respectively) into the gas chromatograph. Plot the heights of the peaks obtained vs. ng of fosetyl-aluminium or phosphorous acid. Also inject 5-ul aliquots of the sample solutions. For the heights of the peaks obtained for the sample solutions, read the appropriate amounts of aluminium-fosetyl or phosphorous acid from the corresponding calibration curve. 7.2 Recoveries and limit of determination The recoveries from untreated control samples, fortified with fosetyl-aluminium and phosphorous acid at levels of 0.1 to 10 mg/kg (hop cones 20 to 150 mg/kg), ranged from 72 to 120% for plant material, wine and tap water, and averaged 97%. Blanks usually were less than 0.1 mg/kg. Strawberries and grapes occasionally gave blanks corresponding to 0.2 and 0.9 mg/kg phosphorous acid, respectively. Hop cones gave blanks corresponding to up to 4 mg/kg for both fosetyl-aluminium and phosphorous acid. The limit of determination was in the range of 0.1 to 1 mg/kg for all materials tested; for hop cones, the limit was approx. 20 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg fosetyl-aluminium or phosphorous acid, is calculated from the following equations: for plant material

R=

W

A • (VEx + x) • VR2 • VEnd V

for wine and water

R=

W

R1 * V R3 * V i * G

A ' VR2' VEnd . Q Vpi * V; • G

93

Fosetyl

215

where G

= sample weight (in g)

x

= water portion of t h e sample weight (plant material) (in g)

V Ex

= volume of dilute sulphuric acid used for extraction of sample (in ml)

V R1

= portion of filtrate (before dilution with isopropanol) used for further processing (in ml)

V R2

= volume of solution after dilution with isopropanol in 6.1 (in ml)

V R3

= p o r t i o n of volume V R2 used for methylation in 6.2 (in m l )

V E n d = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume V End injected into gas chromatograph (in ul)

WA

= amount of fosetyl-aluminium or phosphorous acid, respectively, for V{ read from calibration curve (in ng)

0.93 = factor for conversion of fosetyl-aluminium to fosetyl (not required for phosphorous acid residues)

8 Important points The water content of the sample material can be determined by drying to constant weight at 105 °C. Alternatively, the average water content of the respective material as listed in Table 2, Method S 19 (p. 386, Vol. 1) may be used for calculation. The derivative standard solutions can be stored in a refrigerator for 24 h; for longer storage times, the stability should be checked. Samples to be analyzed should only be stored for short periods even under deep freeze conditions, because breakdown of fosetyl residues was observed during storage at —18 °C (see J. Siebers, H.-G. Nolting and W. D. Weinmann, Initialbelage von Pflanzenschutzmittelwirkstoffen im Gemtisebau, Nachrichtenbl. Dtsch. Pflanzenschutzdienstes Braunschweig 36, 182-189, 1984). Depending on the quality of the hop cones, a routine limit of determination of 2 mg/kg for both fosetyl and phosphorous acid can be attained. Instead of the flame photometric detector, a thermionic phosphorus-specific detector can also be used.

9 Reference A. Bertrand, Determination of residues of phosphorous acid and ethyl phosphonate in lettuces, Method No. 22-79, Rhone-Poulenc Agrochimie, 1979.

216

Fosetyl

10 Authors Rhone-Poulenc Agrochimie, Lyon, France, A. Bertrand and M. A. Muller Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, M. Blacha-Puller and J. Siebers

Glufosinate

651

Almonds, apples, asparagus, bananas, beans, caraway, Chinese cabbage, evening primrose oil, kiwi fruit, lemons, maize (kernels), meat (incl. beef fat, blood, meat broth, kidneys and liver), mirabellas, oranges, peas, plums, potatoes, rape (green matter and seeds), sour cherries, soybeans, sugar beet (foliage and edible root), sunflower (seeds and oil), wheat (grains) Soil, water

Gas-chromatographic determination

(German version published 1991)

1 Introduction Glufosinate-ammonium Chemical name

Ammonium DL-homoalanin-4-yl(methyl)phosphinate (IUPAC)

Structural formula

CH 3 -P—CH 2 —CH 2 —CH-COOH

o O~

Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at 20 °C) Other properties

NH 4 +

NH 2

C 5 H 15 N 2 O 4 P 198.19 215 °C (with decomposition) No data Very readily soluble in water (137 g at 22 °C, pH 5); very sparingly soluble in acetone (16 mg), ethanol (65 mg), ethyl acetate (14 mg) and toluene (14 mg) No data

Glufosinate (free acid) Chemical name

DI^Homoalanin-4-yl(methyl)phosphinic acid (IUPAi

Structural formula

0 II CH 3 -P—CH 2 -CH 2 —CH-COOH 1 1 OH NH2

Empirical formula Molar mass Melting point Boiling point Vapour pressure

C 5 H 12 NO 4 P 181.15 No data No data No data

218

Glufosinate

Solubility (in 100 ml at 20 °C)

Other properties Metabolite Chemical name Structural formula

Readily soluble in water (26.9 g at pH 1, >40 g at pH 7 and pH 10); very sparingly soluble in methanol (12 mg); virtually insoluble in acetone, dichloromethane, ethyl acetate, n-hexane, isopropanol, toluene (each <1 mg), dimethyl sulphoxide (9 mg) and polyethylene glycol (5mg) Commercial formulations contain glufosinate exclusively as the ammonium salt 3-(Methylphosphinico)propionic acid (IUPAC) O II CH3-P-CH2-CH2-COOH OH

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C4H9O4P 152.1 No data No data No data Readily soluble in water (79.4 g; pH of the saturated solution is 1.0); readily soluble in dimethyl sulphoxide (> 40 g) and methanol (>50 g); soluble in isopropanol (9.4 g) and polyethylene glycol (4 g); sparingly soluble in acetone (0.29 g); very sparingly soluble in ethyl acetate (86 mg); virtually insoluble in dichloromethane (2 mg), n-hexane and toluene (each <1 mg) No data

2 Outline of method Residues of glufosinate-ammonium, glufosinate and the metabolite are extracted from plant and animal material and from soil with water. Depending on the type of sample material, the extracts are cleaned up by de-fatting with dichloromethane, by precipitation of carbohydrates and proteins with acetone, or by ion exchange chromatography. From water samples, the residues are concentrated on an anion exchange chromatographic column and eluted with formic acid solution. After evaporation of the cleaned-up extracts or of the eluate, the residues are treated with trimethyl orthoacetate, resulting in the formation of the derivatives: methyl 4-[methoxy(methyl)phosphinoyl]-2-acetamidobutyrate from glufosinate, and methyl 3-[methoxy(methyl)phosphinoyl]propionate from the metabolite. The derivatives are cleaned up on a mini silica gel column and are determined by gas chromatography using a phosphorus-specific flame photometric detector.

Glufosinate

CH3-P—CH2—CH2-CH-COOCH3 OCH3

NH-COCH3

Glufosinate derivative

219

CH 3 -P—CH 2 -CH 2 -COOCH 3 0CH3

Metabolite derivative

3 Apparatus Homogenizer, e. g. Ultra-Turrax (Janke & Kunkel) Meat mincer Erlenmeyer flasks, 500-ml and 300-ml Watch glass, 10 cm dia. Hotplate with magnetic stirrer, incl. stirring rod Graduated cylinders, 1-1, 100-ml, 50-ml and 2-ml Centrifuge, e. g. Labofuge GL (Heraeus-Christ) Volumetric pipets, 20-ml and 10-ml Round-bottomed flasks, 100-ml and 50-ml, with ground joints Rotary vacuum evaporator, 60 °C bath temperature Separatory funnels, 1-1 and 100-ml Filter cartridges for organic solvents, pore size 0.5 \im (e. g. Millex SR Filter, Millipore SLSR 025 NS)

Filter cartidge

Disposable syringe Silica gel

Quartz wool

Figure. Adding the derivatives solution onto the mini silica gel column in step 6.3.

220

Glufosinate

Beaker, 5-1 Chromatographic tube 1: 15 mm i.d., 30 cm long, with stopcock Chromatographic tube 2: 40 mm i.d., 60 cm long, with stopcock Ultrasonic bath Solvent dispensers, 15-ml, 8-ml and 2-ml Reflux condenser, jacketed coil type, 30 cm long, with ground joint Heating mantles, regulated, for 100-ml and 50-ml round-bottomed flasks Disposable syringes, 10-ml, with stainless steel needles (flat tip); needles approx. 15 cm long, straight form and angular bent (see Figure) Pasteur pipets Pear-shaped flask, 10-ml, with ground joint Volumetric flasks, 100-ml, 50-ml, 10-ml and 5-ml Gas chromatograph equipped with phosphorus-specific flame photometric detector Microsyringes, 100-ul and 10-ul

4 Reagents Acetone, p. a. Dichloromethane, p. a. Ethanol, p. a. Ethyl acetate, p. a. Methanol, p. a. Methyl acetate, p. a. Toluene, p. a. Eluting mixture: methyl acetate + methanol 1:1 v/v Solvent mixture 1: methyl acetate + ethanol 1:1 v/v Solvent mixture 2: methyl acetate + toluene 1:1 v/v Solvent mixture 3: methyl acetate + toluene 7 : 3 v/v Glufosinate and metabolite standard solutions for fortification experiments: 5 |xg/ml of each glufosinate-ammonium or 3-(methylphosphinico)propionic acid (Riedel-de Haen) in water or, for fortification of fats and oils, in solvent mixture 1 Glufosinate derivative standard solution: Methyl 4-[methoxy(methyl)phosphinoyl]-2-acetamidobutyrate in eluting mixture, equivalent to 5 pig/ml glufosinate-ammonium. Prepare by weighing glufosinate-ammonium and derivatizing as described in 6.2 Metabolite derivative standard solution: Methyl 3-[methoxy(methyl)phosphinoyl]propionate in eluting mixture, equivalent to 5 ng/ml metabolite. Prepare by weighing 3-(methylphosphinico)propionic acid and derivatizing as described in 6.2 Polyethylene glycol solution: 10 g/100 ml polyethylene glycol 400 p.a. (Serva No. 46170) in acetone Glacial acetic acid, p. a. Hydrochloric acid, 10 g/100 g HC1 p. a. Formic acid, 10 ml/100 ml HCOOH p. a. Ammonia solution, 10 g/100 ml and 1 g/100 ml NH3 Sodium hydroxide solution, 1 mol/1 NaOH Trimethyl orthoacetate (Riedel-de Haen No. 64561)

Glufosinate

221

Cation exchanger, strongly acidic: Ion exchanger I (Merck No. 4765). Preparation: Add 2 1 hydrochloric acid to 1000 g cation exchanger in a beaker, allow to stand for 30 min, decant, wash the resin with water until neutral (pH indicator paper), and further treat, in the following sequence, with: 1 1 ammonia solution (10 g/100 ml), allow to stand for 5 min, wash to neutral with water; 2 1 ethanol-water mixture (1:1 v/v), allow to stand for 30 min, wash with 2 1 water, until free of ethanol; 1 1 hydrochloric acid (conversion to H + form), wash with water until neutral Cation exchange column: Pour 6 g of the damp cation exchanger as prepared above into a chromatographic tube (type 1) Anion exchanger, strongly basic: Dowex 1X8 (Serva No. 41091). Preparation: Allow the resin (in the chloride form as supplied) to swell in water for 1 h. Pour approx. 100 g of the swollen resin into a chromatographic tube (type 2), and wash with 750 ml sodium hydroxide solution to convert it into the OH" form. Wash with water until the eluate has become neutral to pH indicator paper Anion exchange column: Pour 6 g of the damp anion exchanger as prepared above into a chromatographic tube (type 1) pH indicator paper RP-18 disposable cartridge: Sep-Pak Cartridge C18 (Millipore No. 51910) Silica gel, deactivated with 4% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734), for 6 h at 130 °C, allow to cool in a desiccator, and store in a tightly stoppered container in the desiccator. To 100 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 4 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container Quartz wool Air Helium Hydrogen

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material with high water content (apples, asparagus, bananas, beans, Chinese cabbage, kiwi fruit, lemons, mirabellas, oranges, plums, potatoes, sour cherries, sugar beet) and soil

Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer flask. Cover the flask with a watch glass, and magnetically stir the homogenate for 30 min at room temperature. Measure the volume of the homogenate (VEx), and centrifuge a 60-ml

222

Glufosinate

portion at 3000 r.p.m. for 10 min. Pipet 20 ml of the supernatant (VR1) into a 50 ml roundbottomed flask and rotary-evaporate to dryness, using a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.1.2 Plant material with high content of water soluble carbohydrates or proteins (almonds, caraway, maize, peas, soybeans, wheat) Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer flask. Cover the flask with a watch glass, and magnetically stir the homogenate for 30 min at room temperature. Measure the volume of the homogenate (VEx), and centrifuge a 60-ml portion at 3000 r.p.m. for 10 min. Take 40 ml of the supernatant (VR1) and add 40 ml acetone to precipitate carbohydrates and proteins (total volume, VR2). Centrifuge the mixture again. Pipet 40 ml of the supernatant (VR3) into a 100-ml round-bottomed flask and rotaryevaporate to dryness, using firstly room temperature, then a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.1.3 Fatty plant material (rape, sunflower seeds) Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer flask (total volume of the homogenate, VEx). Cover the flask with a watch glass, and magnetically stir the homogenate for 30 min at room temperature. Allow solid particles to settle, then decant 40 ml of the supernatant (VR1) into a centrifuge tube, and add 40 ml acetone (total volume of the mixture, VR2). Mix, and centrifuge the mixture at 3000 r.p.m. for 5 min. Next, transfer 40 ml of the supernatant (VR3) into a 100-ml separatory funnel and shake with 20 ml dichloromethane. Separate the lower organic phase and re-extract it twice with 10-ml portions of water. Combine the aqueous phases in a 100-ml round-bottomed flask and rotary-evaporate to dryness, using firstly room temperature, then a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.1.4 Fats and oils (evening primrose oil, sunflower oil, beef fat) Dissolve 20 g of the analytical sample (G) in 100 ml dichloromethane (use the homogenizer for dissolution of solid fat if required). Add 100 ml water (VEx) and homogenize for 5 min. Decant approx. 60 ml of the aqueous supernatant into a centrifuge tube and centrifuge at 3000 r.p.m. for 5 min. Force 25 ml of the supernatant (VR1) through a RP-18 disposable cartridge that has been previously conditioned with 5 ml methanol and 5 ml water. Wash the cartridge by forcing through 3 ml water. Collect both the aqueous eluate and the wash in a 50-ml round-bottomed flask and rotary-evaporate to dryness, using a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.1.5 Animal matrices (blood, meat, meat broth, liver, kidneys) Add 100 ml water to 20 g of blood (G) and magnetically stir, raising the temperature to 80 °C, and maintain at this temperature for 30 min with further stirring (total volume, VEx). For the

Glufosinate

223

other materials, transfer 20 g of the minced analytical sample or 20 g of meat broth (G) into a 300-ml Erlenmeyer flask, and add 100 ml water (total volume, VEx). Cover the flask with a watch glass and magnetically stir the mixture for 30 min at room temperature. Centrifuge 50-60 ml of the mixture at 3000 r.p.m. for 10 min. To 40 ml of the supernatant (VR1), add 40 ml acetone to precipitate proteins (total volume of this mixture, VR2), and centrifuge the mixture again for 10 min. Carefully rotary-evaporate 40 ml of the clear supernatant (VR3) at room temperature to completely remove acetone; the aqueous solution should not smell of acetone any longer. Transfer the solution onto the anion exchange column at a rate of 3-4 ml/min, wash the column with 100 ml water, and discard the eluate. Next, elute glufosinate and its metabolite with 70 ml dilute formic acid into a 100-ml round-bottomed flask, and rotary-evaporate the eluate to dryness, using a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.1.6 Water

Before concentrating glufosinate and its metabolite on an anion exchange column, the cations present in the water must be exchanged for H + (see 8. Important points). For this purpose, the ion exchange columns are connected in series, so that the eluate from the cation exchanger flows directly onto the anion exchanger. Using a 1-1 separatory funnel, transfer 1 1 of the water sample (G) onto the cation exchange column at a rate of 5-10 ml/min. Wash both columns with 100 ml of distilled or deionized water, and discard the eluates (approx. 1.1 1). Remove the cation exchange column. Next, elute glufosinate and its metabolite from the anion exchange column with 70 ml dilute formic acid into a 100-ml round-bottomed flask. Add 100 \x\ polyethylene glycol solution to the eluate and rotary-evaporate to dryness, using a 60 °C bath temperature. Add 5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required until the residue is absolutely dry. 6.2 Derivatization Suspend the residue derived from 6.1 in 2 ml glacial acetic acid by dipping the flask for 10 min in an ultrasonic bath. Add 8 ml trimethyl orthoacetate, dip the flask for a further 5 min in the ultrasonic bath, and then reflux the mixture for 4 h with occasional swirling to prevent suspended solids from baking onto the walls of the flask. Allow to cool, add 15 ml toluene, and rotary-evaporate to a residual volume of approx. 1 ml (do not evaporate to dryness!) with 40 °C bath temperature. Repeat the evaporation with toluene twice more to completely remove residual acetic acid and derivatization reagent (see 8. Important points). Make up the 0.5-1 ml residue to 3 ml with toluene, dip the flask for 1 min in the ultrasonic bath, and draw up the flask contents, including the suspended solid material, into a 10-ml disposable syringe. Rinse the flask with 5 ml methyl acetate, dip the flask for approx. 2 min in the ultrasonic bath, and also draw up the rinsing into the syringe. Mix the contents well by shaking the syringe.

224

Glufosinate

6.3 Column chromatography Insert a quartz wool plug into the bottom of a Pasteur pipet and add 0.6 g silica gel. Condition the column with 5 ml solvent mixture 2 delivered from a disposable syringe with a 15 cm long stainless steel needle. Using the needle, stir the silica gel-solvent mixture until it is free of air bubbles. Filter the solution derived from 6.2 in the first syringe through a filter cartridge mounted on the syringe and, with the aid of an angular bent needle, transfer the filtrate onto the mini silica gel column (see Figure). Rinse the flask from 6.2 with 10 ml methyl acetate. Draw up the rinsing into the syringe, filter and transfer it onto the column in a similar manner. When the column has run dry, apply gentle suction to dry the packing. Next, elute the derivatives of glufosinate and its metabolite from the column with eluting mixture, collecting exactly 5 ml eluate (VEnd) in a 5-ml volumetric flask. For samples of drinking water, evaporate the 5 ml of eluate to approx. 100 jul, using a 40 °C water bath and a gentle stream of nitrogen, and make up to 1.5 ml (VEnd) with eluting mixture. For most other sample materials, a second cleanup on a mini silica gel column is recommended. Transfer the eluate to a pear-shaped flask using 3 ml toluene and rotary-evaporate (40 °C bath temperature) to approx. 1 ml. The methanol must be removed completely. Make up to 3 ml with toluene, and mix with 5 ml methyl acetate, as described in 6.2. Rechromatograph the mixture on a mini silica gel column as described above, with the exception of the filtration step.

6.4 Gas-chromatographic determination Inject an aliquot of the solution derived from 6.3, e.g. 5 ul (Vj), into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Injection volume Retention times for metabolite derivative glufosinate derivative

Carlo Erba 2150, with capillary injector (Chrompack 8045), split exit closed Fused silica capillary, 0.53 mm i.d., 10 m long; coated with Carbowax 20 M, film thickness 0.25 urn (Restek) Isothermal at 150°C for 2 min, programmed to rise at 39°C/min from 150 to 240 °C, then isothermal at 240 °C for 4 min 225 °C Flame photometric detector, equipped with 526-nm phosphorus filter Temperature 225 °C Helium carrier, 28 ml/min (214 cm/s) Hydrogen, 75 ml/min Air, 125 ml/min 5 ul 42 s 4 min 18 s

Glufosinate

Alternative conditions Column Column temperature Gas flow rates

Retention times for metabolite derivative glufosinate derivative

225

Fused silica capillary, 0.53 mm i.d., 8 m long; coated with RTX-2330, film thickness 0.25 urn (Restek) Isothermal at 140 °C for 2 min, programmed to rise at 39°C/min from 140 to 240 °C, then isothermal at 240 °C for 4 min Helium carrier, 25 ml/min (190 cm/s) Helium purge gas, 10 ml/min Hydrogen, 70 ml/min Air, 120 ml/min 1 min 24 s 5 min

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas or peak heights of the sample solutions and comparing them with the peak areas or peak heights obtained for the derivative standard solutions. Equal volumes of the sample solutions and the derivative standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas or heights. Should pre-analysis checks show that the derivatives with and without matrix give different detector responses, the evaluation is performed with the aid of sample solutions derived from untreated control material which, after the column cleanup (6.3), have been fortified with standard solutions of the derivatives ("fortified calibration solutions"). Evaluation is also possible with the aid of the "Standard Addition Method" if no control material is available. For this purpose, analyze a sample as described above (6.1 to 6.4). To an aliquot of this sample solution, add standard solutions of the derivatives, and measure again. The differences between the peak areas or heights obtained for the two measurements is equivalent to the amounts of the derivatives taken for fortifying the analytical sample.

7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with glufosinate-ammonium or metabolite at levels of 0.05 to 10 mg/kg, ranged from 64 to 116%. The routine limit of determination was 0.05 mg/kg for plant material, fats, oils and soil, 0.05 to 0.1 mg/kg for animal matrices, and 0.05 fig/1 for tap water. 7.3 Calculation of residues The residue R, expressed in mg/kg (for water, ng/1) glufosinate-ammonium or metabolite, is calculated from the following equations:

226

Glufosinate

for plant material, fats, oils, animal matrices, soil R

_ F A -V E x -V R 2 -V E n d -W S t

where G

= sample weight (in g)

VEx

= total volume of homogenate or (for fats and oils) volume of water added to the organic phase for extraction (in ml)

VRI

= portion of volume VEx used for further processing (in ml)

VR2

= total volume of the aqueous solution with acetone added (in ml)

VR3 = portion of volume VR2 used for further processing (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WSt

= amount of glufosinate-ammonium equivalents or metabolite equivalents, respectively, injected with derivative standard solution or "fortified calibration solution" (in ng)

FA

= peak area or height obtained from Vj (in mm 2 or integrator counts)

F st

= peak area or height obtained from WSt (in mm 2 or integrator counts)

for water R = where G

= sample volume (in 1)

VEnd = terminal volume of sample solution from 6.3 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WSt

= amount of glufosinate-ammonium equivalents or metabolite equivalents, respectively, injected with derivative standard solution or "fortified calibration solution" (in ng)

FA

= peak area or height obtained from Yi (in mm 2 or integrator counts)

F st

= peak area or height obtained from WSt (in mm 2 or integrator counts)

To calculate glufosinate as free acid, multiply the result for glufosinate-ammonium by a factor of 0.91, and the result for the metabolite by a factor of 1.19.

Glufosinate

227

8 Important points Cations (more than 10 mg Na + , Mg2+ and Ca2+ in the analytical sample) can drastically reduce the derivatization yields. Mg2+ and Ca2+ are precipitated as hydroxides on the anion exchanger, but are taken into solution again by the formic acid, and are eluted together with glufosinate and its metabolite. The cations must therefore be removed from the water samples with a cation exchange column before the derivatization step. After the derivatization (6.2), the acetic acid must be completely removed because it prevents the concentration of the derivatives in the following silica gel cleanup (6.3). The activity of the silica gel must be checked before each series of analyses. Proceed as follows: Transfer a solution of glufosinate derivative and metabolite derivative in solvent mixture 3 onto the silica gel mini column and elute as described in 6.3. When untreated control material is available, add the derivatives to the sample solution after derivatization and cooling (6.2), but before the solvent evaporating step. The recoveries from the silica gel should be 90 to 100%. If they are less than 80%, repeat the check using a fresh silica gel batch.

9 References No data

10 Author Hoechst AG, Product Development, Division C, Frankfurt/Main, H. Sochor

Glyphosate

405 High-performance liquid chromatographic determination

Beer, bread, cereals (grains, flour and straw), grapes, grass, grass silage, hay, peas, rape (seeds), sugar beet (foliage and edible root) Soil, water (German version published 1989)

1 Introduction Glyphosate

Metabolite (AMPA)

Chemical name

N-Phosphonomethylglycine (IUPAC)

Aminomethylphosphonic acid

Structural formula

HO—P—CH 2 —NH—CH 2 -COOH

0

OH

Empirical formula Molar mass Melting point Vapour pressure Solubility

C3H8NO5P 169.08 230 °C (with decomp.) 3.5-10- 7 mbar at 45 °C Slightly soluble in water (1.2g/100mlat25°C); sparingly soluble in common organic solvents

Other properties

Commercial products contain glyphosate as isopropylamine salt

0 HO—P—CH 2 —NH 2 OH

CH6NO3P 111.04 285 °C (decomp. at 312 °C) 4.0-10" 6 mbar at 75 °C Soluble in water (5.8 g/100 ml at25°C); virtually insoluble in acetone, dichloromethane, diethyl ether, ethanol, n-hexane and toluene

2 Outline of method Residues of glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), are extracted from plant material, bread and flour with dilute hydrochloric adic, and from soil with ammonia solution. Beer and water are filtered. The pH of the extract or filtrate is adjusted to 2.0 ±0.4. The solution is passed through a Chelex 100 ligand exchange resin in the Fe(III) form; the co-eluted ferric ions are removed by chromatography through an anion exchanger. Glyphosate and AMPA are determined separately by high-performance liquid chromatography including post-column derivatization. In the fluorogenic step, glyphosate, which has been oxidized to a primary amine, and AMPA are reacted with o-phthaldialdehyde and 2-mercaptoethanol to yield strongly fluorescent compounds which are measured using a fluorescence detector. For AMPA determination, the oxidation step is omitted.

230

Glyphosate

3 Apparatus Centrifuge, 12000 r.p.m., with 250-ml glass tubes Graduated cylinders, 2-1, 500-ml, 250-ml, 100-ml, 25-ml and 10-ml Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) Glass funnel, 9 cm dia. Fluted filter paper, 18 cm dia. (Schleicher & Schull) Erlenmeyer flasks, 2-1, 250-ml and 100-ml Magnetic stirrer, with stirring rod Vacuum filtration unit fitted with membrane filters, type G S, pore size 0.22 \im (Millipore) Flat-bottomed flask, 10-1 Amber glass bottles, 1-1 Chromatographic tube 1, 8 cm i.d., 40 cm long, with sintered glass disk Chromatographic tube 2, 2.2 cm i.d., 13 cm long, with reservoir 5.5 cm i.d., 9 cm long Chromatographic tube 3, 1.5 cm i.d., 14 cm long, with reservoir 2.5 cm i.d., 6.5 cm long Round-bottomed flask, 100-ml Rotary vacuum evaporator, 20-50 °C bath temperature High-performance liquid chromatograph equipped with post-column derivatization system and fluorescence detector Microsyringe, 100-ul

4 Reagents Note: Use only deionized water for all aqueous solutions and all analytical operations. Dichloromethane, p. a. (Merck No. 6050) Methanol, p. a. (Merck No. 6009) Glyphosate and AMPA standard solutions: 0.05, 0.1, 0.3, 0.5, 0.8 and 1.0 ng/ml glyphosate or AMPA (e. g. Aldrich No. 32,481-7) in water. The solutions can be stored in a refrigerator for about 1 month Boric acid, p. a. (Merck No. 165) Hydrochloric acid, 32%, and 6, 1, 0.2, 0.1 and 0.02 mol/1 HC1 p. a. ortho-Phosphoric acid, p.a., 85% (Merck No. 573) Ammonia solution, 25% (Merck No. 5432) and 1 mol/1 NH3 p. a. Ferric chloride solution, 0.1 mol/1 FeCl3 p. a. Mobile phase (phosphate buffer solution): Dissolve 680 mg potassium dihydrogen phosphate in 1 1 methanol-water mixture (4:96 v/v), and adjust to pH 2.0 with ortho-phosphoric acid. De-gas in a vacuum filtration unit before use Oxidizing solution: Dissolve 11.6 g sodium chloride, 13.6 g potassium dihydrogen phosphate and 5.0 g sodium hydroxide in approx. 900 ml water. Add 4 ml sodium hypochlorite solution and make up to 1 1 with water. The solution is stable for a maximum of 5 days Fluorogenic solution: Dissolve 25 g boric acid and 11 g sodium hydroxide in approx. 900 ml water. Add 30 ml Brij solution, 2 ml 2-mercaptoethanol, and 0.8 g o-phthaldialdehyde, dissolved in 10 ml methanol. Make up to 1 1 with water. The solution is stable for a maximum of 5 days Brij solution: 30 g/100 ml Brij 35 (Merck No. 801962) in water

Glyphosate

231

2-Mercaptoethanol (Merck No. 15433) Ferric chloride hexahydrate, p. a. (Merck No. 3943) Potassium dihydrogen phosphate, p. a. (Merck No. 4873) Sodium chloride, p. a. (Merck No. 6404) Sodium hydroxide, p. a. (Merck No. 6498) Sodium hypochlorite solution, approx. 13% active chlorine (Merck No. 5614) o-Phthaldialdehyde (Aldrich No. P3,940-0) Anion exchange resin: AG 1-X8, particle size 200-400 mesh, Cl form (Bio-Rad No. 140-1451): Slurry 450 g of the resin in 1 1 water. Stir on a magnetic stirrer for 30 min. Allow to settle, decant and discard the water. Wash twice more as described above. Store the resin in an amber bottle under deionized water Ligand exchange resin, Fe(III) form: Slurry 900 g of Chelex 100, particle size 100-200 mesh, Na form (Bio-Rad No. 142-2832) in 3 1 water, add 50 ml hydrochloric acid (6 mol/1) and 1 1 ferric chloride solution. Stir on a magnetic stirrer for 10 min. Allow to settle, decant and discard the aqueous solution. Repeat twice, using a mixture of 2 1 water and 500 ml ferric chloride solution each time. Next transfer the resin into the chromatographic tube 1 and wash with 4 1 hydrochloric acid (0.02 mol/1). The resin can be stored for approx. 3 months under deionized water in an amber bottle Glass wool pH indicator rods: Acilit pH 0-6.0 (Merck No. 9531) and Neutralit pH 5.0-10.0 (Merck No. 9533)

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material (except hay and straw), bread and flour

Weigh 30 g of the analytical sample (G) containing x g water (see 8. Important points) into a centrifuge tube, add 150 ml hydrochloric acid (0.1 mol/1) (VEx) and 50 ml dichloromethane, and homogenize for 1 min. Next centrifuge the homogenate for 20 min at 12000 r.p.m. and filter the supernatant aqueous phase through a glass wool plug. Take an aliquot of 125 ml (VR1) of the filtrate, dilute to approx. 400 ml with water, and mix thoroughly. The pH of the solution should be between 1.6 and 2.4. If the pH is outside this range, dilute further with water, or acidify with hydrochloric acid (6 mol/1). 6.1.2 Hay, straw

Weigh 15 g of the chopped analytical sample (G) containing x g water (see 8. Important points) into a centrifuge tube, add 200 ml hydrochloric acid (6 mol/1), and homogenize for 1 min. Next, centrifuge the suspension for 20 min at 12000 r.p.m. and filter the supernatant through

232

Glyphosate

a glass wool plug. Take an aliquot of 130 ml (VR1) of the filtrate, dilute to approx. 400 ml with water, and mix thoroughly. The pH of the solution should be between 1.6 and 2.4. If the pH is outside this range, dilute further with water, or acidify with hydrochloric acid (6 mol/1). 6.1.3 Beer Filter 100 ml of the analytical sample (G) through a fluted filter paper to remove carbon dioxide. Add 100 ml water and 200 ml hydrochloric acid (0.1 mol/1), and mix thoroughly. 6.1.4 Soil Weigh 25 g of soil (G) into a 250-ml Erlenmeyer flask, add 150 ml ammonia solution, and extract with fast magnetic stirring for 30 min. Centrifuge the suspension for 20 min at 12000 r.p.m. and filter the supernatant through a glass wool plug. Repeat the extraction twice more. Next, adjust the pH of the combined filtrates to 2.0 ±0.4 with 30 ml of hydrochloric acid (32%), followed by 20-30 ml of hydrochloric acid (1 mol/1). Dilute to 1.8 1 with water. Check the pH and re-adjust to pH 2.0 ±0.4, if necessary. If a precipitate forms, leave it to settle for 30 min, or filter the solution through a glass wool plug. 6.1.5 Water Suction-filter 2 1 of the water sample (G) through a membrane filter using the vacuum filtration unit and adjust the pH of the filtrate to 2.0 ±0.4 by adding approx. 5 ml hydrochloric acid (6 mol/1). 6.2 Cleanup 6.2.1 Ligand exchange chromatography Insert a glass wool plug into the bottom of chromatographic tube 2, add 7-8 ml water and 15 ml of the resin in its Fe(III) form. Allow the resin to settle and drain the liquid to the top of the column packing. Next add the filtrate derived from 6.1 onto the column, not disturbing the top layer of the resin, and allow to percolate at a rate of 6-8 ml/min. Open fully the stopcock and wash the column with 50 ml water and 100 ml hydrochloric acid (0.2 mol/1). Cautiously add 3 ml hydrochloric acid (6 mol/1) to the column and allow to percolate at a rate of approx. 4 ml/min. Repeat with a further 4-ml portion of hydrochloric acid. Discard all eluates obtained up to this point. Elute glyphosate and AMPA from the column with twice 5 ml, followed by 7 ml hydrochloric acid (6 mol/1). Collect the eluates in a 100-ml Erlenmeyer flask and add 8 ml hydrochloric acid (32%). 6.2.2 Anion exchange chromatography Insert a glass wool plug into the bottom of chromatographic tube 3, add 7-8 ml water and approx. 7 ml of the anion exchange resin. (In order to ensure that at least 7 ml of the resin is transferred to the column, fill the resin into a 10-ml graduated cylinder first, and allow to settle for approx. 45 min.) Pre-wash the column packing three times with 5-ml portions of hydrochloric acid (6 mol/1) and discard the eluates obtained up to this point.

Glyphosate

233

Cautiously add the solution derived from 6.2.1 onto the column and allow to percolate, the stopcock being fully opened. Be careful not to disturb the top layer of the resin in order to prevent the passage of the yellow-orange ferric chloride through the column. Rinse the Erlenmeyer flask with 2 ml hydrochloric acid (6 mol/1) and add the rinsing to the column. As soon as the solutions have soaked into the resin bed, add a further 8-ml portion of hydrochloric acid (6 mol/1) to the column. Collect the total eluate in a 100-ml round-bottomed flask and carefully rotary-evaporate to dryness, increasing the water bath temperature slowly from 20 °C to 50 °C (Caution! Solution foams readily when evaporated). Dissolve the residue in 5 ml water and check the pH. If it is less than 1.5, add 20 ml water and rotary-evaporate to dryness again. Make up the residue with water to a definite volume (VEnd), e.g. 5.0 ml. 6.3 High-performance liquid chromatographic determination Glyphosate and AMPA are determined by high-performance liquid chromatography using a fluorescence detector. For this purpose, glyphosate is converted in two post-column derivatization steps, first by oxidation with oxidizing solution to a primary amine, then by derivatization to a strongly fluorescent compound with o-phthaldialdehyde (OPA) in the presence of 2mercaptoethanol. In the case of AMPA, the oxidation step is omitted. Switching off reagent pump I (oxidizing solution) permits the separate determination of glyphosate and AMPA (see Diagram below).

Integrator

Detector

1\ Reaction coils

HPLC pump

(^"^

i, /

Injector

Pre-column +analvtical column

Reagent pump II Fluorogenic solution \ Three-way connecting / pieces Reagent pump I <— Oxidizing solution

Diagram of the HPLC system.

For glyphosate determination, inject an aliquot (Vj) of the solution derived from 6.2.2 into the high-performance liquid chromatograph; for AMPA determination, inject a second aliquot of the same sample solution (with reagent pump I switched off). Operating conditions Pump Injector Pre-column

Constant volume pump, model 6000 A (Millipore) Automatic injection system, Model 8055 (Varian), or injection valve with sample loop, Model U6K (Millipore) Stainless steel, 4.6 mm i.d., 2 cm long ("Column Guard Kit", Millipore); packed with Pellicular Media Sax (Alltech No. 2855)

234

Glyphosate

Column Mobile phase Flow rate

Stainless steel, 4.6 mm i.d., 30 cm long; packed with Aminex A9, particle size 11.5 urn (Bio-Rad No. 999-1143-300) Phosphate buffer solution, pH 2.0 0.5 ml/min

Post-column derivatization system: Reagent pump I Model 2150 (LKB Instruments) (for oxidizing solution) Model PCR IB (Varian) Reagent pump II (for fluorogenic solution) Flow rates Reagent pump I: 0.2 ml/min for glyphosate analysis Reagent pump II: 0.3 ml/min Reagent pump I: switched off Reagent pump II: 0.3 ml/min for AMPA analysis Two stainless steel capillary tubes, 0.01 inch i. d., Reaction coils 1/16 inch o.d., 2 m long, with two Valco three way connecting pieces (see Diagram) Fluorescence detector Fluorichrom (Varian) equipped Detector with excitation filters 7-54 and 7-60 (band filters 325 nm and 360 nm) and emission filters 4-76 and 3-72 (band filter 530 nm and cut-off filter 440 nm) SP 4100 (Spectra-Physics); chart speed 5 mm/min Integrator SP410 10-1000 Linearity range 10-100 ng for glyphosate and AMPA Injection volume 100 nl Retention times for glyphosate 16 min AMPA 26 min

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of glyphosate or AMPA standard solutions, respectively, equivalent to 5 to 100 ng glyphosate or AMPA, into the high-performance liquid chromatograph. Plot the areas of the peaks obtained vs. ng glyphosate or AMPA. Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the standard solutions should be injected. For the areas of the peaks obtained for the sample solutions, read the appropriate amounts of glyphosate or AMPA from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples, fortified with glyphosate or AMPA, are given in the Table.

Glyphosate

235

Table. Percent recoveries from plant material, soil and water, fortified with glyphosate and AMPA. Analytical material

Added (mg/kg)

Glyphosate Range Mean

AMPA Range Mean

Beer Bread Cereals Grains Flour Straw Grapes Grass Grass silage Hay Peas Rape (seeds) Sugar beet Foliage Edible root Soil Water

0.02 0.05

83-94 66-90

92 74

62-69 72-100

66 87

0.03-2 0.05-5 0.05-10 0.1-0.2 0.04-40 2-40 2-40 0.05-40 0.06-3.3

71-100 92-114 80-114 57-73 70-120 70-79 84-92 53-92 57-92

90 100 94 65 94 76 88 76 74

64-99 97-103 60-108 64-86 75 70 75-94 50-52

84 100 90 73 75 70 85 51

0.03-0.06 0.03 0.05-1 0.0001-2.5*)

70-100 73-91 66-86 77-138

82 82 80 101

64-93 67-70 69-80 75-95

79 68 73 85

•> Equivalent to 0.1-2500 \xg/\

The limit of detection for glyphosate and AMPA was 0.01 to 0.02 mg/kg for plant material, beer, bread, flour and soil, and the limit of determination was 0.02 to 0.05 mg/kg. Blanks usually did not occur. For tap water, the limit of detection was 0.05 ng/1, and the limit of determination was 0.1 jxg/1. Blanks usually did not occur or, if so, they were less than 0.04 fxg/1.

7.3 Calculation of residues The residue R, expressed in mg/kg glyphosate or AMPA, is calculated from the following equations: for plant material

R=

for beer, soil and water

R=

W

A ' (VBx + x) • VEnd VR1 V G

where G

= sample weight (in g) or volume (in ml)

x

= water portion of the sample weight (plant material) (in g)

VEx

= volume of solvent used for extraction of sample (in ml)

VR1 = portion of volume VEx used for cleanup in 6.2 (in ml)

236

Glyphosate

VEnd = terminal volume of sample solution from 6.2.2 (in ml) Vj

= portion of volume VEnd injected into high-performance liquid chromatograph (in ul)

WA = amount of glyphosate or AMPA, respectively, for V{ read from calibration curve (in ng)

8 Important points The water content of the sample material can be determined by drying to constant weight at 105 °C. Alternatively, the average water content of the respective material as listed in Table 2, Method S 19 (p. 386, Vol. 1) may be used for calculation. (Note that in this Table the figure given for sugar beet root should read 70%.) Two Varian pumps, Model PCR IB, or two high pressure pumps, e. g. Perkin Elmer 10 or LKB 2150, can also be used for the HPLC post-column derivatization system. The solution should exhibit a pH of 8.5 to 9 at the exit of the detector. The Aminex column used for HPLC is very sensitive to metal ions. It is, therefore, very important that the top layer of the anion exchange resin used for removal of the ferric ions should not be disturbed. It is recommended that metal ions in soil samples should be complexed by making up the residue derived from 6.2.2 to a definite volume VEnd, e.g. 5 ml, with EDTA solution (0.001 mol/1; 0.37 g ethylenedinitrilotetraacetic acid disodium salt dihydrate in 1.0 1 water). The determination of glyphosate can also be carried out using a stainless steel column, 4.6 mm i.d., 25 cm long, packed with Zorbax Sax (DuPont No. 880952703) or Partisil Sax (Whatman No. 4226-001). These columns are not suitable for the analysis of the metabolite, AMPA.

9 References H. A. Moye, C. J. Miles and S. J Scherer, A simplified high-performance liquid chromatographic residue procedure for the determination of glyphosate herbicide and (aminomethyl)phosphonic acid in fruits and vegetables employing postcolumn fluorogenic labeling, J. Agric. Food Chem. 31, 69-72 (1983). M. Roth, Fluorescence reaction for amino acids, Anal. Chem. 43, 880-882 (1971). S. S. Simons, Jr. and D. E Johnson, The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines, J. Am. Chem. Soc. 98, 7098-7099 (1976).

10 Authors Monsanto, St. Louis, MO, U.S.A., J E. Cowell and P. J Nord Monsanto, Louvain-la-Neuve, Belgium, M. A. Reding

Glyphosate

a)

b)

c)

Time Fig. 1. Chromatograms of a) standard solution of glyphosate, amount injected equivalent to 80 ng; b) untreated control sample of sugar beet, blank equivalent to < 0.01 mg/kg glyphosate (arrow); c) untreated control sample of sugar beet fortified with 0.2 mg/kg glyphosate, recovery 98%. Column Aminex A9.

a)

b)

Time Fig. 2. Chromatograms of a) standard solution of AMPA, amount injected equivalent to 10 ng; b) untreated control sample of cereal straw, blank equivalent to <0.05 mg/kg AMPA (arrow); c) untreated control sample of cereal straw fortified with 0.067 mg/kg AMPA, recovery 83%. Column Aminex A9.

237

238

Glyphosate

b)

c)

Time Fig. 3. Chromatograms of a) standard solution of glyphosate, amount injected equivalent to 10 ng; b) untreated control sample of tap water, blank equivalent to < 0.05 jig/1 glyphosate (arrow); c) untreated control sample of tap water fortified with 0.1 (ig/1 glyphosate, recovery 83%. Column Zorbax Sax.

Metaldehyde

151-A

Chicoree leaves, Chinese cabbage, corn salad, cut lettuce, endives, lettuce, radishes, spinach, strawberries

Gas-chromatographic determination

(German version published 1987)

1 Introduction

Chemical name

r-2,c-4,c-6,c-8-Tetramethyl-l,3,5,7-tetroxocane (IUPAC) (acetaldehyde tetramer) as main component CHa

CH 3 O

Structural formula Empirical formula Molar mass Melting point Boiling point Solubility

Other properties

CH

C 8 H 16 O 4 176.22 246 °C sealed tube (pure tetramer) Not distillable, sublimes at 110-120 °C Very sparingly soluble in water at 17 °C; soluble in chloroform and dichloromethane; sparingly soluble in benzene (0.12 g/100 ml at 23 °C) Depolymerized in the presence of acids and at elevated temperatures

2 Outline of method Metaldehyde residues are extracted from plant material with dichloromethane. Interfering aldehydes are removed with sodium meta-bisulphite solution. Metaldehyde is depolymerized with hydrochloric acid to acetaldehyde, which is reacted with 2,4-dinitrophenylhydrazine. The resulting acetaldehyde 2,4-dinitrophenylhydrazone is cleaned up on an aluminium oxide column and determined by gas chromatography using a thermionic detector.

3 Apparatus Wide neck bottle, 250-ml, with screw cap Homogenizer, e. g. Ultra-Turrax (Janke & Kunkel) Sintered glass filter funnel, porosity G2, 13 cm dia. Filtration flask, 500-ml Separatory funnels, 250-ml and 100-ml, with ground joints Round-bottomed flasks, 250-ml and 50-ml, with ground joints

240

Metaldehyde

Rotary vacuum evaporator, 40 °C bath temperature Centrifuge, 2000 r.p.m. Laboratory mechanical shaker, suitable for holding separatory funnels Chromatographic tube, 15 mm i.d., 30 cm long Volumetric flasks, 10-ml Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 5-ul

4 Reagents Dichloromethane, for residue analysis; see 8. Important points n-Hexane, for residue analysis Toluene, for residue analysis Eluting mixture: dichloromethane + n-hexane 2:1 v/v Metaldehyde standard solution: 5 (ig/ml toluene. Dissolve metaldehyde first in a minimum of dichloromethane 2,4-Dinitrophenylhydrazine solution: 0.25 g/100 ml 2,4-dinitrophenylhydrazine (Merck No. 3081), recrystallized from toluene, in hydrochloric acid p. a. (6 mol/1) Sodium meta-bisulphite solution, 2 g/100 ml Na2S2O5 p. a. (Merck No. 6528). Prepare fresh daily Sodium sulphate, p.a., anhydrous Aluminium oxide 90, Brockmann standardized, activity grade II-III, 0.063-0.200 mm (Merck No. 1097) Glass wool Air, re-purified Hydrogen, re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

6 Procedure 6.1 Extraction and removal of free aldehydes Homogenize 50 g of the finely comminuted plant material (G) with 100 ml dichloromethane in the wide neck bottle for 5 min. Suction-filter the homogenate through the sintered glass filter funnel (homogenates from strawberries should be centrifuged first). Wash the filter cake three times with 30-ml portions of dichloromethane. Combine the filtrates in a 250-ml separatory funnel and shake for 5 min with 20 ml sodium meta-bisulphite solution. Separate and discard the aqueous phase. Wash the organic phase twice with 50-ml portions of water. Discard the aqueous layers, and rotary-evaporate the organic phase to dryness. Dissolve the residue in about 20 ml of toluene.

Metaldehyde

241

6.2 Depolymerization and derivatization Transfer the toluene solution derived from 6.1 to a 100-ml separatory funnel, add 5 ml 2,4dinitrophenylhydrazine solution, and shake for 60 min on the mechanical shaker. After phase separation, discard the aqueous layer. Wash the organic phase twice with 20-ml portions of water, filter it through sodium sulphate, and rinse the sodium sulphate with approx. 10 ml toluene. Collect the filtrates in a 50-ml round-bottomed flask and rotary-evaporate to dryness. 6.3 Column chromatography Half-fill the chromatographic tube, containing a glass wool plug in the lower end, with hexane. Slowly add 6 g aluminium oxide, gently tapping the tube walls. Allow the adsorbent to settle, and then drain the supernatant hexane until the top of the aluminium oxide is just covered with the solvent. Quantitatively rinse the residue derived from 6.2 onto the prepared column with a total of 4 ml eluting mixture. Allow the liquid to percolate into the column at a rate of 1-2 drops per s. Elute the acetaldehyde derivative with the eluting mixture. Collect a total of 16 ml eluate in a 50-ml round-bottomed flask and rotary-evaporate to dryness. 6.4 Gas-chromatographic determination Rinse the residue derived from 6.3 into a 10-ml volumetric flask (VEnd) with hexane and make up to the mark. Inject an aliquot of this solution (V{) into the gas chromatograph. Carry out a preliminary measurement to estimate approximately the concentration of metaldehyde. If this is higher than 10 fig/ml, dilute the solution. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder Injection volume Retention time for acetaldehyde 2,4dinitrophenylhydrazone

Varian 3700 Glass, 2 mm i.d., 1.3 m long; packed with 6% OV-101 on Gas Chrom Q, 80-100 mesh 200 °C 200 °C Thermionic nitrogen-specific detector Temperature 320 °C Nitrogen carrier, 25 ml/min Hydrogen, 4.5 ml/min Air, 170 ml/min 8 -10~12 1 mV; chart speed 5 mm/min 1 ul 2 min 24 s

242

Metaldehyde

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique using a calibration curve, which must be prepared anew for each series of analyses. Proceed as follows. Pipet each of the following metaldehyde standard solution volumes, 2.0, 5.0, 10.0, and 20.0 ml (corresponding to 10, 25, 50, and 100 \ig metaldehyde, respectively), into 100-ml separatory funnels, and make up to 20 ml with toluene. Process these solutions in the same way as described in 6.2 and 6.3, and finally make up the residues with hexane to 10.0 ml. Inject 1 ul of each of these acetaldehyde derivative standard solutions (equivalent to 1 to 10 ng metaldehyde) into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng metaldehyde. Also inject 1-ul aliquots of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of metaldehyde from the calibration curve. Check the calibration curve by injection of an acetaldehyde derivative standard solution at least after every third injection. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with metaldehyde at levels of 0.2 to 2.0 mg/kg, ranged from 70 to 100%. The routine limit of determination was 0.1 to 0.2 mg/kg, depending on the respective blank values. Blank values were in the range of 0.05 to 0.1 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg metaldehyde, is calculated from the following equation: R

w A -v E n d

where G

= sample weight (in g)

VEnd = terminal volume of sample solution from 6.4 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WA = amount of metaldehyde for Vj read from calibration curve (in ng)

8 Important points In order to avoid interfering reagent blanks, dichloromethane must be shaken with 2,4-dinitrophenylhydrazine solution and further purified on a column with basic aluminium oxide, activity grade I.

Metaldehyde

243

9 Reference S. Selim and J. N. Seiber, Gas chromatographic method for the analysis of metaldehyde in crop tissue, J. Agric. Food Chem. 21, 430-433 (1973).

10 Authors Shell Forschung GmbH, Schwabenheim, D. Eichler RCC Umweltchemie AG, Itingen, Switzerland, W. Vogel

Metribuzin

337

Barley (grains), broad beans (incl. pods), carrots, potatoes (tubers and tops), tomatoes (leaves and fruits), wheat (grains and straw) Soil, water

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

4-Amino-6-tert.butyl-3-methylthio-l,2,4-triazin-5(4H)one (IUPAC) (CH3)3C

Structural formula 1N

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

SCH3

C8H14N4OS 214.29 125.5-126.5 °C Not distillable 1.33-10"5 mbar at 20°C Sparingly soluble in water (0.12 g); readily soluble in acetonitrile, dichloromethane, ethyl acetate (>60 g each), methanol (45 g) and 2-propanol (13 g); slightly soluble in ligroin (1 g); sparingly soluble in petroleum ether (0.48 g) Stable to diluted acids and alkalies (up to pH ~ 12.5) at 20 °C

2 Outline of method Metribuzin residues are extracted from plant material with acetonitrile; from soil samples with a methanol-water mixture, acetonitrile and dichloromethane; from water samples with a dichloromethane-ethyl acetate mixture. The acetonitrile extract from cereal grain samples is washed with petroleum ether. Following evaporation of the extractant, the aqueous residue is extracted with dichloromethane by shaking. After evaporation of dichloromethane, the residue is cleaned up by chromatography on a silica gel column with a mixture of ethanol and dichloromethane. The eluant is removed by evaporation, the residue is dissolved in methanol, and metribuzin is determined by gas chromatography using a thermionic detector.

246

Metribuzin

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Glass filter funnel, e.g. 26D-2 (Schott) Filtration flask, 500-ml Round-bottomed flasks, 1-1, 500-ml and 50-ml, with ground joints Rotary vacuum evaporator, 50 °C bath temperature Ice bath Separatory funnels, 1-1 and 500-ml Glass bottle, 500-ml Laboratory mechanical shaker, 150 r.p.m. Glass funnel Fluted filter paper Chromatographic tube, 25 mm i.d., 35 cm long Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-ul

4 Reagents Acetonitrile, p.a., dist. Dichloromethane, dist. Ethanol, denatured with methyl ethyl ketone Ethyl acetate, dist. Methanol, dist. Petroleum ether, dist., boiling range 40-60°C Eluting mixture: dichloromethane + ethanol 8:2 v/v Dichloromethane + ethyl acetate mixture 9:1 v/v Methanol + water mixture 4:1 v/v Metribuzin standard solution: 5 ng/ml methanol Sodium sulphate, p.a., anhydrous Silica gel 60, 0.063-0.200 mm (Merck No. 7734), heated for 1 h at 100 °C Cottonwool Air, synthetic Helium Hydrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

Metribuzln

247

6 Procedure 6.1 Extraction 6.1.1 Plant material (except barley and wheat)

Weigh 100 g of the analytical sample (G) into the blendor jar, add 200 ml acetonitrile, and homogenize for approx. 2 min. Filter the homogenate with suction through a glass filter funnel. Homogenize the filter cake with 200 ml acetonitrile, and suction-filter the homogenate through the filter funnel. Rinse the blendor jar and the filter funnel with a total of 150 ml acetonitrile, and combine the washings with the filtrate in a 1-1 round-bottomed flask. Carefully rotary-evaporate the combined solutions until they are free of acetonitrile. Then immediately chill the aqueous residue in an ice bath. 6.1.2 Barley, wheat (grains)

Weigh 100 g of the analytical sample (G) into the blendor jar, add 200 ml acetonitrile, and homogenize for approx. 5 min. Filter the homogenate with suction through a glass filter funnel. Homogenize the filter cake with 100 ml acetonitrile, and suction-filter the homogenate through the filter funnel. Rinse the blendor jar and the filter funnel with a total of 150 ml acetonitrile, and combine the washings with the filtrate in a 1-1 separatory funnel. 6.1.3 Wheat (straw) Weigh 10 g of the analytical sample (G) into the blendor jar, add 200 ml acetonitrile, and homogenize for approx. 2 min. Filter the homogenate with suction through a glass filter funnel. Homogenize the filter cake with 200 ml acetonitrile, and suction-filter the homogenate through the filter funnel. Rinse the blendor jar and the filter funnel with a total of 150 ml acetonitrile, and combine the washings with the filtrate in a 1-1 round-bottomed flask. Add 100 ml water, and rotary-evaporate to an aqueous residue. 6.1.4 Soil Weigh 100 g soil (G) into a 500-ml glass bottle, add 200 ml of the methanol-water mixture, tightly stopper the bottle, and shake for 30 min on a mechanical shaker. Decant the supernatant into a 1-1 separatory funnel. Extract the soil sediment with 200 ml acetonitrile for 30 min on the mechanical shaker. Decant the supernatant into the 1-1 separatory funnel, combining it with the first one. Then extract the soil sediment again with 200 ml dichloromethane for 30 min on the mechanical shaker. Decant the supernatant into the separatory funnel, and continue to add water until the phases separate. Shake several times, filter the lower phase through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. Discard the phase left in the separatory funnel. 6.1.5 Water

Extract a water sample of at least 100 ml (G) successively with three equal portions of the dichloromethane-ethyl acetate mixture by shaking in a separatory funnel. Filter the combined lower dichloromethane-ethyl acetate phases through approx. 10 g sodium sulphate, and rotary-evaporate to dryness.

248

Metribuzin

6.2 Liquid-liquid partition 6.2.1 Plant material (except barley and wheat) Filter the chilled aqueous residue derived from 6.1.1 through a fluted filter paper into a 500-ml separatory funnel. Rinse the round-bottomed flask and the fluted filter paper with approx. 100 ml water, and add the washing to the separatory funnel. Extract the combined filtrates successively with 200, 200 and 100-ml portions of dichloromethane. Filter the combined dichloromethane phases through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. 6.2.2 Barley, wheat (grains) Wash the acetonitrile filtrate derived from 6.1.2 twice with 100-ml portions of petroleum ether. Discard the (upper) petroleum ether phases. Add 100 ml water, and rotary-evaporate to an aqueous residue. Extract the residue successively with 150, 100 and 100-ml portions of dichloromethane. Filter the dichloromethane phases through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. 6.2.3 Wheat (straw) Extract the aqueous residue derived from 6.1.3 successively with 150, 100 and 100-ml portions of dichloromethane. Filter the dichloromethane phases through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. 6.3 Column chromatography (plant material, soil) Tamp a plug of cottonwool into the bottom of a chromatographic tube, and half-fill the tube with the eluting mixture. Slurry 10 g silica gel with the eluting mixture into the chromatographic tube, dislodge any trapped air by stirring with a glass rod, and drain the eluting mixture down to the top of the silica gel. Dissolve the residue derived from 6.1.4 or 6.2 in 5 ml of the eluting mixture, and add to the column. Rinse the flask twice with 5-ml portions of the eluting mixture, add the washings separately to the column, allowing each to percolate down to the top of the silica gel layer at a flow rate of approx. 0.7 ml/min. Then elute the column with a further 100 ml of the eluting mixture at the same flow rate until the column has run dry. Rotary-evaporate the eluate to dryness. 6.4 Gas-chromatographic determination Transfer the residue derived from 6.1.5 or 6.3 into a 50-ml round-bottomed flask using methanol as wash. Rotary-evaporate the solution to dryness, and dissolve the residue in 2.0 ml methanol (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature

Hewlett-Packard 5880 A Glass, 2 mm i.d., 1.8 m long; packed with 6% Apiezon L + 3% OV-17 on Chromosorb W-AWDMCS, 80-100 mesh 220 °C 350°C

Metribuzin

Detector Gas flow rates Attenuation Recorder Linearity range Injection volume Retention time for metribuzin

249

Thermionic nitrogen-specific detector Temperature 300 °C Helium carrier, 20-30 ml/min Hydrogen, 1-5 ml/min Air, 40-100 ml/min 25 Chart speed 0.5 inch/min (12.7 mm/min) 5-100 ng 1-3 \i\ approx. 10 min

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for dilutions of the metribuzin standard solution. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. 7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with metribuzin at levels of 0.05 to 0.5 mg/kg. The recoveries are given in the Table, representing the means from 2 to 4 single experiments. The routine limit of determination was 0.05 mg/kg. Table. Percent recoveries from plant material, soil and water, fortified with metribuzin. Analytical material Barley (grains) Broad beans Beans Pods Carrots Potatoes (tubers) Tomatoes Fruits Leaves Wheat Grains Straw Soil Standard soil 2.1 Standard soil 2.2 Standard soil 2.3 Water

Metribuzin added (mg/kg) 0. 05

0.1

0.5

85

83

86

94 75 89 91

96 90 88 89

95 80 82 90

98 98

95 81

86 99

80 80

82 79

85 85

80 80 86 85

80 80 80 89

83 80 84 90

250

Metribuzin

T h e soils used for the recovery experiments h a d the following characteristics:

Organic carbon Soil type

Particles <0.02 mm

%

Standard soil 2.1 *> Standard soil 2.2•> Standard soil 2.3*>

0.31 2.64 1.06

%

8.5 14.5 24.7

„ PH 6.0 6.0 7.0

*> Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

7.3 Calculation of residues The residue R, expressed in mg/kg metribuzin, is calculated from the following equation: p

F A -V End -W st F S t -V r G

where G

= sample weight (in g) or volume (in ml)

V E n d = terminal volume of sample solution from 6.4 (in ml) Vj

= portion of volume V E n d injected into gas chromatograph (in |iil)

W St

= a m o u n t of metribuzin injected with standard solution (in ng)

FA

= peak area obtained from Vj (in m m 2 )

Fst

= peak area obtained from W St (in m m 2 )

8 Important points The entire analytical procedure including the final determinative step should be completed within 2 days to avoid conversion of metribuzin to its metabolites in the extracts. Acetonitrile must be tested for gas-chromatographic purity prior to use. During rotary-evaporation of the extracts, the temperature of the water bath must not exceed 50 °C. Supplementary studies have shown that the following procedure may also be employed for the extraction of soil samples (see step 6.1.4): After extraction of the sample with the methanol-water mixture, acetonitrile and dichloromethane, combine the supernatants in a 1-1 roundbottomed flask, and rotary-evaporate to an aqueous residue. Add 50 ml water, and transfer the solution into a 500-ml separatory funnel. Rinse the round-bottomed flask with a further 50 ml of water, and add the washing to the separatory funnel. Extract the contents three times with dichloromethane (150, 150, 100 ml), shaking the funnel each time. Filter the combined dichloromethane phases through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. Discard the phase left in the separatory funnel. Then proceed to step 6.3.

Metribuzln

251

9 References C. W. Stanley and S. A. Schumann, A gas chromatographic method for the determination of Bay 94337 residues in potatoes, soybeans, and corn, Chemagro Report No. 25838 (1969). E G. von Stryk, Determination of residues of Bay 94337 (4-amino-3-methylthio-6-tert.butyll,2,4-triazin-5-one), J. Chromatogr. 56, 345-348 (1971). J. S. Thornton and C. W. Stanley, Gas chromatographic determination of Sencor and metabolites in crops and soil, J. Agric. Food Chem. 25, 380-386 (1977). H. J. Jarczyk, Methode zur gaschromatographischen Bestimmung von ®Sencor-Ruckstanden in Pflanzenmaterial, Boden und Wasser mit N-spezifischem Detektor, Pflanzenschutz-Nachr. 36, 63-72 (1983).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, H. J. Jarczyk

Nitrothal-isopropyl

416 Gas-chromatographic determination

Apples Soil, water (German version published 1989)

1 Introduction Nitrothal-isopropyl Chemical name

Di-isopropyl 5-nitroisophthalate (IUPAC)

CO-O-CH

Structural formula CO-O-CH

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility

Other properties

C14H17NO6 295.30 65 °C No data <0.1 -10" 6 mbar at 20 °C Very sparingly soluble in water; readily soluble in acetone, benzene, chloroform and ethyl acetate Yellow crystals, steam volatile, hydrolyzed in alkaline media

Metabolite I (Monoester) Chemical name

Isopropyl 5-nitroisophthalate

/

Structural formula

/ CH 3 CO-O-CH

O2N-^J> COOH

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility

C U H U NO 6 253.22 178 °C No data No data No data

254

Nitrothal-isopropyl

Metabolite II (Acid) Chemical name

5-Nitroisophthalic acid /COOH

Structural formula

02NHQ COOH

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility

C8H5NO6 211.13 255 °C No data No data Slightly soluble in water (approx. 2g/100ml at 25 °C); soluble in ethanol and in dilute alkaline solutions

2 Outline of method Nitrothal-isopropyl residues are steam-distilled from the analytical sample in a Bleidner apparatus, simultaneously being extracted from the condensate into isooctane. The metabolites remaining in the aqueous phase are extracted after acidification into tert.butyl methyl ether and methylated with diazomethane. The methylated metabolites are also steam-distilled in the Bleidner apparatus and extracted into isooctane. Nitrothal-isopropyl and its methylated metabolites are determined by electron-capture gas chromatography.

3 Apparatus Round-bottomed flasks, 500-ml and 50-ml, with ground joints Bleidner apparatus, modified by W. Heizler, for distillation and extraction; see Fig. 1, p. 243, Vol. 1 Hotplate with magnetic stirrer, e.g. IKA Combimag RCT (Janke & Kunkel) Aluminium foil Graduated cylinder, 500-ml, with glass stopper Laboratory centrifuge, explosion-proof, with 250-ml glass tubes Water bath, 60 °C temperature Separatory funnel, 500-ml Rotary vacuum evaporator, 20°C, 30°C and 50-60°C bath temperature Methylation apparatus, see Fig. 1, p. 130, Vol. 1 Volumetric flasks, 100-ml, 50-ml and 10-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

N itrothal-isopropy I

255

4 Reagents tert.Butyl methyl ether, dist. 2,2,4-Trimethyl pentane (isooctane), dist. Standard solutions: Mixtures containing 0.2, 0.4 and 0.6 ng/ml each of nitrothal-isopropyl, dimethyl 5-nitroisophthalate and isopropyl methyl 5-nitroisophthalate Compound and metabolite solutions for fortification experiments: Dissolve 10 mg nitrothal-isopropyl, 10 mg metabolite I and 10 mg metabolite II individually in 10 ml methanol. From each of these solutions, pipet 1 ml into a 100-ml volumetric flask and make up to volume with water Diazomethane solution in diethyl ether (for apparatus, see Fig. 1, p. 130, Vol. 1): Dissolve 2 g N-methyl-N-nitroso-p-toluenesulphonamide (Merck No. 808406) in 60 ml diethyl ether and transfer to the dropping funnel. Slowly add this solution dropwise to 10 ml methanolic potassium hydroxide solution contained in the reaction vessel standing in a 60 °C water bath. Distil the generated diazomethane and the diethyl ether through a descending condenser into a receiver which is cooled with an ice + sodium chloride freezing mixture ortho-Phosphoric acid, p.a., 85% (Merck No. 573) Boric acid, p. a. (Merck No. 165) Methanolic potassium hydroxide solution, 10 g/100 ml KOH p. a. in methanol + water mixture 9:1 v/v Sodium chloride solution, saturated Isopropyl 5-nitroisophthalate (metabolite I): Add 4 g nitrothal-isopropyl to 10 ml concentrated sulphuric acid at room temperature and stir (on a magnetic stirrer) until it is dissolved. Add 20 g ice and continue to stir strongly when a white crystalline precipitate containing oily lumps is formed. Add water, and suction-filter the precipitate. Dissolve the precipitate in chloroform and a little acetone and extract the solution with water. With stirring, add sodium hydrogen carbonate solution portionwise to the chloroform phase until the aqueous phase has a pH of 4.5 (pH indicator paper). Shake the mixture vigorously, separate off the organic phase, and extract the aqueous phase with chloroform once more. Wash the combined chloroform phases with water, dry on sodium sulphate, and rotary-evaporate to dryness. Dissolve the residue in a little acetone and dilute the solution with n-hexane to form a precipitate. Suction-filter the precipitate and extract repeatedly with boiling n-hexane to remove unreacted starting material. Dissolve the crystalline residue in acetone, treat with activated charcoal, precipitate with water and suction-filter. Dry over phosphorus pentoxide and paraffin in a desiccator. Yield: approx. 450 mg (m.p. 178°C) Isopropyl methyl 5-nitroisophthalate: Dissolve 200 mg metabolite I in a little diethyl ether and add diazomethane solution until a slight yellow colour remains. Evaporate the solution to dryness. Scratch the remaining oily residue with a glass rod to initiate crystallization, and dry the crystalline mass over phosphorus pentoxide in a desiccator (m.p. 48°C) Dimethyl 5-nitroisophthalate (Aldrich No. 23,736-1) 5-Nitroisophthalic acid (Fluka No. 73450) (metabolite II) Argon + methane mixture 9:1 v/v Helium, pure

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

256

Nitrothal-isopropyl

6 Procedure 6.1 Distillation and extraction (Nitrothal-isopropyl) 6.1.1 Apples, soil Weigh 50 g of the analytical sample (G) into a 500-ml round-bottomed flask and add 2.5 g boric acid, 150 ml water, and a magnetic stirring rod. Half-fill the U-tube of the Bleidner apparatus with water and isooctane, and connect the flask to the lower arm of the Bleidner apparatus. Attach a 50-ml round-bottomed flask containing 25 ml isooctane to the upper arm of the Bleidner apparatus. Wrap both flasks with aluminium foil to exclude light. Heat both flasks for 2 h at a temperature that will ensure condensation of equal amounts of water and isooctane (this can be checked easily from the volumes of the immiscible solvent phases in the capillary of the Bleidner apparatus). On completion of the distillation-extraction step, allow both flasks to cool to room temperature; then proceed to step 6.2.1 for the water phase, and to step 6.4 for the isooctane phase. 6.1.2 Water Transfer 250 ml of the water sample (G) to a 500-ml round-bottomed flask and add 2.5 g boric acid. Attach this flask to the lower arm of the Bleidner apparatus, and a 50-ml round-bottomed flask containing 25 ml isooctane to the upper arm. Proceed as described in 6.1.1.

6.2 Extraction (Metabolites) 6.2.1 Apples, soil Transfer the aqueous phase derived from 6.1.1 into a graduated cylinder and make up to 200 ml with water (VEx). Shake well and centrifuge for 15 min at 3500 r.p.m. Decant an aliquot of 120 ml (VR1) and acidify with 2.5 ml phosphoric acid. Transfer the solution to a separatory funnel and extract twice with tert.butyl methyl ether, shaking successively with 100-ml and 50-ml portions of the solvent. Break any emulsions by centrifuging. Combine the ether extracts in a 500-ml round-bottomed flask and rotary-evaporate to dryness with 30 °C bath temperature. 6.2.2 Water Rotary-evaporate the aqueous phase derived from 6.1.2 to approx. 120 ml with 50-60°C bath temperature. Acidify with 2.5 ml phosphoric acid and extract the solution twice with tert.butyl methyl ether as described in 6.2.1. Rotary-evaporate the ether extracts to dryness with 30 °C bath temperature.

6.3 Methylation (Metabolites) Add 20 ml diazomethane solution to the residue derived from 6.2.1 or 6.2.2, allow to stand for 30 min at room temperature, and rotary-evaporate to a few drops with 20 °C bath temperature.

Nitrothal-isopropyl

257

6.4 Distillation and extraction (Methylated metabolites) To the residue derived from 6.3, add 50 ml water, 50 ml sodium chloride solution and 2.5 g boric acid. Wrap the round-bottomed flask with aluminium foil and attach it to the lower arm of the Bleidner apparatus. Attach the 50-ml round-bottomed flask containing the isooctane extract obtained from 6.1.1 or 6.1.2 (25 ml) to the upper arm of the apparatus. Heat the contents of both flasks to boiling as described in 6.1.1. Allow the isooctane extract to cool to room temperature and make up to a definite volume (VEnd), either by diluting with isooctane or by concentrating on a rotary evaporator with 30 °C bath temperature. 6.5 Gas-chromatographic determination Inject an aliquot (Vj) of the solution derived from 6.4 into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Split ratio Attenuation Recorder Injection volume Retention times for dimethyl 5-nitroisophthalate isopropyl methyl 5-nitroisophthalate nitrothal-isopropyl

Perkin-Elmer 3920 B Glass capillary, 0.28 mm i.d., 25 m long; coated with SE-54, film thickness 0.5 ^m 230 °C 250 °C 63 Ni electron capture detector Temperature 300 °C Helium carrier, inlet pressure 1 bar Argon-methane purge gas, 30 ml/min 1:10 64

1 mV; chart speed 6.7 mm/min lul

2 min 24 s 3 min 9 s 4 min 3 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 1 ul of each standard solution (equivalent to 0.2 to 0.6 ng of each compound) into the gas chromatograph. Plot the heights of the peaks obtained vs. ng of nitrothal-isopropyl, dimethyl 5-nitroisophthalate and isopropyl methyl 5-nitroisophthalate, respectively. Also inject l-|o.l aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of the respective compounds from the corresponding calibration curve.

258

N itrothal-isopropy I

7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with nitrothal-isopropyl and the two metabolites, are given in the Table. The routine limit of determination was 0.1 mg/kg for apples and soil, and 1 u.g/1 for tap water. Table. Percent recoveries for nitrothal-isopropyl, metabolite I and metabolite II from apples and soil, fortified at levels of 0.1 to 1 mg/kg, and from water, fortified at a level of 1 ng/1. Analytical material

n

Apples Soil Tap water

8 8 4

Nitrothal-isopropyl Range Mean 66-83 63-87 57-64

Metabolite I Range Mean

76 75 61

74 85 63

64-83 78-90 55-67

Metabolite II Range Mean 77-80 90-104 109-118

79 97 115

7.3 Calculation of residues The residue R, expressed in mg/kg, of nitrothal-isopropyl and the metabolites (calculated as nitrothal-isopropyl) is calculated from the following equations: for nitrothal-isopropyl

R =

End wA•V •G

Vi

•v E x - V nd v,

WA

for isopropyl methyl 5-nitroisophthalate

for dimethyl 5-nitroisophthalate

E

Rl-Vi •G

WA . y Y R = — ^ — ^ — ^ - • 1.235 VR1 • Vi • G

where G

= sample weight (in g) or volume (in ml)

VEx

= total volume of the water phase from 6.2.1 (in ml)

VR1

= portion of volume VEx used for extraction of metabolites (in ml)

VEnd = terminal volume of sample solution from 6.4 (in ml) ^

= portion of volume VEnd injected into gas chromatograph (in \i\)

WA

= amount of nitrothal-isopropyl, dimethyl 5-nitroisophthalate or isopropyl methyl 5-nitroisophthalate, respectively, for V4 read from calibration curve (in ng)

1.105 = factor for conversion of isopropyl methyl 5-nitroisophthalate to nitrothal-isopropyl 1.235 = factor for conversion of dimethyl 5-nitroisophthalate to nitrothal-isopropyl

Nitrothal-lsopropyl

259

8 Important points The gas-chromatographic determination can also be performed using a packed column under the following conditions: Column Glass, 2.5 mm i.d., 2.6 m long; packed with 1.0% OV-17 + 1.95% OV-210 on Gas Chrom Q, 100-120 mesh Column temperature 200 °C Injection port temperature 250°C 63 Detector Ni electron capture detector Temperature 300 °C Carrier gas flow rate Argon-methane, 40 ml/min Retention times for dimethyl 5-nitroisophthalate 5 min 12 s isopropyl methyl 5-nitroisophthalate 5 min 48 s nitrothal-isopropyl 6 min 48 s The standard solutions as well as the compound and metabolite solutions are stable for at least two weeks when stored in a refrigerator.

9 References No data

10 Authors BASF, Agricultural Research Station, Limburgerhof, W. Keller and P. Beutel

Oxamyl

441

Apples, carrots, celeriac (leaves and bulbs), coffee (raw), cottonseed, grapefruit, grapes, grass, lettuce, oranges, peaches, peanuts (foliage, kernels and shells), potatoes, sweet peppers, tobacco, tomatoes Soil, water

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

N,N-Dimethyl-2-methylcarbamoyloxyimino2-(methylthio)acetamide (IUPAC) H,C

o

o

sf-C-C = N-O-C—NH-CH

Structural formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 25 °C)

Other properties

H,C

S —CH,

219.26 108-110 °C Not distillable without decomposition 3.1-10- 4 mbar at 25 °C 1.0-10- 2 mbar at 70°C Readily soluble in water (28 g); very readily soluble in methanol (144 g); readily soluble in acetone (67 g), ethanol (33 g) and 2-propanol (11 g); slightly soluble in toluene (1 g) Decomposed in alkaline conditions, at elevated temperatures and in ultraviolet light

2 Outline of method Oxamyl residues are extracted from plant material, soil and water with ethyl acetate, partitioned between hexane and water, and cleaned up by extraction with dichloromethane at pH 12 to remove interfering co-extractives. Oxamyl is subjected to alkaline hydrolysis. The resulting, more volatile oximino derivative is determined by gas chromatography using a sulphurspecific flame photometric detector.

262

Oxamyl

X

fi

fi

N—C-C=N-O-C—NHCH3 /

S-CH 3

Oxamyl

|OH-| H 3 C x

°

^N—C-C=N-OH H3C

S-CH 3

Oxamyl oximino derivative

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Centrifuge, 1500 r.p.m., with 250-ml glass tubes Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schull) Vacuum adapter Round-bottomed flasks, 1-1, 500-ml and 100-ml, with ground joints Rotary vacuum evaporator, 35 °C bath temperature Erlenmeyer flask, 250-ml, with ground stopper Laboratory mechanical shaker Separatory funnel, 250-ml Glass funnel, 8 cm dia. Fluted filter paper, 15 cm dia. Water bath, 95 °C temperature Reflux condenser Test tubes, 10-ml, graduated, with ground stoppers Gas chromatograph equipped with sulphur-specific flame photometric detector Microsyringe, 10-ul

4 Reagents Acetone, p. a. Dichloromethane, p. a. Ethyl acetate, p. a. n-Hexane, p. a. Methanol, p. a. Ethyl acetate + methanol mixture 9:1 v/v Derivative standard solutions: 0.1, 0.5, 1.0, 2.0, 3.0, 5.0 and 10.0 ng/ml oxamyl oximino derivative in acetone N,N-Dimethyl-2-hydroxyimino-2-(methylthio)acetamide (oxamyl oximino derivative; DuPont de Nemours) Sodium hydroxide solution, 1 mol/1 NaOH, p. a. Sodium chloride, p. a. Sodium sulphate, p. a., anhydrous Filter aid, e. g. Celite 545 Universal indicator paper

Oxamyl

263

Air, synthetic Helium Hydrogen, re-purified Nitrogen, re-purified Oxygen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material Weigh 25 g of the analytical sample (G) into the blendor jar, add 100 ml ethyl acetate, and homogenize for 5 min. Transfer the homogenate quantitatively into a centrifuge tube and centrifuge at 1500 r.p.m. for 10-15 min. Suction-filter the liquid through a fast flow-rate filter paper covered with 5 g filter aid contained in a Buchner porcelain funnel, and collect in a 1-1 round-bottomed flask. Extract the residue in the centrifuge tube two more times in the same way using ethyl acetate. After the final filtration, wash the filter cake with 50 ml ethyl acetate. Add 50 ml water to the combined filtrates (see Section 8, Important points), and rotary-evaporate to an aqueous residue. 6.1.2 Soil Weigh 25 g of soil (G) into an Erlenmeyer flask, add 100 ml ethyl acetate and 25 ml water, stopper, and shake for 15 min on a mechanical shaker. Suction-filter the extract through a fast flow-rate filter paper contained in a Buchner porcelain funnel, and collect in a 1-1 round-bottomed flask. Repeat the extraction two more times using 100 ml ethyl acetate each time. Wash the filter cake with 50 ml ethyl acetate. Add 50 ml water to the combined filtrates, and rotary-evaporate to an aqueous residue. 6.1.3 Water Place a 50-ml sample of water (G) in a 250-ml separatory funnel, add 100 ml hexane, and shake for 2 min. Allow the phases to separate (centrifuge if necessary to obtain clean separation). Discard the hexane wash. Extract the aqueous phase with three 100-ml portions of ethyl acetate using 2-min shaking periods for each extraction. Allow the phases to separate, and filter the ethyl acetate extract through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask. Wash the sodium sulphate with 50 ml ethyl acetate. Add 50 ml water to the combined filtrates, and rotary-evaporate to an aqueous residue.

264

Oxamyl

6.2 Cleanup Transfer the aqueous residue (approx. 40 ml) derived from 6.1 quantitatively to a 250-ml separatory funnel, washing the 1-1 flask several times with a total of 10 ml water to complete the transfer. Add 50 ml hexane to the separatory funnel, shake gently for 1 min, and allow the phases to separate. Centrifuge, if necessary, to obtain a clean separation. Discard the hexane layer. Repeat the hexane wash two more times using additional 50-ml portions of solvent. Discard the hexane after each wash. Adjust the pH of the aqueous solution to 12 by adding about 3 ml sodium hydroxide solution (check with indicator paper), and then extract with two 50-ml portions of dichloromethane for 1 min each time. Discard the dichloromethane phases, and collect the aqueous phase in a 100-ml round-bottomed flask. 6.3 Hydrolysis Heat the aqueous solution derived from 6.2 on a 95 °C water bath with occasional stirring to remove the residual dichloromethane. Next connect the flask to a reflux condenser, and continue to heat at the same temperature for an additional 15 min to convert oxamyl to the oximino derivative by hydrolysis. Cool and quantitatively transfer the aqueous solution to a 250-ml separatory funnel, washing the flask and the reflux condenser several times with a few ml of water each time to complete the transfer. Extract with two 50-ml portions of dichloromethane, shaking for 1 min each time. Then saturate the aqueous phase by adding about 15 to 25 g of sodium chloride, and extract with four 50-ml portions of ethyl acetate-methanol mixture using 2-min shaking periods for each extraction. Allow the phases to separate. Centrifuge, if necessary, to obtain a clean separation. Dry the combined organic phases on sodium sulphate, and filter through a fluted filter paper into a 500-ml round-bottomed flask. Wash the filter with 50 ml ethyl acetate and rotary-evaporate the filtrate almost to dryness. Remove the residual solvent by swirling the flask in the hand. Transfer the flask contents to a graduated test tube using several acetone washes. Concentrate the solution at room temperature to a given volume (VEnd), using a stream of nitrogen. 6.4 Gas-chromatographic determination Inject an aliquot (Vj) of the solution derived from 6.3 into the gas chromatograph. Operating conditions 6.4.1 Column 1 Gas chromatograph Column Column temperature Injection port temperature Detector

Perkin-Elmer 3920 Glass, 1.6 mm i.d., 0.91 m long; packed with 10% SP1200 + 1% H3PO4 on Chromosorb W-AW, 80-100 mesh Programmed to rise at 16°C/min from 100 to 200 °C, then isothermal at 200 °C for 8 min 230 °C Flame photometric detector (Melpar), equipped with 394-nm sulphur filter Temperature 200 °C

Oxamyl

Gas flow rates

Injection volume Retention time for oxamyl oximino derivative 6.4.2 Column 2 Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Injection volume Retention time for oxamyl oximino derivative

265

Helium carrier, 70 ml/min Hydrogen, 180 ml/min Oxygen, 20 ml/min Air, 40 ml/min 1-3 ul 7 min Per kin-Elmer F 11 Glass, 4 mm i. d., 1.8 m long; packed with 2% Carbowax 20 M + 5% DC-200 on Chromosorb W-AW, 80-100 mesh 205 °C 225 °C Flame photometric detector (Melpar), equipped with 394-nm sulphur filter Temperature 170 °C Nitrogen carrier, 100 ml/min Hydrogen, 150 ml/min Oxygen, 20 ml/min 8-103 1-3 ul 4 min

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject equal volumes of differently concentrated derivative standard solutions into the gas chromatograph. Using log-log paper, plot the heights of the peaks obtained vs. the amounts of oxamyl oximino derivative injected with the standard solutions. Also inject equal volumes of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of oxamyl oximino derivative from the calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with oxamyl at levels of 0.02 to 10 mg/kg, ranged from 70 to 120% and averaged 90%. The routine limit of determination was 0.02 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg oxamyl, is calculated from the following equation:

266

Oxamyl R

=

W

A/^°" Yj • G

. j.352

where G VEnd Vj WA 1.352

= = = = =

sample weight (in g) or volume (in ml) terminal volume of sample solution from 6.3 (in ml) portion of volume VEnd injected into gas chromatograph (in jxl) amount of oxamyl oximino derivative for V{ read from calibration curve (in ng) factor for conversion of oxamyl oximino derivative to oxamyl

8 Important points The determination of oxamyl residues is based on the gas-chromatographic measurement of the hydrolysis product because the oxamyl oximino derivative responds with considerably greater sensitivity to flame photometric detection. During the analysis of highly acidic substrates, e.g. oranges, grapefruit and peaches, somewhat lower recoveries are noted. To improve the recovery rates, the acid must be neutralized. Therefore, prior to evaporation, 50 ml of sodium hydroxide solution is added, instead of 50 ml of water, to the combined filtrates derived from 6.1.1. During studies to validate the method in the Chemistry Division of the Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, it was found that the following gas-chromatographic conditions are also suitable: Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder Injection volume Retention time for oxamyl oximino derivative

Carlo Erba Fractovap 2101 AC Glass, 2 mm i.d., 1.5 m long; packed with 10% DC200 on Chromosorb W-HP, 100-120 mesh 200 °C 275 °C Flame photometric detector (Erba Science, SSD 250), equipped with 394-nm sulphur filter Temperature 200 °C Nitrogen carrier, 37 ml/min Hydrogen, 150 ml/min Air, 200 ml/min 64 10 mV; chart speed 5 mm/min 5—7 pil 1 min 36 s

Oxamyl

267

9 Reference R.E Holt and H.L. Pease, Determination of oxamyl residues using flame photometric gas chromatography, J. Agric. Food Chem. 24, 263-266 (1976).

10 Authors DuPont de Nemours & Co., Wilmington, DE, U.S.A., R.E Holt and H.L. Pease

Phenmedipham

233-B

Fodder beet (foliage and root), mangold, red beet (edible root), strawberries, sugar beet (foliage and edible root), witloof chicory

Gas-chromatographic determination

(German version published 1974)

1 Introduction Chemical name

Methyl 3-(3-methylcarbaniloyloxy)carbanilate (IUPAC)

Structural formula

CH.O-C-NH-

Empirical formula Molar mass Melting point Vapour pressure Solubility (in 100 ml at room temperature)

Other properties

C16H16N2O4 300.32 143-144°C <10~ 8 mbar at 25 °C Virtually insoluble in water; readily soluble in acetone and cyclohexanone (each approx. 20 g), soluble in methanol (approx. 5 g), slightly soluble in chloroform (approx. 2 g), sparingly soluble in benzene (approx. 0.25 g), very sparingly soluble in n-hexane (approx. 50 mg) Largely stable in acid media, rapidly hydrolyzed in alkaline media

2 Outline of method Phenmedipham residues are alkali hydrolyzed in the presence of the analytical material under reflux conditions in a Bleidner apparatus. The resultant 3-methylaniline is simultaneously steam-distilled and extracted from the condensate into isooctane. Following re-extraction with hydrochloric acid, 3-methylaniline is brominated to yield 2,4,6-tribromo-3-methylaniline, which is extracted into toluene and determined by electron-capture gas chromatography.

3 Apparatus High-speed blendor Pyrex round-bottomed flasks, 2-1, 1-1 and 500-ml, with ground joints Round-bottomed flask, 250-ml, with ground joint

270

Phenmedipham

Bleidner apparatus, modified by W. Heizler, for hydrolysis, distillation and extraction; see Fig. 1, p. 243, Vol. 1 Heating mantles, for 2-1, 1-1, 500-ml and 250-ml round-bottomed flasks Erlenmeyer flasks, 50-ml, with ground stoppers Gas chromatograph equipped with electron capture detector Microsyringes, 10-ul and 1-ul

4 Reagents 2,2,4-Trimethyl pentane (isooctane), dist. Toluene, for gas chromatography Derivative standard solution: 1 ng/ml 2,4,6-tribromo-3-methylaniline in toluene Hydrochloric acid, 1 mol/1 HC1 p.a. Sodium hydroxide solution, 2.5 mol/1 and 5 mol/1 Potassium bromate solution, 0.2 g/100 ml KBrO3 2,4,6-Tribromo-3-methylaniline, melting point 98.5-99.5 °C: Prepare from 3-methylaniline, boiling point 203°C, by bromination with a potassium bromate-potassium bromide mixture in hydrochloric acid (1 mol/1) 2,4,6-Trimethylaniline solution, 0.2 g/100 ml in hydrochloric acid (1 mol/1): Prepare from 2,4,6-trimethylaniline purum (Fluka), additionally purified by chromatography on aluminium oxide, basic, activity grade I (e.g. Camag), until the following criterion is met: Dissolve 200 mg 2,4,6-trimethylaniline in 100 ml hydrochloric acid (1 mol/1), brominate 10 ml of this solution as described in 6.2, partition into 5 ml toluene, and inject 2 ul of the toluene solution into the gas chromatograph. At the retention time of 2,4,6-tribromo-3-methylaniline (the "phenmedipham position"), the chromatogram should not exhibit an interference peak exceeding the equivalent of 0.2 ng 2,4,6-tribromo-3-methylaniline Potassium bromide, p.a. Sodium sulphite, p.a. Antifoam liquid, e.g. Antifoam A Argon + methane mixture 95 :5 v/v

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol 1.

6 Procedure 6.1 Hydrolysis, distillation and extraction Weigh 50 g of the comminuted analytical sample (G) into a 500-ml, 1-1 or 2-1 Pyrex round-bottomed flask (choose capacity of flask according to the proneness of the sample material to foaming). Then add 250, 500 or 1000 ml sodium hydroxide solution and 1-4 ml Antifoam. Place the flask in a heating mantle, half-fill the U-tube of the Bleidner apparatus with water

Phenmedipham

271

and isooctane, and connect the flask to the lower arm of the Bleidner apparatus. Attach a 250-ml round-bottomed flask filled with 100 ml isooctane to the upper arm of the Bleidner apparatus. Heat both flasks for 2V2 h at a temperature that will ensure condensation of equal amounts of water and isooctane; this can be checked easily from the volumes of the immiscible solvent phases in the capillary of the Bleidner apparatus. On completion of the distillation-extraction step, let the isooctane cool to room temperature. Next extract the isooctane solution successively with 10-ml, 5-ml, and 5-ml portions of hydrochloric acid and combine the acid extracts in a 50-ml Erlenmeyer flask. 6.2 Bromination Weigh 12 g potassium bromide into a 50-ml Erlenmeyer flask and add 0.5 ml 2,4,6trimethylaniline solution. Transfer the solution derived from 6.1 into the flask, rinsing with 3 ml water to complete the transfer. Stopper the flask and vigorously shake for 5 min. Add 0.5 ml potassium bromate solution, whereby the reaction mixture will become yellow. Should decolourization occur, add a further 0.5 ml of potassium bromate solution. Allow the solution to stand for 30 min; then stop the reaction by adding approx. 100 mg sodium sulphite until decolourization is complete. Next, alkalize the mixture with 6 ml sodium hydroxide solution (5 mol/1), add 5.0 ml toluene (VEnd), and vigorously shake for 1 min. Pipet off the toluene layer after it has fully separated. 6.3 Gas-chromatographic determination Inject an aliquot (Vj) of the solution derived from 6.2 into the gas chromatograph. Before starting the analysis, saturate the gas-chromatographic column with 2,4,6-tribromo-3-methylaniline by multiple injections of 10 ul each of the derivative standard solution. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Injection volume Retention time for 2,4,6tribromo-3-methylaniline

Hewlett-Packard F-M 400 Glass, 3 mm i.d., 1.6 m long; packed with 10% PESE-30 (NPGA terminated) on Chromosorb G, 80-100 mesh 180 °C 235 °C 63 Ni electron capture detector, pulse interval 150 \is Temperature 330 °C Argon-methane carrier, 120 ml/min Argon-methane purge gas, 120 ml/min 2 ul approx. 17 min (see also 8. Important points)

272

Phenmedipham

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject 1 to 10 |il of the derivative standard solution (equivalent to 1 to 10 ng 2,4,6-tribromo3-methylaniline) into the gas chromatograph. Plot the areas of the peaks obtained vs. ng 2,4,6tribromo-3-methylaniline. Also inject 2-ul aliquots of the sample solutions. For the areas of the peaks obtained for these solutions, read the appropriate amounts of 2,4,6-tribromo3-methylaniline from the calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with phenmedipham at levels of 0.05 to 0.3 mg/kg, were as follows: Analytical material

Phenmedipham added (mg/kg)

Fodder beet Foliage Root Red beet, edible root Strawberries Sugar beet Foliage Edible root

Recovery °/o

0.05-0.3 0.05-0.3 0.05-0.3 0.05-0.1

12 19 18 4

103 ± 16 98 ± 9 97 ± 16 85 ± 2

0.1 -0.3 0.05-0.1

16 13

86 ± 14 94 ± 9

The routine limit of determination was approx. 0.05 mg/kg. Blanks varied between 0.005 and 0.03 mg/kg; higher blank values are mostly due to laboratory contamination. 7.3 Calculation of residues The residue R, expressed in mg/kg phenmedipham, is calculated from the following equation: R =

WA VEnd

' VrG

• 0.87

where G

= sample weight (in g)

VEnd = volume of toluene used for extraction of the derivative in 6.2 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WA = amount of derivative, 2,4,6-tribromo-3-methylaniline, for Vj read from calibration curve (in ng) 0.87 = factor for conversion of 2,4,6-tribromo-3-methylaniline to phenmedipham

Phenmedipham

273

8 Important points The addition of 2,4,6-trimethylaniline to the brominating mixture ensures complete bromination of the 3-methylaniline derived from phenmedipham, even at very low concentrations, and also the formation of just one single bromination product. The calibration can therefore be based on 2,4,6-tribromo-3-methylaniline. Following bromination, the extracts of nearly all plant materials produce a most prominent gas-chromatographic peak originating from 2-amino-3,5-dibromo-acetophenone (relative retention time = 0.77, related to 2,4,6-tribromo-3-methylaniline). No interference resulted from the aromatic amines derived from a great number of other herbicides such as barban, chloridazon, chlorpropham, fenuron, monalide, monuron and propham. However, the brominated derivative of 3,4-dichloroaniline as derived from diuron, linuron, neburon and propanil will not be separated satisfactorily from 2,4,6-tribromo3-methylaniline under the gas-chromatographic conditions given in this method.

9 References W.E. Bleidner, Application of chromatography in determination of micro quantities of 3-(p-chlorophenyl)-l,l-dimethylurea, J. Agric. Food Chem. 2, 682-684 (1954). H. Geissbuhler and H. Schredt, Comparison of different procedures used for residue determination of urea herbicides, Weed Research 7, 168-170 (1967). K. Kofimann, Methoden zur Riickstandsbestimmung von Phenmedipham in Pflanzenmaterial, Weed Research 70, 340-348 (1970).

10 Author Schering AG, Berlin, K. Kofimann

Propachlor

310

Cauliflower, head cabbage, leeks, maize (kernels), onions (bulbs), peas, radishes, sugar beet (edible root) Soil, water

Gas-chromatographic determination

(German version published 1987)

1 Introduction 2-Chloro-N-isopropylacetanilide (IUPAC) Chemical name

o II

C-CH2C1 V

Structural formula

CH-CH 3 CH3

Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

CUH14C1NO 211.69 77 °C 110°C at 0.04 mbar 3-10" 4 mbar at 25 °C Very sparingly soluble in water; readily soluble in acetone (31 g), benzene (50 g), carbon tetrachloride (14.8 g), chloroform (38 g), diethyl ether (17.9 g), ethanol (29 g) and xylene (19.3 g); slightly soluble in n-hexane (1.1 g) Decomposed in alkaline and strongly acid media

2 Outline of method Propachlor residues are extracted from plant material and soil with acetone, from water samples with dichloromethane. Extracts from plant material, with the exception of maize extracts, are cleaned up by acid precipitation, followed by partitioning of propachlor from the aqueous residue of the filtrate into a petroleum ether-dichloromethane mixture. The extract is further cleaned up by column chromatography on Florisil. Soil extracts are cleaned up by liquid-liquid partition. Propachlor is determined by gas chromatography using a thermionic or an electron capture detector.

3 Apparatus High-speed blendor fitted with leak-proof steel jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., medium flow rate

276

Propachlor

Vacuum adapter Round-bottomed flasks, 1-1, 500-ml and 250-ml, with ground joints Rotary vacuum evaporator, 30-35 °C bath temperature Erlenmeyer flask, 500-ml, with ground joint Laboratory mechanical shaker Separatory funnels, 2-1, 1-1 and 250-ml Glass funnel, 10 cm dia. Fluted filter paper, 18.5 cm dia. (Schleicher & Schull) Chromatographic tube, 18 mm i.d., 40 cm long Test tubes, 10-ml and 5-ml, graduated, with ground stoppers Gas chromatograph equipped with thermionic nitrogen-specific detector or with electron capture detector Microsyringe, 10-ul

4 Reagents Acetone, high purity, dist. Dichloromethane, high purity, dist. n-Hexane, high purity, dist. Methanol, high purity, dist. Petroleum ether, boiling range 40-60 °C, dist. Eluting mixture 1: n-hexane + acetone 95 :5 v/v Eluting mixture 2: n-hexane + acetone 8:2 v/v Petroleum ether + dichloromethane mixture 8:2 v/v Propachlor standard solutions: 0.01-10 jag/ml n-hexane Hydrochloric acid, 0.1 and 1.0 mol/1 HC1 p.a. Sodium chloride, p.a. Sodium sulphate, p.a., anhydrous Filter aid, e.g. Celite 545 Florisil, 60-100 mesh, heated for 8 h at 130 °C Cottonwool, exhaustively extracted with dichloromethane Air, synthetic Helium Hydrogen, re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

Propachlor

277

6 Procedure 6.1 Extraction 6.1.1 Plant material (except maize)

Homogenize 100 g of the analytical sample (G) with 200 ml acetone and 10 g filter aid for 3 min in the blendor. Suction-filter the homogenate through a filter paper in a Buchner porcelain funnel into a 1-1 round bottomed flask, and wash the filter cake with 100 ml acetone. Rotary-evaporate the filtrate to an aqueous residue. 6.1.2 Maize (kernels)

Grind 50 g maize kernels (G) portionwise in the blendor, then add 200 ml acetone and homogenize for 3 min. Suction-filter the homogenate through a filter paper in a Buchner porcelain funnel containing a layer of filter aid into a 1-1 round-bottomed flask. Wash the filter cake with 100 ml acetone, and rotary-evaporate the filtrate to near dryness. Remove the last traces of solvent by swirling the flask in the hand, and proceed as described in 6.2.3. 6.1.3 Soil Extract 100 g of the soil sample (G) with 30 ml water and 200 ml acetone on the mechanical shaker for 1 h. Suction-filter the extract through a filter paper in a Buchner porcelain funnel into a 1-1 round-bottomed flask, wash the filter cake with 100 ml acetone, and rotary-evaporate the filtrate to an aqueous residue. Proceed as described in 6.2.2. 6.1.4 Water

Add 10 g sodium chloride to 1 1 of the water sample (G) and extract three times with 100-ml portions of dichloromethane. Dry the combined extracts for 30 min on sodium sulphate, filter through a fluted filter paper into a 500-ml round-bottomed flask, and wash the filter with approx. 20 ml dichloromethane. Rotary-evaporate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. Proceed as described in 6.2.3. With water containing only low amounts of organic matter, proceed directly to the gas-chromatographic analysis, as described in 6.3, without further cleanup. 6.2 Cleanup 6.2.1 Acid precipitation To the aqueous residue derived from 6.1.1, add 100 ml water, 100 ml methanol, and 10 ml hydrochloric acid (1 mol/1). Allow the mixture to stand for 10 min for complete precipitation, then suction-filter through a filter paper in a Buchner porcelain funnel containing a layer of filter aid. Wash the filter three times with 20-ml portions of hydrochloric acid (0.1 mol/1).

278

Propachlor

6.2.2 Liquid-liquid partition

Quantitatively transfer the aqueous residue derived from 6.1.3 or the filtrate derived from 6.2.1 into a separatory funnel, add 10 g sodium chloride and shake three times with 100-ml portions of the petroleum ether-dichloromethane mixture. Dry the combined extracts on sodium sulphate, filter through a fluted filter paper into a 500-ml round-bottomed flask, and wash the filter with 30 ml petroleum ether-dichloromethane mixture. Rotary-evaporate the filtrate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. Proceed as described in 6.2.3. For soil extracts, the following column-chromatographic cleanup can usually be omitted. 6.2.3 Column chromatography

Plug the bottom end of the chromatographic tube with cottonwool, and fill about one third of the tube with hexane. Slurry 15 g Florisil with hexane, and pour the slurry into the tube whilst gently tapping the tube walls. After the adsorbent has settled, cover it with an approx. 1-cm layer of sodium sulphate. Drain the hexane until the sodium sulphate layer is just covered with solvent. Next dissolve the residue derived from 6.1.2, 6.1.4, or 6.2.2 in 5 ml eluting mixture 1. Quantitatively transfer the solution onto the column, allow to percolate at a rate of 1-2 drops per s, and rinse the top of the column with approx. 2 ml eluting mixture 1. Elute co-extractives with a total of 70 ml eluting mixture 1, and discard the eluate. Then elute propachlor with 70 ml eluting mixture 2. Collect the eluate in a 250-ml round-bottomed flask, rotary-evaporate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. 6.3 Gas-chromatographic determination Dissolve the residue derived from 6.1.4, 6.2.2, or 6.2.3 in hexane and make up to a definite volume (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. All extracts are measured using a thermionic detector; extracts from water samples can also be measured using an electron capture detector. Operating conditions 6.3.1 Packed column Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder

Carlo Erba Fractovap 2150 Glass, 2 mm i.d., 60 cm long; packed with 5% Carbowax 20M TPA on Gas Chrom Q, 100-120 mesh 200 °C 275 °C Thermionic nitrogen-specific detector (Carlo Erba PN Detector 793) Temperature 320 °C Nitrogen carrier, 30 ml/min Hydrogen, 3 ml/min Air, 200 ml/min 1-32 1 mV; chart speed 5 mm/min

Propachlor

Linearity range Injection volume Retention time for propachlor Parathion had a retention time of 6.3.2 Capillary column Gas chromatograph Column Column temperature Injection technique Detector

Gas flow rates Attenuation Recorder Linearity range Injection volume Retention time for propachlor

279

0.3-75 ng 3 ul 3 min 15 s 20 min using these conditions. Carlo Erba Fractovap 4160 with on-column injector Fused silica capillary, 0.32 mm i.d., 15 m long; coated with OV-1, crossbond, film thickness 0.10-0.15 \xm (Carlo Erba Mega) 60 to 120 °C ballistically, programmed to rise at 2°C/min from 120 to 140 °C Cold on-column at 60°C oven temperature, with secondary cooling 63 Ni electron capture detector ECD HT-25, ECD Control Module 251, constant current, pulse width 1 us (Carlo Erba) Temperature 290 °C Helium carrier, 3 ml/min Nitrogen detector purge gas, 50 ml/min 512 10 mV; chart speed 10 mm/min 0.01-0.2 ng 1 Hi 5 min 6 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject equal volumes of differently concentrated propachlor standard solutions into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng propachlor. Also inject equal volumes of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of propachlor from the calibration curve. 7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples of plant material and soil, fortified with propachlor at levels of 0.02 to 1.0 mg/kg, averaged 89% with a relative standard deviation of 11%. Blanks were usually less than 0.005 mg/kg; for head cabbage, radishes and leeks, however, they could be as high as 0.02 mg/kg. The limit of determination was between 0.02 and 0.05 mg/kg, depending on the sample material; with head cabbage it was at 0.1 mg/kg. The limit of detection was 0.01 mg/kg for most materials. The recoveries from water samples (tap water), fortified with propachlor at levels of 0.001 and 0.1 mg/1, averaged 107% with a relative standard deviation of 18%. The limit of determi-

280

Propachlor

nation was 1 ng/1; the limit of detection was 0.1 \ig/\ (electron capture detector) or 0.5 ng/1 (thermionic detector). Blanks did not exceed 0.03 jig/l. 7.3 Calculation of residues The residue R, expressed in mg/kg propachlor, is calculated from the following equation: R = where G = VEnd = Vj = WA =

WA'V,End V.-G

sample weight (in g) or volume (in ml) terminal volume of sample solution from 6.3 (in ml) portion of volume VEnd injected into gas chromatograph (in \il) amount of propachlor for Vj read from calibration curve (in ng)

8 Important points The use of an electron capture detector for determinations in plant material usually results in high blanks. The electron capture detector is, therefore, only recommended for the analysis of water samples.

9 References J. W. Worley, M.L. Rueppel and EL. Rupel, Determination of a-chloroacetanilides in water by gas chromatography and infrared spectrometry, Anal. Chem. 52, 1845-1849 (1980). A. Ambrus, J. Lantos, E. Visi, I. Csatlos and L. Sdrvdri, General method for determination of pesticide residues in samples of plant origin, soil, and water, I. Extraction and cleanup, J. Assoc. Off. Anal. Chem. 64, 733-742 (1981). A. Ambrus, E. Visi, F. Zakar, E. Hargitai, L. Szabo and A. Pdpa, General method for determination of pesticide residues in samples of plant origin, soil, and water, III. Gas chromatographic analysis and confirmation, J. Assoc. Off. Anal. Chem. 64, 749-768 (1981).

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, J. Siebers and M Blacha-Puller

Propiconazole

624

Barley, rye, wheat (respectively green matter, grains and straw), grapes, wine Soil, water

Gas-chromatographic determination

(German version published 1985)

1 Introduction Chemical name

(± )-l-[2-(2,4-Dichlorophenyl)-4-propyl-l,3-dioxolan2-ylmethyl]-lH-l,2,4-triazole (IUPAC) ci

H

Structural formula Empirical formula Molar mass Boiling point Vapour pressure Solubility (in 100 ml at 20 °C)

Other properties

C15H17C12N3O2 342.22 180 °C at 0.13 mbar 1.3- 10~6 mbar at 20°C Very sparingly soluble in water; miscible with acetone, dichloromethane, methanol, 2-propanol and toluene; soluble in n-hexane (6 g) Very weak base (pKa < 1); thermally stable at up to more than 320 °C

2 Outline of method Propiconazole residues are extracted from cereal green matter and grapes with methanol, and from grains, straw and soil with a mixture of methanol and water. The filtered extract is diluted with water and saturated sodium chloride solution, and propiconazole is partitioned into dichloromethane. Wine and water are diluted with saturated sodium chloride solution, and extracted with dichloromethane. The dichloromethane extracts are rotary-evaporated to dryness. The residue is cleaned up by column chromatography on aluminium oxide. Samples of straw must be additionally cleaned up beforehand by gel permeation chromatography. Propiconazole is determined by gas chromatography using a thermionic detector.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Homogenizer Beater-cross mill

282

Propiconazole

Wide neck bottle, 500-ml, with ground stopper Laboratory mechanical shaker Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., e.g. Macherey-Nagel No. 713 Filtration flask, 1-1 Separatory funnel, 1-1 Round-bottomed flasks, 300-ml, 100-ml and 25-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Test tubes, 10-ml, with ground stoppers Glass syringe, 10-ml, with Luer-lock fitting Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Chromatographic tube, 20 mm i.d., 30 cm long Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-ul

4 Reagents Cyclohexane, p.a. Dichloromethane, p.a. Ethanol, p.a. Ethyl acetate, p.a. n-Hexane, p.a. Methanol, high purity Toluene, p.a. Cyclohexane + ethyl acetate mixture 1:1 v/v Eluting mixture 1: dichloromethane + n-hexane 4:6 v/v Eluting mixture 2: dichloromethane + n-hexane 6:4 v/v Ethanol + n-hexane mixture 1:1 v/v Methanol + water mixture 8:2 v/v Propiconazole standard solutions: 0.25, 0.5, 1.0 and 10.0 ng/ml ethanol-hexane mixture Sodium chloride solution, saturated Aluminium oxide, activity grade V: To 100 g Alumina Woelm B Super I (ICN Biomedicals) in a 300-ml Erlenmeyer flask (with ground joint), add 19 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories, No. 152-2750) Dry ice Cottonwool Compressed air, dried and re-purified Hydrogen, re-purified Nitrogen, re-purified

Propiconazole

283

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Plant material is comminuted and homogenized. Straw is chopped and then ground together with dry ice in a beater-cross mill. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Green plant matter, grains, straw, grapes, soil

Weigh 50 g grapes, 50 g milled grains, 20 g milled straw or 10 g of the other homogenized analytical material (G) into a wide neck bottle. Add 200 ml methanol or 200 ml methanol-water mixture for samples of grains, straw and soil. Tightly stopper the bottle, and shake for 1 h on a mechanical shaker. Suction-filter through a Buchner porcelain funnel, and wash the filter cake with two 25-ml portions of methanol. Transfer the filtrates to a separatory funnel. Add 200 ml water and 50 ml sodium chloride solution, and extract three times with 75-ml portions of dichloromethane. Filter the dichloromethane phases through a cottonwool plug into a 300ml round-bottomed flask, and rotary-evaporate to dryness. Discard the water phase. Proceed to step 6.2.1 for cleanup of the residue from straw and to step 6.2.2 for cleanup of the residues from all other materials. 6.1.2 Wine

Dilute 100 g wine (G) with 100 ml water and 50 ml sodium chloride solution in a separatory funnel, and extract successively with three 75-ml portions of dichloromethane. Filter the dichloromethane phases through a cottonwool plug into a 300-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to step 6.2.2. 6.1.3 Water

Place 500 ml water (G) in a separatory funnel, add 50 ml sodium chloride solution, and extract successively with three 75-ml portions of dichloromethane. Filter the dichloromethane phases through a cottonwool plug into a 300-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to step 6.2.2. 6.2 Cleanup 6.2.1 Gel permeation chromatography (only for straw) Transfer the residue derived from 6.1.1 into a test tube, using a total of 10 ml cyclohexane-ethyl acetate mixture (VEx) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop of the gel permeation chromatograph (VR1) with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with a standard

284

Propiconazole

solution of propiconazole; cf. Cleanup Method 6, pp. 75 ff, Vol. 1 — Elution volumes ranging from 110 to 150 ml were determined for propiconazole on Bio-Beads S-X3 polystyrene gel using the cyclohexane-ethyl acetate mixture as eluant, pumped at a flow rate of 2.5 ml/min. Collect the 110 to 150-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to step 6.2.2. Check the elution range from time to time, and determine anew whenever a new gel column is used. 6.2.2 Column chromatography

Pour 15 ml hexane into the chromatographic tube. Slowly add 30 g aluminium oxide (free from air bubbles). Allow to settle, and then drain the hexane to the top of the column packing. Transfer the residue derived from 6.1.1, 6.1.2, 6.1.3 or 6.2.1 to the column, using three 2-ml portions of toluene to complete the transfer. Drain the toluene to the top of the column packing each time. Elute co-extractives with 50 ml of eluting mixture 1, and then elute propiconazole with 75 ml of eluting mixture 2, using a flow rate of 1 to 2 drops per s. Collect the eluate in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. 6.3 Gas-chromatographic determination Dissolve the residue derived from 6.2.2 in 2 ml ethanol-hexane mixture, and dilute to a suitable volume (VEnd). Inject an aliquot of this solution (V{) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Attenuation Recorder Injection volume Retention time for propiconazole

Hewlett-Packard 5710A/30A Glass, 2 mm i.d., 1.5 m long; packed with 3% CP Wax 40 M on Gas Chrom Q, 80-100 mesh (Chrompack) 245 °C 250 °C Thermionic nitrogen-specific detector Temperature 250 °C Nitrogen carrier, 36 ml/min Hydrogen, 3 ml/min Air, 50 ml/min 16 1 mV; chart speed 10 mm/min 2 ul 2 min 20 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject 2 \x\ of each propiconazole standard solution (equivalent to 0.5 to 20 ng propiconazole) into the gas chromatograph. Plot the heights of the peaks obtained vs. ng propiconazole. Also

Propiconazole

285

inject 2-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of propiconazole from the calibration curve. Prepare a new calibration curve for each sample series.

7.2 Recoveries and lowest determined concentration Recovery experiments were run on different untreated control samples of plant material, wine, soil and water, fortified with propiconazole at levels of 0.002 to 1.0 mg/kg. The recoveries are given in the Table; the values presented in this Table represent the means (± standard deviations) from 3 to 13 single experiments. Table. Percent recoveries from plant material, wine, soil and water, fortified with propiconazole. . . . . . , Analytical material

Added

Recovery n

*

s

0.1 1.0 0.04 0.4 0.1 0.5

6 8 5 6 10 6

98 101 101 87 98 90

11 9 13 5 11 6

Grapes

0.04 0.4

13 13

96 89

10 5

Wine

0.02 -0.4

8

90

10

Soil

0.04 -0.4

4

99

7

0.002 0.02

3 3

111 101

6 4

Cereals Green matter Grains Straw

Water

mg/kg

The routine limit of determination was 0.05 mg/kg for cereal green matter, straw and soil, 0.005 mg/kg for wine, 0.01 mg/kg for grapes and grains, and 1 jj.g/1 for water. 7.3 Calculation of residues The residue R, expressed in mg/kg propiconazole, is calculated from the following equation: £ _ WA • VEx ^VEnd V w -V,-G where G = sample weight (in g) or volume (in ml) VEx = volume of solution prepared for gel permeation chromatography in 6.2.1 (in ml)

286

Propiconazole

VR1 = portion of volume VEx injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) Vj = portion of volume VEnd injected into gas chromatograph (in ul) WA = amount of propiconazole for Vj read from calibration curve (in ng)

8 Important points For the analysis of extracts from water samples containing only small amounts of coextractives, cleanup by column chromatography can be omitted. Gel permeation chromatography can be performed also by the method described on pp. 65 ff, Vol. 1 (Cleanup Method 4).

9 Reference B. Buttler, Gas chromatographic determination of propiconazole and etaconazole in plant material, soil, and water, J. Agric. Food Chem. 31, 762-765 (1983).

10 Authors Ciba-Geigy AG, Agricultural Division, Basle, Switzerland, B. Buttler and W. D. Hormann

Sulphur

184-B

Apples, cucumbers, grapes, hops (foliage and cones), strawberries Soil

High-performance liquid chromatographic determination

(German version published 1985)

1 Introduction Chemical name Empirical formula Molar mass Melting point Boiling point Solubility

Other properties

Sulphur S8 256.51 112.8 °C (a-sulphur) 444.67 °C Insoluble in water; readily soluble in carbon disulphide (42.4 g/100 ml at 20 °C); soluble in carbon tetrachloride; sparingly soluble in commonly used organic solvents Solutions with low content of sulphur are sensitive to light

2 Outline of method Sulphur residues are extracted from the sample material with a mixture of water, methanol and dichloromethane. An aliquot of the separated dichloromethane phase is rotary-evaporated to dryness, and taken up in a mixture of isooctane and isopropanol. The extract is cleaned up by column chromatography on silica gel. Sulphur is determined by high-performance liquid chromatography using a UV detector.

3 Apparatus Erlenmeyer flasks, 500-ml and 250-ml Laboratory mechanical shaker Graduated cylinders, 250-ml, 100-ml and 25-ml Glass funnels, 10 cm and 4 cm dia. Filter paper, 10 cm dia., Whatman 1 PS Rotary vacuum evaporator, 40 °C bath temperature Chromatographic tube, 20 mm i. d., 20 cm long Round-bottomed flasks, 100-ml, with ground joints

288

Sulphur

Ultrasonic bath Volumetric flasks, 5-ml High-performance liquid chromatograph equipped with UV detector for measurement at 260 nm Microsyringe, 100-ul

4 Reagents Dichloromethane, dist. Methanol, dist. 2-Propanol (isopropanol), dist. 2,2,4-Trimethyl pentane (isooctane), dist. Solvent mixture 1: isooctane + isopropanol 8 :2 v/v Solvent mixture 2: dichloromethane + methanol 5 :95 v/v Eluting mixture: isooctane + isopropanol 97 : 3 v/v Sulphur standard solutions: 1.0, 2.0, 5.0 and 10.0 ng/ml solvent mixture 2 Sulphur solutions for recovery experiments: 10 and 100 jig/ml dichloromethane Silica gel 60, 0.05-0.2 mm (Macherey-Nagel No. 81532) Cottonwool

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1.

6 Procedure 6.1 Extraction Weigh 50 g of the analytical sample (5 g for hops) (G) into a 500-ml Erlenmeyer flask, add 25 ml water, 25 ml methanol and 150 ml dichloromethane, stopper the flask tightly, and shake for 1 h on a mechanical shaker. Filter the flask contents through a filter paper in a funnel, and collect in a 250-ml Erlenmeyer flask. The separated dichloromethane phase contains the whole of the sulphur residue (VEx = 150 ml). Take a 75-ml aliquot (VR1) of the dichloromethane phase, and rotary-evaporate to dryness. Dissolve the residue in 2.0 ml of solvent mixture 1. 6.2 Column chromatography Pour 50 ml isooctane into the chromatographic tube, and slurry in 15 g silica gel. Drain the solvent to the top of the silica gel layer. Then add the solution derived from 6.1 to the column. Wash with 3 ml of eluting mixture, and allow the solution to trickle into the column. Next elute the sulphur with the same mixture. From the time of addition, collect a forecut of 25 ml

Sulphur

289

(0-25 ml), and discard. Collect the main eluate of 30 ml (25-55 ml) which contains the sulphur, in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Dissolve the residue in 4 ml of solvent mixture 2; if necessary, use an ultrasonic bath. Filter the solution through a cottonwool pad placed in a small funnel, collect in a 5-ml volumetric flask, rinse with 1 ml of solvent mixture 2, and fill up to the mark with the same solvent mixture (VEnd). 6.3 High-performance liquid chromatographic determination Inject 100 ul (Vj) of the solution derived from 6.2 into the high-performance liquid chromatograph. Operating conditions Pump Injector Column Mobile phase Flow rate Temperature Detector Recorder Injection volume Retention time for sulphur

Orlita No. AE-10-4-4 fitted with pulse dampener Valco injection valve fitted with 100-ul sample loop Stainless steel, 6 mm i.d., 25 cm long; packed with Polygosil 60-5 C18 (Macherey-Nagel) Solvent mixture 2 1.3 ml/min 20 °C Uvikon 722 LC UV detector (Kontron) Wavelength 260 nm 10 mV; chart speed 5 mm/min 100 ul 4 min 12 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Inject 100 ul of each sulphur standard solution (equivalent to 0.1 to 1.0 ug sulphur) into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng sulphur. Also inject 100-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of sulphur from the calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples of hops, fortified with sulphur at levels of 5 to 400 mg/kg, ranged from 85 to 100% with a relative standard deviation of ± 5%. The recoveries from untreated control samples of the other materials, fortified with sulphur at levels of 1 to 10 mg/kg, averaged 100% for cucumbers, 90% for strawberries and apples, 85% for grapes, and 75% for soil. The routine limit of determination was 5 mg/kg for hops, and 1 mg/kg for all other substrates. Relatively high blanks may usually be expected, e.g. 0.2 mg/kg for cucumbers.

290

Sulphur

7.3 Calculation of residues The residue R, expressed in mg/kg sulphur, is calculated from the following equation:

R_

w A -v E x -v E n d VRI-VS-G

where G

= sample weight (in g)

VEx

= volume of dichloromethane used for extraction of analytical sample (in ml)

VR1

= portion of volume VEx used for cleanup in step 6.2 (in ml)

VEnd = terminal volume of sample solution from 6.2 (in ml) Vj = portion of volume VEnd injected into high-performance liquid chromatograph (in Hi) WA

= amount of sulphur for Vj read from calibration curve (in ng)

8 Important points During the analysis, care should be taken not to expose the solutions to sunlight. Extracts with a high content of sulphur and a very low content of co-extractives need not be subjected to the cleanup described in step 6.2 Experience so far gained indicates that sulphur residues can be determined also in crops other than those listed in the heading of the method.

9 References K. Wenzel, Die HPLC-Bestimmung von elementarem Schwefel in Lebensmitteln, Z. Lebensm. Unters. Forsch. 170, 5-6 (1980). R.M. Cassidy, A selective method for elemental sulfur analysis by high-speed liquid chromatography, J. Chromatogr. 117, 71-79 (1976).

10 Author BASF, Agricultural Research Station, Limburgerhof, J. Elzner

Thiabendazole

256-A

Apples (peel), bananas (peel), grapefruit (peel), oranges (peel), potatoes (peel)

Fluorimetric determinination on TLC chromatograms

(German version published 1979)

1 Introduction Chemical name

2-(Thiazol-4-yl)benzimidazole (IUPAC)

Structural formula

Empirical formula Molar mass Melting point Vapour pressure Boiling point Solubility (in 100 ml at room temperature)

Other properties

C10H7N3S 201.25 304-305 °C, sublimes from 250 °C No data No data Slightly soluble in water (approx. 1 g) at pH 2, very sparingly soluble at pH 3-12; soluble in dimethylacetamide (6.47 g), dimethylformamide and dimethylsulphoxide (8 g); slightly to sparingly soluble in acetone, benzene, tert.butanol, chloroform, dichloromethane, diethyl ether, ethyl acetate and methanol (0.93 g) Stable in solution, fluoresces in UV light

2 Outline of method Thiabendazole residues are extracted from plant material with dichloromethane. The extract is concentrated and the concentrate made up to a definite volume with ethanol. In order to separate thiabendazole from plant co-extractives, an aliquot portion of this solution is chromatographed on a silica gel thin-layer plate, together with a series of standards for comparison. Thiabendazole is determined directly on the TLC plate by fluorimetric measurement.

3 Apparatus Soxhlet extractor, capacity of extraction tube approx. 500 ml, fitted with 500-ml round-bottomed flask and reflux condenser Rotary vacuum evaporator

292

Thiabendazole

Volumetric flask, 50-ml Equipment for thin-layer chromatography Micro-capillaries, 2-^1 Spectrophotometric scanner, suitable for fluorimetric measurements on TLC plates

4 Reagents Dichloromethane, p.a. Ethanol, p.a., 96% vol. Ethyl acetate, p.a. Methyl ethyl ketone, p.a. Mobile phase for TLC: ethyl acetate + methyl ethyl ketone + formic acid + water 5 : 3 : 1 : 1 v/v/v/v

Thiabendazole standard solutions: 0.1, 0.2, 0.3 and 0.4 mg/10 ml in ethanol Formic acid, p.a., 98-100% Pre-coated TLC silica gel 60 glass plates, 20 cm x 20 cm, without fluorescent indicator (e.g. Merck No. 5721), or equivalent aluminium sheets Cottonwool

5 Sampling and sample preparation For preparing the analytical sample, weigh 2 to 2.5 kg of the laboratory sample (see pp. 17 ff, Vol. 1), peel the fruits or potatoes, and weigh the peel. Finely chop the peel and mix thoroughly.

6 Procedure 6.1 Extraction Insert a cottonwool plug into the extraction tube of the Soxhlet extractor. Transfer the peel, equivalent to 400 g (G) of fruit or potatoes, into the tube and top with another cottonwool plug. Next, add dichloromethane to the tube until it begins to siphon, allow to drain into the round-bottomed flask, and half-fill the tube with dichloromethane anew. Mount the reflux condenser, heat to reflux, and extract for 3 h. Allow the extract to cool, and rotary-evaporate it to a volume of a few ml. Quantitatively transfer the concentrate to a 50-ml volumetric flask, using ethanol to complete the transfer, and make up to the mark with ethanol (VEnd). 6.2 Thin-layer chromatography Fill a 2-^1 micro-capillary (VA) with the solution derived from 6.1 and let it run out from a vertical position onto the TLC plate, to form a spot at a distance of 25 mm from the bottom edge. Allow the spot to dry without applying any stream of air.

Thiabendazole

293

Onto the same TLC plate, spot additionally 2 \i\ each of the sample solutions derived from 4 other analytical samples, and of the 4 thiabendazole standard solutions, keeping a distance of at least 18 mm between the individual spots. Transfer the plate into the chromatographic tank previously saturated with the vapours of the mobile phase, and allow the chromatogram to develop until the front of the mobile phase has moved to approx. 4 cm from the top edge of the plate (approx. 11/2 to 2 h). Remove the plate from the tank and dry it in a stream of moderately warm air. Carry out the spotting and the development of the chromatograms in a room lit only with normal light bulbs, for fluorescent tubes and daylight may cause thiabendazole losses. 6.3 Fluorimetric measurement For scanning the TLC plate, the exciting radiation at 313 nm (line filter) is directed onto the layer at an angle of 45°. From the emitted radiation, the 355-nm wavelength is selected using a monochromator. Operating conditions Spectrophotometer Light source Monochromator wavelength Slit width Slit height Table speed Plotter speed

Chromatogram spectrophotometer Zeiss KM 3 Mercury lamp fitted with M 313 line filter 355 nm 0.5 mm 15 mm 20 cm/min 60 cm/min

Place the thin-layer plate, by adjusting the position of the table, so that the individual spots give maximum deflection of the recorder. Shift the spot area out of the light beam in the Y direction by hand; then plot the fluorescence curve in Y direction by switching on the table drive. For an alternative arrangement, the TLC plate is exposed to an exciting radiation of 313 nm which is selected by the monochromator. In this case, the emitted radiation is passed through a wide-band filter M 365 positioned between plate and detector. No thiabendazole losses have been observed with this arrangement.

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Measure the fluorescence intensity as described in 6.3 of the four spots of the standard solutions contained on each plate. Calculate the areas of the peaks recorded from the peak heights and the widths at half height. Plot the areas obtained using double log graph paper vs. ng thiabendazole. Also measure the fluorescence intensities for the spots of the sample solutions. For the areas of the peaks obtained for these spots, read the appropriate amounts of thiabendazole from the calibration curve. Plot a new calibration curve for each plate used.

294

Thiabendazole

7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with thiabendazole at levels of 1 to 8 mg/kg, ranged from 89 to 103% and averaged 95% with a relative standard deviation of 2.7%. The routine limit of determination was approx. 0.5 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg thiabendazole, is calculated from the following equation: R = where G = VEnd = VA = WA =

w A -v E n d VAG

sample weight of fruit or potatoes from which the peel was extracted (in g) terminal volume of sample solution from 6.1 (in ml) portion of volume VEnd applied to TLC plate (in ul) amount of thiabendazole for VA read from calibration curve (in ng)

8 Important points With residues of less than 0.5 mg/kg thiabendazole, the results are likely to be affected by interfering co-extractives. For instance, grapefruit peel contains blue fluorescent compounds (emission maxima approx. 390 nm) which are not fully separated from thiabendazole under the given TLC conditions. In the extracts derived from 6.1, residues of biphenyl and o-phenylphenol can also be determined by TLC or gas chromatography, with recoveries ranging from 87 to 100%.

9 References H. Otteneder and U. Hezel, Quantitative routine determination of thiabendazole by fluorimetric evaluation of thin-layer chromatograms, J. Chromatogr. 109, 181-187 (1975). S. Ebel and G. Herold, Quantitative Bestimmung von Thiabendazol durch direkte Fluoreszenzauswertung nach dunnschichtchromatographischer Trennung, Dtsch. Lebensm. Rundsch. 70, 133-136 (1974).

10 Authors Chemisches Untersuchungsamt, Trier, H. Otteneder Carl Zeiss, Oberkochen, Abteilung fur physikalisch-chemische Analyse, U. Hezel

Thiabendazole Grapefruit (peel), oranges (peel)

256-B Spectrophotometric determination

(German version published 1979)

1 Introduction For data on physico-chemical properties of thiabendazole, see the Method on p. 291, this Volume,

2 Outline of method Thiabendazole residues are extracted from citrus peel with dichloromethane. The extract is concentrated and the concentrate made up to a definite volume with ethanol. In order to separate thiabendazole from plant co-extractives, an aliquot portion of this solution is chromatographed on a silica gel thin-layer plate. The thiabendazole band, visualized by exposing the TLC plate to UV light, is scraped off together with the adsorbent. Thiabendazole is eluted with hydrochloric acid and is determined by measuring the absorbance at 302 nm. This spectrophotometric method is less sensitive than the fluorimetric one described on pp. 291 ff, this Volume.

3 Apparatus Soxhlet extractor, capacity of extraction tube approx. 500 ml, fitted with 500-ml round-bottomed flask and reflux condenser Rotary vacuum evaporator Volumetric flasks, 10-ml and 5-ml TLC applicator, e.g. Linomat IV (Camag) Equipment for thin-layer chromatography Pasteur pipets, i.d. 5 mm, 8 cm long UV lamp with 350-nm filter Spectrophotometer fitted with 1-cm quartz cells

4 Reagents Dichloromethane, p.a. Ethanol, p.a., 96% vol. Ethyl acetate, p.a.

296

Thlabendazole

Methyl ethyl ketone, p.a. Mobile phase for T L C : ethyl acetate + methyl ethyl ketone + formic acid + water 5 : 3 : 1 : 1 v/v/v/v

Thiabendazole standard solution: 10 ng/ml in hydrochloric acid (0.1 mol/1) Formic acid, p.a., 98-100% Hydrochloric acid, 0.1 mol/1 HC1 p.a. Pre-coated TLC silica gel 60 glass plates, 20 cm x 20 cm, without fluorescent indicator (e.g. Merck No. 5721), or equivalent aluminium sheets Cottonwool

5 Sampling and sample preparation For preparing the analytical sample, weigh 2 to 2.5 kg of the laboratory sample (see pp. 17 ff, Vol. 1), peel the fruits and weigh the peel. Finely chop the peel and mix thoroughly.

6 Procedure 6.1 Extraction Insert a cottonwool plug into the extraction tube of the Soxhlet extractor. Transfer the peel, equivalent to 400 g (G) of fruit, into the tube and top with another cottonwool plug. Next, add dichloromethane to the tube until it begins to siphon, allow to drain into the round-bottomed flask, and half-fill the tube with dichloromethane anew. Mount the reflux condenser, heat to reflux, and extract for 3 h. Allow the extract to cool, and rotary-evaporate it to a volume of a few ml. Quantitatively transfer the concentrate to a 10-ml volumetric flask, using ethanol to complete the transfer, make up to the mark with ethanol (VEx), and filter. 6.2 Thin-layer chromatography Using the TLC applicator, apply 50 ul (VR1) of the solution derived from 6.1 in the form of a 40 mm long band at a distance of 25 mm from the bottom edge of the TLC plate. The distance between the individual tracks should be 10 mm. A maximum of three samples can be applied to a single 20 cm x 20 cm plate. Transfer the plate into the chromatographic tank previously saturated with the vapours of the mobile phase, and allow the chromatogram to develop until the front of the mobile phase has moved to approx. 4 cm from the top edge of the plate (approx. 11/2 to 2 h). Remove the plate from the tank and dry it in a stream of moderately warm air. View the plate unter a UV lamp, mark the thiabendazole band (Rf = 0.77) as close as possible to its boundary, and scrape the adsorbent layer from the plate using a scalpel to yield a very fine powder. Suck the powder into a Pasteur pipet of which the tapered end has been closed with a cottonwool plug. Elute thiabendazole with 5 ml hydrochloric acid, using light pressure from a pipet ball if necessary. Collect the eluate in a 5-ml or 10-ml volumetric flask and make up to the mark with hydrochloric acid (VEnd).

Thiabendazole

297

6.3 Photometric determination Measure the absorbance of the eluate derived from 6.2 in a spectrophotometer at a wavelength of 302 ± 0.5 nm in a 1-cm quartz cell, using hydrochloric acid for comparison.

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Pipet 0.6, 1.2, 1.8, 2.4 and 3.0 ml each of the thiabendazole standard solution (equivalent to 6, 12, 18, 24 and 30 \xg thiabendazole) into 10-ml volumetric flasks and make up to the mark with hydrochloric acid. Plot the absorbances measured for these solutions vs. thiabendazole concentration (\ig/l0 ml). A linear calibration curve is obtained for the given range where e.g. 18 ng/10 ml showed an absorbance of 0.230. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with thiabendazole at levels of 1 to 10 mg/kg, ranged from 89 to 116% and averaged 103% with a relative standard deviation of 2.1%. The routine limit of determination was approx. 1 mg/kg when G = 600 g. 7.3 Calculation of residues The residue R, expressed in mg/kg thiabendazole, is calculated from the following equation: R=

w A R -v E x -v E n d 10-VR1-G

where G VEx VR1 VEnd WAR

= = = = =

sample weight of fruit from which the peel was extracted (in g) volume of made-up extract from 6.1 (in ml) portion of volume VEx applied to TLC plate (in ml) volume of made-up eluate from TLC layer (in ml) concentration of thiabendazole in the sample solution read from calibration curve (in ng/10 ml)

8 Important points No data

298

Thiabendazole

9 References H. Hey, Spektralanalytik und Gaschromatographie von Thiabendazol. UV-photometrische Bestimmung auf Citrusfriichten und Bananen, Z. Lebensm. Unters. Forsch. 149, 79-86 (1972). H. Otteneder and U. Hezel, Quantitative routine determination of thiabendazole by fluorimetric evaluation of thin-layer chromatograms, J. Chromatogr. 109, 181-187 (1975).

10 Author Chemisches Untersuchungsamt, Trier, H. Otteneder

Part 4 Multiple Pesticide Residue Analytical Methods (contd.)

Pesticides, Chemically Related Compounds and Metabolites Determinable by the Multiresidue Methods in Parts 4 to 6: Supplement to the Table of Compounds, pp. 221 ff, Vol. 1 Common name

Chemical name

Acephate

O,S-dimethyl acetylphosphoroamidothioate

Alachlor

2-chloro-2',6'-diethyl-N-methoxymethylacetanilide

Structural formula

^

Aldicarb

2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime

X

C2H5

CH 2 -O-CH 3

ff

1

CH 3 S-C-CH=N-O-C-NHCH 3 CH, O .C—NHCH 3

Aminocarb

4-dimethylamino-m-tolyl methylcarbamate

N(CH 3 ) 2

3 -amino-1 H-l ,2,4-triazole

Anthraquinone

anthraquinone

Benalaxyl

methyl N-phenylacetylN-2,6-xylyl-DI^alaninate

V

y-CH 2 —C

/CH-COO-CH 3

O ,C-NHCH 3 Bendiocarb

2,2-dimethyl-l,3-benzodioxol4-yl methylcarbamate

NO 2

N-butyl-N-ethyl-2,6-dinitro4-trifluoromethylaniline

\=<

\CH 2 ) 3 -CH 3 NO2

302

Table of Compounds

Common name

Chemical name

Structural formula

Bentazone

3-isopropyl-lH-benzo-2,l,3-thiadiazin-4-one 2,2-dioxide XH(CH 3 ) 2

Bifenox

methyl 5-(2,4-dichlorophenoxy)2-nitrobenzoate

2-methylbiphenyl-3-ylmethyl (Z)-(1RS,3RS)3-(2-chloro-3,3,3-trifluoroprop-l-enyl)2,2-dimethylcyclopropanecarboxylate

Bromopropylate

isopropyl 4,4'-dibromobenzilate

Bromoxynil octanoate

2,6-dibromo-4-cyanophenyl octanoate

Butocarboxim

3 - (methylthio)butanone O-methylcarbamoyloxime

CF3 —C=CH— CH— CH— CO—O—CH2

CH3

O

CH3S-CH-C=N-O-C-NHCH3 CH,

O .C-NHCH3

Carbaryl

1-naphthyl methylcarbamate

Carbendazim

methyl benzimidazol-2-ylcarbamate

O II

C-NHCH 3

Carbofuran

2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate CH 3

Carbophenothionmethyl

S-4-chlorophenylthiomethyl O,O-dimethyl phosphorodithioate

Chinomethionat (Quinomethionate)

6-methyl-l,3-dithiolo[4,5-b]quinoxalin-2-one

CH3O S PCH3O

H 3 C.

Table of Compounds

303

Common name

Chemical name

Chlorbenside

4-chlorobenzyl 4-chlorophenyl sulphide

Chlorbenside sulphone

4-chlorobenzyl 4-chlorophenyl sulphone

C l — ^ V - CH 2 - S -f%—

Chlorfenprop-methyl

methyl ( ± )-2-chloro3 -(4-chloropheny l)propionate

Cl—-CH 2 -CH— COO-CH3

Chlorflurenol

2-chloro-9-hydroxyfluorene-9-carboxylic acid

Chloridazon

5-amino-4-chloro-2-phenylpyridazin-3(2H)-one

Chlormephos

S-chloromethyl O,O-diethyl phosphorodithioate

Chlorobenzilate

ethyl 4,4'-dichlorobenzilate

Structural formula

Cl

COO—C 2 H 5

Chloroneb

l,4-dichloro-2,5-dimethoxybenzene

Chloropropylate

isopropyl 4,4'-dichlorobenzilate

Chlorothalonil

tetrachloroisophthalonitrile

Chlorthal-dimethyl

dimethyl tetrachloroterephthalate

Cl

Cl

CH3-O-CO-£3-COO-CH3

c, M c> C2H5Oxfi Coumaphos

O-3-chloro-4-methyl-2-oxo-2H-chromen-7-yl O,O-diethyl phosphorothioate

C H

2 5° CH 3

304

Table of Compounds

Common name

Chemical name

Structural formula

Crotoxyphos

dimethyl (E)-l-methyl-2(l-phenylethoxycarbonyl)vinyl phosphate

CH 3 O V ° P - O - C = C H — COO—CH— CH3 CH3O ^

Crufomate

4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate

Cyanazine

2-chloro-4-(l-cyano-l-methylethylamino)6-ethylamino-l,3,5-triazine

Cyanophos

O-4-cyanophenyl O,O-dimethyl phosphorothioate

6

p.o/VqcH^

C2H5NH

N-^NH—C-CN CH3

CH3O^

CH3^

Cyfluthrin

Cyhalothrin

(RS)-a-cyano-4-fluoro-3-phenoxybenzyl (lRS,3RS;lRS,3SR)-3-(2,2-dichlorovinyl)2,2-dimethylcyclopropanecarboxylate

(RS)-a-cyano-3-phenoxybenzyl (Z)-(lRS,3RS)-3-(2-chloro-3,3,3-trifluoropropenyl)2,2-dimethylcyclopropanecarboxylate

7CH3

A

?N

C1 2 C=CH—CH—CH—CO—O—CH v

^

o

'T " ci

C

CF3-C=CH—CH—CH—C

Cymoxanil

l-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea

O O NC-C—C-NH—C-NH—C 2 H 5 N—OCH3

2,4-D

(2,4-dichlorophenoxy)acetic acid

Cl—/ S— O—CH2—COOH Cl

2,4-DB

S,S, S-tributyl phosphorotrithioate

Desethylpirimiphos-methyl

O—(CH2)3—COOH

4-(2,4-dichlorophenoxy)butyric acid

O-2-ethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate

Desethyl-simazine see des-tert-butyl-terbuthylazine, p. 226, Vol. 1

(C4H9-S)3PO

CH3O

Table of Compounds

Common name

Chemical name

Desethyl-terbuthylazine

2-amino-4-chloro-6-tert-butylamino1,3,5-triazine

305

Structural formula

N^ N H2N

N"^NH-C(CH 3 ) 3

O .C-N(CH3)2 Desmethylformamidopirimicarb

5,6-dimethyl-2-(N-methylformamido)pyrimidin-4-yl dimethylcarbamate

O C-N(CH3)2 Desmethyl-pirimicarb

5,6-dimethyl-2-methylaminopyrimidin-4-yl dimethylcarbamate

H c

\

x,x ^y

N

H3C

C2H5O S

S-2-chloro-l-phthalimidoethyl O,O-diethyl phosphorodithioate

C2H5O

CH2C1

(CH3)2CH Di-allate

S-2,3-dichloroallyl di-isopropyl(thiocarbamate)

p,p'-Dichlorobenzophenone

4,4'-dichlorobenzophenone

Dichlorprop

( ± )-2-(2,4-dichlorophenoxy)propionic acid

Diclo fop-methyl

methyl (RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionate

(CH3)2CH/

Cl—f

O N-C-S-CH 2 -C=CHC1 ^

Vo-CH-COOH

-CH— coo—CH 3

2,6-dichloro-4-nitroaniline cr

T

Cl

NH 2

H2N

Dinitramine

N l,N 1-diethyl-2,6-dinitro-4-trifluoromethyl-mphenylenediamine

Dinobuton

2-sec-butyl-4,6-dinitrophenyl isopropyl carbonate

NO 2

F,C-/VN

O2N-/~\-O-C-O-CH(CH3)2 CH-C2H5 CH3

306

Table of Compounds

Common name

Chemical name

Structural formula

NO2 2-sec-butyl-4,6-dinitrophenol CH-C2H5 CH3 NO2 Dinoterb

2-tert-butyl-4,6-dinitrophenol

O 2 N-( / \ - °

H

C(CH3)3

O C-NHCH 3 2-(l ,3-dioxolan-2-yl)phenyl methylcarbamate

NO2 DNOC

4,6-dinitro-o-cresol

Edifenphos

O-ethyl S,S-diphenyl phosphorodithioate

VoH

o OC 2 H 5

EPN

O-ethyl O-4-nitrophenyl phenylphosphonothioate

Ethiofencarb

2-ethylthiomethylphenyl methylcarbamate

C2H5O

6

P

o /C-NHCH3

,CH 2 -S-C 2 H 5

Famophos

O-4-dimethylsulphamoylphenyl O,O-dimethyl phosphorothioate

Fenarimol

(± )-2,4'-dichloro-a-(pyrimidin-5-yl)benzhydryl alcohol

Fenoprop

(± )-2-(2,4,5-trichlorophenoxy)propionic acid

Fluazifop-butyl

butyl (RS)-2-[4-(5-trifluoromethyl2-pyridyloxy)phenoxy] propionate

V-O-CH-COOH

°K3~C

Table of Compounds Common name

Chemical name

Flubenzimine

(2Z,4E,5Z)-N2,3-diphenyl-N4,N5-bis(trifluoromethyl)-l,3thiazolidine-2,4,5-triylidenetriamine

307

Structural formula

N-CF 3

s

6

N-CF3

N02 Fluchloralin

N-(2-chloroethyl)-2,6-dinitro-N-propyl4-(trifluoromethyl)aniline

^ ¥•£.-<(

/(CH^-CH, X

V-N

\=<

'CH2-CH2C1 NO2 O-CHF2

Flucythrinate

(RS)-a-cyano-3-phenoxybenzyl (S)2-(4-difluoromethoxyphenyl)-3-methylbutyrate

Fluorodifen

4-nitrophenyl a,a,a-trifluoro2-nitro-p-tolyl ether

Fluroxypyr(1-methylheptyl)

1-methylheptyl 4-amino-3,5-dichloro-6-fluoro-2pyridyloxyacetate

Fluvalinate

(RS)-a-cyano-3-phenoxybenzyl N-(2-chloro-a, a, a-trifluoro-p-tolyl)-D-valinate

I

CH3N

^CH—CH—CO-O-CH CH/

,

NH CN CH-CH—COO-CH

Q

* T1 Genite

2,4-dichlorophenyl benzenesulphonate

Haloxyfop(2-ethoxyethyl)

2-ethoxyethyl (RS)-2-[4-(3-chloro5-trifluoromethyl-2-pyridyloxy)phenoxyjpropionate

o

II .C-NHCH3 3-Hydroxy-carbofuran

2,3-dihydro-3-hydroxy-2,2-dimethylbenzofuran-7-yl methylcarbamate

Ioxynil octanoate

4-cyano-2,6-di-iodophenyl octanoate

C7H15 - C O O - / ~ \ - C N

308

Table of Compounds

Common name

Isobenzan

Chemical name

Structural formula

l,3,4,5,6,7,8,8-octachloro-l,3,3a,4,7,7a-hexahydro-4,7methanoisobenzofuran

Cl

/ \ Isocarbamid

N-isobutyl-2-oxoimidazolidine-l-carboxamide

NO 2 Isopropalin

(CH 3 ) 2 CH-

;CH2)2-CH3 X

4-isopropyl-2,6-dinitro-N,N-dipropylaniline

(CH 2 ) 2 -CH 3

NO 2

.C-NHCH3 3-Keto-carbofuran

2,3-dihydro-2,2-dimethyl-3-oxobenzofuran-7-yl methylcarbamate

8-Keto-endrin

4,5,5,6-exo-7,8-hexachloropentacyclo[7.2.1.02-6. 03-10.04-8]dodecan-12-one

Lenacil

3-cyclohexyl-l,5,6,7-tetrahydrocyclopenta pyrimidine-2,4(3H)-dione

Leptophos

O-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonothioate

CH3OJ

MCPA

(4-chloro-2-methylphenoxy)acetic acid

Cl—<^Vo-CH2-COOH

MCPA-(2-butoxyethyl)

2-butoxyethyl (4-chloro-2-methylphenoxy)acetate

Cl—f^-O-CH2-COO-(CH2)2-O-C4H9

4-(4-chloro-2-methylphenoxy)butyric acid

>-(CH2)3-COOH 'CH3 > S

S-(N-ethoxycarbonyl-N-methylcarbamoylmethyl) O,O-diethyl phosphorodithioate

Mecoprop

( ± )-2-(4-chloro-2-methylphenoxy)propionic acid

O

P-S-CH2-C-N-COO-C2H5

CH3 )-CH-COOH

Table of Compounds Common name

Chemical name

Structural formula

Mephospholan

diethyl 4-methyl-l,3-dithiolan2-ylidenephosphoramidate

C2H5O
309

O C-NHCH3 Mercaptodimethur

4-methylthio-3,5-xylyl methylcarbamate SCH3

Merphos

tributyl phosphorotrithioite

(C4H9-S)3P

NH 2 ,CH3

II

4-amino-3-methyl-6-phenyl-l,2,4-triazin-5(4H)-one

Metazachlor

2-chloro-N-(pyrazol-l-ylmethyl)acet-2',6'-xylidide

Methabenzthiazuron

l-benzothiazol-2-yl-l,3-dimethylurea

£ >-N-C-NH-CH3 CH 3

Methiocarb see Mercaptodimethur

Methomyl

S-methyl N-(methylcarbamoyloxy)thioacetimidate

Metolachlor

2-chloro-6'-ethyl-N-(2-methoxyl-methylethyl)acet-o-toluidide

Mirex

dodecachloropentacyclo[5.3.0.02-6.03'9.04-8]decane

Monocrotophos

dimethyl (E)-l-methyl-2-(methylcarbamoyl)vinyl phosphate

Morphothion

O,O-dimethyl S-morpholinocarbonylmethyl phosphorodithioate

Nitralin

4-methylsulphonyl-2,6-dinitro-N,N-dipropylaniline

CH3S-C=N-0-C-NHCH3

X X

C CH-CH 2 -O-CH 3

perchlorinated

CH3O O

P-O-C=CH-C-NH-CH3

i

^P-S-CH2-CO-N CHgO

O

NO2

MW /(CH2)2-CH3 CH3-S-/ \ - N S 5 W (CH2)2-CH3 NO 2

310

Table of Compounds

Common name

Chemical name

Structural formula

2,4-dichlorophenyl 4-nitrophenyl ether

Nitrothal-isopropyl

-o-(

VNO 2

di-isopropyl 5-nitroisophthalate COOOO-CH(CH3)2

Octachlorodipropyl ether (S 421)

bis-(2,3,3,3-tetrachloropropyl) ether

Octachlorostyrene

octachlorostyrene

C1 3 C-CH-CH 2 -O-CH 2 -CH-CC1 3 Cl Cl

c

kX/ c c l = c c l 2

cr

ci

T

ci

5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-l,3,4-oxadiazol-2(3H)-one

NN

°

Pendimethalin

<3

N-(l-ethylpropyl)-2,6-dinitro-3,4-xylidine

N

CH 3 NO 2

Phenmedipham

3-methoxycarbonylaminophenyl 3'-methylcarbanilate

NH-COO-CH3

P-S-CH-/ Phenthoate

S-a-ethoxycarbonylbenzyl

CHoO

S

0

/Px

O,O-dimethyl S-phthalimidomethyl phosphorodithioate

y

N = / I COO-C 2 H 5

CH3O

O,O-dimethyl phosphorodithioate

Phosmet

C2H5

C.

CH30

S-CH2-

P-O-N=CPhoxim

O,O-diethyl a-cyanobenzylideneamino-oxyphosphonothioate

C2H5O CH3

2-dimethylamino-5,6-dimethylpyrimidin-4-yldimethylcarbamate

O-2-diethylarnino-6-methylpyrimidin-4-yl O,O-diethyl phosphorothioate

3

y H3C'

Pirimiphos-ethyl

^H, •C-N VCH tr nI'

C 2 H 5 OJ C2H5O/

N

N=
C(CH3);

Table of Compounds

Common name

Chemical name

Profluralin

N-cyclopropylmethyl-2,6-dinitro-N-propyl4-trifluoromethylaniline

Promecarb

3 -isopropyl-5 -methylphenyl methylcarbamate

Propachlor

2-chloro-N-isopropylacetanilide

311

Structural formula

O2 / ,(CH 2 ) 2 -CH 3

CH(CH3)2

Propanil

QNHC

N-(3,4-dichlorophenyl)propionamide

Cl

Propiconazole

(± )-l-[2-(2,4-dichlorophenyl)-4-propyll,3-dioxolan-2-ylmethyl]-lH-l,2,4-triazole

Quinalphos

O,O-diethyl O-quinoxalin-2-yl phosphorothioate

°

C2H5O-" Quinomethionate see Chinomethionat

Salithion

2-methoxy-4H-benzo-l,3,2-dioxaphosphorin 2-sulphide

Strobane T

polychloroterpenes (chlorinated mixed terpenes)

Sulprofos

O-ethyl O-4-(methylthio)phenyl S-propyl phosphorodithioate

2,4,5-T

(2,4,5-trichlorophenoxy)acetic acid

3-tert-butyl-5-chloro-6-methyluracil

Terbufos

2 5

O

S-tert-butylthiomethyl O,O-diethyl phosphorodithioate

P-S-CH2-S-C(CH3)3 CHO''

CH3/CH3 Tetramethrin

3,4,5,6-tetrahydrophthalimidomethyl (± )-cis-transchrysanthemate

H 3x

/C\ /-tf C=CH—CH—CH-COO—CH2-N II

312

Table of Compounds

Common name

Tolclofos-methyl

Chemical name

Structural formula

3,3-dimethyl-l-methylthiobutanone O-methylcarbamoyloxime

CH3S-CH2-C=N-O-C-NHCH3 C(CH3)3

O-2,6-dichloro-p-tolyl O,O-dimethyl phosphorothioate

P CH3O

O—T

7-CH3

Cl (CH 3 ) 2 CH S-2,3,3-trichloroallyl di-isopropyl(thiocarbamate) (CH 3 ) 2 CH /

Triamiphos

P-(5-amino-3-phenyl-lH-l,2,4-triazol-l-yl)-N,N,N',N'tetramethylphosphonic diamide

Triazoxide

7-chloro-3 - [(1 H)-imidazol-l-yl] -1,2,4-benzotriazine 1-oxide

Trichloronat

O-ethyl O-2,4,5-trichlorophenyl ethylphosphonothioate

§ N-C-S-CH 2 -C=CC1 2 ^

(CH 3 ) 2

C2H5O. n

Cl

Triclopyr(2-butoxyethyl)

2-butoxyethyl (3,5,6-trichloro2-pyridyloxy)acetate

Cl—(/

VO-CH 2 -COO-(CH 2 ) 2 -O-C 4 H 9 Cl

Organohalogen, Organophosphorus and Triazine Compounds

S 8 (updated)

Since the publication of Multiresidue Method S 8 (see pp. 283 ff, Vol. 1), analytical experience has shown that at least the following 30 pesticides can also be analyzed by this Method: Endosulfan sulphate Fenamiphos Fenarimol Fenson Fluchloralin Fluorodifen Iodofenphos Metazachlor Metolachlor Metribuzin

Bromopropylate Chlorbenside Chlorfenson Chlorflurenol Chlorobenzilate Chloropropylate Cyanazine Cyanophos Dialifos p-Endosulfan

Nitrofen Pendimethalin Profluralin Quinalphos Terbacil Terbufos Tolclofos-methyl Tri-allate Trichloronat Trifluralin

Moreover, the Method was meanwhile extended to include additional food crops such as apricots, aubergines, chillies, honey, mandarin oranges and potatoes. For convenience, the updated Table 2 gives a compilation of the routine limits of determination and the gas-chromatographic retention times for all 121 compounds included in the Method. Table 2 (updated). Routine limits of determination and relative retention times. Relative retention time Compound

Aldrin Ametryn Atrazine Azinphos-ethyl Azinphos-methyl Aziprotryne Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Captafol Captan Carbophenothion

Routine limit of determination mg/kg 0.004 0.1 0.05 0.1 0.2 0.05 0.05 0.02 0.01 0.02 0.05 0.1 0.01 0.03

6.3.1 Aldrin = 1

Column*) 6.3.2.1 6.3.2.2 Parathion = 1

6.3.3

1.00 0.75 0.46 6.4 4.90

5.6 4.6

4.8 4.2 0.59

0.83 1.06 1.38 3.59 1.67 3.95 1.18 2.63

0.51 0.68 1.3

1.23

1.1 1.1

314

Method S 8 (updated)

Table 2. (contd.) Relative retention time

Compound

Chlorbenside Chlorfenson Chlorfenvinphos Chlorflurenol Chlorobenzilate Chloropropylate Chlorpyrifos Chlorpyrifos-methyl Chlorthal Chlorthiophos Cyanazine Cyanofenphos Cyanophos p,p-DDD p,p'-DDE p,p-DDT Desmetryn Dialifos Diazinon Dichlobenil Dichlofenthion Dichlofluanid Dichlorvos Dicofol Dieldrin Dimethachlor Dimethoate Dioxathion Disulfoton Ditalimfos a-Endosulfan p-Endosulfan Endosulfan sulphate Ethion Ethoprophos Etrimfos Fenamiphos Fenarimol Fenchlorphos Fenitrothion Fenson Fensulfothion Fenthion Fluchloralin Fluorodifen

Routine limit of determination mg/kg 0.01 0.08 0.03 0.01 0.2 0.08 0.05 0.01 0.01 0.01 0.04 0.01 0.04 0.01 0.01 0.01 0.02 0.1 0.01 0.01 0.03 0.01 0.02 0.05 0.01 0.05 0.1 0.05 0.01 0.05 0.01 0.01 0.02 0.03 0.02 0.04 0.05 0.01 0.02 0.05 0.04 0.1 0.01 0.02 0.01

6.3.1 Aldrin = 1

Column*) 6.3.2.1 6.3.2.2 Parathion = 1

6.3.3

1.28 1.45 1.28 1.96 1.99 0.93 0.71 1.0 2.46 0.88 2.37 0.55 2.18 1.84 3.1

0.95

0.95

0.48

0.46 0.42

1.25

1.22 2.4

0.66 6.32 0.20

0.28

0.56

0.48 0.84 0.11

0.64 0.31 0.27 1.15

0.61 0.39 0.32 1.18

1.4

1.32 0.27 0.31 1.70

0.41 0.86

0.44 0.88

4.26 0.51

3.37 0.59

0.13 0.87 0.99 1.84 0.70 0.44

1.49 1.56 1.88 2.36 2.4 0.32 0.56 5.11 0.77 0.79 0.99 2.21 0.58 1.44

Method S 8 (updated)

315

Table 2. (contd.) Relative retention time Compound

Folpet Fonofos Formothion a-HCH

3-HCH Heptachlor Heptachlor epoxide Heptenophos Iodofenphos Iprodione Isofenphos Lindane Malaoxon Malathion Metalaxyl Metazachlor Methidathion Methoprotryne Methoxychlor Metolachlor Metribuzin Mevinphos Naled Nitrofen Paraoxon Parathion Parathion-methyl Pendimethalin Perthane Phenkapton Phorate Phosalone Pirimiphos-methyl Procymidone Profenofos Profluralin Prometryn Propazine Propyzamide Prothiofos Pyrazophos Pyrethrum Quinalphos Quintozene Simazine

Routine limit of determination mg/kg 0.01 0.01 0.01 0.01 0.01 0.004 0.005 0.01 0.04 0.05 0.02 0.002 0.025 0.02 0.4 0.04 0.1 0.05 0.1 0.1 0.01 0.1 0.1 0.01 0.1 0.02 0.02 0.04 0.1 0.05 0.01 0.02 0.01 0.02 0.04 0.02 0.01 0.05 0.01 0.01 0.2 0.2 0.02 0.002 0.01

6.3.1 Aldrin = 1 1.27 0.49 0.61 0.39 0.42 0.79 1.22

Column*) 6.3.2.1 6.3.2.2 Parathion = 1 0.28 0.82

0.32 0.80

0.22

0.25

1.52 2.24 1.57 0.47

6.3.3

0.80

0.85

0.94 0.72

0.90 0.72 0.65

1.12 1.3

1.13

1.12 2.0

4.56 0.95 0.69 0.21 0.09

0.20

1.33 1.00 0.79

1.24 1.00 0.80

1.18 0.30 4.65 0.41

1.93 0.23 4.10 0.45 1.47 1.05

1.81 0.96 0.67 1.12 2.15 4.25 6.0 1.30 1.56 0.55

0.77 0.47 0.50 1.61 6.8 1.8; 2.35 1.17 0.49

5.41

0.78 4.67

0.44

316

Method S 8 (updated)

Table 2. (contd.) Relative retention time Compound

Sulfotep Tecnazene Terbacil Terbufos Terbutryn Tetrachlorvinphos Tetradifon Tetrasul Thionazin Tolclofos-methyl Tolylfluanid Triadimefon Tri-allate Triazophos Trichloronat Trifluralin Vinclozolin

Routine limit of determination mg/kg 0.01 0.01 0.1 0.3 0.05 0.1 0.03 0.05 0.01 0.05 0.02 0.04 0.05 0.1 0.02 0.02 0.01

6.3.1 Aldrin = 1

Column*) 6.3.2.1 6.3.2.2 Parathion = 1 0.23

0.26

1.22

1.12

6.3.3

0.24 0.60 0.56 0.88 1.47 4.70 2.45 0.27 0.75 1.20 0.93 0.64

0.19

0.62 2.0

1.06 0.42 0.69

*) For gas-chromatographic conditions, see pp. 290 f, Vol. 1.

Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides

S 19 (updated)

Since the publication of Volume 1 of this Manual, analytical experience has shown that many more pesticides can be analyzed by Multiresidue Method S 19 than those listed in Table 3 on pp. 388ff, Vol. 1. The updated Table 3 shows the data available for more than 220 compounds, as they were published in the 9th Instalment (1987) of the German edition of the Manual. The Table comprises the elution volumes in gel permeation chromatography under the conditions set out in step 5.3 of Cleanup Method 6 (p. 76, Vol. 1), the distribution of the compounds in eluates from silica gel column chromatography under the conditions described in step 6.4.3 of this Method (p. 387, Vol. 1), and the detectors used for the gaschromatographic determination of each compound. Table 3 (updated). Elution volume ranges in gel permeation chromatography, distribution of compounds in eluates from silica gel column chromatography, and detectors used for gas-chromatographic determination.

Compound Acephate Alachlor Aldrin Ametryn Amidithion Anilazinea) Anthraquinone Atrazine Azinphos-ethyl Azinphos-methyl Benfluralin Benzoylprop-ethyl Bifenox Binapacryl Bitertanol Bromacilb) Bromophos Bromophos-ethyl Bromopropylate Bromoxynil octanoate Camphechlor (Toxaphene) Captafolc> Captanc> Carbophenothion

GPC elution volume range

Silica gel eluates *)

ml

1

2

3

4

5

115-145 125-150 120-150 115-190 115-145 105-135 145-185 110-135 130-160 145-180 100-130 125-150 115-150 100-130 100-130 105-140 120-150 110-140 95-135 120-150 110-150 120-150 120-150 120-140

0 0 5 0 0 0 0 0 0 0 5 0 0 0 0 0 4 5 0 0 5 0 0 0

0 0 0 0 0 0 2 0 0 0 0 3 3 5 0 0 2 1 0 5 1 0 0 3

0 5 0 1 0 5 4 4 5 4 0 3 3 0 0 0 0 0 3 1 0 5 5 0

0 0 0 3 4 0 0 3 0 0 0 0 0 0 4 5 0 0 3 0 0 0 0 0

5 0 0 0 3 0 0 0 0 0 0 0 0 0 2 0 0 0 1 0 0 0 0 0

Detectors used for GLC ECD TID FPD +

+

+

+

+ + + + + + + +

+ + +

+

+ 4+

+

+ + + + + + + + + +

-f +

+ +

+

+

318

Method S 19 (updated)

Table 3. (contd.) Compound Carbophenothion-methyl Chinomethionat (Quinomethionate) Chlorbensided) Chlorbenside sulphone a-Chlordane y-Chlordane Chlorfenprop-methyl Chlorfenson Chlorfenvinphos Chloridazon (Pyrazon) Chlormephos Chlorobenzilate Chloroneb Chloropropylate Chlorothalonil Chlorotoluron Chloroxuron Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Chlorthal-dimethyl Chlorthiophos Coumaphos Crotoxyphos Crufomate Cyanazine Cyanofenphos Cyanophos Cymoxanile) Cypermethrin o,p'-DDD p,p'-DDD o,p'-DDE p,p'-DDE o,p'-DDT p,p'-DDT DEF^ Deltamethrin Demeton-S-methyl Demeton-S-methyl sulphone Demeton-S sulphone g) Demeton-S sulphoxideh) N-Desethyl-pirimiphos-methyl Dialifos Di-allate Diazinon

GPC elution volume range

Silica gel eluates *)

ml

1

2

3

4

5

120-160

0

4

0

0

0

170-200 120-155 130-160 110-140 100-130 125-150 120-150 110-140 130-155 115-145 100-135 145-170 100-135 125-165 115-150 130-155 110-135 110-140 120-150 135-160 115-155 135-165 105-145 100-140 110-135 115-145 115-150 110-130 100-135 110-140 100-140 120-150 120-150 120-150 110-140 115-135 100-135 125-155 120-160 115-140 140-170 120-155 110-140 120-150 105-135

0 0 0 5 5 0 1 0 0 3 0 0 0 0 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0 5 5 5 5 5 5 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 5 5 0 0 3 0 5 0 5 0 0 2 4 4 5 4 0 0 0 0 2 0 0 5 0 0 0 0 0 0 0 5 0 0 0 0 0 3 4 0

4 1 5 0 0 0 0 4 0 0 4 0 4 0 0 1 4 0 0 1 0 5 0 0 0 4 4 0 0 0 0 0 0 0 0 5 0 0 0 0 0 1 3 1 5

0 0 0 0 0 0 0 3 4 0 2 0 2 0 5 5 0 0 0 0 0 0 4 3 4 0 0 5 0 0 0 0 0 0 0 1 0 0 2 3 0 5 0 0 0

0 0 0 0 0 0 0 0 1 0 1 0 0 0 2 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 3 0 0 0 0

Detectors used for GLC ECD TID FPD + + + + + + + +

+

+

+

+

+

+ + +

+ +

+ + + +

+ + + +

+ +

+

+

+ +

+ +

+ +

+ + +

+ + + + + + + +

+

+ +

+ + +

+

+

+

+

+

+

Method S 19 (updated)

319

Table 3. (contd.) Compound Dichlobenil Dichlofenthion DichlofluanidJ) p, p'-Dichlorobenzophenonej) Dichlorvos Diclofop-methyl Dicloran Dicofolk) Dicrotophos Dieldrin Dimefox Dimethachlor Dimethoate Dinitramine1) Dinobuton Dinocap Dioxathion Disulfoton m) Disulfoton sulphone Disulfoton sulphoxide Ditalimfos Edifenphos a-Endosulfan p-Endosulfan Endosulfan sulphate Endrin EPN

Ethion Ethoprophos Etrimfos Famophos Fenamiphos Fenchlorphos Fenitrothion Fenson Fensulfothion Fenthion Fenvalerate Flubenzimine^ Fluchloralin Fluotrimazole Fluvalinate Folpet Fonofos Formothion Fuberidazole1* Genite

GPC elution volume range

Silica gel eluates *)

ml

1

2

3

4

5

125-155 110-140 100-140 125-155 115-140 135-165 105-145 100-150 130-160 120-150 120-155 135-165 120-150 105-130 110-140 100-120 110-140 115-150 110-140 120-150 120-150 130-160 110-150 110-150 100-140 130-160 135-160 100-140 120-155 105-140 125-155 105-140 120-150 120-150 130-160 120-150 130-160 105-135 85-120 100-120 100-140 95-120 140-180 120-150 120-150 120-160 135-165

1 3 0 0 0 0 0 2 0 0 0 0 0 4 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0 0

5 3 3 5 0 0 5 4 0 5 0 0 0 1 4 5 3 2 0 0 0 0 4 5 5 5 5 5 0 0 0 0 2 4 5 0 3 4 5 1 0 5 3 4 0 0 5

0 0 3 0 1 5 0 0 0 0 0 4 0 0 2 0 3 0 5 0 4 4 0 0 0 0 0 0 4 5 5 0 0 0 0 0 0 1 0 0 4 0 4 1 4 0 0

0 0 0 0 3 0 0 0 0 0 0 2 3 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 0 4 0 0 0 3 0 0 0 0 2 0 0 0 1 5 0

0 0 0 0 0 0 0 0 5 0 5 0 3 0 0 0 0 3 0 5 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 3 0 0 0 0 0 0 0 0 0 1 0

Detectors used for GLC ECD TID FPD + + + +

+ +

+ +

+ + + + + + +

+ +

+

+ + +

+ + +

+ + +

+

+ + + + + + +

+ +

+ +

+ + + +

+

+ +

+ +

+ +

+ +

+ +

+ +

+

+ + + + + + + + + +

+ +

320

Method S 19 (updated)

Table 3. (contd.) Compound a-HCH (3-HCH 8-HCH 8-HCH Heptachlor cis-Heptachlor epoxide trans-Heptachlor epoxide Heptenophos Hexachlorobenzene Imazalil11'1) Iodofenphos Ioxynil octanoate Iprodione Isobenzan Isocarbamid Isodrin Isopropalin 5-Keto-endrin Lenacil Leptophos Lindane Linuron Malaoxon Malathion MCP A-(2-butoxyethyl) e> Mecarbam Mephosfolan Merphosn) Metalaxyl Methabenzthiazuron Methamidophos Methidathion Methoxychlor Metolachlor Metribuzin Mevinphos Mirex Monocrotophos Monolinuron Morphothion Naled°> Nitralin Nitrofen Nitrothal-isopropyl Octachlorodipropyl ether (S 421) Omethoate

GPC elution volume range

Silica gel eluates*)

Detectors used for GLC ECD TID FPD

ml

1

2

3

4

5

120-150 100-130 100-130 105-135 110-140 125-155 125-155 120-150 140-165 120-150 120-150 125-155 115-145 105-140 130-165 120-150 110-135 135-165 130-160 120-150 110-140 120-140 110-140 110-140 115-145 105-145 140-170 125-145 115-150 150-180 120-150 130-165 125-155 130-160 125-150 120-150 130-160 115-140 125-150 130-170 115-155 115-145 135-165 105-135

5 5 5 5 5 3 3 0 5 0 4 0 0 5 0 5 5 3 0 5 5 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 5 0 0 0 0 0 2 0

0 0 0 0 0 3 3 0 0 0 2 5 0 0 0 0 0 4 0 1 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 1 5 1

0 0 0 0 0 0 0 1 0 0 0 1 5 0 0 0 0 0 0 0 0 4 0 4 5 4 0 0 0 0 0 4 0 5 3 0 0 0 4 0 4 5 0 4

0 0 0 0 0 0 0 4 0 0 0 0 1 0 1 0 0 0 5 0 0 1 4 0 0 0 2 0 5 5 0 0 0 1 1 5 0 0 2 5 1 0 0 1

0 0 0 0 0 0 0 0 0 5 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 4 0 1 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0

+ + + + + + +

110-130 140-160

5 0

0 0

0 0

0 0

0 5

+

+ + + + + + +

+ +

+ + + + + +

+

+ + +

+ +

+ +

+ + + + + + + +

+

+ +

+ +

+

+ + +

+ + +

+ + +

+ +

+

Method S 19 (updated)

321

Table 3. (contd.) Compound Oxadiazon Oxychlordane (Octachlor epoxide) Oxydemeton-methylh) Paraoxon Paraoxon-methyl Parathion Parathion-methyl Pendimethalin Pentachloroaniline Pentachloroanisole Pentachlorobenzene Permethrin Perthane Phenkapton Phenmedipham Phenthoate Phoratep) Phosalone Phosphamidon Phoxim Piperonyl butoxidee) Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Procymidone Profenofos Profluralin Propachlor Propanil Propiconazole Propoxur Propyzamide Prothiofos Pyrazophos Pyrethrins1) Quinalphos Quintozene Rabenzazole1) Resmethrine) Salithion Simazine Strobane T Sulfotep Sulprofos Tecnazene Terbacil

GPC elution volume range

Silica gel eluates *)

ml

1

2

3

4

5

Detectors used for GLC ECD TID FPD

115-145

0

0

5

0

0

+

100-160 135-165 110-140 140-170 110-140 120-150 125-155 110-140 125-160 125-165 115-145 110-140 115-145 105-130 115-150 115-145 110-140 110-145 120-150 100-130 130-170 100-135 105-145 120-150 130-155 100-125 125-150 105-130 120-150 110-130 95-125 105-145 110-140 100-130 115-155 135-165 120-160 100-130 125-165 95-135 125-160 100-130 115-155 130-160 120-145

5 0 0 0 0 0 1 5 5 5 0 5 3 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 5 0 0 0 5 0 0 0 0 5 0 0 5 0

0 0 0 0 4 4 5 0 0 0 5 1 3 0 1 2 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 0 0 4 3 0 0

0 0 1 1 1 1 0 0 0 0 1 0 0 3 4 0 4 0 0 4 0 4 4 5 4 0 5 4 0 4 4 0 5 5 4 0 5 0 0 2 0 1 0 0 0

0 0 4 4 0 0 0 0 0 0 0 0 0 3 0 0 0 1 0 1 5 0 0 0 1 0 0 2 4 2 0 0 0 1 0 0 0 0 0 4 0 0 0 0 5

0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 5 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+

+ +

+ + + + + +

+ + + + +

+

+

+

+ + + +

+ + +

+ +

+ + +

+

+ + +

+ + +

+ +

+ +

+

+

+ +

+

+

+ + +

+

+ +

+ +

+ + + +

+ +

+ +

+ +

+ + +

+

322

Method S 19 (updated)

Table 3. (contd.) Compound Terbufos Terbutryn 2,3,4,5-Tetrachloronitrobenzene Tetrachlorvinphos Tetradifon Tetramethrin Tetrasul Thionazin Tolylfluanid 1 ) Triadimefon Triadimenol Tri-allate Triamiphos Triazophos Triazoxide Trichlorfon h > Trichloronat Trifluralin Vinclozolin

G P C elution volume range ml

Silica gel eluates *)

Detectors used for GLC ECD TID FPD

1

2

3

4

5

0 0

3 0

0 1

0 2

0 0

+

115-175 130-160 120-140 120-150 120-150 125-155 120-150 105-135 100-130 100-130 120-150 125-160 120-140 165-195 100-140 110-140 100-130 100-130

5 0 0 0 5 0 0 0 0 0 0 0 0 0 5 5 0

0 0 5 0 0 1 3 0 0 5 0 0 0 0 1 0 4

0 4 0 5 0 4 3 3 0 0 0 4 0 0 0 0 1

0 1 0 0 0 0 0 3 4 0 0 1 5 0 0 0 0

0 0 0 0 0 0 0 0 2 0 4 0 0 4 0 0 0

+

125-155

+ +

+ +

+

+

+

+

+

+ + +

+

+ +

+

+ +

+ +

+ +

+

+

+

+

+

Footnotes to Table 3 *> Silica gel eluates: 1, n-hexane-toluene 65:35 v / v ; 2, toluene; 3, toluene-acetone 95 :5 v / v ; 4, tolueneacetone 8 : 2 v / v ; 5, acetone. Figures in the Table indicate recovery rates: 5 = more than 9 0 % ; 4 = approx. 6 0 - 9 0 % ; 3 = approx. 3 0 - 6 0 % ; 4 = approx. 1 0 - 3 0 % ; 1 = less than 1 0 % ; 0 = not recovered. a) Extract the analytical sample with the addition of potassium acetate; for G L C , inject an aliquot of the sample solution with the addition of acetic acid. b) Peak height depending on solvent.

Gas-chromatographic column must be well conditioned. When chromatographed on silica gel, an additional peak will be observed with a longer retention time on GLC. e > Analyzed by GC/MS. ^ Also from merphos. 8) Inject standard solution within 2 min after injection of sample solution. h) Improved recovery when eluted with further 8 ml acetone. f) Extract the analytical sample with the addition of citric acid and oxalic acid. j) Degradation product of dicofol. k) Degradation to p,p'-dichlorobenzophenone. J) For analysis, proceed with complete exclusion of light. m) Following chromatography on silica gel, disulfoton sulphoxide is observed in eluate 5. n) Partly oxidized to DEF. o) When chromatographed on silica gel, varying recoveries are observed. p) Partly decomposed during chromatography on silica gel.

Natural Pyrethrins, Piperonyl Butoxide Apples, cucumbers, endives, lettuce, mangold, plums, potatoes, red currants, spinach, sugar beet (edible root), tomatoes

s 22

Gas-chromatographic determination

(German version first published 1985, revised 1991)

1 Introduction The method permits the identification and quantitative determination of the residues of natural pyrethrins and of the synergist piperonyl butoxide which is contained in nearly all pyrethrin formulations. Natural pyrethrins are composed of a mixture of pyrethrin I (main component; highest insecticidal activity) and II, cinerin I and II, and jasmolin I and II. In gas-chromatographic measurements, total residues of the pyrethrins are calculated from the peak of pyrethrin I or, when resolution is poor, from the peaks of pyrethrin I plus jasmolin I.

2 Outline of method The compounds are extracted from plant material with acetone. The extract is filtered, and the compounds are partitioned into dichloromethane. The dichloromethane phase is divided into two, to enable the separate analysis of the pyrethrins and piperonyl butoxide. The portion used for the pyrethrin analysis is cleaned up by chromatography on a Florisil column. The pyrethrins are determined by electron capture gas chromatography. The portion used for the analysis of piperonyl butoxide is cleaned up by gel permeation chromatography. Piperonyl butoxide is brominated to yield 4,5,7-tribromo-6-propyl-l,3benzodioxol, which is partitioned into n-hexane and determined by electron capture gas chromatography.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schtill) Filtration flask, 500-ml Separatory funnels, 1-1 and 50-ml Glass funnels, 8 cm and 5.5 cm dia. Fluted filter paper, 18.5 cm and 12.5 cm dia. Graduated cylinder, 500-ml Round-bottomed flasks, 500-ml, 250-ml and 50-ml, with ground joints Rotary vacuum evaporator, 30 °C bath temperature

324

Method S 22

Chromatographic tube, 15 mm i.d., 30 cm long Equipment for gel permeation chromatography, e.g. chromatographic tube and accessories as described in Cleanup Method 4 (see pp. 65 ff, Vol. 1) Volumetric flasks, 20-ml and 10-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Acetone, p. a. Cyclohexane, p. a. Dichloromethane, p. a. Diethyl ether, p. a. Ethyl acetate, p. a. n-Hexane, p. a. Petroleum ether, boiling range 40-60°C, prepared as follows: Fill a chromatographic tube (2 cm i. d.) to a height of 20 cm with aluminium oxide (Alumina Woelm B, activity grade Super I; ICN Biomedicals), gently tapping the tube walls. Top the packing with a 2-cm layer of sodium sulphate. Add the petroleum ether, and set the flow rate for the solvent to pass through the column at approx. 5 ml/min. Discard the first 90 ml. The column suffices to purify 500 ml solvent Toluene, p. a. Cyclohexane + ethyl acetate mixture 1:1 v/v Eluting mixture 1: diethyl ether + petroleum ether 1:9 v/v Eluting mixture 2: diethyl ether + petroleum ether 3:7 v/v Pyrethrin standard solutions: 1, 10 and 100 ng/ml pyrethrum extract (approx. 35% pyrethrin I content) in toluene Piperonyl butoxide standard solutions: 1, 10 and 100 fig/ml toluene Piperonyl butoxide calibration solution, equivalent to 1.0 p,g/ml piperonyl butoxide in n-hexane: Transfer 2 ml of the 10 [xg/ml piperonyl butoxide standard solution into a 50-ml round-bottomed flask. Remove the solvent by rotating the flask in the hand. Dissolve the residue in 1 ml dichloromethane and add 0.1 ml of the bromine-iodine mixture (see 8. Important points). Swirl the flask, stopper it with a ground stopper, and let it stand for 30 min at room temperature. Add 10 ml sodium acetate solution and 5 ml sodium arsenite solution, and continue to process as described in 6.3 Bromine + iodine mixture: Dissolve 5 g finely powdered iodine p.a. in 95 g bromine p.a. Sodium acetate solution, 20 g/100 ml CH3COONa • 3 H2O p. a. Sodium arsenite solution, 10 g/100 ml NaAsO2 p. a. Sodium sulphate, p.a., anhydrous Florisil, 60-100 mesh, deactivated with 5% water: Heat a weighed sample of Florisil for at least 8 h at 130 °C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared; next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Styrene-divinylbenzene copolymer (2% DVB), e.g. Servachrom XAD-2, 50-100 jim (Serva) or Bio-Beads S-X2; 200-400 mesh (Bio-Rad Laboratories)

Method S 22

325

Glass wool Argon + methane mixture 9:1 v/v Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

6 Procedure 6.1 Extraction Homogenize 100 g of the analytical sample (G) with 150 ml acetone for 3 min. Suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel and wash the filter cake with 80 ml acetone. Transfer the filtrate together with 200 ml dichloromethane into a 1-1 separatory funnel and shake for 2 min. Drain the dichloromethane layer and extract the aqueous phase twice more with 100-ml portions of dichloromethane. Dry the combined dichloromethane phases on sodium sulphate, filter through a fluted filter paper containing a little sodium sulphate into a 500-ml graduated cylinder, rinse flask and filter with a further 80 ml of dichloromethane, and mix the solution thoroughly. Divide the filtrate into two equal halves for the separate pyrethrin and piperonyl butoxide analyses, and rotary-evaporate the two halves to near dryness. Remove the last traces of solvent by swirling the flasks in the hand. 6.2 Cleanup 6.2.1 Column chromatography (pyrethrins) Insert a glass wool plug into the bottom of a chromatographic tube, add 40 ml eluting mixture 1, trickle in 8 g Florisil, allow to settle, and drain the solvent just to the top of the Florisil. Dissolve the residue derived from 6.1 in 5 ml eluting mixture 1, and add to the column. Rinse the flask with 5 ml eluting mixture 1, add the rinsing to the column, and allow to percolate. Repeat this step. Elute the co-extractives with a total of 60 ml eluting mixture 1 and discard this fraction. Next elute the pyrethrins with 50 ml eluting mixture 2 from the column. Rotaryevaporate the eluate to near dryness, and remove the last traces of solvent by swirling the flask in the hand. Dissolve the residue in toluene and make up with toluene to 10 ml (VEnd). 6.2.2 Gel permeation chromatography (piperonyl butoxide)

To carry out the gel permeation chromatographic cleanup, follow the instructions given in Cleanup Method 4 (see pp. 65 ff, Vol. 1), but use cyclohexane-ethyl acetate mixture as eluting solvent. Dissolve the residue derived from 6.1 in 2 ml cyclohexane-ethyl acetate mixture (VEx) and transfer 1 ml of this solution (VR1) to the gel permeation column. Evaporate the resulting eluate to dryness as described in 6.2.1.

326

Method S 22

6.3 Bromination (piperonyl butoxide) Dissolve the residue derived from 6.2.2 in 1 ml dichloromethane, while carefully rotating the flask to wet its walls, and add 5 drops of the bromine-iodine mixture (cf. 8. Important points). Swirl the flask, stopper it with a ground stopper, and let it stand for 10 min at room temperature. Add 10 ml sodium acetate solution and 5 ml sodium arsenite solution, and swirl. Then transfer the solution with 10 ml hexane into a 50-ml separatory funnel and shake for 2 min. Filter the organic phase through a little sodium sulphate into a 20-ml volumetric flask. Extract the aqueous phase once more in the same way, filter, and wash the filter with a little hexane. Make up the combined filtrates to the mark (VEnd) with hexane. 6.4 Gas-chromatographic determination Inject aliquots (Vi) of the solutions derived from 6.2.1 (pyrethrins) or from 6.3 (piperonyl butoxide) into the gas chromatograph. Operating conditions 6.4.1 Pyrethrins Gas chromatograph Column

Attenuation Recorder

Carlo Erba Fractovap 4160 with on-column injector Fused silica capillary, 0.32 mm i.d., 15 m long; coated with OV-1, crossbond, film thickness 0.10-0.15 fim (Carlo Erba Mega) Programmed to rise at 60°C/min from 60 to 180 °C, and at 3°C/min from 180 to 240 °C, then isothermal at 240 °C for 20 min Cold on-column at 60 °C oven temperature with secondary cooling 63 Ni electron capture detector ECD HT-25, ECD Control Module 251, constant current, pulse width 1 us Temperature 290 °C Nitrogen carrier, 3 ml/min Nitrogen purge gas, 50 ml/min 256-2048 10 mV; chart speed 5 mm/min

Injection volume

1 JJLI

Column temperature Injection technique Detector Gas flow rates

Retention times for cinerin I 5 min 41 s jasmolin I 6 min 13 s pyrethrin I 6 min 26 s cinerin II 10 min 4 s jasmolin II 11 min 31 s pyrethrin II 11 min 52 s Aldrin had a retention time of 3 min 35 s under these conditions. 6.4.2 Piperonyl butoxide Gas chromatograph Carlo Erba Fractovap 2150 Column Glass, 2 mm i.d., 2 m long; packed with 29b OV-210 + 1% OV-17 on Gas Chrom Q, 100-120 mesh

Method S 22 Column temperature Injection port temperature Detector

Gas flow rates

327

220 °C 250 °C 63 Ni electron capture detector ECD HT-20, ECD Control Module 250 Temperature 300 °C Nitrogen carrier, 30 ml/min Nitrogen purge gas, 40 ml/min 128 1 mV; chart speed 5 mm/min 3 \i\

Attenuation Recorder Injection volume Retention time for 4,5,7-tribromo6-propyl-l,3-benzodioxol 8 min 12 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of appropriately diluted pyrethrin standard solutions or piperonyl butoxide calibration solutions, respectively, equivalent to 0.1 to 2 ng pyrethrins or piperonyl butoxide, into the gas chromatograph. Plot the heights of the peaks obtained vs. ng pyrethrins or piperonyl butoxide. For chromatograms of the pyrethrins, evaluate the pyrethrin I peak (capillary column) or the pyrethrin I + jasmolin I peak (packed column, see 8. Important points). Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the standard solutions should be injected. For the heights of the peaks obtained for the sample solutions, read the appropriate amounts of pyrethrins or piperonyl butoxide from the corresponding calibration curve.

7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples, fortified with pyrethrins at levels of 0.05 to 1.0 mg/kg, averaged 91% with a standard deviation of 12%. The recoveries from untreated control samples, fortified with piperonyl butoxide at levels of 0.1 to 1.0 mg/kg, averaged 72% with a standard deviation of 18%. The recoveries for the individual compounds and analytical materials are given in the Table. The limit of detection for the pyrethrins was 0.02 mg/kg, and the limit of determination was 0.05 mg/kg. For piperonyl butoxide, the limit of detection was 0.03 mg/kg, and the limit of determination was 0.1 mg/kg.

328

Method S 22

Table. Percent recoveries from plant material fortified with natural pyrethrins at 0.05-1.0 mg/kg, and piperonyl butoxide at 0.1-1.0 mg/kg. ., A . . . Analytical material Apples Cucumbers Endives Lettuce Mangold Plums Potatoes Red currants Spinach Sugar beet (edible root) Tomatoes

„ , . Pyrethrins

Piperonyl , . .,

105 96 85 97 87 94 94 92 77 82 87

105 60 61 70 67 66 84 62 61 70 91

7.3 Calculation of residues The residue R, expressed in mg/kg, of the total pyrethrins is calculated from the following equation:

w A -vEnd where G

= sample weight (in g)

VEnd = terminal volume of sample solution from 6.2.1 (in ml) Vi

= portion of volume VEnd injected into gas chromatograph (in \i\)

WA

= amount of total pyrethrins for Vj read from calibration curve (in ng)

The residue R, expressed in mg/kg piperonyl butoxide, is calculated from the following equation: R

2

w A .y Ex .v End VR1VrG

where G

= sample weight (in g)

VEx

= volume of solvent used to dissolve the evaporation residue from 6.1 (in ml)

VR1

= portion of volume VEx injected for gel permeation chromatography in 6.2.2 (volume of sample loop) (in ml)

VEnd = terminal volume of sample solution from 6.3 (in ml) Vi

= portion of volume VEnd injected into gas chromatograph (in ul)

WA

= amount of piperonyl butoxide for W{ read from calibration curve (in ng)

Method S 22

329

8 Important points The composition of the mixture of natural pyrethrins is practically constant. The main component is pyrethrin I with a proportion of approx. 35%. It is, therefore, chosen to represent the total residues of the pyrethrins. In preparing the calibration curve, the pyrethrin I peak height is plotted vs. the total of the pyrethrins contained in the standard solution aliquot injected. If a gas chromatograph fitted with capillary columns is not available, determination of the pyrethrins can also be performed on packed columns under the following conditions: Gas chromatograph Column Column temperature Injection port temperature Detector Carrier gas flow rate Attenuation Recorder Injection volume Retention times for cinerin I jasmolin I + pyrethrin I cinerin II jasmolin II + pyrethrin II

Hewlett-Packard 5880 A Glass, 2 mm i.d., 0.8 m long; packed with 2% OV-101 on Chromosorb W-HP, 80-100 mesh 190 °C 250 °C 63 Ni electron capture detector Temperature 300 °C Argon-methane, 30 ml/min 2 5 -2 8 Chart speed 5 mm/min 1-3 ul 1 min 27 s 2 min 3 s 4 min 28 s 6 min 26 s

Aldrin had a retention time of 52 s under these conditions. The bromination (cf. 6.3) can also be carried out in acetic acid; however, recoveries tend to be low and extremely variable with cucumbers, lettuce, mangold, plums, potatoes and red currants. Noticeably greater variability of yields has also been recorded for standard solutions when bromination is carried out in acetic acid, as compared to dichloromethane. In order to check the reliability of the bromination reaction, two or three piperonyl butoxide calibration solutions (see 4. Reagents) should be prepared along with each series of sample solutions. Provided a mass selective detector (MSD) being available, it is recommended to analyze piperonyl butoxide underivatized on a capillary column by MSD gas chromatography, in order to circumvent the difficulties encountered with the bromination of the compound. For this end proceed as follows: Dissolve the residue of the cleaned-up eluate derived from 6.2.2 in a suitable volume of hexane (VEnd), and inject an aliquot of this solution (Vj) into the gas chromatograph. The following operating conditions proved to be favourable: Gas chromatograph Column

Hewlett-Packard 5890/5970 B, equipped with cold injection system KAS (Gerstel) Fused silica capillary HP-1 (methyl silicone), 0.2 mm i.d., 12 m long; film thickness 0.33 |am (HewlettPackard)

330

Method S 22

Column temperature Injection technique

Detector Carrier gas flow rate Printer Injection volume Retention time for piperonyl butoxide SIM parameters for piperonyl butoxide

Programmed to rise at 25°C/min from 70 to 250 °C, then isothermal at 250 °C for 5 min Splitless for 48 s Temperature of KAS injection system: Programmed to rise at 12°C/s from 70 to 300 °C, then isothermal at 300 °C for 3 min Mass selective detector HP 5970 El, 70 eV, interface 280 °C Helium, < 1 ml/min Paint Jet (Hewlett-Packard) 10 min 8 s Start 8 min, stop 12 min Dwell time for m/z = 176 and 338: 100 ms each

Perform quantitation in the SIM mode with the aid of external standards. Prepare the piperonyl butoxide standard solutions required by dissolving the compound in cleaned-up extract solutions derived from untreated control samples. Standard solutions and sample solutions should contain comparable concentrations of piperonyl butoxide. The recoveries from untreated control samples of apples and lettuce, fortified with piperonyl butoxide at levels of 0.01 to 1.0 mg/kg and analyzed by MSD gas chromatography, averaged 83% with a standard deviation of 8%. The limit of determination was 0.01 mg/kg.

9 References S.W. Head, The quantitative determination of pyrethrins by gas liquid chromatography, Part I: Detection by electron capture, Pyrethrum Post 8, No. 4, 3-7 (1966). H.-R Thier, Analysengang zur Ermittlung von Pestizid-Riickstanden in Pflanzenmaterial, Dtsch. Lebensm. Rundsch. 68, 345-350 and 397-401 (1972).

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, W.D. Weinmann, H.-G. Nolting and /. Siebers Institute of Food Chemistry, University of Minister, H.-P. Thier

Method S 22

15

331

10

Chromatogram 1. Standard solution of natural pyrethrins, representing 4 ng of the mixture. Conditions as decribed in 6.4.1. Chromatogram 2. Standard solution of natural pyrethrins, representing 8 ng of the mixture, on a packed column (ECD). Conditions as described in 8.

332

Method S 22 TIC of Pip.Butoxid Abundance

7000000 6000000 5000000 4000000 3000000 2000000-\ 1000000 0Time (mln.) Scan 776 (10.143 min) of Pip.Butoxid

200 Mass /Charge

300

Chromatogram 3. Standard solution of piperonyl butoxide. Total ion current (A) and corresponding mass spectrum of piperonyl butoxide (B). Capillary column, MSD; conditions as described in 8.

Pyrethroids Apples, carrots, cauliflower, celeriac (bulbs), cherries, cucumbers, endives, grapes, maize (kernels), onions, potatoes, rape (green matter and seeds), sugar beet (edible root), tomatoes, wheat (grains) Soil, water

s 23 Gas-chromatographic determination

(German version first published 1985, revised 1991)

1 Introduction The method permits the identification and quantitative determination of the residues of the pyrethroids bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, flucythrinate, and permethrin.

2 Outline of method Following homogenization of plant material with a mixture of hexane and acetone, interfering co-extractives are removed on a Florisil column. If required, a further cleanup can be achieved by chromatography on a silica gel-activated charcoal column. With oil-containing plant material, column chromatography is preceded by an acetonitrile-hexane partition. The pyrethroid residues are extracted from water with hexane, and from soil with a mixture of an ammonium chloride solution and acetone. The compounds are determined by electron capture gas chromatography.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schiill) Filtration flask, 1-1 Graduated cylinders, 500-ml and 100-ml Round-bottomed flasks, 500-ml, 250-ml and 100-ml Rotary vacuum evaporator, 30-40 °C bath temperature Separatory funnels, 500-ml and 250-ml Laboratory mechanical shaker Erlenmeyer flask, 500-ml, with ground stopper Glass funnel Fluted filter paper Chromatographic tube 1, 15 mm i.d., 45 cm long

334

Method S 23

Chromatographic tube 2, 12 mm i.d., 20 cm long Volumetric flasks, 5-ml to 500-ml Test tubes, graduated, with ground stoppers Gas chromatograph equipped with electron capture detector Microsyringe, 10-jj.l

4 Reagents Acetone, technically pure, dist. Acetonitrile, p.a. Diethyl ether, technically pure, dist. n-Hexane, technically pure, dist. Petroleum ether, boiling range 40-60°C, prepared as follows: Fill a chromatographic tube (2 cm i.d.) to a height of 20 cm with aluminium oxide (Alumina Woelm B, activity grade Super I; ICN Biomedicals), gently tapping the tube walls. Top the packing with a 2-cm layer of sodium sulphate. Add the petroleum ether, and set the flow rate for the solvent to pass through the column at approx. 5 ml/min. Discard the first 90 ml. The column suffices to purify 500 ml solvent Hexane + acetone mixture 8:2 v/v Petroleum ether + diethyl ether mixture 7:3 v/v Eluting mixture 1: hexane + toluene 8:2 v/v Eluting mixture 2: hexane + toluene 2:8 v/v Eluting mixture 3: hexane + toluene 4:6 v/v Extraction mixture: acetone + ammonium chloride solution 1:1 v/v Pyrethroid standard solutions: 0.1-5 ng/ml of each compound in hexane Ammonium chloride solution, 5 g/100 ml NH4C1 p.a. Sodium chloride solution, 5 g/100 ml NaCl p.a. Phosphate buffer solution (pH 6.7): 68 g/1 KH2PO4 + 87 g/1 K2HPO4 Sodium chloride, p.a. Sodium sulphate, p.a., anhydrous Activated charcoal, p.a. (Merck No. 2186), acetone-washed (150 ml/10 g) and air-dried Filter aid, e.g. Celite 545 Florisil, 60-100 mesh, deactivated with 5% water: Heat a weighed sample of Florisil for at least 8 h at 130 °C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared; next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Silica gel 60, 0.063-0.200 mm (Merck No. 7734) Cottonwool Argon + methane mixture 9:1 v/v Nitrogen, re-purified

Method S 23

335

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material (except rape)

Homogenize 50 g of the analytical sample (G) with 10 g filter aid and 200 ml hexane-acetone mixture for 3 min. Suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake with 50 ml of the same solvent mixture. Transfer the filtrate to a graduated cylinder and make up the organic phase to a total volume of 250 ml (VEx) with hexane-acetone mixture (the lower aqueous phase is not taken into account). Carefully mix the organic phase, then dry an aliquot (VR1) on sodium sulphate and filter into a 250-ml round-bottomed flask. Wash the sodium sulphate with 30 ml acetone, and rotary-evaporate the combined filtrates to near dryness with 30 °C bath temperature. 6.1.2 Rape Homogenize 50 g of the analytical sample (G) with 10 g filter aid and 200 ml hexane-acetone mixture for 3 min. Suction-filter the homogenate and make up the organic phase of the filtrate to a total volume of 250 ml (VEx), as described in 6.1.1. Shake 50 ml of the organic phase (VR1) twice with 50-ml portions of acetonitrile. Combine the acetonitrile phases, add 200 ml sodium chloride solution, and shake with three 50-ml portions of hexane. Dry the combined hexane phases on sodium sulphate and filter into a 500-ml round-bottomed flask. Wash the sodium sulphate with 30 ml hexane, and rotary-evaporate the combined filtrates to near dryness with 30 °C bath temperature. 6.1.3 Soil Shake 50 g soil (G) with 100 ml extraction mixture for 60 min in a 500-ml Erlenmeyer flask on a mechanical shaker. Suction-filter the extract through a fast flow-rate filter paper in a Buchner porcelain funnel. Re-extract the filter cake and filter the extract as described above. Wash the filter cake twice with 30-ml portions of the extraction mixture. Make up the combined filtrates to a volume of 300 ml (VEx) with extraction mixture, and mix carefully. Then transfer 100 ml of the filtrate (VR1) to a separatory funnel, add 50 ml phosphate buffer solution, and shake with three 100-ml portions of hexane. Dry the combined organic phases on sodium sulphate and continue as described in 6.1.2. 6.1.4 Water Add 1 g sodium chloride to 50 g water (G), and shake with three 50-ml portions of hexane. Dry the combined extracts on sodium sulphate and rotary-evaporate as described in 6.1.2. For

336

Method S 23

extracts from water samples containing a low amount of co-extractives, proceed to the gaschromatographic determination without further cleanup. 6.2 Cleanup 6.2.1 Column chromatography on Florisil Pour 40 ml hexane into a chromatographic tube (type 1), sprinkle in 15 g Florisil, and cover the Florisil with an approx. 2-cm layer of sodium sulphate. Drain the supernatant solvent to the top of the sodium sulphate layer. Quantitatively transfer the residue derived from 6.1 onto the column with a total of 5 ml hexane. Let the solution percolate into the column packing, rinse the flask with 20 ml eluting mixture 1, and add the rinsings to the column. Wash the column with a further 80 ml of eluting mixture 1 and discard the eluate. Next elute the pyrethroids from the column with 100 ml eluting mixture 2. Collect this eluate, and rotaryevaporate to near dryness with 40 °C bath temperature. 6.2.2 Column chromatography on silica gel-activated charcoal

For samples of apples, wheat, onions and rape containing pyrethroid residues of less than 0.1 mg/kg, a supplemental cleanup step is recommended. Proceed as follows. Mix 4.5 g silica gel and 0.3 g activated charcoal, slurry the mixture with 15 ml hexane, and pour the slurry into a chromatographic tube (type 2). Prewash the column packing with 50 ml eluting mixture 3. Dissolve the residue derived from 6.2.1 portionwise in a total of 3 ml hexane, add the solutions to the column, and allow to percolate. Rinse the flask with 10 ml eluting mixture 3 and also add this solution to the column. Next elute the pyrethroids with a total of 70 ml eluting mixture 3. Rotary-evaporate the eluate to near dryness with 40 °C bath temperature. 6.3 Gas-chromatographic determination Dissolve the residue derived from 6.1.4 or 6.2 in hexane and make up with hexane to a definite volume, e.g. 5 ml (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection technique Detector Gas flow rates

Carlo Erba Fractovap 4160 with on-column injector Fused silica capillary, 0.32 mm i.d., 15 m long; coated with OV-1, crossbond, film thickness 0.10-0.15 \xm (Carlo Erba Mega) Programmed to rise at 60°C/min from 70 to 180 °C, and at 3°C/min from 180 to 245 °C, then isothermal at 245 °C for 20 min Cold on-column at 70 °C oven temperature with secondary cooling 63 Ni electron capture detector ECD HT-25, ECD Control Module 251, pulse width 1 fis Temperature 290 °C Nitrogen carrier, 3 ml/min Nitrogen detector purge gas, 50 ml/min

Method S 23 Recorder Injection volume Retention times for bifenthrin fenpropathrin cyhalothrin cis-permethrin trans-permethrin cyfluthrin (mixture of isomers) cypermethrin I cypermethrin II cypermethrin III cypermethrin IV flucythrinate I flucythrinate II fenvalerate I fenvalerate II deltamethrin

337

10 mV; chart speed 5 mm/min lul

8 min 36 s 8 min 36 s 10 min 36 s 11 min 55 s 12 min 13 s 13 min 24 s 13 min 48 s 14 min 4 s 14 min 14 s 14 min 24 s 14 min 30 s 15 min 16 min 16 min 36 s 17 min 58 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of each pyrethroid standard solution into the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng of each compound. Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the standard solutions should be injected. For the areas or heights of the peaks obtained for the sample solutions, read the appropriate amounts of the compound identified from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with the pyrethroids at levels of 0.01 to 10 mg/kg. The recoveries ranged from 62 to 122%; see Table. Blanks exceeding 0.001 mg/kg were observed only from extracts of apples, wheat, onions and rape, when the additional cleanup step was omitted. The limit of detection was 0.005 mg/kg, and the limit of determination was 0.03 mg/kg. 7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified pyrethroid is calculated from the following equation: _

WAVFYVR

Added mg/kg 0.03-5 0.05-2 0.1-10 0.1-0.5 0.05-1 0.02-1 0.05-0.1 0.03-0.5 0.03-1 0.1-10 0.1-5 0.03-5 0.05-1 0.03-2 0.05-2 0.05-1 0.03-1 0.01-1

Analytical material

Apples Carrots Cauliflower Celeriac (bulbs) Cherries Cucumbers Endives Grapes Maize (kernels) Onions Potatoes Rape (green matter) Rape (seeds) Sugar beet (edible root) Tomatoes Wheat (grains) Soil Water 66- 69 100-101 92-118 82- 92 68-125 107-111 95-120 104

96-100

86- 90

110-115

87-116 119 115-117

104

Cyfluthrin

101-105 102-115

Bifenthrin

Table. Percent recoveries from plant material, soil and water, fortified with pyrethroids.

74- 78 92- 94 94-108 102-105 67-104 104-109

101-104

79-100 100

89- 94 88-106

Cyhalothrin

74-107 77- 95 85-122 94-112 94-101

85- 95 86-102 80- 83 82- 88 94-110 75- 90 96-103 80-113 85-114 85-118 79-105 91-105

Cypermethrin

70-100 82- 95 86- 95 90-100 95- 97

81-110 85- 96 75- 79 68- 91 94-100 84-107 91-103 87-130 97-115 85-109 79-117 91-103

Deltamethrin

CO

w oo

Apples Carrots Cauliflower Celeriac (bulbs) Cherries Cucumbers Endives Grapes Maize (kernels) Onions Potatoes Rape (green matter) Rape (seeds) Sugar beet (edible root) Tomatoes Wheat (grains) Soil Water

Analytical material

Table, (contd.)

0.03-5 0.05-2 0.1-10 0.1-0.5 0.05-1 0.02-1 0.05-0.1 0.03-0.5 0.03-1 0.1-10 0.1-5 0.03-5 0.05-1 0.03-2 0.05-2 0.05-1 0.03-1 0.01-1

»

77 100 100

77

74 80

91-94

87

97

Fenpropathrin

76-100 76-100 79- 88 90- 97 93- 97

87- 99 85- 98 79- 81 91-100 96-109 74- 95 95-101 84-122 71-111 85-102 79-100 91-130

Fenvalerate

76- 83 95-101 81- 96 81-102 74- 82 90- 98

73- 81

75- 77

Flucythrinate

90-100 90- 92 73-122 84-100 98- 99

84-106 86-115 81- 85 75- 92 92-105 87-105 86- 88 95-128 96- 98 85-114 80-102 62- 83

Permethrin

CO CO CO

N> CO

CO

I

urt

11

Figure. Pyrethroids in tomatoes; conditions as described in 6.3. Chromatogram 1: Standard mixture representing 0.1 ng each of bifenthrin, cyhalothrin, cyfluthrin, and flucythrinate. Chromatogram 2: Untreated control sample. Chromatogram 3: Untreated control sample fortified with 0.1 mg/kg each of bifenthrin, cyhalothrin, cyfluthrin, and flucythrinate.

II

c c

CO

I

Method S 23

341

where G = sample weight (in g) VEx = total volume of organic phase after addition of solvent mixture to filtered extract from plant sample in 6.1.1 or 6.1.2, or total volume of filtered extract from soil sample in 6.1.3 (in ml) VR1 = portion of volume VEx used for cleanup (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) Vj = portion of volume VEnd injected into gas chromatograph (in ul) WA = amount of identified pyrethroid for Vj read from calibration curve (in ng)

8 Important points Emulsions, that may form during the extraction, can be broken with centrifugation. To prevent false results, the elution of the pyrethroids from the chromatographic columns (see 6.2.1 and 6.2.2) should be checked with each new batch of adsorbent. If the compounds are not completely eluted, either the amounts of the eluting mixtures or their polarities must be increased. Additionally, the ranges of the elution volumes of the different pyrethroids can shift according to the activity of the Florisil: When its activity is low, permethrin can appear in the forerun, whereas flucythrinate will elute later (after the main fraction of the eluate) with high Florisil activity. Since the Florisil activity may alter during storage, only freshly prepared adsorbent should be used. Extracts containing residues of fenpropathrin should be evaporated carefully, otherwise low recoveries can be encountered. Several pyrethroids exhibit very similar retention times under the gas-chromatographic operating conditions described in 6.3. It is therefore highly recommended to confirm the results obtained by using a gas-chromatographic column of different polarity or by employing a mass selective detector (MSD). Tetramethrin and pyrethrins can also be analyzed by this method. For this purpose, first elute the pyrethroids from the Florisil column as described in 6.2.1. Then elute tetramethrin and the pyrethrins with an additional 100 ml of petroleum ether-diethyl ether mixture, and proceed with the gas-chromatographic determination as described in 6.3. Resmethrin and phenothrin cannot be analyzed by this method, because these compounds are not detected by the electron capture detector with sufficient sensitivity. If a gas chromatograph fitted with capillary columns is not available, determination of the pyrethroids can also be performed on packed columns, under the following conditions: Gas chromatograph Column Column temperature Injection port temperature

Hewlett-Packard 5880 A Glass, 2 mm i.d., 1.2 m long; packed with 10% SE-30 on Chromosorb W-HP, 80-100 mesh Programmed to rise at 3 °C/min from 200 to 270 °C, then isothermal at 270 °C for 5 min 270 °C

342

Method S 23

Detector Carrier gas flow rate Attenuation Recorder Injection volume Retention times for fenpropathrin permethrin isomer mixture cypermethrin isomer mixture fenvalerate I fenvalerate II deltamethrin

63

Ni electron capture detector Temperature 300 °C Argon-methane, 30 ml/min 28

Chart speed 5 mm/min 1-3 [i\

9 min 12 s 13 min 44 s 16 min 14 s 18 min 10 s 18 min 46 s 20 min 14 s

9 References R.A. Chapman and C.R. Harris, Extraction and liquid-solid chromatography cleanup procedures for the direct analysis of four pyrethroid insecticides in crops by gas-liquid chromatography, J. Chromatogr. 166, 513-518 (1978). K. Naumann, Chemie der synthetischen Pyrethroid-Insektizide, in: R. Wegler, Chemie der Pflanzenschutz- und Schadlingsbekampfungsmittel, Volume 7, Springer-Verlag, BerlinHeidelberg-New York 1981. J. Siebers and H.-G. No Iting, Analysenmethode zur Bestimmung von Pyrethroiden in verschiedenen pflanzlichen Lebensmitteln, Wasser und Boden, Nachrichtenbl. Dtsch. Pflanzenschutzdienstes Braunschweig 34, 166-170 (1982).

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, J. Siebers and H. Ko'hle

Organotin Compounds Apples, aubergines, beans (green), celeriac (leaves and bulbs), grapes, melons, nectarines, peaches, plums, strawberries, sugar beet (foliage and edible root), tomatoes Soil, water

s 24 Gas-chromatographic determination

(German version published 1985)

1 Introduction The method permits the identification and quantitative determination of organotin compounds in which the central tin atom carries three identical organic substituents, such as derivatives from triphenyltin (fentin acetate, fentin chloride, and fentin hydroxide) or tricyclohexyltin (azocyclotin and cyhexatin), and tris(2-methyl-2-phenylpropyl)tin (fenbutatin oxide). Compounds containing the same three substituents are determined simultaneously since they are converted to the same derivatives.

2 Outline of method Residues of organotin compounds are extracted from plant material, soil and water with acetone in the presence of hydrobromic acid. The homogenate is filtered; the filtrate is extracted with n-hexane, and the hexane phase is evaporated to dryness. The compounds are reacted with methyl magnesium chloride in ethereal solution to yield the corresponding methyl derivatives R3Sn-CH3: XMgCl

The derivatives are cleaned up by column chromatography on Florisil and are determined by gas chromatography using a flame photometric detector.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Laboratory mechanical shaker Buchner porcelain funnel, 11 cm dia. Filter paper, 11 cm dia. Filtration flask, 1-1 Separatory funnels, 1-1, 500-ml and 100-ml

344

Method S 24

Round-bottomed flasks, 1-1 and 250-ml Rotary vacuum evaporator, 50 °C bath temperature Chromatographic tube, 20 mm i.d., 30 cm long, with PTFE stopcock Gas chromatograph equipped with sulphur-specific flame photometric detector Microsyringe, 10-ul

4 Reagents Acetone, nanograde (Promochem) Diethyl ether, p.a. n-Hexane, nanograde (Promochem) Derivative standard solutions: Prepare cyhexatin, fentin hydroxide, and fenbutatin oxide solutions with 0.2, 1, 10 and 100 M-g/ml acetone. Evaporate 5 or 10 ml of each to dryness, allow to react with methyl magnesium chloride as described in 6.2, and make up again to 5 or 10 ml with acetone Methyl magnesium chloride solution: methyl magnesium chloride (reagent grade), 20 g/100 ml in tetrahydrofuran (Merck-Schuchardt No. 820 310) Hydrobromic acid, p.a., minimum 47 g/100 g (Merck No. 307) Hydrochloric acid, p.a., cone. Sodium sulphate, p.a., anhydrous Florisil, 60-100 mesh Glass wool Air, synthetic Hydrogen, re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material, soil Homogenize 100 g of plant material (G) with 20 ml hydrobromic acid and 200 ml acetone for 3 min. To 100 g of soil (G), add 100 ml water and extract the mixture in the same manner or by using a mechanical shaker. Suction-filter the homogenate through a filter paper in a Buchner porcelain funnel. Transfer the filter cake back into the blendor jar, extract with 200 ml acetone for a further 3 min and suction-filter again. Shake the combined filtrates twice with 200-ml portions of hexane, dry the combined organic phases on sodium sulphate, and rotary-evaporate to dryness.

Method S 24

345

6.1.2 Water

Transfer 100 ml of water (G) into a separatory funnel, add 200 ml acetone and 20 ml hydrobromic acid, and shake the solution twice with 200-ml portions of hexane. Dry the combined organic phases on sodium sulphate and rotary-evaporate to dryness. 6.2 Derivatization Dissolve the residue derived from 6.1 in 20 ml diethyl ether, add 3 ml methyl magnesium chloride solution, swirl, and allow to stand for approx. 5 min. Next add 10 ml water and 1 ml hydrochloric acid, and transfer the contents of the flask into a 100-ml separatory funnel. Rinse the flask with 10 ml diethyl ether, add this to the separatory funnel, and shake. Discard the lower aqueous phase. Dry the organic phase on sodium sulphate and rotary-evaporate to dryness. 6.3 Column chromatography (not required for water samples) Fill the chromatographic tube with approx. 30 ml hexane, push a glass wool plug through to the lower end, and trickle in 8 g Florisil. Allow air bubbles to escape, and drain the hexane to the top of the Florisil. Dissolve the residue derived from 6.2 in approx. 10 ml hexane, transfer the solution onto the column, and allow to percolate. Rinse the flask several times with 5-ml portions of hexane and transfer each rinsing onto the column before the preceding portion has soaked in. Continue to elute the column with hexane at a rate of 1.5 ml/min until a total of 120 ml eluate has been collected. Rotary-evaporate the eluate to dryness. 6.4 Gas-chromatographic determination Dissolve the residue derived from 6.2 or 6.3 in acetone and make up to 10 ml (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. Operating conditions Gas chromatograph Column 1 Column temperature Column 2 Column temperature

Injection port temperature Detector

Varian 3700 Glass, 2 mm i.d., 1.2 m long; packed with 5% OV-17 on Chromosorb G AW-DMCS, 60-80 mesh 3 min 200 °C, programmed to rise at 15°C/min from 200 to 250 °C, and 17 min 40 s after injection from 250 to 300 °C Glass, 2 mm i.d., 1.2 m long; packed with 5% OV-225 on Chromosorb G AW-DMCS, 60-80 mesh 3 min 150 °C, programmed to rise at 15°C/min from 150 to 180 °C, 8 min after injection from 180 to 200 °C, and 15 min 20 s after injection from 200 to 250 °C 300 °C Flame photometric detector equipped with dual flame system (Varian) and 394-nm sulphur filter (Tracor) Temperature 300 °C

346

Method S 24

Gas flow rates Attenuation Recorder Injection volume Retention times for derivatives from cyhexatin, azocyclotin fentin fenbutatin oxide

Nitrogen carrier, 30 ml/min Hydrogen, 140 ml/min Air, flame 1, 80 ml/min; flame 2, 200 ml/min 8- 10 ~9 1 mV; chart speed 5 mm/min 5 ul Column 1 Column 2 9 min 30 s 11 min 22 min 30 s

7 min 12 min 20 min

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the corresponding derivative standard solutions. Equal volumes of the sample solutions and the derivative standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples of plant material, soil and water, fortified with cyhexatin, azocyclotin, fenbutatin oxide and fentin hydroxide at levels of 0.01 to 0.05 mg/kg, ranged from 80 to 100%. The routine limit of determination was 0.02 to 0.05 mg/kg for plant material and soil, and 0.01 mg/1 for tap water. 7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified organotin compound is calculated from the following equation: R

_ F A -V End -W st

where G VEnd Vj WSt FA F st

= = = = = =

sample weight (in g) or volume (in ml) terminal volume of sample solution from 6.4 (in ml) portion of volume VEnd injected into gas chromatograph (in \i\) amount of compound injected with derivative standard solution (in ng) peak area obtained from Vl (in mm2) peak area obtained from WSt (in mm2)

Method S 24

347

- 200

- 150

16

8

Chromatograms 1 and 2. Untreated control sample of apples fortified with 0.05 mg/kg each of cyhexatin or azocyclotin (1), fentin hydroxide (2), and fenbutatin oxide (3); 50 mg aliquots injected. Upper chromatogram, GLC column 1; lower chromatogram, GLC column 2.

348

Method S 24

8 Important points The method does not differentiate between azocyclotin and cyhexatin or between fentin acetate, chloride and hydroxide. Metabolites of the general formulae R2SnX2 occur only at low levels in foodstuffs. They can also be determined by this method. Unter the conditions given in 6.4, they do not interfere with the determination of the parent compounds. If the recoveries are less than expected, it is advisable to use more methyl magnesium chloride solution.

9 Reference E. Mollhoff Methode zur gaschromatographischen Bestimmung des Akarizids Peropal und seiner Metaboliten in Pflanzen, Boden, Wasser und Kleintierfutter, Pflanzenschutz-Nachr. 30, 249-263 (1977).

10 Author Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, E. Mollhoff

Methyl Carbamate Insecticides Apples, beans (green), carrots, cherries, head cabbage, kohlrabi, leeks, lettuce, radishes, strawberries, tomatoes

s 25 Gas-chromatographic determination

(German version published 1989)

1 Introduction The method permits the identification and quantitative determination of 14 methyl carbamate insecticides and four metabolites, including ten aromatic and four aliphatic N-methyl carbamates as well as one N,N-dimethyl carbamate (see Table 1). The compounds are determined, without derivatization, under conditions by which decomposition of the carbamates is prevented. Table 1. Relative retention times (RRT) of methyl carbamates, relative to lauronitrile (I; retention time 4 min 25 s) and to octadecanonitrile (II; retention time 13 min 46 s). Methyl carbamate

RRT I

Lauronitrile Aldicarb Butocarboxim Methomyl Propoxur Thiofanox Promecarb Bendiocarb Carbofuran Aminocarb Primicarb Ethiofencarb 3-Keto-carbofuran Desmethyl-pirimicarb Dioxacarb Carbaryl Mercaptodimethur (Methiocarb) 3-Hydroxy-carbofuran Desmethylformamido-pirimicarb Octadecanonitrile

1.00 1.28 1.39 1.51 1.51 1.62 1.77 1.77 1.99 2.04 2.27 2.35 2.37 2.39 2.51 2.56 2.67 2.83 3.08

RRT II 0.41 0.45 0.48 0.48 0.52 0.57 0.57 0.64 0.65 0.73 0.75 0.76 0.77 0.81 0.82 0.86 0.91 0.99 1.00

2 Outline of method The methyl carbamate residues are extracted from plant material with ethyl acetate; an aliquot of the extract is evaporated to dryness. The residue is extracted with water and the solution passed through a RP-18 disposable cartridge using an acetonitrile-water mixture as eluant. The

350

Method S 25

eluate is shaken with dichloromethane, and the dichloromethane phase is evaporated. The methyl carbamates are determined by capillary gas chromatography with splitless injection using a thermionic detector.

3 Apparatus Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) Laboratory centrifuge, e.g. Varifuge type 4120 (Heraeus-Christ), with 150-ml stainless steel tubes Pear-shaped flasks, 100-ml and 50-ml, with ground joints and glass stoppers Rotary vacuum evaporator, 30-40 °C bath temperature Glass syringe, 10-ml, with Luer-lock fitting Separatory funnel, 100-ml, with PTFE stopcock Test tubes, 10-ml, with screw cap and PTFE gasket, e.g. SVL45 (Corning No. 611-52) Gas chromatograph equipped with thermionic nitrogen-specific detector Microsyringe, 10-ul Important: All glassware must be rinsed with acetone immediately before use.

4 Reagents Acetone, chemically pure, dist. Acetonitrile, reagent grade (Merck No. 800015), dist. Cyclohexane, chemically pure, distilled from a sodium suspension Dichloromethane, chemically pure, dist. Ethyl acetate (Riedel-de Haen No. 27227), dist. Methanol, chemically pure, distilled from potassium hydroxide Water, deionized and dist. Eluting mixture: acetonitrile + water 1:1 v/v Internal standard solution: 1 ng/ml lauronitrile (Merck-Schuchardt No. 805345) and 2 octadecanonitrile (Aldrich No. 12,258-0) in cyclohexane Methyl carbamate standard solutions: 1-2 ng/ml of each compound in cyclohexane (diluted from a stock solution in ethyl acetate) RP-18 disposable cartridge: Sep-Pak Cartridge C18 (Millipore No. 51910) Sodium hydrogen carbonate, p.a. Air, synthetic, re-purified Hydrogen, re-purified Nitrogen, re-purified

Method S 25

351

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.

6 Procedure 6.1 Extraction Weigh 25 g of the chopped analytical sample (G) into a centrifuge tube and (only in the case of fruit) add 0.5 g sodium hydrogen carbonate. Add 80 ml ethyl acetate and homogenize for 2 min. Rinse the rod of the homogenizer twice with 10-ml portions of ethyl acetate, add the rinsings to the centrifuge tube, and centrifuge the homogenate for 10 min at 5000 r.p.m. Pipet a 40-ml portion (VR1) from the clear supernatant solution (VEx =100 ml) into a 100-ml pearshaped flask and rotary-evaporate to near dryness. Remove the last traces of solvent with a gentle stream of air or nitrogen. 6.2 Cleanup on a RP-18 cartridge Draw 10 ml methanol into the glass syringe, attach a cartridge to the syringe, and force the methanol through the cartridge. Detach the cartridge, pull the plunger out of the syringe, and re-attach the cartridge. Add 10 ml water to the residue derived from 6.1, stopper the flask, and shake well for 10-15 s. The residue does not completely dissolve. Transfer the solution, disregarding floating particles, into the syringe, re-insert the plunger and force the liquid through the cartridge. Detach the cartridge, draw 5 ml water and 5 ml air into the syringe, re-attach the cartridge and force the water and air through the cartridge. Discard the eluates. Add 10 ml eluting mixture to the residue in the pear-shaped flask, stopper, and shake again for 10-15 s. Transfer the solution into the syringe, as described above, the cartridge being attached. Force the liquid through the cartridge and collect the eluate in a separatory funnel. Rinse the pear-shaped flask again with 5 ml eluting mixture, transfer this solution into the syringe, make up to 10 ml with eluting mixture, and force the liquid through the cartridge into the separatory funnel. 6.3 Liquid-liquid partition Add 10 ml dichloromethane to the solution derived from 6.2, shake for 2 min, allow to stand for 30 min, and drain the lower phase into a 50-ml pear-shaped flask. Repeat the extraction with a further 10 ml of dichloromethane. If the upper, aqueous phase is still very turbid after the second extraction, add 5 ml methanol, swirl, allow to stand for 10-30 min, and drain the organic phase into the pearshaped flask. Rotary-evaporate the solution to near dryness. Remove the last traces of solvent with a gentle stream of air or nitrogen.

352

Method S 25

6.4 Gas-chromatographic determination Dissolve the residue derived from 6.3 in 0.1 to 0.2 ml ethyl acetate and add 2.0 ml internal standard solution. If the resulting solution is turbid, transfer it to a test tube, cap the tube, and centrifuge for 5-10 min at 5000 r.p.m. Inject an aliquot of the clear supernatant solution into the gas chromatograph. For splitless injection, cool the column oven down to 50 °C and close the split exit. Open the split exit 30 s after the injection and heat the column oven to the starting temperature of the temperature program (100 °C). Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates

Split ratio Attenuation Recorder Integrator Injection volume Retention times

Varian 3700 Duran glass capillary, 0.3 mm i.d., 0.8 mm o.d., 18 m long; statically coated with 0.1 pirn SE-52 (deactivated with polyethylene glycol PEG 400 and PEG 1000) 50-100°C at maximum heating rate, programmed to rise at 6°C/min from 100 to 230 °C, then isothermal at 230 °C 200 °C Thermionic nitrogen-specific detector Temperature 250 °C Nitrogen carrier, 2.6 ml/min Nitrogen purge gas, 27 ml/min Hydrogen, 4.8 ml/min Air, 175 ml/min 1:10 8-10- 12 1 mV; chart speed 10 mm/min Hewlett-Packard 3380 A, chart speed 5 mm/min 2-2.5 \x\ with closed split exit and at 50 °C column temperature See Table 1

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas obtained for the methyl carbamates and comparing them with the peak areas obtained for the internal standard lauronitrile. When this peak is subject to interference by plant co-extractives, the peak of octadecanonitrile is used instead. Due to the different sensitivities of the detector to the individual methyl carbamates and the internal standard, a correction factor K is applied, derived from injecting a solution containing a definite amount of the respective methyl carbamate and of the internal standard.

Method S 25 10 3,4

353

18 13

15 6,7

16 17 14

II

12

11

ULJ

10

15

mm

Chromatogram 1. Standard mixture representing 2 ng of each methyl carbamate (4 ng each of 8, 12, 14, 15, 16, 17 and II). Conditions as described in 6.4. 1, aldicarb; 2, butocarboxim; 3, methomyl; 4, propoxur; 5, thiofanox; 6, promecarb; 7, bendiocarb; 8, carbofuran; 9, aminocarb; 10, pirimicarb; 11, ethiofencarb; 12, 3-keto-carbofuran; 13, desmethylpirimicarb; 14, dioxacarb; 15, carbaryl; 16, mercaptodimethur (methiocarb); 17, 3-hydroxy-carbofuran; 18, desmethylformamido-pirimicarb. I, lauronitrile; II, octadecanonitrile (internal standards).

354 10

II

^UU—v/N^y

10

15 min

Chromatogram 2. Untreated control sample of lettuce fortified with 0.1 mg/kg (for 14, 15 and 16, each 0.2 mg/kg) of 11 methyl carbamates. Conditions as described in 6.4; for assignment of peaks see Chromatogram 1.

Method S 25

355

7.2 Recoveries, limit of detection and limit of determination The recoveries from untreated control samples fortified with methyl carbamates are given in Tables 2 and 3. At levels of 0.01 to 0.5 mg/kg they ranged, in general, from 70 to 115%; in some cases, however, lower recoveries were observed. Table 2. Percent recoveries from plant material fortified with methyl carbamates; means from quadruplet experiments. Values in brackets: Recovery <70%.

Methyl carbamate Aldicarb Aminocarb Bendiocarb Butocarboxim Carbaryl Carbofuran Dioxacarb Ethiofencarb Mercaptodimethur (Methiocarb) Methomyl Pirimicarb Promecarb Propoxur Thiofanox

a

Added mg/kg

Green Apples Cherries Carrots Lettuce beans

Head cabbagea Leeks

0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1 0.02 0.1 0.2 0.01 0.05 0.1 0.02 0.1 0.2 0.01 0.05 0.1 0.02 0.1 0.2 0.1 0.5 0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1

97 101 100 93 104 95 100 114 120 78 106 112 102 113 116 102 108 103 113 117 114 109 92 104 120 99 112 108 109 98 95 96 114 97 109 93 103 108 79 108 119

59 87 90 (28) 75 70 71 75 82 (48) 75 85 b b b 122 94 95 95 103 106 70 74 73 79 71 76

80 84 92 75 71 95 130 108 112 70 92 87 95 106 107 96 110 115 85 97 96 90 72 77 100 110 112 (31) (63) 85 71 91 90 98 113 100 98 106 69 98 88

93 92 95 89 95 84 91 98 96 78 80 86 105 105 102 105 105 99 108 100 95 88 91 96 106 108 102 (39) 70 104 111 108 115 109 98 93 102 101 93 79 87

72 90 82 125 91 86 74 79 107 70 79 82 87 100 100 78 98 101 88 97 108 70 74 93 73 98 101 89 87 71 82 84 75 95 91 87 98 89 73 87

(52) (68) (36) (52) (52) 119 119 93 (42) (48) 92 85 94 76 94 98 76 75 95 ndc ndc ndc 86 81 91 ndc ndc 102 84 87 76 93 98 75 94 95 (33) (36) (37)

(50) 80 90 94 89 100 96 100 90 93 b b b

ndc ndc ndc (55) 75 73 82 104 ndc ndc ndc 97 90 104 95 98 106 87 92 116 71 79 71 95 96 101 ndc ndc 102 76 78 70 85 84 97 108 (49) (44)

Head cabbage fortified with 0.1, 0.5 and 1 mg/kg or 0.2, 1 and 2 mg/kg; b Interferences from plant co-extractives; ndc Not detectable (no peak observed).

356

Method S 25

Table 3. Percent recoveries from strawberries, tomatoes and radishes, fortified with 0.1 mg/kg of the methyl carbamates; means from duplicate experiments. Methyl carbamate

Strawberries

Tomatoes

Radishes

Aldicarb Aminocarb Bendiocarb Butocarboxim Carbaryla Carbofuran Dioxacarba Ethiofencarb Mercaptodimethura (Methiocarb) Methomyl Pirimicarb Promecarb Propoxur Thiofanox

85 113 89 ndm 109 106 91 77 101 ndm 94 99 97 78

90 99 98 93 103 87 86 69 101 83 100 92 93 85

ndm 111 105 ndm 113 ndm 99 117 109 ndm 92 88 106 63

a

Fortified with 0.2 mg/kg; ndm Not determinable.

The limits of determination of the methyl carbamates from seven plant materials are listed in Table 4. In general, they ranged from 0.01 to 0.05 mg/kg (methomyl, 0.1 to 1 mg/kg). The limits of detection were mostly amounting to one third to one half of the limits of determination. Table 4. Limits of determination (mg/kg) of the methyl carbamates in plant material. Methyl carbamate

Apples

Cherriesa Carrots

Lettuce

Green beans

Head cabbage

Leeksa

Aldicarb Aminocarb Bendiocarb Butocarboxim Carbaryl Carbofuran Dioxacarb Ethiofencarb Mercaptodimethur (Methiocarb) Methomyl Pirimicarb Promecarb Propoxur Thiofanox

0.01 0.01 0.005 0.005 0.02 0.005 0.02 0.01

0.005 0.01 0.005 0.01 0.01 0.005 0.01 0.01

0.01 0.005 0.01 0.01 0.01 0.005 0.01 0.005

0.01 0.01 0.005 0.005 0.01 0.005 0.01 0.005

0.1 b 0.005 b 0.01 0.005 0.01 ndc

0.5 0.5 0.05 0.5 c 0.05 0.1 0.1

ndc 0.05 0.05 d ndc 0.02e 0.01 0.01 e 0.01

0.02 0.1 0.01 0.01 0.01 0.005

0.01 b 0.01 0.01 0.005 0.005

0.01 0.5 0.005 0.005 0.01 0.01

0.01 0.1 0.005 0.01 0.005 0.05

0.01 ndc 0.005 0.005 0.005 b

0.1 1.0 0.05 0.05 0.05 c

0.05d ndc 0.01 e 0.05e 0.01 b,d

a

Evaluated against octadecanonitrile; b Recovery <70%; c Interferences from plant co-extractives; Blanks equivalent to 0.02 mg/kg; e Blanks equivalent to <0.02 mg/kg; ndc Not detectable (no peak observed). d

Method S 25

357

7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified methyl carbamate is calculated from the following equation: R

FA-VEx-WSt FSt-VR1-G

K

where G VEx VR1 Wst FA FSt K

= = = = =

sample weight (in g) volume of ethyl acetate used for extraction (in ml) portion of volume VEx used for cleanup (in ml) amount of internal standard added to sample solution in 6.4 (in \xg) peak area obtained for an identified methyl carbamate (in mm2 or integrator counts) = peak area obtained for the internal standard (in mm2 or integrator counts) = correction factor for the individual methyl carbamate identified

K is calculated from the chromatogram of a standard solution (cf. 7.1) using the following equation: K=

-"a

CsFa

where Ca = concentration of the methyl carbamate in the standard solution (in |ug/ml) Cs = concentration of the internal standard in the standard solution (in u^/ml) Fa = peak area obtained for the methyl carbamate in the standard solution (in mm2 or integrator counts) Fs = peak area obtained for the internal standard in the standard solution (in mm2 or integrator counts)

8 Important points A fused silica capillary column can also be used for the gas-chromatographic determination: DB-1, 30 m long, film thickness 0.1 fxm (J & W Scientific). Keep to the exact conditions as described in 6.4, in order to guarantee optimum sensitivity. Interfering peaks did occur with various methyl carbamate and substrate combinations (see Table 2). For aromatic methyl carbamate insecticides, the results can be confirmed by preparing the 2,4-dinitrophenyl ether of the corresponding phenol and determining the derivative by gas chromatography on the same capillary column using an electron capture detector.

358

Method S 25

It has not been checked if this method is suitable for analysis of the sulphoxides and sulphones of aldicarb, butocarboxim, ethiofencarb, mercaptodimethur (methiocarb), methomyl and thiofanox.

9 Reference S. Brauckhoff and H.-P. Thier, Analysenmethode flir Rtickstande von Carbamat-Insecticiden in pflanzlichen Lebensmitteln, Z. Lebensm. Unters. Forsch. 184, 91-95 (1987).

10 Authors Institute of Food Chemistry, University of Miinster, S. Brauckhoff and H.-P. Thier

Phthalimides Apples, grapes, lettuce, potatoes, wheat (green matter and grains) Soil, water

s 26 Gas-chromatographic determination

(German version published 1989)

1 Introduction The method permits the identification and quantitative determination of the residues of captafol, captan, dialifos, ditalimfos, folpet, and phosmet by capillary gas chromatography.

2 Outline of method Phthalimide residues are extracted from plant material with a hexane-acetone mixture; an aliquot of the extract is evaporated to dryness. Interfering co-extractives are removed on a Florisil cartridge. When this is not sufficient, a further cleanup can be achieved by gel permeation chromatography. Water samples are extracted with dichloromethane, and soil samples with hexane. The phthalimides are determined by electron capture gas chromatography on a fused silica capillary column.

3 Apparatus High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia. Filter paper, 9 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 500-ml Graduated cylinder, 500-ml Volumetric pipet, 100-ml Erlenmeyer flasks, 500-ml and 200-ml, with ground joints Glass funnel, 7 cm dia. Fluted filter paper, 15 cm dia. Round-bottomed flasks, 500-ml, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 30-40 °C bath temperature Pear-shaped flask, 25-ml, with ground joint Separatory funnels Laboratory mechanical shaker Glass syringe, 10-ml, with Luer-lock fitting Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1)

360

Method S 26

Volumetric flasks, 20-ml, 10-ml and 5-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Acetone, fractionally distilled Cyclohexane, for residue analysis Dichloromethane, fractionally distilled Ethyl acetate, for residue analysis n-Hexane, p.a. Toluene, p.a. n-Hexane + acetone mixture 4:1 v/v Eluting mixture : cyclohexane + ethyl acetate 1:1 v/v Phthalimide standard solutions: 0.01—0.1 |ig/ml of each compound in toluene Sodium sulphate, p.a., anhydrous, exhaustively extracted with toluene Florisil disposable cartridge: Sep-Pak Cartridge Florisil (Millipore No. 51960) Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Filter aid, e.g. Celite 545 Helium Nitrogen, re-purified

5 Sampling and sample preparation The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.

6 Procedure 6.1 Extraction 6.1.1 Plant material

Homogenize 50 g of the analytical sample (20 g of cereal green matter) (G) with 10 g filter aid and 200 ml hexane-acetone mixture for 3 min. Suction-filter the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake with a total of 50 ml hexane-acetone mixture. Transfer the filtrate to a graduated cylinder and make up the organic phase to 200 ml (VEx) with the hexane-acetone mixture, ignoring the lower aqueous phase. Mix well; then pipet 100 ml (VR1) of the organic phase into a 200-ml Erlenmeyer flask and dry on sodium sulphate. Filter the solution through a fluted filter paper into a 250-ml round-bottomed flask, and rinse Erlenmeyer flask and filter with 30 ml acetone. Rotary-evaporate the combined filtrates to a volume of 1 to 2 ml, then transfer the concentrated solution to a pear-shaped flask with acetone and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. Proceed to step 6.2.

Method S 26

361

6.1.2 Soil In a separate 10 to 20-g aliquot of the laboratory sample, determine the water content by drying in an open weighing glass at 105 °C to constant weight (approx. 15 h). Discard this aliquot. If the water content is higher than 10%, then use an analytical sample as it is. If the water content is lower than 10%, then add water to raise the water content to at least 10%. Transfer 100 g of the analytical sample (G) with the appropriate water content into a 500-ml Erlenmeyer flask, add 25 g sodium sulphate and 200 ml hexane, and shake on the mechanical shaker for 1 h. Filter the extract through a fast flow-rate filter paper covered with filter aid in a Buchner porcelain funnel, and wash the filter cake twice with 30-ml portions of hexane. Rotary-evaporate the combined filtrates to a volume of 1 to 2 ml, then transfer the concentrated solution to a pear-shaped flask with acetone and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. Proceed to step 6.2. 6.1.3 Water Extract 1 1 of the water sample (G) with 100 ml dichloromethane for 2 min. Filter the organic phase through a fluted filter paper, containing sodium sulphate, into a 500-ml roundbottomed flask. Repeat the extraction twice, shaking each time with 50 ml dichloromethane. Rotary-evaporate the combined filtrates to a volume of 1 to 2 ml, then transfer the concentrated solution to a pear-shaped flask with acetone, and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. In the case of ground and tap water samples, proceed to the gas-chromatographic determination without further cleanup. 6.2 Cleanup 6.2.1 Florisil cartridge Draw 10 ml hexane into the glass syringe, attach a Florisil cartridge to the syringe, and force the hexane through to condition the cartridge packing. Detach the cartridge, pull the plunger out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1 in 0.5 ml hexane and transfer the solution into the syringe with the aid of a Pasteur pipet. Rinse the pear-shaped flask with a further 0.5 ml of hexane and add the rinsings to the syringe. Re-insert the plunger into the syringe, and force the liquid through the cartridge. Detach the cartridge, remove the plunger from the syringe, and re-attach the cartridge. Next elute the compounds with 10 ml hexane-acetone mixture from the Florisil, proceeding in a similar manner as described above. Collect the eluate in a pear-shaped flask and rotary-evaporate to dryness. 6.2.2 Gel permeation chromatography For plant material and soil containing phthalimide residues of less than 0.1 mg/kg, it is recommended to clean up the extract derived from 6.2.1 as follows: Transfer the residue derived from 6.2.1 into a test tube, using a total of 10 ml eluting mixture (VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR3) of the gel permeation chromatograph with 8 to 9 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with standard solutions of

362

Method S 26

the phthalimides; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. - Elution volumes ranging from 100 to 160 ml were determined for the phthalimides on Bio-Beads S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate of 5.0 ml/min. Collect the 100 to 160-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to near dryness. Remove the last traces of solvent by swirling the flask in the hand. Check the elution range from time to time and determine anew whenever a new gel column is used. 6.3 Gas-chromatographic determination Dissolve the residue derived from 6.1.3 or 6.2 in toluene, and make up with toluene to a definite volume, e.g. 5 ml (VEnd). Inject an aliquot of this solution (V{) into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection technique Detector Gas flow rates Attenuation Recorder Injection volume Retention times for captan folpet ditalimfos captafol phosmet dialifos

Carlo Erba Fractovap 4160 with on-column injector Fused silica capillary, 0.32 mm i.d., 15 m long; coated with OV-1, crossbond, film thickness 0.10-0.15 um (Carlo Erba Mega) 100 to 180 °C at maximum heating rate, programmed to rise at 2°C/min from 180 to 215 °C, then isothermal at 215 °C for 2 min Cold on-column at 100 °C oven temperature with secondary cooling 63 Ni electron capture detector ECD HT-25, ECD Control Module 251, pulse width 1 us Temperature 300 °C Helium carrier, 1 ml/min Nitrogen purge gas, 30 ml/min 256-1024 10 mV; chart speed 5 mm/min 1 Hi 5 min 20 s 5 min 32 s 6 min 20 s 9 min 56 s 11 min 20 s 16 min 14 s

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject equal volumes of the phthalimide standard solutions (equivalent to 0.1 to 1.0 ng of each compound) into the gas chrcmatograph. Plot the areas or heights of the peaks obtained vs.

•H

OQ O

15

10

JL

00

0

•P

o

-p

o

min

b)

15

10

CO

P

•H

CD O

-P
o

&

•P


15

10

^ L

cd

o

min

c)

Chromatograms of a) standard mixture representing 0.05 ng of each phthalimide, b) untreated control sample of wheat grains, c) untreated control sample of wheat grains, fortified with 0.1 mg/kg of each phthalimide. Conditions as described in 6.3.

min

a)

tal imfos

Pi i—

P, cd

o

4>

CO

CO

CO

I

364

Method S 26

ng of compound. Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the standard solutions should be injected. For the areas or heights of the peaks obtained for the sample solutions, read the appropriate amounts of the identified compound from the corresponding calibration curve. 7.2 Recoveries, limit of detection and limit of determination Recovery experiments were run on untreated control samples of plant material, soil and water, fortified with phthalimides at different levels. The recoveries are given in the Table. All values presented in the Table represent the means from 2 to 7 single experiments. The recoveries from plant material, fortified at levels of 0.05 to 1 mg/kg (for wheat green matter, 0.05 to 10 mg/kg), ranged from 70 to 108% and averaged 90%. The limit of detection was approx. 0.01 to 0.02 mg/kg, depending on the plant material, and the limit of determination was approx. 0.05 mg/kg. The recoveries from soil samples, fortified at levels of 0.05 to 1 mg/kg, ranged from 68 to 104% and averaged 85%. This limit of detection was approx. 0.01 mg/kg, and the limit of determination was approx. 0.05 mg/kg. The recoveries from ground and tap waters, fortified at levels of 0.5 to 100 ng/1, ranged from 70 to 110% and averaged 92%. The routine limit of determination was approx. 0.1

Table. Percent recoveries from plant material, soil and water, fortified with phthalimides (means of 2 — 7 experiments). Analytical material

Added mg/kg

Apples Grapes Lettuce Potatoes Wheat Green matter Grains Soil Water

0.05-1 0.05-1 0.05-1 0.05-1

fol

0.05-10 0.1-1 0.05-1 0.0005-0.1*)

^

Q

^

Ditalimfos

phosmet

93 98 98 71

91 100 91 80

74 92 112 74

90 82 93 73

92 96 96 78

84 91 99 73

102 92 72 99

91 76 103 96

93 75 58 95

87 94 93 76

91 91 93 94

93 88 75 94

*) Equivalent to 0.5-100 M,g/1.

7.3 Calculation of residues The residue R, expressed in mg/kg, of an identified phthalimide is calculated from the following equation: r

_ w A -v E x -v R 2 -v E n d V R1 -V R3 -V r G

Method S 26

365

where G

= sample weight (in g) or volume (in ml)

VEx

= total volume of organic phase after addition of hexane-acetone mixture to filtered extract from plant sample in 6.1.1 (in ml)

VR1

= portion of volume VEx used for further cleanup (in ml)

VR2 = volume of eluting mixture used to take up the residue from 6.2.1 (in ml) VR3 = portion of volume VR2 injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.3 (in ml) V4

= portion of volume VEnd injected into gas chromatograph (in ul)

WA

= amount of identified phthalimide for Vj read from calibration curve (in ng)

8 Important points A thermionic phosphorus/nitrogen-specific detector is also suitable for the final gas-chromatographic determination. In this case, acetone should be used to prepare the phthalimide standard solutions. After a number of measurements, low recoveries for captafol, captan and folpet can be encountered. In these cases, results can be improved by shortening the inlet end of the capillary column by a few centimeters. The recoveries of dialifos from soil were considerably lower than from other analytical materials, averaging only 58%.

9 References No data

10 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G. Nolting, H. Kohle, J. Siebers and M Blacha-Puller

Part 5 Multiple Pesticide Residue Analytical Methods for Water

Phenoxyalkanoic Acid Herbicides Water

w4 Gas-chromatographic determination

(German version published 1976)

1 Introduction The method permits the identification and quantitative determination of the residues of 2,4-D, dichlorprop (2,4-DP), MCPA, MCPB, mecoprop (CMPP, MCPP), and 2,4,5-T. It is based on the analysis of the derivatives, which are obtained by nitration of the compounds, followed by esterification with methanol. The nitro groups greatly enhance the response of the electron capture detector to the phenoxyalkanoic acid methyl esters. The gas-chromatographic column used permits the separation of all six derivatives.

2 Outline of method The phenoxyalkanoic acid residues are extracted from water with ethyl acetate. Derivatization with nitrating acid and esterification with methanol + sulphuric acid is followed by chromatographic cleanup on an aluminium oxide column. The derivatives formed are shown in the Table. They are determined by electron capture gas chromatography.

3 Apparatus Round-bottomed flasks, 2-1, 500-ml and 100-ml Separatory funnels, 500-ml and 100-ml Volumetric flasks, 10-ml Rotary vacuum evaporator Chromatographic tube, 8 mm i.d., 20 cm long Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul

4 Reagents Benzene, dist. Chloroform, dist. Ethyl acetate, dist. Herbicide standard solutions: 1 and 10 ng/ml of each phenoxyalkanoic acid in methanol

370

Method W 4

Table. Nitro derivatives of the phenoxyalkanoic acid methyl esters. Parent compound

Chemical name of derivative

2,4-D

Methyl (2,4-dichloro-3,6-dinitro-

phenoxy)acetate

N0 2 /T—£

Methyl 2-(2,4-dichloro-3,6-dinitrophenoxy)propionate

/®

C l - f V-O-CH 2 -C-OCH 3 >=< O2N

Dichlorprop

Melting point (°C)

Structural formula

f/

Cl

85

Cl

CH, I // V - O-CH—C-OCH O-

X Cl ON

3

68

2

O2N

MCPA

Methyl (4-chloro-2-methyl-5,6dinitrophenoxy) acetate

2

NO 2

\

/

o

98

C l — \ \ - O - CH 2 - C - OCH3 CH3 O2N

MCPB

Methyl 4-(4-chloro-2-methyl-5,6dinitrophenoxy) butyrate

cl

((

N0 2 y— O—(CH2)3—C—OCH3

52

CH3

Mecoprop

Methyl 2-(4-chloro-2-methyl-5,6dinitrophenoxy) propionate

2,4,5-T

Methyl (2,4,5-trichloro-6-nitrophenoxy)acetate

- C H - C-OCH3

•V

V-O-CH 2 -C-OCH 3

<30

78

Derivative standard solutions: Run a mixture of 1 ml each of the herbicide standard solutions (e.g. 10 ng/ml) through the method from 6.2 (nitration) to and including 6.4. Serially dilute the resulting benzene solution to yield derivative standard solutions containing 25 to 250 ng/ml of phenoxyalkanoic acids equivalents Sulphuric acid, 3 mol/1 H2SO4 p.a. Sodium hydroxide solution, 10 mol/1 NaOH p.a. Nitrating acid: Sulphuric acid, p.a., cone. + nitric acid, fuming (sp.gr. = 1.52) 9:1 v/v Methylating mixture: Methanol p.a. + sulphuric acid, p.a., cone. 9:1 v/v. Prepare fresh daily Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p.a. Sodium chloride, p.a. Sodium sulphate, p.a., anhydrous

Method W 4

371

Aluminium oxide, activity grade IV: To 90 g aluminium oxide 90 basic, activity grade I (Merck No. 1076), in a 300-ml Erlenmeyer flask (with ground joint), add 10 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h Quartz wool Nitrogen, re-purified

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at -20°C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Extraction Place 1 1 of the water sample (G) in a 2-1 round-bottomed flask, add 5 ml sodium hydroxide solution (pH 12-13), and concentrate to a volume of approx. 100 ml in a rotary evaporator (approx. 70 °C bath temperature). Acidify with 30 ml sulphuric acid and add 30 g sodium chloride. Allow the salt to dissolve, transfer the solution into a 500-ml separatory funnel, and shake three times with 100-ml portions of ethyl acetate. Filter the ethyl acetate phases successively through sodium sulphate into a 500-ml round-bottomed flask, and rotary-evaporate to near dryness (approx. 40 °C bath temperature). Transfer the concentrated ethyl acetate solution quantitatively into a 100-ml round bottomed flask and rotary-evaporate to dryness. 6.2 Nitration Add 2 ml nitrating acid to the residue derived from 6.1 in the 100-ml flask, while carefully rotating the flask to wet its walls, and allow to stand for 2 min. Carefully dilute with 20 ml water, transfer into a 100-ml separatory funnel, and shake twice with 20-ml portions of chloroform. Filter the chloroform phases successively through sodium sulphate into a 100-ml round-bottomed flask and wash the filter with a little chloroform. Rotary-evaporate the filtrate to dryness. 6.3 Methylation To the residue derived from 6.2, add 5 ml methylating mixture, and allow to stand with occasional swirling at room temperature for 10 min. Add 15 ml water and extract with 10 ml benzene (VEx). Separate the benzene phase, wash it with 15 ml sodium hydrogen carbonate solution, and dry on sodium sulphate. Rotary-evaporate an aliquot of 5.0 ml (VR1) of the dried solution to approx. 1 ml.

372

Method W 4

6.4 Column chromatography Insert a quartz wool plug into the bottom of a chromatographic tube. Half-fill the tube with benzene, then add 1 g of the aluminium oxide. Top the column with an approx. 0.5-cm layer of sodium sulphate, and drain the benzene until the column packing is covered to a depth of 1 mm. Transfer the concentrated benzene extract derived from 6.3 onto the column and rinse the flask three times with 1-ml portions of benzene. Allow the rinsings to percolate into the column packing each time, collect the eluate in a 10-ml volumetric flask, and continue to elute with benzene until the flask is filled to the mark (VEnd). 6.5 Gas-chromatographic determination Inject an aliquot, e.g. 2 ul (Vj), of the solution derived from 6.4 into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Carrier gas flow rate Attenuation Recorder Injection volume Retention times for the nitro derivatives of 2,4,5-T methyl ester dichlorprop methyl ester mecoprop methyl ester 2,4-D methyl ester MCPA methyl ester MCPB methyl ester

Varian Aerograph 1440 Glass, 2.5 mm i.d., 2.6 m long; packed with 1% OV-17 + 2% OV-210 on Gas Chrom Q, 100-120 mesh 210 °C 250 °C Sc3H electron capture detector Temperature 220 °C Nitrogen, 40 ml/min 4-10"9 1 mV; chart speed 10 mm/min 2 \x\

3 min 5 min 6 min 7 min 8 min 20 min

12 s 42 s 30 s 12 s 48 s 12 s

-I

5

Ir

1

EP4

^

-2 £o«« •c 5 • 2

Time

Chromatogram 1. Control sample of leaching water fortified with 2,4,5-T (0.5 ixg/1); dichlorprop, mecoprop, 2,4-D and MCPA (1 pa.g/1 each); and MCPB (2 ng/1); 2 jn-1 injected.

k

CSl"

CO

Time

Chromatogram 2. Control sample of leaching water; 2 \i\ injected, equivalent to 0.1 ml water.

CD

o

374

Method W 4

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 2 \i\ of each derivative standard solution into the gas chromatograph. Plot the heights of the peaks obtained vs. pg of the corresponding phenoxyalkanoic acid equivalent. Also inject 2-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amount of phenoxyalkanoic acid equivalent from the corresponding calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from untreated control samples, fortified with the phenoxyalkanoic acids at levels of 0.5 to 10 jxg/1, ranged from 70 to 90%. The routine limit of determination was 0.5 n.g/1. 7.3 Calculation of residues The residue R, expressed in u.g/1, of an identified phenoxyalkanoic acid is calculated from the following equation: R

_ WA-VEx-VEnd VR1VrG

where G = sample volume (in ml) VEx = volume of benzene used to extract the phenoxyalkanoic esters from the diluted methylating mixture in 6.3 (in ml) VR1 = portion of volume VEx used for further processing (in ml) VEnd = terminal volume of sample solution from 6.4 (in ml) Vj = portion of volume VEnd injected into gas chromatograph (in ul) WA = amount of identified phenoxyalkanoic acid equivalent for Vj read from calibration curve (in pg)

8 Important points The solutions of the methyl esters of the nitrated phenoxyalkanoic acids are sensitive to light and must, therefore, be kept in the dark. Experiments with the following phenoxyalkanoic acid esters have shown that they are hydrolyzed during the procedure in step 6.1, so they can be included in the method: 2,4-D methyl and isooctyl esters, MCPA isooctyl ester, and 2,4,5-T methyl and isooctyl esters.

Method W 4

375

9 Reference C.A. Bache, D.J. Lisk and M.A. Loos, Electron affinity residue determination of nitrated MCP, MCPB, and NAA, J. Assoc. Off. Agric. Chem. 47, 348-352 (1964).

10 Author BASF, Agricultural Research Station, Limburgerhof, N. Drescher

Fungicides Water

w5 Gas-chromatographic determination

(German version published 1987)

1 Introduction The method permits the identification and quantitative determination of the residues of fungicides (see Table on p. 379) with different chemical structures in water samples of different degrees of purity, using gel permeation chromatography as the essential cleanup step. The compounds are determined by gas chromatography using various specific detectors.

2 Outline of method The fungicide residues are extracted from water samples with dichloromethane. The solvent is evaporated, and the residue is dissolved in a cyclohexane-ethyl acetate mixture and cleaned up by gel permeation chromatography on the polystyrene gel Bio-Beads S-X3. The individual compounds are identified and quantitatively determined by gas chromatography, using either an electron capture, or a thermionic, or a flame photometric detector.

3 Apparatus Separatory funnels, 1-1 and 250-ml, with ground joints Glass funnel, 10 cm dia. Round-bottomed flasks, 1-1, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature Glass syringe, 10-ml, with Luer-lock fitting Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1) Test tubes, 10-ml, graduated, with ground stoppers Gas chromatographs equipped with electron capture detector, thermionic nitrogen and phosphorus-specific detector, and sulphur-specific flame photometric detector Microsyringe, 10-ul

4 Reagents Acetone, for residue analysis Cyclohexane, for residue analysis Dichloromethane, for residue analysis

378

Method W 5

Ethyl acetate, for residue analysis Eluting mixture: cyclohexane + ethyl acetate 1:1 v/v Fungicide standard solutions: CM-20 |ng/ml of each compound in ethyl acetate or acetone Sodium sulphate, p.a., anhydrous Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750) Cottonwool, chemically pure Glass wool Air, synthetic, re-purified Hydrogen 5.0 (< 99.999 vol. %) Nitrogen 4.6 (< 99.996 vol. %)

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Extraction Extract 400 ml water (G) in a 1-1 separatory funnel three times with 200-ml portions of dichloromethane. If only 100-ml water samples are available, reduce the volumes of dichloromethane to 100, 50, and 50 ml. Filter the organic phases successively through a glass funnel fitted with a cottonwool plug, and containing an approx. 3-cm layer of sodium sulphate, into a round-bottomed flask. Wash the sodium sulphate three times with 25-ml portions of dichloromethane, and rotary-evaporate the combined filtrates to dryness. 6.2 Gel permeation chromatography Transfer the residue derived from 6.1 into a test tube, using a total of 10 ml eluting mixture (VR1) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR2) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with fungicide standard solutions (each approx. 20 |ig/ml); cf. Cleanup Method 6, pp. 75 ff, Vol. 1. The following elution volumes were determined for the individual compounds on Bio-Beads S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate of 5.0 ml/min.

Method W 5 _ , Compound Anthr aquinone Bitertanol Captafol Captan Chinomethionat Chlor othalonil Dichlofluanid incl. metabolite DMSA Fluotrimazole Fuberidazole Imazalil Rabenzazole Tolylfluanid incl. metabolite DMST Triadimefon Triadimenol Triazoxide

379

Elution volume , 160 -190 100-130 120-160 120-160 170-190 140 -170 120-150 100-140 120-140 120-150 120-160 120-150 100-130 100-130 160-190

Check the elution volumes every 500 samples, and determine anew whenever a new column is used. For analysis of all compounds mentioned above, collect the total elution volume from 100 to 190 ml in a 250-ml round-bottomed flask. For analysis of an individual compound, collect only the corresponding fraction in a 100-ml round-bottomed flask. Rotary-evaporate the eluate to dryness. Dissolve the residue in an appropriate volume of ethyl acetate or acetone (VEnd)» e-g- 2 or 5 ml, and transfer the solution into a test tube.

6.3 Gas-chromatographic determination Inject an aliquot of the solution derived from 6.2 (Vj) into the gas chromatograph, followed by the same volume of the corresponding standard solution. Repeat each injection as a control. Operating conditions 6.3.1 Anthraquinone, captafol, captan, chinomethionat, chlorothalonil Gas chromatograph Varian 6000 Column 1 Glass, 3 mm i.d., 1.8 m long; packed with 1.5% SP2250 + 1.95% SP-2401 on Supelcoport, 100-120 mesh Column 2 Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE-30 on Chromosorb W-HP, 80-100 mesh Column temperature 200 °C Injection port temperature 200 °C 63 Detector Ni electron capture detector Temperature 250 °C Carrier gas flow rate Nitrogen, 40 ml/min Attenuation 128 Recorder 1 mV; chart speed 5 mm/min Injection volume 2 \i\

380

Method W 5

Column 1 Retention times for chlorothalonil anthraquinone chinomethionat captan captafol capiaiui Chromatogram 1 gives an example

2 min 54 s 4 min 18 s 5 min 36 s 6 min 18 s 18 s 10 min iiiiii 12 iz, & of the separation

Column 2 2 min 3 min 24 s 5 min 48 s 4 min 12 s column suitable i/Uiuiiiii not nut Minauic achieved on column 1

6.3.2 Bitertanol, fluotrimazole, fuberidazole, imazalil, rabenzazole, triadimefon, triadimenol, triazoxide Gas chromatograph Varian 3700 Column 1 same as in 6.3.1, column 1 Column 2 same as in 6.3.1, column 2 Column 3 Glass, 3 mm i.d., 0.9 m long; packed with 5% Carbowax 20M on Chromosorb W-HP, 100-120 mesh Detector Thermionic phosphorus/nitrogen-specific detector Gas flow rates Hydrogen, 4.5 ml/min Air, 175 ml/min Attenuation 10 " u Recorder 1 mV; chart speed 5 mm/min Injection volume 5 nl Column 2 Column 3 Column 1 Injection port temperature 280 °C 250 °C 280 °C 200 °C Column temperature 185 °C 220 °C 350 °C Detector temperature 250 °C 350 °C Nitrogen, Nitrogen, Nitrogen, Carrier gas flow rate 40 ml/min 40 ml/min 30 ml/min Retention times for 2 min 12 s 17 min 30 s fuberidazole 2 min 30 s rabenzazole 3 min 6 min 48 s 2 min 36 s triadimefon 3 min 3 min 42 s 2 min 36 s 3 min 48 s 8 min 12 s triadimenol 3 min 18 s 5 min 18 s 7 min 30 s 4 min 54 s imazalil 9 min 48 s column not suitable 8 min 42 s fluotrimazole 13 min 36 s triazoxide 13 min 12 s 10 min 12 s column not suitable bitertanol 20 min 42 s 21 min Chromatograms 2, 3 and 4 give examples of the separations achieved. 6.3.3 Dichlofluanid, tolylfluanid, and their metabolites DMSA and DMST Tracor 560 Gas chromatograph Glass, 2 mm i.d., 1.2 m long; packed with 5% DC-200 Column 4 on Gas Chrom Q, 80-100 mesh Column temperature 175 °C Injection port temperature 250 °C Detector Flame photometric detector equipped with 394-nm sulphur filter Temperature 200 °C

Method W 5 Gas flow rates Attenuation Recorder Injection volume Retention times for DMSA DMST dichlofluanid tolylfluanid Chromatogram 5 gives an example LV/1J111UU111W

S

381

Nitrogen carrier, 30 ml/min Hydrogen, 50 ml/min Air, 100 ml/min 16- 10 1 mV; chart speed 5 mm/min 5 \i\ 2 min 3 min 6 min 6 s 9 min 12 s of the separation achieved.

111111

J.Z* O

IE

IBI5 I II

6

4

2 0

I

I

I

I

I

0

2

4

6

8

I

I

I

10 12 14

I

16

Chromatogram 1. Standard mixture representing 0.2 ng anthraquinone, 0.05 ng captan, 0.02 ng chinomethionat and 0.02 ng chlorothalonil on column 1, conditions as described in 6.3.1. Chromatogram 2. Standard mixture representing 25 ng fluotrimazole, 12.5 ng fuberidazole, 25 ng imazalil, 12.5 ng rabenzazole, 12.5 ng triadimefon, 25 ng triadimenol and 25 ng triazoxide on column 1. Attenuation 8 • 10 ~ n , other conditions as described in 6.3.2.

Method W 5

382

I

111! /I

11

I

I

I

I

I

I

I

I

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

16

18

20

Chromatogram 3. Standard mixture representing 12.5 ng fluotrimazole, 12.5 ng fuberidazole, 25 ng imazalil, 12.5 ng rabenzazole, 12.5 ng triadimefon, 25 ng triadimenol and 25 ng triazoxide on column 2. Attenuation 16- 10 ~ n , other conditions as described in 6.3.2. Chromatogram 4. Standard mixture, same as shown in Chromatogram 3, on column 3. Attenuation 16 • 10 " n , other conditions as described in 6.3.2.

Method W 5

i-

383

<

i/> to

S

12

10

8

6

4

2>

Chromatogram 5. Standard mixture representing 2 ng each of dichlofluanid, DMSA, tolylfluanid and DMST on column 4, conditions as described in 6.3.3.

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak areas of the sample solutions and comparing them with the peak areas obtained for the standard solutions. If the peaks are well separated and symmetrical, quantitation can be performed using peak heights. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas. 7.2 Recoveries and lowest determined concentration Recovery experiments were run on untreated control samples (mostly leaching water as specified by BBA-Richtlinie IV/4-2 (1987), Braunschweig), fortified with the compounds at levels of 0.005 to 0.5 mg/1 (added in 1 to 2 ml acetone or ethyl acetate). The recoveries ranged from 85 to 114% for all compounds tested, see Table. Experiments with tap, well, lysimeter, and drainage waters as well as water used for fish toxicity studies gave similar results. The routine limits of determination for individual compounds were in the range of 0.001 to 0.01 mg/1, depending on the detector used. As a rule, they were 0.01 mg/1 for the flame pho-

384

Method W 5

tometric detector, 0.005 mg/1 for the thermionic detector, and 0.001 mg/1 for the electron capture detector. At the routine limits of determination, no blank values were observed for the different control waters. Table. Percent recoveries from water fortified with the fungicides; results from duplicate experiments. Compound Anthraquinone Bitertanol

Captafol

Captan

Chinomethionat

Chlorothalonil Dichlofluanid DMSA Fluotrimazole Fuberidazole Imazalil Rabenzazole Tolylfluanid DMST Triadimefon Triadimenol Triazoxide a) = 4 recovery experiments, b) = 6 recovery experiments, c) = 8 recovery experiments.

Added mg/1

Recovery

0.005 0.5 0.005 0.01 0.5 0.005 0.01 0.1 0.005 0.01 0.05 0.005 0.014 0.14 0.005 0.01 0.01 0.01 0.005 0.05 0.005 0.05 0.005 0.01 0.005 0.01 0.01 0.01 0.005 0.01 0.005 0.01 0.01

90-108 b ) 94- 97 91-114 b > 96-105 104 102-103 95-107 105-109 93-103 a > 95-100 96- 99 92- 94 88 84- 86 9 1 - 93 9 3 - 98 100-102 100 96-106 112-116 89-108 b > 96-102 92-113 a> 94- 96 98-100 103-106 100-101 96 88-108 c > 97-112 93-103 b > 94 9 5 - 97

Method W 5

385

7.3 Calculation of residues The residue R, expressed in mg/1, of an identified fungicide is calculated from the following equation: F A -V R 1 -V E n d -W S t Fs,-VR2-VrG where G

= sample volume (in ml)

VR1

= volume of solution from 6.1 prepared for gel permeation chromatography (in ml)

VR2 = portion of volume VR1 injected for gel permeation chromatography (volume of sample loop) (in ml) VEnd = terminal volume of sample solution from 6.2 (in ml) Vj

= portion of volume VEnd injected into gas chromatograph (in ul)

WSt = amount of fungicide injected with standard solution (in ng) FA

= peak area obtained from Vj (in mm 2 )

F St

= peak area obtained from WSt (in mm 2 )

8 Important points If phase separation is hindered by emulsions formed during the extraction of the water samples with dichloromethane (mainly with leaching water from Standard Soil 2.2, cf. BBARichtlinie IV/4-2 (1987), Braunschweig), the total content of the separatory funnel should be filtered through a loose glass wool plug. After transferring the filtrate back to the separatory funnel, the separation of the phases is accomplished. During the following two extraction steps, emulsions did not occur any longer. The solutions of fuberidazole, imazalil and rabenzazole are sensitive to light and must, therefore, be kept in the dark. Also during analysis, the solutions should be protected from light, and all glassware should be wrapped with aluminium foil. If optimum peak separation can not be achieved using the conditions given in 6.3, quantitation of individual compounds can be facilitated by adjusting e.g. the oven temperature. When using the thermionic detector for the identification of the fungicides, two columns of different polarity are required (column 1 or 2, and column 3), because, e.g., rabenzazole and triadimefon are only separated on column 3. On the gas-chromatographic columns described here neither the two diastereoisomers of bitertanol nor those of triadimenol will be separated; one peak will be obtained with a shoulder appearing to a greater or lesser degree. To prevent decomposition of captan and captafol, it is recommended to remove the glass wool from the inlet of the gas-chromatographic columns. When the peaks obtained for the standard solutions become reduced in size, the first few cm of the column should be repacked.

386

Method W 5

An alternative to packed columns are fused silica capillary columns, e.g. 0.3 mm i.d., 15 m long, coated with OV-1, film thickness 0.1-0.15 urn, crossbond (Mega No. 26060300), column temperature programmed to rise from 100 to 160 °C with maximum heating rate, and at 4 °C/min from 160 to 220 °C. Captan and captafol tend to decompose on columns that have been in use for some time. When decomposition is observed, shorten the inlet end of the capillary column by a few cm.

9 References R. Brennecke and K. Vogeler, Methode zur gaschromatographischen Bestimmung von Riickstanden verschiedener Fungizide in Wasser, Pflanzenschutz-Nachr. 37, 44-65 (1984). W. Specht and M. Tillkes, Gaschromatographische Bestimmung von Riickstanden an Pflanzenbehandlungsmitteln na.ch Clean-up iiber Gelchromatographie und Mini-KieselgelSaulenchromatographie. 2. Mitt.: Bestimmung der Fungizide Bitertanol, Fluotrimazol, Fuberidazol, Imazalil, Rabenzazole, Triadimefon und Triadimenol in Pflanzen und Boden, Pflanzenschutz-Nachr. 33, 61-85 (1980).

10 Authors Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Information and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke and K. Vogeler

Organochlorine Insecticides Water

w6 Gas-chromatographic determination

(German version published 1987)

1 Introduction The method permits the identification and quantitative determination of the residues of the organochlorine insecticides given in the Table and their most important analogues and metabolites in water by capillary gas chromatography. Tap water and ground water samples with a low content of organic carbon can be analyzed without cleanup by a miniaturized method. (For general advice on micro methods and equipment see pp. 29 ff, Vol. 1.)

2 Outline of method The organochlorine insecticide residues are extracted from water with a mixture of diisopropyl ether and n-hexane containing aldrin or e-HCH as internal standard. 20 ml of water and 200 \i\ solvent mixture are sufficient for this purpose. The compounds are determined directly from this extract by electron capture gas chromatography using a fused silica capillary column.

3 Apparatus Glass syringe, 20-ml, with Luer-lock fitting, equipped with PTFE tube, glass cap and glass capillary; see Figure Microsyringes, 250-ul and 5-ul Laboratory mechanical shaker, overhead tumbler type Lifting platform ("lab jack") Sample vials, 0.5-ml, with PTFE coated septa and screw or crimp caps Volumetric flask, 10-ml Gas chromatograph equipped with electron capture detector

388

Method W 6 I I — Glass cap PTFE tube — Glass capillary

Figure. All-glass syringe with equipment for extracting water samples.

4 Reagents Note: The prerequisite for trace determinations of organochlorine insecticide residues is the absolute purity of the solvents. Check each new lot carefully: Reduce a portion of the solvent to a tenth of its volume and inject into the gas chromatograph under the conditions given in 6.2. If interfering peaks are observed, fractionally distil the solvent for purification, using a Snyder column. Discard the first 10% of the distillate, and stop distillation when a total of 80% of the solvent has been distilled. Check purity of the distillate as described above; if required, repeat distillation Diisopropyl ether, p.a. n-Hexane, for residue analysis Solvent mixture: diisopropyl ether + n-hexane 53 :47 v/v. Distil as described in the Note, and collect the constant temperature azeotrope Extraction mixture: Add 1 ml of internal standard solution 1 or internal standard solution 2 to 1 1 of the solvent mixture (corresponding to 50 ng/ml internal standard) Compound standard solutions: 100 ng/ml of each insecticide in extraction mixture Internal standard solution 1:50 ng/ml e-HCH in solvent mixture Internal standard solution 2:50 ng/ml aldrin in solvent mixture Argon + methane mixture 9:1 v/v Helium, re-purified

Method W 6

389

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Extraction Transfer 20 ml of the water sample (G) into the glass syringe, close with the glass cap and clamp to a stand in vertical position (tip upward). Remove the cap and suck in approx. 0.5 ml of air. Inject 200 JLXI extraction mixture (VEx), using the 250-ul microsyringe, through the tip of the glass syringe into the water sample. Close the syringe with the cap and shake it well by hand for 3 min. (With larger series of analyses use a suitably equipped mechanical shaker and shake for 15 min.) Next clamp the syringe to the stand again so that the piston rests on the platform of the lab jack. Substitute the glass capillary for the glass cap when the phases have separated, transfer the organic extract to a sample vial by carefully raising the platform, and immediately close the vial. 6.2 Gas-chromatographic determination Inject 1 \i\ of the extract derived from 6.1 into the gas chromatograph. Operating conditions Gas chromatograph 1 Column Column temperature Injection port temperature Detector Gas flow rates Split ratio Integrator Recorder Injection volume Retention times Gas chromatograph 2 Column

Hewlett-Packard 5890 Fused silica capillary DB-1, 0.245 mm i.d., 30 m long; film thickness 0.25 urn (J & W Scientific) 2 min isothermal at 100 °C, programmed to rise at 7°C/min from 100 to 250 °C, then isothermal at 250 °C for 12 min 250 °C 63 Ni electron capture detector Temperature 280 °C Argon-methane carrier, 1 ml/min Argon-methane purge gas, 30 ml/min 1:10 Hewlett-Packard 3390 A 1 mV; chart speed 10 mm/min 1 \i\ see Table Siemens Sichromat 2 Fused silica capillary DB-5, 0.32 mm i.d., 25 m long; film thickness 0.25 u.m (J & W Scientific)

390

Method W 6

Column temperature Injection port temperature Detector Gas flow rates Split ratio Attenuation Integrator Recorder Injection volume Retention times

200 °C 250 °C 63 Ni electron capture detector Temperature 280 °C Helium carrier, 1.5 ml/min Argon-methane purge gas, 30 ml/min 1:15 8 Sichromat 1 mV; chart speed 10 mm/min 1 Hi see Table

Table. Relative retention times (RRT) of the organochlorine insecticides, their analogues and metabolites, relative to e-HCH and to aldrin.

Compound

a-HCH (5-HCH Hexachlorobenzene Y-HCH 5-HCH E-HCH

Heptachlor Aldrin Heptachlor epoxide o,p'-DDE a-Endosulfan Dieldrin p,p'-DDE Endrin fi-Endosulfan o,p'-DDT p,p'-DDT Methoxychlor Absolute retention times »> 13 min 10 s 2 > 14 min 58 s 3 > 15 min 03 s 4 > 16 min 29 s

Gas chromatograph 1 RRT 1IRT E-HCH Aldrin 0.917 0.943 0.953 0.966 0.983 .000 *> .090 .137 .194 .233 1.238 1.276 1.282 1.296 1.301 1.334 1.377 1.449

0.807 0.830 0.839 0.850 0.865 0.880 0.960 11.000 2> 11.051 ]1.085 ]1.090 1.123 1.128 1.141 1.145 1.174 1.212 1.275

Gas chromatograph 2 RRT RRT e-HCH Aldrin 0.930 1 .036 0.905 0.984 1 .062 1 .000 3> 1L.058 1 .095 11.175 11.203 ]1.207 1.252 1.247 1.276 1.309 1.303 1.355 1.431

0.849 0.946 0.827 0.899 0.970 0.914 0.967 1.0004> 1.074 1.099 1.103 1.143 1.140 1.165 1.197 1.190 1.238 1.308

Method W 6

391

7 Evaluation 7.1 Method The chromatograms obtained are evaluated using an internal standard (s-HCH or aldrin). The compound used as internal standard must not be present in the analytical sample. This must be confirmed (in a preliminary examination) by injecting the extract of a parallel sample, using another compound as internal standard or without the addition of a standard. Quantitation is performed by the calibration technique. Prepare a calibration curve as follows. Pipet 1.0, 3.0 and 5.0 ml of a compound standard solution into 10-ml volumetric flasks and make up to the marks with extraction mixture. Inject 1 u.1 of each of these solutions (corresponding to 0.01, 0.03 and 0.05 ng of insecticide and 0.05 ng of internal standard) into the gas chromatograph. Plot the peak areas obtained vs. the concentration of insecticide injected (in ng/^il). In addition, determine the peak areas for the internal standard. Use the same concentration of internal standard in both the preparation of the calibration curve and the analysis. Identification is achieved by determination of the retention times relative to the internal standard. Further information about the identity of the compound can be obtained by GC/MS. 10 [ig/1 of insecticide in the analytical sample is required in order to obtain a complete mass spectrum. For lower concentrations, the SIM (selected ion monitoring) technique is recommended, which — although not furnishing the complete spectrum information — is more sensitive. 7.2 Recoveries and lowest determined concentration The recoveries from tap water, fortified with organochlorine insecticides at levels of 0.02 to 1 M
W A -F 0 V E x F,G

where G VEx WA Fo Ft

= = = =

sample volume (in ml) volume of extraction mixture added (in \i\) concentration of organochlorine insecticide read from calibration curve (in ng/|il) average peak area of internal standard used for calibration curve (in mm2 or integrator counts) = peak area of internal standard from the analysis (in mm2 or integrator counts)

392

Method W 6

8 Important points Hexane alone cannot be used for the extraction because the phases separate incompletely and very slowly, and the extract tends to adhere to the glass syringe walls.

9 References J.F.J. van Rensburg and A.J. Hassett, A low-volume liquid-liquid extraction technique, J. High Resol. Chromatogr. Chromatogr. Commun. 5, 574-576 (1982). L. Weil and K.-E. Quentin, Zur Analytik der Pestizide im Wasser, III. Mitteilung: Extraktion der chlorierten Kohlenwasserstoffe aus dem Wasser, Gas- und Wasserfach 112, 184-185 (1971).

10 Author Institute of Water Chemistry and Chemical Balneology, Technical University of Munich, L. Weil

Phenoxyalkanoic Acid Herbicides Water

wi Gas-chromatographic determination

(German version published 1989)

1 Introduction The method permits the identification and quantitative determination of the residues of 2,4-D, 2,4-DB, dichlorprop (2,4-DP), fenoprop (2,4,5-TP), MCPA, MCPB, mecoprop (CMPP, MCPP), and 2,4,5-T, which may be present as acids, salts or esters. By derivatization to the trichloroethyl esters, it is possible to determine even compounds containing only one chlorine atom with a high degree of sensitivity.

2 Outline of method The phenoxyalkanoic acid residues are extracted from water with dichloromethane. After addition of sodium hydroxide solution the dichloromethane is distilled off, whereby any esters present are hydrolyzed. Neutral and basic co-extractives are removed by acidic and basic partition steps. The phenoxyalkanoic acids are then derivatized with trichloroethanol. The resulting trichloroethyl esters are cleaned up on a silica gel column and are determined by electron capture gas chromatography using a fused silica capillary column.

3 Apparatus Separatory funnels, 2.5-1 and 250-ml Round-bottomed flasks, 500-ml and 250-ml, with ground joints Rotary vacuum evaporator, 30 or 40 °C bath temperature Glass funnel, 7 cm dia. Laboratory mechanical shaker Test tubes, 20-ml, with ground stoppers Glass syringe, 1-ml, gas-tight Chromatographic tube, 1 cm i.d., 30 cm long Volumetric flasks, 20-ml and 10-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-pil

394

Method W 7

4 Reagents Acetone, p.a., fractionally distilled Dichloromethane, p.a., fractionally distilled 2,2,4-Trimethyl pentane (isooctane), p.a., fractionally distilled Toluene, p.a., fractionally distilled Water, bi-distilled Eluting mixture: isooctane + toluene 1:1 v/v Compound standard solution for fortification experiments and for preparing the derivative standard solution: 1 ng/ml of each compound (as acid) in acetone Derivative standard solution (equivalent to 100 ng/ml of each compound) in eluting mixture: The derivatization of the compound standard solution is performed parallel to that of the sample solutions. Pipet 2 ml of the compound standard solution (equivalent to 2 jxg of each acid) into a test tube. Evaporate the solvent, esterify and clean up as described in 6.4 and 6.5. Make up the column eluate to 20 ml Sulphuric acid, ultra pure, cone. (Merck No. 714) Sulphuric acid, 3 mol/1 H2SO4 ultra pure. Prepare in bi-distilled water, and extract twice with isooctane before use Sodium hydroxide solution, 10 mol/1 NaOH p.a. Prepare in bi-distilled water, and extract twice with isooctane before use Sodium hydrogen carbonate solution, 10 g/100 ml NaHCO3 p.a. Prepare in bi-distilled water, and extract twice with isooctane before use Trifluoroacetic anhydride, 99% (Janssen No. 14.781.37), fractionally distilled 2,2,2-Trichloroethanol, reagent grade (Merck No. 808610), fractionally distilled Sodium sulphate, p.a., anhydrous, exhaustively extracted with toluene Silica gel, deactivated with 5% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734) overnight at 200 °C. Allow to cool in a desiccator, and store in a tightly stoppered container. To 95 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml of bi-distilled water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container. Before use, check the separation efficiency with derivative standard solution Universal indicator paper (pH 2-10) Glass wool, high purity, silanized (Serva) Quartz wool, exhaustively extracted with toluene Hydrogen, re-purified Nitrogen, re-purified

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at -20°C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

Method W 7

395

6 Procedure 6.1 Extraction Acidify 2 1 of the water sample (G) with 30 ml sulphuric acid (3 mol/1) and shake for 5 min with 100 ml dichloromethane. Separate the organic phase and extract the water three times more with 50-ml portions of dichloromethane. Collect the organic phases in a 500-ml round-bottomed flask and add 20 ml bi-distilled water. 6.2 Hydrolysis Add 5 ml sodium hydroxide solution to the extract derived from 6.1. Rotary-evaporate to the aqueous residue with 40°C bath temperature; then leave the flask to rotate without suction for 30 to 35 min at 40 °C. 6.3 Liquid-liquid partition Transfer the aqueous residue derived from 6.2 into a 250-ml separatory funnel, using 50 ml bi-distilled water to complete the transfer, and extract once with 50 ml dichloromethane with gentle swirling, avoiding formation of emulsion. Allow the phases to separate and discard the dichloromethane layer. Acidify the aqueous phase with 10 ml sulphuric acid (3 mol/1), check the pH, and extract three times with 20-ml portions of dichloromethane, shaking each time for 5 min. Discard the aqueous phase. Filter the organic phases successively through a silanized glass wool plug covered with an approx. 10-g layer of sodium sulphate contained in the glass funnel. Rinse with 40 ml dichloromethane. Rotary-evaporate the combined filtrates in a 250-ml round-bottomed flask to 1 to 2 ml with 30 °C bath temperature. 6.4 Esterification Transfer the residue derived from 6.3 quantitatively into a test tube, using a few ml of dichloromethane to complete the transfer. Evaporate the solvent at 30-40 °C with a gentle stream of nitrogen. Add to the residue 5 ul concentrated sulphuric acid and, using gas-tight glass syringes, 800 ul trifluoroacetic anhydride and 200 ul trichloroethanol. Stopper the test tube and allow to stand for at least 2 h at room temperature with occasional shaking. Concentrate the solution to 0.2-0.3 ml, using a gentle stream of nitrogen (approx. 10 min), then add 10 ml sodium hydrogen carbonate solution. Extract the solution twice with 2-ml portions of isooctane, shaking each time for 1 min. Separate and combine the organic phases. 6.5 Column chromatography Insert a quartz wool plug into the bottom of a chromatographic tube. Half-fill the tube with isooctane. Trickle in 2 g silica gel, gently tapping the tube walls, and cover with an approx. 1-cm layer of sodium sulphate. Allow the filling to settle; then drain the supernatant solvent just to the top of the sodium sulphate layer. Quantitatively transfer the isooctane solution derived from 6.3 onto the column and allow to soak in at a rate of 1 to 2 drops per s. Rinse the column with 15 ml isooctane and discard the eluate. Next elute the derivatives with 19 ml

396

Method W 7

eluting mixture and collect the eluate in a 20-ml volumetric flask. Make up to the mark with eluting mixture (VEnd). 6.6 Gas-chromatographic determination Inject an aliquot, e.g. 1 \x\ (Vj), of the solution derived from 6.5 into the gas chromatograph. Operating conditions Gas chromatograph Column Column temperature Injection port temperature Detector Gas flow rates Split ratio Attenuation Recorder Injection volume Retention times for the trichloroethyl esters of mecoprop MCPA dichlorprop 2,4-D fenoprop 2,4,5-T MCPB 2,4-DB

Hewlett-Packard 5880 A Fused silica capillary, 0.2 mm i.d., 12.5 m long; coated with dimethyl silicon, cross-linked, film thickness 0.33 urn Programmed to rise at 3°C/min from 120 to 180 °C, next at 6°C/min from 180 to 240 °C, then isothermal at 240 °C for 5 min 150 °C 63 Ni electron capture detector Temperature 320 °C Hydrogen carrier, 2 ml/min Nitrogen purge gas, 25 ml/min Hydrogen septum purge, 5 ml/min 1:30 24 Hewlett-Packard 5880 A GC Terminal 1

10 min 45 s 11 min 30 s 12 min 30 s 13 min 8 s 16 min 2 s 16 min 58 s 17 min 45 s 19 min 25 s

Method W 7

u a a o u u o a HC o<x: oa« o

397

OH tl I PUVA O

(M

PQ

a i

c\T

llll "11

^IVJ

Chromatogram 1. Standard mixture of the trichloroethyl esters of the eight phenoxyalkanoic acid herbicides, representing 0.05 ng of each acid.

398

Method W 7

Chromatogram 2. Tap water. * = Impurity.

Method W 7

m

O

«C

399

g CsJ

il

I!

UL Chromatogram 3. Tap water, fortified with 0.5 |ig/l of each phenoxyalkanoic acid herbicide. * = Impurity.

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Pipet 1.0, 3.0 and 5.0 ml each of the derivative standard solution into 10-ml volumetric flasks and make up to the marks with eluting mixture. Inject 1 ul of each solution (equivalent to

400

Method W 7

0.01, 0.03 and 0.05 ng phenoxyalkanoic acid) into the gas chromatograph. Plot the peak areas or heights of the peaks obtained vs. ng of acid equivalents in the calibration solutions injected. The calibration curves are linear within the above range. Also inject 1-jnl aliquots of the sample solutions. For the areas or heights of the peaks obtained for these solutions, read the appropriate amounts of acid equivalents from the corresponding calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from tap and ground water samples, fortified with the phenoxyalkanoic acids at levels of 0.05 to 5 \xg/\, ranged from 70 to 125% and averaged 85%. The routine limit of determination was 0.05 \xg/\. 7.3 Calculation of residues The residue R, expressed in M-g/1, of an identified phenoxyalkanoic acid is calculated from the following equation: R =

W A V TEnd V,.G

where G

= sample volume (in 1)

VEnd = terminal volume of sample solution from 6.5 (in ml) Vj WA

= portion of volume VEnd injected into gas chromatograph (in fil) = amount of identified phenoxyalkanoic acid equivalent for Vj read from calibration curve (in ng)

8 Important points The method was tested with tap and ground waters. Blank values, mostly lower than 0.05 (xg/1, can occur depending on impurities in the water samples. Interfering peaks can also result from insufficiently pure reagents. In case of doubt, confirm the results by mass spectrometry using multiple ion detection, in order to avoid false positive results when using this very sensitive method. Diclofop is not included in the method because of the unreliability of the recoveries obtained. The retention time for the diclofop trichloroethyl ester was 28 min 6 s, using the conditions given in 6.6.

Method W 7

401

9 References R.V. Smith and S.L. Tsai, Trichloroethyl esters as derivatives for gas chromatography with electron capture detection, J. Chromatogr. 61, 29-34 (1971). S. Mierzwa and S. Witek, Gas-liquid chromatographic method with electron-capture detection for the determination of residues of some phenoxyacetic acid herbicides in water as their 2,2,2trichloroethyl esters, J. Chromatogr. 136, 105-111 (1977).

10 Author Shell Forschung GmbH, Schwabenheim, D. Eichler

Triazine Herbicides Water

w8 High-performance liquid chromatographic determination

(German version published 1989)

1 Introduction The method permits the identification and quantitative determination of ametryn, atrazine, prometryn, propazine, simazine, terbuthylazine, and terbutryn in ground and tap waters.

2 Outline of method The triazine herbicide residues are extracted from water using the solid phase extraction technique. The water sample is passed through a disposable cartridge containing RP-18 reversed-phase material. The retained residues are eluted with a methanol-dichloromethane mixture and determined by high-performance liquid chromatography (HPLC) using a UV detector.

3 Apparatus Solid phase extraction manifold, e.g. Vac Elut (Analytichem International No. AI 6000) Stainless steel tube, 0.8 mm i.d., 1.6 mm o.d., 25 cm long PTFE tube, 1.5 mm i.d., 3 mm o.d., 50 cm long Glass syringe, 50-ml, with PTFE Luer-lock fitting Pear-shaped flask, 100-ml, with ground joint Rotary vacuum evaporator, 40 °C bath temperature High-performance liquid chromatograph equipped with UV detector Glass syringe, 1000-ul

4 Reagents Acetonitrile, for spectroscopy (Merck No. 16) Dichloromethane, for UV spectroscopy (Fluka No. 66745) Ethanol p.a., absolute (Merck No. 983) Methanol, for HPLC (Fluka No. 65541) Water, for HPLC Acetonitrile + water mixture: acetonitrile + water for HPLC 2 : 8 v/v

404

Method W 8

Eluting mixture: dichloromethane + methanol 7:3 v/v Mobile phase: acetonitrile + ammonium acetate solution 45:55 v/v, de-gassed with helium Triazine stock solutions: 1000 ng/ml of each compound in absolute ethanol (except: 100 ng/ml of simazine in methanol). Transfer 500 \ig of each compound (0.5 or 5.0 ml, respectively, of the stock solutions) into a 50-ml volumetric flask and make up to the mark with absolute ethanol. The stock solution mixture then contains 10 ng/ml of each compound Triazine standard solutions: Pipet 2 ml of the stock solution mixture into a 100-ml volumetric flask and make up to the mark with acetonitrile-water mixture. The resulting solution contains 0.2 jxg/ml of each compound. Dilute aliquots of this solution further with acetonitrile-water mixture to obtain solutions containing 0.1, 0.04 and 0.01 fig/ml Triazine solution for recovery experiments: Pipet 1.0 ml of the stock solution mixture into a 50-ml volumetric flask and make up to the mark with water for HPLC. The resulting solution contains 0.2 ing/ml of each compound Ammonium acetate solution, 0.1 mol/1 CH3COONH4 p.a. (Merck No. 1116) in water for HPLC RP-18 disposable cartridge: Bond Elut C-18, 500 mg/2.8 ml, including adapter (Analytichem International No. AI 607303 and AI 636001, respectively) Helium for gas chromatography (for de-gassing the HPLC mobile phase)

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Conditioning of the cartridge Attach the cartridge to the solid phase extraction manifold. Using gentle water jet pump suction, allow to pass through, first 10 ml methanol, followed by 10 ml water for HPLC, at a rate of 5-10 ml/min. 6.2 Sample preparation Measure the volume of the analytical sample (approx. 1 1) (G). Add 2 volumes of methanol for each 1000 volumes of water. 6.3 Sample delivery Connect the conditioned cartridge via an adapter to the PTFE tube. Connect the other end of the PTFE tube to the stainless steel tube, which is dipped in the analytical sample. Pass the sample, with suction, through the cartridge at a rate of 15-20 ml/min. Remove the PTFE tube from the cartridge.

Method W 8

405

6.4 Elution To elute the triazines, pass 30 ml eluting mixture, with suction, through the cartridge, and collect the eluate in a 100-ml pear-shaped flask. Alternatively, connect the cartridge to a 50-ml glass syringe. Fill the syringe with 30 ml eluting mixture, insert the plunger, force the liquid through the cartridge, and collect the eluate in a 100-ml pear-shaped flask. Rotary-evaporate the eluate to dryness. 6.5 High-performance liquid chromatographic determination Dissolve the residue derived from 6.4 in 5.0 ml (VEnd) acetonitrile-water mixture. Inject an aliquot of this solution (Vj) into the sample loop of the high-performance liquid chromatograph. Operating conditions Pump Injector Column Mobile phase Flow rate Detector Attenuation Recorder Injection volume Retention times for simazine atrazine ametryn propazine terbuthylazine prometryn terbutryn

Constant volume pump, model LC 250/1 (Kratos) Injection valve 70-10 fitted with sample loop 70-11 (Rheodyne) Stainless steel, 4.6 mm i.d., 15 cm long; packed with LC 8 DB, particle size 3 |um (Supelco No. 58991) Acetonitrile + ammonium acetate solution 45 :55 v/v, de-gassed with helium 1 ml/min UV detector Spectroflow 783 (Kratos), programmable Wavelength 220 nm Programmed: From start to 6.5 min at 0.07 AUFS, and from 6.5 min to end of chromatogram 0.04 AUFS 10 mV; chart speed 10 mm/min 200 \x\ 4 min 5 min 7 min 8 min 8 min 10 min 11 min

30 s 50 s 20 s 30 s 30 s 30 s

Method W 8

406

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Chromatogram 1. Standard mixture of seven triazine herbicides plus metolachlor, representing 40 ng of each compound.

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Chromatogram 2a. Standard mixture representing 2 ng each of simazine, atrazine, terbuthylazine, terbutryn and metolachlor.

Chromatogram 2b. Water for HPLC. Aliquot injected representing 40 ml water.

408

Method W 8

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Chromatogram 3a. Water for HPLC, fortified with 0.1 jug/l each of simazine, atrazine, terbuthylazine, terbutryn and metolachlor. Aliquot injected representing 40 ml water or 4 ng of each compound added.

Chromatogram 3b. Tap water. Aliquot injected representing 40 ml water.

Method W 8

409

Chromatogram 4a. Standard mixture representing 2 ng each of simazine, atrazine, ametryn, propazine and prometryn.

1

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Chromatogram 4b. Ground water. Aliquot injected representing 40 ml water.

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 200 ul of each triazine standard solution (equivalent to 2, 8, 20 and 40 ng of each triazine herbicide) into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng of each triazine herbicide. Also inject 200-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of each triazine herbicide from the corresponding calibration curve. 7.2 Recoveries and lowest determined concentration The recoveries from water for HPLC, fortified with each triazine herbicide at levels of 0.1 and 0.5 ng/1 (using 0.5 or 2.5 ml of the solution for recovery experiments), ranged from 89 to :

410

Method W 8

and averaged 96% with a standard deviation of 4% (see Table). Blanks usually did not occur. The routine limit of determination was 0.05 ng/1 for each compound. Table. Percent recoveries from water for HPLC fortified with triazine herbicides. Triazine herbicide

0

,

Ametryn Atrazine Prometryn Propazine Simazine Terbuthylazine Terbutryn

101 97, 99, 102 100 89, 92, 93, 99, 92, 93,

Added (ng/1)

99, 110

96, 98 104 98, 99

^ 94 93, 96 92 93, 92, 92,

94, 95, 98

94, 96, 97 95, 97 94, 97, 97

7.3 Calculation of residues The residue R, expressed in \ig/\, of an identified triazine herbicide is calculated from the following equation: p

wA-vEnd

where G

= sample volume (in 1)

VEnd = terminal volume of sample solution from 6.5 (in ml) Vj

= portion of volume VEnd injected into high-performance liquid chromatograph (volume of sample loop) (in ul)

WA

= amount of triazine herbicide for Vj read from calibration curve (in ng)

8 Important points UV detectors other than the one indicated in 6.5 may yield routine limits of determination not as low as reported in 7.2. Metolachlor can also be determined, under the same conditions, with a routine limit of determination of 0.05 ng/1. Recoveries ranged from 83 to 106% at levels of 0.1 and 0.5 u.g/1. It is anticipated that other triazine herbicides can also be determined by this method, with small modifications to the method in individual cases. Triazine herbicides can be determined by both HPLC and GC. The choice of method can only be made after comparative experiments. Often only a combination of HPLC and GC can provide the required separation and allow for the determination of all triazine herbicides in question. In order to prevent false positive results, HPLC results should be confirmed by GC and vice versa.

Method W 8

411

9 References No data

10 Authors Ciba Geigy AG, Agricultural Division, Basle, Switzerland, G. Formica, C. Giannone and W. D. Hormann

Desalkyl Metabolites of Chlorotriazine Herbicides Water

w 13

High performance liquid chromatographic determination

(German version published 1991)

1 Introduction The method permits the identification and quantitative determination of the following three chlorotriazine desalkyl metabolites in ground and tap waters: ci

R =

C2H5

iso-C 3 H 7

tert-C 4 H 9

Code No. Trivial name

G 28279 Desethylsimazine 174-175

G 30033 Desethylatrazine 134-135

GS 26379 Desethylterbuthylazine 143-144

54

270

48

Melting point (°C) Solubility in water at 25 °C (mg/100 ml)

The method is recommended for its simplicity and low routine limit of determination. The desalkyl metabolites cannot be determined by Method W 8 of this Manual because they are only partially retained on the disposable cartridge column. The parent triazine herbicides, however, cannot be analyzed by Method W 13 because they are lost during the evaporation of the analytical samples.

2 Outline of method An aliquot of the analytical sample is concentrated to a few millilitres. The salts present are precipitated with acetonitrile; subsequently, the acetonitrile is removed. The desalkyl metabolites are determined by high-performance liquid chromatography (HPLC) using a UV detector and the column switching technique.

3 Apparatus Round-bottomed flasks, 500-ml and 100-ml, with ground joints Graduated cylinder, 100-ml Rotary vacuum evaporator, 40-50°C and 70-80°C bath temperatures

414

Method W 13

Centrifuge, with buckets for 10-ml tubes Centrifuge glass tubes, 10-ml, with polyethylene caps Pasteur pipets High-performance liquid chromatograph equipped with UV detector and column switching device Glass syringe, 1000-^1

4 Reagents Acetonitrile, for spectroscopy (Merck No. 16) Ethanol p.a., absolute (Merck No. 983) Methanol p.a. (Merck No. 6009) Water, for HPLC Mobile phase 1: water for HPLC + acetonitrile 85:15 v/v (for elution of G 28279 and G 30033) Mobile phase 2: water for HPLC + acetonitrile 70-60:30-40 v/v (for elution of GS 26379) Metabolite stock solutions: 1000 ng/ml each of G 30033 and GS 26379, and 100 ng/ml of G 28279, in absolute ethanol or methanol Metabolite standard solutions: Mix 1.0 ml of the G 30033 and 10.0 ml of the G 28279 stock solutions, equivalent to 1000 \xg of each compound. Progressively dilute both, this mixture and a 1000-jig aliquot of the GS 26379 stock solution, with water for HPLC to yield two series of standard solutions containing 0.02, 0.01, 0.005 and 0.001 ng/ml each of G 30033 and G 28279, or GS 26379, respectively Metabolite solutions for recovery experiments: Mix aliquots of the three stock solutions and progressively dilute with water for HPLC to obtain two solutions containing 0.05 and 0.01 ng/ml of each compound

5 Sampling and sample preparation The analytical samples are taken and prepared as described on pp. 23 ff, Vol. 1. They should be stored in half-full 2-1 bottles at — 20 °C, lying on their sides in order to prevent the bottles from breaking during freezing or thawing.

6 Procedure 6.1 Sample preparation Transfer 100 ml of the analytical sample (G) into a tared 500-ml round-bottomed flask and rotary-evaporate to 2-4 ml with 70-80 °C bath temperature; then add water for HPLC to adjust the weight of the concentrate to 4.0 g. Using a Pasteur pipet, transfer the solution (or suspension) into a 10-ml centrifuge tube previously washed with 2-3 ml acetonitrile. Rinse the flask with 4 ml acetonitrile and pipet the rinsings also into the centrifuge tube. Cap with a polyethylene cap, shake, and centrifuge for 2-4 min at 1000 • g.

Method W 13

415

Rinse a tared 100-ml round-bottomed flask with 2-3 ml acetonitrile, discard the rinsings, and decant the supernatant centrifugate into the flask. Suspend the residue in the centrifuge tube in 2 ml acetonitrile, cap the tube, and centrifuge again. Combine the clear supernatant with the first centrifugate and rotary-evaporate with 40-50 °C bath temperature to less than 4 g. Then adjust the weight of the solution to 5.0 g by adding water for HPLC. This is equivalent to a volume of 5 ml (VEnd). 6.2 High-performance liquid chromatographic determination Inject an aliquot (Vj) of the solution derived from 6.1 into the sample loop of the high-performance liquid chromatograph. A scheme of the HPLC column switching system is given in Fig. 1.

Fig. 1. Set-up of apparatus for HPLC determination using column switching. 1, Filling of sample loop; 2, Dosing and elution of column 1; 3, Transfer from column 1 to column 2; 4, Elution of column 2. Positions of the valves: L, load; I, inject.

416

Method W 13

Operating conditions Pumps 1 and 2 Injector Columns 1 and 2 Column switching valves Mobile phase 1 Mobile phase 2 Flow rates Detector

Attenuation Recorder Injection volume Mobile phases (for columns 1 and 2) Retention times

Constant volume pumps, model LC 250/1 (Kratos) Spark Promis programmed automatic injection (Spark Holland), equipped with a 500-jj.l sample loop in the injection valve (Rheodyne) Stainless steel, 4 mm i.d., 12 cm long; packed with Nucleosil 100 C18, particle size 5 \\m (Knauer No. B7-Y76) MUST (Spark Holland) Water for HPLC + acetonitrile 85:15 v/v Water for HPLC + acetonitrile 70-60:30-40 v/v 1 ml/min UV detector Spectroflow 783 (Kratos), programmable, equipped for the control of the MUST column switching valves Wavelength 220 m Detector range 0.01 AUFS 10 mV; chart speed 5 mm/min 500 ul G 28279 G 30033 GS 26379 Mobile phase 1 Mobile phase 1 Mobile phase 2 10 min 30 s 20 min 12 s 8 min 30 s

Setting the switching times: The switching times must be set anew before the start of each analysis series for GS 26379 and for G 28279 plus G 30033. To do this, connect the exit from column 1 with the detector inlet and inject approx. 500 pi standard solution (0.02 fig/ml) into the high-performance liquid chromatograph under the conditions given for the appropriate substance(s). Follow the elution run with the recorder. Determine the switching intervals, i.e. the times in which the substances are fully eluted. Typical switching times Begin switching 5 min for G 28279 and G 30033: End switching 11 min 12 s Cycle time 22 min Begin switching 4 min 18 s for GS 26379: End switching 5 min 6 s Cycle time 10 min 30 s

Method W 13

417

Fig. 2. G 30033 (desethyl-atrazine) and G 28279 (desethyl-simazine) in water. Chromatogram 1: Standard mixture representing 0.5 ng of each metabolite. Chromatogram 2: Water for HPLC, fortified with 0.1 pig/l of each metabolite. Aliquot injected equivalent to 10 ml water or 1 ng of each compound added. Chromatogram 3: Ground water. Aliquot injected equivalent to 10 ml water.

418

Method W 13

Fig. 3. GS 26379 (desethyl-terbuthylazine) in water. Chromatogram 1: Standard mixture representing 0.5 ng GS 26379. Chromatogram 2: Water for HPLC, fortified with 0.1 ng/1 GS 26379. Aliquot injected equivalent to 10 ml water or 1 ng metabolite. Chromatogram 3: Ground water. Aliquot injected equivalent to 10 ml water.

Method W 13

419

7 Evaluation 7.1 Method Quantitation is performed by the calibration technique. Prepare calibration curves as follows. Inject 500 ul of each standard solution (equivalent to 0.5, 2.5, 5 and 10 ng of each metabolite) into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng of each metabolite. Also inject 500-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of the identified metabolite from the corresponding calibration curve.

7.2 Recoveries and lowest determined concentration The recoveries from water for HPLC, fortified with the metabolites at levels of 0.1, 0.5 and 2 M-g/1, ranged from 84 to 119% and averaged 98% with a standard deviation of 6.2% (see Table). Blanks usually did not occur. The routine limit of determination was 0.05 ng/1 for each compound. Table. Percent recoveries from water for HPLC fortified with G 28279, G 30033 and GS 26379. Added (ng/1)

G 28279

G 30033

G S 26379

0.1

89/90/95/99/99/100/ 101/103/119 89/95/96/98/98/100/ 100/104/105 97/97

94/95/98/101/102/ 102/103/103/105/105 87/89/98/98/99/100/ 102 88/88/98/99/99

84/86/96/102/104/ 109 93/93/95/98/101/101

0.5 2

94/96

7.3 Calculation of residues The residue R, expressed in |ng/l, of an identified metabolite is calculated from the following equation:

p

w A -v E n d

where G

= sample volume (in 1)

VEnd = terminal volume of sample solution from 6.1 (in ml) Vj

= portion of volume VEnd injected into high-performance liquid chromatograph (volume of sample loop) (in ul)

WA

= amount of metabolite for V{ read from calibration curve (in ng)

420

Method W 13

8 Important points Acetonitrile is added to the concentrated analytical sample in 6.1 to precipitate the salts in solution, in order to prevent blockages in the columns and tubes of the high-performance liquid chromatograph.

9 References No data

10 Authors Ciba Geigy AG, Agricultural Division, Basle, Switzerland, G. Formica and C. Giannone

Part 6 Pesticide Residue Analytical Methods for Water Using the AMD Technique

Thin-Layer Chromatographic Analysis of Pesticides and Metabolites Using the Automated Multiple Development (AMD) Technique (German version published 1991)

1 Introduction The technique for the automated multiple development (AMD) of thin-layer chromatograms has been especially developed for the analysis of pesticide residues in drinking water. High-performance thin-layer chromatography (HPTLC) silica gel plates are developed automatically in many (e.g. 20 to 30) individual steps, thereby permitting the mobile phase to advance somewhat farther in each succeeding cycle. Solvent mixtures of different composition can be used for each cycle, so that a reproducible gradient elution is obtained. The final determination is usually based on measuring the UV absorbance in a TLC scanner, using up to six different wavelengths. Applying the AMD technique results in much better separations than would be obtained with conventional thin-layer chromatography. Therefore, several pesticides can be simultaneously determined in the same extract. A high throughput can also be achieved because up to 18 spots can be applied onto a single TLC plate. The AMD technique can be used most successfully if it is preceded by a broad-spectrum cleanup procedure such as described, e.g., in Cleanup Methods 7 and 8 (pp. 37 ff and pp. 41 ff, this Volume).

2 Outline of method An aliquot of the cleaned-up sample solution is applied as a 4 mm long band onto a HPTLC plate, using a TLC applicator. The plate is developed automatically in the AMD unit and dried at 120 °C with a stream of nitrogen. The chromatogram is evaluated by measuring the UV absorbance at six different wavelengths in a TLC scanner. Using a multi-colour plotter, the absorption curves obtained for the individual tracks are plotted, displaying the curves for the six wavelengths superimposed in different colours. Compounds that do not, or only weakly, absorb UV light can be converted to coloured derivatives by immersing the plate into the solution of a suitable reagent. The results are confirmed by chromatographing a second aliquot of the sample solution, using an elution gradient of entirely different selectivity (confirmative gradient) than was used in the first analysis. A further check can be made by recording and comparing the UV spectra of the peaks of the standard and sample solutions on the plate developed with the confirmative gradient.

424

AMD Technique

3 Apparatus Sample vials, 5-ml, with cone-shaped inside, e.g. conic ampoules N 18-5 (Macherey-Nagel No. 702240) TLC applicator, suitable for applying sample solutions in the form of narrow bands to HPTLC plates, e.g. Linomat IV (Camag) Micro syringes, 100-ul, suitable for TLC applicator AMD system, consisting of a development unit, controller and accessories (Camag) Vacuum pump Vacuum manometer Computer-controlled measurement and evaluation system for TLC: Camag TLC scanner II, Computer HP 9816, Multi-colour plotter HP 7550 A, TLC calculation software 88; all obtainable from Camag Glass tank, for simultaneously developing 5 TLC plates 20 cm x 20 cm, with glass cover (Desaga) Laboratory hot plate, electronically controlled (Desaga), equipped with: Plate drying chamber, made from two 23 cm x 23 cm glass plates, placed in a U-shaped aluminium frame at a distance of 2.5 cm. The fourth side of the chamber is open to allow a TLC plate to be inserted. The two opposite sides of the frame have a track each extending to half height to allow the insertion of a second TLC plate. The upper glass plate has a 1-cm dia. hole cut near to its rear edge to allow nitrogen or compressed air to be introduced Chromatogram dipping device for HPTLC plates (only for compounds that do not, or only weakly, absorb UV light)

4 Reagents Acetonitrile, for chromatography (Baker No. 9392) Carbon disulphide, p.a. (Baker No. 8021) Dichloromethane, for chromatography (Baker No. 32222) Diisopropyl ether (Baker No. 8072) n-Heptane, p.a. (Riedel-de Haen No. 32287) Methanol, p.a. (Merck No. 6009) 2-Propanol (isopropanol), for residue analysis (Merck No. 998) Solvent mixture: acetonitrile + methanol + n-heptane 50:50:5 v/v/v Internal standard solution: 0.1 mg/ml diphenyl sulphone in methanol Pesticide standard solutions: 10 |ug/ml of each compound + 10 \xg/m\ diphenyl sulphone in methanol Ammonia solution, p.a., approx. 25% (Riedel-de Haen No. 30501) Formic acid, p.a., 98-100% (Riedel-de Haen No. 33015) Nitrogen, passed through granular activated charcoal (DeguSorb AS IV, Degussa) Pre-coated HPTLC silica gel 60 glass plates, 10 cm x 20 cm, without fluorescent indicator (Merck No. 5641), prepared as follows: Scrape off 5 mm each from the right and left edges of a HPTLC plate. Place this plate and a second, unlayered glass plate of the same size in the

AMD Technique

425

glass tank, previously filled with 2 1 isopropanol, and allow to stand for at least 1 h, or for up to several days. Remove the HPTLC plate and put it, silica gel side up, into the plate drying chamber placed on the hot plate. Insert the second, unlayered glass plate approx. 1 cm above in the frame track. Dry both plates at approx. 120 °C with purified nitrogen or compressed air for 30 min. Immediately after drying, lay the glass plate onto the silica gel layer of the HPTLC plate, and seal with adhesive tape. Wrap the plates in aluminium foil and keep wrapped until used

5 Applying the solutions onto the TLC plates Dissolve the residue obtained, e.g. from Cleanup Method 8, step 6.4 or 6.5, in 200 L| L1 solvent mixture in a sample vial. Pull 100 ul of this solution into a microsyringe. Using the TLC applicator, apply the solution in the form of a 4 mm long band at a distance of 8 mm from the bottom edge of the TLC plate. The distance between the individual tracks should be 6 mm; leaving an empty space of 13 mm from both the left and right edges of the plate, each plate then has room for 18 tracks. Set the application rate at 6 s per ul. For comparison, apply 5 to 50 JLII of the pesticide standard solution (equivalent to 50 to 500 ng of each compound). Continue with the thin-layer chromatographic separation, using the screening gradient for developing the chromatogram (see 6.3).

6 Thin-layer chromatographic determination 6.1 Principle of the AMD technique In AMD, the plate is automatically cycled through a pre-set number of developments in the AMD chamber. In each succeeding development, the mobile phase is permitted to advance somewhat farther by a constant distance. Each time the components of the sample and standard solutions are separated and, at the same time, focused anew. First, the chamber is evacuated to remove the last traces of solvent from the starting lines of the sample or standard solutions. The chamber is flushed with purified nitrogen and, with the addition of eluting solvent, the first cycle is started. A few seconds later, the cycle is terminated by removing the solvent with suction. Then the next cycle is started including drying, ventilating, and a somewhat longer elution, followed by removal of the eluting solvent. This is continued until the whole programme is completed. For each cycle, new mobile phase is introduced into the tank. If its composition differs from the preceding one, this will result in a gradient elution. A gradient for silica gel begins strongly polar (strongly eluting) and ends less polar (weakly eluting). Only when this sequence is followed, a reproducible elution on silica gel can be achieved. The course of a gradient elution with stepwise separation of a mixture is illustrated in the following scheme:

426

AMD Technique

25-[

n:

Fig. 1. Gradient elution by AMD; s, start; n, number of cycles; S, starting line; ch, chromatographable portion of the extract; a to d, components of this portion; , solvent front.

6.2 Chromatographic separation A typical elution gradient for HPTLC silica gel plates that extends from a methanol-dichloromethane mixture via dichloromethane to carbon disulphide is shown in the following diagram: CH 2 CI 2

10080604020-

CH3OH

5

10

15

20

25 n

Fig. 2. Typical concentration profile of an elution gradient; n, number of cycles.

This gradient can also be superimposed with a pH gradient from weakly alkaline to moderately acid, in order to cover acid, basic and neutral substances in the same chromatogram. The determining factor for the direction of the gradient (from alkaline to acid) is the binder of the silica gel, which consists of an alkali carboxylate. The actual chromatographic separation is preceded by a three-step cycle for focusing the spots. 0.01 % ammonia is added to the eluant, which enhances somewhat the alkalinity of the plate. To the following eluants, 0.1% formic acid is added. Due to the buffer effect of alkali salt + ammonia and formic acid, alkaline conditions prevail during the early elution cycles. These conditions are gradually shifted to the acid side in the following cycles.

AMD Technique

427

Six reservoirs are available on the AMD apparatus to produce the gradients. For each cycle, a mixer is filled completely with solvent from the reservoirs. From this, half of the solvent mixture is used as eluant for the next cycle, and the other half remains in the mixer. The mixer is then re-filled with new solvent from one of the reservoirs. So when dichloromethane is used as eluant for one cycle, and the mixer is re-filled with methanol, then the eluant for the next cycle is dichloromethane-methanol 1:1 v/v. After the three runs of the first cycle for focusing the spots, often an entirely different solvent mixture is used as eluant. The mixer must therefore be completely emptied ("Empty mixer after step 3"). A time programme for a 25-cycle gradient elution, the first cycle of three runs serving to focus the spots, is shown in 6.3, Separation conditions. After completion of the last cycle, the HPTLC plate is placed into the plate drying chamber and dried at approx. 120 °C for 3 min in a purified stream of nitrogen. It is then left to cool, and the UV absorption on the individual tracks is measured in the TLC scanner. 6.3 Separation conditions Separation chamber Camag AMD chamber Gradient programmes (Mobile phase components in v/v) Screening gradient Reservoir 2 3 4 5

Solvent Carbon disulphide Dichloromethane Diisopropyl ether Methanol Ammonia solution Formic acid

6

Confirmative gradient Reservoir 1 2 3 4 5 6

100 90

90

10 10 0.01 0.1

100

0.1

50

100

100 100

0.1

0.1

90 90 10 10 0.01 0.1

100

100 50

0.1

0.1

0.1

Time programme for the gradient elution of 25 cycles. Reservoir

Run No.

Cycle No.

1 1 1 2 1 3 Empty mixer

Time min

0.2 0.2 0.2

4 5 6 7

2 3 4 5

0.5 0.8 1.0 1.3

8 9 10 11 12

6 7 8 9 10

1.6 1.9 2.3 2.7 3.1

Reservoir

Run No.

Cycle No.

Time

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

3.5 4.0 4.5 5.0 5.7 6.5 7.2 8.0 9.0 10.0 11.0 11.9 12.8 13.7 14.6

2

4

Abs]

0 10 20 30 40 50 60 70 80 A10G A220 Ad'-l." A260 A280 A300 A650

[mV/10

90 [mm]

Fig. 3. Screening chromatogram of a standard mixture, using the conditions as described in 6.3 and 6.4. IS, diphenyl sulphone (internal standard); 1, omethoate; 2, carbendazim; 3, triadimenol; 4, metamitron; 5, triadimefon; 6, methabenzthiazuron; 7, azinphos-methyl; 8, azinphos-ethyl; 9, parathion. 200 ng of each compound, equivalent to 0.4 |LXg/l in a water sample (after Burger 1988). The remission curve at 650 nm was registered after staining the organophosphate spots by reaction with 4-(4-nitrobenzyl)pyridine and tetraethylenepentamine.

600

CD

CD O IT

o

>

AMD Technique

429

1=

T3

1

o

a o U

430

AMD Technique

Drying period before first cycle after each cycle after final cycle

at least 5 min 3 min 10 min

6.4 Measuring conditions Apparatus and Controller Y position for zeroing Y start position Y track end X start position X track distance Feed Pause No. of values per measuring point Peak optimization Sensitivity Amplification (span) Peak threshold Smoothing Light source Slit Spectral slit width Measuring wavelengths

Camag TLC Scanner II, linked to Computer HP 9816 with Camag 88 TLC evaluation software 4 mm 4 mm 90 mm 15 mm 10 mm 0.1 mm 0.01 s 5 0 Automatic set 7 5 3 Deuterium lamp 0.1 x 3 mm 10 nm 190 or 200 nm, 220, 240, 260, 280, 300 nm

Additional conditions for recording UV spectra: Pilot wavelength According to UV spectrum of compound Measuring range 190-350 nm Resolution 2 nm No. of values per measuring point 20

6.5 Plot parameters To evaluate the sample and standard peaks, for each track the remission curves are drawn, with the six wavelengths superimposed in different colours. By this means each peak is defined not only by its distance from the starting line but also by its absorbance at the various wavelengths (see also Figs. 3 and 4). Scale Y = 200 mAU Baseline correction 1 Plot section Only plot the range containing the compounds of interest 6.6 Confirmation of results Positive results obtained with the screening gradient must be confirmed in any case. For this purpose, apply the second half of the sample solution derived from Section 5 onto another HPTLC plate and chromatograph using the confirmative gradient as described in 6.3. Apply

AMD Technique

431

only those compounds for comparison, for which a signal was obtained in the screening chromatogram of the sample solution.

7 Evaluation 7.1 Method Quantitation is performed by measuring the peak heights on the tracks of the sample solutions and comparing them with the peak heights obtained on the tracks of the standard solutions, both for the chromatograms developed with the confirmative gradient. The results are corrected with the aid of the peak height of the internal standard in the tracks of the sample and standard solutions. The peaks of the solutions should exhibit comparable heights. On no account peak heights obtained at different wavelengths should be compared for quantitation purposes. Depending on the UV absorbancy of the individual compounds, in general approx. 25 ng can be determined from standard solutions.

7.2 Calculation of residues The residue R, expressed in \ig/l, of an identified compound is calculated from the following equation: HA-Wst-HIst-WIA HSfWISt-HIAG where G

= sample volume (in ml)

W st = amount of compound applied onto HPTLC plate with standard solution (in ng) WIA = amount of internal standard applied onto HPTLC plate with sample solution (in ng) WISt = amount of internal standard applied onto HPTLC plate with standard solution (in ng) HA

= peak height obtained for the compound in the sample solution (in mm)

H S t = peak height obtained for the compound in the standard solution (in mm) H I A = peak height obtained for the internal standard in the sample solution (in mm) H I S t = peak height obtained for the internal standard in the standard solution (in mm)

8 Important points For a reproducible gradient elution, an optimal vacuum must be achieved in the AMD chamber during the drying steps. Therefore, make sure that the chamber is vacuum-tight before each gradient run. Proceed as follows: Apply vacuum to the chamber so that the maximum

432

AMD Technique

vacuum (approx. 4 mbar) is reached after 30-60 s. Then turn the three-way valve to a position that the connection between pump and chamber is interrupted. The pressure in the chamber should then not rise above 16 mbar within 1 min. When no sufficient separation of the compounds was achieved, the gradient programme must be changed. For this purpose, measure the distance from the start position to the solvent front (max. 90 mm) and divide it by the number of cycles; e.g., for a distance of 90 mm and 25 cycles this results in 3.6 mm per cycle. If the compound in question travels a distance of 36 mm, corresponding to approx. 10 cycles, then the polarity of the following gradients must be raised to improve the separation. If the confirmative chromatogram gives a positive result, measure and compare the complete UV spectra of both the sample and standard peaks on the plate in the reflection mode (see e.g. Figure 5). For additional confirmation, isolate the compound in question from the adsorbent and identify it by mass spectrometry. Compounds that do not, or only weakly, absorb UV light can be converted to coloured derivatives by immersing the plate into the solution of a suitable reagent before measuring as described in 6.4 (see 9. References). In other cases, they can be converted to fluorescent compounds which are determined at their characteristic wavelengths in the TLC scanner.

(Abs-UV]

190

240

290

340

390

440 nm

Fig. 5. UV spectra of methabenzthiazuron peaks, measured on the HPTLC plate in the reflection mode. from a standard solution, from a sample solution.

AMD Technique

433

9 References K. Burger, Multiple method for ultratrace determination: Pesticide active ingredients in ground and drinking water analyzed by TLC/AMD (Automated Multiple Development), Pflanzenschutz-Nachr. 41, 175-228 (1988). H. Jork, W. Funk, W. Fischer and H. Wimmer, Dunnschicht-Chromatographie. Reagenzien und Nachweismethoden. Band 1 a; Physikalische und chemische Nachweismethoden: Grundlagen, Reagenzien I, VCH Verlagsgesellschaft, Weinheim 1989. K. Burger, J. Kohler and H. Jork, Application of AMD to the determination of crop protection agents in drinking water. Part 1: Fundamentals and method, J. Planar Chromatogr. 3, 504-510 (1990).

10 Author Bayer AG, Analytical Laboratories, Dormagen, K. Burger

Examples for Applying the AMD Technique to the Determination of Pesticide Residues in Ground and Drinking Waters Example 1 Azinphos-ethyl azinphos-methyl bitertanol, carbofuran, metamitron, methabenzthiazuron, parathion, triadimefon, and triadimenol (German version, Method W 9, published 1991). Bromacil, cymoxanil diuron, lenacil, and metribuzin (German version Method W 10, published 1991). 1.1 Apparatus and Reagents See 3. Apparatus and 4. Reagents on pp. 424f, this Volume; for preparing and cleanup of the analytical sample, see Cleanup Method 8, pp. 41 ff, this Volume. Additionally the following standard solutions are required: Compound standard solution 1:10 |ug/ml of each azinphos-ethyl, azinphos-methyl, bitertanol, carbofuran, triadimefon and diphenyl sulphone (internal standard) in methanol Compound standard solution 2:10 pig/ml of each metamitron, methabenzthiazuron, parathion, triadimenol and diphenyl sulphone (internal standard) in methanol Compound standard solution 3:10 jig/ml of each bromacil, cymoxanil, diuron, lenacil, metribuzin and diphenyl sulphone (internal standard) in methanol 1.2 Cleanup Proceed with 1000 ml of the analytical sample (G) as described in Cleanup Method 8, pp. 41 ff, this Volume. 1.3 AMD conditions Proceed as described in Sections 5 and 6 on pp. 425 ff, this Volume, with the following alterations: Gradient programmes for the compounds contained in Compound standard solutions 1 and 2 (mobile phase components in v/v) Screening gradient Confirmative gradient Solvent Reservoir Reservoir

1 Acetonitrile Carbon disulphide Dichloromethane Diisopropyl ether Ammonia solution Formic acid

2

3

4

5

6

1

100 30 70

2

3

4

5

100 25 100

80 100 20

0.01

100

100 75

100 100

100

0.1

0.1

0.1

0.01 0.1

6

0.1

0.1

0.1

436

AMD Technique - Examples Screening gradient

Confirmative gradient

Rf values for Compound standard solution 1 bitertanol triadimefon carbofuran azinphos-methyl azinphos-ethyl diphenyl sulphone

0.16 0.37 0.42 0.55 0.58 0.63

0.23 -*) 0.63 0.71 0.79 0.97

Compound standard solution 2 triadimenol metamitron methabenzthiazuron triadimefon parathion diphenyl sulphone

0.15 0.18 0.40 — 0.80 0.63

0.22 0.19 0.42 0.58**) 0.96 0.97

*) Compound standard solution 1 without triadimefon, **) Compound standard solution 2 with triadimefon added. Gradient programmes for the compounds contained in Compound standard solution 3 (mobile phase components in v/v) Screening gradient Confirmative gradient Solvent Reservoir Reservoir Carbon disulphide Dichloromethane Diisopropyl ether Methanol Ammonia solution Formic acid

Rf values for Compound standard solution 3 lenacil bromacil diuron cymoxanil metribuzin diphenyl sulphone

1

2

90

90

3

4

5

6

1

2

3

4

5

100

10 10 0.01 0.1

100

0.1

6 100

100 100

0.1

100 30 10 70 0.01 0.1

0.1

100 100 100

0.1

0.1

0.1

Screening gradient

Confirmative gradient

0.25 0.33 0.39 0.43 0.55 0.69

0.34 0.41 0.23 0.23 0.60 0.67

1.4 Evaluation For quantitation and calculation of the residues, proceed as described in 7.1 and 7.2, p. 431, this Volume. The recoveries from control samples, fortified with the compounds at levels of 0.05 to 0.4 ng/1, are given in Table 1. The limit of detection was approx. 0.03 fig/1, and the limit of determination was approx. 0.05 ng/1.

AMD Technique - Examples

437

Table 1. Percent recoveries from water, fortified with the compounds contained in Compound standard solutions 1, 2, and 3. Compound

0.05

0.1

0.15

Added (ng/1) 0.2 0.25

0.3

0.4

Standard solutions 1 and 2 Azinphos-ethyl Azinphos-methyl Bitertanol Carbofuran Metamitron Methabenzthiazuron Parathion Triadimefon Triadimenol

92 100 116 128 98 106 108 111 100

85 96 90 94 82 85 83 99 90

94 70 95 87 81 93 98 97 92

91 107 103 98 79 95 89 95 88

90 100 90 100 70 93 93 97 91

89 96 85 85 69 90 87 93 88

100 112 100 91 94 107 81 88 95

Standard solution 3 Bromacil Cymoxanil Diuron Lenacil Metribuzin

114 93 122 97 92

94 93 109 101 78

86 103 96 110 74

95 90 98 107 95

90 103 89 118 88

87 111 93 104 82

95 115 97 104 95

1.5 Important points The recoveries of bitertanol, metribuzin and lenacil can be lower with water samples containing a high proportion of humic acids. 1.6 Author Bayer AG, Analytical Laboratories, Dormagen, K. Burger

Example 2 Benalaxyl bendiocarb, bentazone, dinoseb, dinoterb, DNOQ metalaxyl, nitrothal-isopropyl pendimethalin, and terbumeton (German version Method W 11, published 1991). The esters of fluazifop-P, fluroxypyn haloxyfop, and triclopyr can also be analyzed using this technique. 2.1 Apparatus and Reagents See 3. Apparatus and 4. Reagents on pp. 424f, this Volume; for preparing and cleanup of the analytical sample, see Cleanup Method 8, pp. 41 ff, this Volume. Additionally the following standard solutions are required: Compound standard solution 4: 10 |j,g/ml of each benalaxyl, dinoterb, DNOC, fluazifop-Pbutyl, fluroxypyr-1-methylheptyl, haloxyfop-ethoxyethyl, and metalaxyl in methanol

438

AMD Technique - Examples

Compound standard solution 5: 10 ng/ml of each bendiocarb, bentazone, dinoseb, nitrothal-isopropyl, pendimethalin, terbumeton, and triclopyr-butoxyethyl in methanol An internal standard is not required. 2.2 Cleanup Proceed with 1000 ml of the analytical sample (G) as described in Cleanup Method 8, pp. 41 ff, this Volume. Do not add internal standard to the analytical sample. 2.3 AMD conditions Dissolve the residue derived from Cleanup Method 8, step 6.4 or 6.5, in 200 ul solvent mixture (VEnd) in a sample vial. Apply approx. 100 \i\ of this solution (Vi) onto the HPTLC plate. Also apply 3 to 30 \x\ of the Compound standard solutions 4 or 5 (equivalent to 30 to 300 ng of each compound). Proceed as described in Section 6 on pp. 425 ff, this Volume, with the following alterations: Gradient programmes for the compounds contained in Compound standard solutions 4 and 5 (mobile phase components in v/v) Screening gradient Confirmative gradient Solvent Reservoir Reservoir

1 Acetonitrile Carbon disulphide Dichloromethane Diisopropyl ether Ammonia solution Formic acid

2

3

4

5

6

1

2

3

4

5

100 25

100 30 100 70

100

100

100 100

0.01

75

100 100 100

0.1

0.1

0.01 0.1

6

0.1

0.1

0.1

0.1

0.1

Screening gradient

Confirmative gradient

Rf values for Compound standard solution 4 metalaxyl benalaxyl haloxyfop-ethoxyethyl fluazifop-P-butyl fluroxypyr-1-methylheptyl DNOC dinoterb

0.36 0.64 0.73 0.79 0.84 0.88 0.93

0.30 0.70 0.80 0.79 0.90 0.92 0.98

Compound standard solution 5 terbumeton bendiocarb nitrothal-isopropyl triclopyr-butoxyethyl bentazone dinoseb pendimethalin

0.31 0.73 0.75 0.84 0.88 0.91 0.97

0.32 0.69 0.92 0.91 0.83 0.95 0.97

AMD Technique - Examples

439

2.4 Evaluation 2.4.1 Method Quantitation is performed by measuring the peak heights on the tracks of the sample solutions and comparing them with the peak heights obtained on the tracks of the standard solutions. The peaks from both solutions should exhibit comparable heights. Quantitation can also be performed by the calibration technique. Prepare calibration curves by plotting the peak heights obtained for each compound on the standard solution tracks vs. ng of the respective compound. For the heights of the peaks obtained from the sample solutions, read the appropriate amounts from the corresponding calibration curves. When a suitable software is available (e.g. Camag 88 TLC evaluation software), quantitation can also be performed by computer calculation from the raw data using a calibration curve. 2.4.2 Recoveries, limit of detection and limit of determination The recoveries from control samples, fortified with the compounds at levels of 0.1 to 0.5 |xg/l, ranged from 70 to 110% and averaged 88%; see Table 2. The limit of detection was approx. 0.05 ng/1, a n d the limit of determination was approx. 0.1 \ig/\. Blank values interfering with the determination of the polar compounds metalaxyl and terbumeton were frequently observed in the analysis of waters containing high proportions of humic acids. Moreover, recoveries were reduced in such cases. Table 2. Percent recoveries from water, fortified with the compounds contained in Compound standard solutions 4 and 5 (means of 2-4 experiments). Compound Benalaxyl Bendiocarb Bentazone *> Dinoseb Dinoterb DNOC Fluazifop-P-butyl*0 Fluroxypyr-1-methylheptyl *> Haloxyfop-ethoxyethyl*> Metalaxyl *} Nitrothal-isopropyl Pendimethalin *} Terbumeton *> Triclopyr-butoxyethyl *>

Added ftig/1) 0 1

97 105 89 106 88 95 96 77 84 74 93 111 93 106

Q2

Q5

68 78 93 98 82 76 76 92 91 89 84 78 — 88

82 93 85 94 87 93 87 75 79 77 89 85 69 85

*> The recoveries are not valid for waters with very high humic acid content.

440

AMD Technique - Examples

2.4.3 Calculation of residues The residue R, expressed in jig/1, of an identified compound is calculated from the following equations: when comparing peak heights H A -W St -V End H st • Vi • G when using a calibration curve R

w A -v E n d

where G

= sample volume (in ml)

V E n d = terminal volume of sample solution from 2.3 (in ul) Vt

= portion of volume V E n d applied onto H P T L C plate (in ul)

Wst

= a m o u n t of c o m p o u n d applied onto H P T L C plate with standard solution (in ng)

WA

= a m o u n t of c o m p o u n d for V{ read from calibration curve (in ng)

HA

= peak height obtained from V^ (in m m )

HSt

= peak height obtained from W S t (in m m )

2.5 Important points Using 10 cm x 20 cm pre-coated HPTLC silica gel 60 glass plates with an extra thin layer of 0.1 mm (Merck No. 11764), the intensity of the peaks was increased. The Rf values for the compounds, applied to these plates and run under screening conditions, were as follows (see also Fig. 1 and 2): Rf values for Compound standard solution 4 metalaxyl benalaxyl haloxyfop-ethoxyethyl fluazifop-P-butyl fluroxypyr-1 -methylheptyl DNOC dinoterb

0.32 0.53 0.64 0.72 0.78 0.87 0.91

Compound standard solution 5 terbumeton bendiocarb nitrothal-isopropyl triclopyr-butoxyethyl bentazone dinoseb pendimethalin

0.24 0.61 0.65 0.72 0.76 0.84 0.91

2.6 Authors Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H. Kohle and H.-G. Nolting

AMD Technique - Examples 250

[mV/10

441

Abs]

70

80

90

[mm]

Fig. 1. Screening chromatogram of Compound standard solution 4. 1, metalaxyl; 2, benalaxyl; 3, haloxyfop-ethoxyethyl; 4, fluazifop-P-butyl; 5, fluroxypyr-1-methylheptyl; 6, DNOC; 7, dinoterb. 200 ng of each compound, HPTLC silica gel plate, layer thickness 0.1 mm, measurement wavelengths 200, 220, 240, 260, 280, and 300 nm.

400 [mV/10 Abs]

30

40

50

60

70

80

90 [mm]

Fig. 2. Screening chromatogram of Compound standard solution 5. 8, terbumeton; 9, bendiocarb; 10, nitrothal-isopropyl; 11, triclopyr-butoxyethyl; 12, bentazone; 13, dinoseb; 14, pendimethalin. For conditions, see Fig. 1.

442

AMD Technique - Examples

Example 3 Amitrole (German version Method W 12, published 1991). 3.1 Apparatus and Reagents 3.1.1 Apparatus For cleanup of the analytical sample: pH meter Round-bottomed flask, 50-ml, with ground joint Rotary vacuum evaporator, 45 °C bath temperature Solid phase extraction system, e.g. Visiprep SPE Vacuum Manifold (Supelco No. 5-7030) Disposable column, volume 3 ml, filled with 500 mg silica gel (Baker No. 7086-03) Sample vials, 5-ml, with cone-shaped inside, e.g. conic ampoules N 18-5 (Macherey-Nagel No. 702240) Device for evaporating solvents in a nitrogen stream, suitable to take the 5-ml sample vials, e.g. Silli-Therm heating module with Silli-Vap evaporator and Reacti-Bar heating block (Pierce No. 19793, 19792 and 19785, respectively) For AMD thin-layer chromatography: See p. 424, this Volume. Additionally the following items are required: Double chambered tank, for developing 20 cm x 10 cm TLC plates, with glass cover (Camag No. 022.5253) Second dipping tank for HPTLC plates (e.g. Desaga No. 124152), or additional glass insert suitable for the dipping tank Baron-DC-Tauchfix (Desaga No. 124162) Hot-air blower

3.1.2 Reagents Acetonitrile, for chromatography (Merck No. 30) Dichloromethane, for chromatography (Merck No. 6044) Diisopropyl ether, p.a. (Baker No. 8072) Methanol, for chromatography (Merck No. 6007) Eluting mixture: acetonitrile + methanolic ammonia solution 8:2 v/v Amitrole standard solution: 1 jug/ml methanol Chromogenic solution: 0.1 g/100 ml N-(l-naphthyl)ethylenediamine dihydrochloride p.a. (Merck No. 6237) in dichloromethane + methanol mixture 8:2 v/v Ammonium amidosulphonate solution: 0.1 g/100 ml ammonium amidosulphonate p.a. (Merck No. 1220) in dichloromethane + methanol mixture 75:25 v/v Sulphuric acid, 0.5 mol/1 H 2 SO 4 ; prepared from sulphuric acid, p.a., 95-979/0 (Merck No. 731) Hydrochloric acid, p.a., 32% (Merck No. 319) Formic acid, p.a., 98-100% (Merck No. 264) Ammonia gas (Merck Lecture Bottle No. 823209 with valve No. 823002)

AMD Technique - Examples

443

Methanolic ammonia solution: Saturate 500 ml methanol in a 1-1 two-necked flask, under ice cooling, with gaseous ammonia. Dilute the solution with a further 500 ml of ice-cold methanol, stopper, and store in a deep freeze Sodium nitrite, p.a. (Riedel-de Haen No. 31443) Nitrogen, passed through granular activated charcoal (DeguSorb AS IV, Degussa) Pre-coated HPTLC silica gel 60 glass plates, 10 cm x 20 cm, without fluorescent indicator (Merck No. 5641), prepared as described on pp. 424 f, this Volume 3.2 Cleanup Mount the disposable column onto the vacuum unit of the extraction system and condition it with 5 ml eluting mixture. Do not allow the column to run dry. Acidify 20 ml of the analytical sample (G) in a 50-ml round-bottomed flask to pH 2 with sulphuric acid and rotary-evaporate to dryness. Take up the residue in 2 ml eluting mixture and transfer the solution quantitatively onto the disposable column, then add a further 3 ml eluting mixture to the column. Collect the total eluate (approx. 5 ml) in a sample vial and apply slight suction to the column to drive the remainder of the eluting mixture into the vial. Transfer the vial to the heating block (35 °C) of the evaporation device and evaporate to dryness under a stream of nitrogen. 3.3 Thin-layer chromatographic determination Proceed as described on pp. 425ff., this Volume, with the following alterations: 3.3.1 Applying the solutions onto the TLC plate Dissolve the residue derived from 3.2 in 200 ul methanol (VEnd). Using the TLC applicator, apply approx. 100 ul of this solution (Vj) onto a thin-layer plate, at a distance of 8 mm from the bottom edge, in the form of a 7 mm long band. The distance between the individual tracks should be 3 mm; leaving an empty space of 11.5 mm from both the left and right edges of the plate, each plate then has room for 18 tracks. Set the application rate at 6 s per ul. For comparison, apply 1, 5, 10, and 20 ul of the amitrole standard solution (equivalent to 1 to 20 ng amitrole). 3.3.2 AMD conditions Separation chamber Camag AMD chamber Gradient programmes (Mobile phase components in v/v) Solvent 1 Dichloromethane Diisopropyl ether Methanol Methanolic ammonia solution Formic acid

Screening gradient Reservoir 2 3 4 5 80

90

100 20

10

100

Confirmative gradient Reservoir 1 2 3 4 5 60

72

86

40

28

14

0.1

0.1

0.1

100 100

100

0.1

0.1

444

AMD Technique - Examples

Time programme for the gradient elution of 20 cycles. Reservoir

Run No.

Cycle No.

1 1 2 1 3 1 Empty mixer

Time min 0.2 0.2 0.2

4 5 6 7

2 3 4 5

0.5 0.8 1.0 1.3

8 9 10 11 12

6 7 8 9 10

1.6 1.9 2.3 2.7 3.1

Drying period before first cycle after each cycle after final cycle Rf value for amitrole

Reservoir

at least 5 min 3 min 10 min Screening gradient 0.26

Run No.

Cycle No.

Time min

13 14 15 16 17 18 19 20 21 22

11 12 13 14 15 16 17 18 19 20

3.5 4.0 4.5 5.0 5.7 6.5 7.2 8.0 9.0 10.0

Confirmative gradient 0.32

3.3.3 Visualization Add approx. 10 ml hydrochloric acid dropwise to approx. 12 g sodium nitrite in one of the chambers of the double chambered tank. Close the tank with the glass cover. When the tank is full of brown fumes, allow the dried thin-layer plate to stand in the second, empty chamber of the tank for 10 s. Longer exposure times can lead to oxidations on the plate. Remove the plate from the tank and dry for about 1 min in a fume hood with a hot-air blower to remove nitrous gases. Dip the plate into the chromogenic solution for 3 s, using the chromatogram dipping device, and dry for 20 s with the hot-air blower. Dip the plate into the ammonium amidosulphonate solution for 3 s, using the chromatogram dipping device, and dry for 20 s with the hot-air blower. In the course of this, the pink amitrole spots turn yellow. Then measure the absorbance of the spots immediately! 3.3.4 Measuring conditions and plot parameters

See p. 430, this Volume, with the following alterations: Light source Measuring wavelength Scale Baseline correction Plot section

Tungsten lamp 490 nm Y = 200 mAU 1 Only plot the range containing amitrole

AMD Technique - Examples

445

3.4 Evaluation 3.4.1 Method Quantitation is performed from the chromatograms developed with the confirmative gradient as described for Example 2 on p. 439, this Volume. 3.4.2 Recoveries and limit of determination The recoveries from control water samples, fortified with amitrole at levels of 0.05 to 5 ^g/1, ranged from 77 to 93% and averaged 89%. The limit of determination was approx. 0.05 jug/1. 3.4.3 Calculation of residues The residue R, expressed in u.g/1 amitrole, is calculated from the equations described for Example 2 on p. 440. 3.5 Important points According to step 3.2, the dry residue is taken up in 2 ml eluting mixture. If greater amounts of material remain undissolved, recovery rates of amitrole may be reduced. In these cases, therefore, the analytical sample in 3.2 should be adjusted to pH 2 with hydrochloric acid (1 mol/1) instead of sulphuric acid. When many plates are to be subjected to colour development, the following alteration in the visualization procedure is recommended: Add 20 ml fuming hydrochloric acid p.a. (Merck No. 317) to one of the chambers of the double chambered tank. Place the dried thin-layer plate into the second, empty chamber of the tank for 10 min; then remove excess hydrochloric acid fumes from the plate by exposing it to a hot-air blow for 20 s. Next, transfer the plate into a thin-layer tank (Desaga No. 120101) and introduce nitric oxide from a gas cylinder (Merck Lecture Bottle No. 823317, with valve No. 823009) into the tank, until it is filled with brown fumes. Then remove excess nitrous gases from the plate by exposing it to a hot-air blow for 3 min. Continue as described in 3.3.3, beginning "Dip the plate into the chromogenic solution The amitrole azo-derivative is stable for approx. 1 to 2 h; any measurements must be terminated within this time span.

3.6 Author Bayer AG, Analytical Laboratories, Dormagen, K. Burger

Cumulative Indexes for Volumes 1 and 2

Index of Determinate Pesticides, Metabolites and Related Compounds (Index of Compounds) Numbers in italics refer to Multiple Pesticide Residue Analytical Methods; numbers in italics marked with an asterisk refer to Individual Pesticide Residue Analytical Methods.

Acephate 1: 81*; 2: 25, 31, 301, 317 Acetaldehyde, see Metaldehyde Acetaldehyde 2,4-dinitrophenylhydrazone 2: 239 Acetanilide 1: 252 Acetanilides, substituted 1: 252 3-Acetylamino-l,2,4-triazole, see Monoacetylamitrole N-Acetyl-glufosinate, dimethyl ester 2: 218 Alachlor 2: 25, 31, 301, 317 Aldicarb 1: 87*; 2: 25, 31, 301, 349 Aldicarb sulphone 1: 87; 2: 31, 358 Aldicarb sulphoxide 1: 87; 2: 358 Aldrin 1: 51, 73, 77, 221, 283, 297, 309, 327, 383; 2: 25, 31, 313, 317, 387 Allethrin 2: 25 Allidochlor 2: 31 Ametryn 1: 221, 265, 283; 2: 31, 313, 317, 403 Amidithion 1: 221, 335; 2: 31, 317 Aminocarb 2: 301, 349 5-Amino-4-chloropyridazin-3(2H)-one, see Chloridazon, metabolites 2-Amino-3,5-dibromoacetophenone 2: 273 Aminomethylphosphonic acid, see AMPA Amitraz 2: 31 Amitrole 2: 49* 301, 442 AMPA 2: 229 Anilazine 1: 77, 221, 383; 2: 31, 59* 317 Anilide herbicides 1: 246, 263 Aniline 1: 247 Anilines, substituted 1: 241, 251 Anthraquinone 2: 31, 301, 317, 377 Aroclor 1016-1268, see PCB Atraton 1: 221, 265; 2: 31 Atrazine 1: 221, 265, 283, 347; 2: 25, 31, 313, 317, 403 Azinphos-ethyl 1: 64, 73, 77, 221, 283, 335, 371, 383; 2: 31, 313, 317, 428, 429, 435 Azinphos-methyl 1: 221, 283; 2: 25, 31, 313, 317, 428, 429, 435

Aziprotryne 1: 221, 283; 2: 31, 313 Azocyclotin 1: 222; 2: 343 Azocyclotin, methyl derivative 2: 343 Barban 2: 25, 31, 273 Benalaxyl 2: 301, 437 Benazolin, methyl ester 2: 25 Bendiocarb 2: 25, 31, 301, 349, 437 Benfluralin 2: 31, 301, 317 Benodanil 2: 31 Benomyl 2: 69* 111 Bensulide 2: 31 Bentazone 2: 302, 437 Benzoic acids, chlorinated 1: 171 Benzoylprop-ethyl 2: 31, 317 3,4-Benzpyrene (benzo[a]pyrene) 2: 35 Benzyl thiocyanate 1: 90 Bifenox 2: 31, 302, 317 Bifenthrin 2: 302, 333 Binapacryl 1: 68, 77, 222, 383; 2: 31, 197* 317 Biphenyl 2: 31, 294 Bis(4-chlorophenyl)methanol (DBH) 2: 31 Bisdithiocarbamate fungicides 1: 353, 407 Bitertanol 1: 78, 222, 383; 2: 31, 77* 87* 317, 377, 435 Bromacil 1: 222, 283; 2: 25, 31, 313, 317, 435 Bromacil, N-methyl derivative 2: 25 Bromide, inorganic 1: 377 Bromine-containing fumigants 1: 377 4-Bromoacetanilide 1: 252 4-Bromoaniline 1: 247 4-Bromo-3-chloroacetanilide 1: 252 4-Bromo-3-chloroaniline 1: 247 2-Bromoethanol 1: 377 4-Bromo-l-naphthylacetic acid methyl ester 1:167 Bromophos 1: 64, 77, 222, 283, 297, 309, 335, 371, 383; 2: 25, 31, 313, 317 Bromophos-ethyl 1: 64, 77, 222, 283, 335, 371, 383; 2: 25, 31, 313, 317

450

Index of Compounds

Bromopropylate 2: 31, 302, 575, 317 Bromoxynil 2: 31, 99* Bromoxynil, methyl ether 2: 25, 99 Bromoxynil octanoate 2: 31, 99, 302, 317 Brompyrazon 2: 31 Bupirimate 1: 222, 283; 2: 313 Butocarboxim 2: 302, 349 Butocarboxim, metabolites 2: 358 Buturon 1: 222, 241, 251 Butylhydroxyanisole 2: 35 Butylhydroxytoluene 2: 35 Camphechlor 1: 73, 77, 222, 297, 383; 2: 31, 317 Captafol 1: 78, 93* 99* 222, 283, 383, 401; 2: 25, 31, 313, 317, 359, 377 Captan 1: 78, 105* 222, 283, 327, 383, 401; 2: 25, 31, 313, 317, 359, 377 Carbaryl 2: 25, 31, 302, 349 Carbendazim 2: 25, 69* 107* 302, 428, 429 Carbendazim, pentafluorobenzyl derivative 2:107 Carbetamide 2: 25 Carbofuran 2: 25, 31, 113* 302, 349, 435 Carbofuran, metabolite, see 3-Hydroxycarbofuran Carbon disulphide 1: 353, 359, 411 Carbophenothion 1: 73, 77, 223, 283, 309, 335, 361, 383; 2: 31, 313, 317 Carbophenothion oxygen analogue 2: 31 Carbophenothion sulphone 1: 223, 365, 368, 369 Carbophenothion sulphone, oxygen analogue 1: 369 Carbophenothion-methyl 2: 31, 302, 317 Carbosulfan 2: 775* Chinalphos, see Quinalphos Chinomethionat (quinomethionate) 2: 31, 302, 317, 377 Chloranil 2: 31 Chlorazine 2: 31 Chlorbenside 2: 31, 303, 313, 317 Chlorbenside sulphone 2: 31, 303, 317 Chlorbromuron 1: 223, 241, 251; 2: 25, 31 Chlorbufam 2: 25, 31 a-Chlordane (cis-chlordane) 1: 77, 223, 297, 309, 327, 383; 2: 25, 31, 317 y-Chlordane (trans-chlordane) 1: 73, 77, 223, 297, 309, 327, 383; 2: 25, 31, 317 Chlordecone 1: 295; 2: 32 Chlorfenethol 2: 32 Chlorfenprop-methyl 2: 25, 32, 303, 317 Chlorfenson 1: 73, 77, 223, 383; 2: 32, 313, 317

Chlorfenvinphos 1: 64, 73, 77, 223, 283, 309, 335, 371, 383; 2: 25, 32, 313, 317 Chlorflurenol 2: 127* 303, 313 Chlorflurenol, methyl ester 2: 127 Chloridazon 1: 246, 263; 2: 25, 32, 755* 273, 303, 577 Chloridazon, metabolites 2: 135 Chlormephos 2: 32, 303, 577 Chlormequat 2: 36 4-Chloroacetanilide 1: 252 4-Chloroaniline 1: 247 2-(2-Chloroanilino)-4,6-dimethoxy-l,3,5-triazine, see Dimethoxy anilazine Chlorobenzilate 2: 32, 303, 575, 577 2-Chlorofluorenone 2: 128 2-Chloro-9-hydroxyfluorene 2: 128 3-Chloro-4-methoxyaniline 1: 247 3-Chloro-4-methylacetanilide 1: 252 3-Chloro-4-methylaniline 1: 247; 2: 26 Chloroneb 2: 25, 32, 303, 577 2-Chloro-4-nitroaniline 2: 32 Chloroparaffins (C12 - C18) 2:35 Chlorophenols 2: 35 4-(4'-Chlorophenoxy)acetanilide 1: 252 4-Chlorophenoxyacetic acid 2: 32 Chlorophenoxyalkanoic acids 1: 78, 171; 2: 369, 393 4-(4'-Chlorophenoxy)aniline 1: 247 Chloropropylate 2: 32, 303, 575, 577 Chlorothalonil 2: 32, 303, 577, 377 Chlorotoluron 1: 223, 241, 251; 2: 26, 32, 577 Chlorotriazine herbicides, desalkyl metabolites 1: 59, 347; 2: 413 Chloroxuron 1: 223, 241, 251; 2: 26, 32, 577 Chlorpropham 1: 223, 527; 2: 26, 32, 273, 577 Chlorpropham, dibromo derivative 1: 321 Chlorpyrifos 1: 77, 223, 283, 297, 335, 383; 2: 26, 32, 575, 577 Chlorpyrifos-methyl 1: 223, 283; 2: 32, 575, 577 Chlorsulfuron 2: 145* Chlorthal 1: 223, 283; 2: 575 Chlorthal-dimethyl 2: 26, 32, 303, 577 Chlorthiamid 1: 126; 2: 26 Chlorthion 1: 64, 224, 335, 371 Chlorthiophos 1: 109* 224, 283; 2: 32, 575, 577 Chlorthiophos oxygen analogue 1: 110 Chlorthiophos sulphone 1: 110 Chlorthiophos sulphoxide 1: 110 Cinerins 1: 236; 2: 26, 323 Clophen A 3 0 - A 60, T 64, see PCB CMPP, see Mecoprop Copper 2: 153 Copper oxychloride 2: 755*

Index of Compounds Coumaphos 2: 32, 303, 317 Coumarin 2: 35 Coumithoate 2: 32 Crotoxyphos 2: 32, 304, 317 Crufomate 2: 32, 304, 317 Cyanazine 2: 26, 32, 304, 313, 317 Cyanofenphos 1: 224, 283; 2: 32, 313, 317 Cyanophos 2: 32, 304, 313, 317 Cycluron 2: 32 Cyfluthrin 2: 304, 333 Cyhalothrin 2: 304, 333 Cyhexatin 1: 224; 2: 343 Cyhexatin, methyl derivative 2: 343 Cymoxanil 2: 32, 157* 304, 317, 435 Cypermethrin 1: 224; 2: 26, 32, 317, 333 2,4-D 2: 32, 163% 304, 369, 393 2,4-D, dinitro derivative of methyl ester 2: 369 2,4-D, isooctyl ester 2: 26, 374 2,4-D, methyl ester 2: 26, 32, 374 2,4-D, trichloroethyl ester 2: 393 Dalapon 1: 117*; 2: 26 Dalapon, methyl ester 1: 117 Dazomet 2: 26 2,4-DB 2: 32, 304, 393 2,4-DB, methyl ester 2: 26 2,4-DB, trichloroethyl ester 2: 393 p,p'-DDA 2: 32 o,p'-DDD 1: 73, 77, 224, 297, 383; 2: 32, 317 p,p'-DDD 1: 51, 73, 77, 224, 283, 297, 309, 327, 383; 2: 32, 313, 317 o,p'-DDE 1: 73, 77, 224, 383; 2: 32, 317, 387 p,p'-DDE 1: 51, 73, 77, 224, 283, 297, 309, 327, 383; 2: 32, 313, 317, 387 o,p'-DDT 1: 51, 73, 77, 224, 297, 309, 327, 383; 2: 26, 32, 317, 387 p,p'-DDT 1: 51, 68, 73, 77, 225, 283, 297, 309, 327, 383; 2: 26, 32, 313, 317, 387 Decachlorobiphenyl 2: 32 DEF 2: 304, 317 Deltamethrin 1: 225; 2: 32, 317, 333 Demephion-O 2: 32 Demephion-S 2: 32 Demeton-O 1: 225, 361; 2: 32 Demeton-O sulphone 1: 225, 365, 368, 369 Demeton-O sulphone, oxygen analogue 1: 369 Demeton-S 1: 225, 361; 2: 32 Demeton-S sulphone 1: 225, 365, 368, 369; 2: 32, 317 Demeton-S sulphoxide 1: 225, 368, 369; 2: 32, 317 Demeton-S-methyl 1: 225,335, 361; 2: 26, 32,317 Demeton-S-methyl sulphone 1: 225, 365, 368, 369; 2: 32, 317

451

Demeton-S-methyl sulphoxide (oxydemetonmethyl) 1: 225, 368, 369; 2: 34, 317 Desalkyl triazine metabolites 1: 59, 347; 2: 413 Desethyl-atrazine 1: 226, 347; 2: 413 N-Desethyl-pirimiphos-methyl 1: 192; 2: 32, 304, 317 Desethyl-simazine, see Des-tert-butyl-terbuthylazine bis(Desethyl)-simazine 1: 225, 347 Desethyl-terbuthylazine 2: 305, 413 Desmethyl-formamido-pirimicarb 1: 183, 2: 305, 349 Desmethyl-norflurazon 2: 32 Desmethyl-pirimicarb 1: 183, 2: 305, 349 Desmetryn 1: 226, 265, 283; 2: 26, 32, 313 Des-tert-butyl-desethyl-terbumeton 1: 226, 347 Des-tert-butyl-desethyl-terbutryn 1: 226, 347 Des-tert-butyl-terbumeton 1: 226, 347 Des-tert-butyl-terbuthylazine 1: 226, 347; 2: 413 Des-tert-butyl-terbutryn 1: 226, 347 Dialifos 2: 26, 32, 305, 313, 317, 359 Di-allate 2: 26, 32, 305, 317 Diazinon 1: 64, 73, 77, 226, 283, 309, 335, 371, 383; 2: 26, 32, 313, 317 Diazoxon 1: 226, 371 Dibrom, see Naled Dibutyl phthalate 2: 35 Dicamba, methyl ester 2: 26 Dichlobenil 1: 123* 226, 283; 2: 26, 32, 169* 313, 317 Dichlofenthion 1: 64, 226, 283, 335, 371; 2: 26, 32, 313, 317 Dichlofluanid 1: 78, 227, 283, 327, 383; 2: 26, 32, 777* 191* 313, 317, 377 Dichlofluanid, metabolite, see DMSA Dichlone 2: 32 3,4-Dichloroacetanilide 1: 252 3,4-Dichloroaniline 1: 247; 2: 273 3,5-Dichloroaniline 1: 213 2,6-Dichlorobenzamide 2: 32 Dichlorobenzenes 2: 35 p,p'-Dichlorobenzophenone 2: 32, 305, 317 2,7-Dichlorofluorenone 2: 133 2,7-Dichloro-9-hydroxyfluorene carboxylic acid, methyl ester 2: 133 Dichlorophenols 2: 35 2,4-Dichlorophenoxy-phenoxypropionic acid 2: 32 3,5-Dichlorophenyl-chloroacetamide 1: 213 Dichlorprop (2,4-DP) 2: 32, 163* 305, 369, 393 Dichlorprop, dinitro derivative of methyl ester 2: 369 Dichlorprop, isooctyl ester 2: 26 Dichlorprop, methyl ester 2: 26, 163

452

Index of Compounds

Dichlorprop, trichloroethyl ester 2: 393 Dichlorvos 1: 64, 227, 283, 335, 371; 2: 26, 32, 313, 317 Diclofop 2: 400 Diclofop, trichloroethyl ester 2: 400 Diclofop-methyl 1: 727*; 2: 32, 305, 317 Diclofop-methyl, metabolite 1: 127, 133 Dicloran 1: 399; 2: 32, 305, 317 Dicofol 1: 77, 227, 283, 297, 327, 383; 2: 26, 32, 313, 317 Dicrotophos 1: 77, 227, 383; 2: 32, 317 Dieldrin 1: 51, 73, 77, 227, 283, 297, 309, 327, 383; 2: 26, 32, 313, 317, 387 Dienochlor 2: 32 Dihydrocoumarin 2: 35 Dimefox 1: 77, 227, 335, 383; 2: 32, 317 Dimethachlor 1: 227, 283; 2: 32, 313, 317 Dimethirimol, methyl ether 2: 26 Dimethoate 1: 64, 77, 227, 283, 335, 371, 383; 2: 26, 32, 313, 317 Dimethoate oxygen analogue, see Omethoate Dimethoxy anilazine 2: 32, 59 Dimethylaminosulphanilide, see DMSA Dimethylaminosulphotoluidide, see DMST Dimethyl 5-nitroisophthalate, see Nitrothalisopropyl, methyl esters of metabolites O,O-Dimethyl phosphonate 2: 211 Dinitramine 2: 32, 305, 317 Dinobuton 2: 32, 197* 305, 317 Dinocap 1: 78, 227, 383; 2: 32, 317 Dinoseb 2: 306, 437 Dinoseb acetate 2: 32 Dinoterb 2: 306, 437 Dinoterb, methyl ether 2: 26 Dioxacarb 2: 26, 32, 306, 349 Dioxathion 1: 73, 227, 283, 335; 2: 32, 313, 317 Diphenamid 2: 26, 32 Diphenylamine 2: 32 Diphenyl sulphone 2: 42, 404, 428, 429, 435 Dipropetryn 2: 32 Dipropyl isocinchomeronate 2: 32 Diquat 2: 36 Disulfoton 1: 64, 227, 283, 335, 361, 371; 2: 26, 32, 313, 317 Disulfoton sulphone 1: 77, 227, 335, 365, 368, 369, 371, 383; 2: 32, 317 Disulfoton sulphoxide 1: 77, 228, 368, 369, 383; 2: 32, 317 Ditalimfos 1: 77, 228, 283, 383; 2: 32, 313, 317, 359 Dithianon 2: 33 Dithiocarbamate fungicides 1: 353, 407 Diuron 1: 228, 241, 251; 2: 26, 273, 435 DMSA 2: 32, 177, 195, 377

DMST DNOC DNOC, Dodine 2,4-DP,

2: 32, 177, 195, 377 2: 306, 437 methyl ether 2: 26 2: 26, 36 see Dichlorprop

Edifenphos 2: 33, 306, 317 Endosulfan 1: 228, 327; 2: 26 a-Endosulfan 1: 73, 77, 283, 383; 2: 33, 313, 317, 387 P-Endosulfan 1: 73, 77, 295, 383; 2: 33, 313, 317, 387 Endosulfan sulphate 1: 73, 77, 228, 295, 383; 2: 33, 313, 317 Endrin 1: 73, 77, 228, 297, 309, 327, 383; 2: 26, 33, 317, 387 EPN 2: 33, 306, 317 Ethidimuron 2: 33 Ethiofencarb 2: 26, 306, 349 Ethiofencarb, metabolites 2: 358 Ethion 1: 64, 73, 77, 228, 283, 297, 335, 371, 383; 2: 33, 313, 317 Ethirimol 2: 26, 36 Ethirimol, methyl ether 2: 26 Ethoprophos 1: 228, 283; 2: 33, 313, 317 Ethoxyquin 2: 33 O-Ethyl O-methyl phosphonate, see Fosetyl, methyl ester Ethylene bisdithiocarbamate fungicides 1: 353, 407 Ethylene thiourea 1: 135* 411 Ethylvanillin 2: 35 Etridiazole 2: 33 Etrimfos 1: 228, 283; 2: 27, 33, 313, 317 ETU, see Ethylene thiourea Famophos 2: 33, 306, 317 Fenamiphos 1: 228, 361; 2: 33, 313, 317 Fenamiphos sulphone 1: 228, 365, 368, 369 Fenamiphos sulphoxide 1: 229, 368, 369 Fenarimol 2: 27, 33, 306, 313 Fenazaflor 2: 33 Fenbutatin oxide 1: 229; 2: 343 Fenbutatin oxide, methyl derivative 2: 343 Fenchlorphos 1: 64, 73, 77, 229, 283, 297, 309, 335, 371, 383; 2: 33, 313, 317 Fenitrothion 1: 64, 77, 229, 283, 335, 371, 383; 2: 27, 33, 313, 317 Fenoprop 2: 33, 306, 393 Fenoprop, isooctyl ester 2: 27 Fenoprop, methyl ester 2: 27 Fenoprop, trichloroethyl ester 2: 393 Fenpropathrin 1: 229; 2: 333 Fenson 1: 73, 77, 229, 383; 2: 33, 313, 317

Index of Compounds Fensulfothion 1: 64, 229, 283, 335, 361, 371; 2: 33, 313, 317 Fensulfothion sulphone 1: 229, 365, 368, 369; 2: 33 Fensulfothion sulphone, oxygen analogue 1: 369; 2: 33 Fensulfothion sulphoxide, oxygen analogue 2:33 Fenthion 1: 64, 229, 283, 335, 361, 371; 2 33, 313, 317 Fenthion sulphone 1: 229, 365, 368, 369; 2: 33 Fenthion sulphone, oxygen analogue 1: 369; 2: 33 Fenthion sulphoxide 1: 229, 368, 369; 2: 33 Fenthion sulphoxide, oxygen analogue 2: 33 Fentin, methyl derivative 2: 343 Fentin acetate, chloride, hydroxide 1: 230;

2:343 Fenuron 1: 230, 241, 251; 2: 27, 273 Fenvalerate 1: 229; 2: 33, 317, 333 Flamprop-isopropyl 2: 27 Flamprop-methyl 2: 27 Fluazifop-butyl 2: 33, 306, 437 Flubenzimine 2: 33, 307, 317 Fluchloralin 2: 33, 307, 313, 317 Flucythrinate 2: 307, 333 Fluometuron 1: 230, 241, 251 Fluorenone 2: 128 Fluorodifen 2: 307, 313 Fluotrimazole 1: 78, 230, 383; 2: 33, 317, 377 Flurenol 2: 127* Flurenol, n-butyl ester 2: 127 Fluroxypyr 2: 33 Fluroxypyr, n-butyl ester 2: 33 Fluroxypyr-(l-methylheptyl) 2: 33, 307, 437 Fluvalinate 2: 33, 307, 317 Folpet 1: 143* 230, 283, 327, 401; 2: 33, 313, 317, 359 Fonofos 1: 230, 283; 2: 33, 205* 313, 317 Fonofos oxygen analogue 2: 33 Formothion 1: 77, 230, 283, 383; 2: 27, 33, 313, 317 Fosetyl 2: 211* Fosetyl, metabolite, see Phosphorous acid Fosetyl, methyl ester 2: 211 Fosetyl-aluminium, see Fosetyl Fuberidazole 1: 78, 230, 383; 2: 33, 317, 377 Fumigants, bromine-containing 1: 377 Fungicides, bisdithiocarbamate 1: 353, 407 Fungicides, organotin 2: 343 Fungicides, phthalimide 1: 93, 105, 143, 401; 2: 359, 377 Fungicides, thiuram disulphide 1: 353

453

Genite 2: 33, 307, 317 Glufosinate 2: 217* Glufosinate, metabolite, see 3-(Methylphosphinico)propionic acid Glufosinate-ammonium, see Glufosinate Glyphosate 2: 229* Glyphosate, metabolite, see AMPA Halowax 1000-1051 2: 35 Haloxyfop-(2-ethoxyethyl) 2: 307, 437 HCB 1: 51, 73, 77, 231, 295, 297, 309, 327, 383; 2: 33, 317, 387 HCH isomers 1: 51, 231 a-HCH 1: 51, 73, 77, 283, 297, 309, 327, 383; 2: 33, 313, 317, 387 (3-HCH 1: 51, 73, 77, 283, 297, 309, 327, 383; 2: 33, 313, 317, 387 y-HCH (lindane) 1: 51, 73, 77, 230, 283, 297, 309, 327, 383; 2: 27, 33, 313, 317, 387 5-HCH 1: 73, 77, 297, 309, 327, 383; 2: 33, 317, 387 6-HCH 1: 297; 2: 33, 317, 387 Heptachlor 1: 51, 73, 77, 231, 283, 297, 309, 327, 383; 2: 27, 33, 313, 317, 387 Heptachlor epoxide 1: 51, 73, 77, 231, 283, 297, 309, 327, 383; 2: 33, 313, 317, 387 Heptenophos 1: 149* 231, 283; 2: 33, 313, 317 Herbicides, substituted phenyl urea 1: 241, 251 Herbicides, phenoxyalkanoic acid 1: 171; 2:163* 369, 393 Herbicides, sulphonylurea 2: 145* Herbicides, triazine 1: 57, 265, 283, 347; 2: 313, 403, 413 Hexabromobiphenyl 2: 35 Hexachlorobenzene, see HCB Hexachlorocyclohexane, see HCH Hexazinone 2: 33 Hostatox (chlorinated indene) 2: 36 3-Hydroxy-carbofuran 2: 113, 307, 349 9-Hydroxyfluorene 2: 128 Imazalil 1: 78, 231, 383; 2: 33, 317, 377 Insecticides, methyl carbamate 2: 349 Insecticides, organochlorine 1: 51, 57, 71, 77, 283, 297, 309, 325, 327, 383; 2: 313, 317, 387 Insecticides, organophosphorus 1: 57, 61, 68, 71, 77, 283, 297, 309, 325, 335, 361, 371, 383; 2: 313, 317 Insecticides, pyrethrin 2: 323 Insecticides, pyrethroid 2: 333 Iodofenphos 1: 77, 231, 297, 383; 2: 27, 33, 313, 317 Ioxynil 2: 33, 99* Ioxynil, isooctyl ether 2: 27

454

Index of Compounds

Ioxynil, methyl ether 2: 27, 99 Ioxynil octanoate 2: 33, 99, 307, 317 Ipazine 2: 33 Iprodione 1: 231, 283; 2: 33, 313, 317 Isobenzan 2: 33, 308, 317 Isobumeton, see Secbumeton Isocarbamid 2: 33, 308, 317 Isodrin 1: 77, 231, 383; 2: 33, 317 Isofenphos 1: 232, 283; 2: 33, 313 Isomethiozin 2: 33 Isopropalin 2: 33, 308, 317 Isopropyl methyl 5-nitroisophthalate, see Nitrothal-isopropyl, methyl esters of metabolites Isopropyl 5-nitroisophthalate, see Nitrothalisopropyl, metabolites Isoproturon 2: 27 Jasmolins

1: 237; 2: 27, 323

3-Keto-carbofuran 2: 308, 349 8-Keto-endrin 2: 33, 308, 317 Lauronitrile 2: 349 Lenacil 2: 27, 33, 308, 317, 435 Lenacil, N-methyl derivative 2: 27 Leptophos 2: 33, 308, 317 Leptophos, desbromo derivative 2: 33 Lindane, see y-HCH Linuron 1: 232, 241, 251; 2; 27, 33, 273, 317 Malaoxon 1: 77, 232, 283, 335, 371, 383; 2: 33, 313, 317 Malathion 1: 64, 73, 77, 232, 283, 309, 335, 371, 383; 2: 27, 33, 313, 317 Mancozeb 1: 232, 353, 407; 2: 36 Maneb 1: 232, 353, 407; 2: 36 MCPA 1: 78; 2: 33, 308, 369, 393 MCPA, dinitro derivative of methyl ester 2: 369 MCPA, isooctyl ester 2: 374 MCPA, pentafluorobenzyl ester 2: 33 MCPA, trichloroethyl ester 2: 393 MCPA-(2-butoxyethyl) 2: 33, 308, 317 MCPB 2: 33, 308, 369, 393 MCPB, dinitro derivative of methyl ester 2: 369 MCPB, isooctyl ester 2: 27 MCPB, methyl ester 2: 27 MCPB, trichloroethyl ester 2: 393 MCPP, see Mecoprop Mecarbam 2: 33, 308, 317 Mecoprop 2: 33, 308, 369, 393 Mecoprop, dinitro derivative of methyl ester 2: 369

Mecoprop, isooctyl ester 2: 27 Mecoprop, methyl ester 2: 27 Mecoprop, trichloroethyl ester 2: 393 Mephosfolan 2: 33, 309, 317 Mercaptodimethur 2: 27, 309, 349 Mercaptodimethur, metabolites 2: 358 Merphos 2: 33, 309, 317 Metalaxyl 1: 753* 232, 283; 2: 33, 313, 317, 437 Metaldehyde 2: 239* Metamitron 2: 27, 33, 309, 428, 429, 435 Metazachlor 2: 309, 313 Methabenzthiazuron 2: 27, 33, 309, 577, 428, 429, 435 Methacrifos 2: 33 Methamidophos 1: 77, 81% 232, 383; 2: 33, 317 Metham-sodium 2: 36 Methazole 2: 27 Methidathion 1: 77, 232, 283, 335, 383; 2: 27, 33, 313, 317 Methiocarb, see Mercaptodimethur Methomyl 1: 757*; 2: 27, 309, 349 Methomyl oxime 1: 161 Methomyl sulphone 2: 358 Methomyl sulphoxide 2: 358 Methoprotryne 1: 232, 265, 283; 2: 34, 313 Methoxychlor 1: 73, 77, 232, 283, 297, 309, 327, 383; 2: 34, 313, 317, 387 Methyl bromide 1: 232, 377 Methyl carbamate insecticides 2: 349 Methyl (4-chloro-2-methyl-5,6-dinitrophenoxy)acetate 2: 370 Methyl 4-(4-chloro-2-methyl-5,6-dinitrophenoxy)butyrate 2: 370 Methyl-2-(4-chloro-2-methyl-5,6-dinitrophenoxy)propionate 2: 370 Methyl (2,4-dichloro-3,6-dinitrophenoxy)acetate 2: 370 Methyl 2-(2,4-dichloro-3,6-dinitrophenoxy)propionate 2: 370 Methyl 2,2-dichloropropionate 1: 117 Methyl pentachlorophenyl sulphide 1: 232, 297 Methyl (2,4,5-trichloro-6-nitrophenoxy)acetate 2: 370 3-Methylaniline 2: 269 6-Methylcoumarin 2: 36 Methylmetiram 2: 36 tris(2-Methyl-2-phenylpropyl)tin compound, see Fenbutatin oxide 3-(Methylphosphinico)propionic acid 2: 217 3-(Methylphosphinico)propionic acid dimethyl ester 2: 218 Metiram 1: 233, 407; 2: 36 Metobromuron 1: 233, 241, 251; 2: 27, 34

Index of Compounds Metolachlor 2: 34, 309, 313, 317, 406 Metoxuron 1: 233, 241; 2: 27 Metribuzin 1: 78, 233, 383; 2: 27, 34, 245* 313, 317, 435 Metsulfuron 2: 145* Metsulfuron-methyl, see Metsulfuron Mevinphos 1: 64, 77, 233, 283, 335, 371, 383; 2: 27, 34, 313, 317 Mirex 2: 34, 309, 317 Molinate 2: 34 Monalide 1: 246, 263; 2: 273 Monoacetyl-amitrole 2: 49 Monocrotophos 2: 27, 34, 309, 317 Monolinuron 1: 233, 241, 251; 2: 27, 34, 317 Monuron 1: 233, 241, 251; 2: 273 Morfamquat 2: 36 Morphothion 2: 34, 309, 317 Nabam 1: 233, 407; 2: 36 Naled 1: 64, 233, 283, 335, 371; 2: 34, 313, 317 2-Naphthoxyacetic acid 2: 34 1-Naphthylacetic acid 1: 167* Napropamide 2: 27, 34 Neburon 1: 233, 241, 251; 2: 34, 273 Nicotine 2: 27, 34 Nitralin 2: 34, 309, 317 Nitrapyrin 2: 34 Nitrofen 1: 173*; 2: 27, 34, 310, 313, 317 5-Nitroisophthalic acid, see Nitrothal-isopropyl, metabolites 4-Nitrophenol 2: 34 Nitrothal-isopropyl 2: 34, 253* 310, 317, 437 Nitrothal-isopropyl, metabolites 2: 253 Nitrothal-isopropyl, methyl esters of metabolites 2:254 Norazin 2: 34 Norflurazon 2: 34 Nuarimol 2: 27 Octachlorodipropyl ether (S 421) 2: 34, 310, 317 Octachlorostyrene 2: 36, 310 Octadecanonitrile 2: 349 Omethoate 1: 77, 233, 335, 371, 383; 2: 27, 34, 377, 428, 429 Organochlorine pesticides 1: 51, 57, 71, 77, 283, 297, 309, 325, 327, 383; 2: 313, 317, 387 Organophosphorus pesticides 1: 57, 61, 68, 71, 77, 283, 297, 309, 325, 335, 361, 371, 383; 2:313, 317 Organotin compounds 2: 343 Organotin compounds, metabolites 2: 348 Oxadiazon 2: 27, 34, 310, 317

455

Oxamyl 2: 34, 261* Oxamyl oximino derivative 2: 261 Oxychlordane (octachlor epoxide) 1: 77, 233, 297, 309, 383; 2: 34, 317 Oxydemeton-methyl, see Demeton-S-methyl sulphoxide Paraoxon 1: 64, 77, 234, 283, 335, 371, 383; 2: 34, 313, 317 Paraoxon-methyl 1: 234, 371; 2: 34, 317 Paraquat 1: 177*; 2: 36 Parathion 1: 64, 68, 73, 77, 234, 283, 309, 335, 371, 383; 2: 27, 34, 313, 317, 428, 429, 435 Parathion-methyl 1: 64, 73, 77, 234, 283, 335, 371, 383; 2: 27, 34, 313, 317 PCB 1: 51, 73, 77, 234, 297, 318, 383; 2: 35 Pencycuron, methyl derivative 2: 34 Pendimethalin 2: 27, 34, 310, 313, 317, 437 PCCH, see Pentachlorocyclohex-1-ene Pentachloroaniline 1: 78, 234, 295, 297, 383; 2: 34, 317 Pentachloroanisole 2: 34, 317 Pentachlorobenzene 1: 234, 297; 2: 34, 317 Pentachlorocyclohex-1-ene 1: 234, 297 Pentachloronitrobenzene, see Quintozene Pentachlorophenol 2: 34 Permethrin 1: 234; 2: 27, 34, 317, 333 Perthane 1: 234, 283; 2: 34, 313, 317 Phenkapton 1: 64, 234, 283, 297, 335, 371; 2: 34, 313, 317 Phenmedipham 2: 27, 34, 269% 310, 317 Phenothrin 2: 341 Phenoxyalkanoic acid herbicides 1: 171; 2:163* 369, 393 Phenthoate 2: 34, 310, 317 Phenylcarbamate herbicides 1: 246, 263 o-Phenylphenol 2: 294 Phenylurea herbicides 1: 241, 251 Phorate 1: 64, 235, 283, 335, 361, 371; 2: 34, 313, 317 Phorate oxygen analogue 2: 34 Phorate sulphone 1: 235, 365, 368, 369 Phorate sulphone, oxygen analogue 1: 369 Phorate sulphoxide 2: 34 Phosalone 1: 73, 77, 235, 283, 383; 2: 28, 34, 313, 317 Phosfolan 2: 34 Phosmet 2: 34, 310, 359 Phosphamidon 1: 235, 295, 335; 2: 34, 317 Phosphorous acid 2: 211 Phoxim 2: 34, 310, 317 Phthalimide fungicides 1: 93, 105, 143, 401; 2: 359, 377

456

Index of Compounds

Phthalimides 2: 359 Piperonyl butoxide 1: 78, 235, 383; 2: 34, 317, 323 Piperonyl butoxide, tribromo derivative 2: 323 Pirimicarb 1: 183*; 2: 28, 34, 310, 317, 349 Pirimicarb, metabolites 1: 183 Pirimiphos-ethyl 2: 28, 34, 310, 317 Pirimiphos-methyl 1: 191* 235, 283, 399; 2: 28, 34, 313, 317 Pirimiphos-methyl, metabolite 1: 192 Plifenate 2: 34 Polychlorinated biphenyls, see PCB Polychlorinated terphenyl (50% Cl) 2: 36 Potato sprout suppressants 1: 321 Procymidone 1: 235, 283; 2: 34, 313, 317 Profenofos 1: 235, 283; 2: 34, 313, 317 Profluralin 2: 34, 311, 313, 317 Promecarb 2: 311, 349 Prometon 1: 235, 265; 2: 34 Prometryn 1: 235, 265, 283; 2: 34, 313, 403 Propachlor 2: 28, 34, 275* 311, 317 Propanil 2: 28, 34, 273, 311, 317 Propargite 2: 34 Propazine 1: 235, 265, 283; 2: 34, 313, 403 Propetamphos 2: 34 Propham 1: 235, 246, 263, 321; 2: 28, 273 Propham, dibromo derivative 1: 321 Propiconazole 2: 281* 311, 317 Propineb 1: 236, 353, 407; 2: 36 Propoxur 1: 78, 236, 383; 2: 28, 34, 317, 349 Propylene bisdithiocarbamate fungicides 1: 353, 407 Propylene thiourea 1: 411 Propyzamide 1: 236, 283; 2: 34, 313, 317 Prothiofos 1: 236, 283; 2: 34, 313, 317 Pyrazon, see Chloridazon Pyrazophos 1: 35, 68, 77, 197* 236, 283, 383; 2: 34, 313, 317 Pyrethrins 1: 78, 236, 237, 283, 383; 2: 28, 34, 313, 317, 323, 341 Pyrethroids 2: 333 Quinalphos (chinalphos) 2: 34, 311, 313, 317 Quinomethionate, see Chinomethionat Quintozene 1: 68, 73, 77, 237, 283, 297, 327, 383; 2: 28, 34, 313, 317 Rabenzazole 1: 78, 237, 383; 2: 34, 317, 377 Resmethrin 1: 78, 237, 383; 2: 28, 34, 377, 341 S 421, see Octachlorodipropyl ether Salithion 2: 34, 311, 317 Secbumeton 1: 237, 265, 347; 2: 34 Siduron 1: 237, 241, 251

Simazine 1: 237, 265, 283, 347; 2: 28, 34, 313, 317, 403 Simeton 1: 237, 347; 2: 34 Simetryn 2: 34 Sprout suppressants 1: 321 Strobane T 2: 34, 311, 317 Substituted acetanilides 1: 252 Substituted anilines 1: 241, 251 Substituted phenyl urea herbicides 1: 241, 251 Sulfallate 2: 34 Sulfotep 1: 77, 237, 283, 335, 383; 2: 34, 313, 317 Sulphonylurea herbicides 2: 145* Sulphur 2: 34, 287* Sulprofos 2: 34, 311, 317 2,4,5-T 1: 78; 2: 34, 311, 369, 393 2,4,5-T, amyl ester 2: 34 2,4,5-T, hexyl ester 2: 34 2,4,5-T, methyl ester 2: 374 2,4,5-T, nitro derivative of methyl ester 2: 369 2,4,5-T, trichloroethyl ester 2: 393 2,4,5-T-butyl 2: 34 2,4,5-T-isooctyl 2: 34, 374 TDE, see DDD Tecnazene (TCNB) 1: 77, 238, 283, 327, 383; 2: 28, 34, 313, 317 Terbacil 2: 28, 35, 311, 313, 317 Terbacil, N-methyl derivative 2: 28 Terbufos 2: 35, 311, 313, 317 Terbumeton 1: 238, 347; 2: 437 Terbuthylazine 1: 238, 265, 347; 2: 35, 403 Terbutryn 1: 238, 265, 283, 347; 2: 35, 313, 317, 403 2,3,4,6-Tetrachloroanisole 2: 35 Tetrachlorobenzenes 2: 36 1,2,3,4-Tetrachlorodibenzodioxin (1,2,3,4-TCDD) 2: 36 l,2,4,5-Tetrachloro-3-nitrobenzene, see Tecnazene 2,3,4,5-Tetrachloronitrobenzene 2: 35, 317 Tetrachlorophenols 2: 36 Tetrachlorvinphos 1: 203* 238, 283; 2: 28, 35, 313, 317 Tetradifon 1: 77, 238, 283, 383; 2: 35, 313, 317 Tetramethrin 2: 35, 311, 377, 341 O,O,O',O'-Tetrapropyl dithiopyrophosphate 2: 35 Tetrasul 1: 73, 77, 238, 283, 383; 2: 28, 35, 313, 317 Thiabendazole 2: 28, 35, 291* 295* Thiofanox 2: 28, 312, 349 Thiofanox sulphone 2: 35, 358 Thiofanox sulphoxide 2: 358 Thiometon 1: 238, 335; 2: 28, 35 Thionazin (zinophos) 1: 64, 77, 238, 283, 335, 371, 383; 2: 35, 373, 377

Index of Compounds Thiophanate-methyl 2: 28, 69* 111 Thiram (TMTD) 1: 238, 353; 2: 28 Thiuram disulphide fungicides 1: 353 Tolclofos-methyl 2: 312, 313 Tolylfluanid 1: 238, 283; 2: 35, 177* 191* 313, 317, 377 Tolylfluanid, metabolite, see DMST Toxaphene, see Camphechlor 2,4,5-TP, see Fenoprop Triadimefon 1: 78, 238, 283, 383; 2: 35, 87* 313, 317, 377, 428, 429, 435 Triadimenol 1: 78, 238, 383; 2: 35, 87* 317, 377, 428, 429, 435 Tri-allate 2: 28, 35, 312, 313, 317 Triamiphos 1: 295, 399; 2: 35, 312, 317 Triazine herbicides 1: 57, 265, 283, 347; 2: 313, 403, 413 Triazine herbicides, desalkyl metabolites 1: 57, 347; 2: 413 Triazophos 1: 35, 68, 77, 209* 238, 283, 383; 2: 35, 313, 317 Triazoxide 2: 312, 317, 377 2,4,6-Tribromo-3-methylaniline 2: 269 4,5,7-Tribromo-6-propyl-l,3-benzodioxol, see Piperonyl butoxide, tribromo derivative Trichlorfon 1: 238, 335; 2: 28, 35, 317 Trichlorobenzenes 2: 36

457

Trichloronat 2: 35, 312, 313, 317 Trichlorophenols 2: 36 Tricyclohexyltin compounds 2: 343 Triclopyr-(2-butoxyethyl) 2: 312, 437 Tridemorph 2: 28 Trietazine 2: 28, 35 3-(Trifluormethyl)acetanilide 1: 252 3-(Trifluormethyl)aniline 1: 247 Trifluralin 1: 78, 238, 383; 2: 28, 35, 313, 317 2,4,6-Trimethylaniline 2: 270 Triphenyltin compounds 2: 343 Urea herbicides

1: 241, 251

Vamidothion 1: 64, 238, 371; 2: 28, 35 Vamidothion sulphone 1: 238, 371; 2: 35 Vamidothion sulphoxide 2: 35 Vinclozolin 1: 78, 213* 238, 283, 383; 2: 28, 35, 313, 317 Vinclozolin, metabolites 1: 213 Vondozeb 1: 238, 407 Zineb 1: 238, 353, 407; 2: 36 Zinochlor, see Anilazine Zinophos, see Thionazin Ziram 2: 36

Index of Analytical Materials Alfalfa Triazine herbicides Almonds Glufosinate

1: 265

2: 217

Animal fats Chlorthiophos 1: 109 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Apple juice Tolylfluanid Apple peel Thiabendazole

2: 191

2: 291

Apples Acephate 1: 81 Amitrole 2: 49 Binapacryl 2: 197 Bitertanol 2: 77, 87 Captafol 1: 93 Captan 1: 105 Chlorthiophos 1: 109 Dichlobenil 2: 169 Dichlofluanid 2: 177 Dinobuton 2: 197 Dithiocarbamate fungicides 1: 353, 407 Ethylene thiourea 1: 135 Glufosinate 2: 217 Heptenophos 1: 149 Metalaxyl 1: 153 Methamidophos 1: 81 Methomyl 1: 161 Methyl carbamate insecticides 2: 349 1-Naphthylacetic acid 1: 167 Nitrogen-containing pesticides 1: 383; 2: 317 Nitrothal-isopropyl 2: 253 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317

Apples (contd.) Organophosphorus pesticides 1: 283, 335, 361, 371, 383; 2: 313, 317 Organotin compounds 2: 343 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Piperonyl butoxide 2: 323 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Pyrazophos 1: 197 Pyrethrins 2: 323 Pyrethroids 2: 333 Sulphur 2: 287 Tetrachlorvinphos 1: 203 Thiuram disulphide fungicides 1: 353 Tolylfluanid 2: 177, 191 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213 Applesauce Organophosphorus pesticides Tolylfluanid 2: 191

1: 371

Apricots Bitertanol 2: 87 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 265, 283; 2: 313 Artichokes Anilazine 2: 59 Bitertanol 2: 87 Pyrazophos 1: 197 Asparagus Glufosinate 2: 217 Organochlorine pesticides 1: 327 Organophosphorus pesticides 1: 335 Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265

460

Index of Analytical Materials

Aubergines Anilazine 2: 59 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Organotin compounds 2: 343 Triazine herbicides 1: 283; 2: 313 Banana peel Thiabendazole

2: 291

Bananas Bitertanol 2: 87 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Triadimefon 2: 87 Triadimenol 2: 87 Triazophos 1: 209 Barley, ears Dithiocarbamate fungicides 1: 353 Thiuram disulphide fungicides 1: 353 Barley, grains Anilazine 2: 59 Bitertanol 2: 77, 87 Bromine-containing fumigants 1: 377 Bromoxynil 2: 99 Captafol 1: 99 Chlorflurenol 2: 127 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Flurenol 2: 127 Ioxynil 2: 99 Metalaxyl 1: 153 Metribuzin 2: 245 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241, 251 Phthalimide fungicides 1: 401 Pirimiphos-methyl 1: 191 Propiconazole 2: 281 Pyrazophos 1: 197 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Barley, green matter Anilazine 2: 59 Bitertanol 2: 77, 87 Captafol 1: 99

Barley, green matter (contd.) Dithiocarbamate fungicides 1: 353 Phthalimide fungicides 1: 401 Propiconazole 2: 281 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Barley, straw Anilazine 2: 59 Bitertanol 2: 77, 87 Bromoxynil 2: 99 Captafol 1: 99 Chlorflurenol 2: 127 Dithiocarbamate fungicides 1: 353 Flurenol 2: 127 Ioxynil 2: 99 Phenyl urea herbicides 1: 241, 251 Phthalimide fungicides 1: 401 Propiconazole 2: 281 Pyrazophos 1: 197 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Beans, green Acephate 1: 81 Anilazine 2: 59 Bitertanol 2: 87 Chlorthiophos 1: 109 Dithiocarbamate fungicides 1: 407 Ethylene thiourea 1: 135 Glufosinate 2: 217 Methamidophos 1: 81 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 371, 383; 2: 313, 317 Organotin compounds 2: 343 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Pyrazophos 1: 197 Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213 Beans, pods Vinclozolin 1: 213 Beer Ethylene thiourea 1: 135 Glyphosate 2: 229

Index of Analytical Materials Beer (contd.) Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Vinclozolin 1: 213 Bilberries Dalapon 1: 117

Caraway Glufosinate

Black currants see Currants, black

1: 191

Brassicas* Carbofuran 2: 113 Carbosulfan 2: 113 Fonofos 2: 205 Glufosinate 2: 217 Metalaxyl 1: 153 Metaldehyde 2: 239 Methyl carbamate insecticides 349 Nitrogen-containing pesticides 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 371, 383; 2: 313, 317 Phenyl urea herbicides 1: 241 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Pyrethroids 2: 333 Tetrachlorvinphos 1: 203 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213 Bread Glyphosate 2: 229 Pirimiphos-methyl 1: 191 Broad beans Metribuzin 2: 245 Broccoli Metalaxyl

1: 153

1: 371

Butter Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

Blackberries Vinclozolin 1: 213

Bran Pirimiphos-methyl

Brussels sprouts Chlorthiophos 1: 109 Fonofos 2: 205 Organophosphorus pesticides Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Triazine herbicides 1: 265 Triazophos 1: 209

461

2: 217

Carrots Acephate 1: 81 Dithiocarbamate fungicides 1: 353, 407 Fonofos 2: 205 Heptenophos 1: 149 Methamidophos 1: 81 Methyl carbamate insecticides 2: 349 Metribuzin 2: 245 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 371, 383; 2: 313, 317 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Pyrazophos 1: 197 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265, 283; 2: 313 Cauliflower Chlorthiophos 1: 109 Ethylene thiourea 1: 135 Fonofos 2: 205 Nitrofen 1: 173 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 327, 383; 2: 317 Organophosphorus pesticides 1: 335, 371, 383; 2: 317 Propachlor 2: 275 Pyrethroids 2: 333 Triazophos 1: 209 Celeriac, bulbs Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Organochlorine pesticides 1: 283; 2: 313

462

Index of Analytical Materials

Celeriac, bulbs (contd.) Organophosphorus pesticides 1: 283, 335; 2:313 Organotin compounds 2: 343 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265, 283; 2: 313 Celeriac, leaves Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Organotin compounds 2: 343 Oxamyl 2: 261 Thiuram disulphide fungicides 1: 353 Cereal grains* Anilazine 2: 59 Benomyl 2: 69 Bitertanol 2: 77, 87 Bromine-containing fumigants 1: 377 Bromoxynil 2: 99 Captafol 1: 93, 99 Carbendazim 2: 69, 107 Chlorflurenol 2: 127 Chlorsulfuron 2: 145 2,4-D 2: 163 Dalapon 1: 117 Dichlorprop 2: 163 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Flurenol 2: 127 Glufosinate 2: 217 Glyphosate 2: 229 Heptenophos 1: 149 Ioxynil 2: 99 Metalaxyl 1: 153 Metribuzin 2: 245 Metsulfuron 2: 145 Nitrofen 1: 173 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241, 251 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Pirimiphos-methyl 1: 191 Propiconazole 2: 281 Pyrazophos 1: 197 Pyrethroids 2: 333 Thiophanate-methyl 2: 69

Cereal grains (contd.) Thiuram disulphide fungicides Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Triazophos 1: 209

1: 353

Cereal green matter* Anilazine 2: 59 Bitertanol 2: 77, 87 Bromoxynil 2: 99 Captafol 1: 99 Carbendazim 2: 107 Chlorsulfuron 2: 145 2,4-D 2: 163 Dalapon 1: 117 Dichlorprop 2: 163 Dithiocarbamate fungicides 1: 353 Ioxynil 2: 99 Metsulfuron 2: 145 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Propiconazole 2: 281 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Cereal products, milled Glyphosate 2: 229 Bromine-containing fumigants

1: 377

Cereal straw* Anilazine 2: 59 Benomyl 2: 69 Bitertanol 2: 77, 87 Bromoxynil 2: 99 Captafol 1: 93, 99 Carbendazim 2: 69, 107 Chlorflurenol 2: 127 Chlorsulfuron 2: 145 2,4-D 2: 163 Dalapon 1: 117 Dichlorprop 2: 163 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Flurenol 2: 127 Glyphosate 2: 229 Ioxynil 2: 99 Metribuzin 2: 245 Metsulfuron 2: 145 Nitrofen 1: 173 Phenyl urea herbicides 1: 241, 251 Phthalimide fungicides 1: 401

Index of Analytical Materials Cereal straw (contd.) Propiconazole 2: 281 Pyrazophos 1: 197 Thiophanate-methyl 2: 69 Thiuram disulphide fungicides Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265

Chives Pirimicarb

1: 353

Cheese, cheese spread Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Cherries Amitrole 2: 49 Bitertanol 2: 77, 87 Chlorthiophos 1: 109 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Heptenophos 1: 149 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 383; 2: 313, 317 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213 Cherries: conserves, juice, press pulp Bitertanol 2: 77 Cherries, sour Glufosinate 2: 217 Vinclozolin 1: 213 Chicoree leaves Metaldehyde 2: 239 Chillies Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313 Chinese cabbage Glufosinate 2: 217 Metaldehyde 2: 239 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283, 371; 2: 313 Triazine herbicides 1: 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213

463

1: 183

Chocolate Organochlorine pesticides 1: 297 Organophosphorus pesticides 1: 297 Citrus fruit* Chlorthiophos 1: 109 Glufosinate 2: 217 Heptenophos 1: 149 Metalaxyl 1: 153 Nitrogen-contanining pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 383; 2: 313, 317 Organotin compounds 2: 343 Oxamyl 2: 261 Pirimiphos-methyl 1: 191 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Citrus fruit peel* Thiabendazole 2: 291, 295 Clover Anilazine

2: 59

Cocoa Triazine herbicides

1: 265

Cocoa butter Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Cocoa powder Organochlorine pesticides 1: 297 Organophosphorus pesticides 1: 297 Cocoa products* Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2:317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Coconut oil Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

464

Index of Analytical Materials

Coffee Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Triazine herbicides 1: 265 Triazophos 1: 209 Coffee, raw Anilazine 2: 59 Oxamyl 2: 261 Condensed milk Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Corn, sweet Triazophos 1: 209 Corn salad Metaldehyde 2: 239 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213 Cotton leaves Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Cotton seed Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Oxamyl 2: 261 Cucumbers Binapacryl 2: 197 Bitertanol 2: 77, 87 Chlorflurenol 2: 127 Dinobuton 2: 197 Dithiocarbamate fungicides 1: 353, 407 Flurenol 2: 127 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 383; 2: 313, 317 Piperonyl butoxide 2: 323 Pirimiphos-methyl 1: 191 Pyrazophos 1: 197 Pyrethrins 2: 323 Pyrethroids 2: 333 Sulphur 2: 287 Thiuram disulphide fungicides 1: 353

Cucumbers (contd.) Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213 Curly kale Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 361, 371, 383; 2: 317 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Tetrachlorvinphos 1: 203 Triazine herbicides 1: 265 Currants, black Dithiocarbamate fungicides 1: 353 Heptenophos 1: 149 Pirimiphos-methyl 1: 191 Thiuram disulphide fungicides 1: 353 Vinclozolin 1: 213 Currants, red Chlorthiophos 1: 109 Dichlobenil 2: 169 Dithiocarbamate fungicides 1: 353 Heptenophos 1: 149 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Piperonyl butoxide 2: 323 Pirimiphos-methyl 1: 191 Pyrethrins 2: 323 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265 Cut lettuce Metaldehyde

2: 239

Dairy products* Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Dandelion Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313

Index of Analytical Materials Dried egg products* Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2:317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Dried egg yolk Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Dried fruit Bromine-containing fumigants

1: 377

Dried mushrooms Bromine-containing fumigants

1: 377

Dried skim milk Organochlorine pesticides 1: 297 Organophosphorus pesticides 1: 297 Dried vegetables Bromine-containing fumigants

Egg products, dried* see Dried egg products Egg yolk, dried* see Dried egg yolk Eggs, fresh Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Endives Metaldehyde 2: 239 Organochlorine pesticides 1: 283, 327; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Piperonyl butoxide 2: 323 Pyrethrins 2: 323 Pyrethroids 2: 333 Triazine herbicides 1: 283; 2: 313 Evening primrose oil Glufosinate 2: 217

Fats, animal* Chlorthiophos 1: 109 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Fats, vegetable* Chlorthiophos 1: 109 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Fish Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

1: 377

Dried whole egg Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

465

Fodder beet, foliage and root Phenmedipham 2: 269 Fruit, dried and fresh Bromine-containing fumigants Garlic Anilazine 2: 59 Vinclozolin 1: 213 Gooseberries Vinclozolin 1: 213 Grapefruit Heptenophos 1: 149 Oxamyl 2: 261 Grapefruit peel Thiabendazole 2: 291, 295 Grape juice Ethylene thiourea 1: 135 Vinclozolin 1: 213 Grape must Chlorthiophos

1: 109

1: 377

466

Index of Analytical Materials

Grapes Acephate 1: 81 Amitrole 2: 49 Captafol 1: 93 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Copper oxychloride 2: 153 Cymoxanil 2: 157 2,4-D 2: 163 Dichlobenil 2: 169 Dichlofluanid 2: 177, 191 Dichlorprop 2: 163 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Folpet 1: 143 Fosetyl 2: 211 Glyphosate 2: 229 Metalaxyl 1: 153 Methamidophos 1: 81 Methomyl 1: 161 1-Naphthylacetic acid 1: 167 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 383; 2: 313, 317 Organotin compounds 2: 343 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Propiconazole 2: 281 Pyrazophos 1: 197 Pyrethroids 2: 333 Sulphur 2: 287 Tetrachlorvinphos 1: 203 Thiuram disulphide fungicides 1: 353 Tolylfluanid 2: 177 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Grass 2,4-D 2: 163 Dichlobenil 2: 169 Dichlorprop 2: 163 Glyphosate 2: 229 Oxamyl 2: 261 Triazine herbicides 1: 265 Grass silage Glyphosate 2: 229

Greengages Organochlorine pesticides

1: 327

Groundnuts see Peanuts Hay Dalapon 1: 117 Glyphosate 2: 229 Head cabbage Carbofuran 2: 113 Carbosulfan 2: 113 Fonofos 2: 205 Metalaxyl 1: 153 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 371, 383; 2: 313, 317 Phenyl urea herbicides 1: 241 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Herbs (spices) Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Honey Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313 Hop cones Anilazine 2: 59 Ethylene thiourea 1: 135 Fosetyl 2: 211 Heptenophos 1: 149 Metalaxyl 1: 153 Methomyl 1: 161 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Pyrazophos 1: 197 Sulphur 2: 287 Triadimefon 2: 87 Triadimenol 2: 87 Vinclozolin 1: 213

Index of Analytical Materials Hop foliage Sulphur 2: 287 Instant foods based on milk Organochlorine pesticides 1: 297 Organophosphorus pesticides 1: 297 Jams Bitertanol 2: 77 Vinclozolin 1: 213 Juices* Bitertanol 2: 77, 87 Chlorthiophos 1: 109 Ethylene thiourea 1: 135 Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Tolylfluanid 2: 191 Triadimefon 2: 87 Triadimenol 2: 87 Vinclozolin 1: 213 Kidneys Chlorthiophos 1: 109 Glufosinate 2: 217 Kiwi fruit Glufosinate

2: 217

Kohlrabi Fonofos 2: 205 Heptenophos 1: 149 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 371, 383; 2: 313, 317 Triazine herbicides 1: 283; 2: 313 Lard Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Leeks Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Methyl carbamate insecticides 2: 349 Nitrofen 1: 173 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283, 361; 2: 313 Phenyl urea herbicides 1: 241

467

Leeks (contd.) Pirimiphos-methyl 1: 191 Propachlor 2: 275 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265, 283; 2: 313 Lemons Glufosinate 2: 217 Organochlorine pesticides 1: 327 Triazine herbicides 1: 265 Triazophos 1: 209 Lettuce Acephate 1: 81 Benomyl 2: 69 Bromine-containing fumigants 1: 377 Carbendazim 2: 69 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Dichlofluanid 2: 177 Dithiocarbamate fungicides 1: 353, 407 Ethylene thiourea 1: 135 Folpet 1: 143 Fosetyl 2: 211 Heptenophos 1: 149 Metalaxyl 1: 153 Metaldehyde 2: 239 Methamidophos 1: 81 Methomyl 1: 161 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 371, 383; 2: 313, 317 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Piperonyl butoxide 2: 323 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Pyrethrins 2: 323 Thiophanate-methyl 2: 69 Thiuram disulphide fungicides 1: 353 Tolylfluanid 2: 177 Triazine herbicides 1: 265, 283; 2: 313 Liver Chlorthiophos 1: 109 Glufosinate 2: 217

468

Index of Analytical Materials

Maize cobs Metalaxyl 1: 153 Tetrachlorvinphos 1: 203 Maize, green matter Chlorthiophos 1: 109 Metalaxyl 1: 153 Triazophos 1: 209 Maize, kernels Acephate 1: 81 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Fonofos 2: 205 Glufosinate 2: 217 Heptenophos 1: 149 Methamidophos 1: 81 Phenyl urea herbicides 1: 241 Propachlor 2: 275 Pyrethroids 2: 333 Triazine herbicides 1: 265 Triazophos 1: 209 Maize, leaves Metalaxyl 1: 153 Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Maize, stalks Metalaxyl 1: 153 Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Mandarine oranges Chlorthiophos 1: 109 Organochlorine pesticides 1: 283, 327; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313 Mangold Chloridazon 2: 135 Diclofop-methyl 1: 127 Phenmedipham 2: 269 Piperonyl butoxide 2: 323 Pyrethrins 2: 323 Margarine Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

Meat Chlorthiophos 1: 109 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Meat products* Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1:297, 309, 383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Melons Bitertanol 2: 87 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 327, 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Organotin compounds 2: 343 Pyrazophos 1: 197 Triadimefon 2: 87 Triadimenol 2: 87 Milk, condensed see Condensed milk Milk, fresh, milk powder Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1:297, 309,383; 2: 317 Organophosphorus pesticides 1: 297, 309, 383; 2: 317 Milled cereal products Bromine-containing fumigants Mirabellas Glufosinate

1: 377

2: 217

Mud (sludge) Diclofop-methyl

1: 127

Mushrooms Bromine-containing fumigants 1: 377 Dalapon 1: 117 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Pirimiphos-methyl 1: 191 Triazine herbicides 1: 283; 2: 313 Mushrooms, dried Bromine-containing fumigants

1: 377

Index of Analytical Materials Must Amitrole 2: 49 Cymoxanil 2: 157 Dichlofluanid 2: 177, 191 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Tolylfluanid 2: 177 Triadimefon 2: 87 Triadimenol 2: 87 Nectarines Organotin compounds

2: 343

Nuts Bromine-containing fumigants 1: 377 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Oat, grains Bromine-containing fumigants 1: 377 Heptenophos 1: 149 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241, 251 Triazine herbicides 1: 265 Oat, straw Phenyl urea herbicides 1: 241, 251 Triazine herbicides 1: 265 Oil seeds* Bitertanol 2: 87 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Diclofop-methyl 1: 127 Glufosinate 2: 217 Glyphosate 2: 229 Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Oxamyl 2: 261 Phenyl urea herbicides 1: 241, 251 Pirimiphos-methyl 1: 191 Pyrethroids 2: 333 Triazine herbicides 1: 265 Triazophos 1: 209 Vinclozolin 1: 213

469

Oils, vegetable* see Fats, vegetable Olive oil Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Pirimiphos-methyl 1: 191 Onions, bulbs Anilazine 2: 59 Bromine-containing fumigants 1: 377 Fonofos 2: 205 Metalaxyl 1: 153 Nitrofen 1: 173 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 361, 383; 2: 317 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Pyrethroids 2: 333 Triazine herbicides 1: 265 Vinclozolin 1: 213 Onions, stalks Vinclozolin 1: 213 Orange juice Metalaxyl 1: 153 Orange peel Metalaxyl 1: 153 Thiabendazole 2: 291, 295 Oranges Glufosinate 2: 217 Heptenophos 1: 149 Metalaxyl 1: 153 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Oxamyl 2: 261 Pirimiphos-methyl 1: 191 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Parsley Bromine-containing fumigants 1: 377 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Triazine herbicides 1: 283; 2: 313

470

Index of Analytical Materials

Peaches Bitertanol 2: 87 Captafol 1: 93 Chlorthiophos 1: 109 Dithiocarbamate fungicides 1: 353 1-Naphthylacetic acid 1: 167 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 383; 2: 313, 317 Organotin compounds 2: 343 Oxamyl 2: 261 Pyrazophos 1: 197 Tetrachlorvinphos 1: 203 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Peanut foliage Oxamyl 2: 261 Peanut oil Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Peanut shells Bitertanol 2: 87 Oxamyl 2: 261 Peanuts Bitertanol 2: 87 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Oxamyl 2: 261 Pirimiphos-methyl 1: 191 Pear juice, pear conserves Tolylfluanid 2: 191 Pears Amitrole 2: 49 Bitertanol 2: 77, 87 Captan 1: 105 Dichlobenil 2: 169 Dithiocarbamate fungicides 1: 353 1-Naphthylacetic acid 1: 167 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317

Pears (contd.) Organophosphorus pesticides 1: 283, 335, 383; 2: 313, 317 Phenyl urea herbicides 1: 241 Phthalimide fungicides 1: 401 Pirimiphos-methyl 1: 191 Thiuram disulphide fungicides 1: 353 Tolylfluanid 2: 191 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Peas Ethylene thiourea 1: 135 Glufosinate 2: 217 Glyphosate 2: 229 Metalaxyl 1: 153 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Triazine herbicides 1: 265 Pepper seeds Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Peppers, sweet Anilazine 2: 59 Heptenophos 1: 149 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 383; 2: 313, 317 Oxamyl 2: 261 Pirimiphos-methyl 1: 191 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 283; 2: 313 Pineapples Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 383; 2: 313, 317 Triazine herbicides 1: 265, 283; 2: 313

Index of Analytical Materials Plums Acephate 1: 81 Bitertanol 2: 77, 87 Chlorthiophos 1: 109 Glufosinate 2: 217 Methamidophos 1: 81 1-Naphthylacetic acid 1: 167 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 383; 2: 313, 317 Organotin compounds 2: 343 Piperonyl butoxide 2: 323 Pyrethrins 2: 323 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Plums: jam, puree Bitertanol 2: 77 Poppy seeds Phenyl urea herbicides Potato peel Thiabendazole

1: 251

2: 291

Potato tops Metribuzin 2: 245 Potatoes Anilazine 2: 59 Captafol 1: 93 Chlorpropham 1: 321 Chlorthiophos 1: 109 Cymoxanil 2: 157 2,4-D 2: 163 Dalapon 1: 117 Dichlorprop 2: 163 Dithiocarbamate fungicides 1: 353, 407 Ethylene thiourea 1: 135 Glufosinate 2: 217 Heptenophos 1: 149 Metalaxyl 1: 153 Metribuzin 2: 245 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 383; 2: 313, 317 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Phthalimides 2: 359

471

Potatoes (contd.) Piperonyl butoxide 2: 323 Propham 1: 321 Pyrazophos 1: 197 Pyrethrins 2: 323 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213 Poultry meat Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Radicchio Anilazine 2: 59 Radishes Fonofos 2: 205 Metaldehyde 2: 239 Methyl carbamate insecticides 2: 349 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Propachlor 2: 275 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Radishes, small Captafol 1: 93 Dithiocarbamate fungicides 1: 353 Fonofos 2: 205 Heptenophos 1: 149 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283, 335; 2:313 Pirimiphos-methyl 1: 191 Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 265, 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213 Rape, green matter Anilazine 2: 59 Carbofuran 2: 113 Carbosulfan 2: 113 Dithiocarbamate fungicides 1: 353 Glufosinate 2: 217 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353

472

Index of Analytical Materials

Rape, seeds Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Diclofop-methyl 1: 127 Glufosinate 2: 217 Glyphosate 2: 229 Pyrethroids 2: 333 Triazophos 1: 209 Vinclozolin 1: 213 Rape, straw Vinclozolin 1: 213 Rape oil Chlorthiophos

1: 109

Rye, grains Bromine-containing fumigants 1: 377 Captafol 1: 99 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241 Propiconazole 2: 281 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Rye, green matter Captafol 1: 99 Propiconazole 2: 281 Triadimefon 2: 87 Triadimenol 2: 87

Raspberries Dichlofluanid 2: 177 Pirimiphos-methyl 1: 191 Tolylfluanid 2: 177 Triazine herbicides 1: 265 Vinclozolin 1: 213

Rye, straw Captafol 1: 99 Phenyl urea herbicides 1: 241 Propiconazole 2: 281 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265

Red beet, edible root Chloridazon 2: 135 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Phenmedipham 2: 269 Thiuram disulphide fungicides 1: 353

Sage Bromine-containing fumigants

Red beet, foliage Chloridazon 2: 135 Diclofop-methyl 1: 127

Savoy cabbage Acephate 1: 81 Chlorthiophos 1: 109 Methamidophos 1: 81 Organophosphorus pesticides

Red cabbage Fonofos 2: 205 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283, 335, 361, 371; 2: 313 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Triazine herbicides 1: 265, 283; 2: 313 Red currants see Currants, red Rice, grains Pirimiphos-methyl 1: 191 Pyrazophos 1: 197

1: 377

Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309

1: 335

Skim milk powder Organochlorine pesticides 1: 297 Organophosphorus pesticides 1: 297 Sludge (mud) Diclofop-methyl

1: 127

Small radishes see Radishes, small Soil Aldicarb 1: 87 Amitrole 2: 49 Anilazine 2: 59

Index of Analytical Materials Soil (contd.) Binapacryl 2: 197 Bitertanol 2: 77, 87 Bromoxynil 2: 99 Captafol 1: 93 Carbendazim 2: 107 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorflurenol 2: 127 Chloridazon 2: 135 Chlorsulfuron 2: 145 Chlorthiophos 1: 109 Cymoxanil 2: 157 Dalapon 1: 117 Dichlobenil 1: 123; 2: 169 Diclofop-methyl 1: 127 Dinobuton 2: 197 Dithiocarbamate fungicides 1: 353 Flurenol 2: 127 Fonofos 2: 205 Glufosinate 2: 217 Glyphosate 2: 229 Heptenophos 1: 149 Ioxynil 2: 99 Metalaxyl 1: 153 Methomyl 1: 161 Metribuzin 2: 245 Metsulfuron 2: 145 Nitrothal-isopropyl 2: 253 Organotin compounds 2: 343 Oxamyl 2: 261 Paraquat 1: 177 Phenyl urea herbicides 1: 241, 251 Phthalimide fungicides 1: 401 Phthalimides 2: 359 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Propiconazole 2: 281 Pyrazophos 1: 197 Pyrethroids 2: 333 Sulphur 2: 287 Tetrachlorvinphos 1: 203 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265, 347 Triazine herbicides, desalkyl metabolites 1: 347 Triazophos 1: 209 Vinclozolin 1: 213 Sorghum Triazine herbicides

1: 265

473

Sour cherries Glufosinate 2: 217 Vinclozolin 1: 213 Soybean oil Chlorthiophos 1: 109 Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Soybeans Chlorthiophos 1: 109 Diclofop-methyl 1: 127 Glufosinate 2: 217 Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Spelt, grains Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Spices (herbs) see Herbs (spices) Spinach Anilazine 2: 59 Chlorthiophos 1: 109 Metalaxyl 1: 153 Metaldehyde 2: 239 Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283, 335, 361, 371; 2: 313 Piperonyl butoxide 2: 323 Pyrethrins 2: 323 Triazine herbicides 1: 265, 283; 2: 313 Strawberries Aldicarb 1: 87 Dichlofluanid 2: 177, 191 Dithiocarbamate fungicides 1: 353 Fosetyl 2: 211 Heptenophos 1: 149 Metalaxyl 1: 153 Metaldehyde 2: 239 Methomyl 1: 161 Methyl carbamate insecticides 2: 349 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 383; 2: 313, 317 Organotin compounds 2: 343 Phenmedipham 2: 269 Phenyl urea herbicides 1: 241

474

Index of Analytical Materials

Strawberries (contd.) Pirimiphos-methyl 1: 191 Sulphur 2: 287 Tetrachlorvinphos 1: 203 Thiuram disulphide fungicides 1: 353 Tolylfluanid 2: 177, 191 Triazine herbicides 1: 265, 283; 2: 313 Vinclozolin 1: 213 Strawberry plants Aldicarb 1: 87 Suet (tallow) Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Sugar, raw Vinclozolin

1: 213

Sugar beet, dry chips Vinclozolin 1: 213 Sugar beet, edible root Aldicarb 1: 87 Bitertanol 2: 77, 87 Carbofuran 2: 113 Carbosulfan 2: 113 Chloridazon 2: 135 Chlorthiophos 1: 109 Dithiocarbamate fungicides 1: 353 Glufosinate 2: 217 Glyphosate 2: 229 Heptenophos 1: 149 Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Organotin compounds 2: 343 Phenmedipham 2: 269 Piperonyl butoxide 2: 323 Propachlor 2: 275 Pyrethrins 2: 323 Pyrethroids 2: 333 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Vinclozolin 1: 213 Sugar beet, foliage Bitertanol 2: 77, 87 Chloridazon 2: 135 Chlorthiophos 1: 109

Sugar beet, foliage (contd.) Dithiocarbamate fungicides 1: 353 Glufosinate 2: 217 Glyphosate 2: 229 Metalaxyl 1: 153 Organotin compounds 2: 343 Phenmedipham 2: 269 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Sugar beet, syrup Vinclozolin 1: 213 Sugar cane Triazine herbicides

1: 265

Sunflower oil Glufosinate 2: 217 Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Phenyl urea herbicides 1: 241 Sunflower seeds Glufosinate 2: 217 Metalaxyl 1: 153 Phenyl urea herbicides 1: 241 Triazine herbicides 1: 265 Sweet corn Triazophos

1: 209

Sweet peppers see Peppers, sweet Tallow (suet) see Suet (tallow) Tea, tealike products Bitertanol 2: 77 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Tobacco Ethylene thiourea 1: 135 Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Pyrazophos 1: 197

Index of Analytical Materials Tomato juice Ethylene thiourea

1: 135

Tomato leaves Metribuzin 2: 245 Vinclozolin 1: 213 Tomatoes Acephate 1: 81 Anilazine 2: 59 Bitertanol 2: 77 Carbofuran 2: 113 Carbosulfan 2: 113 Chlorthiophos 1: 109 Cymoxanil 2: 157 Dichlofluanid 2: 177 Dithiocarbamate fungicides 1: 407 Ethylene thiourea 1: 135 Folpet 1: 143 Heptenophos 1: 149 Metalaxyl 1: 153 Methamidophos 1: 81 Methomyl 1: 161 Methyl carbamate insecticides 2: 349 Metribuzin 2: 245 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383; 2: 313, 317 Organophosphorus pesticides 1: 283, 335, 361, 383; 2: 313, 317 Organotin compounds 2: 343 Oxamyl 2: 261 Phenyl urea herbicides 1: 241 Phthalimide fungicides 1: 401 Piperonyl butoxide 2: 323 Pirimiphos-methyl 1: 191 Pyrethrins 2: 323 Pyrethroids 2: 333 Tolylfluanid 2: 177 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 283; 2: 313 Triazophos 1: 209 Vinclozolin 1: 213 Turnips, foliage and edible root Anilazine 2: 59 Vegetable fats and oils* Chlorthiophos 1: 109 Glufosinate 2: 217 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309, 383; 2: 317

475

Vegetable fats and oils* (contd.) Organophosphorus pesticides 1: 297, 309, 383; 2:317 Phenyl urea herbicides 1: 241 Pirimiphos-methyl 1: 191 Vegetables, dried and fresh Bromine-containing fumigants

1: 377

Water

Acephate 1: 81 Amitrole 2: 49, 442 Anilazine 2: 59 Azinphos-ethyl 2: 435 Azinphos-methyl 2: 435 Benalaxyl 2: 437 Bendiocarb 2: 437 Bentazone 2: 437 Binapacryl 2: 197 Bitertanol 2: 77, 87, 435 Bromacil 2: 435 Bromoxynil 2: 99 Captafol 1: 93 Carbofuran 2: 435 Chlorflurenol 2: 127 Chloridazon 2: 135 Chlorotriazine desalkyl metabolites 2: 413 Chlorsulfuron 2: 145 Chlorthiophos 1: 109 Cymoxanil 2: 157, 435 Dalapon 1: 117 Desalkyl metabolites of chlorotriazines 2:413 Dichlobenil 1: 123; 2: 169 Dinobuton 2: 197 Dinoseb 2: 437 Dinoterb 2: 437 Diuron 2: 435 DNOC 2: 437 Ethylene thiourea 1: 135 Fluazifop-P-butyl 2: 437 Flurenol 2: 127 Fluroxypyr-(l-methylheptyl) 2: 437 Fonofos 2: 205 Fosetyl 2: 211 Fungicides 2: 377 Glufosinate 2: 217 Glyphosate 2: 229 Haloxyfop-(2-ethoxyethyl) 2: 437 Ioxynil 2: 99 Lenacil 2: 435 Metalaxyl 1: 153; 2: 437 Metamitron 2: 435

476

Index of Analytical Materials

Water (contd.) Methabenzthiazuron 2: 435 Methamidophos 1: 81 Methomyl 1: 161 Metribuzin 2: 245, 435 Metsulfuron 2: 145 Nitrofen 1: 173 Nitrothal-isopropyl 2: 253, 437 Organochlorine insecticides 2: 387 Organotin compounds 2: 343 Oxamyl 2: 261 Paraquat 1: 177 Parathion 2: 435 Pendimethalin 2: 437 Phenoxyalkanoic acid herbicides 2: 369, 393 Phthalimides 2: 359 Pirimicarb 1: 183 Pirimiphos-methyl 1: 191 Propachlor 2: 275 Propiconazole 2: 281 Pyrethroids 2: 333 Terbumeton 2: 437 Tetrachlorvinphos 1: 203 Triadimefon 2: 87, 435 Triadimenol 2: 87, 435 Triazine herbicides 2: 403 Triazine herbicides, desalkyl metabolites 2: 413 Triclopyr-(2-butoxyethyl) 2: 437 Wheat grains Anilazine 2: 59 Benomyl 2: 69 Bromine-containing fumigants 1: 377 Bromoxynil 2: 99 Captafol 1: 93 Carbendazim 2: 69 Chlorflurenol 2: 127 Dalapon 1: 117 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Flurenol 2: 127 Glufosinate 2: 217 Heptenophos 1: 149 Ioxynil 2: 99 Metribuzin 2: 245 Nitrofen 1: 173 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241, 251 Phthalimides 2: 359 Pirimiphos-methyl 1: 191 Propiconazole 2: 281

Wheat grains (contd.) Pyrazophos 1: 197 Pyrethroids 2: 333 Thiophanate-methyl 2: 69 Thiuram disulphide fungicides Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Triazophos 1: 209

1: 353

Wheat, green matter Anilazine 2: 59 Bromoxynil 2: 99 Dalapon 1: 117 Dithiocarbamate fungicides 1: 353 Ioxynil 2: 99 Phthalimides 2: 359 Propiconazole 2: 281 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Wheat, straw Anilazine 2: 59 Benomyl 2: 69 Captafol 1: 93 Carbendazim 2: 69 Chlorflurenol 2: 127 Dalapon 1: 117 Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135 Flurenol 2: 127 Metribuzin 2: 245 Nitrofen 1: 173 Phenyl urea herbicides 1: 241, 251 Propiconazole 2: 281 Pyrazophos 1: 197 Thiophanate-methyl 2: 69 Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87 Triadimenol 2: 87 Triazine herbicides 1: 265 Whole egg, dried see Dried whole egg Whole milk powder Organochlorine pesticides 1: 297, 309 Organophosphorus pesticides 1: 297, 309 Wine Amitrole 2: 49 Dichlofluanid 2: 177, 191

Index of Analytical Materials Wine (contd.) Ethylene thiourea 1: 135 Folpet 1: 143 Fosetyl 2: 211 Metalaxyl 1: 153 Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1:383; 2:317 Organophosphorus pesticides 1: 383; 2: 317 Propiconazole 2: 281 Tolylfluanid 2: 177 Triadimefon 2: 87 Triadimenol 2: 87 Vinclozolin 1: 213

* see also special entries

477

Witloof chicory Organochlorine pesticides 1: 283; 2: 313 Organophosphorus pesticides 1: 283; 2: 313 Phenmedipham 2: 269 Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213 Youngberries Metalaxyl 1: 153

List of Suppliers Referenced in the Text-Matter of the Manual Aldrich Chemical Co., 1001 West Saint Paul Ave., Milwaukee, WI 53233, USA; Aldrich Europe, Poststr. 56, Postfach 2004, D-4054 Nettetal 2, FRG. Alltech Associates, Inc., 2051 Waukegan Road, Deerfield, IL 60015, USA; Alltech Europe, Begoniastraat 5, B-9731 Eke, Belgium. Alpine AG, Maschinenfabrik, Peter-D6rfler-Str. 13-25, D-8900 Augsburg, FRG. Analytical Bio-Chemistry Laboratories, 7200 ABC Lane, P.O. Box 1097, Columbia, MO 65205, USA; in Europe: N. Foss Electric GmbH, Waidmannstr. 12b, D-2000 Hamburg 50, FRG. Analytichem International, Inc., 24201 Frampton Ave., Harbor City, CA 90710, USA. J. T. Baker Inc., 222 Red School Lane, Phillipsburg, NJ 08865, USA; in Europe: J. T. Baker B.V., Rijsterborgherweg 20, P.O. Box 1, NL-7400 AA Deventer, The Netherlands. Becker: see Packard. Beckman Instruments Inc., 2500 Harbor Blvd., Fullerton, CA 92634, USA. Bender und Hobein GmbH, Lindwurmstr. 71-73, Postfach 150229, D-8000 Miinchen 15, FRG. J. C. Binzer Papierfabrik, Berleburger Str. 71, D-3559 Hatzfeld 1, FRG. Bio-Rad Laboratories, Chemical Division, 3300 Regatta Blvd., Richmond, CA 94804, USA; Heidemannstr. 164, Postfach 450133, D-8000 Miinchen 45, FRG. Bischoff Analysentechnik und -gerate GmbH, Boblinger Str. 23, D-7250 Leonberg, FRG. Boehringer Ingelheim KG, D-6507 Ingelheim, FRG. Brownlee Labs, 2045 Martin Ave., Santa Clara, CA 95050, USA. Camag, Sonnenmattstr. 11, CH-4132 Muttenz, Switzerland; Bismarckstr. 27-29, D-1000 Berlin 41, FRG; Camag Scientific Inc., P.O. Box 563, Wrightsville Beach, NC 28480, USA. Carlo Erba Strumentazione S.P.A., Strada Rivoltana, 1-20090 Rodano (Milano), Italy. Chemie-Mineralien KG, Loningstr. 35, D-2800 Bremen, FRG. Chemie und Filter GmbH Verfahrenstechnik, Im Schumachergewann 7, D-6900 Heidelberg, FRG. Chromatronix Inc., 2300 Leghorn St., Mt. View, CA 94043, USA. Chrompack International B.V., Kuipersweg 6, P.O. Box 8033, NL-4330 EA Middelburg, The Netherlands; Chrompack Inc., 1130 Route 202, Raritan, NJ 08869, USA. Corning Glass Works, Inc., Science Products Division, Corning, NY 14831, USA; Corning Laboratory Products, 1 Prince's Street, Richmond, Surrey TW9 1DZ, U.K. Degussa AG, GB/AC GKA, Postfach 110533, D-6000 Frankfurt 11, FRG. Desaga GmbH, Postfach 101969, D-6900 Heidelberg 1, FRG. Dohrmann Div., Xertex Corp., 3240 Scott Blvd., Santa Clara, CA 95042, USA. Dow Corning Corp., P.O. Box 994, Midland, MI 48686, USA. Du Pont Co., Inc., 1007 Market St., Wilmington, DE 19898, USA; Du Pont de Nemours (Deutschland), Du Pont-Str. 1, D-6380 Bad Homburg, FRG. EGA-Chemie: see Aldrich. Gunther Ehle, Glasinstrumentenfabrik, Holzheimer Str. 56 a, D-6250 Limburg, FRG. F & M Scientific: see Hewlett-Packard.

480

List of Suppliers

Finnigan MAT, 355 River Oaks Pkwy., San Jose, CA 95134, USA; Paradise, Hemel Hempstead, Herts. HP2 4TG, U.K. Fluka A C , CH-9470 Buchs, Switzerland; Fluka Chem. Corp., 980 South Second St., Ronkonkoma, NY 11779, USA. Gerstel GmbH, Aktienstr. 232-234, D-4330 Mulheim/Ruhr 1, FRG. Heraeus-Christ: see Heraeus Sepatech Heraeus Quarzschmelze GmbH, Quarzstrafie, D-6450 Hanau, FRG. Heraeus Sepatech GmbH, Postfach 1220, D-3360 Osterode, FRG. Heraeus-Wittmann GmbH, Englerstr. 11, D-6900 Heidelberg 1, FRG. Hewlett-Packard Co., Mail Stop 2083, 3000 Hanover St., Palo Alto, CA 94304, USA. Hormuth-Vetter: Vetter KG, Malscher Strafle, D-6837 St. Leon-Rot 2, FRG. ICN Biomedicals, Inc., Chromatography Division, 3300 Hyland Ave., Costa Mesa, CA 92626, USA; Miihlgrabenstr. 10, D-5309 Meckenheim, FRG. J & W Scientific, 91 Blue Ravine Rd., Folsom, CA, 95630, USA. Janke & Kunkel GmbH & Co., KG, Neumagenstr. 27, D-7813 Staufen, FRG. Janssen Pharmaceutica N.V., B-2340 Beerse, Belgium. Johns-Manville: Manville, Ken-Caryl Ranch, P.O. Box 5108, Denver, CO 80217, USA. Dr. H. Knauer, Wissenschaftl. Gerate KG, Heuchelheimer Str. 9, D-6380 Bad Homburg, FRG. Kontes Glass Co., Spruce St., P.O. Box 729, Vineland, NJ 08360, USA. Kontron Instruments, Inc., 9 Plymouth St., Everett, MA 02149, USA. Kratos Analytical, Inc., 535 E. Crescent Ave., Ramsey, NJ 07446, USA. Lehmann & Voss & Co., Alsterufer 19, D-2000 Hamburg 36, FRG. LKB Products, Pharmacia LKB, Box 305, S-16126 Bromma, Sweden. Macherey-Nagel, Postfach 101352, D-5160 Diiren, FRG. Mallinckrodt, Inc., 675 McDonnell Blvd., P.O. Box 5840, St. Louis, MO 63134, USA. Mega: see Carlo Erba. Melpar: see Tracor. E. Merck, Frankfurter Str. 250, Postfach 4119, D-6100 Darmstadt, FRG. Messer Griesheim GmbH, Industriegase, Homberger Str. 12, D-4000 Dtisseldorf, FRG. Metrawatt GmbH, Thomas-Mann-Str. 16-20, Postfach 1333, D-8500 Niirnberg, FRG. Micro Tek Instrument: see Tracor. Millipore Corporation, 80 Ashby Rd., Bedford, MA 01730, USA. Nopco Chemical Co., 350 Mt. Kemble Ave., Morristown, NJ 07960, USA. Orlita GmbH & Co. KG, Dosiertechnik, Max-Eyth-Str. 10, D-6300 GieJkn, FRG. Packard Instrument Co., Inc., 2200 Warrenville Rd., Downers Grove, IL 60515, USA. Perkin-Elmer Ltd., Maxwell Rd., Beaconsfield, Bucks. HP9 1QA, U.K.; The Perkin-Elmer Corp., 761 Main Ave., Norwalk, CT 06859, USA. Pharmacia LKB Biotechnology, Bjorkgatan 30, S-75182 Uppsala, Sweden; 800 Centennial Ave., Piscataway, NJ 08854, USA. Phase Separations, Deeside Industrial Estate, Queensferry, Clwyd CH5 2LR, U.K.; 140 Water St., Norwalk CT 06854, USA. Philips, Scientific and Analytical Equipment Div., Lelyweg 1, NL^7602 EA Almelo, The Netherlands; Philips Scientific, Analytical Div., York St., Cambridge CB1 2PX, U.K. Pierce Chemical Co., 3747 N. Meridian Rd., P.O. Box 117, Rockford, IL 61105, USA; Pierce Europe B.V., P.O. Box 1512, NI^3260 BA Oud-Beijerland, The Netherlands.

List of Suppliers

481

Promochem GmbH, Postfach 1246, D-4230 Wesel, FRG. Pye Unicam Ltd., York St., Cambridge CB1 2PX, U.K. Quickfit: see Corning. W. Reiss & Co., Krahenweg 5, D-2000 Hamburg 61, FRG. Restek Corp., 110 Benner Circle, Bellefonte, PA 16823, USA. Retsch GmbH & Co. KG, Rheinische Str. 36, Postfach 1554, D-5657 Haan 1, FRG. Rheodyne, Inc., P.O. Box 996, Cotati, CA 94931, USA. Riedel-de Haen AG, Wunstorfer Str. 40, Postfach 100262, D-3016 Seelze, FRG. Carl Roth GmbH & Co., Schoemperlenstr. 1-5, Postfach 211162, D-7500 Karlsruhe 21, FRG. Sartorius GmbH, Weender Landstr. 94-108, Postfach 3243, D-3400 Gottingen, FRG; Sartorius Corp., 140 Wilbur PL, Bohemia NY 11716, USA. Schleicher & Schull, Postfach 4, D-3354 Dassel, FRG; 10 Optical Ave., Keene, NH 03431, USA. Schoeffel Instrument Corp., 24 Booker St., Westwood, NJ 07675, USA. Schott & Gen., Jenaer Glaswerke, Hattenbergstr. 10, Postfach 2480, D-6500 Mainz, FRG; Schott America, 3 Odell Plaza, Yonkers, NY 10701, USA. Serva Feinbiochemica GmbH & Co., Carl-Benz-Str. 7, Postfach 105260, D-6900 Heidelberg 1, FRG; Serva Biochemicals, 50 A & S Dr., Paramus, NJ 07652, USA. Shimadzu Scientific Instruments, Inc., 7102 Riverwood Dr., Columbia MD 21046, USA; Shimadzu Europa, Albert-Hahn-Str. 6-10, D-4100 Duisburg 29, FRG. Spark Holland B.V., P.O. Box 388, NI^7800 AJ Emmen, The Netherlands. Spectra-Physics, Autolab Div., 3333 North First St., San Jose, CA 95134, USA. Supelco, Inc., Supelco Park, Bellefonte, PA 16823, USA. Tracor Instruments, Inc., 6500 Tracor Lane, Austin, TX 78725, USA. Valco Instruments Co., Inc., P.O. Box 55603, Houston, TX 77255, USA; Valco Europe, Untertannberg 7, CH-6214 Schenkon, Switzerland. Varian Instrument Group, 220 Humboldt C t , Sunnyvale, CA 94089, USA. Waters Chromatography, 34 Maple St., Milford, MA 01757, USA. Weisser: supplied by Hans Otto, Feldstr. 26-28, D-2000 Hamburg 6, FRG. Whatman Inc., 9 Bridewell PL, Clifton, NJ 07014, USA; Whatman Ltd., Springfield Mill, Maidstone, Kent ME14 2LE, U.K. Woelm: see ICN Biomedicals. CarlZeiss, Postfach 1369-1380, D-7082 Oberkochen, FRG; One Zeiss Dr., Thornwood, NY 10541, USA.

Author Index Becker, G. 1: 283; 2: 313 Bertrand, A. 2: 211 Beutel, P. 1: 213; 2: 253 Blacha-Puller, M. 1: 183; 2: 99, 145, 197, 211, 275, 359 Bos, U 1: 37 Brauckhoff, S. 2: 349 Brennecke, R. 2: 59, 77, 87, 177, 377 Bullock, D. J. W. 1: 183, 191 Burger, K. 2: 41, 423, 435, 442 Btittler, B. 1: 99, 105, 143, 401; 2: 281 Cowell, J. E. 2: 229 Dick, J. P. 1: 183 Drescher, N. 1: 21, 135; 2: 369 Ebing, W. 1: 57, 61, 87, 335, 371, 407 Eichler, D. 1: 109; 2: 127, 157, 205, 239, 393 Eichner, M. 1: 57, 327, 335 Elzner, J. 2: 287 Formica, G. 2: 69, 403, 413 Frehse, H. 1: 37; 2: 3 Gaudernack, E. 1: 65 Giannone, C. 2: 69, 403, 413 Gorbach, S. 1: 29, 37, 65, 127, 149; 2: 3 Gottschild, D. 2: 37 Habersaat, B. 1: 51 Hahn, H. 1: 321 Hamann, R. 2: 169 Hershberger, L. W. 2: 145 Herzel, F. 1: 123, 177 Heupt, W. 2: 127, 157 Hezel, U. 2: 291 Hild, J. 1: 361 Holt, R. F. 1: 161; 2: 261 Holtz, K.-H. 2: 3 Hormann, W. D. 1: 99, 105, 143, 153, 265, 401; 2: 69, 281, 403 Ipach, R. 2: 153 Jarczyk, H. J. 2: 49, 245 Karlhuber, B. 1: 57, 347 Keller, W. 1: 117, 135; 2: 135, 163, 253 Kennedy, S. H. 1: 183 Kettrup, A. 2: 169 Kirchhoff, J. 1: 29

Kirkland, J. J. 1: 161 Kohle, H. 2: 37, 113, 145, 333, 359, 437 Koflmann, A. 1: 87 KoBmann, K. 2: 269 Krohn, H. 1: 173 Kunzler, K. 1: 127 Meemken, H.-A. 1: 51 Mollhoff, E. 2: 49, 343 Muller, M. A. 2: 211 Nolting, H.-G. 1: 93, 167, 173, 177, 183, 203; 2: 37, 99, 113, 145, 197, 211, 275, 323, 333, 359, 437 Nord, P.-J. 2: 229 Otteneder, H. 2: 291, 295 Otto, S. 1: 117, 135, 213; 2: 135, 163 Pease, H. L. 1: 161; 2: 261 Pflugmacher, J. 1: 57, 61, 335, 371, 407 Ramsteiner, K. 1: 57, 153, 251, 347 Reding, M. A. 2: 229 Richtarsky, G. W. 1: 167 Schmidt, G. 1: 177 Schmidt, M. 2: 169 Schulte, E. 1: 51 Siebers, J. 1: 81, 183, 191; 2: 99, 113, 145, 197, 211, 275, 323, 333, 359 Slates, R. V. 2: 145 Sochor, H. 2: 217 Specht, W. 1: 71, 75, 309, 383; 2: 31, 107, 317 Stijve, T. 1: 297, 377 Thier, H.-P. 1: 17, 29, 37, 71, 167, 297, 309, 321, 353, 361, 377, 383; 2: 3, 317, 323, 349 Thier, W. 1: 197, 209 Vogel, W. 2: 239 Vogeler, K. 2: 191, 377 Voss, G. 1: 241 Walter, H.-F. 2: 3 Weil, L. 1 : 2 3 ; 2: 387 Weinmann, W. D. 1: 21, 37, 81, 93, 167, 173, 177, 183, 191, 203, 2: 323 Winkler, S. 1: 65 Wolf, A. 1: 93, 203 Zahnow, E. W. 2: 145