Manual of Pesticide Residue Analysis, Volume 2

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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


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


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.


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


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).


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.


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.


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


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).


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).


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-


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


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


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


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 -


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


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.


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


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


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


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


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)


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


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


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


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


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


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




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


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


35



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


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


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


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





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 ( 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 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 "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)


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


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


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


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


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


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


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


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. %)


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).


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 99.999 vol. %) Nitrogen 4.6 (> 99.996 vol. %)

90

Bitertanol, Triadimefon, Triadimenol


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.


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


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


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


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


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


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


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).


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).


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 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 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


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 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 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


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 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


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

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