Manual of Pesticide Residue Analysis Volume II PDF

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)
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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
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All rights reserved (including those of translation into other languages). No part of this book may be
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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 ap-
plicability 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 detec-
tion and determination by the calibration curve technique, thus providing a commendable
alternative to the procedure proposed in Volume 1. Finally, a comprehensive table gives mass-
spectrometric 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 Ger-
man 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 IX
Senate Commission for Pesticides, Deutsche Forschungsgemeinschaft XI
Working Group on Residue Analysis, Senate Commission for Pesticides XV
Part 1: Introduction and Instructions (contd.)

Derivation of the Limits of Detection and Determination Applying the Calibration
Curve Concept 3
Mass-Spectrometric El Data for Confirmation of Results 25
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) 31
Cleanup Method 7. Solid phase extraction of water samples on alkyl-modified silica gel
using disposable columns 37
Cleanup Method 8. Solid phase extraction of water samples on alkyl-modified silica gel 41
Part 3: Individual Pesticide Residue Analytical Methods (contd.)

Amitrole, 4-A*) 49
Anilazine, 186 59
Benomyl, Carbendazim, Thiophanate-methyl, 261-378-370 69
Bitertanol, 613-A 77
Bitertanol, Triadimefon, Triadimenol, 613-425-605 87
Bromoxynil, Ioxynil, 264-212 99
Carbendazim, 378 107
Carbosulfan, Carbofuran, 658-344 113
Chlorflurenol, Flurenol, 275-215 127
Chloridazon, 89-A 135
Chlorsulfuron, Metsulfuron, 664-672 145
Copper Oxychloride, 147-A 153
Cymoxanil, 513 157
2,4-D, Dichlorprop, 27-A-38-A 163
Dichlobenil, 225-A 169
Dichlofluanid, Tolylfluanid, 203-371 177
Dichlofluanid, Tolylfluanid, 203-A-371-A 191
Dinobuton, Binapacryl, 255-8 197
Fonofos, 288 205
Fosetyl, 522 211
*) 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 in-
dividual compound.
VIII Contents
Glufosinate, 651 217

Glyphosate, 405 229
Metaldehyde, 151-A 239
Metribuzin, 337 245
Nitrothal-isopropyl, 416 253
Oxamyl, 441 261
Phenmedipham, 233-B 269
Propachlor, 310 275
Propiconazole, 624 281
Sulphur, 184-B 287
Thiabendazole, 256-A 291
Thiabendazole, 256-B 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. 1 301
S 8 Organohalogen, Organophosphorus and Triazine Compounds (updated) 313
S 19 Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides
(updated) 317
S 22 Natural Pyrethrins, Piperonyl Butoxide 323
S 23 Pyrethroids 333
S 24 Organotin Compounds 343
S 25 Methyl Carbamate Insecticides 349
S 26 Phthalimides 359
Part 5: Multiple Pesticide Residue Analytical Methods for Water

W 4 Phenoxyalkanoic Acid Herbicides 369
W 5 Fungicides 377
W 6 Organochlorine Insecticides 387
W 7 Phenoxyalkanoic Acid Herbicides 393
W 8 Triazine Herbicides 403
W 13 Desalkyl Metabolites of Chlorotriazine Herbicides 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 423
Examples for Applying the AMD Technique to the Determination of Pesticide Residues
in Ground and Drinking Waters 435
Cumulative Indexes for Volumes 1 and 2

Index of Determinable Pesticides, Metabolites and Related Compounds (Index of Com-
pounds) 449
Index of Analytical Materials 459
List of Suppliers Referenced in the Text-Matter of the Manual 479
Author Index 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 co-
distillation
Cleanup Method 3. Cleanup of crude extracts from plant material by gel permeation chro-
matography on Sephadex LH-20
Cleanup Method 4. Cleanup of crude extracts from plant material by gel permeation chro-
matography on polystyrene gels
Cleanup Method 5. Cleanup of large quantities of fats for analysis of residues of organo-
chlorine and organophosphorus compounds
Cleanup Method 6. Cleanup of crude extracts from plant and animal material by gel permea-
tion 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 Com-
pounds)
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 Institut ftir Pflanzenpathologie und Pflanzenschutz der
(Chairman from 1987 to Universitat
1989) 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,
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
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 Institut fur Toxikologie der Universitat

Ohnesorge 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

Senate Commission for Pesticides XIII
Secretaries of the Senate Commission for Pesticides
Frau Dr. Dagmar Weil Institut fur Wasserchemie und Chemische Balneologie
until 1986 der Technischen Universitat
Marchioninistrafie 17, D-8000 Munchen 70
Dr. Friedhelm Dopke Institut fur Pflanzenpathologie und Pflanzenschutz
from 1987 to 1989 der Universitat
Grisebachstr. 6, D-3400 Gottingen-Weende
Assessor Wolfgang Deutsche Forschungsgemeinschaft
Bretschneider t 1990 Kennedyallee 40, D-5300 Bonn 2
Working Group on Residue Analysis,
Senate Commission for Pesticides
Members and Guests

Dr. Hans-Gerd Nolting Biologische Bundesanstalt fur Land- und Forstwirtschaft
(Chairman from 1988 to 1989) Messeweg 11-12, D-3300 Braunschweig
Prof. Dr. Hans-Peter Thier Institut fur Lebensmittelchemie der Universitat
(Chairman from 1976 to 1988) Piusallee 7, D-4400 Munster
Prof. Dr. Hans Zeumer t 1988
(Chairman from 1961 to 1976)
Dr. Gtinther Becker Chemisches Untersuchungsamt
Charlottenstrafie 8, D-6600 Saarbrucken
Prof. Dr. Winfried Ebing Biologische Bundesanstalt fur Land- und Forstwirtschaft
Konigin-Luise-Straite 19, D-1000 Berlin 33
Dr. Siegmund Ehrenstorfer Landesuntersuchungsamt fur das Gesundheitswesen,
Fachabteilung Chemie
Fritz-Hintermayr-Strafle 3, D-8900 Augsburg
Dr. Dietrich Eichler Shell Forschung GmbH
D-6501 Schwabenheim
Dr. Helmut Frehse Bayer AG, PF-A/CE-RA,
Dr. Ing. Siegbert Gorbach Hoechst AG, Analytisches Laboratorium,
Pflanzenschutz-Analyse, G 864
Prof. Dr. Fritz Herzel Bundesgesundheitsamt
Dr. Wolf-Dieter Hormann Division Agrochemie der CIBA-GEIGY AG
CH-4002 Basel/Schweiz
Prof. Dr. Antonius Kettrup Fachbereich Chemie u. Chemietechnik der Universitat
Postfach 1621, D-4790 Paderborn
Dr. Jochen Kirchhoff Institut fur Phytomedizin der Universitat Hohenheim
Otto-Sander-StraBe 5, D-7000 Stuttgart 70
Prof. Dr. Hans Maier-Bode Tannenweg 7, D-7884 Rickenbach b. Sackingen
XVI Working Group on Residue Analysis
Dr. Egon Mollhoff Bayer AG, PF-A/CE-RA,

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 concen-
trations. 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 par-
allel 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 deter-
mination 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 per-
mits 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. Fre-
quently 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 in-
strumental 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 com-
plete 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 for-
tification 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 in-
terval. For an example, see 9.3.
Limits of Detection and Determination 5
2 Calibration curve concept

2.1 Basic considerations
The aim of the concept is to define the limit of detection and, resulting from it, the limit of
determination for the results obtained when a given analyte is determined with a particular
analytical method in an individual laboratory. The definition proceeds from the calibration
curve obtained with the analytical method and employs the upper and lower limits of the
prediction interval of the curve for deriving LDC and LDM. The prediction interval is used
here as the confidence interval.
The operation described for determining LDM permits an appropriate consideration of Re-
quirement I. Requirement III is integrated into the formulae used to calculate LDM. Deter-
mining 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 ex-
it
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 fortifica-
tion 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.
Next, the prediction interval is calculated which symmetrically envelopes the linear calibra-
tion curve (see Figure 1). The curves for the upper (Y + ) and lower (Y_) limits of the predic-
tion 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, in-
dicate the confidence interval for signal values yielded by samples with a "nil" content. Note
that this range is extrapolated from the results of the fortification experiments and is not
derived from the measurement of blank values.
Each signal value Yo > YUP yields three possible points of intersection with the calibration
curve and the limits of its prediction interval. They correspond, respectively, to the values
Xj, X 3 and X 2 (Figure 1) and form the basis for further derivations. When the curvature of
both the upper and lower limits of the prediction interval is negligible, X 3 can be considered
the arithmetic mean of X{ and X 2 with good approximation. X 2 is corresponding to LDC*
(Figure 4) and X v (Figure 5).
Xj and X 2 can be calculated from the formulae IIa and l i b (see 6.6, see also 3.2).
Limits of Detection and Determination
3 Limit of detection (LDC)

3.1 Definition
The limit of detection is defined by the smallest content of the analyte in an analytical sample,
for which the particular analytical method yields signal values which differ, with a selected
level of significance a, from signal values obtained from samples with a "nil" content (blank
signal values).
The level of significance, a, can be arbitrarily chosen. In most cases, values of a between
5 and 1% will allow a sufficient margin of safety. In residue analyses, usually a level of
a = 5%, i.e. a confidence level of S = 1 - a = 95%, is chosen.
Based on the n results (X^Yj) from the fortification experiments and the given
significance level, a, the limit of detection for an analyte is derived from the calibration curve.
It is represented by the smallest value X L D C for which the confidence intervals of the cor-
responding 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.
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 ^ ^ - Y=A+BX
ki
"LDC
V
A
_. -
X=0 LDC 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.
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 for-
tification 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
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 deter-
mined at X = LDM does not extend into the range below the LDC. If Xj denotes that concen-
tration 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
^ Y=A+BX
^ Y_
UP
A
r
x=o 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).
4.3 Consequences of Requirement II

The usual way to obtain a calibration curve is to plot each signal value (Y) versus the cor-
responding given content (X) of the analyte in the sample material. For checking Require-
ment 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%. Re-
quirement 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 Re-
quirements I and II are met as well. This approach corresponds to the procedure recom-
mended 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, for-
tunately, the calibration curve concept offers an elegant possibility to obtain the required in-
formation 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 increas-
ing 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 coor-
dinate of the intersection point (see Figure 5; cf. Figure 9).
-H
C/3
^ ^ ^Y=A+BX
Y(i+tv0) *y
x=o *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-
tification level X follows a Gaussian distribution around the mean value. Moreover, the varia-
tion 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 func-
tions.
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 calcula-
tions 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
H
iv)
i=i\j=i /
k / mi
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
n i = i\j = i '7
In addition, the following square and product sums are needed:
Formula Auxiliary '.Term

k
E2
= K == F -
i= 1 n
k
EG
(Xi-X)E(Yifj-Y) T
=J-
n
G2
=M=H
n
I?
The auxiliary term N=
~K
will also be required.
The terms for the slope, B, and the axis intercept, A, of the calibration straight line are ob-
tained through:
L
B= Slope
A = Y- B X Theoretical Blank Value
6.3 Prediction interval

(X X)2
(I) Y = Y + B ( X - X ) t s , r S R - | / l + ^ + 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
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
(t sR ; )
C = B2 - = 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:
v
C C V \ n K
Substituting YUP for Yo results in X 2 being identical with LDC, and one obtains (IV).
6.7 Calculation of Y c
The expression for Yc is immediately obtained from (I) as:
1 (LDC X) 2
(V) 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 expres-
sion 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 in-
tersection 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
-
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
(ST.)2
D = (B V o ) 2 - ^ L J ^- = auxiliary term; (Vo)2 = 0.04
K.
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 Laborato-
rium 35, 285-291 (1991).
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 Example 2
Concentration [|ig/kg] Concentration [mg/kg]
Added (X) Found (Y) Added (X) Found (Y)
0 12 0.03 0.031
20 26 0.03 0.027
40 45 0.03 0.029
80 63 0.03 0.025
100 105 0.05 0.037
120 115 0.05 0.042
170 153 0.05 0.045
200 180 0.05 0.047
0.1 0.088
0.1 0.080
0.1 0.093
0.1 0.080
0.2 0.159
0.2 0.177
0.2 0.159
0.2 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 calibra-
tion 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.
When proceeding according to 6.1 and 6.2, the following results will be obtained:
E = 730
F = 101700
G = 699
H = 86873
J = 93670
X= 91.25, Y = 87.38
K = 35087.50
L = 29886.25
M= 25797.88
N = 25456.01
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 con-
tinued 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 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 Sec-
tions 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
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:
n = 16 n = 12
(t = 2.145) (t = 2.228)
A 0.0016 0.0026
B 0.8415 0.8240
sR 0.0074 0.0045
YUP 0.0188 0.0146
YLO -0.0156 -0.0094
LDC 0.0402 0.0280
x
i 0.0599 0.0414
Xcv 0.0439 0.0269
YCV 0.0551 0.0358
x
m 0.0635 0.0403
LDM (rounded) 0.06 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 in-
terval 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 predic-
tion 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, per-
missible 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).
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

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.
f t
two-sided two-sided
1 12.71 10 2.228
2 4.303 11 2.201
3 3.182 12 2.179
4 2.776 13 2.160
5 2.571 14 2.145
6 2.447 15 2.131
7 2.365 20 2.086
8 2.306 30 2.042
9 2.262 40 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 , Molar Main fragments m/z (intensities)

mass*) 1 2 3 4 5
Acephate 183 43 (100) 44 (88) 136 (80) 94 (58) 47 (56) 95 (32)

Alachlor 269 45 (100) 188 (23) 160 (18) 77 (7) 146 (6) 224 (6)
Aldicarb 190 41 (100) 86 (89) 58 (85) 85 (61) 87 (50) 44 (50)
Aldrin 362 66 (100) 91 (50) 79 (47) 263 (42) 65 (35) 101 (34)
Allethrin 302 123 (100) 79 (40) 43 (32) 81 (31) 91 (29) 136 (27)
Atrazine 215 43 (100) 58 (84) 44 (75) 200 (69) 68 (43) 215 (40)
Azinphos-methyl 317 77 (100) 160 (77) 132 (67) 44 (30) 105 (29) 104 (27)
Barban 257 51 (100) 153 (76) 87 (66) 222 (44) 52 (43) 63 (43)
Benazolin methyl ester 257 170 (100) 134 (75) 198 (74) 257 (73) 172 (40) 200 (31)
Bendiocarb 223 151 (100) 126 (58) 166 (48) 51 (19) 58 (18) 43 (17)
Bromacil 260 205 (100) 207 (75) 42 (25) 70 (16) 206 (16) 162 (12)
Bromacil N-methyl derivative 274 219 (100) 221 (68) 41 (45) 188 (41) 190 (40) 56 (37)
Bromophos 364 331 (100) 125 (91) 329 (80) 79 (57) 109 (53) 93 (45)
Bromophos-ethyl 392 97 (100) 65 (35) 303 (32) 125 (28) 359 (27) 109 (27)
Bromoxynil methyl ether 289 291 (100) 88 (77) 276 (67) 289 (55) 293 (53) 248 (50)
Captafol 347 79 (100) 80 (42) 77 (28) 78 (19) 151 (17) 51 (13)
Captan 299 79 (100) 80 (61) 77 (56) 44 (44) 78 (37) 149 (34)
Carbaryl 201 144 (100) 115 (82) 116 (48) 57 (31) 58 (20) 63 (20)
Carbendazim 191 159 (100) 191 (57) 103 (38) 104 (37) 52 (32) 51 (29)
Carbetamide 236 119 (100) 72 (54) 91 (44) 45 (38) 64 (37) 74 (29)
Carbofuran 221 164 (100) 149 (70) 41 (27) 58 (25) 131 (25) 122 (25)
Chlorbromuron 292 61 (100) 46 (24) 62 (11) 63 (10) 60 (9) 124 (8)
Chlorbufam 223 53 (100) 127 (20) 51 (13) 164 (13) 223 (13) 70 (10)
cis-Chlordane 406 373 (100) 375 (84) 377 (46) 371 (39) 44 (36) 109 (36)
trans-Chlordane 406 373 (100) 375 (93) 377 (53) 371 (47) 272 (36) 237 (30)
Chlorfenprop-methyl 232 125 (100) 165 (64) 75 (46) 196 (43) 51 (43) 101 (37)
Chlorfenvinphos 358 81 (100) 267 (73) 109 (55) 269 (47) 323 (26) 91 (23)
Chloridazon 221 77 (100) 221 (60) 88 (37) 220 (35) 51 (26) 105 (24)
Chloroneb 206 191 (100) 193 (61) 206 (60) 53 (57) 208 (39) 141 (35)
26 List of Mass-Spectrometric Data
Table, (contd.)
Molar Main fragments m/z (intensities)

Compound
mass*) 1 2 3 4 5 6
Chlorotoluron 212 72 (100) 44 (29) 167 (28) 132 (25) 45 (20) 77(11)
3-Chloro-4-methylaniline
(GLC degradation product of
chlorotoluron) 141 141 (100) 140 (37) 106 (68) 142 (36) 143 (28) 77 (25)
Chloroxuron 290 72 (100) 245 (37) 44 (31) 75 (21) 45 (19) 63 (16)
Chlorpropham 213 43 (100) 127 (49) 41 (35) 45 (20) 44 (18) 129 (16)
Chlorpyrifos 349 97 (100) 195 (59) 199 (53) 65 (27) 47 (23) 314 (21)
Chlorthal-dimethyl 330 301 (100) 299 (81) 303 (47) 332 (29) 142 (26) 221 (24)
Chlorthiamid 205 170 (100) 60 (61) 171 (50) 172 (49) 205 (35) 173 (29)
Cinerin I 316 123 (100) 43 (35) 93 (33) 121 (27) 81 (27) 150 (27)
Cinerin II 360 107 (100) 93 (57) 121 (53) 91 (50) 149 (35) 105 (33)
Cyanazine 240 44 (100) 43 (60) 68 (60) 212 (48) 41 (47) 42 (34)
Cypermethrin 415 163 (100) 181 (79) 165 (68) 91 (41) 77 (33) 51 (29)
2,4-DB methyl ester 262 101 (100) 59 (95) 41 (39) 162 (36) 69 (28) 63 (25)
Dalapon 142 43 (100) 61 (81) 62 (67) 97 (59) 45 (59) 44 (47)
Dazomet 162 162 (100) 42 (87) 89 (79) 44 (73) 76 (59) 43 (53)
Demeton-S-methyl 230 88 (100) 60 (50) 109 (24) 142 (17) 79 (14) 47 (11)
Desmetryn 213 213 (100) 57 (67) 58 (66) 198 (58) 82 (44) 171 (39)
Dialifos 393 208 (100) 210 (31) 76 (20) 173 (17) 209 (12) 357 (10)
Di-allate 269 43 (100) 86 (62) 41 (38) 44 (25) 42 (24) 70 (19)
Diazinon 304 137 (100) 179 (74) 152 (65) 93 (47) 153 (42) 199 (39)
Dicamba methyl ester 234 203 (100) 205 (60) 234 (27) 188 (26) 97 (21) 201 (20)
Dichlobenil 171 171 (100) 173 (62) 100 (31) 136 (24) 75 (24) 50 (19)
Dichlofenthion 314 97 (100) 279 (92) 223 (90) 109 (67) 162 (53) 251 (46)
Dichlofluanid 332 123 (100) 92 (33) 224 (29) 167 (27) 63 (23) 77 (22)
2,4-D isooctyl ester 332 43 (100) 57 (98) 41 (76) 55 (54) 71 (41) 69 (27)
2,4-D methyl ester 234 199 (100) 45 (97) 175 (94) 145 (70) 111 (69) 109 (68)
Dichlorprop isooctyl ester 346 43 (100) 57 (83) 41 (61) 71 (48) 55 (47) 162 (41)
Dichlorprop methyl ester 248 162 (100) 164 (80) 59 (62) 189 (56) 63 (39) 191 (35)
Dichlorvos 220 109 (100) 185 (18) 79 (17) 187 (6) 145 (6) 47 (5)
Dicofol 368 139 (100) 111 (39) 141 (33) 75 (18) 83 (17) 251 (16)
o,p'-DDT 352 235 (100) 237 (59) 165 (33) 236 (16) 199 (12) 75 (12)
p,p'-DDT 352 235 (100) 237 (58) 165 (37) 236 (16) 75 (12) 239 (11)
Dieldrin 378 79 (100) 82 (32) 81 (30) 263 (17) 77 (17) 108 (14)
Dimethirimol methyl ether 223 180 (100) 223 (23) 181 (10) 224 (3) 42 (2) 109 (2)
Dimethoate 229 87 (100) 93 (76) 125 (56) 58 (40) 47 (39) 63 (33)
DNOC methyl ether 212 182 (100) 165 (74) 89 (69) 90 (57) 212 (48) 51 (47)
Dinoterb methyl ether 254 239 (100) 209 (41) 43 (36) 91 (35) 77 (33) 254 (33)
Dioxacarb 223 121 (100) 122 (62) 166 (46) 165 (42) 73 (35) 45 (31)
Diphenamid 239 72 (100) 167 (86) 165 (42) 239 (21) 152 (17) 168 (14)
Disulfoton 274 88 (100) 89 (43) 61 (40) 60 (39) 97 (36) 65 (23)
Diuron 232 72 (100) 44 (34) 73 (25) 42 (20) 232 (19) 187 (13)
Dodine 227 43 (100) 73 (80) 59 (52) 55 (47) 72 (46) 100 (46)
Endosulfan 404 195 (100) 36 (95) 237 (91) 41 (89) 24 (79) 75 (78)
Endrin 378 67 (100) 81 (67) 263 (59) 36 (58) 79 (47) 82 (41)
Ethiofencarb 225 107 (100) 69 (48) 77 (29) 41 (26) 81 (21) 45 (17)
Ethirimol 209 166 (100) 209 (17) 167 (14) 96 (12) 194 (4) 55 (2)
Ethirimol methyl ether 223 180 (100) 223 (23) 85 (14) 181 (12) 55 (10) 96 (9)
List of Mass-Spectrometric Data 27
Table, (contd.)
Molar Main fragments m/z (intensities)

Compound
mass *) 2 3 4 5
Etnmfos 292 125 (100) 292 (91) 181 (90) 47 (84) 153 (84) 56 (73)
Fenarimol 330 139 (100) 107 (95) 111 (40) 219 (39) 141 (33) 251 (31)
Fenitrothion 277 125 (100) 109 (92) 79 (62) 47 (57) 63 (44) 93 (40)
Fenoprop isooctyl ester 380 57 (100) 43 (94) 41 (85) 196 (63) 71 (60) 198 (59)
Fenoprop methyl ester 282 196 (100) 198 (89) 59 (82) 55 (36) 87 (34) 223 (31)
Fenuron 164 72 (100) 164 (27) 119 (24) 91 (22) 42 (14) 44 (11)
Flamprop-isopropyl 363 105 (100) 77 (44) 276 (21) 106 (18) 278 (7) 51 (5)
Flamprop-methyl 335 105 (100) 77 (46) 276 (20) 106 (14) 230 (12) 44 (11)
Formothion 257 93 (100) 125 (89) 126 (68) 42 (49) 47 (48) 87 (40)
Heptachlor 370 100 (100) 272 (81) 274 (42) 237 (33) 102 (33)
Iodofenphos 412 125 (100) 377 (78) 47 (64) 79 (59) 93 (54) 109 (49)
Ioxynil isooctyl ether 483 127 (100) 57 (96) 41 (34) 43 (33) 55 (26) 37 (16)
Ioxynil methyl ether 385 385 (100) 243 (56) 370 (41) 127 (13) 386 (10) 88 (9)
Isoproturon 206 146 (100) 72 (54) 44 (35) 128 (29) 45 (28) 161 (25)
Jasmolin I 330 123 (100) 43 (52) 55 (34) 93 (25) 91 (24) 81 (23)
Jasmolin II 374 107 (100) 91 (69) 135 (69) 93 (67) 55 (66) 121 (58)
Lenacil 234 153 (100) 154 (20) 110 (15) 109 (15) 152 (13) 136 (10)
Lenacil N-methyl derivative 248 167 (100) 166 (45) 168 (12) 165 (12) 124 (9) 123 (6)
Lindane 288 181 (100) 183 (97) 109 (89) 219 (86) 111 (75) 217 (68)
Linuron 248 61 (100) 187 (43) 189 (29) 124 (28) 46 (28) 44 (23)
MCPB isooctyl ester 340 87 (100) 57 (81) 43 (62) 71 (45) 41 (42) 69 (29)
MCPB methyl ester 242 101 (100) 59 (70) 77 (40) 107 (25) 41 (22) 142 (20)
Malathion 330 125 (100) 93 (96) 127 (75) 173 (55) 158 (37) 99 (35)
Mecoprop isooctyl ester 326 43 (100) 57 (94) 169 (77) 41 (70) 142 (69) 55 (52)
Mecoprop methyl ester 228 169 (100) 143 (79) 59 (58) 141 (57) 228 (54) 107 (50)
Metamitron 202 104 (100) 202 (66) 42 (42) 174 (35) 77 (24) 103 (19)
Methabenzthiazuron 221 164 (100) 136 (73) 135 (69) 163 (42) 69 (30) 58 (25)
Methazole 260 44 (100) 161 (44) 124 (36) 187 (31) 159 (24) 163 (23)
Methidathion 302 85 (100) 145 (90) 93 (32) 125 (22) 47 (21) 58 (20)
Methiocarb 225 168 (100) 153 (84) 45 (40) 109 (37) 91 (31) 58 (21)
Methomyl 162 44 (100) 58 (81) 105 (69) 45 (59) 42 (55) 47 (52)
Metobromuron 258 61 (100) 46 (43) 60 (15) 91 (13) 258 (13) 170 (12)
Metoxuron 228 72 (100) 44 (27) 183 (23) 228 (22) 45 (21) 73 (15)
Metribuzin 214 198 (100) 41 (78) 57 (54) 43 (39) 47 (38) 74 (36)
Mevinphos 224 127 (100) 192 (30) 109 (27) 67 (20) 43 (8) 193 (7)
Monocrotophos 223 127 (100) 67 (25) 97 (23) 109 (14) 58 (14) 192 (13)
Monolinuron 214 61 (100) 126 (63) 153 (42) 214 (34) 46 (29) 125 (25)
Napropamide 271 72 (100) 100 (81) 128 (62) 44 (55) 115 (41) 127 (36)
Nicotine 162 84 (100) 133 (21) 42 (18) 162 (17) 161 (15) 105 (9)
Nitrofen 283 283 (100) 285 (67) 202 (55) 50 (55) 139 (37) 63 (37)
Nuarimol 314 107 (100) 235 (91) 203 (85) 139 (60) 123 (46) 95 (35)
Omethoate 213 110 (100) 156 (83) 79 (39) 109 (32) 58 (30) 47 (21)
Oxadiazon 344 43 (100) 175 (92) 57 (84) 177 (60) 42 (35) 258 (22)
Parathion 291 97 (100) 109 (90) 291 (57) 139 (47) 125 (41) 137 (39)
Parathion-methyl 263 109 (100) 125 (80) 263 (56) 79 (26) 63 (18) 93 (18)
Pendimethalin 281 252 (100) 43 (53) 57 (43) 41 (41) 281 (37) 253 (34)
Permethrin 390 183 (100) 163 (100) 165 (25) 44 (15) 184 (15) 91 (13)
Phenmedipham 300 133 (100) 104 (52) 132 (34) 91 (34) 165 (3D 44 (27)
28 List of Mass-Spectrometric Data
Table, (contd.)
, Molar Main fragments m/z (intensities)

P
mass*) 1 2 3 4 5
Phosalone 367 182 (100) 121 (48) 97 (36) 184 (32) 154 (24) 111 (24)
Pirimicarb 238 72 (100) 166 (85) 42 (63) 44 (44) 43 (24) 238 (23)
Pirimiphos-ethyl 333 168 (100) 318 (94) 152 (88) 304 (79) 180 (73) 42 (71)
Pirimiphos-methyl 305 290 (100) 276 (93) 125 (69) 305 (53) 233 (44) 42 (41)
Propachlor 211 120 (100) 77 (66) 93 (36) 43 (35) 51 (30) 41 (27)
Propanil 217 161 (100) 163 (70) 57 (64) 217 (16) 165 (11) 219 (9)
Propham 179 43 (100) 93 (88) 41 (42) 120 (24) 65 (24) 137 (23)
Propoxur 209 110 (100) 152 (47) 43 (28) 58 (27) 41 (21) 111 (20)
Pyrethrin I 328 123 (100) 43 (62) 91 (58) 81 (47) 105 (45) 55 (43)
Pyrethrin II 372 91 (100) 133 (70) 161 (55) 117 (48) 107 (47) 160 (43)
Quintozene 293 142 (100) 237 (96) 44 (75) 214 (67) 107 (62) 212 (61)
Resmethrin 338 123 (100) 171 (67) 128 (52) 143 (49) 81 (38) 91 (28)
Simazine 201 201 (100) 44 (96) 186 (72) 68 (63) 173 (57) 96 (40)
Tecnazene 259 203 (100) 201 (69) 108 (69) 215 (60) 44 (57) 213 (51)
Terbacil 216 160 (100) 161 (99) 117 (69) 42 (45) 41 (41) 162 (37)
Terbacil N-methyl derivative 230 56 (100) 174 (79) 175 (31) 57 (24) 176 (23) 41 (20)
Tetrachlorvinphos 364 109 (100) 329 (48) 331 (42) 79 (20) 333 (14) 93 (9)
Tetrasul 322 252 (100) 254 (67) 324 (51) 108 (49) 75 (40) 322 (40)
Thiabendazole 201 201 (100) 174 (72) 63 (12) 202 (11) 64 (11) 65 (9)
Thiofanox 218 57 (100) 42 (75) 68 (39) 61 (38) 55 (34) 47 (33)
Thiometon 246 88 (100) 60 (63) 125 (56) 61 (52) 47 (49) 93 (47)
Thiophanate-methyl 342 44 (100) 73 (97) 159 (89) 191 (80) 86 (72) 150 (71)
Thiram 240 88 (100) 42 (25) 44 (20) 208 (18) 73 (15) 45 (10)
Tri-allate 303 43 (100) 86 (73) 41 (43) 42 (31) 70 (23) 44 (21)
Trichlorfon 256 109 (100) 79 (34) 47 (26) 44 (20) 185 (17) 80 (8)
Tridemorph 297 128 (100) 43 (26) 42 (18) 44 (13) 129 (11) 55 (5)
Trietazine 229 200 (100) 43 (81) 186 (52) 229 (52) 214 (50) 42 (48)
Trifluralin 335 43 (100) 264 (33) 306 (32) 57 (7) 42 (6) 290 (5)
Vamidothion 287 87 (100) 58 (47) 44 (40) 61 (29) 59 (26) 60 (25)
Vinclozolin 285 54 (100) 53 (93) 43 (82) 124 (65) 212 (63) 187 (61)
*) Molecular ions; chloro and bromo compounds with Cl = 35 and Br = 79.

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).
Elution Elution
Compound volume range Compound volume range
ml ml
Acephate 115-145 Bitertanol 100-130

Alachlor 125-150 Bromacil 105-140
Aldicarb 115-140 Bromophos 120-150
Aldicarb sulphone 110-135 Bromopho s-ethyl 110-140
Aldrin 120-150 Bromopropylate 95-135
Allidochlor 125-160 Bromoxynild) 120-150
Ametryn 115-190 Bromoxynil octanoate 120-150
Amidithion 115-145 Brompyrazon 140-170
Amitraz 125-155 Camphechlor (Toxaphene) 110-150
Anilazine 105-135 Captafol 120-150
Anthraquinone 145-185 Captan 120-150
Atraton 115-140 Carbaryl 125-170
Atrazine 110-135 Carbofuran 125-155
Azinphos-ethyl 130-160 Carbophenothion 120-140
Azinphos-methyl 145-180 Carbophenothion-methyl 120-160
Aziprotryne 120-150 Carbophenothion oxon 130-155
Barban 105-140 Chinalphos see Quinalphos
Bendiocarb 130-160 Chinomethionat
Benfluralin 100-130 (Quinomethionate) 170-200
Benodanil 135-160 Chloranil 140-160
Bensulide 115-135 Chlorazine 115-140
Benzoylprop-ethyl 125-150 Chlorbenside 120-155
Bifenox 115-150 Chlorbenside sulphone 130-160
Binapacryl 100-130 Chlorbromuron 125-150
Biphenyl 155-185 Chlorbufam 115-145
Bis(4-chlorophenyl)methanol a-Chlordane 110-140
(DBH) 125-155 y-Chlordane 100-130
32 Cleanup Method 6 (updated)
Table 1. (contd.)
Elution Elution
ml ml
Chlordecone 110-140 Demeton-S-methyl sulphone 120-160

Chlorfenethol 115-145 Demeton-S sulphone 115-140
Chlorfenprop-methyl 125-150 Demeton-S sulphoxide 140-170
Chlorfenson 120-150 N-Desethyl-pirimiphos-methyl 120-155
Chlorfenvinphos 110-140 Desmethyl-norflurazon 105-130
Chloridazon (Pyrazon) 130-155 Desmetryn 120-175
Chlormephos 115-145 Dialifos 110-140
Chlorobenzilate 100-135 Di-allate 120-150
2-Chloro-4-nitroaniline 105-140 Diazinon 105-135
Chloroneb 145-170 Dichlobenil 125-155
4-Chlorophenoxyacetic acidb) 100-130 Dichlofenthion 110-140
Chloropropylate 100-135 Dichlofluanid 100-140
Chlorothalonil 125-165 Dichlone 155-180
Chlorotoluron 115-150 2,6-Dichlorobenzamide 110-150
Chloroxuron 130-155 p, p'-Dichlorobenzophenone 125-155
Chlorpropham 110-135 2,4-Dichlor ophenoxy-phenoxy-
Chlorpyrifos 110-140 propionic acidb) 100-130
Chlorpyrifos-methyl 120-150 Dichlorprop (2,4-DP)b) 95-130
Chlorthal-dimethyl 135-160 Dichlorvos 115-140
Chlorthiophos 115-155 Diclofop-methyl 135-165
Coumaphos 135-165 Dicloran 105-145
Coumithoate 105-135 Dicofol 100-150
Crotoxyphos 105-145 Dicrotophos 130-160
Crufomate 100-140 Dieldrin 120-150
Cyanazine 110-135 Dienochlor 130-160
Cyanofenphos 115-145 Dimefox 120-155
Cyanophos 115-150 Dimethachlor 135-165
Cycluron 140-160 Dimethoate 120-150
Cymoxanil 110-130 Dimethoxy-anilazine 140-170
Cypermethrin 100-135 Dimethylaminosulphanilide
2,4-Db) 100-130 (DMSA) 125-150
2,4-D methyl ester 135-165 Dimethylaminosulphotoluidide
b
2,4-DB > 100-130 (DMST) 120-145
p,p'-DDA 120-160 Dinitraminea) 105-130
o,p'-DDD 110-140 Dinobuton 110-140
p,p'-DDD 100-140 Dinocap 100-120
o,p'-DDE 120-150 Dinoseb acetate 100-140
p,p'-DDE 120-150 Dioxacarb 140-170
o,p'-DDT 120-150 Dioxathion 110-140
p,p'-DDT 110-140 Diphenamid 145-175
Decachlorobiphenyl 130-165 Diphenylamine 130-160
Deltamethrin 100-135 Dipropetryn 105-130
Demephion (mixture of deme- Dipropyl isocinchomeronate 130-160
phion-O and demephion-S) 125-165 Disulfoton 115-150
Demeton (mixture of Disulfoton sulphone 110-140
demeton-O and demeton-S) 130-155 Disulfoton sulphoxide 120-150
Demeton-S-methyl 125-155 Ditalimfos 120-150
Cleanup Method 6 (updated) 33
Table 1. (contd.)
Elution Elution
ml ml
Dithianon 140-175 Fuberidazolea) 120-160

Edifenphos 130-160 Genite 135-165
a-Endosulfan 110-150 a-HCH 120-150
3-Endosulfan 110-150 (3-HCH 100-130
Endosulfan sulphate 100-140 6-HCH 100-130
Endrin 130-160 8-HCH 105-135
EPN 135-160 Heptachlor 110-140
Ethidimuron 120-150 cis-Heptachlor epoxide 125-155
Ethion 100-140 trans-Heptachlor epoxide 125-155
Ethoprophos 120-155 Heptenophos 120-150
Ethoxyquin 125-150 Hexachlorobenzene 140-165
Etridiazole 140-160 Hexazinone 155-190
Etrimfos 105-140 Imazalila> 120-150
Famophos 125-155 Iodofenphos 120-150
Fenamiphos 105-140 Ioxynild> 120-150
Fenarimol 125-150 Ioxynil octanoate 125-155
Fenazaflor 115-140 Ipazine 105-135
Fenchlorphos 120-150 Iprodione 115-145
Fenitrothion 120-150 Isobenzan 105-140
Fenoprop (2,4,5-TP)b> 95-130 Isocarbamid 130-165
Fenson 130-160 Isodrin 120-150
Fensulfothion 120-150 Isofenphos 95-125
Fensulfothion sulphone 125-155 Isomethiozin 125-150
Fensulfothion sulphone, Isopropalin 110-135
oxygen analogue 125-155 8-Keto-endrin 135-165
Fensulfothion sulphoxide, Lenacil 130-160
oxygen analogue 135-165 Leptophos 120-150
Fenthion 130-160 Leptophos, desbromo
Fenthion sulphone 145-175 derivative 120-150
Fenthion sulphone, oxygen Lindane 110-140
analogue 140-170 Linuron 120-140
Fenthion sulphoxide 155-185 Malaoxon 110-140
Fenthion sulphoxide, oxygen Malathion 110-140
analogue 150-180 MCPAb> 100-130
Fenvalerate 105-135 MCPA-(2-butoxyethyl) 115-145
Fluazifop-butyl 105-130 MCPA pentafluorobenzyl ester 100-125
Flubenzimine 85-120 MCPBb> 100-130
Fluchloralin 100-120 Mecarbam 105-145
Fluotrimazole 100-140 Mecopropb) 95-130
Fluroxypyrc) 95-125 Mephosfolan 140-170
Fluroxypyr n-butyl ester 110-130 Merphos 125-145
Fluroxypyr-( 1 -methylheptyl) 90-120 Metalaxyl 115-150
Fluvalinate 95-120 Metamitron 140-170
Folpet 140-180 Methabenzthiazuron 150-180
Fonofos 120-150 Methacrifos 125-165
Fonofos oxon 145-175 Methamidophos 120-150
Formothion 120-150 Methidathion 130-165
Table 1. (contd.)
Elution Elution
ml ml
Methoprotryne 115-140 Phorate oxon 130-165

Methoxychlor 125-155 Phorate sulphoxide 125-155
Metobromuron 125-150 Phosalone 110-140
Metolachlor 130-160 Phosfolan 150-180
Metribuzin 125-150 Phosmet 145-180
Mevinphos 120-150 Phosphamidon 110-145
Mirex 130-160 Phoxim 120-150
Molinate 150-175 Piperonyl butoxide 100-130
Monocrotophos 115-140 Pirimicarb 130-170
Monolinuron 125-150 Pirimiphos-ethyl 100-135
Morphothion 130-170 Pirimiphos-methyl 105-145
Naled 115-155 Plifenate 125-155
2-Naphthoxyacetic acidb) 110-140 Procymidone 120-150
Napropamide 135-165 Profenofos 130-155
Neburon 110-140 Profluralin 100-125
Nicotine 145-195 Prometon 105-200
Nitralin 115-145 Prometryn 110-140
Nitrapyrin 135-160 Propachlor 125-150
Nitrofen 135-165 Propanil 105-130
4-Nitrophenold) 115-140 Propargite 105-135
Nitrothal-isopropyl 105-135 Propazine 95-125
Norazin 110-140 Propetamphos 110-135
Norflurazon 125-150 Propoxur 110-130
Octachlorodipropyl ether Propyzamide 95-125
(S 421) 110-130 Prothiofos 105-145
Omethoate 140-160 Pyrazophos 110-140
Oxadiazon 115-145 Pyrethrinsa) 100-130
Oxamyl 140-165 Quinalphos (Chinalphos) 115-155
Oxychlordane (Octachlor Quintozene 135-165
epoxide) 100-160 Rabenzazolea) 120-160
Oxydemeton-methyl 135-165 Resmethrin 100-130
Paraoxon 110-140 Salithion 125-165
Paraoxon-methyl 140-170 Secbumeton 115-140
Parathion 110-140 Simazine 95-135
Parathion-methyl 120-150 Simeton 125-190
Pencycuron methyl derivative 130-160 Simetryn 120-150
Pendimethalin 125-155 Strobane T 125-160
Pentachloroaniline 110-140 Sulfallate 145-175
Pentachloroanisole 125-160 Sulfotep 100-130
Pentachlorobenzene 125-165 Sulphur 215-245
Pentachlorophenold) 105-140 Sulprofos 115-155
Permethrin 115-145 2,4,5-Tb> 100-130
Perthane 110-140 2,4,5-T amyl ester 110-140
Phenkapton 115-145 2,4,5-T-butyl 100-130
Phenmedipham 105-130 2,4,5-T hexyl ester 115-145
Phenthoate 115-150 2,4,5-T-(iso-octyl) 105-135
Phorate 115-145 Tecnazene 130-160
Cleanup Method 6 (updated) 35
Table 1. (contd.)
Elution Elution
ml ml
Terbacil 120-145 Thionazin 120-150

Terbufos 125-155 Tolylfluanid 105-135
Terbuthylazine 105-130 Triadimefon 100-130
Terbutryn 115-175 Triadimenol 100-130
2,3,4,6-Tetrachloroanisole 130-160 Tri-allate 120-150
2,3,4,5-Tetrachloronitrobenzene 130-160 Triamiphos 125-160
Tetrachlorvinphos 120-140 Triazophos 120-140
Tetradifon 120-150 Trichlorfon 100-140
Tetramethrin 120-150 Trichloronat 110-140
O,O,O',O'-Tetrapropyl Trietazine 115-150
dithiopyrophosphate 105-135 Trifluralin 100-130
Tetrasul 125-155 Vamidothion 135-165
Thiabendazole 130-160 Vamidothion sulphone 125-155
Thiofanox sulphone 120-150 Vamidothion sulphoxide 150-180
Thiometon 145-185 Vinclozolin 100-130
a)
with complete exclusion of light; b) gel-chromatographed as acid, determined as methyl ester; c) gel-
chromatographed 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).
Elution Elution
ml ml
Ethylvanillin 115-150 Clophen A 50 120-165

Aroclor 1016 140-165 Clophen A 60 120-160
Aroclor 1221 140-170 Clophen T 64 135-160
Aroclor 1232 120-170 Coumarin 135-170
Aroclor 1242 120-170 Dibutyl phthalate 120-150
Aroclor 1248 120-170 1,2-Dichlorobenzene 140-165
Aroclor 1268 120-165 2,3-Dichlorophenold) 120-145
3,4-Benzpyrene 2,4-Dichlorophenold) 120-145
(Benzo[a]pyrene) 195-240 2,5 -Dichlorophenold) 120-140
Butylhydroxyanisole 110-140 2,6-Dichlorophenold) 120-145
Butylhydroxytoluene 105-135 3,4-Dichlorophenold) 120-140
Chloroparaffins (C{2 C{8) 105-130 3,5-Dichlorophenold) 120-145
2-Chlorophenold) 120-140 Dihydrocoumarin 135-170
3-Chlorophenold) 115-145 Halowax 1000 140-180
4-Chlorophenold) 120-140 Halowax 1013 140-180
Clophen A 30 120-165 Halowax 1051 140-200
Clophen A 40 120-165 Hexabromobiphenyl 160-190
Table 2. (contd.)
Elution Elution
ml ml
d)
Hostatox (chlorinated indene) 110-150 2,3,4,6-Tetrachlorophenol 120-145
6-Methylcoumarin 140-160 2,3,5,6-Tetrachlorophenold) 105-140
Octachlorostyrene 140-160 1,2,3-Trichlor obenzene 125-155
Polychlorinated terphenyl 1,2,4-Trichlorobenzene 125-150
(5O7o Cl) 110-140 1,3,5-Trichlorobenzene 125-150
1,2,3,4-Tetrachlorodibenzo- 2,3,4-Trichlorophenold) 120-140
dioxin (1,2,3,4-TCDD) 145-175 2,3,5-Trichlorophenold) 115-145
1,2,3,4-Tetrachlorobenzene 125-165 2,3,6-Trichlorophenold) 115-145
1,2,3,5 -Tetrachlor obenzene 140-165 2,4,5-Trichlorophenold) 100-130
1,2,4,5-Tetrachlorobenzene 125-165 2,4,6-Trichlor ophenold) 115-145
2,3,4,5 -Tetr achlorophenold) 105-140 3,4,5-Trichlorophenold) 115-135
d)
gel-chromatographed as phenol, determinated as acetyl derivative.
Table 3. Pesticides not determinable using gel permeation chromatography.
Chlormequat Metiram
Diquat Morfamquat
Dodine Nabam
Ethirimol Paraquat
Mancozeb Propineb
Maneb Zineb
Metham-sodium Ziram
Methylmetiram
Cleanup Method 7
Solid phase extraction of water samples on alkyl-modified silica gel using disposable
columns
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 dichloro-
methane-methanol mixture. The eluate is evaporated to a definite small volume and used
directly for the final chromatographic determination. Heavily contaminated water samples re-
quire 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 ac-
tivated 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 respec-
tive chapters of the Multiresidue Methods S 8 or S 19 (see pp. 283 ff and pp. 383 ff, respec-
tively, 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 hydroxylgrup-
penhaltigen 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
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 high-
performance 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 char-
coal 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 char-
coal 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

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 com-
pletely 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 col-
umn 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 respec-
tive chapters of the Multiresidue Methods S 8 or S 19 (see pp. 283 ff and pp. 383 ff, respec-
tively, 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 suc-
cessively 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 45
9 Author
Bayer AG, Analytical Laboratories, Dormagen, K. Burger
Part 3
Individual Pesticide Residue
Analytical Methods (contd.)
Amitrole 4-A
Apples, cherries, grapes, must, pears, wine Gas-chromatographic

Soil, water determination
1 Introduction
Chemical name 3-Amino-lH-l,2,4-triazole (IUPAC)

N CNH2
II II
Structural formula
H
Empirical formula C2H4N4
Molar mass 84.1
Melting point 157-159C
Boiling point No data
Vapour pressure 3.3 10"7 mbar at 20 C
Solubility (in 100ml at 20C) Readily soluble in water (30 g);
slightly soluble in isopropanol (2.7 g),
very sparingly soluble in dichloromethane (11 mg),
virtually insoluble in toluene (2 mg) and n-hexane
(<1 mg)
Other properties Stable in acid, neutral and alkaline media
2 Outline of method
Amitrole residues are extracted from plant material with aqueous ethanol. The ethanol is
evaporated from the extract and the aqueous residue, after the addition of acetic acid, is
shaken with dichloromethane. From soil samples, amitrole residues are also extracted with
aqueous ethanol. Pre-cleaned extracts from plant material, extracts from soil, and water
samples are concentrated. Subsequent addition of ethanol and acetic anhydride results in the
conversion of amitrole to its monoacetyl derivative. The derivative is partitioned into
dichloromethane and cleaned up by both gel permeation chromatography and column
chromatography on silica gel. Monoacetyl-amitrole is determined by gas chromatography
using a thermionic detector.
3 Apparatus
High-speed blendor, e.g. Waring Blendor fitted with 2-1 metal jar
Glass funnels, 16 cm and 8 cm dia.
Fluted filter paper
50 Amitrole
Centrifuge
Graduated cylinder, 500-ml
Round-bottomed flasks, 1-1, 500-ml, 250-ml, 100-ml and 50-ml, with ground joints
Rotary vacuum evaporator, 40 C bath temperature
Separatory funnels, 500-ml and 250-ml
Polyethylene bottle, 500-ml, with screw cap
Laboratory mechanical shaker, 150 r.p.m.
Buchner porcelain funnel, with round filter paper
Graduated test tubes, 10-ml, with ground stoppers
Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A
(Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1)
Glass syringe, 10-ml, with Luer-lock fitting
Chromatographic tube, 18 mm i.d., 35 cm long, with extended outlet and PTFE stopcock
Gas chromatograph equipped with thermionic nitrogen-specific detector
Microsyringe, 10-ul
4 Reagents
Cyclohexane, for residue analysis
Dichloromethane, for residue analysis
Ethanol 96%, denatured with ethyl methyl ketone
Ethyl acetate, for residue analysis
Eluting mixture: cyclohexane + ethyl acetate 1:1 v/v
Extraction mixture 1: ethanol + water 2:1 v/v
Extraction mixture 2: ethanol + water 3:2 v/v
Amitrole standard solution: 10 jag/ml ethanol
Derivative standard solution: 1.5 ng/ml monoacetyl-amitrole in ethanol, prepared as follows:
Derivatize 5 \xg amitrole as described in 6.2.2, clean up the acetylated product as described
in 6.3 and 6.4, and dissolve it in 5 ml ethanol
Glacial acetic acid, p.a.
Acetic anhydride, p.a.
Sodium sulphate, p.a., anhydrous
Filter aid, e.g. Celite 545
Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750)
Silica gel, deactivated with 4% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734),
for 5 h at 130 C, allow to cool in a desiccator, and store in a tightly stoppered container in
the desiccator. To 100 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add
4 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with
ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for
2 h on a mechanical shaker, and then store in a tightly stoppered container
Cottonwool, chemically pure
Air, synthetic, re-purified
Helium 4.6 (> 99.996 vol. %)
Hydrogen 5.0 (> 99.999 vol. /o)
Nitrogen 4.6 (> 99.996 vol. %)
Amitrole 51

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, correspond-
ing 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 round-
bottomed 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 extrac-
tion 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 round-
bottomed 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 round-
bottomed flask, ignoring a slight turbidity. Rinse the filter with approx. 40 ml dichloro-
methane. 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 im-
mediately 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 syr-
inge, 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 monoacetyl-
amitrole 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 Varian 3700
Column 1 Fused silica capillary, 0.53 mm i.d., 25 m long; coated
with SE-54, CB 2.0 (Macherey-Nagel)
Column temperature 1 Programmed to rise at 10 C/min from 80 to 280 C,
then isothermal at 280 C for 10 min
Injection port temperature 280 C, splitless
Detector Thermionic nitrogen-specific detector
Temperature 300 C
Gas flow rates Helium carrier, 8 ml/min
Nitrogen purge gas, 30 ml/min
Hydrogen, 4 ml/min
Air, 170 ml/min
Linearity range 1-20 ng
Injection volume 2 nl
Retention time for
monoacetyl-amitrole 4 min 35 s
Alternative conditions
Gas chromatograph Hewlett-Packard 5880 A
Column 2 Capillary column, 0.5 mm i.d., 20 m long; coated
with SE-30, film thickness 2.5 u.m
Column temperature 2 90 C
Column 3 Capillary column, 0.5 mm i.d., 50 m long; coated
with SE-54, film thickness 2.5 \im
Column temperature 3 140 C
Injection port temperature 150 C
Split ratio 1:10
Temperature 225 C
Gas flow rates Helium carrier, 7.5 ml/min
Helium purge gas, 12 ml/min
Hydrogen, 3 ml/min
Air, 45 ml/min
Injection volume 1-2 u.1
Column 2 Column 3
Retention times for
monoacetyl-amitrole 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
0.00 2.00 4.01 6.01 8.02 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 solu-
tions 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 stan-
dard solutions should be injected; additionally, the peaks of the solutions should exhibit com-
parable 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 , . , . , Added Recovery
Analytical material mg/kg %
Apples 0,01 82 (5)
Cherries 0,01 98 (5)
Grapes 0,01 83 (5)
Must 0,01 77 (2)
Pears 0,01 79 (3)
Wine 0,01 76 (2)
Soil
Standard soil 2.1 0,01 82 (3)
Tap water 0,001 *) 78 (2)
0,01 *) 103 (4)
0,1*) 79(1)
Gas-chromatographic measurement with column 1. Numbers in brackets: number of recovery ex-

periments.
*) mg/1.
The soils used for the recovery experiments had the following characteristics:
Organic Particles
Soil type carbon <0.02 mm pH
Standard soil 2.1*) 0.31 8.5 6.0

Standard soil 2.2*) 2.64 14.5 6.0
Standard soil 2.3*) 1.06 24.7 7.0
*) Standard soils as specified by Biologische Bundesanstalt fiir Land- und Forstwirtschaft (BBA), cf.
BBA-Richtlinie IV/4-2 (1987), Braunschweig.
Amitrole 57
Blank values for plant material and soils did not occur or, if so, they were less than
0.001 mg/kg.
The routine limit of determination was 0.01 mg/kg for plant material, must, wine and soil,
and 0.001 mg/1 for water.
7.3 Calculation of residues

The residue R, expressed in mg/kg amitrole, is calculated from the following equation:
FsfVRrVR3-VrG
where
G = sample weight (in g) or volume (in ml)
VEx = total volume of plant material homogenate from 6.1.1 (in ml)
VR1 = portion of filtrate from 6.1.1 used for further clean-up (in ml)
VR2 = volume of solution prepared for gel permeation chromatography in 6.3 (before filtra-
tion) (in ml)
VR3 = portion of volume VR2 injected for gel permeation chromatography (volume of sam-
ple loop) (in ml)
V E n d = terminal volume of sample solution from 6.4 (in ml)
Vj = portion of volume V E n d injected into gas chromatograph (in ul)
W St = a m o u n t of amitrole equivalent injected with derivative standard solution (in ng)
FA = peak area or height obtained from Vj (in m m 2 or integrator counts)
F St = peak area or height obtained from W St (in m m 2 or integrator counts)
8 Important points
The water content and the maximum water capacity of soil samples must be determined
before the soil extraction is performed as described in 6.1.2. To determine the water content,
dry 10 to 20 g of the soil to constant weight in an oven at 105 C (approx. 15 h). To determine
the maximum water capacity, weigh 20 g of the soil into a plastic cylinder (26 mm i. d.) per-
forated with 12 holes at the junction of the side wall with the bottom (bored so that no water
can collect in the bottom of the cylinder). Allow the cylinder to stand in a Petri dish containing
just as much water as can be absorbed through the holes. After approx. 1 h remove the
cylinder, wipe it with filter paper, and let it stand on a pad of filter paper for 10 min to allow
excess water to flow out. Wipe the cylinder again and re-weigh it. Calculate the maximum
water capacity from 5 such determinations, considering the actual water content.
The analysis can only be interrupted after the acetylation of the extracts or the standards.
The final determination should be performed without delay because acetylated amitrole in
solution is stable only for a short time even in a refrigerator.
58 Amltrole
The aqueous phases derived from 6.1.1, 6.1.2 or 6.1.3 must not be evaporated to dryness,
and the water bath temperature must not exceed 40 C.
Gas chromatography with columns 2 or 3 under the conditions given in 6.5 requires at least
30 min between injections to allow time for interfering peaks from the previous chromato-
gram to elute. Gas chromatography with column 1 requires only 20 min between injections
because of the temperature programme. The peak areas of monoacetyl-amitrole on the
capillary columns used are dependent on the sample matrix (compare Chromatograms 2 and
3). Therefore, the derivative standard solution should be added to the cleaned-up extract from
an untreated control sample to yield a reference standard solution suitable for quantitation
according to 7.1.
For preparing monoacetyl-amitrole in larger amounts, 1 g amitrole is acetylated as
described in 6.2, but using 5 times the volumes of the solvents and of acetic anhydride. The
derivative can also be synthesized according to Staab and Seel who give its formula as 3-acetyl-
amino-1,2,4-triazole.
When preparing standard solutions from monoacetyl-amitrole analytical standard material,
consider that its molar mass (126.1) is 1.5 times greater than that of amitrole; accordingly,
1.5 |iig monoacetyl-amitrole is equivalent to 1 |ug amitrole.
9 References
W. Jaggi, Die Bestimmung der CO2-Bildung als Mass der bodenbiologischen Aktivitat,
Schweizerische landwirtschaftliche Forschung 75, 371-380 (1976).
H. J. Jarczyk, Methode zur gaschromatographischen Bestimmung von 3-Amino-l,2,4-triazol-
Riickstanden in Apfeln, Birnen, Kirschen, Weintrauben und Wasser mit N-spezifischem
Detektor, Bayer AG, Report No. RA-988, 16.10.1985 (unpublished).
H. 1 Jarczyk and E. Mollhoff, Methode zur gaschromatographischen Bestimmung von 3-
Amino-l,2,4-triazol-Ruckstanden in Pflanzen, Boden und Wasser mit Stickstoff-spezifischem
Detektor, Bayer AG, Report No. RA-785, 7.7.1988 (unpublished).
J. M. van der Poll, M. Vink and J. K. Quirijns, Capillary gas chromatographic determination
of amitrole in water with alkali flame ionization detection, Chromatographia 25, 511-514
(1988).
H.A. Staab and G. Seel, Uber N-Acyl-Derivate des 3-Amino-l,2,4-triazols, Chem. Ber. 92,
1302-1306 (1959).
10 Authors
Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Informa-
tion and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, H. J.
Jarczyk and E. Mollhoff
Anilazine 186
Artichokes, aubergines, barley (green matter, grains and Gas-chromatographic
straw), beans (green), clover, coffee (raw), garlic, hop determination
cones, onions, potatoes, radicchio, rape (green matter),
spinach, sweet peppers, tomatoes, turnips (foliage and
edible root), wheat (green matter, grains and straw)
Soil, water
1 Introduction
2,4-Dichloro-6-(2-chloroanilino)-l,3,5-triazine (IUPAC)
Chemical name
H
Structural formula
Cl
Empirical formula C9H5C13N4

Molar mass 275.53
Melting point 159 C
Vapour pressure 9 10-8 mbar at 20 C (extrapolated)
Solubility Virtually insoluble in water;
(in 100 ml at 20 C) soluble in dichloromethane (5-10 g),
slightly soluble in toluene (2-5 g),
sparingly soluble in n-hexane (0.1-0.2 g) and
isopropanol (0.5-1 g)
Other properties Colourless crystals;
stability to hydrolysis (half lives at 22 C):
730 h at pH 4, 790 h at pH 7, 22 h at pH 9
2 Outline of method
Anilazine residues are extracted with methanolic sodium hydroxide solution, whereby they are
converted to dimethoxy anilazine.
The extract is filtered and the methanol evaporated. After the addition of sodium chloride
solution, the aqueous residue is shaken with a dichloromethane-acetone mixture. The organic
phase is dried on sodium sulphate and evaporated to dryness.
60 Anilazine
Anilazine Dimethoxy anilazine

With plant material, the subsequent cleanup includes chromatography on silica gel,
followed by gel permeation chromatography on a polystyrene gel. The silica gel column
chromatography can be omitted for soil and water samples. In the concentrated eluates,
dimethoxy anilazine is determined by gas chromatography using a thermionic detector.
3 Apparatus
Homogenizer
Wide neck glass bottle, 1-1 or 500-ml, with ground joint
Buchner porcelain funnel, 11 cm dia.
Filter paper, 11 cm dia., fast flow rate (Schleicher & Schull)
Filtration flask, 1-1
Round-bottomed flasks, 1-1, 500-ml, 250-ml and 100-ml, with ground joints
Rotary vacuum evaporator, 40 C and 55 C bath temperature
Separatory funnels, 1-1 and 500-ml, with ground stoppers
Polyethylene bottle, 500-ml, with cap
Laboratory mechanical shaker
Chromatographic tube, 17.5 mm i.d., 30 cm long, with PTFE-stopcock
Test tubes, 10-ml, with ground stoppers
Microsyringe, 10-jul
4 Reagents
Acetone, for residue analysis
Cyclohexane, p. a.
Methanol, for residue analysis
Toluene, p. a.
Dichloromethane + acetone mixture 4:1 v/v
Eluting mixture 1: cyclohexane + ethyl acetate 85 :15 v/v
Eluting mixture 2: cyclohexane + ethyl acetate 7:3 v/v
Anilazine 61
Anilazine standard solutions: 1.0-1000 ng/ml ethyl acetate

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

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. Rotary-
evaporate 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. Pro-
ceed 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.

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 col-
umn, 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 40C bath temperature; then proceed as
described in 6.3.

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.

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 in-
jection. When using a capillary column, inject 1 \i\.
Gas chromatograph Varian 3700 or Varian 6000
Column 1 Glass, 3 mm i.d., 1.8 m long; packed with 1.5% SP-
2250 + 1.95% SP-2401 on Supelcoport, 100-120 mesh
Column 2 Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE-
30 on Chromosorb W-HP, 80-100 mesh
Gas flow rates Nitrogen carrier, approx. 40 ml/min
Hydrogen, 4.5 ml/min
Air, 175 ml/min
Attenuation 1 10"11, plot attenuation 1.0
Recorder 1 mV; chart speed 5 mm/min
Linearity range 0.5-50 ng
Injection volume 5 Hi
Column 1 Column 2
Column temperatures 220 C 210 C
Injection port temperatures 250 C 260 C
Detector temperatures 250 C 35OC
Retention times for
dimethoxy anilazine 3 min 24 s 2 min 42 s
Column 3 Fused silica capillary, 0.53 mm i.d., 15 m long;
coated with OV-17 RTX 50, crossbond, film thick-
ness 0.5 M-m (Restek No. 10537)
Column temperature Isothermal at 100 C for 2 min, programmed to rise
at 20C/min from 100 to 230 C, then isothermal
at 230 C for 10 min
64 Anilazine

Temperature 280 C
Gas flow rates Nitrogen carrier, 14.6 ml/min
Air, 175 ml/min
Attenuation 1 10~n
Injection volume 1 ul
Retention time for
dimethoxy anilazine 8 min 9 s
7 Evaluation
7.1 Method
Quantitation is performed by measuring the peak areas of the sample solutions and compar-
ing 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 ex-
periments.
Analytical material Added (mg/kg) Range (%)

Artichokes 0.02 86-94
0.2 81-85
Aubergines 0.02 89
0.2 84-97
2.0 107-108
20 94-99
Barley
Green matter 0.04 84-93
1.0 87-95
Grains 0.02 96-97
0.2 83-91
Straw 0.04 92-95
0.4 86-87
Beans 0.02 109-111
0.2 87-95
Anilazine 65
Table, (contd.)
Clover 0.04 79-81

Coffee 0.04 92-96
1.04 72-80
Garlic 0.04 84-85
Hop cones 0.4 82-91
4.0 82-87
Onions 0.04 90-91
Potatoes 0.02 85-98
0.1 72-82
0.2 73-81
1.0 75-79
Radicchio 0.02 91-103
0.2 93-96
2.0 114
Rape green matter 0.04 79-86
Spinach 0.02 97
0.2 94-99
2.0 98-103
20 94-99
Sweet peppers 0.02 86
0.2 85-91
2.0 100-104
20 98-100
Tomatoes 0.02 75-76
0.2 81
2.0 76-79
Turnips
Foliage 0.04 64-70
Edible root 0.04 90-91
Wheat
Green matter 0.04 86-95
0.4 84-90
1.0 76-88c>
10 74-88 a>
Grains 0.02 85-91
0.2 78-90
Straw 0.04 77-86a>
0.4 81-87a>
Soil
Standard soil 2.1 0.02 92-101
1.0 91-92
1.0 88-91
"Laacherhof West' 0.02 87-90
1.0 71-72
66 Anilazine
Table, (contd.)
Water 0.005 82-93 b>

0.01 88-94a>
0.05 87-94
0.5 90-94
a) b) c)
Different number of recovery experiments: 4, 5, 6.
Organic carbon Particles <0.02 mm
Soil type % PH
%
Standard soil 2.1 * } 0.31 8.5 6.0
Standard soil 2.2*> 2.64 14.5 6.0
"Laacherhof West" 1.78 20.5 6.1
*) 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.

The residue R, expressed in mg/kg anilazine, is calculated from the following equation:
R
R L033
Fst.VR2.Vi.G
where
V R1 = volume of solution prepared for gel permeation chromatography in 6.3 (in ml)
VR2 = 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)
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 Infor-
mation and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk,
R. Brennecke
Benomyl, Carbendazim,
Thiophanate-methyl 261-378-370
Lettuce, wheat (grains and straw) High-performance

liquid chromato-
graphic determination
1 Introduction
Benomyl
Chemical name Methyl l-(butylcarbamoyl)benzimidazol-2-ylcarbamate
(IUPAC)
CONH(CH2)3CH3
Structural formula
Empirical formula
a>
C14H18N4O3
Molar mass 290.32
Melting point Undergoes decomposition on heating
Boiling point Not distillable
Solubility Virtually insoluble in water
Other properties Decomposed in acid and alkaline media
Carbendazim
Chemical name Methyl benzimidazol-2-ylcarbamate (IUPAC)
N
Structural formula /V-NHCOOCH3
Empirical formula C9H9N3O2

Molar mass 191.19
Melting point 307-312C (with decomposition)
Solubility Virtually insoluble in water at pH 7;
(in 100 ml at 20 C) readily soluble in acetic acid and dimethylformamide;
sparingly soluble in ethanol (30 mg);
70 Benomyl, Carbendazim, Thiophanate-methyl
virtually insoluble in benzene (3.6 mg) and

dichloromethane (6.8 mg)
Other properties 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 C12H14N4O4S2

Molar mass 342.40
Melting point 178 C (with decomposition)
(in 100 ml at 20 C) 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)
Other properties 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 con-
centrated in the presence of dimethylformamide, and any residual thiophanate-methyl is con-
verted 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 col-
umn chromatography on acidic aluminium oxide, and carbendazim is determined by high-
performance 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
Benomyl, Carbendazim, Thiophanate-methyl 71

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, 40-45C, 58-65 C and 80-85C bath temperature
Water bath, 80 C temperature
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
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

The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Let-
tuce 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 beater-
cross mill in the presence of dry ice.
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 mix-
ture 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 (ap-
prox. 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
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.

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 man-
ner 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.
Pump Constant volume pump, model LC 250/1 (Kratos)
Injector Injection valve 70-10 fitted with sample loop 70-11
(Rheodyne)
Column Stainless steel, 4 mm i.d., 12 cm long (Knauer);
packed with LiChrospher Si 100, medium particle size
10 urn (Merck No. 9312)
Mobile phase Ethanol + n-hexane + phosphoric acid 700:300:0.2
v/v/v
Flow rate 1.5 ml/min
Detector UV detector Spectroflow 773 (Kratos)
Wavelength 285 nm
Attenuation Detector range 0.009 AUFS
Retention time for carbendazim 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
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 carbend-
azim from the calibration curve.

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, carbend-
azim and thiophanate-methyl, expressed as carbendazim equivalents.
Compound added (mg/kg)

Analytical material Recovery
Thiophanate- Carbendazim
Carbendazim methyl Benomyl
equiv. *)
Lettuce 0.04 78
0.4
0.06 0.034 78
0.6 0.34 73
0.06 0.04 81
0.6 0.40 85
0.02 0.02 0.02 0.044 77/85/88
0.2 0.2 0.2 0.44 85/88/88
Wheat grains 0.1 82
0.5 86
0.2 0.12 79/82
1.0 0.56 81/82/89/91
0.05 0.05 0.05 0.11 84
0.2 0.2 0.2 0.44 83
Wheat straw 0.2 93
1.0 84
0.4 0.22 79
1.0 0.56 73
0.1 0.1 0.1 0.22 78/90/94
0.5 0.5 0.5 1.11 79/83/86
*) The factors for conversion of thiophanate-methyl and benomyl to carbendazim are 0.558 and 0.659,
respectively.

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
where
G = sample weight (in g)
V Ex = volume of extract solution from 6.1 (in ml)
VR1 = portion of volume V Ex used for further processing (in ml)
V End = terminal volume of sample solution from 6.5 (in ml)
Vj = portion of volume V End injected into high-performance liquid chromatograph
(volume of sample loop) (in \i\)
WA = 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 clean-
up 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 613-A
Apples, barley (green matter, grains and straw), cherries High-performance
(fruit, conserves, juice and press pulp), cucumbers, pears, liquid chromato-
plums (fruit, jam and puree), sugar beet (foliage and graphic determination
edible root), tea (dry leaf, liquor and infused leaf),
tomatoes
Soil, water
1 Introduction
For data on physico-chemical properties of bitertanol, see Method for Bitertanol, Triadi-
mefon, 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 high-
performance liquid chromatography using a fluorescence detector.
3 Apparatus
Homogenizer
Wide neck glass bottles, 1-1 and 500-ml, with ground joints
Filter paper, 11 cm dia., fast flow rate
Round-bottomed flasks, 1-1, 500-ml and 250 ml, with ground joints
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
Acetonitrile, for chromatography
Water, ultrapure
Acetone + water mixture 2:1 v/v
Acetonitrile + water mixture 1:1 v/v
Methanol + water mixture 7:3 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
Disposable extraction columns, 100-ml and 50-ml (Chem Elut CE 20100 and CE 2050;
Analytichem)
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.
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.
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 col-
umn 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 round-
bottomed 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.

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.
Chromatograph Spectra-Physics 8100 fitted with autosampler
Column Stainless steel, 4 mm i.d., 12.5 cm long; packed with
LiChrospher RP 18, particle size 5 |nm (Merck)
Column temperature 40 C
Mobile phase Acetonitrile + water 55:45 v/v
Column inlet pressure Approx. 60 bar
Flow rate 1 ml/min
Detector Fluorescence detector RF 530 (Shimadzu)
Wavelengths: excitation 254 nm, emission 322 nm
Attenuation Detector range 2 (for water, range 1)
Sensitivity high
Integrator Integrator Spectra-Physics SP 4270 or Laboratory
Data System LAS on a Hewlett-Packard HP 1000 A
Injection volume 10 ul (for water, 25 \i\)
Retention time for bitertanol 4 min 36 s
Bitertanol 81
0 00 i.25 2.50 3.75 5.00 6.25 7.50 8.75
Fig. 1. Bitertanol in apples (VEnd = 5 ml each).

Chromatogram A: Extract from untreated control sam-
ple, 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 Bitertanol
0 00 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.
with 0.02 mg/kg bitertanol.
^ 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
ing 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.

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.
. . . . . . Added Range
e
Apples 0.02 > 80-110a>
0.2 96-98
2.0 96-99
Barley
Green matter 0.02 105
0.2 96-98
2.0 93-97
Grains 0.02 104-105
0.2 97-101
2.0 86-98
Straw 0.02 90a>
0.2 81-93
2.0 87-91
Cherries
Fruit 0.02 95-99
0.2 95-97
2.0 93-97
20 98-102
Conserves 0.02 98-100 a)
0.2 93-103
1.0 94-96
Juice 0.002b> 100
0.02b> 100
0.2b> 95-101
1.0b> 101-103
Press pulp 0.02 81-87a>
0.2 92-99
1.0 89-101
84 Bitertanol
Table, (contd.)
, . , . . Added Range
Cucumbers 0.02 93-94

0.2 89-95
2.0 93-95
Pears 0.02 95-97
0.2 97
2.0 97-98
Plums
Fruit 0.02 85-88
0.2 81-93
2.0 82-84
Jam 0.02d> 70-77
0.2d> 75-80
Puree 0.02d> 66-71
0.2d> 65-78
Tea
Liquor 0.002b> 100
0.02 b> 100-105
Dry leaf 0.02 75-80a>
0.2 83-86
2.0 84
10 79-86
Infused leaf 2.0 99-100
Tomatoes 0.02 101-104
0.2 98
2.0 100-102
Sugar beet
Foliage 0.02 96
0.2 93-97
2.0 99-100
Edible root 0.02 97-99
0.2 92-98
2.0 96-101
Soil
0.1 86-88
1.0 90-95
0.1 94-98
1.0 93-94
Pisserfeld soil 0.01 91-94
0.1 92-96
1.0 81-90
Laacherhof soil 0.01 89-90
0.1 83-89
1.0 94-98
Bitertanol 85
Table, (contd.)
. . . . ^ . . Added Range
Water
Tap water 0.1 c> 91-99
Process water 0.1 c> 91-97
1.0c> 85-88
10 c> 99-100
Leaching water 0.1c'e> 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.
_ ., Organic carbon Particles < 0.02 mm

Soil type % % pH
Standard soil 2.1*> 0.79 7.4 5.7

Standard soil 2.2* > 2.06 11.0 6.0
"Pisserfeld" 1.65 55.0 6.7
"Laacherhof, Miete A " 1.45 20.2 5.8
*) Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf.
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.

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
VEx = volume of concentrated extract after dilution with water (in ml)
VRI = portion of volume VEx used for further cleanup (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 Fluoreszenz-
Detektion, Pflanzenschutz-Nachr. 41, 113-135 (1988).
M. C. S. Mendes, A gas chromatographic method for the determination of residues of bitert-
anol, 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-Kieselgel-
Saulenchromatographie. 2. Mitt.: Bestimmung der Fungizide Bitertanol, Fluotrimazol,
Fuberidazol, Imazalil, Rabenzazole, Triadimefon und Triadimenol in Pflanzen und Boden,
Pflanzenschutz-Nachr. 33, 61-85 (1980).
10 Author
tion and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk,
R. Brennecke
Bitertanol, Triadimefon, Triadimenol 613-425-605
Apples, bananas, barley (green matter, grains and straw), Gas-chromatographic
cucumbers, fruit juices, melons, peaches, pears, sugar determination
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
Bitertanol
Chemical name a//-rac-l-(Biphenyl-4-yloxy)-3,3-dimethyl-l-(lH-l,2,4-
triazol-l-yl)butan-2-ol (IUPAC)
OH CH33
I I
- O - C H - C H - C C H 33
Structural formula I I
CH
A 3
(l N
N U
Empirical formula C2oH23N302
Molar mass 337.42
Melting point 139.8 C (diastereoisomer A)
146.3 C (diastereoisomer B)
118.0C (eutectic)
Vapour pressure <10~ 5 mbar at 20 C (extrapolated)
(in 100 ml at 20 C) readily soluble in dichloromethane (20-50 g [A] and
[B]);
soluble to slightly soluble in 2-propanol (2-5 g [A]
and [B]);
slightly to sparingly soluble in toluene (1-2 g [A],
0.1-0.2 g[B]);
sparingly soluble in n-hexane (0.2 g [A] and [B])
88 Bltertanol, Triadimefon, Triadimenol
Other properties Colourless crystals. Stability to hydrolysis: No

breakdown after 12 months in acid (pH 3), neutral,
and alkaline (pH 10) media
Triadimefon Triadimenol
Chemical name l-(4-Chlorophenoxy)-3,3- l-(4-Chlorophenoxy)-3,3-
dimethyl-l-(lH-l,2,4-triazol- dimethyl-l-(lH-l,2,4-triazol-
l-yl)butanone (IUPAC) l-yl)butan-2-ol (IUPAC)
OH CH3
I I
Structural formula O - C H - C H - C - C H 33
I I
N CH3
(I N
N U
Empirical formula C14H16C1N3O2 C14H18C1N3O2

Molar mass 293.76 295.77
Melting point 82.3 C 110C (eutectic mixture of the
two diastereoisomers)
Vapour pressure < 1 0 " 5 mbar at 20 C < 1 0 " 5 mbar at 20 C
Solubility Virtually insoluble in water; Very sparingly soluble in water;
readily soluble in readily soluble in dichloro-
dichloromethane, 2-propanol methane and 2-propanol;
and toluene; soluble in toluene;
soluble in n-hexane very sparingly soluble in
n-hexane
Other properties Colourless crystals, resistant Colourless crystals, resistant to
to hydrolysis hydrolysis
2 Outline of method
Bitertanol, triadimefon and triadimenol residues are extracted from plant material of low
water content with an acetone-water mixture, and from other plant material with acetone. The
extracts are saturated with sodium chloride and shaken with dichloromethane. Soil samples
are refluxed in aqueous methanol to extract the residues. The methanol is evaporated, and the
aqueous residue is shaken with dichloromethane. Dichloromethane is used for direct extrac-
tion from water samples.
The dichloromethane phases are evaporated to dryness. With plant material and soil, the
extracts are cleaned up on a silica gel column, followed by gel permeation chromatography
on a Bio-Beads S-X3 column. For water samples, and in the case of triadimefon and
triadimenol residues also for soil samples, the silica gel cleanup step can be omitted. The com-
pounds are determined by gas chromatography using a thermionic detector.
Bitertanol, Triadimefon, Triadimenol 89
3 Apparatus
Homogenizer
Wide neck glass bottle, 1-1, with ground joint
Separatory funnels, 1-1, 500-ml and 250-ml, with ground stoppers
Reflux condenser
Heating mantle for 1-1 round-bottomed flask
Chromatographic tube, 17.5 mm i.d., 30 cm long, extended outlet fitted with PTFE-stopcock
Micro syringe, 10-ul
4 Reagents
Toluene, for residue analysis
Compound standard solutions: 0.2-250 îg/ml bitertanol, 0.1-100 fig/ml triadimefon and
0.2-200 M-g/nil triadimenol in ethyl acetate
Glass wool
Hydrogen 5.0 (> 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 acetone-
water 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. Rotary-
evaporate 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.
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 dichloro-
methane 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 cor-
respondingly 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.

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.

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 deter-
mined 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
determined for triadimefon and triadimenol, and from 120 to 140 ml for bitertanol, 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 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.

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.
Column 2 Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE-
Temperature 35OC
Gas flow rates Hydrogen, 4.5 ml/min
Air, 175 ml/min
Attenuation 10- 11
Linearity range Bitertanol 1-50 ng
Triadimefon 0.5-50 ng
Triadimenol 1-100 ng
Injection volume 5ul
Column 1 Column 2
Conditions for bitertanol:
Column temperature 245 C 255 C
Carrier gas flow rates Nitrogen, 55 ml/min Nitrogen, 30 ml/min
Retention times for
bitertanol 3 min 54 s 2 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
7 Evaluation
7.1 Method
ing them with the peak areas obtained for the compound standard solutions. Equal volumes

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 ,, Bitertanol Triadimefon Triadimenol
Apples
Fruit 0.02-0.05 89-110e> 84-91 94-99
0.4-0.5 80-100c> 92-99
1.0-5.0 102-107 85-86
Juice 0.02-0.05 87-105c> 84-87 99-100
Pulp 0.02-0.05 96-108b> 80-81 92-93
0.05 89-92
Apricots 0.5 87-92
0.05 95
Artichokes 0.5 80-86
Bananas 0.01-0.05 83-89b)

Fruit 0.01-0.05
0.4-0.5 83-89b)
82-90 87-91 91-93
1.0 85-103
0.01-0.05 88-95b> 89-108
Peel 0.01-0.05
0.4-0.5 88-95b>
85-92 95-99 92
1.0 82-85
92-96
Barley 0.04-0.08 82-89
Green matter 0.2-0.4
0.04-0.08 82-89 90-105b> 90-104b>
1.0-4.0 92-93 87-97 87-89
0.04-0.08 87-91 86-87 87-91
Grains 0.4-0.8
0.04-0.08 86-91
87-91 84-91b> 90-93 b>
1.0-2.0 92-93 95
0.04-0.08 95-104 77-83 85-89
Straw 0.4-0.8
0.04-0.08 86-97
95-104 89-99b> 92-104b>
1.0-4.0 90-102 87-95
0.05 85-92 8 5_ 89 b) 93-95b>
Beans 0.5 83-86
Table, (contd.)
Added
Analytical material Bitertanol Triadimefon Triadimenol
mg/kg
Cherries
Fruit 0.05 82-90b>
0.5 89-102b>
Juice 0.05 87-91
0.5 93-97
Cucumbers 0.02-0.05 89-105b> 90-94 84-87
0.1-0.5 85-98b> 88-92
1.0 93-95
Grapes 0.02-0.05 95 107-110
0.4 86-88
1.0 92-100
Hop cones 0.3-0.5 84-88 101-104
3.0-5.0 81-86 85-93
Melons 0.02-0.05 93-99 88-97 93-102
0.4 90-92
1.0 86-96 97-98
Must 0.02-0.05 86-93 85-86
Peaches 0.02-0.05 93-100 86 100-103
0.25-0.5 85-94b> 83
1.0 96-97
Peanuts
Kernels 0.02-0.05 83-92b>
0.5 88-89
Shells 0.05 91-92
0.5 86-90
Pears
Fruit 0.02-0.05 90-104^ 81-91 87-95
0.1-0.5 87-110) 100-101
1.0 85
Juice 0.02-0.05 86-100b> 80-81 89-90
Plums 0.05 93-105
0.5 82-83
Rye
Green matter 0.04-0.08 75_98b) 81-97b>
0.4-0.8 83-89 91-96
1.0-2.0 86-90 94-102
Grains 0.04-0.08 82-103 92-106
1.0-2.0 92-96 94-98
Straw 0.04-0.08 90-103b> 91-101b>
1.0-2.0 78-79 78-86
Sugar beet
Foliage 0.02-0.05 98-104b> 80 90-93
0.4-0.5 84-94 85-86
1.0 86-88 96-98
Edible root 0.02-0.05 91-104b> 88-91 82-87
0.4-0.5 86-88 89-100
1.0 83-93 94-105
Table, (contd.)
Added
Analytical material Bitertanol Triadimefon Triadimenol
mg/kg
Sweet peppers 0.02-0.05 88-91 97-105

0.4 82-87
1.0 95-100
Tomatoes 0.02-0.05 90-97 90-96
0.4 89-94
1.0 100-106
Wheat
Green matter 0.04-0.08 90-110b) 83-98b)
0.4-0.8 85-91 90-99
1.0-2.0 80-87 83-101b>
Grains 0.04-0.08 90-94 93-99
0.4-0.8 89 98-99
Straw 0.04-0.08 84-104a) 93-1093)
0.4-0.8 96-105 103-112
Wine 0.02-0.05 102-109a) 92-1033)
0.4 87-94a)
1.0 87-1033)
Soil 0.04-0.08 83-107d) 96-103) 104-108c)
0.4-0.8 79-101d> 94-108) 94_1 nc)
Water 0.005*) 91-114e> 88-108e) 93-103)
o.oi*) 96-105 97-112 94
0.5*> 104
*) 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.
Organic carbon Particles < 0.02 mm

Soil type p H
/o %
Standard soil 2.1*) 0.31 8.5 6.0

Standard soil 2.2* > 2.64 14.5 6.0
Standard soil 2.3* ) 1.06 24.7 7.0
Alluvial soil 1.47 26.6 7.0
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.

The residue R, expressed in mg/kg, of an identified compound is calculated from the follow-
ing equation:
R _ F A -V R1 -V End -W St
Fs,-VR2-VrG
where
VR1 = volume of solution prepared for gel permeation chromatography in 6.3 (in ml)
ple 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, Pflanzen-
schutz-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).
W. Specht and M. Tillkes, Gaschromatographische Bestimmung von Riickstanden an

Pflanzenbehandlungsmitteln nach Clean-up iiber Gelchromatographie und Mini-Kieselgel-
10 Author
R. Brennecke
Bromoxynil, Ioxynil 264-212
Barley (grains and straw), wheat (grains and green matter) Gas-chromatographic
1 Introduction
Bromoxynil Ioxynil
Chemical name 3,5-Dibromo-4- 3,5-Diiodo-4-hydroxybenzonitrile
hydroxybenzonitrile (IUPAC) (IUPAC)
Structural formula
Br
Empirical formula C7H3Br2NO C7H3I2NO
Molar mass 276.93 370.92
Melting point 194-195C, 212-213 C,
octanoate 45-46C octanoate 59-60 C
Boiling point Not distillable Not distillable
Vapour pressure <10" 5 mbar at 20C <10" 5 mbar at 20 C
Solubility Very sparingly soluble in Virtually insoluble in water;
(in 100 ml at 20 C) water; soluble in acetone (7 g) and
readily soluble in acetone methanol (2 g)
(17 g),
soluble in methanol (9 g)
Other properties Commercial products contain bromoxynil and ioxynil mostly as
octanoates or alkali metal salts
2 Outline of method
After refluxing the analytical sample with methanolic potassium hydroxide solution, brom-
oxynil and ioxynil residues are partitioned into dichloromethane. The extract is cleaned up by
acid-base partitioning, whereupon the compounds are methylated with diazomethane. The
methyl ethers are chromatographed on a Florisil column and are determined by electron cap-
ture gas chromatography.
3 Apparatus
High-speed blendor, e.g. Waring Blendor
100 Bromoxynil, loxynil
Heating mantles, 1-1 and 500-ml

Reflux condenser (Dimroth type)
Glass funnel
Fluted filter paper, 15 cm dia. (Schleicher & Schull)
Separatory funnels, 1-1 and 500-ml
Membrane filter, e.g. SM 11605-025 N, 0.65-u.m (Sartorius), and Millex-HV4 filter unit
(Millipore)
Chromatographic tube, 15 mm i.d., 40 cm long
Methylation apparatus, see Fig. 1, p. 130, Vol. 1
Graduated cylinders, 1-1, 500-ml, 250-ml, 100-ml and 50-ml
Gas chromatograph equipped with electron capture detector
Microsyringe, 10-ul
4 Reagents
Acetone, high purity
Dichloromethane, techn. pure, dist.
Diethyl ether, high purity, dried over calcium chloride
n-Hexane, high purity
Methanol, high purity
Toluene, high purity
2,2,4-Trimethyl pentane (isooctane), p. a.
Eluting mixture 2: n-hexane + toluene 8:2 v/v
Eluting mixture 3: n-hexane + toluene 2:8 v/v
Compound standard solutions: 1.46, 14.6, 146 and 1460 u.g/ml bromoxynil octanoate or 1.34,
13.4, 134 and 1340 |iig/ml ioxynil octanoate (equivalent to 1, 10, 100 and 1000 ng/ml brom-
oxynil or ioxynil) in methanol
Derivative standard solutions: bromoxynil methyl ether or ioxynil methyl ether (equivalent to
0.01 to 0.1 ng/ml bromoxynil or ioxynil) in isooctane.
Reflux 728 [ig bromoxynil octanoate or 670 |ug ioxynil octanoate (equivalent to 500 fig
bromoxynil or ioxynil) for 1 h in 200 ml methanolic potassium hydroxide solution. Allow to
cool and rinse the condenser with 20 ml methanol. Filter through a fluted filter paper and
wash with 20 ml methanol. Add 20 ml water and rotary-evaporate to an aqueous residue.
Transfer the residue into a 250-ml separatory funnel, using 100 ml water to complete the
transfer, acidify with 3 ml sulphuric acid, and extract the compound with dichloromethane
(see 6.2.1). Perform the methylation as described in 6.2.3. Remove the solvent, dissolve the
residue in isooctane, and dilute this solution to the concentrations given above
Bromoxynil, loxynil 101
Glacial acetic acid, p. a.

Sulphuric acid, p.a., cone.
Ethanolic potassium hydroxide solution: Dissolve 7 g KOH p.a. in 10 ml water and make up
to 100 ml with ethanol
Methanolic potassium hydroxide solution: 3 g/100 ml KOH p.a.
Florisil, 60-100 mesh, deactivated with 5% water: Heat a weighed sample of Florisil for at
least 8 h at 130 C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml
Erlenmeyer flask (with ground joint), add 5 ml water dropwise from a burette, with con-
tinuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min
until all lumps have disappeared; next shake for at least 20 min on a mechanical shaker, and
then store in a tightly stoppered container for at least 24 h with occasional swirling
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 hydrox-
ide solution contained in the reaction vessel, and sweep the generated diazomethane into 20 ml
diethyl ether, using a gentle stream of nitrogen, while the receiver containing the ether is
cooled in an ice + sodium chloride freezing mixture
Cottonwool, exhaustively extracted with dichloromethane
Helium
Nitrogen, re-purified

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

Reflux 50 g of finely ground grains or 50 g soil (G) for 1 h with 200 ml methanolic potassium
hydroxide solution. Then proceed as described in 6.1.1.
6.1.3 Water
Reflux 500 ml of the water sample (G) with 200 ml methanolic potassium hydroxide solution
for 1 h. Allow to cool and rinse the condenser with 20 ml methanol. Filter the solution
through a fluted filter paper, wash the filter with 30 ml methanol, and rotary-evaporate the
filtrate to an aqueous residue. Then proceed as described in 6.2.1.
6.2 Cleanup
6.2.1 Acid-base partition
Quantitatively transfer the residue derived from 6.1.1 into a 500-ml separatory funnel, using
100 ml water to complete the transfer. Quantitatively transfer the residue derived from 6.1.3
into a 1-1 separatory funnel. Acidify to pH 1-2 with 3-7 ml of sulphuric acid. Allow to cool
to room temperature, and shake three times with dichloromethane (50, 25, 25 ml) for 2 min
each time. Dry the combined dichloromethane phases on sodium sulphate and filter through
a fluted filter paper covered with sodium sulphate. Wash the filter with 30 ml dichloro-
methane, and rotary-evaporate the filtrate to near dryness. Remove the last traces of solvent
by swirling the flask in the hand.
6.2.2 Gel permeation chromatography (see 8. Important points)

Dissolve the residue derived from 6.2.1 in 10 ml eluting mixture 1 (VEx) and filter the solution
through a membrane filter. Using a 10-ml syringe, load the 5-ml sample loop (VR1) of the gel
permeation chromatograph with 8 to 9 ml of the filtrate. Set the gel permeation chromato-
graph at the eluting conditions determined beforehand with standard solutions of bromoxynil
and ioxynil; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. Elution volumes ranging from 80 to
140 ml were determined for bromoxynil and ioxynil on Bio-Beads S-X3 polystyrene gel, using
eluting mixture 1 as eluant, pumped at a flow rate of 5.0 ml/min.
to near dryness. Remove the last traces of solvent by swirling the flask in the hand.
Check the elution range from time to time, and determine anew whenever a new gel column
is used.
6.2.3 Methylation
Dissolve the residue derived from 6.2.1 or 6.2.2 in 1 ml methanol, add 5 ml of the
diazomethane solution, and allow to stand for 15 min with occasional swirling. Remove excess
diazomethane and solvent with a gentle stream of nitrogen.
6.2.4 Column chromatography

Insert a cottonwool plug into the bottom of the chromatographic tube and add 5 ml hexane.
Fill the chromatographic tube with a slurry of 15 g Florisil in 35 ml hexane, while gently tap-
ping the tube walls. As soon as the Florisil has settled, trickle in 5 g sodium sulphate. Drain
the supernatant to the top of the sodium sulphate layer. Dissolve the residue derived from 6.2.3
in 1 ml eluting mixture 2 and transfer the solution quantitatively onto the column. Rinse the
flask portionwise with a total of 20 ml eluting mixture 2, add the rinsings to the column, and
Bromoxynil, loxynil 103
allow to percolate. Wash the column, the stopcock being opened a little, with a further 80 ml
eluting mixture 2, and discard this eluate. Next elute the derivatives with 100 ml eluting mix-
ture 3. Collect the eluate in a 250-ml round-bottomed flask and rotary-evaporate to near
dryness.

Dissolve the residue derived from 6.2.4 in isooctane and dilute to an appropriate volume
(VEnd), e.g. 10 ml. Inject an aliquot of this solution (Vj) into the gas chromatograph.
6.3.1 Packed column
Gas chromatograph Carlo Erba Fractovap 230
Column Glass, 2 mm i.d., 1.5 m long; packed with 7.5% DC-
200 + 7.5% QF-1 on Gas Chrom Q, 80-100 mesh
63
Detector Ni electron capture detector ECD HT-25,
ECD Control Module 250
Temperature 275 C
Gas flow rates Nitrogen carrier, 25 ml/min
Injection volume 1-3 ul
Retention times for
bromoxynil methyl ether 1 min 24 s
ioxynil methyl ether 3 min 36 s
6.3.2 Capillary column
Gas chromatograph Carlo Erba Fractovap 4160 with on-column injector
Column Fused silica capillary, 0.32 mm i.d., 15 m long; coated
with OV-1, crossbond, film thickness 0.10-0.15 |im
(Carlo Erba Mega)
Column temperature 90 to 170 C at maximum heating rate, programmed to
rise at 2C/min from 170 to 200 C
Injection technique Cold on-column at 90 C oven temperature with
secondary cooling
63
Detector Ni electron capture detector ECD HT-25, ECD Con-
trol Module 251
Temperature 300 C
Retention times for
bromoxynil methyl ether 2 min 20 s
ioxynil methyl ether 4 min
7 Evaluation
7.1 Method
Inject equal volumes of each derivative standard solution (equivalent to 0.01 to 0.1 ng brom-
oxynil or ioxynil, respectively) into the gas chromatograph. Plot the areas or heights of the
peaks obtained vs. ng bromoxynil or ioxynil. Also inject equal volumes of the sample solu-
tions. For the areas or heights of the peaks obtained for these solutions, read the appropriate
amounts of bromoxynil or ioxynil from the corresponding calibration curve.
7.2 Recoveries, limit of detection and limit of determination

The recoveries from untreated control samples, fortified with bromoxynil and ioxynil (as oc-
tanoates) at levels of 0.05 to 10 mg/kg, ranged from 68 to 99% and averaged 85%. The limit
of detection was 0.01 mg/kg, and the limit of determination was 0.05 mg/kg.
The recoveries from tap and pond waters ranged from 71 to 93% and averaged 83%; the
limit of detection was 0.05 jxg/1, and the limit of determination was 1 fxg/1.

The residue R, expressed in mg/kg bromoxynil or ioxynil, is calculated from the following
equation:
r w A -v E x -v E n d
VR,-V,-G
where
VEX = volume of solvent used to dissolve the residue from 6.2.1 (in ml)
VR1 = portion of volume VEx injected for gel permeation chromatography (volume of
WA = amount of bromoxynil or ioxynil, respectively, for Vj read from calibration curve
(in ng)
8 Important points
The gel permeation chromatographic cleanup (6.2.2) is only required when interfering peaks
are observed. In some cases, e.g. for ground and tap waters, the column chromatographic
cleanup (6.2.4) can be omitted.
BromoxynJI, loxynil 105
With straw samples, sometimes more dichloromethane will be required to wash the
precipitate formed during the acidification in 6.2.1. Centrifugation is sometimes required to
obtain separation of the dichloromethane phase during the partitioning steps.
9 Reference
G. Zweig, Analytical methods for pesticides, plant growth regulators and food additives,
Bromoxynil, Vol. V, 347-362, and Vol. VI, 605-610, Academic Press, New York and London,
1967 and 1972.
10 Authors
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H.-G.
Nolting, J. Siebers and M. Blacha-Puller
Carbendazim 378
Cereals (green matter, grains and straw) Gas-chromatographic

Soil determination
1 Introduction
Chemical name Methyl benzimidazol-2-ylcarbamate (IUPAC)
Structural formula o

Molar mass 191.19
Melting point 307-312 C (decomp.)
Vapour pressure 6.5-10- 10 mbar at 20C
(in 100 ml at 20 C) readily soluble in dimethylformamide and glacial
acetic acid;
very sparingly soluble in ethanol (30 mg) and ethyl
acetate (13.5 mg);
virtually insoluble in benzene (3.6 mg),
dichloromethane (6.8 mg) and n-hexane (0.05 mg)
Other properties Stable in weakly acid media; slowly decomposed
under alkaline conditions; soluble in diluted acids,
with salt formation
2 Outline of method
Carbendazim residues are extracted from the sample material by adding sodium hydrogen car-
bonate to the substrate and homogenizing it with ethyl acetate. Neutral and acidic co-
extractives are separated by acid-base partition. An aliquot of the extract is derivatized with
pentafluorobenzyl bromide. The pentafluorobenzyl derivative of carbendazim is cleaned up
by chromatography on a silica gel minicolumn and determined by electron capture gas
chromatography.
3 Apparatus
High-speed blendor fitted with leak-proof glass or stainless steel jar and explosion-proof
motor
Buchner porcelain funnel, 13.5 cm dia.
108 Carbendazim
Filter paper, 12.5 cm dia., fast flow rate (Schleicher & Schtill), extracted with dichloromethane
Separatory funnels, 1-1 and 250-ml, with ground stoppers and FIFE stopcocks
Volumetric flasks, 100-ml, with ground joints
Round-bottomed flask, 100-ml, with ground joint and 9-cm long neck
Drying cabinet, 50 C temperature
Chromatographic tube, 7.5 mm i. d., 23 cm long, with extended outlet
Test tubes, 12 to 15 ml, with ground stoppers and graduation mark at 10.0 ml
Microsyringes, 10-ul and 100-ul
4 Reagents
Prepare all aqueous solutions with distilled water, not with deionized water
n-Hexane, for residue analysis
Water, distilled, not deionized
Eluting mixture: toluene + acetone 8:2 v/v
Derivative standard solution, equivalent to 25 ng/ml carbendazim, in eluting mixture: Pipet
0.5 ml of a solution containing 10 ng/ml carbendazim in ethyl acetate into a 100-ml long neck
flask, rotary-evaporate to dryness, and process as described in steps 6.2 and 6.3. Make up the
column eluate to a volume of 10.0 ml, and dilute 1.0 ml of the solution wih 19.0 ml of eluting
mixture
Sulphuric acid, 0.5 mol/1 H2SO4 p.a.
Potassium carbonate solution, 30 g/100 ml K2CO3 p. a.
Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p. a.
Pentafluorobenzyl bromide, 99 + % (Aldrich No. 10,105-2)
Sodium hydrogen carbonate, p.a.
Sodium sulphate, p.a., heated at 550C for at least 2 h
Glass wool, extracted exhaustively with dichloromethane
Silica gel, deactivated with 1.5% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734),
for at least 5 h at 130 C, allow to cool in a desiccator, and store in a tightly stoppered con-
tainer in the desiccator. To 98.5 g dried silica gel in a 300-ml Erlenmeyer flask (with ground
joint), add 1.5 ml water dropwise from a burette, with continuous swirling. Immediately stop-
per flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared,
next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container.
Before use, the deactivated silica gel must be checked for its separating efficiency by testing
it with the derivative standard solution as described in 6.3
Universal indicator paper (pH 2-10)
Cottonwool, extracted with dichloromethane
Argon + methane mixture 9:1 v/v
Carbendazim 109

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

Pack the chromatographic column successively with a glass wool plug, 1.0 g deactivated silica
gel, a 5 to 10-mm layer of sodium sulphate, and another glass wool plug. Immediately before
use, rinse the column with 5 ml hexane, and discard the eluate. As soon as the hexane has
drained to the top of the silica gel, add the solution derived from 6.2 using a pipet, and allow
110 CarbendazJm
it to trickle in. Pour 2.0 ml toluene into the flask, swirl, and transfer the solution in the same
way to the column. Allow the toluene to drain to the top of the silica gel, rinse the column
with 6.0 ml toluene, and discard the eluate. Then place a graduated test tube under the column,
pour 2.0 ml eluting mixture into the flask, swirl, and transfer the solution quantitatively to
the column using a pipet. As soon as the solution has drained to the top of the silica gel, elute
the column with 6.0 ml eluting mixture, and make up the eluate with the mixture to a volume
of 10.0 ml (VEnd).

Inject an aliquot (Vj) of the solution derived from 6.3 into the gas chromatograph.
Gas chromatograph Hewlett-Packard 5710 G
Column Glass, 4 mm i.d., 1.8 m long; packed with 3% OV-61
+ 7.5 % QF-1 + 3% XE-60 on Chromosorb W-HP,
100-120 mesh
63
Detector Ni electron capture detector
Temperature 300 C
Carrier gas flow rate Argon-methane, 46 ml/min
Attenuation 64
Recorder 1 mV; chart speed 15 inch/h (38.1 cm/h)
Retention time for carbendazim
pentafluorobenzyl derivative 8 min 15 s
7 Evaluation
7.1 Method
tions and comparing them with the peak areas or peak heights obtained for the derivative
standard solution or dilutions thereof. Equal volumes of the sample solutions and the stan-
dard solutions should be injected; additionally, the peaks of the solutions should exhibit com-
parable areas or heights.

The recoveries from untreated control samples, fortified with carbendazim at levels of 0.05 to
0.5 mg/kg, ranged from 65 to 90% and averaged 75% for cereals (green matter, grains and
straw), and ranged from 56 to 64% for soil. The limit of determination was 0.05 mg/kg.
Carbendazlm 111

The residue R, expressed in m g / k g carbendazim, is calculated from the following equation:
R _ FA-VEx-VR2-VEnd-WSt
FSfVR1-VR3-VrG
where
=
VEX volume of solvent used for extraction of analytical sample (in ml)
VRI = portion of volume V E x used for acid-base partition (in ml)
V R2 = volume of extract after acid-base partition in step 6.1 (in ml)
V R3 = portion of volume V R 2 used for derivatization in step 6.2 (in ml)
W St = a m o u n t of carbendazim injected with standard solution (in ng)
FA = peak area or height obtained from V{ (in m m 2 or m m )
Fst = peak area or height obtained from W S t (in m m 2 or m m )
8 Important points
Any residues of benomyl that may be present are completely converted to carbendazim during
the extraction and cleanup steps, and are co-determined. Thiophanate-methyl, however, is
converted to carbendazim to a much lesser degree (less than 10% at a level of 5 mg/kg).
The method is applicable also to the analysis of tap water. Extraction is performed by ad-
ding 10 g sodium hydrogen carbonate to 100 g of the analytical sample, and shaking the mix-
ture with 200 ml ethyl acetate for 3 min. Then a 100-ml aliquot of the organic phase is pro-
cessed as described in 6.1.1. However, considerable variations (60 to 95%) in recovery rates
were observed.
9 Reference
G. H. Tjan and J. T. A. Jansen, Gas-liquid chromatographic determination of thiabendazole
and methyl 2-benzimidazole carbamate in fruits and crops, J. Assoc. Off. Anal. Chem. 62,
769-773 (1979).
112 Carbendazim
10 Author
Institute for Residue Analysis Dr. Specht & Partner, Hamburg, W. Specht
Carbosulfan, Carbofuran 658-344
Grapes, head cabbage, lettuce, maize (kernels), rape Gas-chromatographic
(green matter and seeds), sugar beet (edible root), determination
tomatoes
Soil
1 Introduction
Carbosulfan Carbofuran
Chemical name 2,3-Dihydro-2,2- 2,3-Dihydro-2,2-
dimethylbenzofuran-7-yl dimethylbenzofuran-7-yl
(dibutylaminothio)methyl- methylcarbamate (IUPAC)
carbamate (IUPAC)
O=C-NH-CH 3
CH 2 -CH 2 -CH 2 -CH 3
Structural formula
CH,
Empirical formula C20H32N2O3S C12H15NO3

Molar mass 380.5 221.3
Melting point 96C (flashpoint) 153-154C
Vapour pressure 4.1 10"7 mbar (no temp. 2.6-10"5 mbar at 33 C
data)
Solubility Virtually insoluble in water; Virtually insoluble in water;
(in 100 ml at 20 C) soluble in most organic readily soluble in acetone (15 g),
solvents, e.g. acetone, acetonitrile (14 g) and
dichloromethane, hexane, dichloromethane (12 g);
methanol and xylene soluble in benzene (4 g) and
ethanol (4 g);
sparingly soluble in petroleum
ether and xylene
Other properties Hydrolyzed in aqueous Unstable in alkaline media
media depending on pH,
more stable at higher pH
values. Half lives in water at
25 C:
pH 4: lh; pH 6: 22 h;
pH 7: 7.6 d; pH 9: 58.3 d
3-Hydroxy-carbofuran (main metabolite)
Chemical name 2,3-Dihydro-3-hydroxy-2,2-dimethylbenzofuran-7-yl
methylcarbamate
114 Carbosulfan, Carbofuran
O=C-NH-CH 3
O
Structural formula
OH
Empirical formula C 12 H 15 NO 4
Molar mass 237.1
Melting point No data
Vapour pressure No data
Solubility No data
Other properties No data
2 Outline of method
Carbosulfan and carbofuran residues are extracted from plant material and soil with acetone,
and from rape with a hexane-isopropanol mixture. Water and sodium chloride are added to
an aliquot of the extract, followed by shaking with hexane. Interfering co-extractives are
removed by chromatography on activated charcoal-silica gel and Florisil columns. Car-
bosulfan and carbofuran are determined by gas chromatography using a thermionic detector.
Residues of the main metabolite, 3-hydroxy-carbofuran, are extracted from plant material
with hydrochloric acid after conjugates have been hydrolyzed. The extract is shaken with ethyl
acetate. Interfering co-extractives are removed by chromatography on a silica gel column.
3-Hydroxy-carbofuran is determined by gas chromatography using a mass-selective detector
operated in the SIM mode.
3 Apparatus
High-speed blendor fitted with glass jar
Beaker, 1-1
Filtration flask, 500-ml
Graduated cylinder, 500-ml, with ground joint
Round-bottomed flasks, 500-ml, 250-ml and 100-ml, with ground joints
Erlenmeyer flask, 500-ml
Erlenmeyer flask, 100-ml, with ground joint
Centrifuge, 2800 r. p. m., with 200-ml glass tubes
Reflux condenser, with ground joint
Heating mantle, for 500-ml round-bottomed flask
Carbosulfan, Carbofuran 115
Chromotographic tube 1: 25 mm i.d., 40 cm long, with sintered glass disk and stopcock
Chromatographic tube 2: 10 mm i.d., 40 cm long
Test tubes, graduated, with ground stoppers
Gas chromatograph equipped with mass-selective detector
Micro syringe, 10- \i\
4 Reagents
Acetone, p. a.
Dichloromethane, p. a.
Diisopropyl ether, HPLC quality
Ethanol, p. a.
Ethyl acetate, p. a.
n-Hexane, fractionally distilled
2-Propanol (isopropanol), p. a.
Toluene, p. a.
2,2,4-Trimethyl pentane (isooctane), fractionally distilled
Solvent mixture 1: n-hexane + isopropanol 2:1 v/v
Solvent mixture 2: dichloromethane + acetone + toluene 10:2:2 v/v/v
Solvent mixture 3: n-hexane + ethyl acetate 97:3 v/v
Solvent mixture 6: n-hexane + ethanol 7:3 v/v
Standard solutions: 0.3-3.0 ng/ml each of carbosulfan, carbofuran and 3-hydroxy-carbofuran
in isooctane
Internal standard solutions: 0.3-3.0 ng/ml carbofuran in diisopropyl ether
Hydrochloric acid, 0.25 mol/1 HC1 p. a.
Sodium chloride, p.a., exhaustively extracted with dichloromethane and dried for 8 h at
120 C
Sodium sulphate, p. a., anhydrous, exhaustively extracted with dichloromethane and dried for
8 h at 120 C
until all lumps have disappeared, next shake for at least 20 min on a mechanical shaker, and
Activated charcoal, p. a. (Merck No. 2186)
Glass wool (Merck No. 4086)
Sea sand, p. a. (Merck No. 7712)
Air, synthetic
Helium
Hydrogen, re-purified

The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Rape
seeds are ground in a high-speed blendor.
6 Procedure
6.1 Extraction (carbosulfan, carbofuran)
6.1.1 Plant material (except rape green matter)
Weigh 100 g of the analytical sample (G) into a beaker, add 150 ml acetone, and homogenize
for 3 min. Rinse the homogenizer with 50 ml acetone. Suction-filter the homogenate through
a fast flow-rate filter paper in a Buchner porcelain funnel. Rinse the beaker and wash the filter
cake with 60 ml acetone. Transfer the filtrate to a graduated cylinder and make up the volume
to 300 ml (VEx) with acetone. Mix thoroughly, and pipet off one fifth (60 ml, VR1) of the
filtrate into a separatory funnel. Add 10 g sodium chloride, 50 ml water and 100 ml hexane,
and shake for 2 min. Separate the layers, and extract the aqueous phase twice more with
100-ml portions of hexane. Dry the combined organic extracts for 30 min in an Erlenmeyer
flask on sodium sulphate; then filter the solution through a fluted filter paper into a 500-ml
round-bottomed flask. Wash the Erlenmeyer flask and the filter with 50 ml hexane. Rotary-
evaporate the combined filtrates to 1-2 ml and remove the last traces of solvent with a gentle
stream of nitrogen.
6.1.2 Rape (green matter)

Weigh 20 g of the analytical sample (G) into a centrifuge tube, add 100 ml solvent mixture
1 and homogenize for 3 min. Rinse the homogenizer with 40 ml solvent mixture 1. Centrifuge
the suspension for 15 min at 2800 r.p.m. Decant the supernatant liquid through a glass wool
plug into a graduated cylinder. Repeat the extraction. Combine the extracts and make up the
organic phase to a total volume of 240 ml (VEx) with solvent mixture 1. After carefully mix-
ing, pipet off a quarter (60 ml, VR1) of the organic phase into a separatory funnel, and pro-
ceed as described in 6.1.1.
6.1.3 Soil
Weigh 50 g of the analytical sample (G) into a 100-ml Erlenmeyer flask, add 25 ml water, stop-
per, and shake the mixture thoroughly for 5 min to distribute the water evenly. Add 50 ml
acetone, and shake for 30 min on the mechanical shaker. Decant the supernatant liquid
through a fast flow-rate filter paper covered with a layer of filter aid in a Buchner porcelain
funnel. Repeat the extraction with a further 50 ml of acetone; then suction-filter the
homogenate and wash the flask and the filter cake with a total of 70 ml acetone. Transfer the
combined filtrates to a graduated cylinder and make up the volume to 200 ml (VEx) with
acetone. Mix thoroughly, and pipet off a quarter (50 ml, VR1) of the filtrate into a separatory
funnel. Proceed as described in 6.1.1.
6.2 Extraction (3-hydroxy-carbofuran)

6.2.1 Rape (green matter), tomatoes
Weigh 50 g of the analytical sample (G) into a 500-ml round-bottomed flask, add 200 ml
hydrochloric acid, and heat to reflux for 1 h. Allow the mixture to cool, and suction-filter
through a Buchner porcelain funnel containing a layer of glass wool covered with sea sand.
Rinse the flask and the filter cake with 50 ml hydrochloric acid. Transfer the combined filtrates
to a graduated cylinder and make up the volume to 300 ml (VEx) with water. Mix well, then
transfer a quarter of the solution (75 ml, VR1) into a separatory funnel. Extract the aqueous
phase three times with 90-ml portions of ethyl acetate. Dry the combined organic phases for
30 min on sodium sulphate in an Erlenmeyer flask. Filter the solution through a fluted filter
paper into a 500-ml round-bottomed flask and rinse the Erlenmeyer flask and the filter with
50 ml ethyl acetate. Rotary-evaporate the combined filtrates to 1-2 ml and remove the last
traces of solvent with a gentle stream of nitrogen.
6.2.2 Rape (seeds)

Weigh 20 g of the ground analytical sample (G) into a 250-ml round-bottomed flask, add
150 ml hydrochloric acid, and heat to reflux for 1 h. Allow the mixture to cool, and suction-
filter through a Buchner porcelain funnel containing a layer of glass wool covered with sea
sand. Rinse the flask and the filter cake with 50 ml hydrochloric acid. Transfer the combined
filtrates to a graduated cylinder and make up the volume to 200 ml (VEx) with hydrochloric
acid. Mix well, then transfer a quarter of the solution (50 ml, VR1) into a separatory funnel,
and continue as described in 6.2.1.
6.3 Cleanup (carbosulfan, carbofuran)

6.3.1 Activated charcoal-silica gel column (see Method S 8, pp. 283 ff, Vol. 1)
Fill a chromatographic tube (type 1) with dichloromethane to a level of 1 cm. Slurry 5 g silica
gel in 15 ml solvent mixture 2, and pour the slurry into the tube. Drain off the supernatant.
Next, thoroughly mix 15 g silica gel and 1 g activated charcoal in a 50-ml beaker, and slowly
add, with stirring, 35 ml solvent mixture 2 (Caution! Generation of heat!). Do not add more
than 35 ml solvent mixture otherwise the suspension will become separated into phases,
resulting in poor passage of material through the column.
Add the activated charcoal-silica gel mixture onto the silica gel in the chromatographic col-
umn, by pouring it through a funnel, at first slowly and then in a gush, at the same time stir-
ring constantly and with the column stopcock open. Use any eluate that has already passed
through column for rinsing the flask. Drain the solvent mixture to a level of 2 cm above the
packing, and top the column with a total of 5 g sodium sulphate added in small portions. Next
pre-wash the column with 50 ml solvent mixture 2.
Quantitatively transfer the residue derived from 6.1 to the column, using a total of 15 ml
dichloromethane to complete the transfer. Collect liquid already flowing through the column
and subsequent eluate in a 250-ml round-bottomed flask. Elute carbosulfan and carbofuran
with 140 ml solvent mixture 2. Rotary-evaporate the eluate to approx. 2 ml, and remove the
last traces of solvent with a gentle stream of nitrogen. Immediately dissolve the residue in 5
ml hexane.
6.3.2 Florisil column

Slurry 10 g Florisil in 30 ml hexane, and pour the slurry into a chromatographic tube (type
2). Allow to settle, then top the Florisil with 5 g sodium sulphate. Pre-wash the column pack-
ing with 50 ml hexane. Quantitatively transfer the residue derived from 6.3.1 onto the column,
using a total of 30 ml hexane to complete the transfer. Wash the column with 55 ml solvent
mixture 3, and discard all eluates obtained up to this point.
Next, elute carbosulfan and carbofuran with 210 ml solvent mixture 4, collecting the eluate
in a 250-ml round-bottomed flask. Rotary-evaporate the eluate to near dryness, add 5 ml acetone
to the residue, and rotary-evaporate to near dryness again. Transfer the residue to a test tube with
a total of 5 ml acetone, and evaporate the solution to dryness in a nitrogen stream.
If required, carbosulfan and carbofuran can be separated by collecting carbosulfan in the
first 90 ml of the eluate in a separate flask. Carbofuran will appear in the following 120 ml
eluate.
6.4 Cleanup (3-hydroxy-carbofuran)

Slurry 10 g silica gel in 30 ml hexane, and pour the slurry into a chromatographic tube (type
2). Allow to settle, then top the silica gel with 5 g sodium sulphate. Pre-wash the column pack-
ing with 50 ml hexane. Quantitatively transfer the residue derived from 6.2 onto the column,
using a total of 10 ml solvent mixture 5 to complete the transfer. Wash the column with 80 ml
solvent mixture 5, followed by 25 ml hexane. Discard all eluates obtained up to this point.
Next, elute 3-hydroxy-carbofuran with 55 ml solvent mixture 6, collecting the eluate in a
100-ml round-bottomed flask. Rotary-evaporate the eluate to near dryness. Transfer the
residue to a test tube with a total of 5 ml diisopropyl ether, and evaporate the solution to near
dryness using a gentle stream of nitrogen.

Dissolve the residue derived from 6.3.2 in isooctane and make up to a definite volume (VEnd),
e.g. 1 ml. Inject an aliquot of this solution (Vj) into the gas chromatograph equipped with
a thermionic detector.
To the residue derived from 6.4, add a definite volume of the internal standard solution,
and make up with diisopropyl ether to a definite volume (VEnd), e.g. 1 ml. Inject an aliquot
of this solution (Vj) into the gas chromatograph equipped with a mass-selective detector.
6.5.1 Thermionic detector

Gas chromatograph Hewlett-Packard 5890, equipped with cold injection
system KAS (Gerstel)
Column Fused silica capillary HP-1 (methyl silicone), 0.32 mm
i.d., 25 m long; film thickness 0.17 urn (Hewlett-
Packard)
Column temperature Programmed to rise at 20C/min from 100 to 160 C,
and at 9C/min from 160 to 240 C, then isothermal
at 240 C for 3 min
Injection technique Splitless for 48 s

Temperature of KAS injection system: Programmed to
rise at 10C/s from 70 to 250 C, then isothermal at
250 C for 1 min
Temperature 300 C
Hydrogen, 5 ml/min
Air, 85 ml/min
Attenuation 21
Integrator Hewlett-Packard 3396 A, chart speed 5 mm/min
Injection volume I nl
Retention times for
carbofuran 5 min 8 s
3 -hydroxy-carbofuran 6 min 27 s
carbosulfan II min 53 s
6.5.2 Mass-selective detector

Gas chromatograph Hewlett-Packard 5890/5970 B, equipped with cold in-
jection system KAS (Gerstel)
i.d., 12 m long; film thickness 0.33 |nm (Hewlett-
Packard)
250 C for 1 min
Detector Mass selective detector HP 5970
El, 70 eV, interface 280 C
Carrier gas flow rate Helium, < 1 ml/min
Printer Paint Jet (Hewlett-Packard)
Injection volume
Retention times for
carbofuran 8 min 13 s
3-hydroxy-carbofuran 9 min 4 s
carbosulfan 11 min 59 s
SIM parameters for 3-hydroxy-
carbofuran Group 1 (internal standard carbofuran):
Start 7 min, stop 8 min 40 s
Dwell time for m/z = 164 and 221:100 ms each
Group 2 (3-hydroxy-carbofuran):
Start 8 min 40 s, stop 9 min 40 s
Dwell time for m/z = 137 and 180:100 ms each
When using carbofuran as an internal standard, make sure that residues of carbofuran present
have been quantitatively removed by the column cleanup in 6.4.
Carbofuran
3-Hydroxy-carbofuran
Carbosulfan
10 15 min
Fig. 1. Gas chromatogram of a standard mixture representing 1 ng each of carbofuran, 3-hydroxy-
carbofuran, and carbosulfan. Conditions as described in 6.5.1.
Abundance Carbofuran
6000000 - J 3-Hydroxy-carbofuran
5000000 -
, Carbosulfan
4000000 -
3000000 -
I
2000000 -
1000000 -
n. A JL__J
9.0 12.0 min
Fig. 2. Total ion chromatogram of a standard mixture representing 50 ng each of carbofuran, 3-hydroxy-
carbofuran, and carbosulfan. Conditions as described in 6.5.2.
a) Scan 55 (8.216 min)

Abundance
Coxbofuran 164
1000000 -
800000 -
149
600000 -
400000 -
122
2J0000- 15 33 58 c 221
V 103 I,
191 [
0 1 ,!. ll. J...J ,h. I. ... LI. III. , I, til I
40 80 120 160 200
Mass/Charge
b) Scan 92 (9.007 min)

Abundance
3-Hydroxy-carbofuran 137
280000 -
240000-
200000 -
180
160000-
120000 -
151
60000 -
28 4- 58
40000 - | Z / 91 123 237
: 1, ill,.. 1,1.1. ,1,1 hi. ..1. u1 III i..i.. ...i,U 1. 1, i. i i
40 80 120 160 200 240
Mass/Charge
c] Scan 231 (11.979 min)

Abundance
Carbosulfan J60
1000000-
118
800000 -
600000
400000- 323
76
li
200000 -
221 252
0
200
Mass /Charge
Fig. 3. Mass spectra of a) carbofuran, b) 3-hydroxy-carbofuran, c) carbosulfan. Conditions as described

in 6.5.2.
7 Evaluation
7.1 Method
7.1.1 Determination with the thermionic detector
Quantitation is performed by the calibration technique. Prepare calibration curves as follows.
Inject equal volumes of the standard solutions (equivalent to 0.3 to 3.0 ng of carbosulfan, car-
bofuran and 3-hydroxy-carbofuran, respectively) into the gas chromatograph. Plot the areas
or heights of the peaks obtained vs. ng of compound. Also inject aliquots of the sample solu-
tions. Equal volumes of the sample solutions and the standard solutions should be injected.
For the areas or heights of the peaks obtained for the sample solutions, read the appropriate
amounts of the identified compound from the corresponding calibration curve.
7.1.2 Determination with the mass-selective detector

Quantitation is performed using an internal standard. For this purpose, prepare a sample solu-
tion from an untreated control sample. Fortify this solution, in the same order of magnitude
as the anticipated residue, with equal amounts of 3-hydroxy-carbofuran and internal standard
carbofuran. The volume of this measuring solution should be equal to that of the analytical
sample solution. Inject an aliquot of the measuring solution into the gas chromatograph and
determine the ratio, f, of the peak areas (or heights) obtained for the metabolite and the inter-
nal standard, f must be determined separately for each sample material.
Add internal standard to the analytical sample solution in the same order of magnitude as
the anticipated residue. Inject an aliquot of this solution into the gas chromatograph and per-
form the evaluation using the ratio of the peak areas (or heights) obtained for the metabolite
and the internal standard.

The recoveries from untreated control samples of plant material, fortified with carbosulfan
and carbofuran at levels of 0.05 to 5 mg/kg, ranged from 65 to 110% and averaged 88%. The
limit of detection was 0.02 to 0.03 mg/kg, depending on the sample material, and the limit
of determination was 0.05 mg/kg. Carbosulfan and carbofuran in rape seeds were not ex-
amined.
The recoveries from soil samples, fortified with carbosulfan and carbofuran at levels of 0.05
to 1 mg/kg, ranged from 86 to 113% and averaged 99%. The limit of detection was 0.02
mg/kg, and the limit of determination was 0.05 mg/kg.
The recoveries from untreated control samples of rape (green matter and seeds) and
tomatoes, fortified with 3-hydroxy-carbofuran at levels of 0.1 to 1 mg/kg, ranged from 68 to
115% and averaged 87%. The limit of detection was 0.01 to 0.02 mg/kg, depending on the
sample material, and the limit of determination was 0.05 mg/kg.
The average recoveries are given in the Table.
Table. Percent recoveries from plant material and soil, fortified with carbosulfan, carbofuran and
3-hydroxy-carbofuran; means from 2 to 5 experiments.
Added 3-Hydroxy-
Analytical material mg/kg Carbosulfan Carbofuran carbofuran
Grapes 0.05-5 65 88
Head cabbage 0.05-0.5 95 65 -
Lettuce 0.05-0.5 80 86 -
Maize 0.05-5 103 83
Rape
Green matter 0.05-1 91 103 79
Seeds 0.1-1 77
Tomatoes 0.05-1 103 88 111
Sugar beet 0.05-5 86 80
Soil 0.05-1 92 106 -

The residue R, expressed in mg/kg carbosulfan, carbofuran or 3-hydroxy-carbofuran, is
calculated from the following equations:
WA VF v
End
for carbosulfan and carbofuran: R =
V
R1 Y - G
where
VEx = total volume of organic phase after addition of solvent or solvent mixture, respec-
tively, to filtered extract (in ml)
VR1 = portion of volume VEx used for further cleanup (in ml)
VEnd = terminal volume of sample solution from 6.3.2 (in ml)
Y = portion of volume VEnd injected into gas chromatograph (in ul)
reac
WA = amount of carbosulfan or carbofuran, respectively, for Y ^ from calibration
curve (in ng)
S t - E v - YEx * VEnd
for 3-hydroxy-carbofuran: R =
F st .f-v Rr v r G
where f = Ik
and
VEx = total volume of filtered extract from 6.2 after addition of hydrochloric acid or water,
respectively (in ml)
V R1 = p o r t i o n of volume V E x used for further cleanup (in ml)
Vj = portion of volume V End injected into gas chromatograph (in ul)
St = amount of internal standard injected with sample solution (in ng)
FA = peak area or height for 3-hydroxy-carbofuran obtained from Vj (in integrator
counts)
F St = peak area or height for internal standard carbofuran obtained from Vj (in integrator
counts)
'A = peak area or height for 3-hydroxy-carbofuran (in integrator counts), and
Fs t = peak area or height for internal standard carbofuran (in integrator counts), both
from injection of the measuring solution (see 7.1.2)
8 Important points
Carbosulfan, carbofuran and 3-hydroxy-carbofuran have different stabilities in solution. The
stability of the standard solutions must therefore be checked at least once a week.
Extracts from sugar beet obtained in 6.1.1 should be suction-filtered through a layer of filter
aid on the fast flow-rate filter paper in order to prevent the filter from being clogged.
The elution range in 6.3.2 should be checked from time to time, as the activity of the Florisil
can change on storage.
Blank value problems can occur with 3-hydroxy-carbofuran in rape using a thermionic
detector; it is, therefore, preferably determined with a mass-selective detector in the SIM mode
using carbofuran as internal standard. For carbosulfan and carbofuran, mass-selective detec-
tion is used only to confirm results obtained. The respective mass spectra are given in Fig. 3.
9 References
E. Mollhoff, Uber die Riickstandsanalyse von N-Methylcarbamat-Insektiziden, Pflanzen-
schutz-Nachr. 28, 388-395 (1975).
E. Mollhoff, Methode zur gaschromatographischen Bestimmung der Riickstande von Cura-
terr in Pflanzen- und Bodenproben unter Beriicksichtigung von Metaboliten, Pflanzenschutz-
Nachr. 28, 370-381 (1975).
R. F. Cook, R. P. Stanovick and C. C. Cassil, Determination of carbofuran and its carbamate
metabolite residues in corn using a nitrogen-specific gas chromatographic detector, J. Agric.
Food Chem. 17, 277- 282 (1969).
FMC Europe SA Agriculture, Chemical Group, Determination of 3-hydroxy-carbofuran crop

residues by nitrogen selective gas chromatography, Internal Report INT 35001-2, Brussels
6/79.
FMC Europe SA Agriculture, Chemical Group, Determination of FMC 35001 and carbofuran
crop residues by nitrogen selective gas chromatography, Internal Report INT 35001-4, Brussels
6/79.
10 Authors
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, J. Siebers,
H. Kohle and H.-G. Nolting
Chlorflurenol, Flurenol 275-215
Barley (grains and straw), cucumbers, wheat (grains Gas-chromatographic

and straw) determination
Soil, water
1 Introduction
Chlorflurenol Flurenol
Chemical name 2-Chloro-9-hydroxyfluorene- 9-Hydroxyfluorene-9-carboxylic
9-carboxylic acid (IUPAC) acid (IUPAC)
Structural formula
COOH
Empirical formula C14H9C1O3 C14H10O3

Molar mass 260.68 226.23
Data for Chlorflurenol methyl ester Flurenol n-butyl ester

Melting point 152 C 71 C
Vapour pressure 6.7-10" 5 mbar at 25 C 10" 6 mbar at 25 C
Solubility Virtually insoluble in water; Virtually insoluble in water;
(in 100 ml at 20 C) readily soluble to soluble in very readily soluble to readily
most organic solvents, e.g. soluble in most organic
readily soluble in acetone solvents, e.g.
(26 g) and methanol (15 g); very readily soluble in acetone
soluble in benzene (7 g) and (145 g) and methanol (150 g);
ethanol (8 g); readily soluble in benzene (95 g)
sparingly soluble in and ethanol (70 g);
petroleum ether (0.16 g) sparingly soluble in petroleum
ether (0.7 g)
Other properties Cream coloured, crystalline, Beige, crystalline, practically
odourless odourless
Commercial products contain chlorflurenol as methyl ester and flurenol as n-butyl ester,
sometimes as amine salt. As the acids are relatively unstable and the esters are of greater im-
portance, the physical data for the esters have been given.
2 Outline of method
Residues, which can be present as esters (I), or in the case of flurenol as salts, are extracted
under acid conditions with acetone or acetonitrile. If present, the conversion products II and
128 Chlorflurenol, Flurenol
IV, as well as the relatively unstable product III, are also extracted. Next, the extract is washed
with toluene or petroleum ether. I is hydrolyzed with potassium hydroxide in the concentrated
extract. II is decarboxylated with sulphuric acid to yield compound III which is subsequently
oxidized to 2-chlorofluorenone or fluorenone (IV) with chromium trioxide. After column
chromatographic cleanup, the oxidation products IV are determined by electron capture gas
chromatography.
Chlorflurenol: R2 = Cl; R2 = CH 3
Flurenol: R2 = H; R2 = C 4 H 9
3 Apparatus
High-speed blendor fitted with leak-proof glass jar and explosion-proof motor
Sintered glass filter funnels, porosity 2, 13 cm and 6 cm dia.
Volumetric flasks, 500-ml, 250-ml, 10-ml and 5-ml
Separatory funnels, 2.5-1, 500-ml and 250-ml
Laboratory mechanical shaker, suitable for holding separatory funnels
Round-bottomed flasks, 1-1, 250-ml and 100-ml, with ground joints
Centrifuge, with 250-ml glass tubes (may be required)
Microsyringe, 10-ul
4 Reagents
Acetone, p.a., fractionally distilled
Acetronitrile, p.a., fractionally distilled
Petroleum ether, saturated with acetonitrile
Petroleum ether, technical grade, fractionally distilled, boiling range 40-70 C
Toluene, p.a., fractionally distilled
Eluting mixture: petroleum ether + toluene 1:1 v/v
2-Chlorofluorenone standard solutions: 0.01, 0.02, 0.04, 0.08 and 0.1 [ig/ml petroleum ether
Fluorenone standard solutions: 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 ng/ml petroleum ether
Sulphuric acid, 1 mol/1 H2SO4 p. a.
Chlorflurenol, Flurenol 129
Potassium hydroxide solution, 10 g/100 ml KOH p. a.

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

6 Procedure
6.1 Extraction
6.1.1 Cereal grains, straw
Homogenize 50 g of the comminuted grains or 20 g straw (G) with 5 ml sulphuric acid and
150 ml acetonitrile in the blendor for 3 min. Suction-filter the homogenate through the 13-cm
glass filter funnel and wash the filter cake with approx. 50 ml acetonitrile. Repeat the extrac-
tion with a further 150 ml acetonitrile. Wash the filter with 50 ml acetonitrile. Combine the
filtrates in a 500-ml volumetric flask and make up to the mark with acetonitrile (VEx). Shake
a 100-ml aliquot (VR1) twice in a 250-ml separatory funnel, each time for 5 min, with 50-ml
portions of petroleum ether saturated with acetonitrile. Collect the lower acetonitrile phase
in a 250-ml round-bottomed flask and rotary-evaporate to 1 -2 ml. Proceed as described in 6.2.
6.1.2 Cucumbers
Homogenize 50 g of the comminuted material (G) with 5 ml sulphuric acid and 100 ml acetone
for 3 min with the homogenizer. Suction-filter the homogenate through the 6-cm glass filter
funnel. Wash the filter cake three times with 30-ml portions of acetone. Combine the filtrates
in a 250-ml volumetric flask and make up to the mark with acetone (VEx). Transfer a 100-ml
aliquot (VR1) into a 500-ml separatory funnel, dilute with 150 ml water and 75 ml sodium
chloride solution and shake twice, each time for 5 min, with 50-ml portions of toluene. Filter
the upper toluene layers through sodium sulphate and wash the filter with 10 ml toluene. Com-
bine the filtrates in a 250-ml round-bottomed flask and rotary-evaporate to 1-2 ml. Proceed
as described in 6.2.
6.1.3 Soil
Acidify 100 g soil with 50 ml sulphuric acid and add 100 ml acetone. Allow to stand for 1 h,
and homogenize for 3 min in the blendor. Suction-filter through the 13-cm glass filter funnel
and repeat the extraction twice, each time with 100 ml acetone-water mixture. Collect the
filtrates in a 500-ml volumetric flask and make up to the mark with acetone (VEx). Transfer
a 100-ml aliquot (VR1) into a 500-ml separatory funnel, dilute with 150 ml water and 75 ml
sodium chloride solution and shake twice, each time for 3 min, with 50-ml portions of toluene.
Filter the upper toluene layers through sodium sulphate and wash the filter with 10 ml toluene.
Collect the filtrates in a 250-ml round-bottomed flask and rotary-evaporate to 1-2 ml. Proceed
as described in 6.2.
6.1.4 Water
Acidify a 2-1 water sample (G) in a 2.5-1 separatory funnel with 5 ml sulphuric acid and extract
twice, each time for 5 min, with 250-ml portions of toluene on the mechanical shaker. Filter
the separated toluene layers through sodium sulphate into a 1-1 round-bottomed flask and
rotary-evaporate to about 50 ml. Transfer into a 250-ml round-bottomed flask and concentrate
further to 1-2 ml. Proceed as described in 6.2.
6.2 Hydrolysis of the esters

Treat the residual solution derived from 6.1 with 20 ml potassium hydroxide solution. Allow
the flask to rotate without suction on a rotary evaporator for 20 min in a boiling water bath.
6.3 Decarboxylation and oxidation

Transfer the cooled solution derived from 6.2 with 40 ml sulphuric acid into a 250-ml
separatory funnel. Add 5 ml chromium trioxide solution and 30 ml petroleum ether. Shake
the mixture for 1 h on the mechanical shaker. Separate the petroleum ether layer, using a cen-
trifuge if required. Shake the aqueous phase for 5 min with a further 30 ml petroleum ether.
Filter the organic layers through sodium sulphate, wash the filter with 10 ml petroleum ether,
and rotary-evaporate to 1-2 ml.

Place a quartz wool plug in the bottom end of a chromatographic tube half-filled with the
eluting mixture. Add 1.5 g aluminium oxide and then, after settling, 4 g Florisil whilst gently
tapping the tube walls. Wash the prepared column with 30 ml eluting mixture. Drain the sol-
vent to the top of the adsorbent. Transfer the petroleum ether solution derived from 6.3 quan-
titatively onto the column with 5 ml eluting mixture. Allow to percolate, and elute with 180 ml
eluting mixture. Discard the first 40 ml, collect the subsequent eluate in a 250-ml round-
bottomed flask and rotary-evaporate to approx. 2 ml.

Transfer the solution derived from 6.4 into a 5 or 10-ml volumetric flask with petroleum ether
and make up to the mark (VEnd). Inject 1 JLXI of this solution (Vj) (if necessary, after dilution
with petroleum ether) into the gas chromatograph.
Column Glass, 2 mm i.d., 1.3 m long; packed with 1.5% OV-
17 + 2% OV-210 on Gas Chrom Q, 100-120 mesh
63
Temperature 320 C
Carrier gas flow rate Nitrogen, 15 ml/min
Attenuation 16-10"11
Linearity range 2-Chlorofluorenone 0.01-0.1 ng
Fluorenone 0.005-0.1 ng
Retention times for
fluorenone 2 min 24 s
2-chlorofluorenone 4 min 6 s
7 Evaluation
7.1 Method
Inject 1 JLLI of each 2-chlorofluorenone or fluorenone standard solution, respectively, (equiv-
alent to 0.01 to 0.1 ng 2-chlorofluorenone or 0.005 to 0.05 ng fluorenone) into the gas
chromatograph. Plot the areas or heights of the peaks obtained vs. ng of compound. Also in-
ject 1-ul aliquots of the sample solutions. For the areas or heights of the peaks obtained for
these solutions, read the appropriate amounts of compound from the corresponding calibra-
tion curve.

Recovery experiments were run on different untreated control samples fortified with
chlorflurenol methyl ester, flurenol n-butyl ester, or the conversion products II and IV. The
recoveries and the routine limits of determination were as follows:
Added Recovery Routine limit of determination

Analytical material mg/kg 7o mg/kg
Cereal grains 0.02-0.1 70-85 0.02
Cereal straw 0.2-1.0 70-90 0.1
Cucumbers 0.02-0.2 75-95 0.02
Soil 0.05-0.5 70-85 0.02
Blanks usually did not occur or, if so, they were less than 0.01 mg/kg (grains and soil) or
0.05 mg/kg (straw).
From water, the recoveries were 70 to 90% when fortified with 0.05 to 20 ng/1. The routine
limit of determination was 0.05 fxg/1.
The residue R, expressed in mg/kg chlorflurenol or flurenol, is calculated from the following
equations:
W V n d
for chlorflurenol R= ^' ?'^ 1.214
V
R1
V
Ex ' V End
for flurenol 1.255
V
R1 V r G
where

V Ex = total volume of extract (in ml)
VRI = portion of volume V Ex used for t h e analysis (in ml)
WA = a m o u n t of 2-chlorofluorenone or fluorenone, respectively, for Vj read from calibra-
tion curve
1.214 = factor for conversion of 2-chlorofluorenone t o chlorflurenol
1.255 = factor for conversion of fluorenone to flurenol
For conversion of 2-chlorofluorenone to chlorflurenol methyl ester, t h e conversion factor

is 1.280; for conversion of fluorenone t o flurenol n-butyl ester, t h e factor is 1.567.
8 Important points
2,7-Dichloro-9-hydroxyfluorenecarboxylic acid methyl ester, which is present as an impurity
in technical chlorflurenol methyl ester, and the corresponding 2,7-dichlorofluorenone can also
be determined by this method. The retention time for 2,7-dichlorofluorenone was 7 min 12 s.
9 References
H. Sieper, Residue analysis and degradation of morphactins, Proc. 2nd Intern. IUPAC Congr.
Pestic. Chem., Vol. 6, pp. 157-174. Gordon and Breach, New York 1972.
E. Amadori and W. Heupt, Chlorflurecol-methyl, in: G. Zweig, Analytical methods for
pesticides and plant growth regulators, Vol. X, Academic Press, New York-San Francisco-
London 1978.
E. Amadori and W. Heupt, Flurecol, in: G. Zweig, Analytical methods for pesticides and
plant growth regulators, Vol. XI, Academic Press, New York-San Francisco-London 1980.
D. Eichler, W. Heupty IRE. Anderson, K. H. Domsch and G. Jagnow, Chlorflurecol-methyl
in soil: Degradation, leaching and effects on microbiological processes, Arch. Environm.
Toxicol. 11 185-193 (1982).
10 Authors
Shell Forschung GmbH, Schwabenheim, D. Eichler and W. Heupt
Chloridazon 89-A
Mangold, red beet (foliage and edible root), sugar beet Gas-chromatographic
(foliage and edible root) and high-perfor-
Soil, water mance liquid
chromatographic
determination
1 Introduction
1.1 Chloridazon
Chemical name 5-Amino-4-chloro-2-phenylpyridazin-3(2H)-one
(IUPAC)
Structural formula NH9
Empirical formula C 10 H 8 ClN 3 O

Molar mass 221.65
Vapour pressure 10 ~ 7 mbar at 20 C
Solubility Very sparingly soluble in water;
(in 100 ml at 20 C) slightly soluble in acetone (2.8 g) and methanol
(3.4 g);
sparingly soluble in dichloromethane (0.33 g) and
ethyl acetate (0.6 g);
very sparingly soluble in benzene (0.07 g)
Other properties Rapidly photolyzed in sunlight
1.2 Metabolite A
Chemical name 5-(N-Glucosyl)amino-4-chloro-2-phenylpyridazin-
3(2H)-one
o Cl CH 2 0H
r H
N . /
0-
1P\ H
Structural formula \ ^
-J X
N= H \ H ÂOH
y ^\
OH H
Empirical formula C l 6 H18C1N 3 o6
Molar mass 383 .79
136 Chloridazon
Melting point Approx. 220 C with decomposition

Solubility Readily soluble in water at 20 C
1.3 Metabolite B
Chemical name 5-Amino-4-chloropyridazin-3(2H)-one
formula H-N NH2
Empirical formula C4H4CIN3O

Molar mass 145.55
Melting point Approx. 315 C with sublimation
Solubility Very sparingly soluble in water at 20 C
Other properties Readily soluble in dilute alkali
2 Outline of method
Residues of chloridazon, metabolite A and metabolite B are extracted from plant material and
soil with methanol. The methanol extract is divided in two. One half, or a water sample, serves
for the combined determination of chloridazon and metabolite A (as chloridazon), the other
half, or a second water sample, for the determination of metabolite B.
The half of the plant extract used for the chloridazon analysis is purified from plant
pigments and sugars by precipitation; then metabolite A is converted to chloridazon by acid
hydrolysis. The extract so prepared, half of a soil extract, or a water sample is evaporated in
acid medium, and cleaned up by partition between water and dichloromethane on an Extrelut
column, and by column chromatography on aluminium oxide. Chloridazon is determined by
electron capture gas chromatography using a fused silica capillary column.
The second half of the extract, or a second water sample, is used to determine metabolite
B. After a partition step on an Extrelut column, followed by gel permeation chromatography
on Sephadex LH-20 and column chromatography on silica gel, determination in performed
by high-performance liquid chromatography using a UV detector at 286 nm.
3 Apparatus
Homogenizer, e.g. Ultra-Turrax T 45 N (Janke & Kunkel)
Ice bath
Chloridazon 137

Filter paper, 9 cm dia., fast flow rate (Schleicher & Schiill)
Allihn reflux condenser
Hotplate with magnetic stirrer, e. g. Ika-Combimag RTC (Janke & Kunkel), with PTFE stirring
rod
Rotary vacuum evaporator, 40-60 C bath temperature
Fluted filter paper, 24 cm dia.
Round-bottomed flasks, 1-1, 500-ml and 250-ml
Erlenmeyer flask, 1-1
Ultrasonic bath
Membrane filter holder, i.d. 25 mm, with filter membranes, 25 mm dia. (e.g. SM 13400, Sar-
torius)
Automated instrument for gel permeation chromatography, e. g. GPC Autoprep 1002 A
(Analytical Bio-Chemistry Laboratories), equipped with 5-ml sample loops and chromato-
graphic tube, 25 mm i.d., 45 cm long; column filling 75 g Sephadex LH-20 pre-swelled in
methanol
High-performance liquid chromatograph equipped with autosampler, pump, and variable
wavelength detector
4 Reagents
Acetone, dist.
Chloroform, dist.
Dichloromethane, dist.
Ethanol, dist.
Methanol, dist.
2-Propanol (isopropanol), dist.
2,2,4-Trimethyl pentane (isooctane), dist.
Dissolving mixture: dichloromethane + methanol + triethylamine 91:8:1 v/v/v
Eluting mixture 1: chloroform + ethanol 98:2 v/v
Eluting mixture 2: dichloromethane + isopropanol 85:15 v/v
Eluting mixture 3: chloroform + ethanol 95:5 v/v
Eluting mixture 4: chloroform + methanol 8:2 v/v
Injection mixture: isooctane + isopropanol 9:1 v/v
Mobile phase: dichloromethane + methanol + triethylamine 940:60:1 v/v/v
Chloridazon standard solutions: 0.5, 1, and 2 ng/ml injection mixture
Metabolite B standard solutions: 0.5, 1, and 2 ng/ml dissolving mixture
138 Chloridazon
Hydrochloric acid, 1 mol/1 HC1 p. a.

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

6 Procedure
6.1 Extraction
Homogenize 50 g of the analytical sample (G) with 300 ml methanol for 10 min, immersing
the flask in an ice bath. Suction-filter the homogenate through a fast flow-rate filter paper
in a Buchner porcelain funnel, and wash the filter cake twice with 50-ml portions of methanol.
Transfer the combined filtrates to a 500-ml volumetric flask, make up to the mark with
methanol (VEx), and shake well.
6.1.2 Soil
Reflux 50 g of the soil sample (G) with 250 ml methanol in an Erlenmeyer flask, fitted with
an Allihn condenser, on the stirrer-hotplate with magnetic stirring for 60 min. Allow to cool,
and rinse the condenser with 20 ml methanol. Filter the mixture through a fast flow-rate filter
paper in a Buchner porcelain funnel, and wash the filter cake twice with 50-ml portions of
methanol. Transfer the combined filtrates to a 500-ml volumetric flask, make up to the mark
with methanol (VEx), and shake well.
Chloridazon 139
6.1.3 Water
Treat each 500 g of water sample (G) as described in 6.2.1.4 or 6.3.1.3.
6.2 Cleanup for chloridazon and metabolite A

6.2.1 Preparation
6.2.1.1 Mangold and beet foliage
Transfer 250 ml of the methanolic extract derived from 6.1.1 (VR1) into a 1-1 round-bottomed
flask, add 100 ml coagulating solution, and rotary-evaporate to approx. 100 ml, with 50 C
bath temperature. Filter the solution through a fluted filter paper into a tared 500-ml round-
bottomed flask. Wash the filter with 10 ml water, and discard the filter residue. Reflux the
aqueous filtrate for 15 min on the stirrer-hotplate, whereby metabolite A is quantitatively con-
verted to chloridazon. Allow to cool, rinse the condenser with 10 ml water, and rotary-
evaporate to exactly 27 g of aqueous residue (equivalent to 25 ml, VR2), with 60 C bath
temperature.
6.2.1.2 Red beet and sugar beet (edible roots)

Rotary-evaporate 250 ml of the methanolic extract derived from 6.1.1 (VR1) in a 1-1 round-
bottomed flask to about 10 ml, with 50 C bath temperature. Add 20 ml sodium chloride solu-
tion and give the solution a good swirl to dissolve the residue. Add 400 ml acetone and shake
well for 1 min. Leave to stand for a short time, and then decant the acetone solution. Extract
the semi-crystalline aqueous residue again with 150 ml acetone. Combine both acetone solu-
tions in a 1-1 Erlenmeyer flask and leave to stand for at least 60 min to allow complete
precipitation of sugars and sodium chloride to take place. Filter the suspension through a
fluted filter paper into a tared 1-1 round-bottomed flask. Wash the filter with about 20 ml
acetone. Rotary-evaporate the filtrate to 5-10 ml with 40 C bath temperature. Add 50 ml
hydrochloric acid and reflux for 15 min on the stirrer-hotplate, whereby metabolite A is quan-
titatively converted to chloridazon. Allow to cool, rinse the condenser with about 10 ml water,
and rotary-evaporate to exactly 25 g of aqueous residue (equivalent to 25 ml, VR2), with 60 C
bath temperature.
6.2.1.3 Soil
Transfer 250 ml of the methanolic extract derived from 6.1.2 (VR1) into a tared 500-ml round-
bottomed flask, add 25 ml hydrochloric acid, and rotary-evaporate to exactly 25 g (equivalent
to 25 ml, VR2), with 50 C bath temperature.
6.2.1.4 Water
Transfer 500 g of the analytical sample (G) into a tared 1-1 round-bottomed flask, add 25 ml
hydrochloric acid, and rotary-evaporate to exactly 25 g (equivalent to 25 ml, VR2), with 60 C
bath temperature.
140 Chloridazon
6.2.2 Partition on an Extrelut column

Fit an empty Extrelut tube on its lower end with a 10-mm dia. filter disc. Pour 3 g Extrelut
into the tube and cover the Extrelut with 3 g sea sand to give an approx. 2 mm deep layer.
Add 4 ml sodium hydroxide solution and allow it to soak in. Next add the contents of an Ex-
trelut refill pack (about 12 g). Hold the filling in place with the basket insert and a 24-mm
dia. filter disc. Transfer 20.0 ml of the aqueous extract from 6.2.1 (VR3) onto the Extrelut col-
umn, using a pipet, and allow the liquid to soak into the column. After 15 min, elute with
four 25-ml portions of dichloromethane. Combine the eluates in a 250-ml round-bottomed
flask and rotary-evaporate to dryness with 40 C bath temperature.
6.2.3 Column chromatography on aluminium oxide

Insert a cottonwool plug into the lower end of the chromatographic tube, half-fill the tube
with chloroform, and trickle in 10 g aluminium oxide. Drain the chloroform until the adsorb-
ent is just covered with liquid.
Dissolve the residue derived from 6.2.2 in 5 ml chloroform and transfer the solution onto
the column. Allow to percolate, and rinse the flask three times with 5-ml portions of
chloroform. Successively add the rinsings to the column, allowing each portion to drain to
the top of the aluminium oxide before the next one is added. Next elute chloridazon with 100
ml eluting mixture 1. Collect the total eluate in a 250-ml round-bottomed flask and rotary-
evaporate to 1-2 ml with 40 C bath temperature. Transfer the solution quantitatively with a
few ml of dichloromethane into the pear-shaped flask and rotary-evaporate to dryness.
6.3 Cleanup for metabolite B

6.3.1 Preparation
6.3.1.1 Plant material
Transfer 250 ml of the methanolic extract derived from 6.1.1 (VR1) into a tared 1-1 round-
bottomed flask, add 6.5 g sodium chloride, and rotary-evaporate to an aqueous residue with
50 C bath temperature. Make up to 24.5 g with water.
6.3.1.2 Soil
Transfer 250 ml of the methanolic extract derived from 6.1.2 (VR1) into a tared 1-1 round-
bottomed flask, add 6.5 g sodium chloride and 25 ml hydrochloric acid, and rotary-evaporate
to a residue of 24.5 g with 50 C bath temperature.
6.3.1.3 Water
Transfer 500 g of the analytical sample (G) into a tared 1-1 round-bottomed flask, add 6.5 g
sodium chloride and 25 ml hydrochloric acid, and rotary-evaporate to a residue of 24.5 g with
60 C bath temperature.
6.3.2 Partition on an Extrelut column

Transfer the aqueous solution derived from 6.3.1 onto an Extrelut column and allow the solu-
tion to soak in. After 15 min, pre-elute the column four times with 25-ml portions of
Chloridazon 141
dichloromethane, which have been used beforehand to wash the flask in order to transfer
residues of water from the flask to the column. Discard this eluate. Next, elute metabolite B
with six 25-ml portions of eluting mixture 2. Combine the eluates in a 250-ml round-bottomed
flask and rotary-evaporate to dryness with 40 C bath temperature. Dissolve the residue in
10.0 ml methanol (VR4), immersing the flask in an ultrasonic bath.
6.3.3 Gel permeation chromatography

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

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

Dissolve the residue derived from 6.2.3 in 2.0 ml (VEnd) injection mixture. Inject an aliquot
of this solution (Y{) if necessary, after dilution with injection mixture into the gas
chromatograph.
Column Fused silica capillary, i.d. 0.28 mm, 25 m long;
coated with SE-54, film thickness 0.5 |xm
63
Temperature 300 C
142 Chloridazon
Gas flow rates Helium carrier, inlet pressure 1 bar

Helium septum purge gas, 170 ml/min
Helium split gas, 25 ml/min
Attenuation 32-10" 12
Retention time for chloridazon 7 min 30 s
6.5 High-performance liquid chromatographic determination of metabolite B

Dissolve the residue derived from 6.3.4 in 2.0 ml (VEnd) dissolving mixture, immersing the
flask in an ultrasonic bath (no layer of solid material should remain on the walls). Inject an
aliquot of this solution (Vj) into the high-performance liquid chromatograph.
Chromatograph HPLC instrument fitted with pump LC-410 and
autosampler MSI 660 (Kontron)
Columns Two stainless steel columns, each 3.2 mm i. d. and
10 cm long; packed with Silica Spheri-5 (Brownlee
Labs); connected in series
Mobile phase Dichloromethane + methanol + triethylamine
940:60:1 v/v/v
Column inlet pressure Approx. 80 bar
Detector UV detector Uvikon LC-720 (Kontron)
Wavelength 286 nm
Attenuation 0.01 E
Retention time for 5-amino-4-
chloropyridazin-3(2H)-one
(metabolite B) 3 min 13 s
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Prepare two calibration curves prior
to each series of measurements as follows. Dilute aliquots of the chloridazon standard solu-
tions with appropriate amounts of the injection mixture to yield a set of chloridazon measur-
ing standards. Inject 1 ul each of these solutions (equivalent to 0.025 to 0.2 ng chloridazon)
into the gas chromatograph. Likewise inject 100 ul of each metabolite B standard solution
(equivalent to 50 to 200 ng metabolite B) into the high-performance liquid chromatograph.
Plot the heights of the peaks obtained vs. ng chloridazon or ng metabolite B, respectively. Also
inject 1-ul or 100-ul aliquots, respectively, of the sample solutions. For the peaks obtained for
Chloridazon 143
these solutions, read the appropriate amounts of chloridazon or metabolite B from the respec-
tive calibration curve.

and water, fortified with chloridazon and metabolite B at levels of 0.05 to 0.5 mg/kg (water
0.5 to 5 |ig/l). The recoveries are given in the Table. The routine limit of determination was
0.05 mg/kg for plant material and soil, and 0.5 |ig/l for water.
Table. Percent recoveries from plant material, soil and water, fortified with chloridazon and metabolite B.
. . . , .. Chloridazon Metabolite B
Analytical material
Range Mean Range Mean
Mangold 63-106 85 81-91 86
Red beet
Foliage 71- 76 74 62- 85 74
Edible root 81-109 96 70- 78 73
Sugar beet
Foliage 86-100 92 77- 83 79
Edible root 86-104 95 67-104 86
Soil 96-103 98 73- 91 81
Water 83- 87 85 80- 92 84

The residue R, expressed in mg/kg chloridazon or metabolite B, is calculated from the follow-
ing equations:
W VR 2
for chloridazon R= */ \ ' ,/ > "
VR4
for metabolite B R= ^ % ' "VEnd
V R 5 Vi G
where
G = s a m p l e weight (in g)
VEx = total v o l u m e o f extract o b t a i n e d in 6.1.1 o r 6.1.2 (in m l )
VR1 = p o r t i o n of v o l u m e V E x used for c l e a n u p (in m l )
VR2 = v o l u m e o f a q u e o u s residue o b t a i n e d from evaporation in 6.2.1 (in m l )
VR3 = p o r t i o n o f v o l u m e V R 2 used for c l e a n u p o n t h e Extrelut c o l u m n in step 6.2.2 (in m l )
VR4 = v o l u m e of m e t h a n o l used t o dissolve t h e d r y residue in 6.3.2 (in m l )
144 Chloridazon
ple loop) (in ml)
VEnd = terminal volume of sample solution from 6.4 or 6.5 (in ml)
Vj = portion of volume VEnd injected into gas chromatograph or high-performance liquid
chromatograph (in \i\)
WA = amount of chloridazon or metabolite B, respectively, for V{ read from calibration
curve (in ng)
8 Important points
Broad peaks with strong tailing and decreased sensitivity can occur during the gas
chromatography of metabolite A (as chloridazon). Shortening the inlet end of the column by
approx. 2 cm can greatly restore the sensitivity (see 9. Reference).
Due to the presence of hydrochloric acid in the aqueous solutions from 6.3.1, ferric chloride
can be released from the Extrelut column during the cleanup in step 6.3.2. In order to prevent
any precipitation in the HPLC column, which would adversely affect the separation efficiency,
the ferric chloride is removed by the preceding gel permeation chromatography as described
in 6.3.3.
If a gas chromatograph fitted with capillary columns is not available, chloridazon deter-
mination can also be performed on packed columns, under the following conditions: Glass
column, i.d. 2.5 mm, 90 cm long; packed with 5% Silar-5 CP on Gas Chrom Q, 100-120
mesh; column temperature 240C; injection port temperature 260C; 63Ni electron capture
detector, temperature 260C; carrier gas argon + methane 9:1 v/v, 120 ml/min; retention
time for chloridazon about 11 min.
In most cases, chloridazon residues can also be determined by HPLC. Operating conditions
then differ from those given in 6.5 in the following points: Mobile phase isooctane +
isopropanol + methanol 88:10:2 v/v/v; flow rate 2 ml/min, column inlet pressure 20 bar;
attenuation 0.03 E; retention time for chloridazon 6 min 36 s.
9 Reference
F. Kuhlmann, Ruckstandsbestimmung von Pyrazon und seinen Metaboliten in Zuckerriiben,
Z. Lebensm. Unters. Forsch. 775, 35-39 (1981).
10 Authors
BASF, Agricultural Research Station, Limburgerhof, W. Keller and S. Otto
Chlorsulfuron, Metsulfuron 664-672
Cereals (green matter, grains and straw) High-performance
Soil, water liquid chromato-
1 Introduction
Chlorsulfuron Metsulfuron
(as metsulfuron-methyl)
Chemical name l-(2-Chlorophenylsulphonyl)- Methyl 2-[3-(4-methoxy-6-

3-(4-methoxy-6-methyl-l,3,5- methyl-l,3,5-triazin-2-
triazin-2-yl)urea (IUPAC) yl)ureidosulphonyl]benzoate
(IUPAC)
CH, CH,
C1 CO2CH3 O ^
ff N- SO2-NH-C-NH(/
N
N
SO2-NH-C-NH(/
Structural formula
OCH3 OCH,
Empirical formula C12H12C1N5O4S C14H15N5O6S

Molar mass 357.78 381.37
Melting point 174-178C 163-166C
Vapour pressure 6.1-10 " 6 mbar at 25 C 7.7-10" 5 mbar at 25 C
Solubility Very sparingly soluble in Virtually insoluble in water at
water at 25 C; 25 C;
slightly soluble in readily soluble in
dichloromethane (1-2 g), dichloromethane (10-20 g),
sparingly soluble in acetone soluble in acetone (2-5 g),
(0.5-1 g) and methanol sparingly soluble in ethanol and
(0.1-0.2 g), methanol (0.2-0.5 g),
very sparingly soluble in very sparingly soluble in xylene
toluene (0.01-0.1 g), (0.01-0.1 g),
virtually insoluble in virtually insoluble in n-hexane
n-hexane (<0.01 g), (<0.01 g),
in 100 ml each at 22 C in 100 ml each at 20 C
Other properties Hydrolyzed slowly in acid Stable in acid and alkaline
media, heat sensitive, little media, little sensitivity to
sensitivity to sunlight and sunlight and UV radiation.
UV radiation Metsulfuron is present as the
methyl ester in commercial
preparations
146 Chlorsulfuron, Metsulfuron
2 Outline of method
Chlorsulfuron and metsulfuron residues are extracted from cereal and soil samples with a
slightly alkaline buffer solution. The extract is washed with dichloromethane; the aqueous
phase is acidified, and the compounds are partitioned into toluene. Water samples are acidi-
fied and extracted with dichloromethane. The extracts are cleaned up using a disposable silica
gel cartridge. Chlorsulfuron and metsulfuron are determined by high-performance liquid
chromatography using a photoconductivity detector.
3 Apparatus
Laboratory centrifuge, 3800 r.p.m.
Centrifuge tubes, 500-ml and 250-ml
Graduated cylinders, 500-ml, 250-ml, 100-ml, 50-ml and 10-ml
Magnetic stirrer, with stirring rod
Beakers, 400-ml and 250-ml
pH Meter
Volumetric pipets, 50-ml, 25-ml, 2-ml and 1-ml
Vacuum filtration unit, with 0.45 fim membrane filter
Ultrasonic bath
High-performance liquid chromatograph equipped with photoconductivity detector
Microsyringe, 100-ul
4 Reagents
Acetone, p. a.
Cyclohexane, HPLC quality
Diethyl ether, p. a.
Glacial acetic acid, HPLC quality
Chlorsulfuron, Metsulfuron 147
Methanol, HPLC quality

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

6 Procedure
6.1 Extraction
6.1.1 Cereals
Homogenize 10 g of the analytical sample (G) (25 g of grains) with 150 ml extraction solution
in the mixer for 2 min. Suction-filter the supernatant liquid through a fast-flow rate filter
paper in a Buchner porcelain funnel. Homogenize the residue in the glass jar and suction-filter
twice more as above, each time using 120 ml extraction solution (for cereal grains, use 100 ml
each time). Finally, rinse the glass jar and the filter with a further 80 ml of extraction solution.
Transfer the filtrate to a volumetric flask and make up to a definite volume, e.g. 500 ml
(VEx). Next transfer a tenth of this solution (VR1) to a 500-ml separatory funnel, add 100 ml
buffer solution A, and proceed to step 6.2.1.
6.1.2 Soil
Weigh 50 g of the analytical sample (G) into a 500-ml centrifuge tube, add 100 ml buffer solu-
tion A, and vigorously shake on a mechanical shaker for 1 h. Centrifuge the suspension at
3800 r.p.m. for 15 min, and transfer the supernatant liquid into a 500-ml separatory funnel.
Repeat the extraction once more, combine the extracts, and proceed to step 6.2.1.
6.1.3 Water
Transfer 2 1 water (G) into a separatory funnel, adjust the pH to 3-4 with hydrochloric acid
(1 mol/1), and extract the water three times with 100-ml portions of dichloromethane. Filter
the combined dichloromethane phases through a layer of sodium sulphate, contained in a fun-
nel, into a 500-ml round-bottomed flask. Rinse the funnel with 50 ml dichloromethane. Add
1 ml glacial acetic acid and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml
round-bottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and
rotary-evaporate to 1-2 ml. Evaporate the solution to dryness, using a gentle stream of
nitrogen, and proceed to step 6.2.2.
6.2 Cleanup
6.2.1 Liquid-liquid partition (only cereals and soil)
Shake the extracts derived from 6.1.1 or 6.1.2 twice with 50-ml portions of dichloromethane
(for soil extracts, use two 50-ml portions of diethyl ether) for 3 min. Discard the organic
phases. Break emulsions, if required, by centrifugation. Transfer the aqueous phase to a
beaker and acidify, with vigorous stirring, to pH 5-6 (pH meter) with concentrated
hydrochloric acid. Further, adjust the pH to 3.5 using hydrochloric acid (10% w/w) (see
8. Important points). Transfer the solution back to the separatory funnel, rinse the beaker
with 5 ml water and 50 ml toluene, also add the rinsings to the separatory funnel, and shake
for 3 min. Separate the aqueous layer, drain the organic phase into a 250-ml centrifuge tube,
rinse the separatory funnel with 5 ml water, and add the rinsings to the centrifuge tube. Repeat
the extraction twice, each time using 50 ml toluene and adding the organic phases and water
rinsings to the centrifuge tube. Add a further 10 ml of water to the centrifuge tube if the phase
boundary is difficult to see after the third extraction.
Centrifuge for 15 min at 3800 r.p.m., then transfer the upper toluene phase into a 250-ml
round-bottomed flask, using a 50-ml volumetric pipet. Add 30 ml toluene to the aqueous
phase remaining in the centrifuge tube, mix, centrifuge for 5 min, and likewise pipet off the
top layer into the 250-ml round-bottomed flask. Add 1 ml glacial acetic acid to the combined
toluene phases, and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml round-
bottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and rotary-
evaporate to 1 -2 ml. Evaporate the solution to dryness, using a gentle stream of nitrogen, and
proceed to step 6.2.2.
6.2.2 Silica gel cartridge

Draw 10 ml of the mobile phase into the glass syringe, attach a silica gel cartridge to the syr-
inge, and force the mobile phase through to condition the cartridge packing. Repeat the condi-
tioning with a further 10-ml portion of mobile phase. Next detach the cartridge, pull the
plunger out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1.3
or 6.2.1 in 1 ml mobile phase and transfer the solution quantitatively into the syringe with the
aid of a Pasteur pipet. Rinse the 50-ml flask with 1 ml mobile phase and also add the rinsings
to the syringe. Re-insert the plunger into the syringe and force the liquid through the cartridge,
collecting the eluate in a 10-ml test tube. Detach the cartridge, remove the plunger from the
syringe, and re-attach the cartridge. Force a further 5 ml mobile phase through the cartridge,
proceeding in a similar manner as described above, and collect the eluate in the same test tube.
Evaporate the solution to dryness, using a gentle stream of nitrogen.

Dissolve the residue derived from 6.2.2 in mobile phase and dilute to an appropriate volume,
e.g. 10 ml (V End ). Inject an aliquot of this solution (Vj) into the high-performance liquid
chromatograph.
Chromatograph Spectra-Physics SP 8700
Injector Injection valve 7125 with sample loop (Rheodyne)
Column Zorbax Sil, 4.6 mm i. d., 25 cm long (Du Pont No.
880952701)
Mobile phase Cyclohexane-isopropanol-methanol-acetic acid-water
Conditioning solution Isopropanol-methanol-acetic acid-water
Detector Photoconductivity detector (Tracor 965), operated with
a mercury lamp at 254 nm, ATT = 5
Injection volume 20 JLAI
Retention times for
chlorsulfuron 13 min
metsulfuron-methyl 15 min
The Zorbax Sil column must be conditioned before use. For this end, pump conditioning solu-
tion through the column for 4 h at a flow rate of 0.7 ml/min. Next equilibrate the column
for 3 h with mobile phase at the same flow rate. Moreover, pump conditioning solution at a
flow rate of 0.15 ml/min through the system over night, changing to mobile phase at a flow
rate of 0.5 ml/min for 1 h before beginning a new series of measurements the next morning.
7 Evaluation
7.1 Method
Inject equal volumes of each chlorsulfuron and metsulfuron-methyl standard solution into the
high-performance liquid chromatograph. Plot the areas or heights of the peaks obtained vs.
ng chlorsulfuron or metsulfuron-methyl, respectively. Also inject equal volumes of the sample

solutions. For the areas or heights of the peaks obtained for these solutions, read the ap-
propriate amounts of compound from the corresponding calibration curve.

The recoveries from untreated cereal control samples, fortified with chlorsulfuron and
metsulfuron-methyl at levels of 0.05 to 10 mg/kg, ranged from 68 to 103% and averaged 80%.
The limit of detection was 0.02 mg/kg, and the limit of determination was 0.05 mg/kg or, for
cereal green matter, 0.1 mg/kg. Blanks usually did not occur or, if so, they were less than
0.02 mg/kg.
The recoveries from soils, fortified at levels of 0.01 to 1 mg/kg, ranged from 70 to 112%
and averaged 91%. The limit of detection was 0.005 mg/kg, and the limit of determination
was 0.01 mg/kg. Blanks were less than 0.0025 mg/kg. The recoveries from tap water fortified
at 1 |ig/l ranged from 70 to 100%, and the limit of determination was 1 u.g/1.

The residue R, expressed in mg/kg chlorsulfuron or metsulfuron-methyl, is calculated from
the following equations:
WA
for soil and water R = ' VEnd
VrG
where
VEx = total volume of solution after addition of extraction solution to filtered cereal extract
from 6.1.1 (in ml)
Vj = portion of volume VEnd injected into high-performance liquid chromatograph
(volume of sample loop) (in \i\)
WA = amount of chlorsulfuron or metsulfuron-methyl for Vj read from calibration curve
(in ng)
8 Important points
The pH adjustment in 6.2.1 takes longer than one would expect and must be carried out very
carefully. Only at pH 3-4, chlorsulfuron and metsulfuron-methyl are existing in the unionized
form which can be transferred into the organic phase. If the pH is too low, decomposition
is likely to occur; if it is too high, both compounds are partially ionized and will not be ex-
tracted quantitatively.
A LiChrosorb column can also be used for the HPLC measurement: 4 mm i. d., 25 cm long;
packed with LiChrosorb Si 60, particle size 7 urn; other conditions as described in 6.3.
When evaporated to dryness, the cleaned-up extract can be stored in a freezer for a max-
imum of four days if immediate measurement is not possible.
In order to make allowance for baseline drifting, the balance is best set to approx. 30%
above the zero point of the recorder prior to each measurement. The balance must be re-
adjusted after each measurement, if necessary. If possible, leave the mercury lamp switched
on over night.
9 References
E.W. Zahnow, Analysis of the herbicide chlorsulfuron in soil by liquid chromatography,
J. Agric. Food Chem. 30, 854-857 (1982).
R. V. Slates, Determination of chlorsulfuron residues in grain, straw, and green plants of
cereals by high-performance liquid chromatography, J. Agric. Food Chem. 31, 113-117 (1983).
10 Authors
DuPont de Nemours & Co., Biochemicals Department, Research Division, Experimental Sta-
tion, Wilmington, DE, U.S.A., L. W. Hershberger, R. V. Slates and E. W. Zahnow
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, M. Blacha-
Puller, H. Kohle, H.-G. Nolting and J. Siebers
Copper Oxychloride (as copper) 147-A
Grapes Atomic absorption

spectrophotometric
determination
1 Introduction
Chemical name Dicopper chloride trihydroxide (IUPAC)

Structural formula Cu2(OH)3Cl or CuCl2 3Cu(OH)2
Empirical formula Cu2H3ClO3 or Cu4H6Cl2O6
Molar mass 213.56 or 427.12
Melting point Above 220 C, decomposition with elimination of
hydrochloric acid to oxides of copper
Solubility Virtually insoluble in water and organic solvents;
soluble in mineral acids yielding the corresponding
copper salts;
soluble in ammonia, amine and EDTA solutions,
under complex formation
Other properties Largely stable in neutral media, decomposed by warm-
ing in alkaline media, yielding oxides
2 Outline of method
Copper containing residues are stripped from the grapes with a lead-doped aqueous solution
of ethylenedinitrilo tetraacetic acid (EDTA, disodium salt). The copper content of this solu-
tion is determined by flame atomic absorption spectrophotometry (FAAS) at 324.7 nm.
3 Apparatus
Volumetric flasks, 1-1 and 100-ml

Wide neck bottle, 500-ml, with ground stopper
Flame atomic absorption spectrophotometer
154 Copper Oxychloride (as copper)
4 Reagents
Cu standard solution: A solution of 2.116 g cupric chloride (CuCl2) in water (Merck No.
9987) is made up to 1 1 in a volumetric flask. The solution contains 1 mg/ml Cu
Pb standard solution: A solution of 1.598 g lead nitrate [Pb(NO3)2] in water (Merck No.
9969) is made up to 1 1 in a volumetric flask. The solution contains 1 mg/ml Pb
EDTA solution: 1 g/100 ml of ethylenedinitrilo tetraacetic acid disodium salt dihydrate p. a.
(Merck No. 8418)
Stripping solution: 10 g ethylenedinitrilo tetraacetic acid disodium salt dihydrate p.a. and
20 ml Pb standard solution are made up to 1 1 in a volumetric flask. The solution contains
20 mg/1 Pb
Acetylene, 99.6 vol. /o
Air, re-purified

The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1.
6 Procedure
6.1 Extraction
Transfer 100 g of the unchopped analytical sample (G) into the wide neck bottle, add 100 ml
stripping solution (VEx), and allow to stand for 1 h with occasional swirling. With deep-
frozen grapes, warm the solution in a water bath at 40 C. Filter the solution through a fluted
filter paper.
6.2 Atomic absorption measurement

Measure the absorbance at 324.7 nm (Cu) and at 217.0 nm (Pb), using the filtrate derived from
6.1.
Atomic absorption photometer Perkin-Elmer 420
Light source Hollow cathode lamp (Perkin-Elmer), operating cur-
rent Cu 15 mA, Pb 10 mA
Wavelength Cu 324.7 nm, Pb 217.0 nm
Slit width 0.7 nm
Burner Standard burner head
Burner gases Acetylene, input pressure 0.6 bar
Air, input pressure 3.2 bar
Copper Oxychloride (as copper) 155
Deuterium background compen-

sation Only with Pb measurement
Measurement value reading Digital
Measurement time 3s
7 Evaluation
7.1 Method
The evaluation is based on instrument internal standardization or on a calibration curve. For
either procedure, take 0.5, 1.5 and 3.0 ml of the Cu standard solution, respectively, and make
up to 100 ml with stripping solution (equivalent to 5, 15 and 30 mg/1 Cu). Measure the absor-
bances of these solutions at 324.7 nm. In order to take into account an eventual dilution of
the analytical solution during the extraction step by some grape juice and/or condensate,
measure the absorbance of the 20 mg/1 Pb containing stripping solution at 217.0 nm, and the
blank of the EDTA solution without the addition of lead ions. Next, determine the Cu content
(in mg/1) of the analytical solution from the instrument internal standardization or from the
calibration curve, and ascertain the percent deviation of the Pb concentration from the ex-
pected value (20 mg/1 = 100%). Assume the blank of the EDTA solution without the addition
of lead ions as 0%.

The recoveries from untreated control samples, fortified with copper oxychloride at levels of 2
to 20 mg/kg Cu, ranged from 97 to 100%. The routine limit of determination was 0.2 mg/kg.
Blanks were approx. 0.1 mg/kg.

The residue R, expressed in mg/kg Cu, is calculated from the following equation:
wAvEx
R = 100
G F
where
VEx = volume of stripping solution used to extract copper containing residues from the sur-
face of the grapes (in ml)
WA = copper concentration of analytical solution, derived from internally generated stan-
dard, or read from calibration curve (in mg/1)
F = recovery rate of lead ions (in /o)
156 Copper Oxychloride (as copper)
8 Important points
No data
9 Reference
R. Ipach, B. Altmayer and K. W. Eichhorn, Neue Methode zur AAS-Bestimmung von Kupfer-
riickstanden auf Weintrauben, Fresenius Z. Anal. Chem. 314, 157-158 (1983).
10 Author
Landes-Lehr- und Forschungsanstalt fiir Landwirtschaft, Weinbau und Gartenbau, Abteilung
Phytomedizin, Neustadt/W., R. Ipach
Cymoxanil 513
Grapes (berries and must), potatoes, tomatoes Gas-chromatographic

1 Introduction
Chemical name l-(2-Cyano-2-methoxyiminoacetyl)-3-ethylurea (IUPAC)
Structural formula
NOCH3

Molar mass 198.18
Melting point 160-161 C
Vapour pressure 8 10 ~7 mbar at 25 C (extrapolated)
Solubility Sparingly soluble in water (approx. 0.1 g);
(in 100 ml at 25 C) readily soluble in dichloromethane (approx. 15 g);
soluble in acetone (approx. 10 g) and methanol (ap-
prox. 4 g);
very sparingly soluble in n-hexane (<0.1 g)
Other properties White crystals, odourless; stable under normal storage
conditions
2 Outline of method
Cymoxanil residues are extracted from crop or soil samples with ethyl acetate. The aqueous
extract is initially washed with hexane. The aqueous solution is then extracted with
dichloromethane. Water samples are directly extracted with dichloromethane. In both cases,
the dichloromethane phase is rotary-evaporated, the residue is dissolved in ethyl acetate, and
the solution is cleaned up on a silica gel column. Cymoxanil is determined by gas chromato-
graphy using a thermionic detector.
3 Apparatus
Homogenizer, e. g. Ultra-Turrax (Janke & Kunkel)

Laboratory centrifuge, Type UJ 3 (Heraeus-Christ), with 340-ml glass tubes
158 Cymoxanil

Volumetric flasks, 10-ml
Microsyringe, 10-ul
4 Reagents
Acetone, p. a.
n-Hexane, p. a.
Eluting mixture 1: ethyl acetate + n-hexane 1:9 v/v
Eluting mixture 2: ethyl acetate + n-hexane 4:6 v/v
Cymoxanil standard solutions: 0.5-5 ng/ml acetone
Silica gel, deactivated with 10% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734)
Sodium sulphate, p.a., anhydrous, washed with dichloromethane
Glass wool
Compressed air, re-purified
Helium, re-purified

Cymoxanil 159
6 Procedure
6.1 Extraction
6.1.1 Plant material, soil
Weigh 50 g of the analytical sample (G) into a 340-ml centrifuge tube, and add 100 ml ethyl
acetate and 5 g filter aid. Homogenize the mixture for 5 min, and then centrifuge. Decant the
supernatant liquid into a 500-ml round-bottomed flask. Extract the residue with 100 ml ethyl
acetate for 5 min on a mechanical shaker. Centrifuge and decant the liquid phase. Combine
the extracts, add 50 ml water, and rotary-evaporate until the water begins to condense. Quan-
titatively transfer the aqueous phase into a 250-ml separatory funnel, and wash successively
with three 30-ml portions of hexane, with gentle swirling. Discard the organic phases. Extract
the aqueous solution successively with three 50-ml portions of dichloromethane for 5 min on
a mechanical shaker. Separate the lower organic phases (centrifuge, if necessary), and filter
through sodium sulphate into a 250-ml round-bottomed flask. Wash the sodium sulphate with
dichloromethane. Rotary-evaporate the solution almost to dryness.
6.1.2 Water
Extract 50 to 200 ml water (G) successively with three 50-ml portions of dichloromethane for
5 min on a mechanical shaker. Separate the organic phases (centrifuge, if necessary), and filter
dichloromethane. Rotary-evaporate the solution almost to dryness.

Plug the bottom end of the chromatographic tube with glass wool and add about 5 ml hexane.
Slurry 10 g silica gel in 20 ml hexane, and slowly pour the slurry into the column, gently tap-
ping the glass walls. Allow to settle, and add 3 g sodium sulphate. Drain the hexane to the
top of the sodium sulphate.
Dissolve the residue derived from 6.1 in 1 ml ethyl acetate, and transfer quantitatively to the
prepared column, using three 3-ml portions of eluting mixture 1 to complete the transfer. Let
the solution percolate into the column packing (flow rate of 1-2 drops per s).
Elute the column firstly with 200 ml of eluting mixture 1. Discard this fraction. Then elute
cymoxanil with 200 ml of eluting mixture 2. Collect the eluate in a 250-ml flask, and rotary-
evaporate almost to dryness.

Quantitatively transfer the residue derived from 6.2 into a volumetric flask, using acetone to
complete the transfer, and dilute the solution with acetone to a given volume, e. g. 10 ml
(VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph.
Gas chromatograph Hewlett Packard 5880 A
Column Glass, 2 mm i.d., 50 cm long; packed with 2% OV-101
on Chromosorb W-HP, 100-120 mesh
160 Cymoxanil

Temperature 320 C
Air, 70 ml/min
Attenuation 4
Injection volume 1 \x\
Retention time for cymoxanil 2 min 24 s
7 Evaluation
7.1 Method
Inject 1 ul of each cymoxanil standard solution (equivalent to 0.5 to 5.0 ng cymoxanil) into
the gas chromatograph. Plot the areas or heights of the peaks obtained vs. ng cymoxanil. Also
inject 1-ul aliquots of the sample solutions. For the areas or heights of the peaks obtained for
these solutions, read the appropriate amounts of cymoxanil from the calibration curve. For
control, repeat each injection.

The recoveries from untreated control samples, fortified with cymoxanil at levels of 0.05 to
0.5 mg/kg, ranged from 85 to 95%. Blanks usually did not occur or, if so, they were less than
0.03 mg/kg (however, blank values were higher for samples of unripe tomatoes). The routine
limit of determination was 0.05 mg/kg.

The residue R, expressed in mg/kg cymoxanil, is calculated from the following equation:
p WA-VEnd
where
Vj = portion of volume VEnd injected into gas chromatograph (in jul)
WA = amount of cymoxanil for V{ read from calibration curve (in ng)
Cymoxanil 161
8 Important points
No data
9 Reference
R. R Holt, Determination of residues of l-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea
(DPX-3217) by gas-liquid chromatography, Pestic. Sci. 70, 455-459 (1979).
10 Authors
Shell Forschung GmbH, Schwabenheim, D. Eichler and W. Heupt
2,4-D, Dichlorprop (2,4-DP) 27-A-38-A
Cereals (green matter, grains and straw), grapes, grass, Gas-chromatographic

potatoes determination
1 Introduction
2,4-D Dichlorprop
Chemical name (2,4-Dichlorophenoxy)- 2-(2,4-Dichlorphenoxy)-
acetic acid (IUPAC) propionic acid (IUPAC)
Cl
P CH3
Structural formula Cl/ V - O-CH2-COOH C l ^ V - O - C H COOH
Empirical formula C8H6C12O3 C9H8C12O3

Molar mass 221.04 235.07
Melting point 139.5-140.5 C 117-118C
Vapour pressure 0.53 mbar at 160 C; No data
93 mbar at 251 C
Solubility Very sparingly soluble in Very sparingly soluble in water
water; readily soluble in acetone,
readily soluble in acetone, chloroform, diethyl ether,
chloroform, diethyl ether, ethanol and ethyl acetate
ethanol and ethyl acetate
Other properties No data No data
2 Outline of method
Residues of 2,4-D and dichlorprop, which may be present as free acids, salts or esters, are ex-
tracted from the plant material with aqueous methanol. The methanol is evaporated from the
extract in alkaline medium, resulting in hydrolysis of the esters. The extracts are partitioned
between dichloromethane and water, and then the compounds are esterified with a mixture
of methanol and sulphuric acid. The resultant methyl esters of 2,4-D and dichlorprop are
determined by electron capture gas chromatography on a fused silica capillary column.
3 Apparatus
Wide neck bottle, 500-ml
Homogenizer, e. g. Ultra-Turrax T 45 N (Janke & Kunkel)
164 2,4-D, Dichlorprop (2,4-DP)

Round-bottomed flasks, 1-1 and 100-ml
Rotary vacuum evaporator, 30 to 40 C or 60 C bath temperature
Centrifuge, with 250-ml glass tubes
Microsyringe, 10-ul
4 Reagents
n-Hexane, dist.
Methanol, dist.
2,4-D and dichlorprop solutions for recovery experiments: 5 |ug/ml 2,4-D or dichlorprop in
methanol
Derivative standard solutions: 0.05, 0.1 and 0.2 |ng/ml 2,4-D methyl ester or dichlorprop
methyl ester in n-hexane
Sulphuric acid, cone, and 3 mol/1 H2SO4 p. a.
Methylating mixture: Methanol + sulphuric acid, cone. 9:1 v/v. Prepare fresh daily
Sodium hydroxide solution, 10 mol/1 NaOH p. a.
Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p. a.
Helium, 99.996%

The analytical sample is taken and prepared as described on pp. 17 ff, Vol. 1. Straw must be
finely chopped.
6 Procedure
6.1 Extraction
Place 50 g of the analytical sample (20 g for straw) (G) in a wide neck bottle. Add 300 ml
methanol-water mixture and homogenize for 10 min, with ice water cooling. Suction-filter the
homogenate through a filter paper in a Buchner porcelain funnel. Wash the filter cake suc-
cessively with three 30-ml portions of methanol-water mixture. Combine the extracts in a 1-1
round-bottomed flask, alkalize with 10 ml sodium hydroxide solution, and rotary-evaporate
to an aqueous residue at 60 C bath temperature.
2,4-D, Dichlorprop (2,4-DP) 165

Transfer the aqueous residue derived from 6.1 to a 250-ml separatory funnel, using 50 ml water
to complete the transfer. Extract with two 25-ml portions of dichloromethane by shaking.
Allow the phases to separate (centrifuge, if necessary), and discard the dichloromethane
phase. Acidify the water phase with 25 ml sulphuric acid (3 mol/1) (test with pH indicator
paper), and extract successively with three 25-ml portions of dichloromethane by shaking
(centrifuge if phases separate poorly). Filter the combined dichloromethane phases through
sodium sulphate. Wash the sodium sulphate with 10 ml dichloromethane. Collect the whole
filtrate in a 100-ml round-bottomed flask, and rotary-evaporate to dryness at 30 C bath
temperature.
6.3 Methylation
To the residue derived from 6.2, add 5 ml methylating mixture, and allow to stand for 10 min
at room temperature with occasional swirling. Then add 15 ml water, and extract with 10.0 ml
hexane (VEnd). Allow the phases to separate (centrifuge, if necessary), and discard the
aqueous phase. Wash the hexane phase with 15 ml sodium hydrogen carbonate solution, and
dry with a little sodium sulphate.

Inject an aliquot of the hexane solution derived from 6.3, e.g. 1 ul (Vj), into the gas chroma-
tograph.
Gas chromatograph Perkin-Elmer F 22
with SE-54, film thickness 0.5 \xm
63
Temperature 300 C
Carrier gas Helium, regulator pressure 1 bar
Detector purge gas Argon-methane, 30 ml/min
Septum purge gas Helium, 0.5 ml/min
Split ratio 1:10
Splitter exit Helium, 15 ml/min
Attenuation 32-10" 4
Injection volume 1 L| L1
Retention times for
dichlorprop methyl ester 5 min 18 s
2,4-D methyl ester 5 min 48 s
166 2,4-D, Dichlorprop (2,4-DP)
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Prior to each set of measurements,
prepare a calibration curve as follows. Inject 1 ul of each derivative standard solution
(equivalent to 0.05 to 0.2 ng 2,4-D methyl ester or dichlorprop methyl ester) into the gas
chromatograph. Plot the heights of the peaks obtained vs. ng derivative. Also inject 1-ul ali-
quots of the sample solutions. For the heights of the peaks obtained for these solutions, read
the appropriate amounts of 2,4-D methyl ester or dichlorprop methyl ester from the calibra-
tion curve.

The recoveries from untreated control samples, fortified with 2,4-D and dichlorprop at
levels of 0.05 to 5 mg/kg, ranged from 70 to 100%. The routine limit of determination was
0.05 mg/kg.

The residue R, expressed in mg/kg 2,4-D or dichlorprop, is calculated from the following
equations:
WA VEnd
for 2,4-D R= ' 0.940
WA
for dichlorprop R= ' VEnd 0.944
VrG
where
VEnd = volume of hexane used for extraction in step 6.3 (in ml)
WA = a m o u n t of derivative for Vj read from calibration curve (in ng)
0.940 = factor for conversion of 2,4-D methyl ester to 2,4-D
0.944 = factor for conversion of dichlorprop methyl ester to dichlorprop
8 Important points
Laboratories equipped with a combined GC-MS system can confirm the results by mass spec-
trometry, with the aid of the multiple ion detection technique, as follows:
2,4-D, Dichlorprop (2,4-DP) 167
Gas chromatograph Finnigan 9500
Mass spectrometer Finnigan 3300 equipped with
Finnigan Glass Jet Separator System and 6000 Com-
puter Data System
Column Glass, 2.5 mm i.d., 1.5 m long; packed with 3% OV-
17 on Gas Chrom Q, 100-120 mesh
Separator temperature 250 C
Ionization mode El (MID)
Electron voltage 70 eV
Carrier gas Helium, 25 ml/min
Fragment ions usable for quan-
titation of
2,4-D methyl ester m/e 175, 177, 234, 236
dichlorprop methyl ester m/e 162, 164, 248, 250
9 References
No data
10 Authors
BASF, Agricultural Research Station, Limburgerhof, W. Keller and S. Otto
Dichlobenil 225-A
Apples, grapes, grass, pears, red currants High-performance

1 Introduction
2,6-Dichlorobenzonitrile (IUPAC)
Chemical name
CN
Cl Cl
Structural formula
Empirical formula C7H3C12N

Molar mass 172.02
Boiling point 270 C
Vapour pressure 7.33 10 " 4 mbar at 20 C
2.0-10" 2 mbar at 50 C
(in 100 ml at 20 C) readily soluble in acetone (11.1 g) and dimethylform-
amide (15.8 g);
soluble in dichloromethane (9.2 g), dimethyl sulph-
oxide (7.7 g) and xylene (4.3 g);
slightly soluble in methanol (2.2 g)
Other properties Hydrolyzed in strong acid and alkaline media, ther-
mally very stable, steam volatile
2 Outline of method
Dichlobenil residues are separated by steam distillation from plant material, soil, and water
samples containing high amounts of organic and inorganic matter. The distillate is filtered
through a membrane filter. Dichlobenil in the filtrate is concentrated on a short HPLC column
that is connected to the analytical column with a 10-port valve. After elution from the column,
dichlobenil is determined by HPLC using a UV detector at 210 nm.
Water samples with a dichlobenil content greater than 0.1 mg/1 are filtered through a mem-
brane filter, and the compound is determined directly by high-performance liquid chromato-
graphy. If the concentration is less than 0.1 mg/1, dichlobenil must be concentrated using the
short HPLC column.
170 Dichlobenil
3 Apparatus
High-speed blendor, e.g. Waring Blendor
Beater-cross mill
Round-bottomed flasks, 1-1 and 500-ml, with ground joints
Glass tube, 15 cm long, with standard ground joints, used as fractionating column
Distillation head, with ground joints, fitted with Liebig condenser (jacket length 20 cm) with
vertical outlet
Gas inlet tube, glass or FIFE, with sintered bottom, for steam inlet
Aluminium foil
Heating mantles, for 1-1 and 500-ml round-bottomed flasks
Steam generator
Glass syringes, 20-ml and 5-ml, with Luer-lock fittings (Waters)
Swinny filter holder, stainless steel (Millipore)
Filter membranes, for filtration of aqueous solutions, pore size 0.45 \im (Millipore)
Sample vials, 5-ml, with septum caps
Amber glass bottle, 50-ml, with glass stopper
High-performance liquid chromatograph equipped with variable wavelength UV detector for
measurement at 210 nm
Injection set-up for sample volumes of 0-2000 \i\
Recording integrator, HP 3385 A (Hewlett-Packard)
Sample loop, 0.5-ml
10-port valve (Valco Instruments)
2 HPLC pumps, M 6000 A (Waters)
4 Reagents
Acetonitrile, LiChrosolv (Merck No. 30)
Methanol, LiChrosolv (Merck No. 6007)
Mobile phase 1: methanol + water 7:3 v/v
Mobile phase 2: acetonitrile + water 54:46 v/v
Water, bi-distilled
Dichlobenil standard solutions, 0.002, 0.004, 0.008, 0.02, 0.08, 0.1, 0.2, 0.4, 0.8 and 1.0 fig/ml
water
Calcium chloride solution, saturated

Dichlobenil 171
6 Procedure
6.1 Extraction by steam distillation
6.1.1 Apples, grapes, pears, soil
Transfer 100 g of comminuted plant material or air dried soil (free from large particles and
finely ground) (G) into a 500-ml round-bottomed flask and add 150 ml bi-distilled water. Place
the flask into a heating mantle, attach the 15-cm glass tube to the flask, and connect the
distillation head incl. Liebig condenser to the glass tube. Next slip the gas inlet tube through
the distillation head and the glass tube into the flask, so that the gas distribution frit is posi-
tioned near to the bottom of the flask. Wrap aluminium foil around the vertical glass tube,
then steam distil, using bi-distilled water in the steam generator. Maintain a constant water
level in the flask during steam distillation by adjusting the heat on the heating mantle control.
Collect the distillate in an ice-cooled 250-ml volumetric flask. Terminate the steam distilla-
tion when a little less than 250 ml of distillate have been collected, and make up to the mark
(VEx) with bi-distilled water at 20 C. Proceed as described in 6.2.3.
6.1.2 Red currants

Transfer 100 g of finely comminuted red currants (G), together with 15 ml calcium chloride
solution as a foam suppressant, and 150 ml bi-distilled water into a 500-ml round-bottomed
flask. Steam distil as described in 6.1.1.
6.1.3 Grass
Transfer 100 g of finely comminuted grass (G) into a 1-1 round-bottomed flask, add 400 ml
bi-distilled water, and steam distil as described in 6.1.1.
6.1.4 Water (clean water samples, dichlobenil content > 0.1 mg/1)
Subject the sample directly to the cleanup as described in 6.2.1.
6.1.5 Water (clean water samples, dichlobenil content < 0.1 mg/1)
Subject the sample directly to the cleanup as described in 6.2.2.
6.1.6 Water (highly contaminated water samples, e. g. river water)

Transfer 250 ml of the water sample (G) into a 500-ml round-bottomed flask, and steam distil
as described in 6.1.1.
6.2 Cleanup by filtration

6.2.1 Water (dichlobenil content > 0.1 mg/1)
Filter approx. 13 ml of the water sample, using the 20-ml Luer-lock syringe fitted with the
Swinny filter holder, through the filter membrane. Discard the first 10 ml, then filter a few
172 Dichlobenil
ml into a sample vial with an air-tight septum cap. HPLC determination should be performed
immediately afterwards. If this is not possible, the filtrate can be stored in a refrigerator for
1-2 d.
6.2.2 Water (dichlobenil content <0.1 mg/1)

Filter approx. 30 ml of the water sample as described in 6.2.1 and collect in an amber glass
bottle. HPLC determination should be performed immediately afterwards. If this is not possi-
ble, the filtrate can be stored in a refrigerator for 1-2 d.
6.2.3 Plant material, soil, and highly contaminated water

Filter approx. 30 ml of the steam distillate derived from 6.1.1, 6.1.2, 6.1.3, or 6.1.6 as described
in 6.2.2.

6.3.1 Determination by direct injection
Inject an aliquot of the filtrate derived from 6.2.1 (Vj) into the high-performance liquid
chromatograph.
Chromatograph Hewlett-Packard HP 1084 A
Column Stainless steel, 4 mm i.d., 12.5 cm long; packed with
LiChrosorb RP-18, 5 p (Merck No. 9333)
Mobile phase 1 Methanol + water 7:3 v/v
Detector Variable wavelength UV detector for measurement at
210 nm
Injection volume 20-40 JLXI
Retention time for dichlobenil 3 min 40 s
6.3.2 Determination after enrichment

See Diagrams 1 and 2 for the required apparatus set-up and the column switching. Use 1 to
1.5 ml of the filtrate derived from 6.2.2 or 6.2.3 to fill the sample loop (volume 0.5 ml, V{),
see Diagram 1.
Switch the 10-port valve (see Diagram 2). Flush the sample loop with 1.5 to 2 ml of bi-
distilled water, delivered by pump 2, thereby transferring the sample solution onto the short
enrichment column. Switch the valve to the starting position again (Diagram 1), and pump
mobile phase 2, delivered by pump 1, in the opposite direction through the enrichment column
to the analytical column. Dichlobenil is thereby eluted from the enrichment column and
chromatographed on the analytical column.
The enrichment step for the next determination can be started 2 min after the last valve
switching, i.e. before the chromatographic separation is terminated.
Dichlobenil 173
Glass syringe
Sample loop
Waste
Pump 2
(bi-dist. water)
Waste
Pump 1
(mobile phase)
Diagram 1
Jl
Diagram 2
Diagrams 1 and 2. Set-up of apparatus for HPLC determination of dichlobenil using enrichment column
with column switching (for explanation, see step 6.3.2).
174 Dichlobenil
Pumps Two pumps M 6000 A (Waters)
Injector 10-port valve (Valco No. C. 10UN60), fitted with 0.5-
ml sample loop
Enrichment column Stainless steel, 4 mm i.d., 17 mm long; packed with
Spherisorb ODS (RP-18) (Phase Separations)
Enrichment flow rate Bi-distilled water, 1.5 ml/min
Analytical column Stainless steel, 4 mm i.d., 12.5 cm long; packed with
LiChrosorb RP-18, 5 \xm (Merck No. 9333)
Mobile phase 2 Acetonitrile + water 54:46 v/v
Detector Variable wavelength UV detector for measurement at
210 nm
Retention time for dichlobenil 4 min 30 s
7 Evaluation
7.1 Method
ing them with the peak areas obtained for the dichlobenil standard solutions. Equal volumes

Recovery experiments were run on different untreated control samples of plant material and
soil, fortified with dichlobenil at levels of 0.01 to 0.1 mg/kg. The percent recoveries were as
follows:
Dichlobenil added (mg/kg)

Analytical material 0.01 0.05 0.1
Apples 83 91 93
Grapes 88 98 99
Grass 84 90 88
Pears 91 93 95
Red currants 73 88 92
Soil 95 97 94
Dichlobenil 175
_ .. Organic carbon Particles <0.02 mm

p H
Soil type or 07
Standard soil 2.1 *) 0.48 8.3 6.5

Standard soil 2.2*) 2.19 15.2 5.6
BBA-Richtlinie IV/4-2 (1987), Braunschweig
Recoveries ranging from 98 to 100% were obtained for dichlobenil from clean water, fortified
at levels of 0.01 to 0.1 mg/1, and from river water, fortified at levels of 0.05 to 1.0 mg/1.
The routine limit of determination was 0.01 mg/kg for plant material and soil, 0.001 mg/1
for clean water, and 0.002 mg/1 for river water.

The residue R, expressed in mg/kg dichlobenil, is calculated from the following equation:
R _ F A -V E x -W s t
where
VEx = volume of steam distillate (in ml)
Vj = portion of volume VEx injected into the high-performance liquid chromatograph or,
when enriched as described in 6.3.2, volume of sample loop (in ul)
WSt = amount of dichlobenil injected with standard solution (in ng)
FA = peak area obtained from Vj (in mm 2 )
F st = peak area obtained from WSt (in mm 2 )
8 Important points
The routine limit of determination for dichlobenil in water can be reduced by using a larger
sample loop, e.g. 2.0 ml, for enrichment.
176 Dichlobenil
9 References
F. Herzel, Zur Ruckstandsbestimmung von 2.6-Dichlorbenzonitril aus Boden und Wasser,
J. Chromatogr. 193, 320-321 (1980).
M. Schmidt, A. Hiltawki, W. Maasfeld and A. Kettrup, Chromatographic investigations of the
behaviour of dichlobenil and 2,4,5-T in water during slow sand filtration, Int. J. Environ.
Anal. Chem. 13, 289-307 (1983).
10 Authors
Department of Analytical Chemistry, University of Paderborn, A. Kettrup, M. Schmidt and
R. Hamann
Dichlofluanid, Tolylfluanid 203-371
Apples, grapes, lettuce, must, raspberries, strawberries, Gas-chromatographic
tomatoes, wine determination
1 Introduction
Dichlofluanid Metabolite (DMSA)
Chemical name N-Dichlorofluoromethylthio- Dimethylaminosulphanilide
N\N'-dimethyl-N-phenyl-
sulphamide (IUPAC)
Structural formula (CH3)2N S O 2 N - / (CH3)2N SO2P

I
S-CC12F H
Empirical formula C9HUC12FN2O2S2 C8H12N2O2S

Molar mass 333.23 200.2
Vapour pressure <10" 6 mbar at 20 C, 2.2-10- 6 mbar at 20C
3-1O"5 mbar at 50 C
Solubility Virtually insoluble in water; Sparingly soluble in water
(in 100 ml at 20 C) readily soluble in dichloro- (0.13 g);
methane (>20 g) and readily soluble in acetone,
toluene (10-20 g); acetonitrile, dichloromethane
soluble in xylene (7 g); (each > 20 g) and
slightly soluble in isopropanol (13 g);
isopropanol (1-2 g) and soluble in toluene (4.3 g)
methanol (1.5 g); and octanol (3.7 g);
sparingly soluble in n-hexane very sparingly soluble in
(0.1-0.2 g) n-hexane (24 mg)
Other properties Hydrolyzed in aqueous Stable between pH 4 and pH 9;
media depending on pH; half life in water at 22 C >
more stable at lower pH lyear
values. Stability to hydrolysis
(half lives at 22 C): approx.
15 d at pH 4, approx. 19 h
at pH 7, ^10 min at pH 9
178 Dichlofluanid, Tolylfluanid
Tolylfluanid Metabolite (DMST)

Chemical name N-Dichlorofluoromethylthio- Dimethylaminosulphotoluidide
N',N'-dimethyl-N-p-tolyl-
sulphamide (IUPAC)
(CH3)2N SO 2 N- -CH 3 (CH 3 ) 2 NS0 2 N- CH-,

Structural formula
S-CC1 2 F H
Empirical formula C 10 H 13 Cl 2 FN 2 O 2 S 2 C 9 H 14 N 2 O 2 S
Molar mass 347.26 214.2
Vapour pressure 1.6-lO" 6 mbar at 20C, 8.8-10" 7 mbar at 20 C
9-10" 5 mbar at 50 C
(in 100 ml at 20 C) readily soluble in benzene readily soluble in acetone,
(54 g), dichloromethane, acetonitrile, dichloromethane
toluene (each > 20 g) and isopropanol (each >20 g);
and xylene (23 g); soluble in toluene (9.8 g)
soluble in isopropanol and octanol (5.4 g);
(2-5 g) and methanol (4.6 g); very sparingly soluble in
sparingly soluble in n-hexane (53 mg)
n-hexane (0.5-1 g)
Other properties Hydrolyzed in aqueous Stable between pH 4 and pH 9;
media depending on pH; half life in water at 22 C
more stable at lower pH > 1 year
values. Stability to hydrolysis
(half lives at 22 C): approx.
12 d at pH 4, approx. 29 h
at pH 7, < 10 min at pH 9
2 Outline of method
Dichlofluanid, DMSA, tolylfluanid and DMST residues are extracted from plant material with
acetone. The extract is concentrated to an aqueous residue, which is made up to a definite
volume. An aliquot of this solution is transferred onto a disposable extraction column.
Beverages such as must and wine are transferred directly onto the disposable extraction col-
umn. The column is eluted with a cyclohexane-ethyl acetate mixture, and the eluate is cleaned
up on a small silica gel-activated charcoal column. The parent compounds and the metabolites
are determined by gas chromatography using a thermionic or a sulphur-specific flame photo-
metric detector.
3 Apparatus
Homogenizer
Dichlofluanid, Tolylfluanid 179

Round-bottomed flasks, 1-1 and 250-ml, with ground joints
Solvent dispensers, 50-ml, 10-ml and 5-ml
Graduated cylinders, 1-1, 500-ml, 250-ml and 100-ml
Volumetric flasks, 100-ml, 50-ml and 25-ml, with ground joints
Volumetric pipets, 50-ml, 10-ml, 5-ml, 2-ml and 1-ml
Gas chromatograph equipped with thermionic nitrogen-specific detector or sulphur-specific
flame photometric detector
Microsyringe, 10-ul
4 Reagents
Eluting mixture 1: cyclohexane + ethyl acetate 85 :15 v/v
Compound stock solution: 1000 pig/ml of each dichlofluanid, DMSA, tolylfluanid and
DMST in ethyl acetate. The solution can be stored in a refrigerator for approx. 6 months
Compound standard solutions: 0.2-100 M-g/ml of each dichlofluanid, DMSA, tolylfluanid
and DMST in ethyl acetate. The solutions can be stored in a refrigerator for approx. 6 months
Disposable extraction column, 50-ml (Chem Elut CE 2050; Analytichem)
Mini silica gel column: Disposable column, volume 6 ml, filled with 1 g silica gel (Baker
No. 7086-07), with adapter and funnel column (reservoir) 75-ml (Baker No. 7122-00 and
7120-03)
Activated charcoal, p. a. (Riedel-de Haen No. 31616)
Glass wool
Helium 4.6 (> 99.996 vol. %)
Hydrogen 5.0 (> 99.999 vol. %)
Nitrogen 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) to the 1-1 glass bottle with 200 ml acetone, and
homogenize for approx. 3 min. For tomatoes, grapes, and other material from which it is dif-
ficult to take a representative 100-g sample, homogenize 200 g with 200 ml acetone as well.
aqueous residue (approx. 170 ml for a 100-g sample, approx. 270 ml for a 200-g sample),
without evaporating any water, which would result in compound losses. When a precipitation
occurs as a result of the evaporation, re-dissolve it using a maximum of 10% acetone.
Make up the aqueous residue with water to 250 ml (100-g sample) or 500 ml (200-g sample)
in a graduated cylinder (VEx). Pipet 50 ml (VR1) of this solution onto a dry disposable extrac-
tion column and allow the solution to soak in. Elute the column three times with 50-ml por-
tions of eluting mixture 1 (see 8. Important points). Collect the eluate in a 250-ml round-
bottomed flask and rotary-evaporate to dryness. Proceed to step 6.2.
6.1.2 Beverages (must, wine)

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

Dissolve the residue derived from 6.1 in 3 ml dichloromethane. Using a Pasteur pipet, transfer
the solution onto the prepared column and allow it to soak into the charcoal layer. Rinse the
flask twice with 3-ml portions of dichloromethane and also add the rinsings successively to
the column. Fasten the reservoir with adapter to the column, and continue to elute with a
further 10 ml of dichloromethane (see 8. Important points). Collect the entire eluate (approx.
19 ml) in a pear-shaped flask and rotary-evaporate to dryness. Dissolve the residue in 5-10 ml
ethyl acetate and rotary-evaporate to dryness again to remove the last traces of dichloro-
methane.

Dissolve the residue derived from 6.2 in a definite volume of ethyl acetate, e. g. 5 ml (VEnd),
and transfer the solution to a test tube. Inject an aliquot of this solution (Vj) into the gas
chromatograph.
Gas chromatograph 1 Varian 3700
on Supelcoport, 80-100 mesh
Column temperature Isothermal at 180 C for 4 min, programmed to rise at
10C/min from 180 to 225 C, then isothermal at
225 C for 18 min
Temperature 320 C
Air, 175 ml/min
Attenuation 10"11
Retention times for
DMSA 2 min 24 s
DMST 3 min 30 s
dichlofluanid 5 min 24 s
tolylfluanid 6 min 36 s
Gas chromatograph 2 Varian 6000

with OV-1 CB, film thickness 2.0 \xm (Macherey-Nagel
No. 723520)
220 C for 15 min
Temperature 300 C
Air, 175 ml/min
0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00
IO.OO nun
Fig. 1. Dichlofluanid (DCFA), DMSA, Tolylfluanid

(TYFA) and DMST in strawberries (VEnd = 5 ml each,
packed column, TSD).
Chromatogram A: Standard solution, representing 1 ng
of each compound.
with 0.05 mg/kg of each compound.
o.oo min
Dlchlofluanid, Tolylfluanid 183
0.00 1.25 2.50 3.75 5.00

(TYFA) and DMST in strawberries (VEnd = 5 ml each,
capillary column, FPD).
ple, fortified with standard solution; peaks represent-
ing 0.4 ng of each compound.
3.73 3.00 7.50 6.75 IO.OO min

7.00 8.25 9.50 10.75 12.00 13.25 14.50 15.75 17.00
7.00 8.25 min

(TYFA) and DMST in grapes (VEnd = 5 ml each,
capillary column, TSD).
7.00 8.25 17.00 min

0,00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00
IO.OO min

(TYFA) and DMST in grapes (VEnd = 5 ml each,
capillary column, FPD).
io.oo min
Attenuation 10-12
Injection volume 1 1*1
Retention times for
DMSA 10 min 18 s
DMST 11 min 24 s
Gas chromatograph 3 Carlo Erba Mega 5300

with CP-SIL 5 CB, film thickness 5.0 \xm (Chrompack
No. 7645)
220 C for 12 min
Detector Flame photometric detector (Melpar), equipped with
394-nm sulphur filter
Temperature 250 C
Hydrogen, 150 ml/min
Air, 200 ml/min
Attenuation 1-16; excitation 600
Retention times for
DMSA 2 min 30 s
DMST 3 min 30 s
7 Evaluation
7.1 Method
Quantitation is performed within the recommended measuring ranges (see below) by measur-
ing 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 com-
parable areas.
When using capillary columns, perform the evaluation with the aid of sample solutions
derived from untreated control material which, after the column chromatographic cleanup
(6.2.2), have been fortified with the compound standard solutions to yield "fortified calibra-
tion solutions".
Using the thermionic nitrogen detector, a linear relationship was observed between peak
area and amount of compound injected. The flame photometric detector, however, was linear
only over a very small range (from 0.4 to 2 ng for the model used).
The recommended ranges for quantitative evaluation of the compounds are 1 to 10 ng for
gas-chromatographic system 1, 0.2 to 2 ng for gas-chromatographic system 2, and 0.4 to 2 ng
for gas-chromatographic system 3.

The recoveries from untreated control samples, fortified with dichlofluanid, tolylfluanid,
DMSA and DMST at levels of 0.05, 0.5 and 5 mg/kg, are given in the Table.
Table. Percent recoveries from plant material, must, and wine, fortified with dichlofluanid, DMSA,
tolylfluanid and DMST.
Analytical Added Dichlofluanid DMSA Tolylfluanid DMST

material mg/kg n X s X s X s X s
Apples 0.05 8 95 4.5 92 5.6 96 5.6 92 7.9
0.5 8 96 6.4 93 7.0 97 5.5 97 8.2
5.0 8 98 4.8 94 4.4 95 2.2 96 4.8
Grapes 0.05 8 94 8.1 92 9.6 91 6.0 94 4.9
0.5 8 89 1.4 87 8.6 88 3.6 89 3.8
5.0 8 85 3.8 88 7.1 87 4.1 88 6.0
Lettuce 0.05 8 101 9.3 101 4.3 99 7.6 97 4.3
0.5 8 97 4.7 97 9.0 97 5.1 99 6.7
5.0 6 101 1.9 99 2.6 102 2.5 99 2.6
Must 0.05 *> 6 96 5.1 101 5.0 99 4.7 99 7.0
0.5 > 6 99 3.1 100 3.8 101 2.7 100 6.8
5.0*) 4 99 1.7 100 1.7 103 1.5 101 0.8
Raspberries 0.05 8 99 8.6 95 5.0 98 8.4 98 7.1
0.5 8 95 8.5 89 11.1 95 9.1 93 8.9
5.0 4 92 9.3 83 6.2 95 14.2 86 5.6
Strawberries 0.05 8 93 8.5 98 9.8 92 7.8 96 6.0
0.5 6 93 6.5 95 4.2 96 5.4 94 8.6
5.0 6 84 2.5 88 6.0 87 1.9 88 7.9
Tomatoes 0.05 6 89 4.4 92 6.9 92 4.9 102 6.3
0.5 6 85 7.8 94 7.9 87 6.9 96 7.7
5.0 6 88 8.9 86 5.5 90 6.1 90 2.9
Wine 0.05*) 6 97 4.1 91 3.6 97 3.6 87 2.3
0.5 *> 6 97 1.3 97 1.8 98 1.6 94 2.5
5.0*) 6 100 4.5 95 3.7 98 3.9 96 3.3
*) mg/1
The routine limit of determination for the compounds was 0.05 mg/kg. Using the flame
photometric detector, blanks usually did not occur or, if so, they were less than 0.01 mg/kg.
Using the thermionic detector, blanks were also clearly less than 0.01 mg/kg, except for
DMSA (maximum 0.01 mg/kg) and DMST (0.01 to 0.1 mg/kg).

The residue R, expressed in mg/kg, of an identified compound is calculated from the follow-
ing equation:
p F A -V E x -V E n d -W s t
FsfVR1-VrG
where
VEx = volume of concentrated extract after dilution with water (in ml)
Vi = portion of volume VEnd injected into gas chromatograph (in ul)
W st = amount of compound injected with standard solution or "fortified calibration solu-
tion" (in ng)
FA = peak area obtained from Y{ (in mm 2 or integrator counts)
FSt = peak area obtained from WSt (in mm 2 or integrator counts)
8 Important points
The pH of the homogenate in step 6.1.1 should be measured using a pH meter. If it is higher
than pH 5, adjust it to pH 4-5 with hydrochloric acid (10 g/100 g HC1 p.a.) in order to pre-
vent hydrolysis of dichlofluanid and tolylfluanid.
In a few cases, the complete elution of the compounds from the disposable extraction col-
umn required four 50-ml portions of eluting mixture 1 instead of three portions. Therefore,
the conditions given in 6.1.1 and 6.1.2 should be checked before a new series of analyses is
started or whenever a new batch of the disposable extraction columns is used (observe batch
number); if required, increase the number of elutions to four.
To obtain complete elution of DMSA and DMST, do not use more than 150 mg activated
charcoal for preparing the silica gel-charcoal column as described in 6.2.1.
The eluting conditions given in 6.2.2 should be checked before starting a new series of
analyses, or whenever a new batch of the mini silica gel columns is used (observe batch
number); if required, re-define them.
Using the flame photometric detector, the signal-to-noise ratio can be improved by dissolv-
ing the residue derived from 6.2 in n-hexane instead of ethyl acetate. However, not all sample
material residue will dissolve completely in hexane; additionally, DMSA and DMST are only
very sparingly soluble in hexane. The analyst must therefore balance the chance of a better
signal-to-noise ratio against the risk of not completely determining the residues present.
9 References
T. Aizawa, Improved analytical method for the determination of Euparen [N-(dichloro-
fluoromethylthio)-N',N'-dimethyl-N-phenylsulfamide] in vegetables and fruits, Nihon
Tokushu Noyaku Seizo K.K. (Nitokuno), Tokyo, Report No. 1092, 7.2.1979 (unpublished).
R. Brennecke, Methode zur gaschromatographischen Bestimmung von Riickstanden der
Fungizide Euparen, Euparen M und Folicur in Pflanzenmaterial und Getranken, Pflanzen-
schutz-Nachr. 42, 237-298 (1989).
K. Vogeler, Methode zur gaschromatographischen Bestimmung von Riickstanden von
Dichlofluanid, Tolylfluanid sowie deren Abbauprodukten DMSA und DMST und von
Chinomethionat in Pflanzenmaterial, Bayer AG, Report No. RA-524, 25.6.1984 (un-
published).
K. Vogeler and W. Piorr, Gaschromatographische Methode zur Bestimmung von Dichlo-
fluanid- bzw. Tolylfluanid-Ruckstanden sowie deren Abbauprodukten Dimethylaminosulf-
anilid bzw. Dimethylaminosulftoluidid in Pflanzenmaterial und Boden, Bayer AG, Report
No. RA-956, 19.12.1977 (unpublished).
10 Author
R. Brennecke
Dichlofluanid, Tolylfluanid 203-A-371-A
Strawberries Gas-chromatographic
Dichlofluanid (additionally): Grapes, must, wine determination
Tolylfluanid (additionally): Apples, apple juice,
apple pulp, pears, pear conserves, pear juice
(German versions published 1989)
1 Introduction
For data on physico-chemical properties of dichlofluanid and tolylfluanid, see the Method on
p. 177, this Volume.
2 Outline of method
Dichlofluanid and tolylfluanid residues are extracted from plant material with acetone. The
extract is concentrated to an aqueous residue. The compounds are partitioned into dichloro-
methane, and the dichloromethane phase is evaporated to dryness. From fruit juice, must and
wine, dichlofluanid and tolylfluanid are directly extracted with dichloromethane; the
dichloromethane phase is evaporated to dryness.
The residue is dissolved in a cyclohexane-ethyl acetate mixture and cleaned up by gel
permeation chromatography on a polystyrene gel. The compounds are determined by gas
chromatography using a sulphur-specific flame photometric detector. If interfering peaks
occur, a supplemental cleanup on a silica gel column is necessary.
3 Apparatus
Homogenizer
Separatory funnel, 250-ml, with ground stopper
Fluted filter paper
Chromatographic tube, 17.5 mm i.d., 30 cm long, with PTFE stopcock
Gas chromatograph equipped with sulphur-specific flame photometric detector
Microsyringe, 5-ul
4 Reagents
Compound standard solutions: 1-100 fig/ml of each dichlofluanid and tolylfluanid in
n-hexane
Silver nitrate solution, 0.1 mol/1 AgNO3 p. a.
Hydrogen 5.0 (> 99.999 vol.%)
Nitrogen 4.6 (> 99.996 vol.%)

6 Procedure
6.1 Extraction
6.1.1 Apples, apple pulp, grapes, pears, pear conserves, strawberries
To 100 g of the analytical material (G) add 200 ml acetone; for pear conserves, also add 50 ml
silver nitrate solution (see 8. Important points). Homogenize the mixture for 3 min. Add ap-
prox. 20 g filter aid, mix well, and suction-filter the homogenate through a fast flow-rate filter
paper in a Buchner porcelain funnel. Wash the filter cake with approx. 100 ml acetone. Deter-
mine the total volume of the filtrate (VEx), and rotary-evaporate an aliquot (e. g. half this
Dlchlofluanid, Tolylfluanid 193
volume) (VR1) in a 500-ml round-bottomed flask to an aqueous residue. Transfer the residue
to a separatory funnel, add 20 ml saturated sodium chloride solution, and extract the aqueous
phase with 100 ml dichloromethane used earlier for rinsing the flask. Repeat rinsing and ex-
traction twice more, using 100-ml portions of dichloromethane each time. Dry the combined
dichloromethane phases on sodium sulphate and rotary-evaporate the solution to dryness.
6.1.2 Fruit juice, must, wine

Filter 50 g of the analytical sample (G) through a fluted filter paper into a separatory funnel,
and extract the filtrate three times with 100-ml portions of dichloromethane. Dry the combined
dichloromethane phases on sodium sulphate and rotary-evaporate the solution to dryness.

Transfer the residue derived from 6.1.1 or 6.1.2 into a test tube, using a total of 10 ml eluting
mixture (VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop
(VR3) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel
permeation chromatograph at the eluting conditions determined beforehand with standard
solutions of dichlofluanid or tolylfluanid; cf. Cleanup Method 6, pp. 75ff, Vol. 1. Elution
volumes ranging from 110 to 150 ml were determined for both dichlofluanid and tolylfluanid
on Bio-Beads S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate
of 5.0 ml/min.
to dryness. Then proceed to step 6.3 or 6.4.
Check the elution ranges every 500 samples, and determine anew whenever a new gel col-
umn is used.

Insert a cottonwool plug into the bottom of the chromatographic tube, and introduce a slurry
of 15 g silica gel suspended in toluene. Insert another cottonwool plug onto the top of the
silica gel, trickle in approx. 3 g sodium sulphate and drain the toluene to the top of the sodium
sulphate layer. Dissolve the residue derived from 6.2 in 5 ml toluene. Add the solution to the
column and allow to percolate. Rinse the flask twice with 5-ml portions of toluene, add these
rinsings successively to the column and also allow to percolate. Next elute dichlofluanid or
tolylfluanid with 130 ml toluene. Collect the eluate in a 250-ml round-bottomed flask and
rotary-evaporate to dryness.

Dissolve the residue derived from 6.2 or 6.3 in 2 ml hexane (VEnd). Inject 5 |j,l of this solution
(Vj) into the gas chromatograph.
Gas chromatograph Tracor 560
Column Glass, 2 mm i.d., 1.2 m long; packed with 5% DC-
200 on Gas Chrom Q, 80-100 mesh
Column temperature 180C

Injection port temperature 250C
Detector Flame photometric detector equipped with 394-nm
sulphur filter
Temperature 200 C
Air, 170 ml/min
Injection volume 5 \i\
Retention times for
7 Evaluation
7.1 Method
ing them with the peak areas obtained for the compound standard solutions. Equal volumes

The recoveries from untreated control samples, fortified with dichlofluanid and tolylfluanid
at levels of 0.1 to 1.0 mg/kg, ranged from 77 to 118% for dichlofluanid, and from 75 to 105%
for tolylfluanid (see Table). The limit of determination was 0.1 mg/kg for each compound.

The residue R, expressed in mg/kg dichlofluanid or tolylfluanid, is calculated from the follow-
ing equation:
R _ FA-VEx-VR2-VEnd-WSt
F s ,-V R1 -V R 3.V r G
where
VEx = total volume of filtrate from 6.1.1 (in ml)
VRI = portion of volume VEx used for further processing (in ml)
VR2 = volume of solution prepared for gel permeation chromatography in 6.2 (in ml)
ple loop) (in ml)
Table. Percent recoveries from fruits, fruit conserves, fruit juice, and wine, fortified with dichlofluanid,
tolylfluanid, and DMST; duplicate experiments.
Analytical material Added (mg/kg) Dichlofluanid Tolylfluanid DMST

Apples
Fruit 0.1 97- 98 92-104
1.0 98-105 101-106
Juice 0.1 85- 89 88
Pulp 0.1 84- 94 98-109
Grapes
Fruit 0.1 100-105
1.0 93- 97
Must 0.1 107-108
1.0 88- 90
Wine 0.1 116-118
Pears
Fruit 0.1 87- 88 98-108
1.0 80- 81 99-103
Conserves 0.1 76- 78 92- 93
Juice 0.1 85- 86 96-103
Strawberries 0.1 77-104 75- 95

Wst = amount of dichlofluanid or tolylfluanid, respectively, injected with standard solution
(in ng)
FA = peak area obtained from Vj (in mm2)
FSt = peak area obtained from WSt (in mm2)
8 Important points
Lower recoveries were obtained from processed fruits, especially pear conserves, as compared
to fresh fruits. This probably results from thiol containing plant constituents. Therefore, silver
nitrate is added to the pear conserves, as recommended by Aizawa (1979) for dichlofluanid
analysis.
The method permits the simultaneous determination of the metabolites dimethylamino-
sulphanilide (DMSA) and dimethylaminosulphotoluidide (DMST). For that purpose, it is
necessary to carry out the column chromatography on silica gel as described in 6.3. After
dichlofluanid (or tolylfluanid) has been eluted with 130 ml toluene, change the receiver and
elute the respective metabolite with 100 ml of a mixture of acetone + toluene, 1:1 v/v. Rotary-
evaporate both fractions to dryness, and proceed with each residue as described in 6.4. The
retention time for DMSA was about 2 min under the operating conditions given in 6.4; for
DMST it was 2 min 24 s at a column temperature of 185 C. The recoveries from untreated
control samples, fortified with DMST at levels of 0.1 to 1.0 mg/kg, ranged from 88 to 109%
(see Table). The limit of determination for each metabolite was approx. 0.1 mg/kg.
A thermionic nitrogen-specific detector can be used instead of the flame photometric detec-
tor. For the detection of dichlofluanid or tolylfluanid alone, an electron capture detector can
also be used.
9 References
T. Aizawa, Improved analytical method for the determination of Euparen [N-(dichloro-
fluoromethylthio)-N',N'-dimethyl-N-phenylsulfamide] in vegetables and fruits, Nihon
Tokushu Noyaku Seizo K.K. (Nitokuno), Tokyo, Report No. 1092, 7.2.1979 (unpublished).
K. Vogeler, Methode zur gaschromatographischen Bestimmung von Ruckstanden von
Dichlofluanid, Tolylfluanid sowie deren Abbauprodukten DMSA und DMST und von Chino-
methionat in Pflanzenmaterial, Bayer AG, Report No. RA-524, 4.6.1984 (unpublished).
10 Author
K. Vogeler
Dinobuton, Binapacryl 255-8
Apples, cucumbers High-performance

1 Introduction
Dinobuton Binapacryl
Chemical name 2-sec.Butyl-4,6-dinitrophenyl 2-sec.Butyl-4,6-dinitrophenyl
isopropyl carbonate (IUPAC) 3-methylcrotonate (IUPAC)
CH 3
Structural formula O-CH
CH-C 2 H 5 CH-C 2 H 5
CH 3 CH 3
Empirical formula C 14 H 18 N 2 O 7 C 15 H 18 N 2 O 6
Molar mass 326.30 322.32
Boiling point Not distillable Not distillable
Vapour pressure <10" 5 mbar at 20 C 4.2-10- 7 mbar at 20 C
(in 100 ml at 20 C) very readily soluble in readily soluble in acetone
acetone (120 g); (78 g), ethanol (11 g),
readily soluble in xylene dichloromethane (75 g) and
(89 g); xylene (70 g)
soluble in ethanol (8.3 g);
slightly soluble in n-hexane
(1.9 g)
Other properties Hydrolyzed in alkaline Hydrolyzed in concentrated
media acids and dilute bases;
long exposure to UV light
causes decomposition
2 Outline of method
Dinobuton and binapacryl residues are extracted from plant material with a hexane-methanol
mixture. An aliquot of the organic extract is shaken with water to remove interfering polar
co-extractives, and cleaned up further by both gel permeation chromatography and adsorption
chromatography on a Florisil cartridge. From soil samples, the residues are extracted with
methanol. An aliquot of the extract is diluted with water, and the compounds are partitioned
198 Dinobuton, Binapacryl
into dichloromethane. Further cleanup is as with plant material extracts, by gel permeation
and adsorption chromatography. From water samples, the residues are extracted with
dichloromethane; the extract is cleaned up on a Florisil cartridge.
Dinobuton and binapacryl are determined by high-performance liquid chromatography
using a UV detector at 254 nm.
3 Apparatus
High-speed blendor fitted with leak-proof stainless steel jar and explosion-proof motor
Filter paper, 9 cm dia., medium flow rate
Round-bottomed flasks, 500-ml, 250-ml, 100-ml and 25-ml, with ground joints
Erlenmeyer flask, 500-ml, with ground stopper
Test tubes, 10-ml and 3-ml, graduated
Glass syringes, 10-ml and 2-ml, with Luer-lock fitting
Membrane filter, e.g. SM 11605-025 N, 0.65 nm (Sartorius), and Millex HV4 filter unit
(Millipore)
4 Reagents
Acetone, p. a.
Methanol, p. a.
Methanol, HPLC quality (only for mobile phase)
Water, HPLC quality (only for mobile phase)
Extraction mixture: n-hexane + methanol 95:5 v/v
Eluting mixture 2: n-hexane + acetone 98 :2 v/v
Mobile phase: methanol + water 8:2 v/v
Standard solutions for recovery experiments: 1, 10, 100 and 1000 M<g/ml dinobuton and
binapacryl in methanol
Dinobuton, Binapacryl 199
Dinobuton and binapacryl standard solutions for HPLC measurement: 0.1-2 ng/ml of each
in mobile phase
Sodium sulphate, p.a., anhydrous, washed with dichloromethane and heated for 4 h at 500C
Florisil disposable cartridge: Sep-Pak Cartridge Florisil (Millipore No. 51960)

6 Procedure
6.1 Extraction
Homogenize 50 g of the analytical sample (G) with 5 g filter aid and 100 ml extraction mix-
ture for 3 min. Suction-filter the homogenate through a medium flow-rate filter paper in a
Buchner porcelain funnel, and wash the filter cake with a further 80 ml extraction mixture.
Transfer the filtrate to a graduated cylinder, and make up the organic phase to a definite
volume (VEx) (max. 200 ml), ignoring the lower aqueous phase. Transfer an aliquot (VR1) of
the organic phase, equivalent to 20 g of the analytical sample, into a 250-ml separatory funnel
and wash twice with 20-ml portions of water. Discard the aqueous phases. Dry the organic
phase on sodium sulphate, filter through cottonwool, and wash the filter residue with 10 ml
hexane. Rotary-evaporate the filtrate to near dryness, and remove the last traces of solvent by
swirling the flask in the hand.
6.1.2 Soil
In a separate 10 to 20-g aliquot of the laboratory sample, determine the water content by dry-
ing in an open weighing glass at 105 C to constant weight (approx. 15 h). Discard this aliquot.
Adjust the water content of 100 g soil (G) to 30% in a 500-ml Erlenmeyer flask (see 8. Im-
portant points), add 150 ml methanol, and shake for 30 min on the mechanical shaker.
Suction-filter the extract through a medium flow-rate filter paper in a Buchner porcelain fun-
nel. Re-extract the filter cake with a further 150 ml methanol and filter as described above.
Wash the filter cake with 50 ml methanol and make up the combined filtrates to 350 ml (VEx)
with methanol in a graduated cylinder. Transfer a 70-ml aliquot (VR1), equivalent to 20 g of
the analytical sample, into a 250-ml separatory funnel, add 30 ml water, and extract three
times with 50-ml portions of dichloromethane. Wash the combined dichloromethane solutions
twice with 40-ml portions of water. Dry the organic phase on sodium sulphate, filter through
cottonwool, and wash the filter residue with 10 ml hexane. Rotary-evaporate the filtrate to near
dryness, and remove the last traces of solvent by swirling the flask in the hand.
6.1.3 Water
Extract 1 1 of the water sample (G) three times with 100-ml portions of dichloromethane. Dry
the combined organic phases on sodium sulphate, filter through cottonwool, and wash the
filter residue with 10 ml hexane. Rotary-evaporate the filtrate to near dryness, and remove the
last traces of solvent by swirling the flask in the hand. Proceed to step 6.2.2.
6.2 Cleanup
Transfer the residue derived from 6.1.1 or 6.1.2 into a test tube, using a total of 10 ml eluting
mixture 1 (VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop
(VR3) of the gel permeation chromatograph with 8 to 9 ml of the solution. Set the gel
permeation chromatograph at the eluting conditions determined beforehand with standard
solutions of dinobuton and binapacryl; cf. Cleanup Method 6, pp. 75ff, Vol. 1. Elution
volumes ranging from 80 to 100 ml were determined for both compounds on Bio-Beads S-X3
polystyrene gel, using eluting mixture 1 as eluant, pumped at a flow rate of 5.0 ml/min.
is used.
6.2.2 Florisil cartridge

Draw 10 ml hexane into the glass syringe, attach a Florisil cartridge to the syringe, and force
the hexane through to condition the cartridge packing. Repeat the conditioning with a further
10-ml portion of hexane. Next detach the cartridge, pull the plunger out of the syringe, and
re-attach the cartridge. Dissolve the residue derived from 6.1.3 or 6.2.1 in 1 ml hexane and
transfer the solution quantitatively into the syringe with the aid of a Pasteur pipet. Re-insert
the plunger into the syringe and force the liquid through the cartridge. Detach the cartridge,
remove the plunger from the syringe, and re-attach the cartridge. Force 10 ml hexane through
the cartridge, proceeding in a similar manner as described above, and discard the eluates. Next
elute the compounds with 10 ml eluting mixture 2 from the Florisil and collect the eluate in
a 25-ml round-bottomed flask. Rotary-evaporate to near dryness and remove the last traces
of solvent by swirling the flask in the hand.

Dissolve the residue derived from 6.2.2 in mobile phase and dilute to an appropriate volume
(VEnd). Filter the solution through a membrane filter. Inject an aliquot of this solution (Vj)
into the high-performance liquid chromatograph.
Chromatograph Spectra-Physics SP 8700
Injector Injection valve 7125 with sample loop (Rheodyne)
Column 1 Cartridge column, 4 mm i. d., 25 cm long, and pre-

column, 4 mm i. d., 3 cm long (Knauer)
Column 2 Hyperchrome NC column, 4.6 mm i. d., 25 cm long
(Bischoff)
Column packing LiChrosorb RP-18, 5-um (Merck)
Mobile phase Methanol + water 8 :2 v/v
Detector UV/VIS detector Uvicon 720 LC (Kontron)
Wavelength 254 nm
Retention times for Column 1 Column 2

dinobuton 8 min 48 s 8 min 48 s
binapacryl 10 min 42 s 9 min 48 s
7 Evaluation
7.1 Method
Inject equal volumes of each dinobuton and binapacryl standard solution into the high-per-
formance liquid chromatograph. Plot the heights of the peaks obtained vs. ng dinobuton or
binapacryl, respectively. Also inject equal volumes of the sample solutions. For the heights
of the peaks obtained for these solutions, read the appropriate amounts of compound from
the corresponding calibration curve.

The recoveries from untreated control samples, fortified with dinobuton and binapacryl at
levels of 0.05 to 5.0 mg/kg, ranged from 70 to 116% (average 82%) for plant material, and
from 78 to 98% (average 90%) for soil. The recoveries for tap water, fortified at levels of
0.5 u.g/1 to 0.5 mg/1, ranged from 73 to 112% and averaged 92%.
The limit of detection was 0.03 mg/kg for apples and cucumbers, and 0.01 mg/kg for soil.
Due to reagent blanks, the limit of detection for tap water was 0.2 ng/1; a lower limit of detec-
tion of 0.05 |ig/l can possibly be achieved by using reagents of higher purity.
The limit of determination was 0.05 mg/kg for plant material and soil, and 0.5 |ig/l for tap
water.

The residue R, expressed in mg/kg dinobuton or binapacryl, is calculated from the following
equations:
WA VEFx VR2 VEnd
for plant material and soil R= * _x_R_2_ J; nd
V
R1 * VR3 ' V i * U
for water R=
where
VEx = total volume of organic phase after addition of extraction mixture to filtered extract
from plant sample in 6.1.1 (in ml), or total volume of filtered soil extract from 6.1.2
after addition of methanol (in ml)
VR2 = volume of eluting mixture 1 used to take up the residue from 6.1.1 or 6.1.2 (in ml)
ple loop) (in ml)
Vj = portion of volume VEnd injected into high-performance liquid chromatograph (in ul)
WA = amount of dinobuton or binapacryl for Vj read from calibration curve (in ng)
8 Important points
Before beginning a series of analyses, check the reagents for purity, particularly n-hexane and
sodium sulphate.
When the filtration of extracts from plant material causes difficulties, slurry the filter aid
onto the medium flow-rate filter paper with water, instead of adding it to the sample.
When soil samples containing less than 30 percent water are taken for analysis, the phases
will generally not separate after the soil extract has been shaken with dichloromethane. In this
case, phase separation will occur when water is added.
The HPLC measurement can also be performed on a n-Bondapak Column C-18 (Millipore),
4 mm i. d., 30 cm long, substituting the mobile phase with a methanol + water mixture
75:25 v/v.
For the quantitative determination, a wavelength of 254 nm is used; fewer interfering peaks
occur at this wavelength than at the absorption maxima of dinobuton and binapacryl at
242 nm.
9 Reference
H. Roseboom and H. A. Herbold, Determination and confirmation of binapacryl and
dinobuton residues on apples and cucumbers by high-performance liquid chromatography,
J. Chromatogr. 208, 137-140 (1981).
10 Authors
Nolting, M. Blacha-Puller and J. Siebers
Fonofos 288
Brussels sprouts, carrots, cauliflower, head cabbage, Gas-chromatographic
kohlrabi, maize (kernels), onions, radishes (large and determination
small types), red cabbage
Soil, water
1 Introduction
O-Ethyl S-phenyl (RS)-ethylphosphonodithioate
Chemical name (IUPAC)
Structural formula
Empirical formula C10H15OPS2

Molar mass 246.34
Boiling point 130C at 0.133 mbar
Vapour pressure 2.8-10"4 mbar at 25 C
miscible with organic solvents such as acetone, diethyl
ether, n-hexane, methanol and xylene
Other properties Light yellow liquid; hydrolyzed in acid and
alkaline media
2 Outline of method
Fonofos residues are extracted from plant material or soil with acetone. The extract is diluted
with water, and fonofos is partitioned into petroleum ether. Petroleum ether is used for direct
extraction from water samples. The organic phase is rotary-evaporated and cleaned up on a
Florisil column. Fonofos is determined by gas chromatography using a thermionic detector.
3 Apparatus
Ball grinding mill
Glass filter funnel, porosity G2, 13 cm dia.
206 Fonofos

Round-bottomed flasks, 500-ml, with ground joints
Gas chromatograph equipped with thermionic phosphorus-specific detector
Microsyringe, 10-ul
4 Reagents
Acetone, dist.
Acetonitrile, dist., saturated with petroleum ether
Diethyl ether, dist.
n-Hexane, p. a.
Petroleum ether, dist., boiling range 40-80C
Eluting mixture: petroleum ether + diethyl ether 98:2 v/v
Fonofos standard solutions: 0.5-5 M-g/ml n-hexane
Fenchlorphos solution (internal standard): 5 mg/ml n-hexane
Florisil, 60-100 mesh, 6% water content: Determine the water content of commercial Florisil
by heating a given amount for 1 h in a muffle furnace at 400 C, followed by re-weighing. To
100 g commercial Florisil in a 300-ml Erlenmeyer flask (with ground joint), add an ap-
propriate amount of water dropwise from a burette to adjust the water content to 6%. Shake
vigorously for 5 min until all lumps have disappeared, next shake for at least 20 min on a
mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occa-
sional swirling
Glass wool
Compressed air, re-purified

Fonofos 207
6 Procedure
6.1 Extraction
6.1.1 Plant material and soil
Weigh 100 g of the analytical sample (G) into the blendor jar, and homogenize with 200 ml
acetone for 3 min. Suction-filter the mixture, and thoroughly wash the blendor jar and filter
cake with a total of 75 ml acetone. Transfer the extract into a 1-1 separatory funnel, and add
150 ml sodium chloride solution and 250 ml water. Extract the mixture twice with 100-ml por-
tions of petroleum ether by shaking on a mechanical shaker for 5 min each time. Combine
the organic phases, and filter through sodium sulphate into a 500-ml round-bottomed flask.
Wash the sodium sulphate with petroleum ether. Rotary-evaporate the solution to a volume
of 5 ml; during the analysis of maize kernels, concentrate the solution to a residual volume
of approx. 50 ml. Then proceed to 6.2.2; for maize kernel samples, however, first clean up the
solution as described in 6.2.1.
6.1.2 Water
Extract a water sample (G) twice with equal volumes of petroleum ether (at least 100 ml) by
shaking for 5 min each time on a mechanical shaker. Combine the organic phases, and filter
petroleum ether. Rotary-evaporate the solution to a volume of approx. 5 ml. With extracts
from water samples containing only small amounts of co-extractives, proceed directly to gas-
chromatographic analysis in 6.3, otherwise proceed firstly to 6.2.2.
6.2 Cleanup
6.2.1 Liquid-liquid partition (maize kernels)
Transfer the solution derived from 6.1.1 to a 250-ml separatory funnel, and extract successively
with four 50-ml portions of acetonitrile. Combine the acetonitrile phases in a 1-1 separatory
funnel, dilute with 125 ml sodium chloride solution and 350 ml water, and extract twice with
100-ml portions of petroleum ether by shaking for 5 min each time. Combine the organic
phases, filter through sodium sulphate into a 500-ml round-bottomed flask, and rotary-
evaporate to a volume of approx. 5 ml.

Place a glass wool plug in the bottom end of a chromatographic tube, and half-fill the tube
with petroleum ether. Slowly add 20 g Florisil to the column, gently tapping the tube walls.
Allow the adsorbent to settle, and drain the petroleum ether until the top of the Florisil is just
covered with liquid. Then quantitatively rinse the concentrated solution derived from 6.1.1,
6.1.2 or 6.2.1 onto the prepared column with a little petroleum ether. Allow the solution to
percolate into the column at a rate of 1-2 drops per s, and then elute the column with 200 ml
eluting mixture. Rotary-evaporate the eluate almost to dryness.
208 Fonofos

Quantitatively rinse the residue derived from 6.2.2 into a 10-ml volumetric flask with hexane,
and dilute to the mark with hexane. Carry out a preliminary measurement to estimate approx-
imately the concentration of fonofos. If its concentration is higher than 5 ng/ul, dilute with
hexane. On the other hand, if the solution has a very low content of fonofos, it can be concen-
trated to a lower volume. Using a microsyringe, add to the thus prepared sample solution a
pl-amount of the internal standard fenchlorphos solution (shake!) equivalent to the ml-
volume (VEnd) of the sample solution.
Inject an aliquot of this solution into the gas chromatograph.
Gas chromatograph Varian Aerograph 2740
Column Glass, 2 mm i.d., 1.5 m long; packed with 5% SE-30
on Chromosorb W-AW-DMCS, 80-100 mesh
Detector Thermionic phosphorus-specific detector
Temperature 240 C
Hydrogen, 15 ml/min
Air, 170 ml/min
Attenuation 64 10~u
Retention times for
fonofos 2 min 42 s
fenchlorphos 4 min 24 s
7 Evaluation
7.1 Method
To 10 ml of each fonofos standard solution (equivalent to 5 to 50 |ug fonofos), add 10 JLLI fen-
chlorphos solution (equivalent to 50 \ig fenchlorphos) as internal standard (shake!). Inject
1-ul aliquots of these solutions into the gas chromatograph. For graphic representation of the
calibration curve, divide the peak heights of fonofos by the peak heights of the internal stan-
dard, and plot each quotient vs. the concentration of fonofos in the standard solutions
(ng/îl). From the gas chromatogram of each injected sample solution, measure the peak
heights, calculate the quotients as described above, and read the concentration of fonofos in
ng/|nl (WA) from the calibration curve.
Fonofos 209

The recoveries from untreated control samples, fortified with fonofos at levels of 0.02 to
0.5 mg/kg, ranged from 85 to 95%. The routine limit of determination was 0.02 mg/kg.

The residue R, expressed in mg/kg fonofos, is calculated from the following equation:
w A -v E n d
R=
where
WA = concentration of fonofos read from calibration curve (in ng/ul)
8 Important points
No data
9 References
No data
10 Author
Shell Forschung GmbH, Schwabenheim, D. Eichler
Fosetyl 522
Grapes, hop cones, lettuce, strawberries, wine Gas-chromatographic

Water determination
1 Introduction
Fosetyl (as fosetyl- Main metabolite

aluminium)
Chemical name Aluminium tris(O-ethyl Phosphorous acid
phosphonate) (IUPAC)
o OH o
Structural formula C 2 H 5 O-P-O- HO-P-OH HO-P-OH
I
H H
Empirical formula C6H18A1O9P3 H3PO3

Molar mass 354.10 81.99
Melting point 200 C with decomposition 73.6C
Boiling point Not distillable 200 C with decomposition
Vapour pressure <10" 5 mbar at 20 C No data
Solubility Readily soluble in water Very readily soluble in water
(12 g/100 ml at 20 C); (309 g/100 ml at 0C,
virtually insoluble in organic 694 g/100 ml at 40 C);
solvents, e.g. acetonitrile soluble in ethanol
(8 mg), methyl glycol (8 mg),
in 100 ml each at 20 C
Other properties Stability to hydrolysis: Half Forms a tautomeric equilibrium
life in aqueous solution predominantly in favour of
(1 g/1) > 100 d; hydrolyzed phosphonic acid
in strong acid and alkaline
media
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 chromato-
graphy using a phosphorus-specific flame photometric detector.
212 Fosetyl
3 Apparatus
Laboratory centrifuge with 250-ml glass tubes
Volumetric flasks, 100-ml, 50-ml and 5-ml
Round-bottomed flask, 50-ml, with ground joint
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 fosetyl-
aluminium 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 solu-
tions progressively to obtain solutions containing O-ethyl O-methyl phosphonate or O,O-
dimethyl phosphonate equivalent to 0.01, 0.02, 0.05, 0.08, 0.1, 0.15, and 0.2 ng/ml of fosetyl-
aluminium 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 hydrox-
ide 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
Fosetyl 213

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.

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.
Gas chromatograph Carlo Erba Fractovap 2101 AC
Column Glass, 2 mm i.d., 2 m long; packed with 15% Carbo-
wax 20M on Chromosorb 750, 100-120 mesh
214 Fosetyl
Detector Flame photometric detector, Model SSD 250, equipped

with 526-nm phosphorus filter
Temperature 175 C
Hydrogen, 60 ml/min
Air, 200 ml/min
Attenuation 1 32
Retention times for
dimethyl phosphonate 3 min
ethyl methyl phosphonate 3 min 36 s
7 Evaluation
7.1 Method
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 ap-
propriate amounts of aluminium-fosetyl or phosphorous acid from the corresponding calibra-
tion curve.

The recoveries from untreated control samples, fortified with fosetyl-aluminium and phos-
phorous 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.

The residue R, expressed in mg/kg fosetyl-aluminium or phosphorous acid, is calculated from
the following equations:
W
for plant material R= A (VEx + x) VR2 VEnd
V
R1 * V R3 * V i * G
W
for wine and water R= A ' VR2' VEnd . Q 93
Vpi * V; G
Fosetyl 215
where
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 )
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 con-
ditions, because breakdown of fosetyl residues was observed during storage at 18 C (see
J. Siebers, H.-G. Nolting and W. D. Weinmann, Initialbelage von Pflanzenschutzmittelwirk-
stoffen 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 let-
tuces, Method No. 22-79, Rhone-Poulenc Agrochimie, 1979.
216 Fosetyl
10 Authors
Rhone-Poulenc Agrochimie, Lyon, France, A. Bertrand and M. A. Muller
Nolting, M. Blacha-Puller and J. Siebers
Glufosinate 651
Almonds, apples, asparagus, bananas, beans, caraway, Gas-chromatographic
Chinese cabbage, evening primrose oil, kiwi fruit, determination
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
1 Introduction
Glufosinate-ammonium
Chemical name Ammonium DL-homoalanin-4-yl(methyl)phosphinate
(IUPAC)
o
Structural formula CH 3 -PCH 2 CH 2 CH-COOH NH 4 +
O~ NH 2
Empirical formula C 5 H 15 N 2 O 4 P
Molar mass 198.19
Melting point 215 C (with decomposition)
Solubility Very readily soluble in water (137 g at 22 C, pH 5);
(in 100 ml at 20 C) very sparingly soluble in acetone (16 mg), ethanol
(65 mg), ethyl acetate (14 mg) and toluene (14 mg)
Glufosinate (free acid)

Chemical name DI^Homoalanin-4-yl(methyl)phosphinic acid (IUPAi
0
II
Structural formula CH 3 -PCH 2 -CH 2 CH-COOH
1 1
OH NH2
Empirical formula C 5 H 12 NO 4 P
Molar mass 181.15
218 Glufosinate
Solubility Readily soluble in water (26.9 g at pH 1, >40 g at

(in 100 ml at 20 C) pH 7 and pH 10);
very sparingly soluble in methanol (12 mg);
virtually insoluble in acetone, dichloromethane, ethyl
acetate, n-hexane, isopropanol, toluene (each <1 mg),
dimethyl sulphoxide (9 mg) and polyethylene glycol
(5mg)
Other properties Commercial formulations contain glufosinate ex-
clusively as the ammonium salt
Metabolite
Chemical name 3-(Methylphosphinico)propionic acid (IUPAC)
O
II
Structural formula CH3-P-CH2-CH2-COOH
OH
Empirical formula C4H9O4P
Molar mass 152.1
Solubility Readily soluble in water (79.4 g; pH of the saturated
(in 100 ml at 20 C) solution is 1.0);
readily soluble in dimethyl sulphoxide (> 40 g) and
methanol (>50 g);
soluble in isopropanol (9.4 g) and polyethylene glycol
(4 g);
sparingly soluble in acetone (0.29 g);
very sparingly soluble in ethyl acetate (86 mg);
virtually insoluble in dichloromethane (2 mg),
n-hexane and toluene (each <1 mg)
2 Outline of method
Residues of glufosinate-ammonium, glufosinate and the metabolite are extracted from plant
and animal material and from soil with water. Depending on the type of sample material, the
extracts are cleaned up by de-fatting with dichloromethane, by precipitation of carbohydrates
and proteins with acetone, or by ion exchange chromatography. From water samples, the
residues are concentrated on an anion exchange chromatographic column and eluted with
formic acid solution. After evaporation of the cleaned-up extracts or of the eluate, the
residues are treated with trimethyl orthoacetate, resulting in the formation of the derivatives:
methyl 4-[methoxy(methyl)phosphinoyl]-2-acetamidobutyrate from glufosinate, and methyl
3-[methoxy(methyl)phosphinoyl]propionate from the metabolite. The derivatives are cleaned
up on a mini silica gel column and are determined by gas chromatography using a
phosphorus-specific flame photometric detector.
Glufosinate 219
CH3-PCH2CH2-CH-COOCH3 CH 3 -PCH 2 -CH 2 -COOCH 3

OCH3 NH-COCH3
0CH3
Glufosinate derivative
Metabolite derivative
3 Apparatus
Meat mincer
Erlenmeyer flasks, 500-ml and 300-ml
Watch glass, 10 cm dia.
Hotplate with magnetic stirrer, incl. stirring rod
Graduated cylinders, 1-1, 100-ml, 50-ml and 2-ml
Centrifuge, e. g. Labofuge GL (Heraeus-Christ)
Volumetric pipets, 20-ml and 10-ml
Filter cartridges for organic solvents, pore size 0.5 \im (e. g. Millex SR Filter, Millipore SLSR
025 NS)
Filter cartidge
Disposable
syringe
Silica gel
Quartz wool
Figure. Adding the derivatives solution onto the mini silica gel column in step 6.3.
220 Glufosinate
Beaker, 5-1
Chromatographic tube 1: 15 mm i.d., 30 cm long, with stopcock
Chromatographic tube 2: 40 mm i.d., 60 cm long, with stopcock
Ultrasonic bath
Solvent dispensers, 15-ml, 8-ml and 2-ml
Reflux condenser, jacketed coil type, 30 cm long, with ground joint
Heating mantles, regulated, for 100-ml and 50-ml round-bottomed flasks
Disposable syringes, 10-ml, with stainless steel needles (flat tip); needles approx. 15 cm long,
straight form and angular bent (see Figure)
Pasteur pipets
Pear-shaped flask, 10-ml, with ground joint
Volumetric flasks, 100-ml, 50-ml, 10-ml and 5-ml
Gas chromatograph equipped with phosphorus-specific flame photometric detector
4 Reagents
Acetone, p. a.
Ethanol, p. a.
Methanol, p. a.
Methyl acetate, p. a.
Toluene, p. a.
Eluting mixture: methyl acetate + methanol 1:1 v/v
Solvent mixture 1: methyl acetate + ethanol 1:1 v/v
Solvent mixture 2: methyl acetate + toluene 1:1 v/v
Solvent mixture 3: methyl acetate + toluene 7 : 3 v/v
Glufosinate and metabolite standard solutions for fortification experiments: 5 |xg/ml of each
glufosinate-ammonium or 3-(methylphosphinico)propionic acid (Riedel-de Haen) in water or,
for fortification of fats and oils, in solvent mixture 1
Glufosinate derivative standard solution: Methyl 4-[methoxy(methyl)phosphinoyl]-2-acet-
amidobutyrate in eluting mixture, equivalent to 5 pig/ml glufosinate-ammonium. Prepare by
weighing glufosinate-ammonium and derivatizing as described in 6.2
Metabolite derivative standard solution: Methyl 3-[methoxy(methyl)phosphinoyl]propionate
in eluting mixture, equivalent to 5 ng/ml metabolite. Prepare by weighing 3-(methylphos-
phinico)propionic acid and derivatizing as described in 6.2
Polyethylene glycol solution: 10 g/100 ml polyethylene glycol 400 p.a. (Serva No. 46170) in
acetone
Glacial acetic acid, p. a.
Hydrochloric acid, 10 g/100 g HC1 p. a.
Formic acid, 10 ml/100 ml HCOOH p. a.
Ammonia solution, 10 g/100 ml and 1 g/100 ml NH3
Sodium hydroxide solution, 1 mol/1 NaOH
Trimethyl orthoacetate (Riedel-de Haen No. 64561)
Glufosinate 221
Cation exchanger, strongly acidic: Ion exchanger I (Merck No. 4765).

Preparation: Add 2 1 hydrochloric acid to 1000 g cation exchanger in a beaker, allow to stand
for 30 min, decant, wash the resin with water until neutral (pH indicator paper), and further
treat, in the following sequence, with:
1 1 ammonia solution (10 g/100 ml), allow to stand for 5 min, wash to neutral with water;
2 1 ethanol-water mixture (1:1 v/v), allow to stand for 30 min, wash with 2 1 water, until free
of ethanol;
1 1 hydrochloric acid (conversion to H + form), wash with water until neutral
Cation exchange column: Pour 6 g of the damp cation exchanger as prepared above into a
chromatographic tube (type 1)
Anion exchanger, strongly basic: Dowex 1X8 (Serva No. 41091).
Preparation: Allow the resin (in the chloride form as supplied) to swell in water for 1 h. Pour
approx. 100 g of the swollen resin into a chromatographic tube (type 2), and wash with 750 ml
sodium hydroxide solution to convert it into the OH" form. Wash with water until the eluate
has become neutral to pH indicator paper
Anion exchange column: Pour 6 g of the damp anion exchanger as prepared above into a
chromatographic tube (type 1)
pH indicator paper
RP-18 disposable cartridge: Sep-Pak Cartridge C18 (Millipore No. 51910)
Silica gel, deactivated with 4% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734),
Quartz wool
Air
Helium
Hydrogen

6 Procedure
6.1 Extraction
6.1.1 Plant material with high water content (apples, asparagus, bananas, beans, Chinese
cabbage, kiwi fruit, lemons, mirabellas, oranges, plums, potatoes, sour cherries, sugar
beet) and soil
Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer
flask. Cover the flask with a watch glass, and magnetically stir the homogenate for 30 min
at room temperature. Measure the volume of the homogenate (VEx), and centrifuge a 60-ml
222 Glufosinate
portion at 3000 r.p.m. for 10 min. Pipet 20 ml of the supernatant (VR1) into a 50 ml round-
bottomed flask and rotary-evaporate to dryness, using a 60 C bath temperature. Add 5-10 ml
ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual traces of
water. Repeat if required until the residue is absolutely dry.
6.1.2 Plant material with high content of water soluble carbohydrates or proteins (almonds,
caraway, maize, peas, soybeans, wheat)
Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer
flask. Cover the flask with a watch glass, and magnetically stir the homogenate for 30 min
at room temperature. Measure the volume of the homogenate (VEx), and centrifuge a 60-ml
portion at 3000 r.p.m. for 10 min. Take 40 ml of the supernatant (VR1) and add 40 ml
acetone to precipitate carbohydrates and proteins (total volume, VR2). Centrifuge the mixture
again. Pipet 40 ml of the supernatant (VR3) into a 100-ml round-bottomed flask and rotary-
evaporate to dryness, using firstly room temperature, then a 60 C bath temperature. Add
5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual
traces of water. Repeat if required until the residue is absolutely dry.
6.1.3 Fatty plant material (rape, sunflower seeds)

Homogenize 25 g of the analytical sample (G) with 200 ml water in a 500-ml Erlenmeyer flask
(total volume of the homogenate, VEx). Cover the flask with a watch glass, and magnetically
stir the homogenate for 30 min at room temperature. Allow solid particles to settle, then de-
cant 40 ml of the supernatant (VR1) into a centrifuge tube, and add 40 ml acetone (total
volume of the mixture, VR2). Mix, and centrifuge the mixture at 3000 r.p.m. for 5 min. Next,
transfer 40 ml of the supernatant (VR3) into a 100-ml separatory funnel and shake with 20 ml
dichloromethane. Separate the lower organic phase and re-extract it twice with 10-ml portions
of water. Combine the aqueous phases in a 100-ml round-bottomed flask and rotary-evaporate
to dryness, using firstly room temperature, then a 60 C bath temperature. Add 5-10 ml ethyl
acetate to the residue and rotary-evaporate to dryness again to remove residual traces of water.
Repeat if required until the residue is absolutely dry.
6.1.4 Fats and oils (evening primrose oil, sunflower oil, beef fat)
Dissolve 20 g of the analytical sample (G) in 100 ml dichloromethane (use the homogenizer
for dissolution of solid fat if required). Add 100 ml water (VEx) and homogenize for 5 min.
Decant approx. 60 ml of the aqueous supernatant into a centrifuge tube and centrifuge at
3000 r.p.m. for 5 min. Force 25 ml of the supernatant (VR1) through a RP-18 disposable car-
tridge that has been previously conditioned with 5 ml methanol and 5 ml water. Wash the car-
tridge by forcing through 3 ml water. Collect both the aqueous eluate and the wash in a 50-ml
round-bottomed flask and rotary-evaporate to dryness, using a 60 C bath temperature. Add
5-10 ml ethyl acetate to the residue and rotary-evaporate to dryness again to remove residual
traces of water. Repeat if required until the residue is absolutely dry.
6.1.5 Animal matrices (blood, meat, meat broth, liver, kidneys)

Add 100 ml water to 20 g of blood (G) and magnetically stir, raising the temperature to 80 C,
and maintain at this temperature for 30 min with further stirring (total volume, VEx). For the
Glufosinate 223
other materials, transfer 20 g of the minced analytical sample or 20 g of meat broth (G) into
a 300-ml Erlenmeyer flask, and add 100 ml water (total volume, VEx). Cover the flask with
a watch glass and magnetically stir the mixture for 30 min at room temperature.
Centrifuge 50-60 ml of the mixture at 3000 r.p.m. for 10 min. To 40 ml of the supernatant
(VR1), add 40 ml acetone to precipitate proteins (total volume of this mixture, VR2), and cen-
trifuge the mixture again for 10 min. Carefully rotary-evaporate 40 ml of the clear supernatant
(VR3) at room temperature to completely remove acetone; the aqueous solution should not
smell of acetone any longer.
Transfer the solution onto the anion exchange column at a rate of 3-4 ml/min, wash the
column with 100 ml water, and discard the eluate. Next, elute glufosinate and its metabolite
with 70 ml dilute formic acid into a 100-ml round-bottomed flask, and rotary-evaporate the
eluate to dryness, using a 60 C bath temperature. Add 5-10 ml ethyl acetate to the residue
and rotary-evaporate to dryness again to remove residual traces of water. Repeat if required
until the residue is absolutely dry.
6.1.6 Water
Before concentrating glufosinate and its metabolite on an anion exchange column, the cations
present in the water must be exchanged for H + (see 8. Important points). For this purpose,
the ion exchange columns are connected in series, so that the eluate from the cation exchanger
flows directly onto the anion exchanger.
Using a 1-1 separatory funnel, transfer 1 1 of the water sample (G) onto the cation exchange
column at a rate of 5-10 ml/min. Wash both columns with 100 ml of distilled or deionized
water, and discard the eluates (approx. 1.1 1). Remove the cation exchange column. Next, elute
glufosinate and its metabolite from the anion exchange column with 70 ml dilute formic acid
into a 100-ml round-bottomed flask. Add 100 \x\ polyethylene glycol solution to the eluate and
rotary-evaporate to dryness, using a 60 C bath temperature. Add 5-10 ml ethyl acetate to the
residue and rotary-evaporate to dryness again to remove residual traces of water. Repeat if re-
quired until the residue is absolutely dry.
6.2 Derivatization
Suspend the residue derived from 6.1 in 2 ml glacial acetic acid by dipping the flask for 10 min
in an ultrasonic bath. Add 8 ml trimethyl orthoacetate, dip the flask for a further 5 min in
the ultrasonic bath, and then reflux the mixture for 4 h with occasional swirling to prevent
suspended solids from baking onto the walls of the flask.
Allow to cool, add 15 ml toluene, and rotary-evaporate to a residual volume of approx. 1 ml
(do not evaporate to dryness!) with 40 C bath temperature. Repeat the evaporation with
toluene twice more to completely remove residual acetic acid and derivatization reagent (see
8. Important points).
Make up the 0.5-1 ml residue to 3 ml with toluene, dip the flask for 1 min in the ultrasonic
bath, and draw up the flask contents, including the suspended solid material, into a 10-ml
disposable syringe. Rinse the flask with 5 ml methyl acetate, dip the flask for approx. 2 min
in the ultrasonic bath, and also draw up the rinsing into the syringe. Mix the contents well
by shaking the syringe.
224 Glufosinate

Insert a quartz wool plug into the bottom of a Pasteur pipet and add 0.6 g silica gel. Condition
the column with 5 ml solvent mixture 2 delivered from a disposable syringe with a 15 cm long
stainless steel needle. Using the needle, stir the silica gel-solvent mixture until it is free of air
bubbles. Filter the solution derived from 6.2 in the first syringe through a filter cartridge
mounted on the syringe and, with the aid of an angular bent needle, transfer the filtrate onto
the mini silica gel column (see Figure). Rinse the flask from 6.2 with 10 ml methyl acetate.
Draw up the rinsing into the syringe, filter and transfer it onto the column in a similar manner.
When the column has run dry, apply gentle suction to dry the packing. Next, elute the
derivatives of glufosinate and its metabolite from the column with eluting mixture, collecting
exactly 5 ml eluate (VEnd) in a 5-ml volumetric flask.
For samples of drinking water, evaporate the 5 ml of eluate to approx. 100 jul, using a 40 C
water bath and a gentle stream of nitrogen, and make up to 1.5 ml (VEnd) with eluting
mixture.
For most other sample materials, a second cleanup on a mini silica gel column is recom-
mended. Transfer the eluate to a pear-shaped flask using 3 ml toluene and rotary-evaporate
(40 C bath temperature) to approx. 1 ml. The methanol must be removed completely. Make
up to 3 ml with toluene, and mix with 5 ml methyl acetate, as described in 6.2. Re-
chromatograph the mixture on a mini silica gel column as described above, with the exception
of the filtration step.

Inject an aliquot of the solution derived from 6.3, e.g. 5 ul (Vj), into the gas chromatograph.
Gas chromatograph Carlo Erba 2150, with capillary injector (Chrompack
8045), split exit closed
with Carbowax 20 M, film thickness 0.25 urn (Restek)
Column temperature Isothermal at 150C for 2 min, programmed to rise
at 39C/min from 150 to 240 C, then isothermal
at 240 C for 4 min
Detector Flame photometric detector, equipped with 526-nm
phosphorus filter
Temperature 225 C
Gas flow rates Helium carrier, 28 ml/min (214 cm/s)
Hydrogen, 75 ml/min
Air, 125 ml/min
Retention times for
metabolite derivative 42 s
glufosinate derivative 4 min 18 s
Glufosinate 225
with RTX-2330, film thickness 0.25 urn (Restek)
Column temperature Isothermal at 140 C for 2 min, programmed to rise
at 39C/min from 140 to 240 C, then isothermal at
240 C for 4 min
Gas flow rates Helium carrier, 25 ml/min (190 cm/s)
Hydrogen, 70 ml/min
Air, 120 ml/min
Retention times for
metabolite derivative 1 min 24 s
glufosinate derivative 5 min
7 Evaluation
7.1 Method
tions and comparing them with the peak areas or peak heights obtained for the derivative
standard solutions. Equal volumes of the sample solutions and the derivative standard solu-
tions should be injected; additionally, the peaks of the solutions should exhibit comparable
areas or heights.
Should pre-analysis checks show that the derivatives with and without matrix give different
detector responses, the evaluation is performed with the aid of sample solutions derived from
untreated control material which, after the column cleanup (6.3), have been fortified with
standard solutions of the derivatives ("fortified calibration solutions"). Evaluation is also
possible with the aid of the "Standard Addition Method" if no control material is available.
For this purpose, analyze a sample as described above (6.1 to 6.4). To an aliquot of this sample
solution, add standard solutions of the derivatives, and measure again. The differences be-
tween the peak areas or heights obtained for the two measurements is equivalent to the
amounts of the derivatives taken for fortifying the analytical sample.

The recoveries from untreated control samples, fortified with glufosinate-ammonium or
metabolite at levels of 0.05 to 10 mg/kg, ranged from 64 to 116%. The routine limit of deter-
mination was 0.05 mg/kg for plant material, fats, oils and soil, 0.05 to 0.1 mg/kg for animal
matrices, and 0.05 fig/1 for tap water.

The residue R, expressed in mg/kg (for water, ng/1) glufosinate-ammonium or metabolite, is
calculated from the following equations:
226 Glufosinate
for plant material, fats, oils, animal matrices, soil
R _ F A -V E x -V R 2 -V E n d -W S t
where
VEx = total volume of homogenate or (for fats and oils) volume of water added to the
organic phase for extraction (in ml)
VRI = portion of volume VEx used for further processing (in ml)
VR2 = total volume of the aqueous solution with acetone added (in ml)
VR3 = portion of volume VR2 used for further processing (in ml)
WSt = amount of glufosinate-ammonium equivalents or metabolite equivalents, respectively,
injected with derivative standard solution or "fortified calibration solution" (in ng)
FA = peak area or height obtained from Vj (in mm 2 or integrator counts)
F st = peak area or height obtained from WSt (in mm 2 or integrator counts)
for water
R =
where
G = sample volume (in 1)
WSt = amount of glufosinate-ammonium equivalents or metabolite equivalents, respectively,
injected with derivative standard solution or "fortified calibration solution" (in ng)
FA = peak area or height obtained from Yi (in mm 2 or integrator counts)
F st = peak area or height obtained from WSt (in mm 2 or integrator counts)
To calculate glufosinate as free acid, multiply the result for glufosinate-ammonium by a fac-
tor of 0.91, and the result for the metabolite by a factor of 1.19.
Glufosinate 227
8 Important points
Cations (more than 10 mg Na + , Mg2+ and Ca2+ in the analytical sample) can drastically
reduce the derivatization yields. Mg2+ and Ca2+ are precipitated as hydroxides on the anion
exchanger, but are taken into solution again by the formic acid, and are eluted together with
glufosinate and its metabolite. The cations must therefore be removed from the water samples
with a cation exchange column before the derivatization step.
After the derivatization (6.2), the acetic acid must be completely removed because it
prevents the concentration of the derivatives in the following silica gel cleanup (6.3).
The activity of the silica gel must be checked before each series of analyses. Proceed as
follows: Transfer a solution of glufosinate derivative and metabolite derivative in solvent mix-
ture 3 onto the silica gel mini column and elute as described in 6.3. When untreated control
material is available, add the derivatives to the sample solution after derivatization and cool-
ing (6.2), but before the solvent evaporating step. The recoveries from the silica gel should be
90 to 100%. If they are less than 80%, repeat the check using a fresh silica gel batch.
9 References
No data
10 Author
Hoechst AG, Product Development, Division C, Frankfurt/Main, H. Sochor
Glyphosate 405
Beer, bread, cereals (grains, flour and straw), grapes, High-performance

grass, grass silage, hay, peas, rape (seeds), sugar beet liquid chromato-
(foliage and edible root) graphic determination
Soil, water
1 Introduction
Glyphosate Metabolite (AMPA)
Chemical name N-Phosphonomethylglycine Aminomethylphosphonic acid
(IUPAC)
0 0
Structural formula HOPCH 2 NHCH 2 -COOH HOPCH 2 NH 2
OH OH
Empirical formula C3H8NO5P CH6NO3P

Molar mass 169.08 111.04
Melting point 230 C (with decomp.) 285 C (decomp. at 312 C)
Vapour pressure 3.5-10- 7 mbar at 45 C 4.0-10" 6 mbar at 75 C
Solubility Slightly soluble in water Soluble in water (5.8 g/100 ml
(1.2g/100mlat25C); at25C);
sparingly soluble in common virtually insoluble in acetone,
organic solvents dichloromethane, diethyl ether,
ethanol, n-hexane and toluene
Other properties Commercial products con-
tain glyphosate as isopropyl-
amine salt
2 Outline of method
Residues of glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), are
extracted from plant material, bread and flour with dilute hydrochloric adic, and from soil
with ammonia solution. Beer and water are filtered. The pH of the extract or filtrate is ad-
justed to 2.0 0.4. The solution is passed through a Chelex 100 ligand exchange resin in the
Fe(III) form; the co-eluted ferric ions are removed by chromatography through an anion ex-
changer. Glyphosate and AMPA are determined separately by high-performance liquid
chromatography including post-column derivatization. In the fluorogenic step, glyphosate,
which has been oxidized to a primary amine, and AMPA are reacted with o-phthaldialdehyde
and 2-mercaptoethanol to yield strongly fluorescent compounds which are measured using a
fluorescence detector. For AMPA determination, the oxidation step is omitted.
230 Glyphosate
3 Apparatus
Centrifuge, 12000 r.p.m., with 250-ml glass tubes
Graduated cylinders, 2-1, 500-ml, 250-ml, 100-ml, 25-ml and 10-ml
Erlenmeyer flasks, 2-1, 250-ml and 100-ml
Magnetic stirrer, with stirring rod
Vacuum filtration unit fitted with membrane filters, type G S, pore size 0.22 \im (Millipore)
Flat-bottomed flask, 10-1
Amber glass bottles, 1-1
Chromatographic tube 1, 8 cm i.d., 40 cm long, with sintered glass disk
Chromatographic tube 2, 2.2 cm i.d., 13 cm long, with reservoir 5.5 cm i.d., 9 cm long
Chromatographic tube 3, 1.5 cm i.d., 14 cm long, with reservoir 2.5 cm i.d., 6.5 cm long
Round-bottomed flask, 100-ml
High-performance liquid chromatograph equipped with post-column derivatization system
and fluorescence detector
4 Reagents
Note: Use only deionized water for all aqueous solutions and all analytical operations.
Dichloromethane, p. a. (Merck No. 6050)
Methanol, p. a. (Merck No. 6009)
Glyphosate and AMPA standard solutions: 0.05, 0.1, 0.3, 0.5, 0.8 and 1.0 ng/ml glyphosate
or AMPA (e. g. Aldrich No. 32,481-7) in water. The solutions can be stored in a refrigerator
for about 1 month
Boric acid, p. a. (Merck No. 165)
Hydrochloric acid, 32%, and 6, 1, 0.2, 0.1 and 0.02 mol/1 HC1 p. a.
ortho-Phosphoric acid, p.a., 85% (Merck No. 573)
Ammonia solution, 25% (Merck No. 5432) and 1 mol/1 NH3 p. a.
Ferric chloride solution, 0.1 mol/1 FeCl3 p. a.
Mobile phase (phosphate buffer solution): Dissolve 680 mg potassium dihydrogen phosphate
in 1 1 methanol-water mixture (4:96 v/v), and adjust to pH 2.0 with ortho-phosphoric acid.
De-gas in a vacuum filtration unit before use
Oxidizing solution: Dissolve 11.6 g sodium chloride, 13.6 g potassium dihydrogen phosphate
and 5.0 g sodium hydroxide in approx. 900 ml water. Add 4 ml sodium hypochlorite solution
and make up to 1 1 with water. The solution is stable for a maximum of 5 days
Fluorogenic solution: Dissolve 25 g boric acid and 11 g sodium hydroxide in approx. 900 ml
water. Add 30 ml Brij solution, 2 ml 2-mercaptoethanol, and 0.8 g o-phthaldialdehyde,
dissolved in 10 ml methanol. Make up to 1 1 with water. The solution is stable for a maximum
of 5 days
Brij solution: 30 g/100 ml Brij 35 (Merck No. 801962) in water
Glyphosate 231
2-Mercaptoethanol (Merck No. 15433)

Ferric chloride hexahydrate, p. a. (Merck No. 3943)
Potassium dihydrogen phosphate, p. a. (Merck No. 4873)
Sodium chloride, p. a. (Merck No. 6404)
Sodium hydroxide, p. a. (Merck No. 6498)
Sodium hypochlorite solution, approx. 13% active chlorine (Merck No. 5614)
o-Phthaldialdehyde (Aldrich No. P3,940-0)
Anion exchange resin: AG 1-X8, particle size 200-400 mesh, Cl form (Bio-Rad No. 140-1451):
Slurry 450 g of the resin in 1 1 water. Stir on a magnetic stirrer for 30 min. Allow to settle,
decant and discard the water. Wash twice more as described above. Store the resin in an amber
bottle under deionized water
Ligand exchange resin, Fe(III) form: Slurry 900 g of Chelex 100, particle size 100-200 mesh,
Na form (Bio-Rad No. 142-2832) in 3 1 water, add 50 ml hydrochloric acid (6 mol/1) and 1 1
ferric chloride solution. Stir on a magnetic stirrer for 10 min. Allow to settle, decant and
discard the aqueous solution. Repeat twice, using a mixture of 2 1 water and 500 ml ferric
chloride solution each time. Next transfer the resin into the chromatographic tube 1 and wash
with 4 1 hydrochloric acid (0.02 mol/1). The resin can be stored for approx. 3 months under
deionized water in an amber bottle
Glass wool
pH indicator rods: Acilit pH 0-6.0 (Merck No. 9531) and Neutralit pH 5.0-10.0 (Merck
No. 9533)

6 Procedure
6.1 Extraction
6.1.1 Plant material (except hay and straw), bread and flour
Weigh 30 g of the analytical sample (G) containing x g water (see 8. Important points) into
a centrifuge tube, add 150 ml hydrochloric acid (0.1 mol/1) (VEx) and 50 ml dichloromethane,
and homogenize for 1 min. Next centrifuge the homogenate for 20 min at 12000 r.p.m. and
filter the supernatant aqueous phase through a glass wool plug. Take an aliquot of 125 ml
(VR1) of the filtrate, dilute to approx. 400 ml with water, and mix thoroughly. The pH of the
solution should be between 1.6 and 2.4. If the pH is outside this range, dilute further with
water, or acidify with hydrochloric acid (6 mol/1).
6.1.2 Hay, straw

Weigh 15 g of the chopped analytical sample (G) containing x g water (see 8. Important points)
into a centrifuge tube, add 200 ml hydrochloric acid (6 mol/1), and homogenize for 1 min.
Next, centrifuge the suspension for 20 min at 12000 r.p.m. and filter the supernatant through
232 Glyphosate
a glass wool plug. Take an aliquot of 130 ml (VR1) of the filtrate, dilute to approx. 400 ml
with water, and mix thoroughly. The pH of the solution should be between 1.6 and 2.4. If the
pH is outside this range, dilute further with water, or acidify with hydrochloric acid (6 mol/1).
6.1.3 Beer
Filter 100 ml of the analytical sample (G) through a fluted filter paper to remove carbon diox-
ide. Add 100 ml water and 200 ml hydrochloric acid (0.1 mol/1), and mix thoroughly.
6.1.4 Soil
Weigh 25 g of soil (G) into a 250-ml Erlenmeyer flask, add 150 ml ammonia solution, and
extract with fast magnetic stirring for 30 min. Centrifuge the suspension for 20 min at
12000 r.p.m. and filter the supernatant through a glass wool plug. Repeat the extraction twice
more. Next, adjust the pH of the combined filtrates to 2.0 0.4 with 30 ml of hydrochloric
acid (32%), followed by 20-30 ml of hydrochloric acid (1 mol/1). Dilute to 1.8 1 with water.
Check the pH and re-adjust to pH 2.0 0.4, if necessary. If a precipitate forms, leave it to
settle for 30 min, or filter the solution through a glass wool plug.
6.1.5 Water
Suction-filter 2 1 of the water sample (G) through a membrane filter using the vacuum filtra-
tion unit and adjust the pH of the filtrate to 2.0 0.4 by adding approx. 5 ml hydrochloric
acid (6 mol/1).
6.2 Cleanup
6.2.1 Ligand exchange chromatography
Insert a glass wool plug into the bottom of chromatographic tube 2, add 7-8 ml water and
15 ml of the resin in its Fe(III) form. Allow the resin to settle and drain the liquid to the top
of the column packing. Next add the filtrate derived from 6.1 onto the column, not disturbing
the top layer of the resin, and allow to percolate at a rate of 6-8 ml/min. Open fully the stop-
cock and wash the column with 50 ml water and 100 ml hydrochloric acid (0.2 mol/1).
Cautiously add 3 ml hydrochloric acid (6 mol/1) to the column and allow to percolate at a
rate of approx. 4 ml/min. Repeat with a further 4-ml portion of hydrochloric acid. Discard
all eluates obtained up to this point.
Elute glyphosate and AMPA from the column with twice 5 ml, followed by 7 ml hydro-
chloric acid (6 mol/1). Collect the eluates in a 100-ml Erlenmeyer flask and add 8 ml hydro-
chloric acid (32%).
6.2.2 Anion exchange chromatography

Insert a glass wool plug into the bottom of chromatographic tube 3, add 7-8 ml water and
approx. 7 ml of the anion exchange resin. (In order to ensure that at least 7 ml of the resin
is transferred to the column, fill the resin into a 10-ml graduated cylinder first, and allow to
settle for approx. 45 min.) Pre-wash the column packing three times with 5-ml portions of
hydrochloric acid (6 mol/1) and discard the eluates obtained up to this point.
Glyphosate 233
Cautiously add the solution derived from 6.2.1 onto the column and allow to percolate, the
stopcock being fully opened. Be careful not to disturb the top layer of the resin in order to
prevent the passage of the yellow-orange ferric chloride through the column. Rinse the
Erlenmeyer flask with 2 ml hydrochloric acid (6 mol/1) and add the rinsing to the column.
As soon as the solutions have soaked into the resin bed, add a further 8-ml portion of
hydrochloric acid (6 mol/1) to the column. Collect the total eluate in a 100-ml round-bottomed
flask and carefully rotary-evaporate to dryness, increasing the water bath temperature slowly
from 20 C to 50 C (Caution! Solution foams readily when evaporated). Dissolve the residue
in 5 ml water and check the pH. If it is less than 1.5, add 20 ml water and rotary-evaporate
to dryness again. Make up the residue with water to a definite volume (VEnd), e.g. 5.0 ml.

Glyphosate and AMPA are determined by high-performance liquid chromatography using a
fluorescence detector. For this purpose, glyphosate is converted in two post-column derivatiza-
tion steps, first by oxidation with oxidizing solution to a primary amine, then by derivatization
to a strongly fluorescent compound with o-phthaldialdehyde (OPA) in the presence of 2-
mercaptoethanol. In the case of AMPA, the oxidation step is omitted. Switching off reagent
pump I (oxidizing solution) permits the separate determination of glyphosate and AMPA (see
Diagram below).
Reagent
Integrator pump II
Detector Fluorogenic
1\ solution
\ Three-way
Reaction coils (^"^ connecting
/ pieces
HPLC
pump Injector
i, / Reagent
pump I
< Oxidizing
Pre-column solution
+analvtical column
Diagram of the HPLC system.
For glyphosate determination, inject an aliquot (Vj) of the solution derived from 6.2.2 into
the high-performance liquid chromatograph; for AMPA determination, inject a second ali-
quot of the same sample solution (with reagent pump I switched off).
Pump Constant volume pump, model 6000 A (Millipore)
Injector Automatic injection system, Model 8055 (Varian), or in-
jection valve with sample loop, Model U6K (Millipore)
Pre-column Stainless steel, 4.6 mm i.d., 2 cm long ("Column
Guard Kit", Millipore); packed with Pellicular Media
Sax (Alltech No. 2855)
234 Glyphosate
Column Stainless steel, 4.6 mm i.d., 30 cm long; packed

with Aminex A9, particle size 11.5 urn (Bio-Rad
No. 999-1143-300)
Mobile phase Phosphate buffer solution, pH 2.0
Post-column derivatization system:
Reagent pump I Model 2150 (LKB Instruments)
(for oxidizing solution)
Reagent pump II Model PCR IB (Varian)
(for fluorogenic solution)
Flow rates
Reagent pump I: 0.2 ml/min
for glyphosate analysis Reagent pump II: 0.3 ml/min
Reagent pump I: switched off
for AMPA analysis Reagent pump II: 0.3 ml/min
Two stainless steel capillary tubes, 0.01 inch i. d.,
Reaction coils 1/16 inch o.d., 2 m long, with two Valco three way
connecting pieces (see Diagram)
Detector Fluorescence detector Fluorichrom (Varian) equipped
with excitation filters 7-54 and 7-60 (band filters
325 nm and 360 nm) and emission filters 4-76 and
3-72 (band filter 530 nm and cut-off filter 440 nm)
Integrator SP 4100 (Spectra-Physics); chart speed 5 mm/min
SP410
Linearity range 10-1000
10-100 ng for glyphosate and AMPA
Retention times for
glyphosate 16 min
AMPA 26 min
7 Evaluation
7.1 Method
Inject equal volumes of glyphosate or AMPA standard solutions, respectively, equivalent to
5 to 100 ng glyphosate or AMPA, into the high-performance liquid chromatograph. Plot the
areas of the peaks obtained vs. ng glyphosate or AMPA. Also inject aliquots of the sample
solutions. Equal volumes of the sample solutions and the standard solutions should be in-
jected. For the areas of the peaks obtained for the sample solutions, read the appropriate
amounts of glyphosate or AMPA from the corresponding calibration curve.

The recoveries from untreated control samples, fortified with glyphosate or AMPA, are given
in the Table.
Glyphosate 235
Table. Percent recoveries from plant material, soil and water, fortified with glyphosate and AMPA.
Glyphosate AMPA
Analytical material Added (mg/kg)
Range Mean Range Mean
Beer 0.02 83-94 92 62-69 66
Bread 0.05 66-90 74 72-100 87
Cereals
Grains 0.03-2 71-100 90 64-99 84
Flour 0.05-5 92-114 100 97-103 100
Straw 0.05-10 80-114 94 60-108 90
Grapes 0.1-0.2 57-73 65 64-86 73
Grass 0.04-40 70-120 94 75 75
Grass silage 2-40 70-79 76 70 70
Hay 2-40 84-92 88 - -
Peas 0.05-40 53-92 76 75-94 85
Rape (seeds) 0.06-3.3 57-92 74 50-52 51
Sugar beet
Foliage 0.03-0.06 70-100 82 64-93 79
Edible root 0.03 73-91 82 67-70 68
Soil 0.05-1 66-86 80 69-80 73
Water 0.0001-2.5*) 77-138 101 75-95 85
> Equivalent to 0.1-2500 \xg/\
The limit of detection for glyphosate and AMPA was 0.01 to 0.02 mg/kg for plant material,
beer, bread, flour and soil, and the limit of determination was 0.02 to 0.05 mg/kg. Blanks
usually did not occur.
For tap water, the limit of detection was 0.05 ng/1, and the limit of determination was
0.1 jxg/1. Blanks usually did not occur or, if so, they were less than 0.04 fxg/1.

The residue R, expressed in mg/kg glyphosate or AMPA, is calculated from the following
equations:
W
for plant material R= A ' (VBx + x) VEnd
VR1 V G
for beer, soil and water R=
where
x = water portion of the sample weight (plant material) (in g)
VEx = volume of solvent used for extraction of sample (in ml)
VR1 = portion of volume VEx used for cleanup in 6.2 (in ml)
236 Glyphosate

Vj = portion of volume VEnd injected into high-performance liquid chromatograph (in ul)
WA = amount of glyphosate or AMPA, respectively, for V{ read from calibration curve
(in ng)
8 Important points
The water content of the sample material can be determined by drying to constant weight at
105 C. Alternatively, the average water content of the respective material as listed in Table 2,
Method S 19 (p. 386, Vol. 1) may be used for calculation. (Note that in this Table the figure
given for sugar beet root should read 70%.)
Two Varian pumps, Model PCR IB, or two high pressure pumps, e. g. Perkin Elmer 10 or
LKB 2150, can also be used for the HPLC post-column derivatization system. The solution
should exhibit a pH of 8.5 to 9 at the exit of the detector.
The Aminex column used for HPLC is very sensitive to metal ions. It is, therefore, very im-
portant that the top layer of the anion exchange resin used for removal of the ferric ions
should not be disturbed.
It is recommended that metal ions in soil samples should be complexed by making up
the residue derived from 6.2.2 to a definite volume VEnd, e.g. 5 ml, with EDTA solu-
tion (0.001 mol/1; 0.37 g ethylenedinitrilotetraacetic acid disodium salt dihydrate in 1.0 1
water).
The determination of glyphosate can also be carried out using a stainless steel column,
4.6 mm i.d., 25 cm long, packed with Zorbax Sax (DuPont No. 880952703) or Partisil Sax
(Whatman No. 4226-001). These columns are not suitable for the analysis of the metabolite,
AMPA.
9 References
H. A. Moye, C. J. Miles and S. J Scherer, A simplified high-performance liquid chromato-
graphic residue procedure for the determination of glyphosate herbicide and (amino-
methyl)phosphonic acid in fruits and vegetables employing postcolumn fluorogenic labeling,
J. Agric. Food Chem. 31, 69-72 (1983).
M. Roth, Fluorescence reaction for amino acids, Anal. Chem. 43, 880-882 (1971).
S. S. Simons, Jr. and D. E Johnson, The structure of the fluorescent adduct formed in the
reaction of o-phthalaldehyde and thiols with amines, J. Am. Chem. Soc. 98, 7098-7099
(1976).
10 Authors
Monsanto, St. Louis, MO, U.S.A., J E. Cowell and P. J Nord
Monsanto, Louvain-la-Neuve, Belgium, M. A. Reding
Glyphosate 237
a) b) c)
Time
Fig. 1. Chromatograms of
a) standard solution of glyphosate, amount injected equivalent to 80 ng;
b) untreated control sample of sugar beet, blank equivalent to < 0.01 mg/kg glyphosate (arrow);
c) untreated control sample of sugar beet fortified with 0.2 mg/kg glyphosate, recovery 98%.
Column Aminex A9.
a) b)
Time
a) standard solution of AMPA, amount injected equivalent to 10 ng;
b) untreated control sample of cereal straw, blank equivalent to <0.05 mg/kg AMPA (arrow);
c) untreated control sample of cereal straw fortified with 0.067 mg/kg AMPA, recovery 83%.
Column Aminex A9.
238 Glyphosate
b) c)
Time
a) standard solution of glyphosate, amount injected equivalent to 10 ng;
b) untreated control sample of tap water, blank equivalent to < 0.05 jig/1 glyphosate (arrow);
c) untreated control sample of tap water fortified with 0.1 (ig/1 glyphosate, recovery 83%.
Column Zorbax Sax.
Metaldehyde 151-A
Chicoree leaves, Chinese cabbage, corn salad, cut lettuce, Gas-chromatographic

endives, lettuce, radishes, spinach, strawberries determination
1 Introduction
r-2,c-4,c-6,c-8-Tetramethyl-l,3,5,7-tetroxocane (IUPAC)
(acetaldehyde tetramer) as main component
Chemical name
CHa CH 3
Structural formula CH
Empirical formula C 8 H 16 O 4
Molar mass 176.22
Melting point 246 C sealed tube (pure tetramer)
Boiling point Not distillable, sublimes at 110-120 C
Solubility Very sparingly soluble in water at 17 C;
soluble in chloroform and dichloromethane;
sparingly soluble in benzene (0.12 g/100 ml at 23 C)
Other properties Depolymerized in the presence of acids and at elevated
temperatures
2 Outline of method
Metaldehyde residues are extracted from plant material with dichloromethane. Interfering
aldehydes are removed with sodium meta-bisulphite solution. Metaldehyde is depolymerized
with hydrochloric acid to acetaldehyde, which is reacted with 2,4-dinitrophenylhydrazine. The
resulting acetaldehyde 2,4-dinitrophenylhydrazone is cleaned up on an aluminium oxide col-
umn and determined by gas chromatography using a thermionic detector.
3 Apparatus
Wide neck bottle, 250-ml, with screw cap

Sintered glass filter funnel, porosity G2, 13 cm dia.
Separatory funnels, 250-ml and 100-ml, with ground joints
240 Metaldehyde

Centrifuge, 2000 r.p.m.
Laboratory mechanical shaker, suitable for holding separatory funnels
Microsyringe, 5-ul
4 Reagents
Dichloromethane, for residue analysis; see 8. Important points
Eluting mixture: dichloromethane + n-hexane 2:1 v/v
Metaldehyde standard solution: 5 (ig/ml toluene. Dissolve metaldehyde first in a minimum of
dichloromethane
2,4-Dinitrophenylhydrazine solution: 0.25 g/100 ml 2,4-dinitrophenylhydrazine (Merck No.
3081), recrystallized from toluene, in hydrochloric acid p. a. (6 mol/1)
Sodium meta-bisulphite solution, 2 g/100 ml Na2S2O5 p. a. (Merck No. 6528). Prepare fresh
daily
Aluminium oxide 90, Brockmann standardized, activity grade II-III, 0.063-0.200 mm (Merck
No. 1097)
Glass wool
Air, re-purified

6 Procedure
6.1 Extraction and removal of free aldehydes
Homogenize 50 g of the finely comminuted plant material (G) with 100 ml dichloromethane
in the wide neck bottle for 5 min. Suction-filter the homogenate through the sintered glass
filter funnel (homogenates from strawberries should be centrifuged first). Wash the filter cake
three times with 30-ml portions of dichloromethane. Combine the filtrates in a 250-ml
separatory funnel and shake for 5 min with 20 ml sodium meta-bisulphite solution. Separate
and discard the aqueous phase. Wash the organic phase twice with 50-ml portions of water.
Discard the aqueous layers, and rotary-evaporate the organic phase to dryness. Dissolve the
residue in about 20 ml of toluene.
Metaldehyde 241
6.2 Depolymerization and derivatization

Transfer the toluene solution derived from 6.1 to a 100-ml separatory funnel, add 5 ml 2,4-
dinitrophenylhydrazine solution, and shake for 60 min on the mechanical shaker. After phase
separation, discard the aqueous layer. Wash the organic phase twice with 20-ml portions of
water, filter it through sodium sulphate, and rinse the sodium sulphate with approx. 10 ml
toluene. Collect the filtrates in a 50-ml round-bottomed flask and rotary-evaporate to dryness.

Half-fill the chromatographic tube, containing a glass wool plug in the lower end, with hexane.
Slowly add 6 g aluminium oxide, gently tapping the tube walls. Allow the adsorbent to settle,
and then drain the supernatant hexane until the top of the aluminium oxide is just covered
with the solvent. Quantitatively rinse the residue derived from 6.2 onto the prepared column
with a total of 4 ml eluting mixture. Allow the liquid to percolate into the column at a rate
of 1-2 drops per s. Elute the acetaldehyde derivative with the eluting mixture. Collect a total
of 16 ml eluate in a 50-ml round-bottomed flask and rotary-evaporate to dryness.

Rinse the residue derived from 6.3 into a 10-ml volumetric flask (VEnd) with hexane and make
up to the mark. Inject an aliquot of this solution (V{) into the gas chromatograph.
Carry out a preliminary measurement to estimate approximately the concentration of
metaldehyde. If this is higher than 10 fig/ml, dilute the solution.
on Gas Chrom Q, 80-100 mesh
Temperature 320 C
Air, 170 ml/min
Attenuation 8 -10~12
Retention time for
acetaldehyde 2,4-
dinitrophenylhydrazone 2 min 24 s
242 Metaldehyde
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique using a calibration curve, which must
be prepared anew for each series of analyses. Proceed as follows. Pipet each of the following
metaldehyde standard solution volumes, 2.0, 5.0, 10.0, and 20.0 ml (corresponding to 10, 25,
50, and 100 \ig metaldehyde, respectively), into 100-ml separatory funnels, and make up to
20 ml with toluene. Process these solutions in the same way as described in 6.2 and 6.3, and
finally make up the residues with hexane to 10.0 ml. Inject 1 ul of each of these acetaldehyde
derivative standard solutions (equivalent to 1 to 10 ng metaldehyde) into the gas chromato-
graph. Plot the areas or heights of the peaks obtained vs. ng metaldehyde. Also inject 1-ul ali-
quots of the sample solutions. For the areas or heights of the peaks obtained for these solu-
tions, read the appropriate amounts of metaldehyde from the calibration curve. Check the
calibration curve by injection of an acetaldehyde derivative standard solution at least after
every third injection.

The recoveries from untreated control samples, fortified with metaldehyde at levels of 0.2 to
2.0 mg/kg, ranged from 70 to 100%. The routine limit of determination was 0.1 to 0.2 mg/kg,
depending on the respective blank values. Blank values were in the range of 0.05 to 0.1 mg/kg.

The residue R, expressed in mg/kg metaldehyde, is calculated from the following equation:
R w A -v E n d
where
WA = amount of metaldehyde for Vj read from calibration curve (in ng)
8 Important points
In order to avoid interfering reagent blanks, dichloromethane must be shaken with 2,4-dinitro-
phenylhydrazine solution and further purified on a column with basic aluminium oxide, ac-
tivity grade I.
Metaldehyde 243
9 Reference
S. Selim and J. N. Seiber, Gas chromatographic method for the analysis of metaldehyde in
crop tissue, J. Agric. Food Chem. 21, 430-433 (1973).
10 Authors
RCC Umweltchemie AG, Itingen, Switzerland, W. Vogel
Metribuzin 337
Barley (grains), broad beans (incl. pods), carrots, Gas-chromatographic
potatoes (tubers and tops), tomatoes (leaves and fruits), determination
wheat (grains and straw)
Soil, water
1 Introduction
Chemical name 4-Amino-6-tert.butyl-3-methylthio-l,2,4-triazin-5(4H)-
one (IUPAC)
(CH3)3C
Structural formula
1N
SCH3
Empirical formula C8H14N4OS

Molar mass 214.29
Melting point 125.5-126.5 C
Vapour pressure 1.33-10"5 mbar at 20C
Solubility Sparingly soluble in water (0.12 g);
(in 100 ml at 20 C) readily soluble in acetonitrile, dichloromethane,
ethyl acetate (>60 g each), methanol (45 g) and
2-propanol (13 g);
slightly soluble in ligroin (1 g);
sparingly soluble in petroleum ether (0.48 g)
Other properties 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 dichloro-
methane 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
Glass filter funnel, e.g. 26D-2 (Schott)
Ice bath
Glass bottle, 500-ml
Laboratory mechanical shaker, 150 r.p.m.
Glass funnel
Fluted filter paper
Microsyringe, 10-ul
4 Reagents
Acetonitrile, p.a., dist.
Ethanol, denatured with methyl ethyl ketone
Ethyl acetate, dist.
Methanol, dist.
Petroleum ether, dist., boiling range 40-60C
Eluting mixture: dichloromethane + ethanol 8:2 v/v
Dichloromethane + ethyl acetate mixture 9:1 v/v
Metribuzin standard solution: 5 ng/ml methanol
Silica gel 60, 0.063-0.200 mm (Merck No. 7734), heated for 1 h at 100 C
Cottonwool
Air, synthetic
Helium

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 fun-
nel. 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 im-
mediately chill the aqueous residue in an ice bath.
6.1.2 Barley, wheat (grains)

acetonitrile, and combine the washings with the filtrate in a 1-1 separatory funnel.
6.1.3 Wheat (straw)

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 superna-
tant 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 con-
tinue 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
248 Metribuzin

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 di-
chloromethane. 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 chromato-
graphic tube, dislodge any trapped air by stirring with a glass rod, and drain the eluting mix-
ture 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.

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.
Column Glass, 2 mm i.d., 1.8 m long; packed with
6% Apiezon L + 3% OV-17 on Chromosorb W-AW-
DMCS, 80-100 mesh
Metribuzin 249

Temperature 300 C
Gas flow rates Helium carrier, 20-30 ml/min
Hydrogen, 1-5 ml/min
Air, 40-100 ml/min
Attenuation 25
Recorder Chart speed 0.5 inch/min (12.7 mm/min)
Injection volume 1-3 \i\
Retention time for metribuzin approx. 10 min
7 Evaluation
7.1 Method
ing 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.

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 deter-
mination was 0.05 mg/kg.
Table. Percent recoveries from plant material, soil and water, fortified with metribuzin.
Metribuzin added (mg/kg)
Analytical material
0. 05 0.1 0.5
Barley (grains) 85 83 86
Broad beans
Beans 94 96 95
Pods 75 90 80
Carrots 89 88 82
Potatoes (tubers) 91 89 90
Tomatoes
Fruits 98 95 86
Leaves 98 81 99
Wheat
Grains 80 82 85
Straw 80 79 85
Soil
Standard soil 2.1 80 80 83
Water 85 89 90
250 Metribuzin
T h e soils used for the recovery experiments h a d the following characteristics:
Organic carbon Particles <0.02 mm

Soil type % % PH
Standard soil 2.1 *> 0.31 8.5 6.0

Standard soil 2.2> 2.64 14.5 6.0
Standard soil 2.3*> 1.06 24.7 7.0
*> Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA),
cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.

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
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 ex-
ceed 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 metha-
nol-water mixture, acetonitrile and dichloromethane, combine the supernatants in a 1-1 round-
bottomed 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.butyl-
l,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
H. J. Jarczyk
Nitrothal-isopropyl 416
Apples Gas-chromatographic
1 Introduction
Nitrothal-isopropyl
Chemical name Di-isopropyl 5-nitroisophthalate (IUPAC)
CO-O-CH
Structural formula
CO-O-CH
Empirical formula C14H17NO6

Molar mass 295.30
Melting point 65 C
Vapour pressure <0.1 -10" 6 mbar at 20 C
Solubility Very sparingly soluble in water;
readily soluble in acetone, benzene, chloroform and
ethyl acetate
Other properties Yellow crystals, steam volatile, hydrolyzed in alkaline
media
Metabolite I (Monoester)
Chemical name Isopropyl 5-nitroisophthalate
/ CH 3
CO-O-CH
/
Structural formula O2N-^J>
COOH
Empirical formula C U H U NO 6
Molar mass 253.22
Melting point 178 C
Solubility No data
254 Nitrothal-isopropyl
Metabolite II (Acid)
Chemical name 5-Nitroisophthalic acid
/COOH
Structural formula 02NHQ

COOH
Empirical formula C8H5NO6

Molar mass 211.13
Melting point 255 C
Solubility Slightly soluble in water (approx. 2g/100ml at 25 C);
soluble in ethanol and in dilute alkaline solutions
2 Outline of method
Nitrothal-isopropyl residues are steam-distilled from the analytical sample in a Bleidner appa-
ratus, simultaneously being extracted from the condensate into isooctane. The metabolites re-
maining in the aqueous phase are extracted after acidification into tert.butyl methyl ether and
methylated with diazomethane. The methylated metabolites are also steam-distilled in the
Bleidner apparatus and extracted into isooctane. Nitrothal-isopropyl and its methylated me-
tabolites are determined by electron-capture gas chromatography.
3 Apparatus
Bleidner apparatus, modified by W. Heizler, for distillation and extraction; see Fig. 1, p. 243,
Vol. 1
Hotplate with magnetic stirrer, e.g. IKA Combimag RCT (Janke & Kunkel)
Aluminium foil
Graduated cylinder, 500-ml, with glass stopper
Laboratory centrifuge, explosion-proof, with 250-ml glass tubes
Rotary vacuum evaporator, 20C, 30C and 50-60C bath temperature
Microsyringe, 10-ul
N itrothal-isopropy I 255
4 Reagents
tert.Butyl methyl ether, dist.
Standard solutions: Mixtures containing 0.2, 0.4 and 0.6 ng/ml each of nitrothal-isopropyl,
dimethyl 5-nitroisophthalate and isopropyl methyl 5-nitroisophthalate
Compound and metabolite solutions for fortification experiments: Dissolve 10 mg nitro-
thal-isopropyl, 10 mg metabolite I and 10 mg metabolite II individually in 10 ml methanol.
From each of these solutions, pipet 1 ml into a 100-ml volumetric flask and make up to vol-
ume with water
Diazomethane solution in diethyl ether (for apparatus, see Fig. 1, p. 130, Vol. 1):
Dissolve 2 g N-methyl-N-nitroso-p-toluenesulphonamide (Merck No. 808406) in 60 ml diethyl
ether and transfer to the dropping funnel. Slowly add this solution dropwise to 10 ml
methanolic potassium hydroxide solution contained in the reaction vessel standing in a 60 C
water bath. Distil the generated diazomethane and the diethyl ether through a descending con-
denser into a receiver which is cooled with an ice + sodium chloride freezing mixture
ortho-Phosphoric acid, p.a., 85% (Merck No. 573)
Boric acid, p. a. (Merck No. 165)
Methanolic potassium hydroxide solution, 10 g/100 ml KOH p. a. in methanol + water mix-
ture 9:1 v/v
Isopropyl 5-nitroisophthalate (metabolite I): Add 4 g nitrothal-isopropyl to 10 ml concen-
trated sulphuric acid at room temperature and stir (on a magnetic stirrer) until it is dissolved.
Add 20 g ice and continue to stir strongly when a white crystalline precipitate containing oily
lumps is formed. Add water, and suction-filter the precipitate. Dissolve the precipitate in chlo-
roform and a little acetone and extract the solution with water. With stirring, add sodium hy-
drogen carbonate solution portionwise to the chloroform phase until the aqueous phase has
a pH of 4.5 (pH indicator paper). Shake the mixture vigorously, separate off the organic
phase, and extract the aqueous phase with chloroform once more. Wash the combined chloro-
form phases with water, dry on sodium sulphate, and rotary-evaporate to dryness. Dissolve
the residue in a little acetone and dilute the solution with n-hexane to form a precipitate. Suc-
tion-filter the precipitate and extract repeatedly with boiling n-hexane to remove unreacted
starting material. Dissolve the crystalline residue in acetone, treat with activated charcoal, pre-
cipitate with water and suction-filter. Dry over phosphorus pentoxide and paraffin in a desic-
cator. Yield: approx. 450 mg (m.p. 178C)
Isopropyl methyl 5-nitroisophthalate: Dissolve 200 mg metabolite I in a little diethyl ether and
add diazomethane solution until a slight yellow colour remains. Evaporate the solution to dry-
ness. Scratch the remaining oily residue with a glass rod to initiate crystallization, and dry the
crystalline mass over phosphorus pentoxide in a desiccator (m.p. 48C)
Dimethyl 5-nitroisophthalate (Aldrich No. 23,736-1)
5-Nitroisophthalic acid (Fluka No. 73450) (metabolite II)
Helium, pure

256 Nitrothal-isopropyl
6 Procedure
6.1 Distillation and extraction (Nitrothal-isopropyl)
6.1.1 Apples, soil
Weigh 50 g of the analytical sample (G) into a 500-ml round-bottomed flask and add 2.5 g
boric acid, 150 ml water, and a magnetic stirring rod. Half-fill the U-tube of the Bleidner
apparatus with water and isooctane, and connect the flask to the lower arm of the Bleidner
apparatus. Attach a 50-ml round-bottomed flask containing 25 ml isooctane to the upper arm
of the Bleidner apparatus. Wrap both flasks with aluminium foil to exclude light. Heat both
flasks for 2 h at a temperature that will ensure condensation of equal amounts of water and
isooctane (this can be checked easily from the volumes of the immiscible solvent phases in the
capillary of the Bleidner apparatus). On completion of the distillation-extraction step, allow
both flasks to cool to room temperature; then proceed to step 6.2.1 for the water phase, and
to step 6.4 for the isooctane phase.
6.1.2 Water
Transfer 250 ml of the water sample (G) to a 500-ml round-bottomed flask and add 2.5 g boric
acid. Attach this flask to the lower arm of the Bleidner apparatus, and a 50-ml round-bot-
tomed flask containing 25 ml isooctane to the upper arm. Proceed as described in 6.1.1.
6.2 Extraction (Metabolites)

6.2.1 Apples, soil
Transfer the aqueous phase derived from 6.1.1 into a graduated cylinder and make up to
200 ml with water (VEx). Shake well and centrifuge for 15 min at 3500 r.p.m. Decant an ali-
quot of 120 ml (VR1) and acidify with 2.5 ml phosphoric acid. Transfer the solution to a
separatory funnel and extract twice with tert.butyl methyl ether, shaking successively with
100-ml and 50-ml portions of the solvent. Break any emulsions by centrifuging. Combine the
ether extracts in a 500-ml round-bottomed flask and rotary-evaporate to dryness with 30 C
bath temperature.
6.2.2 Water
Rotary-evaporate the aqueous phase derived from 6.1.2 to approx. 120 ml with 50-60C bath
temperature. Acidify with 2.5 ml phosphoric acid and extract the solution twice with tert.butyl
methyl ether as described in 6.2.1. Rotary-evaporate the ether extracts to dryness with 30 C
bath temperature.
6.3 Methylation (Metabolites)

Add 20 ml diazomethane solution to the residue derived from 6.2.1 or 6.2.2, allow to stand
for 30 min at room temperature, and rotary-evaporate to a few drops with 20 C bath tempera-
ture.
Nitrothal-isopropyl 257
6.4 Distillation and extraction (Methylated metabolites)

To the residue derived from 6.3, add 50 ml water, 50 ml sodium chloride solution and 2.5 g
boric acid. Wrap the round-bottomed flask with aluminium foil and attach it to the lower arm
of the Bleidner apparatus. Attach the 50-ml round-bottomed flask containing the isooctane
extract obtained from 6.1.1 or 6.1.2 (25 ml) to the upper arm of the apparatus. Heat the con-
tents of both flasks to boiling as described in 6.1.1. Allow the isooctane extract to cool to room
temperature and make up to a definite volume (VEnd), either by diluting with isooctane or by
concentrating on a rotary evaporator with 30 C bath temperature.

Gas chromatograph Perkin-Elmer 3920 B
Column Glass capillary, 0.28 mm i.d., 25 m long; coated with
SE-54, film thickness 0.5 ^m
63
Temperature 300 C
Gas flow rates Helium carrier, inlet pressure 1 bar
Argon-methane purge gas, 30 ml/min
Split ratio 1:10
Attenuation 64
Recorder 1 mV; chart speed 6.7 mm/min
Injection volume lul
Retention times for
dimethyl 5-nitroisophthalate 2 min 24 s
isopropyl methyl 5-nitro-
isophthalate 3 min 9 s
nitrothal-isopropyl 4 min 3 s
7 Evaluation
7.1 Method
Inject 1 ul of each standard solution (equivalent to 0.2 to 0.6 ng of each compound) into the
gas chromatograph. Plot the heights of the peaks obtained vs. ng of nitrothal-isopropyl,
dimethyl 5-nitroisophthalate and isopropyl methyl 5-nitroisophthalate, respectively. Also in-
ject l-|o.l aliquots of the sample solutions. For the heights of the peaks obtained for these solu-
tions, read the appropriate amounts of the respective compounds from the corresponding cali-
bration curve.
258 N itrothal-isopropy I

The recoveries from untreated control samples, fortified with nitrothal-isopropyl and the two
metabolites, are given in the Table.
The routine limit of determination was 0.1 mg/kg for apples and soil, and 1 u.g/1 for tap
water.
Table. Percent recoveries for nitrothal-isopropyl, metabolite I and metabolite II from apples and soil, for-
tified at levels of 0.1 to 1 mg/kg, and from water, fortified at a level of 1 ng/1.
Analytical Nitrothal-isopropyl Metabolite I Metabolite II

n
material Range Mean Range Mean Range Mean
Apples 8 66-83 76 64-83 74 77-80 79

Soil 8 63-87 75 78-90 85 90-104 97
Tap water 4 57-64 61 55-67 63 109-118 115

The residue R, expressed in mg/kg, of nitrothal-isopropyl and the metabolites (calculated as
nitrothal-isopropyl) is calculated from the following equations:
for nitrothal-isopropyl R = wAV

G
End
Vi
for isopropyl methyl 5-nitroisophthalate

WA v E x - V nd
E
v, Rl-Vi G
WA . y Y
for dimethyl 5-nitroisophthalate R = ^ ^ ^ - 1.235
VR1 Vi G
where
VEx = total volume of the water phase from 6.2.1 (in ml)
VR1 = portion of volume VEx used for extraction of metabolites (in ml)
^ = portion of volume VEnd injected into gas chromatograph (in \i\)
WA = amount of nitrothal-isopropyl, dimethyl 5-nitroisophthalate or isopropyl methyl
5-nitroisophthalate, respectively, for V4 read from calibration curve (in ng)
1.105 = factor for conversion of isopropyl methyl 5-nitroisophthalate to nitrothal-iso-
propyl
1.235 = factor for conversion of dimethyl 5-nitroisophthalate to nitrothal-isopropyl
Nitrothal-lsopropyl 259
8 Important points
The gas-chromatographic determination can also be performed using a packed column under
the following conditions:
Column Glass, 2.5 mm i.d., 2.6 m long; packed with 1.0%
OV-17 + 1.95% OV-210 on Gas Chrom Q,
100-120 mesh
63
Temperature 300 C
Retention times for
dimethyl 5-nitroisophthalate 5 min 12 s
isopropyl methyl 5-nitro-
isophthalate 5 min 48 s
nitrothal-isopropyl 6 min 48 s
The standard solutions as well as the compound and metabolite solutions are stable for at least
two weeks when stored in a refrigerator.
9 References
No data
10 Authors
BASF, Agricultural Research Station, Limburgerhof, W. Keller and P. Beutel
Oxamyl 441
Apples, carrots, celeriac (leaves and bulbs), coffee (raw), Gas-chromatographic
cottonseed, grapefruit, grapes, grass, lettuce, oranges, determination
peaches, peanuts (foliage, kernels and shells), potatoes,
sweet peppers, tobacco, tomatoes
Soil, water
1 Introduction
N,N-Dimethyl-2-methylcarbamoyloxyimino-
Chemical name 2-(methylthio)acetamide (IUPAC)
H,C o o
sf-C-C = N-O-CNH-CH
H,C S CH,
Structural formula
Empirical formula
Molar mass 219.26
Melting point 108-110 C
Boiling point Not distillable without decomposition
Vapour pressure 3.1-10- 4 mbar at 25 C
1.0-10- 2 mbar at 70C
Solubility (in 100 ml at 25 C) Readily soluble in water (28 g);
very readily soluble in methanol (144 g);
readily soluble in acetone (67 g), ethanol (33 g) and
2-propanol (11 g);
slightly soluble in toluene (1 g)
Other properties Decomposed in alkaline conditions, at elevated
temperatures and in ultraviolet light
2 Outline of method
Oxamyl residues are extracted from plant material, soil and water with ethyl acetate, parti-
tioned between hexane and water, and cleaned up by extraction with dichloromethane at pH
12 to remove interfering co-extractives. Oxamyl is subjected to alkaline hydrolysis. The result-
ing, more volatile oximino derivative is determined by gas chromatography using a sulphur-
specific flame photometric detector.
262 Oxamyl
X fi fi |OH-| H 3 C x
NC-C=N-O-CNHCH3 ^NC-C=N-OH
/ H3C
S-CH 3 S-CH 3
Oxamyl Oxamyl oximino derivative
3 Apparatus
Centrifuge, 1500 r.p.m., with 250-ml glass tubes
Vacuum adapter
Reflux condenser
Microsyringe, 10-ul
4 Reagents
Acetone, p. a.
n-Hexane, p. a.
Methanol, p. a.
Ethyl acetate + methanol mixture 9:1 v/v
Derivative standard solutions: 0.1, 0.5, 1.0, 2.0, 3.0, 5.0 and 10.0 ng/ml oxamyl oximino deriv-
ative in acetone
N,N-Dimethyl-2-hydroxyimino-2-(methylthio)acetamide (oxamyl oximino derivative; DuPont
de Nemours)
Sodium hydroxide solution, 1 mol/1 NaOH, p. a.
Sodium sulphate, p. a., anhydrous
Universal indicator paper
Oxamyl 263
Air, synthetic
Helium
Oxygen, re-purified

6 Procedure
6.1 Extraction
Weigh 25 g of the analytical sample (G) into the blendor jar, add 100 ml ethyl acetate, and
homogenize for 5 min. Transfer the homogenate quantitatively into a centrifuge tube and cen-
trifuge at 1500 r.p.m. for 10-15 min. Suction-filter the liquid through a fast flow-rate filter
paper covered with 5 g filter aid contained in a Buchner porcelain funnel, and collect in a 1-1
round-bottomed flask. Extract the residue in the centrifuge tube two more times in the same
way using ethyl acetate. After the final filtration, wash the filter cake with 50 ml ethyl acetate.
Add 50 ml water to the combined filtrates (see Section 8, Important points), and rotary-evap-
orate to an aqueous residue.
6.1.2 Soil
Weigh 25 g of soil (G) into an Erlenmeyer flask, add 100 ml ethyl acetate and 25 ml water,
stopper, and shake for 15 min on a mechanical shaker. Suction-filter the extract through a fast
flow-rate filter paper contained in a Buchner porcelain funnel, and collect in a 1-1 round-bot-
tomed flask. Repeat the extraction two more times using 100 ml ethyl acetate each time. Wash
the filter cake with 50 ml ethyl acetate. Add 50 ml water to the combined filtrates, and
rotary-evaporate to an aqueous residue.
6.1.3 Water
Place a 50-ml sample of water (G) in a 250-ml separatory funnel, add 100 ml hexane, and
shake for 2 min. Allow the phases to separate (centrifuge if necessary to obtain clean separa-
tion). Discard the hexane wash. Extract the aqueous phase with three 100-ml portions of ethyl
acetate using 2-min shaking periods for each extraction. Allow the phases to separate, and fil-
ter the ethyl acetate extract through approx. 10 g sodium sulphate into a 1-1 round-bottomed
flask. Wash the sodium sulphate with 50 ml ethyl acetate. Add 50 ml water to the combined
filtrates, and rotary-evaporate to an aqueous residue.
264 Oxamyl
6.2 Cleanup
Transfer the aqueous residue (approx. 40 ml) derived from 6.1 quantitatively to a 250-ml
separatory funnel, washing the 1-1 flask several times with a total of 10 ml water to complete
the transfer. Add 50 ml hexane to the separatory funnel, shake gently for 1 min, and allow
the phases to separate. Centrifuge, if necessary, to obtain a clean separation. Discard the hex-
ane layer. Repeat the hexane wash two more times using additional 50-ml portions of solvent.
Discard the hexane after each wash. Adjust the pH of the aqueous solution to 12 by adding
about 3 ml sodium hydroxide solution (check with indicator paper), and then extract with two
50-ml portions of dichloromethane for 1 min each time. Discard the dichloromethane phases,
and collect the aqueous phase in a 100-ml round-bottomed flask.
6.3 Hydrolysis
Heat the aqueous solution derived from 6.2 on a 95 C water bath with occasional stirring to
remove the residual dichloromethane. Next connect the flask to a reflux condenser, and con-
tinue to heat at the same temperature for an additional 15 min to convert oxamyl to the
oximino derivative by hydrolysis. Cool and quantitatively transfer the aqueous solution to a
250-ml separatory funnel, washing the flask and the reflux condenser several times with a few
ml of water each time to complete the transfer. Extract with two 50-ml portions of
dichloromethane, shaking for 1 min each time. Then saturate the aqueous phase by adding
about 15 to 25 g of sodium chloride, and extract with four 50-ml portions of ethyl ace-
tate-methanol mixture using 2-min shaking periods for each extraction. Allow the phases to
separate. Centrifuge, if necessary, to obtain a clean separation.
Dry the combined organic phases on sodium sulphate, and filter through a fluted filter pa-
per into a 500-ml round-bottomed flask. Wash the filter with 50 ml ethyl acetate and ro-
tary-evaporate the filtrate almost to dryness. Remove the residual solvent by swirling the flask
in the hand. Transfer the flask contents to a graduated test tube using several acetone washes.
Concentrate the solution at room temperature to a given volume (VEnd), using a stream of
nitrogen.

6.4.1 Column 1
Gas chromatograph Perkin-Elmer 3920
Column Glass, 1.6 mm i.d., 0.91 m long; packed with 10% SP-
1200 + 1% H3PO4 on Chromosorb W-AW, 80-100
mesh
Temperature 200 C
Oxamyl 265

Oxygen, 20 ml/min
Air, 40 ml/min
Retention time for oxamyl
oximino derivative 7 min
6.4.2 Column 2
Gas chromatograph Per kin-Elmer F 11
Column Glass, 4 mm i. d., 1.8 m long; packed with 2% Car-
bowax 20 M + 5% DC-200 on Chromosorb W-AW,
80-100 mesh
Temperature 170 C
Oxygen, 20 ml/min
Attenuation 8-103
oximino derivative 4 min
7 Evaluation
7.1 Method
Inject equal volumes of differently concentrated derivative standard solutions into the gas
chromatograph. Using log-log paper, plot the heights of the peaks obtained vs. the amounts
of oxamyl oximino derivative injected with the standard solutions. Also inject equal volumes
of the sample solutions. For the heights of the peaks obtained for these solutions, read the
appropriate amounts of oxamyl oximino derivative from the calibration curve.

The recoveries from untreated control samples, fortified with oxamyl at levels of 0.02 to
10 mg/kg, ranged from 70 to 120% and averaged 90%. The routine limit of determination
was 0.02 mg/kg.

The residue R, expressed in mg/kg oxamyl, is calculated from the following equation:
266 Oxamyl
W
R = A/^" . j.352
Yj G
where
Vj = portion of volume VEnd injected into gas chromatograph (in jxl)
WA = amount of oxamyl oximino derivative for V{ read from calibration curve (in ng)
1.352 = factor for conversion of oxamyl oximino derivative to oxamyl
8 Important points
The determination of oxamyl residues is based on the gas-chromatographic measurement of
the hydrolysis product because the oxamyl oximino derivative responds with considerably
greater sensitivity to flame photometric detection.
During the analysis of highly acidic substrates, e.g. oranges, grapefruit and peaches, some-
what lower recoveries are noted. To improve the recovery rates, the acid must be neutralized.
Therefore, prior to evaporation, 50 ml of sodium hydroxide solution is added, instead of 50 ml
of water, to the combined filtrates derived from 6.1.1.
During studies to validate the method in the Chemistry Division of the Federal Biological
Research Centre for Agriculture and Forestry, Braunschweig, it was found that the following
gas-chromatographic conditions are also suitable:
Gas chromatograph Carlo Erba Fractovap 2101 AC
Column Glass, 2 mm i.d., 1.5 m long; packed with 10% DC-
Detector Flame photometric detector (Erba Science, SSD 250),
equipped with 394-nm sulphur filter
Temperature 200 C
Air, 200 ml/min
Attenuation 64
Injection volume 57 pil
oximino derivative 1 min 36 s
Oxamyl 267
9 Reference
R.E Holt and H.L. Pease, Determination of oxamyl residues using flame photometric gas
chromatography, J. Agric. Food Chem. 24, 263-266 (1976).
10 Authors
DuPont de Nemours & Co., Wilmington, DE, U.S.A., R.E Holt and H.L. Pease
Phenmedipham 233-B
Fodder beet (foliage and root), mangold, red beet (edible Gas-chromatographic
root), strawberries, sugar beet (foliage and edible root), determination
witloof chicory
1 Introduction
Chemical name Methyl 3-(3-methylcarbaniloyloxy)carbanilate (IUPAC)
Structural formula CH.O-C-NH-

Molar mass 300.32
Vapour pressure <10~ 8 mbar at 25 C
Solubility (in 100 ml at room Virtually insoluble in water;
temperature) readily soluble in acetone and cyclohexanone (each
approx. 20 g),
soluble in methanol (approx. 5 g),
slightly soluble in chloroform (approx. 2 g),
sparingly soluble in benzene (approx. 0.25 g),
very sparingly soluble in n-hexane (approx. 50 mg)
Other properties Largely stable in acid media, rapidly hydrolyzed in
alkaline media
2 Outline of method
Phenmedipham residues are alkali hydrolyzed in the presence of the analytical material under
reflux conditions in a Bleidner apparatus. The resultant 3-methylaniline is simultaneously
steam-distilled and extracted from the condensate into isooctane. Following re-extraction with
hydrochloric acid, 3-methylaniline is brominated to yield 2,4,6-tribromo-3-methylaniline,
which is extracted into toluene and determined by electron-capture gas chromatography.
3 Apparatus
High-speed blendor
Pyrex round-bottomed flasks, 2-1, 1-1 and 500-ml, with ground joints
270 Phenmedipham
Bleidner apparatus, modified by W. Heizler, for hydrolysis, distillation and extraction; see
Fig. 1, p. 243, Vol. 1
Heating mantles, for 2-1, 1-1, 500-ml and 250-ml round-bottomed flasks
Erlenmeyer flasks, 50-ml, with ground stoppers
4 Reagents
Toluene, for gas chromatography
Derivative standard solution: 1 ng/ml 2,4,6-tribromo-3-methylaniline in toluene
Hydrochloric acid, 1 mol/1 HC1 p.a.
Sodium hydroxide solution, 2.5 mol/1 and 5 mol/1
Potassium bromate solution, 0.2 g/100 ml KBrO3
2,4,6-Tribromo-3-methylaniline, melting point 98.5-99.5 C: Prepare from 3-methylaniline,
boiling point 203C, by bromination with a potassium bromate-potassium bromide mixture
in hydrochloric acid (1 mol/1)
2,4,6-Trimethylaniline solution, 0.2 g/100 ml in hydrochloric acid (1 mol/1): Prepare from
2,4,6-trimethylaniline purum (Fluka), additionally purified by chromatography on aluminium
oxide, basic, activity grade I (e.g. Camag), until the following criterion is met: Dissolve 200 mg
2,4,6-trimethylaniline in 100 ml hydrochloric acid (1 mol/1), brominate 10 ml of this solution
as described in 6.2, partition into 5 ml toluene, and inject 2 ul of the toluene solution into
the gas chromatograph. At the retention time of 2,4,6-tribromo-3-methylaniline (the "phen-
medipham position"), the chromatogram should not exhibit an interference peak exceeding
the equivalent of 0.2 ng 2,4,6-tribromo-3-methylaniline
Potassium bromide, p.a.
Sodium sulphite, p.a.
Antifoam liquid, e.g. Antifoam A
Argon + methane mixture 95 :5 v/v

The analytical sample is taken and prepared as described on pp. 17 ff, Vol 1.
6 Procedure
6.1 Hydrolysis, distillation and extraction
Weigh 50 g of the comminuted analytical sample (G) into a 500-ml, 1-1 or 2-1 Pyrex round-bot-
tomed flask (choose capacity of flask according to the proneness of the sample material to
foaming). Then add 250, 500 or 1000 ml sodium hydroxide solution and 1-4 ml Antifoam.
Place the flask in a heating mantle, half-fill the U-tube of the Bleidner apparatus with water
Phenmedipham 271
and isooctane, and connect the flask to the lower arm of the Bleidner apparatus. Attach a
250-ml round-bottomed flask filled with 100 ml isooctane to the upper arm of the Bleidner
apparatus. Heat both flasks for 2V2 h at a temperature that will ensure condensation of equal
amounts of water and isooctane; this can be checked easily from the volumes of the immis-
cible solvent phases in the capillary of the Bleidner apparatus. On completion of the distilla-
tion-extraction step, let the isooctane cool to room temperature.
Next extract the isooctane solution successively with 10-ml, 5-ml, and 5-ml portions of hy-
drochloric acid and combine the acid extracts in a 50-ml Erlenmeyer flask.
6.2 Bromination
Weigh 12 g potassium bromide into a 50-ml Erlenmeyer flask and add 0.5 ml 2,4,6-
trimethylaniline solution. Transfer the solution derived from 6.1 into the flask, rinsing with
3 ml water to complete the transfer. Stopper the flask and vigorously shake for 5 min. Add
0.5 ml potassium bromate solution, whereby the reaction mixture will become yellow. Should
decolourization occur, add a further 0.5 ml of potassium bromate solution.
Allow the solution to stand for 30 min; then stop the reaction by adding approx. 100 mg
sodium sulphite until decolourization is complete. Next, alkalize the mixture with 6 ml sodium
hydroxide solution (5 mol/1), add 5.0 ml toluene (VEnd), and vigorously shake for 1 min.
Pipet off the toluene layer after it has fully separated.

Inject an aliquot (Vj) of the solution derived from 6.2 into the gas chromatograph. Before
starting the analysis, saturate the gas-chromatographic column with 2,4,6-tribromo-3-me-
thylaniline by multiple injections of 10 ul each of the derivative standard solution.
Gas chromatograph Hewlett-Packard F-M 400
Column Glass, 3 mm i.d., 1.6 m long; packed with 10% PE-
SE-30 (NPGA terminated) on Chromosorb G,
80-100 mesh
63
Detector Ni electron capture detector, pulse interval 150 \is
Temperature 330 C
Gas flow rates Argon-methane carrier, 120 ml/min
Retention time for 2,4,6-
tribromo-3-methylaniline approx. 17 min (see also 8. Important points)
272 Phenmedipham
7 Evaluation
7.1 Method
Inject 1 to 10 |il of the derivative standard solution (equivalent to 1 to 10 ng 2,4,6-tribromo-
3-methylaniline) into the gas chromatograph. Plot the areas of the peaks obtained vs. ng 2,4,6-
tribromo-3-methylaniline. Also inject 2-ul aliquots of the sample solutions. For the areas of
the peaks obtained for these solutions, read the appropriate amounts of 2,4,6-tribromo-
3-methylaniline from the calibration curve.

The recoveries from untreated control samples, fortified with phenmedipham at levels of 0.05
to 0.3 mg/kg, were as follows:
Analytical Phenmedipham Recovery

material added (mg/kg) /o
Fodder beet
Foliage 0.05-0.3 12 103 16
Root 0.05-0.3 19 98 9
Red beet, edible root 0.05-0.3 18 97 16
Strawberries 0.05-0.1 4 85 2
Sugar beet
Foliage 0.1 -0.3 16 86 14
Edible root 0.05-0.1 13 94 9
The routine limit of determination was approx. 0.05 mg/kg. Blanks varied between 0.005
and 0.03 mg/kg; higher blank values are mostly due to laboratory contamination.

The residue R, expressed in mg/kg phenmedipham, is calculated from the following equation:
WA VEnd
R = ' 0.87
VrG
where
VEnd = volume of toluene used for extraction of the derivative in 6.2 (in ml)
WA = amount of derivative, 2,4,6-tribromo-3-methylaniline, for Vj read from calibration
curve (in ng)
0.87 = factor for conversion of 2,4,6-tribromo-3-methylaniline to phenmedipham
Phenmedipham 273
8 Important points
The addition of 2,4,6-trimethylaniline to the brominating mixture ensures complete bromina-
tion of the 3-methylaniline derived from phenmedipham, even at very low concentrations, and
also the formation of just one single bromination product. The calibration can therefore be
based on 2,4,6-tribromo-3-methylaniline.
Following bromination, the extracts of nearly all plant materials produce a most prominent
gas-chromatographic peak originating from 2-amino-3,5-dibromo-acetophenone (relative re-
tention time = 0.77, related to 2,4,6-tribromo-3-methylaniline).
No interference resulted from the aromatic amines derived from a great number of other
herbicides such as barban, chloridazon, chlorpropham, fenuron, monalide, monuron and pro-
pham. However, the brominated derivative of 3,4-dichloroaniline as derived from diuron,
linuron, neburon and propanil will not be separated satisfactorily from 2,4,6-tribromo-
3-methylaniline under the gas-chromatographic conditions given in this method.
9 References
W.E. Bleidner, Application of chromatography in determination of micro quantities of
3-(p-chlorophenyl)-l,l-dimethylurea, J. Agric. Food Chem. 2, 682-684 (1954).
H. Geissbuhler and H. Schredt, Comparison of different procedures used for residue determi-
nation of urea herbicides, Weed Research 7, 168-170 (1967).
K. Kofimann, Methoden zur Riickstandsbestimmung von Phenmedipham in Pflanzen-
material, Weed Research 70, 340-348 (1970).
10 Author
Schering AG, Berlin, K. Kofimann
Propachlor 310
Cauliflower, head cabbage, leeks, maize (kernels), onions Gas-chromatographic
(bulbs), peas, radishes, sugar beet (edible root) determination
Soil, water
1 Introduction
2-Chloro-N-isopropylacetanilide (IUPAC)
Chemical name o
II
C-CH2C1
V
CH-CH 3
Structural formula
CH3
Empirical formula CUH14C1NO
Molar mass 211.69
Melting point 77 C
Boiling point 110C at 0.04 mbar
Vapour pressure 3-10" 4 mbar at 25 C
Solubility (in 100 ml at 20 C) Very sparingly soluble in water;
readily soluble in acetone (31 g), benzene (50 g), car-
bon tetrachloride (14.8 g), chloroform (38 g), diethyl
ether (17.9 g), ethanol (29 g) and xylene (19.3 g);
slightly soluble in n-hexane (1.1 g)
Other properties Decomposed in alkaline and strongly acid media
2 Outline of method
Propachlor residues are extracted from plant material and soil with acetone, from water sam-
ples with dichloromethane. Extracts from plant material, with the exception of maize extracts,
are cleaned up by acid precipitation, followed by partitioning of propachlor from the aqueous
residue of the filtrate into a petroleum ether-dichloromethane mixture. The extract is further
cleaned up by column chromatography on Florisil. Soil extracts are cleaned up by liquid-liquid
partition. Propachlor is determined by gas chromatography using a thermionic or an electron
capture detector.
3 Apparatus
High-speed blendor fitted with leak-proof steel jar and explosion-proof motor
Filter paper, 9 cm dia., medium flow rate
276 Propachlor
Vacuum adapter
Separatory funnels, 2-1, 1-1 and 250-ml
Fluted filter paper, 18.5 cm dia. (Schleicher & Schull)
Test tubes, 10-ml and 5-ml, graduated, with ground stoppers
Gas chromatograph equipped with thermionic nitrogen-specific detector or with electron cap-
ture detector
Microsyringe, 10-ul
4 Reagents
Acetone, high purity, dist.
Dichloromethane, high purity, dist.
n-Hexane, high purity, dist.
Methanol, high purity, dist.
Petroleum ether, boiling range 40-60 C, dist.
Eluting mixture 1: n-hexane + acetone 95 :5 v/v
Eluting mixture 2: n-hexane + acetone 8:2 v/v
Petroleum ether + dichloromethane mixture 8:2 v/v
Propachlor standard solutions: 0.01-10 jag/ml n-hexane
Hydrochloric acid, 0.1 and 1.0 mol/1 HC1 p.a.
Sodium chloride, p.a.
Florisil, 60-100 mesh, heated for 8 h at 130 C
Air, synthetic
Helium

The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol 1. For
Propachlor 277
6 Procedure
6.1 Extraction
6.1.1 Plant material (except maize)
Homogenize 100 g of the analytical sample (G) with 200 ml acetone and 10 g filter aid for
3 min in the blendor. Suction-filter the homogenate through a filter paper in a Buchner porce-
lain funnel into a 1-1 round bottomed flask, and wash the filter cake with 100 ml acetone.
Rotary-evaporate the filtrate to an aqueous residue.
6.1.2 Maize (kernels)

Grind 50 g maize kernels (G) portionwise in the blendor, then add 200 ml acetone and homog-
enize for 3 min. Suction-filter the homogenate through a filter paper in a Buchner porcelain
funnel containing a layer of filter aid into a 1-1 round-bottomed flask. Wash the filter cake
with 100 ml acetone, and rotary-evaporate the filtrate to near dryness. Remove the last traces
of solvent by swirling the flask in the hand, and proceed as described in 6.2.3.
6.1.3 Soil
Extract 100 g of the soil sample (G) with 30 ml water and 200 ml acetone on the mechanical
shaker for 1 h. Suction-filter the extract through a filter paper in a Buchner porcelain funnel
into a 1-1 round-bottomed flask, wash the filter cake with 100 ml acetone, and rotary-evaporate
the filtrate to an aqueous residue. Proceed as described in 6.2.2.
6.1.4 Water
Add 10 g sodium chloride to 1 1 of the water sample (G) and extract three times with 100-ml
portions of dichloromethane. Dry the combined extracts for 30 min on sodium sulphate, filter
through a fluted filter paper into a 500-ml round-bottomed flask, and wash the filter with ap-
prox. 20 ml dichloromethane. Rotary-evaporate to near dryness, and remove the last traces of
solvent by swirling the flask in the hand. Proceed as described in 6.2.3. With water containing
only low amounts of organic matter, proceed directly to the gas-chromatographic analysis, as
described in 6.3, without further cleanup.
6.2 Cleanup
6.2.1 Acid precipitation
To the aqueous residue derived from 6.1.1, add 100 ml water, 100 ml methanol, and 10 ml
hydrochloric acid (1 mol/1). Allow the mixture to stand for 10 min for complete precipitation,
then suction-filter through a filter paper in a Buchner porcelain funnel containing a layer of
filter aid. Wash the filter three times with 20-ml portions of hydrochloric acid (0.1 mol/1).
278 Propachlor
6.2.2 Liquid-liquid partition

Quantitatively transfer the aqueous residue derived from 6.1.3 or the filtrate derived from 6.2.1
into a separatory funnel, add 10 g sodium chloride and shake three times with 100-ml portions
of the petroleum ether-dichloromethane mixture. Dry the combined extracts on sodium sul-
phate, filter through a fluted filter paper into a 500-ml round-bottomed flask, and wash the
filter with 30 ml petroleum ether-dichloromethane mixture. Rotary-evaporate the filtrate to
near dryness, and remove the last traces of solvent by swirling the flask in the hand. Proceed
as described in 6.2.3. For soil extracts, the following column-chromatographic cleanup can
usually be omitted.

Plug the bottom end of the chromatographic tube with cottonwool, and fill about one third
of the tube with hexane. Slurry 15 g Florisil with hexane, and pour the slurry into the tube
whilst gently tapping the tube walls. After the adsorbent has settled, cover it with an approx.
1-cm layer of sodium sulphate. Drain the hexane until the sodium sulphate layer is just covered
with solvent. Next dissolve the residue derived from 6.1.2, 6.1.4, or 6.2.2 in 5 ml eluting mix-
ture 1. Quantitatively transfer the solution onto the column, allow to percolate at a rate of
1-2 drops per s, and rinse the top of the column with approx. 2 ml eluting mixture 1. Elute
co-extractives with a total of 70 ml eluting mixture 1, and discard the eluate. Then elute propa-
chlor with 70 ml eluting mixture 2. Collect the eluate in a 250-ml round-bottomed flask,
rotary-evaporate to near dryness, and remove the last traces of solvent by swirling the flask
in the hand.

Dissolve the residue derived from 6.1.4, 6.2.2, or 6.2.3 in hexane and make up to a definite
volume (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph. All extracts
are measured using a thermionic detector; extracts from water samples can also be measured
using an electron capture detector.
6.3.1 Packed column
Column Glass, 2 mm i.d., 60 cm long; packed with 5% Car-
bowax 20M TPA on Gas Chrom Q, 100-120 mesh
Detector Thermionic nitrogen-specific detector (Carlo Erba PN
Detector 793)
Temperature 320 C
Hydrogen, 3 ml/min
Air, 200 ml/min
Attenuation 1-32
Propachlor 279

Retention time for propachlor 3 min 15 s
Parathion had a retention time of 20 min using these conditions.
6.3.2 Capillary column
with OV-1, crossbond, film thickness 0.10-0.15 \xm
(Carlo Erba Mega)
Column temperature 60 to 120 C ballistically, programmed to rise at
2C/min from 120 to 140 C
Injection technique Cold on-column at 60C oven temperature, with sec-
ondary cooling
63
trol Module 251, constant current, pulse width 1 us
(Carlo Erba)
Temperature 290 C
Nitrogen detector purge gas, 50 ml/min
Attenuation 512
Linearity range 0.01-0.2 ng
Retention time for propachlor 5 min 6 s
7 Evaluation
7.1 Method
Inject equal volumes of differently concentrated propachlor standard solutions into the gas
chromatograph. Plot the areas or heights of the peaks obtained vs. ng propachlor. Also inject
equal volumes of the sample solutions. For the areas or heights of the peaks obtained for these
solutions, read the appropriate amounts of propachlor from the calibration curve.

The recoveries from untreated control samples of plant material and soil, fortified with pro-
pachlor at levels of 0.02 to 1.0 mg/kg, averaged 89% with a relative standard deviation of
11%. Blanks were usually less than 0.005 mg/kg; for head cabbage, radishes and leeks, how-
ever, they could be as high as 0.02 mg/kg. The limit of determination was between 0.02 and
0.05 mg/kg, depending on the sample material; with head cabbage it was at 0.1 mg/kg. The
limit of detection was 0.01 mg/kg for most materials.
The recoveries from water samples (tap water), fortified with propachlor at levels of 0.001
and 0.1 mg/1, averaged 107% with a relative standard deviation of 18%. The limit of determi-
280 Propachlor
nation was 1 ng/1; the limit of detection was 0.1 \ig/\ (electron capture detector) or 0.5 ng/1
(thermionic detector). Blanks did not exceed 0.03 jig/l.

The residue R, expressed in mg/kg propachlor, is calculated from the following equation:
WA'V,End
R =
V.-G
where
Vj = portion of volume VEnd injected into gas chromatograph (in \il)
WA = amount of propachlor for Vj read from calibration curve (in ng)
8 Important points
The use of an electron capture detector for determinations in plant material usually results
in high blanks. The electron capture detector is, therefore, only recommended for the analysis
of water samples.
9 References
J. W. Worley, M.L. Rueppel and EL. Rupel, Determination of a-chloroacetanilides in water
by gas chromatography and infrared spectrometry, Anal. Chem. 52, 1845-1849 (1980).
A. Ambrus, J. Lantos, E. Visi, I. Csatlos and L. Sdrvdri, General method for determination
of pesticide residues in samples of plant origin, soil, and water, I. Extraction and cleanup, J.
Assoc. Off. Anal. Chem. 64, 733-742 (1981).
A. Ambrus, E. Visi, F. Zakar, E. Hargitai, L. Szabo and A. Pdpa, General method for determi-
nation of pesticide residues in samples of plant origin, soil, and water, III. Gas chromato-
graphic analysis and confirmation, J. Assoc. Off. Anal. Chem. 64, 749-768 (1981).
10 Authors
Nolting, J. Siebers and M Blacha-Puller
Propiconazole 624
Barley, rye, wheat (respectively green matter, grains and Gas-chromatographic
straw), grapes, wine determination
Soil, water
1 Introduction
( )-l-[2-(2,4-Dichlorophenyl)-4-propyl-l,3-dioxolan-
2-ylmethyl]-lH-l,2,4-triazole (IUPAC)
Chemical name
ci
H
Structural formula
Empirical formula C15H17C12N3O2

Molar mass 342.22
Boiling point 180 C at 0.13 mbar
Vapour pressure 1.3- 10~6 mbar at 20C
Solubility (in 100 ml at 20 C) Very sparingly soluble in water;
miscible with acetone, dichloromethane, methanol,
2-propanol and toluene;
soluble in n-hexane (6 g)
Other properties Very weak base (pKa < 1);
thermally stable at up to more than 320 C
2 Outline of method
Propiconazole residues are extracted from cereal green matter and grapes with methanol, and
from grains, straw and soil with a mixture of methanol and water. The filtered extract is
diluted with water and saturated sodium chloride solution, and propiconazole is partitioned
into dichloromethane. Wine and water are diluted with saturated sodium chloride solution,
and extracted with dichloromethane. The dichloromethane extracts are rotary-evaporated to
dryness. The residue is cleaned up by column chromatography on aluminium oxide. Samples
of straw must be additionally cleaned up beforehand by gel permeation chromatography.
Propiconazole is determined by gas chromatography using a thermionic detector.
3 Apparatus
Homogenizer
Beater-cross mill
282 Propiconazole
Wide neck bottle, 500-ml, with ground stopper

Filter paper, 9 cm dia., e.g. Macherey-Nagel No. 713
Separatory funnel, 1-1
Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Ana-
lytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1)
Microsyringe, 10-ul
4 Reagents
Cyclohexane, p.a.
Dichloromethane, p.a.
Ethanol, p.a.
Ethyl acetate, p.a.
n-Hexane, p.a.
Methanol, high purity
Toluene, p.a.
Cyclohexane + ethyl acetate mixture 1:1 v/v
Eluting mixture 1: dichloromethane + n-hexane 4:6 v/v
Eluting mixture 2: dichloromethane + n-hexane 6:4 v/v
Ethanol + n-hexane mixture 1:1 v/v
Propiconazole standard solutions: 0.25, 0.5, 1.0 and 10.0 ng/ml ethanol-hexane mixture
Aluminium oxide, activity grade V: To 100 g Alumina Woelm B Super I (ICN Biomedicals)
in a 300-ml Erlenmeyer flask (with ground joint), add 19 ml water dropwise from a burette,
with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously
until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h
Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories, No. 152-2750)
Dry ice
Cottonwool
Compressed air, dried and re-purified
Propiconazole 283

The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Plant
material is comminuted and homogenized. Straw is chopped and then ground together with
dry ice in a beater-cross mill. For water samples, observe the guidelines given on pp. 23 ff,
Vol. 1.
6 Procedure
6.1 Extraction
6.1.1 Green plant matter, grains, straw, grapes, soil
Weigh 50 g grapes, 50 g milled grains, 20 g milled straw or 10 g of the other homogenized ana-
lytical material (G) into a wide neck bottle. Add 200 ml methanol or 200 ml methanol-water
mixture for samples of grains, straw and soil. Tightly stopper the bottle, and shake for 1 h
on a mechanical shaker. Suction-filter through a Buchner porcelain funnel, and wash the filter
cake with two 25-ml portions of methanol. Transfer the filtrates to a separatory funnel. Add
200 ml water and 50 ml sodium chloride solution, and extract three times with 75-ml portions
of dichloromethane. Filter the dichloromethane phases through a cottonwool plug into a 300-
ml round-bottomed flask, and rotary-evaporate to dryness. Discard the water phase. Proceed
to step 6.2.1 for cleanup of the residue from straw and to step 6.2.2 for cleanup of the residues
from all other materials.
6.1.2 Wine
Dilute 100 g wine (G) with 100 ml water and 50 ml sodium chloride solution in a separatory
funnel, and extract successively with three 75-ml portions of dichloromethane. Filter the
dichloromethane phases through a cottonwool plug into a 300-ml round-bottomed flask, and
rotary-evaporate to dryness. Then proceed to step 6.2.2.
6.1.3 Water
Place 500 ml water (G) in a separatory funnel, add 50 ml sodium chloride solution, and ex-
tract successively with three 75-ml portions of dichloromethane. Filter the dichloromethane
phases through a cottonwool plug into a 300-ml round-bottomed flask, and rotary-evaporate
to dryness. Then proceed to step 6.2.2.
6.2 Cleanup
6.2.1 Gel permeation chromatography (only for straw)
Transfer the residue derived from 6.1.1 into a test tube, using a total of 10 ml cyclohexane-ethyl
acetate mixture (VEx) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample
loop of the gel permeation chromatograph (VR1) with 7 to 8 ml of the solution. Set the gel
permeation chromatograph at the eluting conditions determined beforehand with a standard
284 Propiconazole
solution of propiconazole; cf. Cleanup Method 6, pp. 75 ff, Vol. 1 Elution volumes ranging
from 110 to 150 ml were determined for propiconazole on Bio-Beads S-X3 polystyrene gel
using the cyclohexane-ethyl acetate mixture as eluant, pumped at a flow rate of 2.5 ml/min.
to dryness. Then proceed to step 6.2.2.
is used.
Pour 15 ml hexane into the chromatographic tube. Slowly add 30 g aluminium oxide (free
from air bubbles). Allow to settle, and then drain the hexane to the top of the column packing.
Transfer the residue derived from 6.1.1, 6.1.2, 6.1.3 or 6.2.1 to the column, using three 2-ml
portions of toluene to complete the transfer. Drain the toluene to the top of the column pack-
ing each time. Elute co-extractives with 50 ml of eluting mixture 1, and then elute pro-
piconazole with 75 ml of eluting mixture 2, using a flow rate of 1 to 2 drops per s. Collect
the eluate in a 100-ml round-bottomed flask, and rotary-evaporate to dryness.

Dissolve the residue derived from 6.2.2 in 2 ml ethanol-hexane mixture, and dilute to a suitable
volume (VEnd). Inject an aliquot of this solution (V{) into the gas chromatograph.
Gas chromatograph Hewlett-Packard 5710A/30A
Column Glass, 2 mm i.d., 1.5 m long; packed with 3% CP
Wax 40 M on Gas Chrom Q, 80-100 mesh (Chrom-
pack)
Temperature 250 C
Hydrogen, 3 ml/min
Air, 50 ml/min
Attenuation 16
Retention time for propiconazole 2 min 20 s
7 Evaluation
7.1 Method
Inject 2 \x\ of each propiconazole standard solution (equivalent to 0.5 to 20 ng propiconazole)
into the gas chromatograph. Plot the heights of the peaks obtained vs. ng propiconazole. Also
Propiconazole 285
inject 2-ul aliquots of the sample solutions. For the heights of the peaks obtained for these
solutions, read the appropriate amounts of propiconazole from the calibration curve. Prepare
a new calibration curve for each sample series.

Recovery experiments were run on different untreated control samples of plant material, wine,
soil and water, fortified with propiconazole at levels of 0.002 to 1.0 mg/kg. The recoveries are
given in the Table; the values presented in this Table represent the means ( standard devia-
tions) from 3 to 13 single experiments.
Table. Percent recoveries from plant material, wine, soil and water, fortified with propiconazole.
. . . . . , Added Recovery
Analytical material mg/kg n * s
Cereals
Green matter 0.1 6 98 11
1.0 8 101 9
Grains 0.04 5 101 13
0.4 6 87 5
Straw 0.1 10 98 11
0.5 6 90 6
Grapes 0.04 13 96 10
0.4 13 89 5
Wine 0.02
-0.4 8 90 10
Soil 0.04
-0.4 4 99 7
Water 0.002 3 111 6
0.02 3 101 4
The routine limit of determination was 0.05 mg/kg for cereal green matter, straw and soil,
0.005 mg/kg for wine, 0.01 mg/kg for grapes and grains, and 1 jj.g/1 for water.

The residue R, expressed in mg/kg propiconazole, is calculated from the following equation:
_ WA VEx ^VEnd
V w -V,-G
where
VEx = volume of solution prepared for gel permeation chromatography in 6.2.1 (in ml)
286 Propiconazole
VR1 = portion of volume VEx injected for gel permeation chromatography (volume of
WA = amount of propiconazole for Vj read from calibration curve (in ng)
8 Important points
For the analysis of extracts from water samples containing only small amounts of co-
extractives, cleanup by column chromatography can be omitted.
Gel permeation chromatography can be performed also by the method described on
pp. 65 ff, Vol. 1 (Cleanup Method 4).
9 Reference
B. Buttler, Gas chromatographic determination of propiconazole and etaconazole in plant
material, soil, and water, J. Agric. Food Chem. 31, 762-765 (1983).
10 Authors
Ciba-Geigy AG, Agricultural Division, Basle, Switzerland, B. Buttler and W. D. Hormann
Sulphur 184-B
Apples, cucumbers, grapes, hops (foliage and cones), High-performance

strawberries liquid chromato-
Soil graphic determination
1 Introduction
Chemical name Sulphur

Empirical formula S8
Molar mass 256.51
Melting point 112.8 C (a-sulphur)
Boiling point 444.67 C
Solubility Insoluble in water;
readily soluble in carbon disulphide (42.4 g/100 ml at
20 C);
soluble in carbon tetrachloride;
sparingly soluble in commonly used organic solvents
Other properties Solutions with low content of sulphur are sensitive to
light
2 Outline of method
Sulphur residues are extracted from the sample material with a mixture of water, methanol
and dichloromethane. An aliquot of the separated dichloromethane phase is rotary-evap-
orated to dryness, and taken up in a mixture of isooctane and isopropanol. The extract is
cleaned up by column chromatography on silica gel. Sulphur is determined by high-perfor-
mance liquid chromatography using a UV detector.
3 Apparatus
Erlenmeyer flasks, 500-ml and 250-ml

Graduated cylinders, 250-ml, 100-ml and 25-ml
Glass funnels, 10 cm and 4 cm dia.
Filter paper, 10 cm dia., Whatman 1 PS
Chromatographic tube, 20 mm i. d., 20 cm long
Round-bottomed flasks, 100-ml, with ground joints
288 Sulphur
Ultrasonic bath
High-performance liquid chromatograph equipped with UV detector for measurement at
260 nm
4 Reagents
Methanol, dist.
2-Propanol (isopropanol), dist.
Solvent mixture 1: isooctane + isopropanol 8 :2 v/v
Solvent mixture 2: dichloromethane + methanol 5 :95 v/v
Eluting mixture: isooctane + isopropanol 97 : 3 v/v
Sulphur standard solutions: 1.0, 2.0, 5.0 and 10.0 ng/ml solvent mixture 2
Sulphur solutions for recovery experiments: 10 and 100 jig/ml dichloromethane
Silica gel 60, 0.05-0.2 mm (Macherey-Nagel No. 81532)
Cottonwool

6 Procedure
6.1 Extraction
Weigh 50 g of the analytical sample (5 g for hops) (G) into a 500-ml Erlenmeyer flask, add
25 ml water, 25 ml methanol and 150 ml dichloromethane, stopper the flask tightly, and shake
for 1 h on a mechanical shaker. Filter the flask contents through a filter paper in a funnel,
and collect in a 250-ml Erlenmeyer flask. The separated dichloromethane phase contains the
whole of the sulphur residue (VEx = 150 ml). Take a 75-ml aliquot (VR1) of the dichloro-
methane phase, and rotary-evaporate to dryness. Dissolve the residue in 2.0 ml of solvent mix-
ture 1.

Pour 50 ml isooctane into the chromatographic tube, and slurry in 15 g silica gel. Drain the
solvent to the top of the silica gel layer. Then add the solution derived from 6.1 to the column.
Wash with 3 ml of eluting mixture, and allow the solution to trickle into the column. Next
elute the sulphur with the same mixture. From the time of addition, collect a forecut of 25 ml
Sulphur 289
(0-25 ml), and discard. Collect the main eluate of 30 ml (25-55 ml) which contains the sul-
phur, in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Dissolve the residue
in 4 ml of solvent mixture 2; if necessary, use an ultrasonic bath. Filter the solution through
a cottonwool pad placed in a small funnel, collect in a 5-ml volumetric flask, rinse with 1 ml
of solvent mixture 2, and fill up to the mark with the same solvent mixture (VEnd).

Inject 100 ul (Vj) of the solution derived from 6.2 into the high-performance liquid
chromatograph.
Pump Orlita No. AE-10-4-4 fitted with pulse dampener
Injector Valco injection valve fitted with 100-ul sample loop
Column Stainless steel, 6 mm i.d., 25 cm long; packed with
Polygosil 60-5 C18 (Macherey-Nagel)
Mobile phase Solvent mixture 2
Temperature 20 C
Detector Uvikon 722 LC UV detector (Kontron)
Wavelength 260 nm
Retention time for sulphur 4 min 12 s
7 Evaluation
7.1 Method
Inject 100 ul of each sulphur standard solution (equivalent to 0.1 to 1.0 ug sulphur) into the
high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng sul-
phur. Also inject 100-ul aliquots of the sample solutions. For the heights of the peaks obtained
for these solutions, read the appropriate amounts of sulphur from the calibration curve.

The recoveries from untreated control samples of hops, fortified with sulphur at levels of 5
to 400 mg/kg, ranged from 85 to 100% with a relative standard deviation of 5%. The recov-
eries from untreated control samples of the other materials, fortified with sulphur at levels
of 1 to 10 mg/kg, averaged 100% for cucumbers, 90% for strawberries and apples, 85% for
grapes, and 75% for soil. The routine limit of determination was 5 mg/kg for hops, and
1 mg/kg for all other substrates. Relatively high blanks may usually be expected, e.g.
0.2 mg/kg for cucumbers.
290 Sulphur

The residue R, expressed in mg/kg sulphur, is calculated from the following equation:
R_ w A -v E x -v E n d
VRI-VS-G
where
VEx = volume of dichloromethane used for extraction of analytical sample (in ml)
VR1 = portion of volume VEx used for cleanup in step 6.2 (in ml)
Vj = portion of volume VEnd injected into high-performance liquid chromatograph
(in Hi)
WA = amount of sulphur for Vj read from calibration curve (in ng)
8 Important points
During the analysis, care should be taken not to expose the solutions to sunlight.
Extracts with a high content of sulphur and a very low content of co-extractives need not
be subjected to the cleanup described in step 6.2
Experience so far gained indicates that sulphur residues can be determined also in crops
other than those listed in the heading of the method.
9 References
K. Wenzel, Die HPLC-Bestimmung von elementarem Schwefel in Lebensmitteln, Z. Lebensm.
Unters. Forsch. 170, 5-6 (1980).
R.M. Cassidy, A selective method for elemental sulfur analysis by high-speed liquid chroma-
tography, J. Chromatogr. 117, 71-79 (1976).
10 Author
BASF, Agricultural Research Station, Limburgerhof, J. Elzner
Thiabendazole 256-A
Apples (peel), bananas (peel), grapefruit (peel), oranges Fluorimetric
(peel), potatoes (peel) determinination on
TLC chromatograms
1 Introduction
Chemical name 2-(Thiazol-4-yl)benzimidazole (IUPAC)
Structural formula
Empirical formula C10H7N3S

Molar mass 201.25
Melting point 304-305 C, sublimes from 250 C
Solubility (in 100 ml at Slightly soluble in water (approx. 1 g) at pH 2,
room temperature) very sparingly soluble at pH 3-12;
soluble in dimethylacetamide (6.47 g), dimethylform-
amide and dimethylsulphoxide (8 g);
slightly to sparingly soluble in acetone, benzene,
tert.butanol, chloroform, dichloromethane, diethyl
ether, ethyl acetate and methanol (0.93 g)
Other properties Stable in solution, fluoresces in UV light
2 Outline of method
Thiabendazole residues are extracted from plant material with dichloromethane. The extract
is concentrated and the concentrate made up to a definite volume with ethanol. In order to
separate thiabendazole from plant co-extractives, an aliquot portion of this solution is
chromatographed on a silica gel thin-layer plate, together with a series of standards for com-
parison. Thiabendazole is determined directly on the TLC plate by fluorimetric measurement.
3 Apparatus
Soxhlet extractor, capacity of extraction tube approx. 500 ml, fitted with 500-ml round-bot-
tomed flask and reflux condenser
Rotary vacuum evaporator
292 Thiabendazole

Equipment for thin-layer chromatography
Micro-capillaries, 2-^1
Spectrophotometric scanner, suitable for fluorimetric measurements on TLC plates
4 Reagents
Ethanol, p.a., 96% vol.
Ethyl acetate, p.a.
Methyl ethyl ketone, p.a.
Mobile phase for TLC: ethyl acetate + methyl ethyl ketone + formic acid + water 5 : 3 : 1 : 1
v/v/v/v
Thiabendazole standard solutions: 0.1, 0.2, 0.3 and 0.4 mg/10 ml in ethanol
Formic acid, p.a., 98-100%
Pre-coated TLC silica gel 60 glass plates, 20 cm x 20 cm, without fluorescent indicator (e.g.
Merck No. 5721), or equivalent aluminium sheets
Cottonwool

For preparing the analytical sample, weigh 2 to 2.5 kg of the laboratory sample (see pp. 17 ff,
Vol. 1), peel the fruits or potatoes, and weigh the peel. Finely chop the peel and mix
thoroughly.
6 Procedure
6.1 Extraction
Insert a cottonwool plug into the extraction tube of the Soxhlet extractor. Transfer the peel,
equivalent to 400 g (G) of fruit or potatoes, into the tube and top with another cottonwool
plug. Next, add dichloromethane to the tube until it begins to siphon, allow to drain into the
round-bottomed flask, and half-fill the tube with dichloromethane anew. Mount the reflux
condenser, heat to reflux, and extract for 3 h. Allow the extract to cool, and rotary-evaporate
it to a volume of a few ml. Quantitatively transfer the concentrate to a 50-ml volumetric flask,
using ethanol to complete the transfer, and make up to the mark with ethanol (VEnd).
6.2 Thin-layer chromatography

Fill a 2-^1 micro-capillary (VA) with the solution derived from 6.1 and let it run out from a
vertical position onto the TLC plate, to form a spot at a distance of 25 mm from the bottom
edge. Allow the spot to dry without applying any stream of air.
Thiabendazole 293
Onto the same TLC plate, spot additionally 2 \i\ each of the sample solutions derived from
4 other analytical samples, and of the 4 thiabendazole standard solutions, keeping a distance
of at least 18 mm between the individual spots. Transfer the plate into the chromatographic
tank previously saturated with the vapours of the mobile phase, and allow the chromatogram
to develop until the front of the mobile phase has moved to approx. 4 cm from the top edge
of the plate (approx. 11/2 to 2 h). Remove the plate from the tank and dry it in a stream of
moderately warm air.
Carry out the spotting and the development of the chromatograms in a room lit only with
normal light bulbs, for fluorescent tubes and daylight may cause thiabendazole losses.
6.3 Fluorimetric measurement

For scanning the TLC plate, the exciting radiation at 313 nm (line filter) is directed onto the
layer at an angle of 45. From the emitted radiation, the 355-nm wavelength is selected using
a monochromator.
Spectrophotometer Chromatogram spectrophotometer Zeiss KM 3
Light source Mercury lamp fitted with M 313 line filter
Monochromator wavelength 355 nm
Slit width 0.5 mm
Slit height 15 mm
Table speed 20 cm/min
Plotter speed 60 cm/min
Place the thin-layer plate, by adjusting the position of the table, so that the individual spots
give maximum deflection of the recorder. Shift the spot area out of the light beam in the Y
direction by hand; then plot the fluorescence curve in Y direction by switching on the table
drive.
For an alternative arrangement, the TLC plate is exposed to an exciting radiation of 313 nm
which is selected by the monochromator. In this case, the emitted radiation is passed through
a wide-band filter M 365 positioned between plate and detector. No thiabendazole losses have
been observed with this arrangement.
7 Evaluation
7.1 Method
Measure the fluorescence intensity as described in 6.3 of the four spots of the standard solu-
tions contained on each plate. Calculate the areas of the peaks recorded from the peak heights
and the widths at half height. Plot the areas obtained using double log graph paper vs. ng
thiabendazole. Also measure the fluorescence intensities for the spots of the sample solutions.
For the areas of the peaks obtained for these spots, read the appropriate amounts of thiabend-
azole from the calibration curve. Plot a new calibration curve for each plate used.
294 Thiabendazole

The recoveries from untreated control samples, fortified with thiabendazole at levels of 1 to
8 mg/kg, ranged from 89 to 103% and averaged 95% with a relative standard deviation of
2.7%. The routine limit of determination was approx. 0.5 mg/kg.

The residue R, expressed in mg/kg thiabendazole, is calculated from the following equation:
w A -v E n d
R =
VAG
where
G = sample weight of fruit or potatoes from which the peel was extracted (in g)
VA = portion of volume VEnd applied to TLC plate (in ul)
WA = amount of thiabendazole for VA read from calibration curve (in ng)
8 Important points
With residues of less than 0.5 mg/kg thiabendazole, the results are likely to be affected by in-
terfering co-extractives. For instance, grapefruit peel contains blue fluorescent compounds
(emission maxima approx. 390 nm) which are not fully separated from thiabendazole under
the given TLC conditions.
In the extracts derived from 6.1, residues of biphenyl and o-phenylphenol can also be deter-
mined by TLC or gas chromatography, with recoveries ranging from 87 to 100%.
9 References
H. Otteneder and U. Hezel, Quantitative routine determination of thiabendazole by
fluorimetric evaluation of thin-layer chromatograms, J. Chromatogr. 109, 181-187 (1975).
S. Ebel and G. Herold, Quantitative Bestimmung von Thiabendazol durch direkte Fluores-
zenzauswertung nach dunnschichtchromatographischer Trennung, Dtsch. Lebensm.
Rundsch. 70, 133-136 (1974).
10 Authors
Chemisches Untersuchungsamt, Trier, H. Otteneder
Carl Zeiss, Oberkochen, Abteilung fur physikalisch-chemische Analyse, U. Hezel
Thiabendazole 256-B
Grapefruit (peel), oranges (peel) Spectrophotometric
determination
1 Introduction
For data on physico-chemical properties of thiabendazole, see the Method on p. 291, this
Volume,
2 Outline of method
Thiabendazole residues are extracted from citrus peel with dichloromethane. The extract is
concentrated and the concentrate made up to a definite volume with ethanol. In order to sepa-
rate thiabendazole from plant co-extractives, an aliquot portion of this solution is chromato-
graphed on a silica gel thin-layer plate. The thiabendazole band, visualized by exposing the
TLC plate to UV light, is scraped off together with the adsorbent. Thiabendazole is eluted
with hydrochloric acid and is determined by measuring the absorbance at 302 nm.
This spectrophotometric method is less sensitive than the fluorimetric one described on
pp. 291 ff, this Volume.
3 Apparatus
Soxhlet extractor, capacity of extraction tube approx. 500 ml, fitted with 500-ml round-bot-
tomed flask and reflux condenser
Volumetric flasks, 10-ml and 5-ml
TLC applicator, e.g. Linomat IV (Camag)
Equipment for thin-layer chromatography
Pasteur pipets, i.d. 5 mm, 8 cm long
UV lamp with 350-nm filter
Spectrophotometer fitted with 1-cm quartz cells
4 Reagents
Ethanol, p.a., 96% vol.
Ethyl acetate, p.a.
296 Thlabendazole
Methyl ethyl ketone, p.a.

Mobile phase for T L C : ethyl acetate + methyl ethyl ketone + formic acid + water 5 : 3 : 1 : 1
v/v/v/v
Thiabendazole standard solution: 10 ng/ml in hydrochloric acid (0.1 mol/1)
Formic acid, p.a., 98-100%
Hydrochloric acid, 0.1 mol/1 HC1 p.a.
Pre-coated TLC silica gel 60 glass plates, 20 cm x 20 cm, without fluorescent indicator (e.g.
Merck No. 5721), or equivalent aluminium sheets
Cottonwool

For preparing the analytical sample, weigh 2 to 2.5 kg of the laboratory sample (see pp. 17 ff,
Vol. 1), peel the fruits and weigh the peel. Finely chop the peel and mix thoroughly.
6 Procedure
6.1 Extraction
Insert a cottonwool plug into the extraction tube of the Soxhlet extractor. Transfer the peel,
equivalent to 400 g (G) of fruit, into the tube and top with another cottonwool plug. Next,
add dichloromethane to the tube until it begins to siphon, allow to drain into the round-bot-
tomed flask, and half-fill the tube with dichloromethane anew. Mount the reflux condenser,
heat to reflux, and extract for 3 h. Allow the extract to cool, and rotary-evaporate it to a vol-
ume of a few ml. Quantitatively transfer the concentrate to a 10-ml volumetric flask, using
ethanol to complete the transfer, make up to the mark with ethanol (VEx), and filter.
6.2 Thin-layer chromatography

Using the TLC applicator, apply 50 ul (VR1) of the solution derived from 6.1 in the form of
a 40 mm long band at a distance of 25 mm from the bottom edge of the TLC plate. The dis-
tance between the individual tracks should be 10 mm. A maximum of three samples can be
applied to a single 20 cm x 20 cm plate.
Transfer the plate into the chromatographic tank previously saturated with the vapours of
the mobile phase, and allow the chromatogram to develop until the front of the mobile phase
has moved to approx. 4 cm from the top edge of the plate (approx. 11/2 to 2 h). Remove the
plate from the tank and dry it in a stream of moderately warm air. View the plate unter a UV
lamp, mark the thiabendazole band (Rf = 0.77) as close as possible to its boundary, and
scrape the adsorbent layer from the plate using a scalpel to yield a very fine powder. Suck the
powder into a Pasteur pipet of which the tapered end has been closed with a cottonwool plug.
Elute thiabendazole with 5 ml hydrochloric acid, using light pressure from a pipet ball if nec-
essary. Collect the eluate in a 5-ml or 10-ml volumetric flask and make up to the mark with
hydrochloric acid (VEnd).
Thiabendazole 297
6.3 Photometric determination

Measure the absorbance of the eluate derived from 6.2 in a spectrophotometer at a wavelength
of 302 0.5 nm in a 1-cm quartz cell, using hydrochloric acid for comparison.
7 Evaluation
7.1 Method
Pipet 0.6, 1.2, 1.8, 2.4 and 3.0 ml each of the thiabendazole standard solution (equivalent to
6, 12, 18, 24 and 30 \xg thiabendazole) into 10-ml volumetric flasks and make up to the mark
with hydrochloric acid. Plot the absorbances measured for these solutions vs. thiabendazole
concentration (\ig/l0 ml). A linear calibration curve is obtained for the given range where e.g.
18 ng/10 ml showed an absorbance of 0.230.

The recoveries from untreated control samples, fortified with thiabendazole at levels of 1 to
10 mg/kg, ranged from 89 to 116% and averaged 103% with a relative standard deviation of
2.1%. The routine limit of determination was approx. 1 mg/kg when G = 600 g.

The residue R, expressed in mg/kg thiabendazole, is calculated from the following equation:
R= w A R -v E x -v E n d
10-VR1-G
where
G = sample weight of fruit from which the peel was extracted (in g)
VEx = volume of made-up extract from 6.1 (in ml)
VR1 = portion of volume VEx applied to TLC plate (in ml)
VEnd = volume of made-up eluate from TLC layer (in ml)
WAR = concentration of thiabendazole in the sample solution read from calibration curve
(in ng/10 ml)
8 Important points
No data
298 Thiabendazole
9 References
H. Hey, Spektralanalytik und Gaschromatographie von Thiabendazol. UV-photometrische
Bestimmung auf Citrusfriichten und Bananen, Z. Lebensm. Unters. Forsch. 149, 79-86 (1972).
H. Otteneder and U. Hezel, Quantitative routine determination of thiabendazole by fluori-
metric evaluation of thin-layer chromatograms, J. Chromatogr. 109, 181-187 (1975).
10 Author
Chemisches Untersuchungsamt, Trier, H. Otteneder
Part 4
Multiple Pesticide Residue Analytical Methods
(contd.)
Pesticides, Chemically Related Compounds and Metabolites
Determinable by the Multiresidue Methods in Parts 4 to 6:
Supplement to the Table of Compounds, pp. 221 ff, Vol. 1
Common name Chemical name Structural formula
Acephate O,S-dimethyl acetylphosphoroamidothioate
Alachlor 2-chloro-2',6'-diethyl-N-methoxymethylacetanilide
X
^ CH 2 -O-CH 3
C2H5
Aldicarb 2-methyl-2-(methylthio)propionaldehyde 1 ff
O-methylcarbamoyloxime CH 3 S-C-CH=N-O-C-NHCH 3
CH,
O
.CNHCH 3
Aminocarb 4-dimethylamino-m-tolyl methylcarbamate
N(CH 3 ) 2
3 -amino-1 H-l ,2,4-triazole
Anthraquinone anthraquinone
methyl N-phenylacetyl- V y-CH 2 C /CH-COO-CH 3

Benalaxyl
N-2,6-xylyl-DIâlaninate
O
,C-NHCH 3
Bendiocarb 2,2-dimethyl-l,3-benzodioxol-
4-yl methylcarbamate
NO 2
N-butyl-N-ethyl-2,6-dinitro-
4-trifluoromethylaniline \=< \CH 2 ) 3 -CH 3
NO2
302 Table of Compounds
Bentazone 3-isopropyl-lH-benzo-2,l,3-thiadiazin-4-one 2,2-dioxide

XH(CH 3 ) 2
Bifenox methyl 5-(2,4-dichlorophenoxy)-

2-nitrobenzoate
2-methylbiphenyl-3-ylmethyl (Z)-(1RS,3RS)-
3-(2-chloro-3,3,3-trifluoroprop-l-enyl)-
CF3 C=CH CH CH COOCH2
2,2-dimethylcyclopropanecarboxylate
Bromopropylate isopropyl 4,4'-dibromobenzilate
Bromoxynil octanoate 2,6-dibromo-4-cyanophenyl octanoate
CH3 O
Butocarboxim 3 - (methylthio)butanone
O-methylcarbamoyloxime CH3S-CH-C=N-O-C-NHCH3
CH,
O
.C-NHCH3
Carbaryl 1-naphthyl methylcarbamate
Carbendazim methyl benzimidazol-2-ylcarbamate
O
II
C-NHCH 3
Carbofuran 2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate

CH 3
CH3O S
Carbophenothion- S-4-chlorophenylthiomethyl O,O-dimethyl P-
methyl phosphorodithioate CH3O
H 3 C.
Chinomethionat 6-methyl-l,3-dithiolo[4,5-b]quinoxalin-2-one
(Quinomethionate)
Table of Compounds 303
Chlorbenside 4-chlorobenzyl 4-chlorophenyl sulphide
Chlorbenside sulphone 4-chlorobenzyl 4-chlorophenyl sulphone C l ^ V - CH 2 - S -f% Cl
Chlorfenprop-methyl methyl ( )-2-chloro- Cl<Q>-CH 2 -CH COO-CH3

3 -(4-chloropheny l)propionate
Chlorflurenol 2-chloro-9-hydroxyfluorene-9-carboxylic acid
Chloridazon 5-amino-4-chloro-2-phenylpyridazin-3(2H)-one
Chlormephos S-chloromethyl O,O-diethyl phosphorodithioate
Chlorobenzilate ethyl 4,4'-dichlorobenzilate

COOC 2 H 5
Chloroneb l,4-dichloro-2,5-dimethoxybenzene
Chloropropylate isopropyl 4,4'-dichlorobenzilate
Chlorothalonil tetrachloroisophthalonitrile
Cl Cl
Chlorthal-dimethyl dimethyl tetrachloroterephthalate CH3-O-CO-3-COO-CH3

c, M c>
C2H5Oxfi
C H
Coumaphos O-3-chloro-4-methyl-2-oxo-2H-chromen-7-yl O,O-diethyl 2 5
phosphorothioate
CH 3
Crotoxyphos dimethyl (E)-l-methyl-2- CH 3 O V

P - O - C = C H COOCH CH3
(l-phenylethoxycarbonyl)vinyl phosphate
6
CH3O ^
Crufomate 4-tert-butyl-2-chlorophenyl p.o/VqcH^

methyl methylphosphoramidate
Cyanazine 2-chloro-4-(l-cyano-l-methylethylamino)- C2H5NH N-^NHC-CN

6-ethylamino-l,3,5-triazine CH3
Cyanophos O-4-cyanophenyl O,O-dimethyl phosphorothioate

CH3O^
CH3^ 7CH3
Cyfluthrin (RS)-a-cyano-4-fluoro-3-phenoxybenzyl
(lRS,3RS;lRS,3SR)-3-(2,2-dichlorovinyl)-
A ?N
C1 2 C=CHCHCHCOOCH v ^ o
'T "
Cyhalothrin (RS)-a-cyano-3-phenoxybenzyl ci C
(Z)-(lRS,3RS)-3-(2-chloro-3,3,3-trifluoropropenyl)- CF3-C=CHCHCHC
O O
Cymoxanil l-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea NC-CC-NHC-NHC 2 H 5
NOCH3
2,4-D (2,4-dichlorophenoxy)acetic acid Cl/ S OCH2COOH
Cl
2,4-DB 4-(2,4-dichlorophenoxy)butyric acid O(CH2)3COOH
S,S, S-tributyl phosphorotrithioate (C4H9-S)3PO
Desethyl- O-2-ethylamino-6-methylpyrimidin-4-yl
pirimiphos-methyl O,O-dimethyl phosphorothioate
CH3O
Desethyl-simazine
see des-tert-butyl-terbuthylazine, p. 226, Vol. 1
Desethyl-terbuthylazine 2-amino-4-chloro-6-tert-butylamino- N^ N
1,3,5-triazine
H2N N"^NH-C(CH 3 ) 3
O
.C-N(CH3)2
Desmethylformamido- 5,6-dimethyl-2-(N-methylformamido)-
pirimicarb pyrimidin-4-yl dimethylcarbamate
O
C-N(CH3)2
Desmethyl-pirimicarb 5,6-dimethyl-2-methylaminopyrimidin-4-yl dimethylcarb- H c \
amate
H3C
x,x
^y N
C2H5O S
S-2-chloro-l-phthalimidoethyl
O,O-diethyl phosphorodithioate C2H5O CH2C1
(CH3)2CH O
Di-allate S-2,3-dichloroallyl N-C-S-CH 2 -C=CHC1
di-isopropyl(thiocarbamate) (CH3)2CH/ ^
p,p'-Dichlorobenzophenone 4,4'-dichlorobenzophenone
Clf Vo-CH-COOH
Dichlorprop ( )-2-(2,4-dichlorophenoxy)propionic acid
-CH cooCH 3
Diclo fop-methyl methyl (RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionate
2,6-dichloro-4-nitroaniline
cr T Cl
NH 2
H2N NO 2
Dinitramine N l,N 1-diethyl-2,6-dinitro-4-trifluoromethyl-m- F,C-/VN

phenylenediamine
O2N-/~\-O-C-O-CH(CH3)2
Dinobuton 2-sec-butyl-4,6-dinitrophenyl isopropyl
carbonate CH-C2H5
CH3
NO2
2-sec-butyl-4,6-dinitrophenol
CH-C2H5
CH3
NO2
Dinoterb 2-tert-butyl-4,6-dinitrophenol O 2 N-( / \ - H
C(CH3)3
O
C-NHCH 3
2-(l ,3-dioxolan-2-yl)phenyl methylcarbamate
NO2
DNOC 4,6-dinitro-o-cresol
VoH
o
Edifenphos O-ethyl S,S-diphenyl phosphorodithioate
OC 2 H 5
C2H5O
EPN O-ethyl O-4-nitrophenyl phenylphosphonothioate P
6
o
Ethiofencarb 2-ethylthiomethylphenyl methylcarbamate /C-NHCH3
,CH 2 -S-C 2 H 5
Famophos O-4-dimethylsulphamoylphenyl
O,O-dimethyl phosphorothioate
Fenarimol ( )-2,4'-dichloro-a-(pyrimidin-5-yl)benzhydryl alcohol
Fenoprop ( )-2-(2,4,5-trichlorophenoxy)propionic acid V-O-CH-COOH
Fluazifop-butyl butyl (RS)-2-[4-(5-trifluoromethyl-

2-pyridyloxy)phenoxy] propionate K3~C
s N-CF 3
Flubenzimine (2Z,4E,5Z)-N2,3-diphenyl-N4,N5-bis(trifluoromethyl)-l,3-
thiazolidine-2,4,5-triylidenetriamine N-CF3
6
N02
^ /(CH^-CH,
Fluchloralin N-(2-chloroethyl)-2,6-dinitro-N-propyl- .-<( X
V-N
4-(trifluoromethyl)aniline \=< 'CH2-CH2C1
NO2
O-CHF2
Flucythrinate (RS)-a-cyano-3-phenoxybenzyl (S)- CH3N

I
2-(4-difluoromethoxyphenyl)-3-methylbutyrate ^CHCHCO-O-CH
CH/
Fluorodifen 4-nitrophenyl a,a,a-trifluoro-

2-nitro-p-tolyl ether
Fluroxypyr- 1-methylheptyl 4-amino-3,5-dichloro-6-fluoro-2-

(1-methylheptyl) pyridyloxyacetate
Fluvalinate (RS)-a-cyano-3-phenoxybenzyl
, NH CN
N-(2-chloro-a, a, a-trifluoro-p-tolyl)-D-valinate
CH-CHCOO-CH Q
* T1
Genite 2,4-dichlorophenyl benzenesulphonate
Haloxyfop- 2-ethoxyethyl (RS)-2-[4-(3-chloro-

5-trifluoromethyl-2-pyridyloxy)-
(2-ethoxyethyl)
phenoxyjpropionate
oII
.C-NHCH3
3-Hydroxy-carbofuran 2,3-dihydro-3-hydroxy-2,2-dimethylbenzofuran-7-yl
methylcarbamate
Ioxynil octanoate 4-cyano-2,6-di-iodophenyl octanoate C7H15 - C O O - / ~ \ - C N

Isobenzan l,3,4,5,6,7,8,8-octachloro-l,3,3a,4,7,7a-hexahydro-4,7-
methanoisobenzofuran
Cl
/ \
Isocarbamid N-isobutyl-2-oxoimidazolidine-l-carboxamide
NO 2
;CH2)2-CH3
(CH 3 ) 2 CH-
Isopropalin X
4-isopropyl-2,6-dinitro-N,N-dipropylaniline (CH 2 ) 2 -CH 3
NO 2
.C-NHCH3
2,3-dihydro-2,2-dimethyl-3-oxobenzofuran-7-yl methyl-
3-Keto-carbofuran
carbamate
8-Keto-endrin 4,5,5,6-exo-7,8-hexachloropentacyclo-
[7.2.1.02-6. 03-10.04-8]dodecan-12-one
Lenacil 3-cyclohexyl-l,5,6,7-tetrahydrocyclopenta
pyrimidine-2,4(3H)-dione
Leptophos O-4-bromo-2,5-dichlorophenyl CH3OJ

O-methyl phenylphosphonothioate
MCPA (4-chloro-2-methylphenoxy)acetic acid Cl<^Vo-CH2-COOH
MCPA-(2-butoxyethyl) 2-butoxyethyl (4-chloro-2-methylphenoxy)- Clf^-O-CH2-COO-(CH2)2-O-C4H9

acetate
4-(4-chloro-2-methylphenoxy)butyric acid >-(CH2)3-COOH

'CH3
> S O
S-(N-ethoxycarbonyl-N-methylcarbamoylmethyl) P-S-CH2-C-N-COO-C2H5
O,O-diethyl phosphorodithioate
CH3
)-CH-COOH
Mecoprop ( )-2-(4-chloro-2-methylphenoxy)propionic acid
C2H5O<? S
Mephospholan diethyl 4-methyl-l,3-dithiolan-
P-N=<
2-ylidenephosphoramidate
CaHBo' S
O
C-NHCH3
Mercaptodimethur 4-methylthio-3,5-xylyl methylcarbamate
SCH3
Merphos tributyl phosphorotrithioite (C4H9-S)3P
NH 2
,CH3
4-amino-3-methyl-6-phenyl-l,2,4-triazin-5(4H)-one II
Metazachlor 2-chloro-N-(pyrazol-l-ylmethyl)acet-2',6'-xylidide
Methabenzthiazuron l-benzothiazol-2-yl-l,3-dimethylurea >-N-C-NH-CH3

CH 3
Methiocarb
see Mercaptodimethur
Methomyl S-methyl N-(methylcarbamoyl- CH3S-C=N-0-C-NHCH3

oxy)thioacetimidate
Metolachlor 2-chloro-6'-ethyl-N-(2-methoxy-
X
X
l-methylethyl)acet-o-toluidide CH-CH 2 -O-CH 3
C
Mirex dodecachloropentacyclo- perchlorinated

[5.3.0.02-6.03'9.04-8]decane
Monocrotophos dimethyl (E)-l-methyl-2-(methylcarbamoyl)-

vinyl phosphate P-O-C=CH-C-NH-CH3
O
CH3O i
Morphothion O,O-dimethyl S-morpholinocarbonylmethyl

phosphorodithioate ^P-S-CH2-CO-N O
CHgO
NO2
MW /(CH2)2-CH3
Nitralin 4-methylsulphonyl-2,6-dinitro-N,N-dipropylaniline CH3-S-/ \ - N
S
5 W (CH2)2-CH3
NO 2
2,4-dichlorophenyl 4-nitrophenyl ether -o-( VNO 2
Nitrothal-isopropyl di-isopropyl 5-nitroisophthalate
COO-
OO-CH(CH3)2
Octachlorodipropyl bis-(2,3,3,3-tetrachloropropyl) ether C1 3 C-CH-CH 2 -O-CH 2 -CH-CC1 3

ether (S 421) Cl Cl
c
kX/ c c l = c c l 2
Octachlorostyrene octachlorostyrene
cr T ci
ci
5-tert-butyl-3-(2,4-dichloro-5-isopropoxy-
NN
phenyl)-l,3,4-oxadiazol-2(3H)-one
C(CH3);
<3
Pendimethalin N-(l-ethylpropyl)-2,6-dinitro-3,4-xylidine N
C2H5
CH 3 NO 2
Phenmedipham 3-methoxycarbonylaminophenyl
3'-methylcarbanilate NH-COO-CH3
P-S-CH-/ y
N = /
Phenthoate S-a-ethoxycarbonylbenzyl CH3O I
O,O-dimethyl phosphorodithioate COO-C 2 H 5
CHoO S 0
Phosmet O,O-dimethyl S-phthalimidomethyl phosphorodithioate /Px C.
CH30 S-CH2-
P-O-N=C-
Phoxim O,O-diethyl a-cyanobenzylideneamino-oxyphosphonothioate C2H5O
CH3 ^H,
C-N
2-dimethylamino-5,6-dimethylpyrimidin-4-yl- nI' VCH
tr
3
dimethylcarbamate y
N
H3C'
C 2 H 5 OJ N=<NN
Pirimiphos-ethyl O-2-diethylarnino-6-methylpyrimidin-4-yl O,O-diethyl
phosphorothioate C2H5O/
CH3
O2
/ ,(CH 2 ) 2 -CH 3
Profluralin N-cyclopropylmethyl-2,6-dinitro-N-propyl-
4-trifluoromethylaniline
Promecarb 3 -isopropyl-5 -methylphenyl methylcarbamate
Propachlor 2-chloro-N-isopropylacetanilide
CH(CH3)2
Propanil N-(3,4-dichlorophenyl)propionamide QNHC

Cl
Propiconazole ( )-l-[2-(2,4-dichlorophenyl)-4-propyl-
l,3-dioxolan-2-ylmethyl]-lH-l,2,4-triazole
Quinalphos O,O-diethyl O-quinoxalin-2-yl phosphorothioate

C2H5O-"
Quinomethionate
see Chinomethionat
Salithion 2-methoxy-4H-benzo-l,3,2-dioxaphosphorin 2-sulphide
Strobane T polychloroterpenes (chlorinated mixed terpenes)
Sulprofos O-ethyl O-4-(methylthio)phenyl S-propyl

phosphorodithioate
2,4,5-T (2,4,5-trichlorophenoxy)acetic acid
3-tert-butyl-5-chloro-6-methyluracil
2 5 O
Terbufos S-tert-butylthiomethyl O,O-diethyl phosphorodithioate P-S-CH2-S-C(CH3)3
CHO''
CH3/CH3
H 3x /C\ /-tf
Tetramethrin 3,4,5,6-tetrahydrophthalimidomethyl ( )-cis-trans- C=CHCHCH-COOCH2-N II
chrysanthemate
3,3-dimethyl-l-methylthiobutanone CH3S-CH2-C=N-O-C-NHCH3
O-methylcarbamoyloxime C(CH3)3
Tolclofos-methyl O-2,6-dichloro-p-tolyl O,O-dimethyl phosphorothioate P

CH3O OT 7-CH3
Cl
(CH 3 ) 2 CH
S-2,3,3-trichloroallyl di-isopropyl(thiocarbamate) N-C-S-CH 2 -C=CC1 2
(CH 3 ) 2 CH / ^
Triamiphos P-(5-amino-3-phenyl-lH-l,2,4-triazol-l-yl)-N,N,N',N'-
tetramethylphosphonic diamide (CH 3 ) 2
Triazoxide 7-chloro-3 - [(1 H)-imidazol-l-yl] -1,2,4-benzotriazine 1-oxide
C2H5O. n
Trichloronat O-ethyl O-2,4,5-trichlorophenyl ethylphosphonothioate
Cl
Triclopyr- 2-butoxyethyl (3,5,6-trichloro-

(2-butoxyethyl) 2-pyridyloxy)acetate Cl(/ VO-CH 2 -COO-(CH 2 ) 2 -O-C 4 H 9
Cl
Organohalogen, Organophosphorus
and Triazine Compounds S 8 (updated)
Since the publication of Multiresidue Method S 8 (see pp. 283 ff, Vol. 1), analytical experience
has shown that at least the following 30 pesticides can also be analyzed by this Method:
Bromopropylate Endosulfan sulphate Nitrofen

Chlorbenside Fenamiphos Pendimethalin
Chlorfenson Fenarimol Profluralin
Chlorflurenol Fenson Quinalphos
Chlorobenzilate Fluchloralin Terbacil
Chloropropylate Fluorodifen Terbufos
Cyanazine Iodofenphos Tolclofos-methyl
Cyanophos Metazachlor Tri-allate
Dialifos Metolachlor Trichloronat
p-Endosulfan Metribuzin Trifluralin
Moreover, the Method was meanwhile extended to include additional food crops such as
apricots, aubergines, chillies, honey, mandarin oranges and potatoes.
For convenience, the updated Table 2 gives a compilation of the routine limits of determina-
tion and the gas-chromatographic retention times for all 121 compounds included in the
Method.
Table 2 (updated). Routine limits of determination and relative retention times.
Relative retention time

Routine limit
Compound of determination Column*)
mg/kg 6.3.1 6.3.2.1 6.3.2.2 6.3.3
Aldrin = 1 Parathion = 1
Aldrin 0.004 1.00

Ametryn 0.1 0.75
Atrazine 0.05 0.46
Azinphos-ethyl 0.1 6.4 5.6 4.8
Azinphos-methyl 0.2 4.90 4.6 4.2
Aziprotryne 0.05 0.59
Bromacil 0.05 0.83
Bromophos 0.02 1.06 0.51
Bromophos-ethyl 0.01 1.38 0.68
Bromopropylate 0.02 3.59
Bupirimate 0.05 1.67 1.3
Captafol 0.1 3.95
Captan 0.01 1.18 1.1
Carbophenothion 0.03 2.63 1.23 1.1
314 Method S 8 (updated)
Table 2. (contd.)

Routine limit
mg/kg 6.3.1 6.3.2.1 6.3.2.2 6.3.3
Chlorbenside 0.01 1.28

Chlorfenson 0.08 1.45
Chlorfenvinphos 0.03 0.95 0.95
Chlorflurenol 0.01 1.28
Chlorobenzilate 0.2 1.96
Chloropropylate 0.08 1.99
Chlorpyrifos 0.05 0.93 0.48 0.46
Chlorpyrifos-methyl 0.01 0.71 0.42
Chlorthal 0.01 1.0
Chlorthiophos 0.01 2.46 1.25 1.22
Cyanazine 0.04 0.88
Cyanofenphos 0.01 2.37 2.4
Cyanophos 0.04 0.55
p,p-DDD 0.01 2.18
p,p'-DDE 0.01 1.84
p,p-DDT 0.01 3.1
Desmetryn 0.02 0.66
Dialifos 0.1 6.32
Diazinon 0.01 0.20 0.28
Dichlobenil 0.01 0.13
Dichlofenthion 0.03 0.56 0.48
Dichlofluanid 0.01 0.87 0.84
Dichlorvos 0.02 0.11
Dicofol 0.05 0.99
Dieldrin 0.01 1.84
Dimethachlor 0.05 0.70
Dimethoate 0.1 0.44 0.64 0.61
Dioxathion 0.05 0.31 0.39
Disulfoton 0.01 0.27 0.32
Ditalimfos 0.05 1.49 1.15 1.18
a-Endosulfan 0.01 1.56
p-Endosulfan 0.01 1.88
Endosulfan sulphate 0.02 2.36
Ethion 0.03 2.4 1.4 1.32
Ethoprophos 0.02 0.32 0.27
Etrimfos 0.04 0.56 0.31
Fenamiphos 0.05 1.70
Fenarimol 0.01 5.11
Fenchlorphos 0.02 0.77 0.41 0.44
Fenitrothion 0.05 0.79 0.86 0.88
Fenson 0.04 0.99
Fensulfothion 0.1 2.21 4.26 3.37
Fenthion 0.01 0.51 0.59
Fluchloralin 0.02 0.58
Fluorodifen 0.01 1.44
Method S 8 (updated) 315
Table 2. (contd.)

Routine limit
mg/kg 6.3.1 6.3.2.1 6.3.2.2 6.3.3
Folpet 0.01 1.27

Fonofos 0.01 0.49 0.28 0.32
Formothion 0.01 0.61 0.82 0.80
a-HCH 0.01 0.39
3-HCH 0.01 0.42
Heptachlor 0.004 0.79
Heptachlor epoxide 0.005 1.22
Heptenophos 0.01 0.22 0.25
Iodofenphos 0.04 1.52
Iprodione 0.05 2.24
Isofenphos 0.02 1.57 0.80
Lindane 0.002 0.47
Malaoxon 0.025 0.94 0.90
Malathion 0.02 0.85 0.72 0.72
Metalaxyl 0.4 0.65
Metazachlor 0.04 1.12
Methidathion 0.1 1.3 1.13 1.12
Methoprotryne 0.05 2.0
Methoxychlor 0.1 4.56
Metolachlor 0.1 0.95
Metribuzin 0.01 0.69
Mevinphos 0.1 0.21 0.20
Naled 0.1 0.09
Nitrofen 0.01 1.81
Paraoxon 0.1 1.33 1.24
Parathion 0.02 0.96 1.00 1.00
Parathion-methyl 0.02 0.67 0.79 0.80
Pendimethalin 0.04 1.12
Perthane 0.1 2.15
Phenkapton 0.05 4.25 1.18 1.93
Phorate 0.01 0.30 0.23
Phosalone 0.02 6.0 4.65 4.10
Pirimiphos-methyl 0.01 0.41 0.45
Procymidone 0.02 1.30 1.47
Profenofos 0.04 1.56 1.05
Profluralin 0.02 0.55
Prometryn 0.01 0.77
Propazine 0.05 0.47
Propyzamide 0.01 0.50
Prothiofos 0.01 1.61 0.78
Pyrazophos 0.2 6.8 5.41 4.67
Pyrethrum 0.2 1.8; 2.35
Quinalphos 0.02 1.17
Quintozene 0.002 0.49
Simazine 0.01 0.44
Table 2. (contd.)

Routine limit
mg/kg 6.3.1 6.3.2.1 6.3.2.2 6.3.3
Sulfotep 0.01 0.23 0.26

Tecnazene 0.01 0.24
Terbacil 0.1 0.60
Terbufos 0.3 0.56
Terbutryn 0.05 0.88
Tetrachlorvinphos 0.1 1.47 1.22 1.12
Tetradifon 0.03 4.70
Tetrasul 0.05 2.45
Thionazin 0.01 0.27 0.19
Tolclofos-methyl 0.05 0.75
Tolylfluanid 0.02 1.20
Triadimefon 0.04 0.93 0.62
Tri-allate 0.05 0.64
Triazophos 0.1 2.0
Trichloronat 0.02 1.06
Trifluralin 0.02 0.42
Vinclozolin 0.01 0.69
*) For gas-chromatographic conditions, see pp. 290 f, Vol. 1.

Organochlorine, Organophosphorus,
Nitrogen-Containing and Other
Pesticides S 19 (updated)
Since the publication of Volume 1 of this Manual, analytical experience has shown that many
more pesticides can be analyzed by Multiresidue Method S 19 than those listed in Table 3 on
pp. 388ff, Vol. 1. The updated Table 3 shows the data available for more than 220 com-
pounds, as they were published in the 9th Instalment (1987) of the German edition of the
Manual. The Table comprises the elution volumes in gel permeation chromatography under
the conditions set out in step 5.3 of Cleanup Method 6 (p. 76, Vol. 1), the distribution of the
compounds in eluates from silica gel column chromatography under the conditions described
in step 6.4.3 of this Method (p. 387, Vol. 1), and the detectors used for the gas-
chromatographic determination of each compound.
Table 3 (updated). Elution volume ranges in gel permeation chromatography, distribution of compounds
in eluates from silica gel column chromatography, and detectors used for gas-chromatographic determina-
tion.
GPC elution Silica gel Detectors

Compound volume range eluates *) used for GLC
ml 1 2 3 4 5 ECD TID FPD
Acephate 115-145 0 0 0 0 5 +
Alachlor 125-150 0 0 5 0 0 + +
Aldrin 120-150 5 0 0 0 0 +
Ametryn 115-190 0 0 1 3 0 +
Amidithion 115-145 0 0 0 4 3 + +
Anilazinea) 105-135 0 0 5 0 0 + +
Anthraquinone 145-185 0 2 4 0 0 +
Atrazine 110-135 0 0 4 3 0 +
Azinphos-ethyl 130-160 0 0 5 0 0 + 4- +
Azinphos-methyl 145-180 0 0 4 0 0 + +
Benfluralin 100-130 5 0 0 0 0 +
Benzoylprop-ethyl 125-150 0 3 3 0 0 +
Bifenox 115-150 0 3 3 0 0 +
Binapacryl 100-130 0 5 0 0 0 +
Bitertanol 100-130 0 0 0 4 2 +
Bromacilb) 105-140 0 0 0 5 0 +
Bromophos 120-150 4 2 0 0 0 + -f +
Bromophos-ethyl 110-140 5 1 0 0 0 + + +
Bromopropylate 95-135 0 0 3 3 1 +
Bromoxynil octanoate 120-150 0 5 1 0 0 +
Camphechlor (Toxaphene) 110-150 5 1 0 0 0 +
Captafolc> 120-150 0 0 5 0 0 +
Captanc> 120-150 0 0 5 0 0 +
Carbophenothion 120-140 0 3 0 0 0 + + +
Table 3. (contd.)

Carbophenothion-methyl 120-160 0 4 0 0 0 +
Chinomethionat
(Quinomethionate) 170-200 0 1 4 0 0 +
Chlorbensided) 120-155 0 0 1 0 0 +
Chlorbenside sulphone 130-160 0 0 5 0 0 +
a-Chlordane 110-140 5 0 0 0 0 +
y-Chlordane 100-130 5 0 0 0 0 +
Chlorfenprop-methyl 125-150 0 5 0 0 0 +
Chlorfenson 120-150 1 5 0 0 0 +
Chlorfenvinphos 110-140 0 0 4 3 0 + +
Chloridazon (Pyrazon) 130-155 0 0 0 4 1 +
Chlormephos 115-145 3 3 0 0 0 + +
Chlorobenzilate 100-135 0 0 4 2 1 +
Chloroneb 145-170 0 5 0 0 0 +
Chloropropylate 100-135 0 0 4 2 0 +
Chlorothalonil 125-165 0 5 0 0 0 +
Chlorotoluron 115-150 0 0 0 5 2 +
Chloroxuron 130-155 0 0 1 5 0 + +
Chlorpropham 110-135 0 2 4 0 0 + +
Chlorpyrifos 110-140 2 4 0 0 0 + + +
Chlorpyrifos-methyl 120-150 1 4 0 0 0 + + +
Chlorthal-dimethyl 135-160 0 5 1 0 0 +
Chlorthiophos 115-155 0 4 0 0 0 + +
Coumaphos 135-165 0 0 5 0 0 +
Crotoxyphos 105-145 0 0 0 4 0 +
Crufomate 100-140 0 0 0 3 4 +
Cyanazine 110-135 0 0 0 4 0 + +
Cyanofenphos 115-145 0 2 4 0 0 + +
Cyanophos 115-150 0 0 4 0 0 +
Cymoxanile) 110-130 0 0 0 5 0
Cypermethrin 100-135 0 5 0 0 0 +
o,p'-DDD 110-140 5 0 0 0 0 +
p,p'-DDD 100-140 5 0 0 0 0 +
o,p'-DDE 120-150 5 0 0 0 0 +
p,p'-DDE 120-150 5 0 0 0 0 +
o,p'-DDT 120-150 5 0 0 0 0 +
p,p'-DDT 110-140 5 0 0 0 0 +
DEF^ 115-135 0 0 5 1 0
Deltamethrin 100-135 0 5 0 0 0 +
Demeton-S-methyl 125-155 0 0 0 0 0 + +
Demeton-S-methyl sulphone 120-160 0 0 0 2 3 + +
Demeton-S sulphone g) 115-140 0 0 0 3 3 +
Demeton-S sulphoxideh) 140-170 0 0 0 0 3 +
N-Desethyl-pirimiphos-methyl 120-155 0 0 1 5 0 + +
Dialifos 110-140 0 3 3 0 0
Di-allate 120-150 0 4 1 0 0 +
Diazinon 105-135 0 0 5 0 0 + + +
Table 3. (contd.)

Dichlobenil 125-155 1 5 0 0 0 + +
Dichlofenthion 110-140 3 3 0 0 0 + + +
DichlofluanidJ) 100-140 0 3 3 0 0 +
p, p'-Dichlorobenzophenonej) 125-155 0 5 0 0 0 +
Dichlorvos 115-140 0 0 1 3 0 +
Diclofop-methyl 135-165 0 0 5 0 0 +
Dicloran 105-145 0 5 0 0 0 +
Dicofolk) 100-150 2 4 0 0 0 +
Dicrotophos 130-160 0 0 0 0 5 +
Dieldrin 120-150 0 5 0 0 0 +
Dimefox 120-155 0 0 0 0 5 +
Dimethachlor 135-165 0 0 4 2 0 +
Dimethoate 120-150 0 0 0 3 3 + + +
Dinitramine1) 105-130 4 1 0 0 0 +
Dinobuton 110-140 0 4 2 0 0 +
Dinocap 100-120 0 5 0 0 0 +
Dioxathion 110-140 0 3 3 1 0 + + +
Disulfoton m) 115-150 0 2 0 0 3 + +
Disulfoton sulphone 110-140 0 0 5 0 0 + +
Disulfoton sulphoxide 120-150 0 0 0 0 5 +
Ditalimfos 120-150 0 0 4 1 0 +
Edifenphos 130-160 0 0 4 0 0 +
a-Endosulfan 110-150 2 4 0 0 0 +
p-Endosulfan 110-150 0 5 0 0 0 +
Endosulfan sulphate 100-140 0 5 0 0 0 +
Endrin 130-160 0 5 0 0 0 +
EPN 135-160 0 5 0 0 0 + + +
Ethion 100-140 0 5 0 0 0 + + +
Ethoprophos 120-155 0 0 4 1 0 +
Etrimfos 105-140 0 0 5 0 0 +
Famophos 125-155 0 0 5 0 0 + +
Fenamiphos 105-140 0 0 0 4 2 +
Fenchlorphos 120-150 4 2 0 0 0 + + +
Fenitrothion 120-150 0 4 0 0 0 + + +
Fenson 130-160 0 5 0 0 0 +
Fensulfothion 120-150 0 0 0 3 3 + +
Fenthion 130-160 0 3 0 0 0 + +
Fenvalerate 105-135 0 4 1 0 0 +
Flubenzimine^ 85-120 0 5 0 0 0 +
Fluchloralin 100-120 5 1 0 0 0 +
Fluotrimazole 100-140 0 0 4 2 0 +
Fluvalinate 95-120 0 5 0 0 0 +
Folpet 140-180 0 3 4 0 0 +
Fonofos 120-150 0 4 1 0 0 + +
Formothion 120-150 0 0 4 1 0 + +
Fuberidazole1* 120-160 0 0 0 5 1 +
Genite 135-165 0 5 0 0 0 +
Table 3. (contd.)

Compound volume range eluates*) used for GLC
a-HCH 120-150 5 0 0 0 0 +
(3-HCH 100-130 5 0 0 0 0 +
8-HCH 100-130 5 0 0 0 0 +
8-HCH 105-135 5 0 0 0 0 +
Heptachlor 110-140 5 0 0 0 0 +
cis-Heptachlor epoxide 125-155 3 3 0 0 0 +
trans-Heptachlor epoxide 125-155 3 3 0 0 0 +
Heptenophos 120-150 0 0 1 4 0 +
Hexachlorobenzene 140-165 5 0 0 0 0 +
Imazalil11'1) 120-150 0 0 0 0 5 + +
Iodofenphos 120-150 4 2 0 0 0 + +
Ioxynil octanoate 125-155 0 5 1 0 0 +
Iprodione 115-145 0 0 5 1 0 +
Isobenzan 105-140 5 0 0 0 0 +
Isocarbamid 130-165 0 0 0 1 5 +
Isodrin 120-150 5 0 0 0 0 +
Isopropalin 110-135 5 0 0 0 0 +
5-Keto-endrin 135-165 3 4 0 0 0 +
Lenacil 130-160 0 0 0 5 0 +
Leptophos 120-150 5 1 0 0 0 + +
Lindane 110-140 5 0 0 0 0 +
Linuron 120-140 0 0 4 1 0 +
Malaoxon 110-140 0 0 0 4 0 + +
Malathion 110-140 0 0 4 0 0 + + +
MCP A-(2-butoxyethyl) e> 115-145 0 0 5 0 0
Mecarbam 105-145 0 0 4 0 0 +
Mephosfolan 140-170 0 0 0 2 4 +
Merphosn) 125-145 5 0 0 0 0 +
Metalaxyl 115-150 0 0 0 5 1 +
Methabenzthiazuron 150-180 0 0 0 5 0 +
Methamidophos 120-150 0 0 0 0 4 +
Methidathion 130-165 0 0 4 0 0 + +
Methoxychlor 125-155 0 5 0 0 0 +
Metolachlor 130-160 0 0 5 1 0 +
Metribuzin 125-150 0 0 3 1 0 + +
Mevinphos 120-150 0 0 0 5 0 + +
Mirex 130-160 5 0 0 0 0 +
Monocrotophos 115-140 0 0 0 0 5 +
Monolinuron 125-150 0 0 4 2 0 + +
Morphothion 130-170 0 0 0 5 0 +
Naled> 115-155 0 0 4 1 0 +
Nitralin 115-145 0 1 5 0 0 + +
Nitrofen 135-165 2 5 0 0 0 +
Nitrothal-isopropyl 105-135 0 1 4 1 0 + +
Octachlorodipropyl ether
(S 421) 110-130 5 0 0 0 0 +
Omethoate 140-160 0 0 0 0 5 +
Table 3. (contd.)

Oxadiazon 115-145 0 0 5 0 0 + +
Oxychlordane (Octachlor
epoxide) 100-160 5 0 0 0 0 +
Oxydemeton-methylh) 135-165 0 0 0 0 0 + +
Paraoxon 110-140 0 0 1 4 0 + +
Paraoxon-methyl 140-170 0 0 1 4 1 + +
Parathion 110-140 0 4 1 0 0 + + +
Parathion-methyl 120-150 0 4 1 0 0 + + +
Pendimethalin 125-155 1 5 0 0 0 +
Pentachloroaniline 110-140 5 0 0 0 0 + +
Pentachloroanisole 125-160 5 0 0 0 0 +
Pentachlorobenzene 125-165 5 0 0 0 0 +
Permethrin 115-145 0 5 1 0 0 +
Perthane 110-140 5 1 0 0 0 +
Phenkapton 115-145 3 3 0 0 0 + + +
Phenmedipham 105-130 0 0 3 3 1 + +
Phenthoate 115-150 0 1 4 0 0 + +
Phoratep) 115-145 0 2 0 0 0 +
Phosalone 110-140 0 0 4 0 0 + +
Phosphamidon 110-145 0 0 0 1 5 +
Phoxim 120-150 0 5 0 0 0 +
Piperonyl butoxidee) 100-130 0 0 4 1 0 +
Pirimicarb 130-170 0 0 0 5 0 +
Pirimiphos-ethyl 100-135 0 0 4 0 0 +
Pirimiphos-methyl 105-145 0 0 4 0 0 + + +
Procymidone 120-150 0 0 5 0 0 + +
Profenofos 130-155 0 0 4 1 0 +
Profluralin 100-125 5 0 0 0 0 + +
Propachlor 125-150 0 0 5 0 0 +
Propanil 105-130 0 0 4 2 0 + +
Propiconazole 120-150 0 0 0 4 2 +
Propoxur 110-130 0 0 4 2 0 +
Propyzamide 95-125 0 0 4 0 0 +
Prothiofos 105-145 5 0 0 0 0 + +
Pyrazophos 110-140 0 0 5 0 0 + +
Pyrethrins1) 100-130 0 0 5 1 0 +
Quinalphos 115-155 0 0 4 0 0 +
Quintozene 135-165 5 0 0 0 0 +
Rabenzazole1) 120-160 0 0 5 0 0 +
Resmethrine) 100-130 0 5 0 0 0
Salithion 125-165 0 5 0 0 0 + +
Simazine 95-135 0 0 2 4 0 + +
Strobane T 125-160 5 0 0 0 0 +
Sulfotep 100-130 0 4 1 0 0 +
Sulprofos 115-155 0 3 0 0 0 + +
Tecnazene 130-160 5 0 0 0 0 +
Terbacil 120-145 0 0 0 5 0 +
Table 3. (contd.)
G P C elution Silica gel Detectors

Terbufos 125-155 0 3 0 0 0 + + +
Terbutryn 115-175 0 0 1 2 0 +
2,3,4,5-Tetrachloronitro-
benzene 130-160 5 0 0 0 0 +
Tetrachlorvinphos 120-140 0 0 4 1 0 + + +
Tetradifon 120-150 0 5 0 0 0 +
Tetramethrin 120-150 0 0 5 0 0 +
Tetrasul 125-155 5 0 0 0 0 +
Thionazin 120-150 0 1 4 0 0 + +
Tolylfluanid 1 ) 105-135 0 3 3 0 0 +
Triadimefon 100-130 0 0 3 3 0 + +
Triadimenol 100-130 0 0 0 4 2 +
Tri-allate 120-150 0 5 0 0 0 +
Triamiphos 125-160 0 0 0 0 4 + +
Triazophos 120-140 0 0 4 1 0 + +
Triazoxide 165-195 0 0 0 5 0 +
Trichlorfon h > 100-140 0 0 0 0 4 +
Trichloronat 110-140 5 1 0 0 0 + +
Trifluralin 100-130 5 0 0 0 0 +
Vinclozolin 100-130 0 4 1 0 0 + +
Footnotes to Table 3
*> Silica gel eluates: 1, n-hexane-toluene 65:35 v / v ; 2, toluene; 3, toluene-acetone 95 :5 v / v ; 4, toluene-
acetone 8 : 2 v / v ; 5, acetone.
Figures in the Table indicate recovery rates: 5 = more than 9 0 % ; 4 = approx. 6 0 - 9 0 % ;
3 = approx. 3 0 - 6 0 % ; 4 = approx. 1 0 - 3 0 % ; 1 = less than 1 0 % ; 0 = not recovered.
a)
Extract the analytical sample with the addition of potassium acetate; for G L C , inject an aliquot of
the sample solution with the addition of acetic acid.
b)
Peak height depending on solvent.
Gas-chromatographic column must be well conditioned.
When chromatographed on silica gel, an additional peak will be observed with a longer retention time
on GLC.
e
> Analyzed by GC/MS.
^ Also from merphos.
8)
Inject standard solution within 2 min after injection of sample solution.
h)
Improved recovery when eluted with further 8 ml acetone.
f)
Extract the analytical sample with the addition of citric acid and oxalic acid.
j)
Degradation product of dicofol.
k)
Degradation to p,p'-dichlorobenzophenone.
J)
For analysis, proceed with complete exclusion of light.
m)
Following chromatography on silica gel, disulfoton sulphoxide is observed in eluate 5.
n)
Partly oxidized to DEF.
o)
When chromatographed on silica gel, varying recoveries are observed.
p)
Partly decomposed during chromatography on silica gel.
Natural Pyrethrins, Piperonyl Butoxide s 22
Apples, cucumbers, endives, lettuce, mangold, plums, Gas-chromatographic
potatoes, red currants, spinach, sugar beet (edible root), determination
tomatoes
(German version first published 1985, revised 1991)
1 Introduction
The method permits the identification and quantitative determination of the residues of
natural pyrethrins and of the synergist piperonyl butoxide which is contained in nearly all
pyrethrin formulations. Natural pyrethrins are composed of a mixture of pyrethrin I (main
component; highest insecticidal activity) and II, cinerin I and II, and jasmolin I and II.
In gas-chromatographic measurements, total residues of the pyrethrins are calculated from
the peak of pyrethrin I or, when resolution is poor, from the peaks of pyrethrin I plus jasmolin I.
2 Outline of method
The compounds are extracted from plant material with acetone. The extract is filtered, and
the compounds are partitioned into dichloromethane. The dichloromethane phase is divided
into two, to enable the separate analysis of the pyrethrins and piperonyl butoxide.
The portion used for the pyrethrin analysis is cleaned up by chromatography on a Florisil
column. The pyrethrins are determined by electron capture gas chromatography.
The portion used for the analysis of piperonyl butoxide is cleaned up by gel permeation
chromatography. Piperonyl butoxide is brominated to yield 4,5,7-tribromo-6-propyl-l,3-
benzodioxol, which is partitioned into n-hexane and determined by electron capture gas
chromatography.
3 Apparatus
Filter paper, 9 cm dia., fast flow rate (Schleicher & Schtill)
Glass funnels, 8 cm and 5.5 cm dia.
Fluted filter paper, 18.5 cm and 12.5 cm dia.
324 Method S 22

Equipment for gel permeation chromatography, e.g. chromatographic tube and accessories as
described in Cleanup Method 4 (see pp. 65 ff, Vol. 1)
Microsyringe, 10-ul
4 Reagents
Acetone, p. a.
Cyclohexane, p. a.
Diethyl ether, p. a.
n-Hexane, p. a.
Petroleum ether, boiling range 40-60C, prepared as follows: Fill a chromatographic tube
(2 cm i. d.) to a height of 20 cm with aluminium oxide (Alumina Woelm B, activity grade
Super I; ICN Biomedicals), gently tapping the tube walls. Top the packing with a 2-cm layer
of sodium sulphate. Add the petroleum ether, and set the flow rate for the solvent to pass
through the column at approx. 5 ml/min. Discard the first 90 ml. The column suffices to purify
500 ml solvent
Toluene, p. a.
Cyclohexane + ethyl acetate mixture 1:1 v/v
Eluting mixture 1: diethyl ether + petroleum ether 1:9 v/v
Eluting mixture 2: diethyl ether + petroleum ether 3:7 v/v
Pyrethrin standard solutions: 1, 10 and 100 ng/ml pyrethrum extract (approx. 35% pyrethrin
I content) in toluene
Piperonyl butoxide standard solutions: 1, 10 and 100 fig/ml toluene
Piperonyl butoxide calibration solution, equivalent to 1.0 p,g/ml piperonyl butoxide in
n-hexane: Transfer 2 ml of the 10 [xg/ml piperonyl butoxide standard solution into a 50-ml
round-bottomed flask. Remove the solvent by rotating the flask in the hand. Dissolve the
residue in 1 ml dichloromethane and add 0.1 ml of the bromine-iodine mixture (see 8. Impor-
tant points). Swirl the flask, stopper it with a ground stopper, and let it stand for 30 min at
room temperature. Add 10 ml sodium acetate solution and 5 ml sodium arsenite solution, and
continue to process as described in 6.3
Bromine + iodine mixture: Dissolve 5 g finely powdered iodine p.a. in 95 g bromine p.a.
Sodium acetate solution, 20 g/100 ml CH3COONa 3 H2O p. a.
Sodium arsenite solution, 10 g/100 ml NaAsO2 p. a.
Styrene-divinylbenzene copolymer (2% DVB), e.g. Servachrom XAD-2, 50-100 jim (Serva) or
Bio-Beads S-X2; 200-400 mesh (Bio-Rad Laboratories)
Method S 22 325
Glass wool

6 Procedure
6.1 Extraction
Homogenize 100 g of the analytical sample (G) with 150 ml acetone for 3 min. Suction-filter
the homogenate through a fast flow-rate filter paper in a Buchner porcelain funnel and wash
the filter cake with 80 ml acetone. Transfer the filtrate together with 200 ml dichloromethane
into a 1-1 separatory funnel and shake for 2 min. Drain the dichloromethane layer and extract
the aqueous phase twice more with 100-ml portions of dichloromethane. Dry the combined
dichloromethane phases on sodium sulphate, filter through a fluted filter paper containing a
little sodium sulphate into a 500-ml graduated cylinder, rinse flask and filter with a further
80 ml of dichloromethane, and mix the solution thoroughly.
Divide the filtrate into two equal halves for the separate pyrethrin and piperonyl butoxide
analyses, and rotary-evaporate the two halves to near dryness. Remove the last traces of sol-
vent by swirling the flasks in the hand.
6.2 Cleanup
6.2.1 Column chromatography (pyrethrins)
Insert a glass wool plug into the bottom of a chromatographic tube, add 40 ml eluting mix-
ture 1, trickle in 8 g Florisil, allow to settle, and drain the solvent just to the top of the Florisil.
Dissolve the residue derived from 6.1 in 5 ml eluting mixture 1, and add to the column. Rinse
the flask with 5 ml eluting mixture 1, add the rinsing to the column, and allow to percolate.
Repeat this step. Elute the co-extractives with a total of 60 ml eluting mixture 1 and discard
this fraction. Next elute the pyrethrins with 50 ml eluting mixture 2 from the column. Rotary-
evaporate the eluate to near dryness, and remove the last traces of solvent by swirling the flask
in the hand. Dissolve the residue in toluene and make up with toluene to 10 ml (VEnd).
6.2.2 Gel permeation chromatography (piperonyl butoxide)

To carry out the gel permeation chromatographic cleanup, follow the instructions given in
Cleanup Method 4 (see pp. 65 ff, Vol. 1), but use cyclohexane-ethyl acetate mixture as eluting
solvent.
Dissolve the residue derived from 6.1 in 2 ml cyclohexane-ethyl acetate mixture (VEx) and
transfer 1 ml of this solution (VR1) to the gel permeation column. Evaporate the resulting
eluate to dryness as described in 6.2.1.
326 Method S 22
6.3 Bromination (piperonyl butoxide)

Dissolve the residue derived from 6.2.2 in 1 ml dichloromethane, while carefully rotating the
flask to wet its walls, and add 5 drops of the bromine-iodine mixture (cf. 8. Important points).
Swirl the flask, stopper it with a ground stopper, and let it stand for 10 min at room
temperature. Add 10 ml sodium acetate solution and 5 ml sodium arsenite solution, and swirl.
Then transfer the solution with 10 ml hexane into a 50-ml separatory funnel and shake for
2 min. Filter the organic phase through a little sodium sulphate into a 20-ml volumetric flask.
Extract the aqueous phase once more in the same way, filter, and wash the filter with a little
hexane. Make up the combined filtrates to the mark (VEnd) with hexane.

Inject aliquots (Vi) of the solutions derived from 6.2.1 (pyrethrins) or from 6.3 (piperonyl
butoxide) into the gas chromatograph.
6.4.1 Pyrethrins
with OV-1, crossbond, film thickness 0.10-0.15 fim
(Carlo Erba Mega)
and at 3C/min from 180 to 240 C, then isothermal
at 240 C for 20 min
secondary cooling
63
trol Module 251, constant current, pulse width 1 us
Temperature 290 C
Attenuation 256-2048
Injection volume 1 JJLI
Retention times for
cinerin I 5 min 41 s
jasmolin I 6 min 13 s
pyrethrin I 6 min 26 s
cinerin II 10 min 4 s
jasmolin II 11 min 31 s
pyrethrin II 11 min 52 s
Aldrin had a retention time of 3 min 35 s under these conditions.
6.4.2 Piperonyl butoxide
Column Glass, 2 mm i.d., 2 m long; packed with 29b OV-210
+ 1% OV-17 on Gas Chrom Q, 100-120 mesh
Method S 22 327

63
trol Module 250
Temperature 300 C
Attenuation 128
Retention time for 4,5,7-tribromo-
6-propyl-l,3-benzodioxol 8 min 12 s
7 Evaluation
7.1 Method
Inject equal volumes of appropriately diluted pyrethrin standard solutions or piperonyl but-
oxide calibration solutions, respectively, equivalent to 0.1 to 2 ng pyrethrins or piperonyl but-
oxide, into the gas chromatograph. Plot the heights of the peaks obtained vs. ng pyrethrins
or piperonyl butoxide. For chromatograms of the pyrethrins, evaluate the pyrethrin I peak
(capillary column) or the pyrethrin I + jasmolin I peak (packed column, see 8. Important
points).
Also inject aliquots of the sample solutions. Equal volumes of the sample solutions and the
standard solutions should be injected. For the heights of the peaks obtained for the sample
solutions, read the appropriate amounts of pyrethrins or piperonyl butoxide from the cor-
responding calibration curve.

The recoveries from untreated control samples, fortified with pyrethrins at levels of 0.05 to
1.0 mg/kg, averaged 91% with a standard deviation of 12%. The recoveries from untreated
control samples, fortified with piperonyl butoxide at levels of 0.1 to 1.0 mg/kg, averaged 72%
with a standard deviation of 18%. The recoveries for the individual compounds and analytical
materials are given in the Table.
The limit of detection for the pyrethrins was 0.02 mg/kg, and the limit of determination
was 0.05 mg/kg. For piperonyl butoxide, the limit of detection was 0.03 mg/kg, and the limit
of determination was 0.1 mg/kg.
328 Method S 22
Table. Percent recoveries from plant material fortified with natural pyrethrins at 0.05-1.0 mg/kg, and
piperonyl butoxide at 0.1-1.0 mg/kg.
A . . . ., , . Piperonyl
Analytical material Pyrethrins , . .,
Apples 105 105

Cucumbers 96 60
Endives 85 61
Lettuce 97 70
Mangold 87 67
Plums 94 66
Potatoes 94 84
Red currants 92 62
Spinach 77 61
Sugar beet (edible root) 82 70
Tomatoes 87 91

The residue R, expressed in mg/kg, of the total pyrethrins is calculated from the following
equation:
w A -vEnd
where
Vi = portion of volume VEnd injected into gas chromatograph (in \i\)
WA = amount of total pyrethrins for Vj read from calibration curve (in ng)
The residue R, expressed in mg/kg piperonyl butoxide, is calculated from the following equa-
tion:
R 2 w A .y Ex .v End
VR1VrG
where
VEx = volume of solvent used to dissolve the evaporation residue from 6.1 (in ml)
VR1 = portion of volume VEx injected for gel permeation chromatography in 6.2.2 (volume
of sample loop) (in ml)
Vi = portion of volume VEnd injected into gas chromatograph (in ul)
WA = amount of piperonyl butoxide for W{ read from calibration curve (in ng)
Method S 22 329
8 Important points
The composition of the mixture of natural pyrethrins is practically constant. The main compo-
nent is pyrethrin I with a proportion of approx. 35%. It is, therefore, chosen to represent the
total residues of the pyrethrins. In preparing the calibration curve, the pyrethrin I peak height
is plotted vs. the total of the pyrethrins contained in the standard solution aliquot injected.
If a gas chromatograph fitted with capillary columns is not available, determination of the
pyrethrins can also be performed on packed columns under the following conditions:

63
Temperature 300 C
Attenuation 2 5 -2 8
Recorder Chart speed 5 mm/min
Retention times for
cinerin I 1 min 27 s
jasmolin I + pyrethrin I 2 min 3 s
cinerin II 4 min 28 s
jasmolin II + pyrethrin II 6 min 26 s
Aldrin had a retention time of 52 s under these conditions.

The bromination (cf. 6.3) can also be carried out in acetic acid; however, recoveries tend
to be low and extremely variable with cucumbers, lettuce, mangold, plums, potatoes and red
currants. Noticeably greater variability of yields has also been recorded for standard solutions
when bromination is carried out in acetic acid, as compared to dichloromethane.
In order to check the reliability of the bromination reaction, two or three piperonyl butoxide
calibration solutions (see 4. Reagents) should be prepared along with each series of sample
solutions.
Provided a mass selective detector (MSD) being available, it is recommended to analyze
piperonyl butoxide underivatized on a capillary column by MSD gas chromatography, in order
to circumvent the difficulties encountered with the bromination of the compound. For this end
proceed as follows: Dissolve the residue of the cleaned-up eluate derived from 6.2.2 in a
suitable volume of hexane (VEnd), and inject an aliquot of this solution (Vj) into the gas
chromatograph. The following operating conditions proved to be favourable:
Gas chromatograph Hewlett-Packard 5890/5970 B, equipped with cold in-

jection system KAS (Gerstel)
i.d., 12 m long; film thickness 0.33 |am (Hewlett-
Packard)
330 Method S 22

300 C for 3 min
Detector Mass selective detector HP 5970
El, 70 eV, interface 280 C
Carrier gas flow rate Helium, < 1 ml/min
Printer Paint Jet (Hewlett-Packard)
Injection volume
Retention time for piperonyl
butoxide 10 min 8 s
SIM parameters for piperonyl
butoxide Start 8 min, stop 12 min
Dwell time for m/z = 176 and 338: 100 ms each
Perform quantitation in the SIM mode with the aid of external standards. Prepare the
piperonyl butoxide standard solutions required by dissolving the compound in cleaned-up ex-
tract solutions derived from untreated control samples. Standard solutions and sample solu-
tions should contain comparable concentrations of piperonyl butoxide.
The recoveries from untreated control samples of apples and lettuce, fortified with
piperonyl butoxide at levels of 0.01 to 1.0 mg/kg and analyzed by MSD gas chromatography,
averaged 83% with a standard deviation of 8%. The limit of determination was 0.01 mg/kg.
9 References
S.W. Head, The quantitative determination of pyrethrins by gas liquid chromatography,
Part I: Detection by electron capture, Pyrethrum Post 8, No. 4, 3-7 (1966).
H.-R Thier, Analysengang zur Ermittlung von Pestizid-Riickstanden in Pflanzenmaterial,
Dtsch. Lebensm. Rundsch. 68, 345-350 and 397-401 (1972).
10 Authors
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, W.D. Wein-
mann, H.-G. Nolting and /. Siebers
Institute of Food Chemistry, University of Minister, H.-P. Thier
Method S 22 331
15 10
Chromatogram 1. Standard solution of natural pyrethrins, representing 4 ng of the mixture. Conditions

as decribed in 6.4.1.
Chromatogram 2. Standard solution of natural pyrethrins, representing 8 ng of the mixture, on a packed

column (ECD). Conditions as described in 8.
332 Method S 22
TIC of Pip.Butoxid
Abundance
7000000 -
6000000 -
5000000 -
4000000 -
3000000 -
2000000-\
1000000 -
0-
Time (mln.)
Scan 776 (10.143 min) of Pip.Butoxid
200 300
Mass /Charge
Chromatogram 3. Standard solution of piperonyl butoxide. Total ion current (A) and corresponding mass
spectrum of piperonyl butoxide (B). Capillary column, MSD; conditions as described in 8.
Pyrethroids s 23
Apples, carrots, cauliflower, celeriac (bulbs), cherries, Gas-chromatographic
cucumbers, endives, grapes, maize (kernels), onions, determination
potatoes, rape (green matter and seeds), sugar beet
(edible root), tomatoes, wheat (grains)
Soil, water
(German version first published 1985, revised 1991)
1 Introduction
The method permits the identification and quantitative determination of the residues of the
pyrethroids bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, fenpropathrin,
fenvalerate, flucythrinate, and permethrin.
2 Outline of method
Following homogenization of plant material with a mixture of hexane and acetone, interfering
co-extractives are removed on a Florisil column. If required, a further cleanup can be achieved
by chromatography on a silica gel-activated charcoal column. With oil-containing plant
material, column chromatography is preceded by an acetonitrile-hexane partition. The
pyrethroid residues are extracted from water with hexane, and from soil with a mixture of an
ammonium chloride solution and acetone. The compounds are determined by electron capture
gas chromatography.
3 Apparatus
Round-bottomed flasks, 500-ml, 250-ml and 100-ml
Glass funnel
Fluted filter paper
Chromatographic tube 1, 15 mm i.d., 45 cm long
334 Method S 23
Chromatographic tube 2, 12 mm i.d., 20 cm long

Volumetric flasks, 5-ml to 500-ml
Test tubes, graduated, with ground stoppers
Microsyringe, 10-jj.l
4 Reagents
Acetone, technically pure, dist.
Acetonitrile, p.a.
Diethyl ether, technically pure, dist.
n-Hexane, technically pure, dist.
Petroleum ether, boiling range 40-60C, prepared as follows: Fill a chromatographic tube
(2 cm i.d.) to a height of 20 cm with aluminium oxide (Alumina Woelm B, activity grade
Super I; ICN Biomedicals), gently tapping the tube walls. Top the packing with a 2-cm layer
of sodium sulphate. Add the petroleum ether, and set the flow rate for the solvent to pass
through the column at approx. 5 ml/min. Discard the first 90 ml. The column suffices to
purify 500 ml solvent
Hexane + acetone mixture 8:2 v/v
Petroleum ether + diethyl ether mixture 7:3 v/v
Eluting mixture 1: hexane + toluene 8:2 v/v
Extraction mixture: acetone + ammonium chloride solution 1:1 v/v
Pyrethroid standard solutions: 0.1-5 ng/ml of each compound in hexane
Ammonium chloride solution, 5 g/100 ml NH4C1 p.a.
Sodium chloride solution, 5 g/100 ml NaCl p.a.
Phosphate buffer solution (pH 6.7): 68 g/1 KH2PO4 + 87 g/1 K2HPO4
Activated charcoal, p.a. (Merck No. 2186), acetone-washed (150 ml/10 g) and air-dried
Cottonwool
Method S 23 335

6 Procedure
6.1 Extraction
6.1.1 Plant material (except rape)
Homogenize 50 g of the analytical sample (G) with 10 g filter aid and 200 ml hexane-acetone
mixture for 3 min. Suction-filter the homogenate through a fast flow-rate filter paper in a
Buchner porcelain funnel, and wash the filter cake with 50 ml of the same solvent mixture.
Transfer the filtrate to a graduated cylinder and make up the organic phase to a total volume
of 250 ml (VEx) with hexane-acetone mixture (the lower aqueous phase is not taken into ac-
count). Carefully mix the organic phase, then dry an aliquot (VR1) on sodium sulphate and
filter into a 250-ml round-bottomed flask. Wash the sodium sulphate with 30 ml acetone, and
rotary-evaporate the combined filtrates to near dryness with 30 C bath temperature.
6.1.2 Rape
Homogenize 50 g of the analytical sample (G) with 10 g filter aid and 200 ml hexane-acetone
mixture for 3 min. Suction-filter the homogenate and make up the organic phase of the filtrate
to a total volume of 250 ml (VEx), as described in 6.1.1. Shake 50 ml of the organic phase
(VR1) twice with 50-ml portions of acetonitrile. Combine the acetonitrile phases, add 200 ml
sodium chloride solution, and shake with three 50-ml portions of hexane. Dry the combined
hexane phases on sodium sulphate and filter into a 500-ml round-bottomed flask. Wash the
sodium sulphate with 30 ml hexane, and rotary-evaporate the combined filtrates to near
dryness with 30 C bath temperature.
6.1.3 Soil
Shake 50 g soil (G) with 100 ml extraction mixture for 60 min in a 500-ml Erlenmeyer flask
on a mechanical shaker. Suction-filter the extract through a fast flow-rate filter paper in a
Buchner porcelain funnel. Re-extract the filter cake and filter the extract as described above.
Wash the filter cake twice with 30-ml portions of the extraction mixture. Make up the com-
bined filtrates to a volume of 300 ml (VEx) with extraction mixture, and mix carefully. Then
transfer 100 ml of the filtrate (VR1) to a separatory funnel, add 50 ml phosphate buffer solu-
tion, and shake with three 100-ml portions of hexane. Dry the combined organic phases on
sodium sulphate and continue as described in 6.1.2.
6.1.4 Water
Add 1 g sodium chloride to 50 g water (G), and shake with three 50-ml portions of hexane.
Dry the combined extracts on sodium sulphate and rotary-evaporate as described in 6.1.2. For
336 Method S 23
extracts from water samples containing a low amount of co-extractives, proceed to the gas-
chromatographic determination without further cleanup.
6.2 Cleanup
6.2.1 Column chromatography on Florisil
Pour 40 ml hexane into a chromatographic tube (type 1), sprinkle in 15 g Florisil, and cover
the Florisil with an approx. 2-cm layer of sodium sulphate. Drain the supernatant solvent to
the top of the sodium sulphate layer. Quantitatively transfer the residue derived from 6.1 onto
the column with a total of 5 ml hexane. Let the solution percolate into the column packing,
rinse the flask with 20 ml eluting mixture 1, and add the rinsings to the column. Wash the
column with a further 80 ml of eluting mixture 1 and discard the eluate. Next elute the
pyrethroids from the column with 100 ml eluting mixture 2. Collect this eluate, and rotary-
evaporate to near dryness with 40 C bath temperature.
6.2.2 Column chromatography on silica gel-activated charcoal

For samples of apples, wheat, onions and rape containing pyrethroid residues of less than
0.1 mg/kg, a supplemental cleanup step is recommended. Proceed as follows. Mix 4.5 g silica
gel and 0.3 g activated charcoal, slurry the mixture with 15 ml hexane, and pour the slurry
into a chromatographic tube (type 2). Prewash the column packing with 50 ml eluting mix-
ture 3. Dissolve the residue derived from 6.2.1 portionwise in a total of 3 ml hexane, add the
solutions to the column, and allow to percolate. Rinse the flask with 10 ml eluting mixture 3
and also add this solution to the column. Next elute the pyrethroids with a total of 70 ml
eluting mixture 3. Rotary-evaporate the eluate to near dryness with 40 C bath temperature.

Dissolve the residue derived from 6.1.4 or 6.2 in hexane and make up with hexane to a definite
volume, e.g. 5 ml (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph.
with OV-1, crossbond, film thickness 0.10-0.15 \xm
(Carlo Erba Mega)
Column temperature Programmed to rise at 60C/min from 70 to 180 C, and
at 3C/min from 180 to 245 C, then isothermal at
245 C for 20 min
Injection technique Cold on-column at 70 C oven temperature with second-
ary cooling
63
trol Module 251, pulse width 1 fis
Temperature 290 C
Nitrogen detector purge gas, 50 ml/min
Method S 23 337

Injection volume lul
Retention times for
bifenthrin 8 min 36 s
fenpropathrin 8 min 36 s
cyhalothrin 10 min 36 s
cis-permethrin 11 min 55 s
trans-permethrin 12 min 13 s
cyfluthrin (mixture of isomers) 13 min 24 s
cypermethrin I 13 min 48 s
cypermethrin II 14 min 4 s
cypermethrin III 14 min 14 s
cypermethrin IV 14 min 24 s
flucythrinate I 14 min 30 s
flucythrinate II 15 min
fenvalerate I 16 min
fenvalerate II 16 min 36 s
deltamethrin 17 min 58 s
7 Evaluation
7.1 Method
Inject equal volumes of each pyrethroid standard solution into the gas chromatograph. Plot
the areas or heights of the peaks obtained vs. ng of each compound. Also inject aliquots of
the sample solutions. Equal volumes of the sample solutions and the standard solutions
should be injected. For the areas or heights of the peaks obtained for the sample solutions,
read the appropriate amounts of the compound identified from the corresponding calibration
curve.

and water, fortified with the pyrethroids at levels of 0.01 to 10 mg/kg. The recoveries ranged
from 62 to 122%; see Table. Blanks exceeding 0.001 mg/kg were observed only from extracts
of apples, wheat, onions and rape, when the additional cleanup step was omitted. The limit
of detection was 0.005 mg/kg, and the limit of determination was 0.03 mg/kg.

The residue R, expressed in mg/kg, of an identified pyrethroid is calculated from the following
equation:
_ WAVFYVR
Table. Percent recoveries from plant material, soil and water, fortified with pyrethroids. w
oo
Added
Analytical material Bifenthrin Cyfluthrin Cyhalothrin Cypermethrin Deltamethrin
mg/kg
Apples 0.03-5 101-105 104 89- 94 85- 95 81-110

Carrots 0.05-2 102-115 88-106 86-102 85- 96
Cauliflower 0.1-10 80- 83 75- 79
Celeriac (bulbs) 0.1-0.5 82- 88 68- 91
Cherries 0.05-1 87-116 110-115 79-100 94-110 94-100 CO
Cucumbers 0.02-1 119 100 75- 90 84-107
Endives 0.05-0.1 96-103 91-103
Grapes 0.03-0.5 115-117 101-104 80-113 87-130
Maize (kernels) 0.03-1 85-114 97-115
Onions 0.1-10 85-118 85-109
Potatoes 0.1-5 79-105 79-117
Rape (green matter) 0.03-5 91-105 91-103
Rape (seeds) 0.05-1 66- 69 86- 90 74- 78
Sugar beet (edible root) 0.03-2 100-101 92- 94 74-107 70-100
Tomatoes 0.05-2 92-118 96-100 94-108 77- 95 82- 95
Wheat (grains) 0.05-1 82- 92 102-105 85-122 86- 95
Soil 0.03-1 68-125 95-120 67-104 94-112 90-100
Water 0.01-1 107-111 104 104-109 94-101 95- 97
Table, (contd.)
Analytical material Fenpropathrin Fenvalerate Flucythrinate Permethrin
Apples 0.03-5 97 87- 99 84-106

Carrots 0.05-2 85- 98 75- 77 86-115
Cauliflower 0.1-10 79- 81 81- 85
Celeriac (bulbs) 0.1-0.5 91-100 75- 92
Cherries 0.05-1 87 96-109 73- 81 92-105
Cucumbers 0.02-1 91-94 74- 95 87-105
Endives 0.05-0.1 74 95-101 86- 88
Grapes 0.03-0.5 80 84-122 95-128
Maize (kernels) 0.03-1 71-111 96- 98
Onions 0.1-10 85-102 85-114
Potatoes 0.1-5 79-100 80-102
Rape (green matter) 0.03-5 77 91-130 62- 83
Rape (seeds) 0.05-1 76- 83
Sugar beet (edible root) 0.03-2 76-100 95-101 90-100
Tomatoes 0.05-2 76-100 81- 96 90- 92
Wheat (grains) 0.05-1 77 79- 88 81-102 73-122
Soil 0.03-1 100 90- 97 74- 82 84-100
Water 0.01-1 100 93- 97 90- 98 98- 99
I
CO
N>
CO
CO
CO
CO
I
CO
11
c c
II
urt
Figure. Pyrethroids in tomatoes; conditions as described in 6.3.

Chromatogram 1: Standard mixture representing 0.1 ng each of bifenthrin, cyhalothrin, cyfluthrin, and flucythrinate.
Chromatogram 2: Untreated control sample.
Chromatogram 3: Untreated control sample fortified with 0.1 mg/kg each of bifenthrin, cyhalothrin, cyfluthrin, and flucythrinate.
Method S 23 341
where
VEx = total volume of organic phase after addition of solvent mixture to filtered extract from
plant sample in 6.1.1 or 6.1.2, or total volume of filtered extract from soil sample in
6.1.3 (in ml)
VR1 = portion of volume VEx used for cleanup (in ml)
WA = amount of identified pyrethroid for Vj read from calibration curve (in ng)
8 Important points
Emulsions, that may form during the extraction, can be broken with centrifugation.
To prevent false results, the elution of the pyrethroids from the chromatographic columns
(see 6.2.1 and 6.2.2) should be checked with each new batch of adsorbent. If the compounds
are not completely eluted, either the amounts of the eluting mixtures or their polarities must
be increased.
Additionally, the ranges of the elution volumes of the different pyrethroids can shift accord-
ing to the activity of the Florisil: When its activity is low, permethrin can appear in the
forerun, whereas flucythrinate will elute later (after the main fraction of the eluate) with high
Florisil activity. Since the Florisil activity may alter during storage, only freshly prepared ad-
sorbent should be used.
Extracts containing residues of fenpropathrin should be evaporated carefully, otherwise low
recoveries can be encountered.
Several pyrethroids exhibit very similar retention times under the gas-chromatographic
operating conditions described in 6.3. It is therefore highly recommended to confirm the
results obtained by using a gas-chromatographic column of different polarity or by employing
a mass selective detector (MSD).
Tetramethrin and pyrethrins can also be analyzed by this method. For this purpose, first
elute the pyrethroids from the Florisil column as described in 6.2.1. Then elute tetramethrin
and the pyrethrins with an additional 100 ml of petroleum ether-diethyl ether mixture, and
proceed with the gas-chromatographic determination as described in 6.3.
Resmethrin and phenothrin cannot be analyzed by this method, because these compounds
are not detected by the electron capture detector with sufficient sensitivity.
If a gas chromatograph fitted with capillary columns is not available, determination of the
pyrethroids can also be performed on packed columns, under the following conditions:

Column Glass, 2 mm i.d., 1.2 m long; packed with 10% SE-30 on
Chromosorb W-HP, 80-100 mesh
Column temperature Programmed to rise at 3 C/min from 200 to 270 C, then
isothermal at 270 C for 5 min
342 Method S 23
63
Temperature 300 C
Attenuation 28
Recorder Chart speed 5 mm/min
Injection volume 1-3 [i\
Retention times for
fenpropathrin 9 min 12 s
permethrin isomer mixture 13 min 44 s
cypermethrin isomer mixture 16 min 14 s
fenvalerate I 18 min 10 s
fenvalerate II 18 min 46 s
deltamethrin 20 min 14 s
9 References
R.A. Chapman and C.R. Harris, Extraction and liquid-solid chromatography cleanup pro-
cedures for the direct analysis of four pyrethroid insecticides in crops by gas-liquid
chromatography, J. Chromatogr. 166, 513-518 (1978).
K. Naumann, Chemie der synthetischen Pyrethroid-Insektizide, in: R. Wegler, Chemie der
Pflanzenschutz- und Schadlingsbekampfungsmittel, Volume 7, Springer-Verlag, Berlin-
Heidelberg-New York 1981.
J. Siebers and H.-G. No Iting, Analysenmethode zur Bestimmung von Pyrethroiden in
verschiedenen pflanzlichen Lebensmitteln, Wasser und Boden, Nachrichtenbl. Dtsch.
Pflanzenschutzdienstes Braunschweig 34, 166-170 (1982).
10 Authors
Nolting, J. Siebers and H. Ko'hle
Organotin Compounds s 24
Apples, aubergines, beans (green), celeriac (leaves and Gas-chromatographic
bulbs), grapes, melons, nectarines, peaches, plums, determination
strawberries, sugar beet (foliage and edible root),
tomatoes
Soil, water
1 Introduction
The method permits the identification and quantitative determination of organotin com-
pounds in which the central tin atom carries three identical organic substituents, such as
derivatives from triphenyltin (fentin acetate, fentin chloride, and fentin hydroxide) or
tricyclohexyltin (azocyclotin and cyhexatin), and tris(2-methyl-2-phenylpropyl)tin (fenbutatin
oxide). Compounds containing the same three substituents are determined simultaneously
since they are converted to the same derivatives.
2 Outline of method
Residues of organotin compounds are extracted from plant material, soil and water with
acetone in the presence of hydrobromic acid. The homogenate is filtered; the filtrate is ex-
tracted with n-hexane, and the hexane phase is evaporated to dryness. The compounds are
reacted with methyl magnesium chloride in ethereal solution to yield the corresponding methyl
derivatives R3Sn-CH3:
XMgCl
The derivatives are cleaned up by column chromatography on Florisil and are determined by
gas chromatography using a flame photometric detector.
3 Apparatus
Filter paper, 11 cm dia.
Separatory funnels, 1-1, 500-ml and 100-ml
344 Method S 24
Round-bottomed flasks, 1-1 and 250-ml

Chromatographic tube, 20 mm i.d., 30 cm long, with PTFE stopcock
Microsyringe, 10-ul
4 Reagents
Acetone, nanograde (Promochem)
Diethyl ether, p.a.
n-Hexane, nanograde (Promochem)
Derivative standard solutions: Prepare cyhexatin, fentin hydroxide, and fenbutatin oxide solu-
tions with 0.2, 1, 10 and 100 M-g/ml acetone. Evaporate 5 or 10 ml of each to dryness, allow
to react with methyl magnesium chloride as described in 6.2, and make up again to 5 or 10 ml
with acetone
Methyl magnesium chloride solution: methyl magnesium chloride (reagent grade),
20 g/100 ml in tetrahydrofuran (Merck-Schuchardt No. 820 310)
Hydrobromic acid, p.a., minimum 47 g/100 g (Merck No. 307)
Hydrochloric acid, p.a., cone.
Florisil, 60-100 mesh
Glass wool
Air, synthetic

6 Procedure
6.1 Extraction
6.1.1 Plant material, soil
Homogenize 100 g of plant material (G) with 20 ml hydrobromic acid and 200 ml acetone for
3 min. To 100 g of soil (G), add 100 ml water and extract the mixture in the same manner or
by using a mechanical shaker. Suction-filter the homogenate through a filter paper in a
Buchner porcelain funnel. Transfer the filter cake back into the blendor jar, extract with
200 ml acetone for a further 3 min and suction-filter again. Shake the combined filtrates twice
with 200-ml portions of hexane, dry the combined organic phases on sodium sulphate, and
Method S 24 345
6.1.2 Water
Transfer 100 ml of water (G) into a separatory funnel, add 200 ml acetone and 20 ml
hydrobromic acid, and shake the solution twice with 200-ml portions of hexane. Dry the com-
bined organic phases on sodium sulphate and rotary-evaporate to dryness.
6.2 Derivatization
Dissolve the residue derived from 6.1 in 20 ml diethyl ether, add 3 ml methyl magnesium
chloride solution, swirl, and allow to stand for approx. 5 min. Next add 10 ml water and 1 ml
hydrochloric acid, and transfer the contents of the flask into a 100-ml separatory funnel. Rinse
the flask with 10 ml diethyl ether, add this to the separatory funnel, and shake. Discard the
lower aqueous phase. Dry the organic phase on sodium sulphate and rotary-evaporate to
dryness.
6.3 Column chromatography (not required for water samples)

Fill the chromatographic tube with approx. 30 ml hexane, push a glass wool plug through to
the lower end, and trickle in 8 g Florisil. Allow air bubbles to escape, and drain the hexane
to the top of the Florisil. Dissolve the residue derived from 6.2 in approx. 10 ml hexane,
transfer the solution onto the column, and allow to percolate. Rinse the flask several times
with 5-ml portions of hexane and transfer each rinsing onto the column before the preceding
portion has soaked in. Continue to elute the column with hexane at a rate of 1.5 ml/min until
a total of 120 ml eluate has been collected. Rotary-evaporate the eluate to dryness.

Dissolve the residue derived from 6.2 or 6.3 in acetone and make up to 10 ml (VEnd). Inject
an aliquot of this solution (Vj) into the gas chromatograph.
Column 1 Glass, 2 mm i.d., 1.2 m long; packed with 5% OV-17
on Chromosorb G AW-DMCS, 60-80 mesh
Column temperature 3 min 200 C, programmed to rise at 15C/min from
200 to 250 C, and 17 min 40 s after injection from
250 to 300 C
Column 2 Glass, 2 mm i.d., 1.2 m long; packed with 5% OV-225
on Chromosorb G AW-DMCS, 60-80 mesh
Column temperature 3 min 150 C, programmed to rise at 15C/min from
150 to 180 C, 8 min after injection from 180 to
200 C, and 15 min 20 s after injection from 200 to
250 C
Detector Flame photometric detector equipped with dual flame
system (Varian) and 394-nm sulphur filter (Tracor)
Temperature 300 C
346 Method S 24

Air, flame 1, 80 ml/min; flame 2, 200 ml/min
Attenuation 8- 10 ~9
Column 1 Column 2
Retention times for derivatives
from
cyhexatin, azocyclotin 9 min 30 s 7 min
fentin 11 min 12 min
fenbutatin oxide 22 min 30 s 20 min
7 Evaluation
7.1 Method
ing them with the peak areas obtained for the corresponding derivative standard solutions.
Equal volumes of the sample solutions and the derivative standard solutions should be in-
jected; additionally, the peaks of the solutions should exhibit comparable areas.

The recoveries from untreated control samples of plant material, soil and water, fortified with
cyhexatin, azocyclotin, fenbutatin oxide and fentin hydroxide at levels of 0.01 to 0.05 mg/kg,
ranged from 80 to 100%. The routine limit of determination was 0.02 to 0.05 mg/kg for plant
material and soil, and 0.01 mg/1 for tap water.

The residue R, expressed in mg/kg, of an identified organotin compound is calculated from
the following equation:
R _ F A -V End -W st
where
Vj = portion of volume VEnd injected into gas chromatograph (in \i\)
WSt = amount of compound injected with derivative standard solution (in ng)
FA = peak area obtained from Vl (in mm2)
F st = peak area obtained from WSt (in mm2)
Method S 24 347
- 200
- 150
16 8
Chromatograms 1 and 2. Untreated control sample of apples fortified with 0.05 mg/kg each of cyhexatin
or azocyclotin (1), fentin hydroxide (2), and fenbutatin oxide (3); 50 mg aliquots injected. Upper
chromatogram, GLC column 1; lower chromatogram, GLC column 2.
348 Method S 24
8 Important points
The method does not differentiate between azocyclotin and cyhexatin or between fentin
acetate, chloride and hydroxide.
Metabolites of the general formulae R2SnX2 occur only at low levels in foodstuffs. They
can also be determined by this method. Unter the conditions given in 6.4, they do not interfere
with the determination of the parent compounds.
If the recoveries are less than expected, it is advisable to use more methyl magnesium
chloride solution.
9 Reference
E. Mollhoff Methode zur gaschromatographischen Bestimmung des Akarizids Peropal und
seiner Metaboliten in Pflanzen, Boden, Wasser und Kleintierfutter, Pflanzenschutz-Nachr. 30,
249-263 (1977).
10 Author
E. Mollhoff
Methyl Carbamate Insecticides s 25
Apples, beans (green), carrots, cherries, head cabbage, Gas-chromatographic
kohlrabi, leeks, lettuce, radishes, strawberries, tomatoes determination
1 Introduction
The method permits the identification and quantitative determination of 14 methyl carbamate
insecticides and four metabolites, including ten aromatic and four aliphatic N-methyl carb-
amates as well as one N,N-dimethyl carbamate (see Table 1). The compounds are determined,
without derivatization, under conditions by which decomposition of the carbamates is
prevented.
Table 1. Relative retention times (RRT) of methyl carbamates, relative to lauronitrile (I; retention time
4 min 25 s) and to octadecanonitrile (II; retention time 13 min 46 s).
Methyl carbamate RRT I RRT II
Lauronitrile 1.00
Aldicarb 1.28 0.41
Butocarboxim 1.39 0.45
Methomyl 1.51 0.48
Propoxur 1.51 0.48
Thiofanox 1.62 0.52
Promecarb 1.77 0.57
Bendiocarb 1.77 0.57
Carbofuran 1.99 0.64
Aminocarb 2.04 0.65
Primicarb 2.27 0.73
Ethiofencarb 2.35 0.75
3-Keto-carbofuran 2.37 0.76
Desmethyl-pirimicarb 2.39 0.77
Dioxacarb 2.51 0.81
Carbaryl 2.56 0.82
Mercaptodimethur (Methiocarb) 2.67 0.86
3-Hydroxy-carbofuran 2.83 0.91
Desmethylformamido-pirimicarb 3.08 0.99
Octadecanonitrile 1.00
2 Outline of method
The methyl carbamate residues are extracted from plant material with ethyl acetate; an aliquot
of the extract is evaporated to dryness. The residue is extracted with water and the solution
passed through a RP-18 disposable cartridge using an acetonitrile-water mixture as eluant. The
350 Method S 25
eluate is shaken with dichloromethane, and the dichloromethane phase is evaporated. The
methyl carbamates are determined by capillary gas chromatography with splitless injection
using a thermionic detector.
3 Apparatus
Laboratory centrifuge, e.g. Varifuge type 4120 (Heraeus-Christ), with 150-ml stainless steel
tubes
Pear-shaped flasks, 100-ml and 50-ml, with ground joints and glass stoppers
Separatory funnel, 100-ml, with PTFE stopcock
Test tubes, 10-ml, with screw cap and PTFE gasket, e.g. SVL45 (Corning No. 611-52)
Microsyringe, 10-ul
Important: All glassware must be rinsed with acetone immediately before use.
4 Reagents
Acetone, chemically pure, dist.
Acetonitrile, reagent grade (Merck No. 800015), dist.
Cyclohexane, chemically pure, distilled from a sodium suspension
Dichloromethane, chemically pure, dist.
Ethyl acetate (Riedel-de Haen No. 27227), dist.
Methanol, chemically pure, distilled from potassium hydroxide
Water, deionized and dist.
Eluting mixture: acetonitrile + water 1:1 v/v
Internal standard solution: 1 ng/ml lauronitrile (Merck-Schuchardt No. 805345) and 2
octadecanonitrile (Aldrich No. 12,258-0) in cyclohexane
Methyl carbamate standard solutions: 1-2 ng/ml of each compound in cyclohexane (diluted
from a stock solution in ethyl acetate)
RP-18 disposable cartridge: Sep-Pak Cartridge C18 (Millipore No. 51910)
Sodium hydrogen carbonate, p.a.
Method S 25 351

6 Procedure
6.1 Extraction
Weigh 25 g of the chopped analytical sample (G) into a centrifuge tube and (only in the case
of fruit) add 0.5 g sodium hydrogen carbonate. Add 80 ml ethyl acetate and homogenize for
2 min. Rinse the rod of the homogenizer twice with 10-ml portions of ethyl acetate, add the
rinsings to the centrifuge tube, and centrifuge the homogenate for 10 min at 5000 r.p.m. Pipet
a 40-ml portion (VR1) from the clear supernatant solution (VEx =100 ml) into a 100-ml pear-
shaped flask and rotary-evaporate to near dryness. Remove the last traces of solvent with a
gentle stream of air or nitrogen.
6.2 Cleanup on a RP-18 cartridge

Draw 10 ml methanol into the glass syringe, attach a cartridge to the syringe, and force the
methanol through the cartridge. Detach the cartridge, pull the plunger out of the syringe, and
re-attach the cartridge.
Add 10 ml water to the residue derived from 6.1, stopper the flask, and shake well for
10-15 s. The residue does not completely dissolve. Transfer the solution, disregarding floating
particles, into the syringe, re-insert the plunger and force the liquid through the cartridge.
Detach the cartridge, draw 5 ml water and 5 ml air into the syringe, re-attach the cartridge
and force the water and air through the cartridge. Discard the eluates.
Add 10 ml eluting mixture to the residue in the pear-shaped flask, stopper, and shake again
for 10-15 s. Transfer the solution into the syringe, as described above, the cartridge being at-
tached. Force the liquid through the cartridge and collect the eluate in a separatory funnel.
Rinse the pear-shaped flask again with 5 ml eluting mixture, transfer this solution into the syr-
inge, make up to 10 ml with eluting mixture, and force the liquid through the cartridge into
the separatory funnel.

Add 10 ml dichloromethane to the solution derived from 6.2, shake for 2 min, allow to stand
for 30 min, and drain the lower phase into a 50-ml pear-shaped flask. Repeat the extraction
with a further 10 ml of dichloromethane.
If the upper, aqueous phase is still very turbid after the second extraction, add 5 ml
methanol, swirl, allow to stand for 10-30 min, and drain the organic phase into the pear-
shaped flask. Rotary-evaporate the solution to near dryness. Remove the last traces of solvent
with a gentle stream of air or nitrogen.
352 Method S 25

Dissolve the residue derived from 6.3 in 0.1 to 0.2 ml ethyl acetate and add 2.0 ml internal
standard solution. If the resulting solution is turbid, transfer it to a test tube, cap the tube,
and centrifuge for 5-10 min at 5000 r.p.m. Inject an aliquot of the clear supernatant solution
into the gas chromatograph.
For splitless injection, cool the column oven down to 50 C and close the split exit. Open
the split exit 30 s after the injection and heat the column oven to the starting temperature of
the temperature program (100 C).
Column Duran glass capillary, 0.3 mm i.d., 0.8 mm o.d., 18 m
long; statically coated with 0.1 pirn SE-52 (deactivated
with polyethylene glycol PEG 400 and PEG 1000)
Column temperature 50-100C at maximum heating rate, programmed to
rise at 6C/min from 100 to 230 C, then isothermal
at 230 C
Temperature 250 C
Gas flow rates Nitrogen carrier, 2.6 ml/min
Air, 175 ml/min
Split ratio 1:10
Attenuation 8-10- 12
Integrator Hewlett-Packard 3380 A, chart speed 5 mm/min
Injection volume 2-2.5 \x\ with closed split exit and at 50 C column
temperature
Retention times See Table 1
7 Evaluation
7.1 Method
Quantitation is performed by measuring the peak areas obtained for the methyl carbamates
and comparing them with the peak areas obtained for the internal standard lauronitrile. When
this peak is subject to interference by plant co-extractives, the peak of octadecanonitrile is
used instead.
Due to the different sensitivities of the detector to the individual methyl carbamates and
the internal standard, a correction factor K is applied, derived from injecting a solution con-
taining a definite amount of the respective methyl carbamate and of the internal standard.
Method S 25 353
10
18
3,4 13
15
6,7 16 17
14
12 II
11
ULJ
10 15 mm
Chromatogram 1. Standard mixture representing 2 ng of each methyl carbamate (4 ng each of 8, 12, 14,
15, 16, 17 and II). Conditions as described in 6.4.
1, aldicarb; 2, butocarboxim; 3, methomyl; 4, propoxur; 5, thiofanox; 6, promecarb; 7, bendiocarb;
8, carbofuran; 9, aminocarb; 10, pirimicarb; 11, ethiofencarb; 12, 3-keto-carbofuran; 13, desmethyl-
pirimicarb; 14, dioxacarb; 15, carbaryl; 16, mercaptodimethur (methiocarb); 17, 3-hydroxy-carbofuran;
18, desmethylformamido-pirimicarb.
I, lauronitrile; II, octadecanonitrile (internal standards).
354
10 II
ÛUv/N^y
10 15 min
Chromatogram 2. Untreated control sample of lettuce fortified with 0.1 mg/kg (for 14, 15 and 16, each
0.2 mg/kg) of 11 methyl carbamates. Conditions as described in 6.4; for assignment of peaks see
Chromatogram 1.
Method S 25 355

The recoveries from untreated control samples fortified with methyl carbamates are given in
Tables 2 and 3. At levels of 0.01 to 0.5 mg/kg they ranged, in general, from 70 to 115%; in
some cases, however, lower recoveries were observed.
Table 2. Percent recoveries from plant material fortified with methyl carbamates; means from quadruplet
experiments. Values in brackets: Recovery <70%.
Added Green Head

Methyl carbamate mg/kg Apples Cherries Carrots Lettuce beans cabbagea Leeks
Aldicarb 0.01 97 80 93 72 59 ndc
0.05 101 84 92 90 (52) 87 ndc
0.1 100 92 95 82 (68) 90 ndc
Aminocarb 0.01 93 75 89 125 (36) (28) (55)
0.05 104 71 95 91 (52) 75 75
0.1 95 95 84 86 (52) 70 73
Bendiocarb 0.01 100 130 91 74 119 71
0.05 114 108 98 79 119 75 82
0.1 120 112 96 107 93 82 104
Butocarboxim 0.01 78 70 78 70 (48) ndc
0.05 106 92 80 79 (42) 75 ndc
0.1 112 87 86 82 (48) 85 ndc
Carbaryl 0.02 102 95 105 87 92 b 97
0.1 113 106 105 100 85 b 90
0.2 116 107 102 100 94 b 104
Carbofuran 0.01 102 96 105 78 76 122 95
0.05 108 110 105 98 94 94 98
0.1 103 115 99 101 98 95 106
Dioxacarb 0.02 113 85 108 88 76 95 87
0.1 117 97 100 97 75 103 92
0.2 114 96 95 108 95 106 116
Ethiofencarb 0.01 109 90 88 70 ndc 70 71
0.05 92 72 91 74 ndc 74 79
0.1 104 77 96 93 ndc 73 71
Mercaptodimethur 0.02 120 100 106 73 86 79 95
(Methiocarb) 0.1 99 110 108 98 81 71 96
0.2 112 112 102 101 91 76 101
Methomyl 0.1 108 (31) (39) 89 ndc ndc
0.5 109 (63) 70 87 ndc (50) ndc
Pirimicarb 0.01 98 85 104 71 102 80 102
0.05 95 71 111 82 84 90 76
0.1 96 91 108 84 87 94 78
Promecarb 0.01 114 90 115 75 76 89
0.05 97 98 109 95 93 100 70
0.1 109 113 98 91 98 96 85
Propoxur 0.01 93 100 93 87 75 100 84
0.05 103 98 102 98 94 90 97
0.1 108 106 101 89 95 93 108
Thiofanox 0.01 79 69 93 (33) b
0.05 108 98 79 73 (36) b (49)
0.1 119 88 87 87 (37) b (44)
a
Head cabbage fortified with 0.1, 0.5 and 1 mg/kg or 0.2, 1 and 2 mg/kg; b Interferences from plant
co-extractives; ndc Not detectable (no peak observed).
356 Method S 25
Table 3. Percent recoveries from strawberries, tomatoes and radishes, fortified with 0.1 mg/kg of the
methyl carbamates; means from duplicate experiments.
Methyl carbamate Strawberries Tomatoes Radishes

Aldicarb 85 90 ndm
Aminocarb 113 99 111
Bendiocarb 89 98 105
Butocarboxim ndm 93 ndm
Carbaryla 109 103 113
Carbofuran 106 87 ndm
Dioxacarba 91 86 99
Ethiofencarb 77 69 117
Mercaptodimethura (Methiocarb) 101 101 109
Methomyl ndm 83 ndm
Pirimicarb 94 100 92
Promecarb 99 92 88
Propoxur 97 93 106
Thiofanox 78 85 63
a
Fortified with 0.2 mg/kg; ndm Not determinable.
The limits of determination of the methyl carbamates from seven plant materials are listed
in Table 4. In general, they ranged from 0.01 to 0.05 mg/kg (methomyl, 0.1 to 1 mg/kg). The
limits of detection were mostly amounting to one third to one half of the limits of deter-
mination.
Table 4. Limits of determination (mg/kg) of the methyl carbamates in plant material.
Green Head
Methyl carbamate Apples Cherriesa Carrots Lettuce Leeksa
beans cabbage
Aldicarb 0.01 0.005 0.01 0.01 0.1 0.5 ndc

Aminocarb 0.01 0.01 0.005 0.01 b 0.5 0.05
Bendiocarb 0.005 0.005 0.01 0.005 0.005 0.05 0.05 d
Butocarboxim 0.005 0.01 0.01 0.005 b 0.5 ndc
Carbaryl 0.02 0.01 0.01 0.01 0.01 c 0.02e
Carbofuran 0.005 0.005 0.005 0.005 0.005 0.05 0.01
Dioxacarb 0.02 0.01 0.01 0.01 0.01 0.1 0.01 e
Ethiofencarb 0.01 0.01 0.005 0.005 ndc 0.1 0.01
Mercaptodimethur
(Methiocarb) 0.02 0.01 0.01 0.01 0.01 0.1 0.05d
Methomyl 0.1 b 0.5 0.1 ndc 1.0 ndc
Pirimicarb 0.01 0.01 0.005 0.005 0.005 0.05 0.01 e
Promecarb 0.01 0.01 0.005 0.01 0.005 0.05 0.05e
Propoxur 0.01 0.005 0.01 0.005 0.005 0.05 0.01
Thiofanox 0.005 0.005 0.01 0.05 b c b,d
a
Evaluated against octadecanonitrile; b Recovery <70%; c Interferences from plant co-extractives;
d
Blanks equivalent to 0.02 mg/kg; e Blanks equivalent to <0.02 mg/kg; ndc Not detectable (no
peak observed).
Method S 25 357

The residue R, expressed in mg/kg, of an identified methyl carbamate is calculated from the
following equation:
R FA-VEx-WSt
K
FSt-VR1-G
where
VEx = volume of ethyl acetate used for extraction (in ml)
VR1 = portion of volume VEx used for cleanup (in ml)
Wst = amount of internal standard added to sample solution in 6.4 (in \xg)
FA = peak area obtained for an identified methyl carbamate (in mm2 or integrator
counts)
FSt = peak area obtained for the internal standard (in mm2 or integrator counts)
K = correction factor for the individual methyl carbamate identified
K is calculated from the chromatogram of a standard solution (cf. 7.1) using the following
equation:
-"a
K=
CsFa
where
Ca = concentration of the methyl carbamate in the standard solution (in |ug/ml)
Cs = concentration of the internal standard in the standard solution (in u^/ml)
Fa = peak area obtained for the methyl carbamate in the standard solution (in mm2 or
integrator counts)
Fs = peak area obtained for the internal standard in the standard solution (in mm2 or
integrator counts)
8 Important points
A fused silica capillary column can also be used for the gas-chromatographic determination:
DB-1, 30 m long, film thickness 0.1 fxm (J & W Scientific). Keep to the exact conditions as
described in 6.4, in order to guarantee optimum sensitivity.
Interfering peaks did occur with various methyl carbamate and substrate combinations (see
Table 2).
For aromatic methyl carbamate insecticides, the results can be confirmed by preparing the
2,4-dinitrophenyl ether of the corresponding phenol and determining the derivative by gas
chromatography on the same capillary column using an electron capture detector.
358 Method S 25
It has not been checked if this method is suitable for analysis of the sulphoxides and
sulphones of aldicarb, butocarboxim, ethiofencarb, mercaptodimethur (methiocarb), meth-
omyl and thiofanox.
9 Reference
S. Brauckhoff and H.-P. Thier, Analysenmethode flir Rtickstande von Carbamat-Insecticiden
in pflanzlichen Lebensmitteln, Z. Lebensm. Unters. Forsch. 184, 91-95 (1987).
10 Authors
Institute of Food Chemistry, University of Miinster, S. Brauckhoff and H.-P. Thier
Phthalimides s 26
Apples, grapes, lettuce, potatoes, wheat (green matter Gas-chromatographic
and grains) determination
Soil, water
1 Introduction
The method permits the identification and quantitative determination of the residues of cap-
tafol, captan, dialifos, ditalimfos, folpet, and phosmet by capillary gas chromatography.
2 Outline of method
Phthalimide residues are extracted from plant material with a hexane-acetone mixture; an ali-
quot of the extract is evaporated to dryness. Interfering co-extractives are removed on a Florisil
cartridge. When this is not sufficient, a further cleanup can be achieved by gel permeation
chromatography. Water samples are extracted with dichloromethane, and soil samples with
hexane. The phthalimides are determined by electron capture gas chromatography on a fused
silica capillary column.
3 Apparatus
Volumetric pipet, 100-ml
Erlenmeyer flasks, 500-ml and 200-ml, with ground joints
Separatory funnels
360 Method S 26

Microsyringe, 10-ul
4 Reagents
Acetone, fractionally distilled
Dichloromethane, fractionally distilled
n-Hexane, p.a.
Toluene, p.a.
n-Hexane + acetone mixture 4:1 v/v
Eluting mixture : cyclohexane + ethyl acetate 1:1 v/v
Phthalimide standard solutions: 0.010.1 |ig/ml of each compound in toluene
Sodium sulphate, p.a., anhydrous, exhaustively extracted with toluene
Florisil disposable cartridge: Sep-Pak Cartridge Florisil (Millipore No. 51960)
Helium

6 Procedure
6.1 Extraction
Homogenize 50 g of the analytical sample (20 g of cereal green matter) (G) with 10 g filter
aid and 200 ml hexane-acetone mixture for 3 min. Suction-filter the homogenate through a
fast flow-rate filter paper in a Buchner porcelain funnel, and wash the filter cake with a total
of 50 ml hexane-acetone mixture. Transfer the filtrate to a graduated cylinder and make up
the organic phase to 200 ml (VEx) with the hexane-acetone mixture, ignoring the lower
aqueous phase. Mix well; then pipet 100 ml (VR1) of the organic phase into a 200-ml
Erlenmeyer flask and dry on sodium sulphate. Filter the solution through a fluted filter paper
into a 250-ml round-bottomed flask, and rinse Erlenmeyer flask and filter with 30 ml acetone.
Rotary-evaporate the combined filtrates to a volume of 1 to 2 ml, then transfer the concen-
trated solution to a pear-shaped flask with acetone and rotary-evaporate to near dryness.
Remove the last traces of solvent by swirling the flask in the hand. Proceed to step 6.2.
Method S 26 361
6.1.2 Soil
In a separate 10 to 20-g aliquot of the laboratory sample, determine the water content by dry-
ing in an open weighing glass at 105 C to constant weight (approx. 15 h). Discard this aliquot.
If the water content is higher than 10%, then use an analytical sample as it is. If the water
content is lower than 10%, then add water to raise the water content to at least 10%.
Transfer 100 g of the analytical sample (G) with the appropriate water content into a 500-ml
Erlenmeyer flask, add 25 g sodium sulphate and 200 ml hexane, and shake on the mechanical
shaker for 1 h. Filter the extract through a fast flow-rate filter paper covered with filter aid
in a Buchner porcelain funnel, and wash the filter cake twice with 30-ml portions of hexane.
trated solution to a pear-shaped flask with acetone and rotary-evaporate to near dryness.
Remove the last traces of solvent by swirling the flask in the hand. Proceed to step 6.2.
6.1.3 Water
Extract 1 1 of the water sample (G) with 100 ml dichloromethane for 2 min. Filter the organic
phase through a fluted filter paper, containing sodium sulphate, into a 500-ml round-
bottomed flask. Repeat the extraction twice, shaking each time with 50 ml dichloromethane.
trated solution to a pear-shaped flask with acetone, and rotary-evaporate to near dryness.
Remove the last traces of solvent by swirling the flask in the hand. In the case of ground and
tap water samples, proceed to the gas-chromatographic determination without further
cleanup.
6.2 Cleanup
6.2.1 Florisil cartridge
Draw 10 ml hexane into the glass syringe, attach a Florisil cartridge to the syringe, and force
the hexane through to condition the cartridge packing. Detach the cartridge, pull the plunger
out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1 in 0.5 ml
hexane and transfer the solution into the syringe with the aid of a Pasteur pipet. Rinse the
pear-shaped flask with a further 0.5 ml of hexane and add the rinsings to the syringe. Re-insert
the plunger into the syringe, and force the liquid through the cartridge. Detach the cartridge,
remove the plunger from the syringe, and re-attach the cartridge. Next elute the compounds
with 10 ml hexane-acetone mixture from the Florisil, proceeding in a similar manner as
described above. Collect the eluate in a pear-shaped flask and rotary-evaporate to dryness.

For plant material and soil containing phthalimide residues of less than 0.1 mg/kg, it is recom-
mended to clean up the extract derived from 6.2.1 as follows:
Transfer the residue derived from 6.2.1 into a test tube, using a total of 10 ml eluting mixture
(VR2) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR3) of the
gel permeation chromatograph with 8 to 9 ml of the solution. Set the gel permeation
chromatograph at the eluting conditions determined beforehand with standard solutions of
362 Method S 26
the phthalimides; cf. Cleanup Method 6, pp. 75 ff, Vol. 1. - Elution volumes ranging from
100 to 160 ml were determined for the phthalimides on Bio-Beads S-X3 polystyrene gel, using
the eluting mixture as eluant, pumped at a flow rate of 5.0 ml/min.
Check the elution range from time to time and determine anew whenever a new gel column
is used.

Dissolve the residue derived from 6.1.3 or 6.2 in toluene, and make up with toluene to a definite
volume, e.g. 5 ml (VEnd). Inject an aliquot of this solution (V{) into the gas chromatograph.
with OV-1, crossbond, film thickness 0.10-0.15 um
(Carlo Erba Mega)
Column temperature 100 to 180 C at maximum heating rate, programmed
to rise at 2C/min from 180 to 215 C, then isother-
mal at 215 C for 2 min
secondary cooling
63
trol Module 251, pulse width 1 us
Temperature 300 C
Attenuation 256-1024
Retention times for
captan 5 min 20 s
folpet 5 min 32 s
ditalimfos 6 min 20 s
captafol 9 min 56 s
phosmet 11 min 20 s
dialifos 16 min 14 s
7 Evaluation
7.1 Method
Inject equal volumes of the phthalimide standard solutions (equivalent to 0.1 to 1.0 ng of each
compound) into the gas chrcmatograph. Plot the areas or heights of the peaks obtained vs.
a) b) c)
o
-p Pi
i
o
o
<H
ClJ
P
tal imfos
&
4>
P,
cd
o
OQ
O P
CO
0 -P
00
o <D
H CD
cd O
H
P
JL ^ L
I
CO
min 15 10 min 15 10 min 15 10
Chromatograms of
a) standard mixture representing 0.05 ng of each phthalimide, b) untreated control sample of wheat grains,
c) untreated control sample of wheat grains, fortified with 0.1 mg/kg of each phthalimide. CO
Conditions as described in 6.3. CO

364 Method S 26
ng of compound. Also inject aliquots of the sample solutions. Equal volumes of the sample
solutions and the standard solutions should be injected. For the areas or heights of the peaks
obtained for the sample solutions, read the appropriate amounts of the identified compound
from the corresponding calibration curve.

Recovery experiments were run on untreated control samples of plant material, soil and water,
fortified with phthalimides at different levels. The recoveries are given in the Table. All values
presented in the Table represent the means from 2 to 7 single experiments.
The recoveries from plant material, fortified at levels of 0.05 to 1 mg/kg (for wheat green
matter, 0.05 to 10 mg/kg), ranged from 70 to 108% and averaged 90%. The limit of detection
was approx. 0.01 to 0.02 mg/kg, depending on the plant material, and the limit of determina-
tion was approx. 0.05 mg/kg.
The recoveries from soil samples, fortified at levels of 0.05 to 1 mg/kg, ranged from 68 to
104% and averaged 85%. This limit of detection was approx. 0.01 mg/kg, and the limit of
determination was approx. 0.05 mg/kg.
The recoveries from ground and tap waters, fortified at levels of 0.5 to 100 ng/1, ranged
from 70 to 110% and averaged 92%. The routine limit of determination was approx. 0.1
Table. Percent recoveries from plant material, soil and water, fortified with phthalimides (means of 2 7
experiments).
Analytical Added fol Q ^ ^ Ditalim- phosmet

material mg/kg fos
Apples 0.05-1 93 91 74 90 92 84
Grapes 0.05-1 98 100 92 82 96 91
Lettuce 0.05-1 98 91 112 93 96 99
Potatoes 0.05-1 71 80 74 73 78 73
Wheat
Green matter 0.05-10 102 91 93 87 91 93
Grains 0.1-1 92 76 75 94 91 88
Soil 0.05-1 72 103 58 93 93 75
Water 0.0005-0.1*) 99 96 95 76 94 94
*) Equivalent to 0.5-100 M,g/1.

The residue R, expressed in mg/kg, of an identified phthalimide is calculated from the follow-
ing equation:
r _ w A -v E x -v R 2 -v E n d
V R1 -V R3 -V r G
Method S 26 365
where
VEx = total volume of organic phase after addition of hexane-acetone mixture to filtered
extract from plant sample in 6.1.1 (in ml)
VR2 = volume of eluting mixture used to take up the residue from 6.2.1 (in ml)
VR3 = portion of volume VR2 injected for gel permeation chromatography (volume of
V4 = portion of volume VEnd injected into gas chromatograph (in ul)
WA = amount of identified phthalimide for Vj read from calibration curve (in ng)
8 Important points
A thermionic phosphorus/nitrogen-specific detector is also suitable for the final gas-chroma-

tographic determination. In this case, acetone should be used to prepare the phthalimide
standard solutions.
After a number of measurements, low recoveries for captafol, captan and folpet can be en-
countered. In these cases, results can be improved by shortening the inlet end of the capillary
column by a few centimeters.
The recoveries of dialifos from soil were considerably lower than from other analytical
materials, averaging only 58%.
9 References
No data
10 Authors
Nolting, H. Kohle, J. Siebers and M Blacha-Puller
Part 5
Multiple Pesticide Residue
Analytical Methods for Water
Phenoxyalkanoic Acid Herbicides w4
Water Gas-chromatographic
determination
1 Introduction
The method permits the identification and quantitative determination of the residues of 2,4-D,
dichlorprop (2,4-DP), MCPA, MCPB, mecoprop (CMPP, MCPP), and 2,4,5-T. It is based on
the analysis of the derivatives, which are obtained by nitration of the compounds, followed
by esterification with methanol. The nitro groups greatly enhance the response of the electron
capture detector to the phenoxyalkanoic acid methyl esters. The gas-chromatographic column
used permits the separation of all six derivatives.
2 Outline of method
The phenoxyalkanoic acid residues are extracted from water with ethyl acetate. Derivatization
with nitrating acid and esterification with methanol + sulphuric acid is followed by chroma-
tographic cleanup on an aluminium oxide column. The derivatives formed are shown in the
Table. They are determined by electron capture gas chromatography.
3 Apparatus
Round-bottomed flasks, 2-1, 500-ml and 100-ml
Microsyringe, 10-ul
4 Reagents
Benzene, dist.
Chloroform, dist.
Ethyl acetate, dist.
Herbicide standard solutions: 1 and 10 ng/ml of each phenoxyalkanoic acid in methanol
370 Method W 4
Table. Nitro derivatives of the phenoxyalkanoic acid methyl esters.
Parent Chemical name Melting

Structural formula
compound of derivative point (C)
N0 2
2,4-D Methyl (2,4-dichloro-3,6-dinitro- /T / 85
phenoxy)acetate C l - f V-O-CH 2 -C-OCH 3
>=<
O2N Cl
CH,
f/ I //
Dichlorprop Methyl 2-(2,4-dichloro-3,6-dinitro- Cl V - O-CHC-OCH
O- 68
phenoxy)propionate
ON
X Cl
2
3
O2N NO 2
2
MCPA Methyl (4-chloro-2-methyl-5,6- \ / o 98
dinitrophenoxy) acetate C l \ \ - O - CH 2 - C - OCH3
CH3
O2N N0 2
MCPB Methyl 4-(4-chloro-2-methyl-5,6- cl (( y O(CH2)3COCH3 52
dinitrophenoxy) butyrate
CH3
Mecoprop Methyl 2-(4-chloro-2-methyl-5,6- - C H - C-OCH3 <30

dinitrophenoxy) propionate
2,4,5-T Methyl (2,4,5-trichloro-6-nitro- 78

V V-O-CH 2 -C-OCH 3
phenoxy)acetate
Derivative standard solutions: Run a mixture of 1 ml each of the herbicide standard solutions
(e.g. 10 ng/ml) through the method from 6.2 (nitration) to and including 6.4. Serially dilute
the resulting benzene solution to yield derivative standard solutions containing 25 to
250 ng/ml of phenoxyalkanoic acids equivalents
Sulphuric acid, 3 mol/1 H2SO4 p.a.
Sodium hydroxide solution, 10 mol/1 NaOH p.a.
Nitrating acid: Sulphuric acid, p.a., cone. + nitric acid, fuming (sp.gr. = 1.52) 9:1 v/v
Methylating mixture: Methanol p.a. + sulphuric acid, p.a., cone. 9:1 v/v. Prepare fresh daily
Sodium hydrogen carbonate solution, 4 g/100 ml NaHCO3 p.a.
Method W 4 371
Aluminium oxide, activity grade IV: To 90 g aluminium oxide 90 basic, activity grade I (Merck
No. 1076), in a 300-ml Erlenmeyer flask (with ground joint), add 10 ml water dropwise from
a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake
vigorously until all lumps have disappeared, and then store in a tightly stoppered container
for at least 2 h
Quartz wool

be stored in half-full 2-1 bottles at -20C, lying on their sides in order to prevent the bottles
6 Procedure
6.1 Extraction
Place 1 1 of the water sample (G) in a 2-1 round-bottomed flask, add 5 ml sodium hydroxide
solution (pH 12-13), and concentrate to a volume of approx. 100 ml in a rotary evaporator
(approx. 70 C bath temperature). Acidify with 30 ml sulphuric acid and add 30 g sodium
chloride. Allow the salt to dissolve, transfer the solution into a 500-ml separatory funnel, and
shake three times with 100-ml portions of ethyl acetate. Filter the ethyl acetate phases suc-
cessively through sodium sulphate into a 500-ml round-bottomed flask, and rotary-evaporate
to near dryness (approx. 40 C bath temperature). Transfer the concentrated ethyl acetate solu-
tion quantitatively into a 100-ml round bottomed flask and rotary-evaporate to dryness.
6.2 Nitration
Add 2 ml nitrating acid to the residue derived from 6.1 in the 100-ml flask, while carefully
rotating the flask to wet its walls, and allow to stand for 2 min. Carefully dilute with 20 ml
water, transfer into a 100-ml separatory funnel, and shake twice with 20-ml portions of
chloroform. Filter the chloroform phases successively through sodium sulphate into a 100-ml
round-bottomed flask and wash the filter with a little chloroform. Rotary-evaporate the
filtrate to dryness.
6.3 Methylation
To the residue derived from 6.2, add 5 ml methylating mixture, and allow to stand with occa-
sional swirling at room temperature for 10 min. Add 15 ml water and extract with 10 ml
benzene (VEx). Separate the benzene phase, wash it with 15 ml sodium hydrogen carbonate
solution, and dry on sodium sulphate. Rotary-evaporate an aliquot of 5.0 ml (VR1) of the
dried solution to approx. 1 ml.
372 Method W 4

Insert a quartz wool plug into the bottom of a chromatographic tube. Half-fill the tube with
benzene, then add 1 g of the aluminium oxide. Top the column with an approx. 0.5-cm layer
of sodium sulphate, and drain the benzene until the column packing is covered to a depth of
1 mm.
Transfer the concentrated benzene extract derived from 6.3 onto the column and rinse the
flask three times with 1-ml portions of benzene. Allow the rinsings to percolate into the col-
umn packing each time, collect the eluate in a 10-ml volumetric flask, and continue to elute
with benzene until the flask is filled to the mark (VEnd).

Inject an aliquot, e.g. 2 ul (Vj), of the solution derived from 6.4 into the gas chromatograph.
Gas chromatograph Varian Aerograph 1440
Column Glass, 2.5 mm i.d., 2.6 m long; packed with 1% OV-17
+ 2% OV-210 on Gas Chrom Q, 100-120 mesh
Detector Sc3H electron capture detector
Temperature 220 C
Attenuation 4-10"9
Retention times for
the nitro derivatives of
2,4,5-T methyl ester 3 min 12 s
dichlorprop methyl ester 5 min 42 s
mecoprop methyl ester 6 min 30 s
2,4-D methyl ester 7 min 12 s
MCPA methyl ester 8 min 48 s
MCPB methyl ester 20 min 12 s
-I
-2 o
CO c 5 2
CSl" 5 EP4 ^
1
Ir
CD
k o
Time Time
Chromatogram 1. Control sample of leaching water fortified with 2,4,5-T Chromatogram 2. Control sample of leaching water; 2 \i\ injected,
(0.5 ixg/1); dichlorprop, mecoprop, 2,4-D and MCPA (1 pa.g/1 each); and MCPB equivalent to 0.1 ml water.
(2 ng/1); 2 jn-1 injected.
374 Method W 4
7 Evaluation
7.1 Method
Inject 2 \i\ of each derivative standard solution into the gas chromatograph. Plot the heights
of the peaks obtained vs. pg of the corresponding phenoxyalkanoic acid equivalent. Also in-
ject 2-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solu-
tions, read the appropriate amount of phenoxyalkanoic acid equivalent from the correspond-
ing calibration curve.

The recoveries from untreated control samples, fortified with the phenoxyalkanoic acids at
levels of 0.5 to 10 jxg/1, ranged from 70 to 90%. The routine limit of determination was
0.5 n.g/1.

The residue R, expressed in u.g/1, of an identified phenoxyalkanoic acid is calculated from the
following equation:
R _ WA-VEx-VEnd
VR1VrG
where
G = sample volume (in ml)
VEx = volume of benzene used to extract the phenoxyalkanoic esters from the diluted
methylating mixture in 6.3 (in ml)
VR1 = portion of volume VEx used for further processing (in ml)
WA = amount of identified phenoxyalkanoic acid equivalent for Vj read from calibration
curve (in pg)
8 Important points
The solutions of the methyl esters of the nitrated phenoxyalkanoic acids are sensitive to light
and must, therefore, be kept in the dark.
Experiments with the following phenoxyalkanoic acid esters have shown that they are
hydrolyzed during the procedure in step 6.1, so they can be included in the method: 2,4-D
methyl and isooctyl esters, MCPA isooctyl ester, and 2,4,5-T methyl and isooctyl esters.
Method W 4 375
9 Reference
C.A. Bache, D.J. Lisk and M.A. Loos, Electron affinity residue determination of nitrated
MCP, MCPB, and NAA, J. Assoc. Off. Agric. Chem. 47, 348-352 (1964).
10 Author
BASF, Agricultural Research Station, Limburgerhof, N. Drescher
Fungicides w5
determination
1 Introduction
The method permits the identification and quantitative determination of the residues of fungi-
cides (see Table on p. 379) with different chemical structures in water samples of different
degrees of purity, using gel permeation chromatography as the essential cleanup step. The
compounds are determined by gas chromatography using various specific detectors.
2 Outline of method
The fungicide residues are extracted from water samples with dichloromethane. The solvent
is evaporated, and the residue is dissolved in a cyclohexane-ethyl acetate mixture and cleaned
up by gel permeation chromatography on the polystyrene gel Bio-Beads S-X3. The individual
compounds are identified and quantitatively determined by gas chromatography, using either
an electron capture, or a thermionic, or a flame photometric detector.
3 Apparatus
Separatory funnels, 1-1 and 250-ml, with ground joints
Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Ana-
lytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1)
Gas chromatographs equipped with electron capture detector, thermionic nitrogen and phos-
phorus-specific detector, and sulphur-specific flame photometric detector
Microsyringe, 10-ul
4 Reagents
378 Method W 5

Fungicide standard solutions: CM-20 |ng/ml of each compound in ethyl acetate or acetone
Glass wool
Hydrogen 5.0 (< 99.999 vol. %)
Nitrogen 4.6 (< 99.996 vol. %)

6 Procedure
6.1 Extraction
Extract 400 ml water (G) in a 1-1 separatory funnel three times with 200-ml portions of
dichloromethane. If only 100-ml water samples are available, reduce the volumes of
dichloromethane to 100, 50, and 50 ml. Filter the organic phases successively through a glass
funnel fitted with a cottonwool plug, and containing an approx. 3-cm layer of sodium sul-
phate, into a round-bottomed flask. Wash the sodium sulphate three times with 25-ml por-
tions of dichloromethane, and rotary-evaporate the combined filtrates to dryness.

Transfer the residue derived from 6.1 into a test tube, using a total of 10 ml eluting mixture
(VR1) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR2) of the
gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation
chromatograph at the eluting conditions determined beforehand with fungicide standard solu-
tions (each approx. 20 |ig/ml); cf. Cleanup Method 6, pp. 75 ff, Vol. 1.
The following elution volumes were determined for the individual compounds on Bio-Beads
S-X3 polystyrene gel, using the eluting mixture as eluant, pumped at a flow rate of
5.0 ml/min.
Method W 5 379
_ , Elution volume
Compound ,
Anthr aquinone 160 -190
Bitertanol 100-130
Captafol 120-160
Captan 120-160
Chinomethionat 170-190
Chlor othalonil 140 -170
Dichlofluanid incl. metabolite DMSA 120-150
Fluotrimazole 100-140
Fuberidazole 120-140
Imazalil 120-150
Rabenzazole 120-160
Tolylfluanid incl. metabolite DMST 120-150
Triadimefon 100-130
Triadimenol 100-130
Triazoxide 160-190
Check the elution volumes every 500 samples, and determine anew whenever a new column
is used.
For analysis of all compounds mentioned above, collect the total elution volume from 100
to 190 ml in a 250-ml round-bottomed flask. For analysis of an individual compound, collect
only the corresponding fraction in a 100-ml round-bottomed flask. Rotary-evaporate the
eluate to dryness. Dissolve the residue in an appropriate volume of ethyl acetate or acetone
(VEnd) e-g- 2 or 5 ml, and transfer the solution into a test tube.

Inject an aliquot of the solution derived from 6.2 (Vj) into the gas chromatograph, followed by
the same volume of the corresponding standard solution. Repeat each injection as a control.
6.3.1 Anthraquinone, captafol, captan, chinomethionat, chlorothalonil
Column 2 Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE-30
63
Temperature 250 C
Attenuation 128
380 Method W 5
Column 1 Column 2
Retention times for
chlorothalonil 2 min 54 s 2 min
anthraquinone 4 min 18 s 3 min 24 s
chinomethionat 5 min 36 s 5 min 48 s
captan 6 min 18 s 4 min 12 s
captafol
capiaiui 18
10 min
iiiiii 12 s
iz, & column
i/Uiuiiiii not suitable
nut Minauic
Chromatogram 1 gives an example of the separation achieved on column 1

6.3.2 Bitertanol, fluotrimazole, fuberidazole, imazalil, rabenzazole, triadimefon, triadimenol,
triazoxide
Column 1 same as in 6.3.1, column 1
Column 2 same as in 6.3.1, column 2
Column 3 Glass, 3 mm i.d., 0.9 m long; packed with 5% Car-
bowax 20M on Chromosorb W-HP, 100-120 mesh
Detector Thermionic phosphorus/nitrogen-specific detector
Gas flow rates Hydrogen, 4.5 ml/min
Air, 175 ml/min
Attenuation 10 " u
Column 1 Column 2 Column 3
Injection port temperature 280 C 280 C 250 C
Column temperature 220 C 200 C 185 C
Detector temperature 350 C 350 C 250 C
Carrier gas flow rate Nitrogen, Nitrogen, Nitrogen,
40 ml/min 40 ml/min 30 ml/min
Retention times for
fuberidazole 2 min 30 s 2 min 12 s 17 min 30 s
rabenzazole 2 min 36 s 3 min 6 min 48 s
triadimefon 2 min 36 s 3 min 3 min 42 s
triadimenol 3 min 18 s 3 min 48 s 8 min 12 s
imazalil 4 min 54 s 5 min 18 s 7 min 30 s
fluotrimazole 8 min 42 s 9 min 48 s column not suitable
triazoxide 13 min 12 s 10 min 12 s 13 min 36 s
bitertanol 20 min 42 s 21 min column not suitable
Chromatograms 2, 3 and 4 give examples of the separations achieved.
6.3.3 Dichlofluanid, tolylfluanid, and their metabolites DMSA and DMST
Gas chromatograph Tracor 560
Column 4 Glass, 2 mm i.d., 1.2 m long; packed with 5% DC-200
on Gas Chrom Q, 80-100 mesh
Detector Flame photometric detector equipped with 394-nm sul-
phur filter
Temperature 200 C
Method W 5 381

Hydrogen, 50 ml/min
Air, 100 ml/min
Attenuation 16- 10
Retention times for
DMSA 2 min
DMST 3 min
tolylfluanid
LV/1J111UU111W S 9 min 12 s
111111 J.Z* O
Chromatogram 5 gives an example of the separation achieved.
IE
IBI5
I II
I I I I I I I I I
6 4 2 0 0 2 4 6 8 10 12 14 16
Chromatogram 1. Standard mixture representing 0.2 ng anthraquinone, 0.05 ng captan, 0.02 ng

chinomethionat and 0.02 ng chlorothalonil on column 1, conditions as described in 6.3.1.
Chromatogram 2. Standard mixture representing 25 ng fluotrimazole, 12.5 ng fuberidazole, 25 ng

imazalil, 12.5 ng rabenzazole, 12.5 ng triadimefon, 25 ng triadimenol and 25 ng triazoxide on column 1.
Attenuation 8 10 ~ n , other conditions as described in 6.3.2.
382 Method W 5
I 111!
/I
11
I I I I I I I I
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 16 18 20
Chromatogram 3. Standard mixture representing 12.5 ng fluotrimazole, 12.5 ng fuberidazole, 25 ng

imazalil, 12.5 ng rabenzazole, 12.5 ng triadimefon, 25 ng triadimenol and 25 ng triazoxide on column 2.
Attenuation 16- 10 ~ n , other conditions as described in 6.3.2.
Chromatogram 4. Standard mixture, same as shown in Chromatogram 3, on column 3. Attenuation

16 10 " n , other conditions as described in 6.3.2.
Method W 5 383
i- <
i/> to
S
12 10 8 6 4 2>
Chromatogram 5. Standard mixture representing 2 ng each of dichlofluanid, DMSA, tolylfluanid and

DMST on column 4, conditions as described in 6.3.3.
7 Evaluation
7.1 Method
ing them with the peak areas obtained for the standard solutions. If the peaks are well sepa-
rated and symmetrical, quantitation can be performed using peak heights. Equal volumes of
the sample solutions and the standard solutions should be injected; additionally, the peaks

Recovery experiments were run on untreated control samples (mostly leaching water as speci-
fied by BBA-Richtlinie IV/4-2 (1987), Braunschweig), fortified with the compounds at levels
of 0.005 to 0.5 mg/1 (added in 1 to 2 ml acetone or ethyl acetate). The recoveries ranged from
85 to 114% for all compounds tested, see Table. Experiments with tap, well, lysimeter, and
drainage waters as well as water used for fish toxicity studies gave similar results.
The routine limits of determination for individual compounds were in the range of 0.001
to 0.01 mg/1, depending on the detector used. As a rule, they were 0.01 mg/1 for the flame pho-
384 Method W 5
tometric detector, 0.005 mg/1 for the thermionic detector, and 0.001 mg/1 for the electron cap-
ture detector. At the routine limits of determination, no blank values were observed for the
different control waters.
Table. Percent recoveries from water fortified with the fungicides; results from duplicate experiments.
Added
Compound Recovery
mg/1
Anthraquinone 0.005 90-108 b )

0.5 94- 97
Bitertanol 0.005 91-114 b >
0.01 96-105
0.5 104
Captafol 0.005 102-103
0.01 95-107
0.1 105-109
Captan 0.005 93-103 a >
0.01 95-100
0.05 96- 99
Chinomethionat 0.005 92- 94
0.014 88
0.14 84- 86
Chlorothalonil 0.005 9 1 - 93
0.01 9 3 - 98
Dichlofluanid 0.01 100-102
DMSA 0.01 100
Fluotrimazole 0.005 96-106
0.05 112-116
Fuberidazole 0.005 89-108 b >
0.05 96-102
Imazalil 0.005 92-113 a>
0.01 94- 96
Rabenzazole 0.005 98-100
0.01 103-106
Tolylfluanid 0.01 100-101
DMST 0.01 96
0.005 88-108 c >
Triadimefon 0.01 97-112
0.005 93-103 b >
Triadimenol 0.01 94
0.01 9 5 - 97
Triazoxide
a)
= 4 recovery experiments,
b)
= 6 recovery experiments,
c)
= 8 recovery experiments.
Method W 5 385

The residue R, expressed in mg/1, of an identified fungicide is calculated from the following
equation:
F A -V R 1 -V E n d -W S t
Fs,-VR2-VrG
where
VR1 = volume of solution from 6.1 prepared for gel permeation chromatography (in ml)
VR2 = portion of volume VR1 injected for gel permeation chromatography (volume of
WSt = amount of fungicide injected with standard solution (in ng)
FA = peak area obtained from Vj (in mm 2 )
F St = peak area obtained from WSt (in mm 2 )
8 Important points
If phase separation is hindered by emulsions formed during the extraction of the water sam-
ples with dichloromethane (mainly with leaching water from Standard Soil 2.2, cf. BBA-
Richtlinie IV/4-2 (1987), Braunschweig), the total content of the separatory funnel should be
filtered through a loose glass wool plug. After transferring the filtrate back to the separatory
funnel, the separation of the phases is accomplished. During the following two extraction
steps, emulsions did not occur any longer.
The solutions of fuberidazole, imazalil and rabenzazole are sensitive to light and must,
therefore, be kept in the dark. Also during analysis, the solutions should be protected from
light, and all glassware should be wrapped with aluminium foil.
If optimum peak separation can not be achieved using the conditions given in 6.3, quantita-
tion of individual compounds can be facilitated by adjusting e.g. the oven temperature.
When using the thermionic detector for the identification of the fungicides, two columns
of different polarity are required (column 1 or 2, and column 3), because, e.g., rabenzazole
and triadimefon are only separated on column 3.
On the gas-chromatographic columns described here neither the two diastereoisomers of
bitertanol nor those of triadimenol will be separated; one peak will be obtained with a shoul-
der appearing to a greater or lesser degree.
To prevent decomposition of captan and captafol, it is recommended to remove the glass
wool from the inlet of the gas-chromatographic columns. When the peaks obtained for the
standard solutions become reduced in size, the first few cm of the column should be repacked.
386 Method W 5
An alternative to packed columns are fused silica capillary columns, e.g. 0.3 mm i.d., 15 m
long, coated with OV-1, film thickness 0.1-0.15 urn, crossbond (Mega No. 26060300), column
temperature programmed to rise from 100 to 160 C with maximum heating rate, and at
4 C/min from 160 to 220 C. Captan and captafol tend to decompose on columns that have
been in use for some time. When decomposition is observed, shorten the inlet end of the capil-
lary column by a few cm.
9 References
R. Brennecke and K. Vogeler, Methode zur gaschromatographischen Bestimmung von
Riickstanden verschiedener Fungizide in Wasser, Pflanzenschutz-Nachr. 37, 44-65 (1984).
W. Specht and M. Tillkes, Gaschromatographische Bestimmung von Riickstanden an
Pflanzenbehandlungsmitteln na.ch Clean-up iiber Gelchromatographie und Mini-Kieselgel-
10 Authors
R. Brennecke and K. Vogeler
Organochlorine Insecticides w6
determination
1 Introduction
The method permits the identification and quantitative determination of the residues of the
organochlorine insecticides given in the Table and their most important analogues and metab-
olites in water by capillary gas chromatography. Tap water and ground water samples with a
low content of organic carbon can be analyzed without cleanup by a miniaturized method.
(For general advice on micro methods and equipment see pp. 29 ff, Vol. 1.)
2 Outline of method
The organochlorine insecticide residues are extracted from water with a mixture of diisopropyl
ether and n-hexane containing aldrin or e-HCH as internal standard. 20 ml of water and
200 \i\ solvent mixture are sufficient for this purpose. The compounds are determined directly
from this extract by electron capture gas chromatography using a fused silica capillary
column.
3 Apparatus
Glass syringe, 20-ml, with Luer-lock fitting, equipped with PTFE tube, glass cap and glass
capillary; see Figure
Laboratory mechanical shaker, overhead tumbler type
Lifting platform ("lab jack")
Sample vials, 0.5-ml, with PTFE coated septa and screw or crimp caps
388 Method W 6
I I Glass cap
PTFE tube
Glass capillary
Figure. All-glass syringe with equipment for extracting water samples.
4 Reagents
Note: The prerequisite for trace determinations of organochlorine insecticide residues is the
absolute purity of the solvents. Check each new lot carefully: Reduce a portion of the solvent
to a tenth of its volume and inject into the gas chromatograph under the conditions given in
6.2. If interfering peaks are observed, fractionally distil the solvent for purification, using a
Snyder column. Discard the first 10% of the distillate, and stop distillation when a total of
80% of the solvent has been distilled. Check purity of the distillate as described above; if re-
quired, repeat distillation
Diisopropyl ether, p.a.
Solvent mixture: diisopropyl ether + n-hexane 53 :47 v/v. Distil as described in the Note, and
collect the constant temperature azeotrope
Extraction mixture: Add 1 ml of internal standard solution 1 or internal standard solution 2
to 1 1 of the solvent mixture (corresponding to 50 ng/ml internal standard)
Compound standard solutions: 100 ng/ml of each insecticide in extraction mixture
Internal standard solution 1:50 ng/ml e-HCH in solvent mixture
Internal standard solution 2:50 ng/ml aldrin in solvent mixture
Helium, re-purified
Method W 6 389

6 Procedure
6.1 Extraction
Transfer 20 ml of the water sample (G) into the glass syringe, close with the glass cap and
clamp to a stand in vertical position (tip upward). Remove the cap and suck in approx. 0.5 ml
of air. Inject 200 JLXI extraction mixture (VEx), using the 250-ul microsyringe, through the tip
of the glass syringe into the water sample. Close the syringe with the cap and shake it well
by hand for 3 min. (With larger series of analyses use a suitably equipped mechanical shaker
and shake for 15 min.) Next clamp the syringe to the stand again so that the piston rests on
the platform of the lab jack. Substitute the glass capillary for the glass cap when the phases
have separated, transfer the organic extract to a sample vial by carefully raising the platform,
and immediately close the vial.

Inject 1 \i\ of the extract derived from 6.1 into the gas chromatograph.
Gas chromatograph 1 Hewlett-Packard 5890
Column Fused silica capillary DB-1, 0.245 mm i.d., 30 m long;
film thickness 0.25 urn (J & W Scientific)
Column temperature 2 min isothermal at 100 C, programmed to rise at
250 C for 12 min
63
Temperature 280 C
Gas flow rates Argon-methane carrier, 1 ml/min
Split ratio 1:10
Integrator Hewlett-Packard 3390 A
Retention times see Table
Gas chromatograph 2 Siemens Sichromat 2
Column Fused silica capillary DB-5, 0.32 mm i.d., 25 m long;
film thickness 0.25 u.m (J & W Scientific)
390 Method W 6

63
Temperature 280 C
Split ratio 1:15
Attenuation 8
Integrator Sichromat
Retention times see Table
Table. Relative retention times (RRT) of the organochlorine insecticides, their analogues and metabolites,
relative to e-HCH and to aldrin.
Gas chromatograph 1 Gas chromatograph 2

Compound 1IRT RRT RRT RRT
E-HCH Aldrin e-HCH Aldrin
a-HCH 0.917 0.807 0.930 0.849

(5-HCH 0.943 0.830 1 .036 0.946
Hexachlorobenzene 0.953 0.839 0.905 0.827
Y-HCH 0.966 0.850 0.984 0.899
5-HCH 0.983 0.865 1 .062 0.970
E-HCH .000 *> 0.880 1 .000 3> 0.914
Heptachlor .090 0.960 1L.058 0.967
Aldrin .137 11.000 2> 1 .095 1.0004>
Heptachlor epoxide .194 11.051 11.175 1.074
o,p'-DDE .233 ]1.085 11.203 1.099
a-Endosulfan 1.238 ]1.090 ]1.207 1.103
Dieldrin 1.276 1.123 1.252 1.143
p,p'-DDE 1.282 1.128 1.247 1.140
Endrin 1.296 1.141 1.276 1.165
fi-Endosulfan 1.301 1.145 1.309 1.197
o,p'-DDT 1.334 1.174 1.303 1.190
p,p'-DDT 1.377 1.212 1.355 1.238
Methoxychlor 1.449 1.275 1.431 1.308
Absolute retention times
> 13 min 10 s
2
> 14 min 58 s
3
> 15 min 03 s
4
> 16 min 29 s
Method W 6 391
7 Evaluation
7.1 Method
The chromatograms obtained are evaluated using an internal standard (s-HCH or aldrin). The
compound used as internal standard must not be present in the analytical sample. This must
be confirmed (in a preliminary examination) by injecting the extract of a parallel sample, using
another compound as internal standard or without the addition of a standard.
Quantitation is performed by the calibration technique. Prepare a calibration curve as fol-
lows. Pipet 1.0, 3.0 and 5.0 ml of a compound standard solution into 10-ml volumetric flasks
and make up to the marks with extraction mixture. Inject 1 u.1 of each of these solutions (cor-
responding to 0.01, 0.03 and 0.05 ng of insecticide and 0.05 ng of internal standard) into the
gas chromatograph. Plot the peak areas obtained vs. the concentration of insecticide injected
(in ng/îl). In addition, determine the peak areas for the internal standard. Use the same con-
centration of internal standard in both the preparation of the calibration curve and the
analysis.
Identification is achieved by determination of the retention times relative to the internal
standard. Further information about the identity of the compound can be obtained by
GC/MS. 10 [ig/1 of insecticide in the analytical sample is required in order to obtain a com-
plete mass spectrum. For lower concentrations, the SIM (selected ion monitoring) technique
is recommended, which although not furnishing the complete spectrum information is
more sensitive.

The recoveries from tap water, fortified with organochlorine insecticides at levels of 0.02 to
1 M<g/1, ranged from 80 to 110%. The routine limit of determination was 0.05 ng/1.

The residue R, expressed in \ig/\, of an identified insecticide is calculated from the following
equation:
R = W A -F 0 V E x
F,G
where
VEx = volume of extraction mixture added (in \i\)
WA = concentration of organochlorine insecticide read from calibration curve (in ng/|il)
Fo = average peak area of internal standard used for calibration curve (in mm2 or inte-
grator counts)
Ft = peak area of internal standard from the analysis (in mm2 or integrator counts)
392 Method W 6
8 Important points
Hexane alone cannot be used for the extraction because the phases separate incompletely and
very slowly, and the extract tends to adhere to the glass syringe walls.
9 References
J.F.J. van Rensburg and A.J. Hassett, A low-volume liquid-liquid extraction technique, J. High
Resol. Chromatogr. Chromatogr. Commun. 5, 574-576 (1982).
L. Weil and K.-E. Quentin, Zur Analytik der Pestizide im Wasser, III. Mitteilung: Extraktion
der chlorierten Kohlenwasserstoffe aus dem Wasser, Gas- und Wasserfach 112, 184-185 (1971).
10 Author
Institute of Water Chemistry and Chemical Balneology, Technical University of Munich,
L. Weil
Phenoxyalkanoic Acid Herbicides wi
determination
1 Introduction
The method permits the identification and quantitative determination of the residues of 2,4-D,
2,4-DB, dichlorprop (2,4-DP), fenoprop (2,4,5-TP), MCPA, MCPB, mecoprop (CMPP,
MCPP), and 2,4,5-T, which may be present as acids, salts or esters. By derivatization to the
trichloroethyl esters, it is possible to determine even compounds containing only one chlorine
atom with a high degree of sensitivity.
2 Outline of method
The phenoxyalkanoic acid residues are extracted from water with dichloromethane. After ad-
dition of sodium hydroxide solution the dichloromethane is distilled off, whereby any esters
present are hydrolyzed. Neutral and basic co-extractives are removed by acidic and basic parti-
tion steps. The phenoxyalkanoic acids are then derivatized with trichloroethanol. The result-
ing trichloroethyl esters are cleaned up on a silica gel column and are determined by electron
capture gas chromatography using a fused silica capillary column.
3 Apparatus
Separatory funnels, 2.5-1 and 250-ml
Rotary vacuum evaporator, 30 or 40 C bath temperature
Glass syringe, 1-ml, gas-tight
Chromatographic tube, 1 cm i.d., 30 cm long
Microsyringe, 10-pil
394 Method W 7
4 Reagents
Acetone, p.a., fractionally distilled
Dichloromethane, p.a., fractionally distilled
2,2,4-Trimethyl pentane (isooctane), p.a., fractionally distilled
Toluene, p.a., fractionally distilled
Water, bi-distilled
Eluting mixture: isooctane + toluene 1:1 v/v
Compound standard solution for fortification experiments and for preparing the derivative
standard solution: 1 ng/ml of each compound (as acid) in acetone
Derivative standard solution (equivalent to 100 ng/ml of each compound) in eluting mixture:
The derivatization of the compound standard solution is performed parallel to that of the
sample solutions. Pipet 2 ml of the compound standard solution (equivalent to 2 jxg of each
acid) into a test tube. Evaporate the solvent, esterify and clean up as described in 6.4 and 6.5.
Make up the column eluate to 20 ml
Sulphuric acid, ultra pure, cone. (Merck No. 714)
Sulphuric acid, 3 mol/1 H2SO4 ultra pure. Prepare in bi-distilled water, and extract twice with
isooctane before use
Sodium hydroxide solution, 10 mol/1 NaOH p.a. Prepare in bi-distilled water, and extract
twice with isooctane before use
Sodium hydrogen carbonate solution, 10 g/100 ml NaHCO3 p.a. Prepare in bi-distilled water,
and extract twice with isooctane before use
Trifluoroacetic anhydride, 99% (Janssen No. 14.781.37), fractionally distilled
2,2,2-Trichloroethanol, reagent grade (Merck No. 808610), fractionally distilled
Sodium sulphate, p.a., anhydrous, exhaustively extracted with toluene
Silica gel, deactivated with 5% water: Heat silica gel 60, 0.063-0.200 mm (Merck No. 7734)
overnight at 200 C. Allow to cool in a desiccator, and store in a tightly stoppered container.
To 95 g dried silica gel in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml of bi-dis-
tilled water dropwise from a burette, with continuous swirling. Immediately stopper flask with
2 h on a mechanical shaker, and then store in a tightly stoppered container. Before use, check
the separation efficiency with derivative standard solution
Universal indicator paper (pH 2-10)
Glass wool, high purity, silanized (Serva)
Quartz wool, exhaustively extracted with toluene

be stored in half-full 2-1 bottles at -20C, lying on their sides in order to prevent the bottles
Method W 7 395
6 Procedure
6.1 Extraction
Acidify 2 1 of the water sample (G) with 30 ml sulphuric acid (3 mol/1) and shake for 5 min
with 100 ml dichloromethane. Separate the organic phase and extract the water three times
more with 50-ml portions of dichloromethane. Collect the organic phases in a 500-ml
round-bottomed flask and add 20 ml bi-distilled water.
6.2 Hydrolysis
Add 5 ml sodium hydroxide solution to the extract derived from 6.1. Rotary-evaporate to the
aqueous residue with 40C bath temperature; then leave the flask to rotate without suction
for 30 to 35 min at 40 C.

Transfer the aqueous residue derived from 6.2 into a 250-ml separatory funnel, using 50 ml
bi-distilled water to complete the transfer, and extract once with 50 ml dichloromethane with
gentle swirling, avoiding formation of emulsion. Allow the phases to separate and discard the
dichloromethane layer. Acidify the aqueous phase with 10 ml sulphuric acid (3 mol/1), check
the pH, and extract three times with 20-ml portions of dichloromethane, shaking each time
for 5 min. Discard the aqueous phase. Filter the organic phases successively through a
silanized glass wool plug covered with an approx. 10-g layer of sodium sulphate contained in
the glass funnel. Rinse with 40 ml dichloromethane. Rotary-evaporate the combined filtrates
in a 250-ml round-bottomed flask to 1 to 2 ml with 30 C bath temperature.
6.4 Esterification
Transfer the residue derived from 6.3 quantitatively into a test tube, using a few ml of
dichloromethane to complete the transfer. Evaporate the solvent at 30-40 C with a gentle
stream of nitrogen. Add to the residue 5 ul concentrated sulphuric acid and, using gas-tight
glass syringes, 800 ul trifluoroacetic anhydride and 200 ul trichloroethanol. Stopper the test
tube and allow to stand for at least 2 h at room temperature with occasional shaking. Concen-
trate the solution to 0.2-0.3 ml, using a gentle stream of nitrogen (approx. 10 min), then add
10 ml sodium hydrogen carbonate solution. Extract the solution twice with 2-ml portions of
isooctane, shaking each time for 1 min. Separate and combine the organic phases.

Insert a quartz wool plug into the bottom of a chromatographic tube. Half-fill the tube with
isooctane. Trickle in 2 g silica gel, gently tapping the tube walls, and cover with an approx.
1-cm layer of sodium sulphate. Allow the filling to settle; then drain the supernatant solvent
just to the top of the sodium sulphate layer. Quantitatively transfer the isooctane solution de-
rived from 6.3 onto the column and allow to soak in at a rate of 1 to 2 drops per s. Rinse
the column with 15 ml isooctane and discard the eluate. Next elute the derivatives with 19 ml
396 Method W 7
eluting mixture and collect the eluate in a 20-ml volumetric flask. Make up to the mark with
eluting mixture (VEnd).

Inject an aliquot, e.g. 1 \x\ (Vj), of the solution derived from 6.5 into the gas chromatograph.
Column Fused silica capillary, 0.2 mm i.d., 12.5 m long; coated
with dimethyl silicon, cross-linked, film thickness
0.33 urn
next at 6C/min from 180 to 240 C, then isothermal
at 240 C for 5 min
63
Temperature 320 C
Gas flow rates Hydrogen carrier, 2 ml/min
Hydrogen septum purge, 5 ml/min
Split ratio 1:30
Attenuation 24
Recorder Hewlett-Packard 5880 A GC Terminal 1
Injection volume
Retention times for the
trichloroethyl esters of
mecoprop 10 min 45 s
MCPA 11 min 30 s
dichlorprop 12 min 30 s
2,4-D 13 min 8 s
fenoprop 16 min 2 s
2,4,5-T 16 min 58 s
MCPB 17 min 45 s
2,4-DB 19 min 25 s
Method W 7 397
u
a a
o u OH
u o
a HC tl I PQ
a
o<x: PUVA
oa o O i
(M c\T
llll "11
ÎVJ
Chromatogram 1. Standard mixture of the trichloroethyl esters of the eight phenoxyalkanoic acid herbi-
cides, representing 0.05 ng of each acid.
398 Method W 7
Chromatogram 2. Tap water.

* = Impurity.
Method W 7 399
m O C g
CsJ
il
I!
UL
Chromatogram 3. Tap water, fortified with 0.5 |ig/l of each phenoxyalkanoic acid herbicide.
* = Impurity.
7 Evaluation
7.1 Method
Pipet 1.0, 3.0 and 5.0 ml each of the derivative standard solution into 10-ml volumetric flasks
and make up to the marks with eluting mixture. Inject 1 ul of each solution (equivalent to
400 Method W 7
0.01, 0.03 and 0.05 ng phenoxyalkanoic acid) into the gas chromatograph. Plot the peak areas
or heights of the peaks obtained vs. ng of acid equivalents in the calibration solutions injected.
The calibration curves are linear within the above range. Also inject 1-jnl aliquots of the sample
solutions. For the areas or heights of the peaks obtained for these solutions, read the appro-
priate amounts of acid equivalents from the corresponding calibration curve.

The recoveries from tap and ground water samples, fortified with the phenoxyalkanoic acids
at levels of 0.05 to 5 \xg/\, ranged from 70 to 125% and averaged 85%. The routine limit of
determination was 0.05 \xg/\.

The residue R, expressed in M-g/1, of an identified phenoxyalkanoic acid is calculated from the
following equation:
W A V TEnd
R =
V,.G
where
Vj = portion of volume VEnd injected into gas chromatograph (in fil)
WA = amount of identified phenoxyalkanoic acid equivalent for Vj read from calibration
curve (in ng)
8 Important points
The method was tested with tap and ground waters. Blank values, mostly lower than 0.05 (xg/1,
can occur depending on impurities in the water samples. Interfering peaks can also result from
insufficiently pure reagents. In case of doubt, confirm the results by mass spectrometry using
multiple ion detection, in order to avoid false positive results when using this very sensitive
method.
Diclofop is not included in the method because of the unreliability of the recoveries ob-
tained. The retention time for the diclofop trichloroethyl ester was 28 min 6 s, using the condi-
tions given in 6.6.
Method W 7 401
9 References
R.V. Smith and S.L. Tsai, Trichloroethyl esters as derivatives for gas chromatography with
electron capture detection, J. Chromatogr. 61, 29-34 (1971).
S. Mierzwa and S. Witek, Gas-liquid chromatographic method with electron-capture detection
for the determination of residues of some phenoxyacetic acid herbicides in water as their 2,2,2-
trichloroethyl esters, J. Chromatogr. 136, 105-111 (1977).
10 Author
Triazine Herbicides w8
Water High-performance
liquid chromato-
1 Introduction
The method permits the identification and quantitative determination of ametryn, atrazine,
prometryn, propazine, simazine, terbuthylazine, and terbutryn in ground and tap waters.
2 Outline of method
The triazine herbicide residues are extracted from water using the solid phase extraction tech-
nique. The water sample is passed through a disposable cartridge containing RP-18 re-
versed-phase material. The retained residues are eluted with a methanol-dichloromethane mix-
ture and determined by high-performance liquid chromatography (HPLC) using a UV de-
tector.
3 Apparatus
Solid phase extraction manifold, e.g. Vac Elut (Analytichem International No. AI 6000)
Stainless steel tube, 0.8 mm i.d., 1.6 mm o.d., 25 cm long
PTFE tube, 1.5 mm i.d., 3 mm o.d., 50 cm long
Glass syringe, 50-ml, with PTFE Luer-lock fitting
Glass syringe, 1000-ul
4 Reagents
Acetonitrile, for spectroscopy (Merck No. 16)
Dichloromethane, for UV spectroscopy (Fluka No. 66745)
Ethanol p.a., absolute (Merck No. 983)
Methanol, for HPLC (Fluka No. 65541)
Water, for HPLC
Acetonitrile + water mixture: acetonitrile + water for HPLC 2 : 8 v/v
404 Method W 8
Eluting mixture: dichloromethane + methanol 7:3 v/v

Mobile phase: acetonitrile + ammonium acetate solution 45:55 v/v, de-gassed with helium
Triazine stock solutions: 1000 ng/ml of each compound in absolute ethanol (except:
100 ng/ml of simazine in methanol). Transfer 500 \ig of each compound (0.5 or 5.0 ml,
respectively, of the stock solutions) into a 50-ml volumetric flask and make up to the mark
with absolute ethanol. The stock solution mixture then contains 10 ng/ml of each compound
Triazine standard solutions: Pipet 2 ml of the stock solution mixture into a 100-ml volumetric
flask and make up to the mark with acetonitrile-water mixture. The resulting solution contains
0.2 jxg/ml of each compound. Dilute aliquots of this solution further with acetonitrile-water
mixture to obtain solutions containing 0.1, 0.04 and 0.01 fig/ml
Triazine solution for recovery experiments: Pipet 1.0 ml of the stock solution mixture into a
50-ml volumetric flask and make up to the mark with water for HPLC. The resulting solution
contains 0.2 ing/ml of each compound
Ammonium acetate solution, 0.1 mol/1 CH3COONH4 p.a. (Merck No. 1116) in water for
HPLC
RP-18 disposable cartridge: Bond Elut C-18, 500 mg/2.8 ml, including adapter (Analytichem
International No. AI 607303 and AI 636001, respectively)
Helium for gas chromatography (for de-gassing the HPLC mobile phase)

6 Procedure
6.1 Conditioning of the cartridge
Attach the cartridge to the solid phase extraction manifold. Using gentle water jet pump suc-
tion, allow to pass through, first 10 ml methanol, followed by 10 ml water for HPLC, at a
rate of 5-10 ml/min.
6.2 Sample preparation

Measure the volume of the analytical sample (approx. 1 1) (G). Add 2 volumes of methanol
for each 1000 volumes of water.
6.3 Sample delivery

Connect the conditioned cartridge via an adapter to the PTFE tube. Connect the other end
of the PTFE tube to the stainless steel tube, which is dipped in the analytical sample. Pass
the sample, with suction, through the cartridge at a rate of 15-20 ml/min. Remove the PTFE
tube from the cartridge.
Method W 8 405
6.4 Elution
To elute the triazines, pass 30 ml eluting mixture, with suction, through the cartridge, and col-
lect the eluate in a 100-ml pear-shaped flask. Alternatively, connect the cartridge to a 50-ml
glass syringe. Fill the syringe with 30 ml eluting mixture, insert the plunger, force the liquid
through the cartridge, and collect the eluate in a 100-ml pear-shaped flask. Rotary-evaporate
the eluate to dryness.

Dissolve the residue derived from 6.4 in 5.0 ml (VEnd) acetonitrile-water mixture. Inject an ali-
quot of this solution (Vj) into the sample loop of the high-performance liquid chromatograph.
Pump Constant volume pump, model LC 250/1 (Kratos)
Injector Injection valve 70-10 fitted with sample loop 70-11
(Rheodyne)
Column Stainless steel, 4.6 mm i.d., 15 cm long; packed with
LC 8 DB, particle size 3 |um (Supelco No. 58991)
Mobile phase Acetonitrile + ammonium acetate solution 45 :55 v/v,
de-gassed with helium
Flow rate 1 ml/min
Detector UV detector Spectroflow 783 (Kratos), programmable
Wavelength 220 nm
Attenuation Programmed: From start to 6.5 min at 0.07 AUFS,
and from 6.5 min to end of chromatogram 0.04 AUFS
Retention times for
simazine 4 min 30 s
atrazine 5 min 50 s
ametryn 7 min 20 s
propazine 8 min
terbuthylazine 8 min 30 s
prometryn 10 min 30 s
terbutryn 11 min 30 s
406 Method W 8
f iZ~: ; s-'-jo, p 3--*U .. N3 g:~:

B-
. S | ' ' j g '"' * ' ' * ' j g V ' |PH' ) ' ' ' ^ ' " ft i < " " : ' ' ; <| CD ! _^
Chromatogram 1. Standard mixture of seven triazine herbicides plus metolachlor, representing 40 ng of

each compound.
Method W 8 407
"-?.; . . . i .
g:; :: r i.'''.!'
j
. . . . . . . . . . . . . i..:.
* i .
. i . j :
i ja r id) i S:!: ':j
o . ' ; " i5 !
^.!:.oi;.;: ;{; ; .:. |
' '
:::: : [<_ i. i i
I '
1 ' . j : j : :: : . u
4 ~"-L
J ... !J:.
... . .:. . I.1 . j!. . . . . . 1j :
1 i: :
"el , T
1 ' i
1
r 1 '
i { '

i 1
i
1

*
i

; !il i ' - " ^
1. . : . T J
: . T.!
. . ..... . j . .^ . . . . . i W
; / ,
. ; . . I 1'
A
i
-
* f \ ; . ; ! ; : ; ! : : ; j ; . . . ! ... |
: : : : ! ' r: i : !?
; . ; ! : :
Chromatogram 2a. Standard mixture representing 2 ng each of simazine, atrazine, terbuthylazine, ter-
butryn and metolachlor.
Chromatogram 2b. Water for HPLC. Aliquot injected representing 40 ml water.

408 Method W 8
3a!:: :. :|.:.;:.. : :
f. ::::|.::|-i:-
. ^ : .:M: : :-:
.!.- 1 .;:' '7f1
II: ;-$};
-El-
! 5 l o !
:
"SI ::;pj: !:::! t 1' :
7
1 .
1 ;;; j;;;' ^
!:;|;:::
It !
. . . . - !i . . .
Chromatogram 3a. Water for HPLC, fortified with 0.1 jug/l each of simazine, atrazine, terbuthylazine,
terbutryn and metolachlor. Aliquot injected representing 40 ml water or 4 ng of each compound added.
Chromatogram 3b. Tap water. Aliquot injected representing 40 ml water.

Method W 8 409
Chromatogram 4a. Standard mixture representing 2 ng each of simazine, atrazine, ametryn, propazine
and prometryn.
-
. ; : . : : ; i ; ; ; ;
: . : :
1 '
t~T!"Tr~ri""':"
;:: I : '
:
- ::!' i : : :
:::
. : 4*
CO
yL-!
i
i'
I :
Chromatogram 4b. Ground water. Aliquot injected representing 40 ml water.
7 Evaluation
7.1 Method
Inject 200 ul of each triazine standard solution (equivalent to 2, 8, 20 and 40 ng of each
triazine herbicide) into the high-performance liquid chromatograph. Plot the heights of the
peaks obtained vs. ng of each triazine herbicide. Also inject 200-ul aliquots of the sample solu-
tions. For the heights of the peaks obtained for these solutions, read the appropriate amounts
of each triazine herbicide from the corresponding calibration curve.

The recoveries from water for HPLC, fortified with each triazine herbicide at levels of 0.1 and
0.5 ng/1 (using 0.5 or 2.5 ml of the solution for recovery experiments), ranged from 89 to :
410 Method W 8
and averaged 96% with a standard deviation of 4% (see Table). Blanks usually did not occur.
The routine limit of determination was 0.05 ng/1 for each compound.
Table. Percent recoveries from water for HPLC fortified with triazine herbicides.
Triazine herbicide 0 , Added (ng/1) ^
Ametryn 101 94
Atrazine 97, 99, 99, 110 93, 94, 95, 98
Prometryn 102 96
Propazine 100 92
Simazine 89, 92, 96, 98 93, 94, 96, 97
Terbuthylazine 93, 99, 104 92, 95, 97
Terbutryn 92, 93, 98, 99 92, 94, 97, 97

The residue R, expressed in \ig/\, of an identified triazine herbicide is calculated from the fol-
lowing equation:
p wA-vEnd
where
Vj = portion of volume VEnd injected into high-performance liquid chromatograph (vol-
ume of sample loop) (in ul)
WA = amount of triazine herbicide for Vj read from calibration curve (in ng)
8 Important points
UV detectors other than the one indicated in 6.5 may yield routine limits of determination
not as low as reported in 7.2.
Metolachlor can also be determined, under the same conditions, with a routine limit of de-
termination of 0.05 ng/1. Recoveries ranged from 83 to 106% at levels of 0.1 and 0.5 u.g/1.
It is anticipated that other triazine herbicides can also be determined by this method, with
small modifications to the method in individual cases.
Triazine herbicides can be determined by both HPLC and GC. The choice of method can
only be made after comparative experiments. Often only a combination of HPLC and GC can
provide the required separation and allow for the determination of all triazine herbicides in
question. In order to prevent false positive results, HPLC results should be confirmed by GC
and vice versa.
Method W 8 411
9 References
No data
10 Authors
Ciba Geigy AG, Agricultural Division, Basle, Switzerland, G. Formica, C. Giannone and
W. D. Hormann
Desalkyl Metabolites of Chlorotriazine
Herbicides w 13
Water High performance
liquid chromato-
1 Introduction
The method permits the identification and quantitative determination of the following three
chlorotriazine desalkyl metabolites in ground and tap waters:
ci R = C2H5 iso-C 3 H 7 tert-C 4 H 9
Code No. G 28279 G 30033 GS 26379

Trivial name Desethyl- Desethyl- Desethyl-
simazine atrazine terbuthylazine
Melting point (C) 174-175 134-135 143-144
Solubility in water
at 25 C (mg/100 ml) 54 270 48
The method is recommended for its simplicity and low routine limit of determination.
The desalkyl metabolites cannot be determined by Method W 8 of this Manual because they
are only partially retained on the disposable cartridge column. The parent triazine herbicides,
however, cannot be analyzed by Method W 13 because they are lost during the evaporation
of the analytical samples.
2 Outline of method
An aliquot of the analytical sample is concentrated to a few millilitres. The salts present are
precipitated with acetonitrile; subsequently, the acetonitrile is removed. The desalkyl metabo-
lites are determined by high-performance liquid chromatography (HPLC) using a UV detector
and the column switching technique.
3 Apparatus
Rotary vacuum evaporator, 40-50C and 70-80C bath temperatures
414 Method W 13
Centrifuge, with buckets for 10-ml tubes

Centrifuge glass tubes, 10-ml, with polyethylene caps
Pasteur pipets
High-performance liquid chromatograph equipped with UV detector and column switching
device
Glass syringe, 1000-^1
4 Reagents
Acetonitrile, for spectroscopy (Merck No. 16)
Ethanol p.a., absolute (Merck No. 983)
Methanol p.a. (Merck No. 6009)
Water, for HPLC
Mobile phase 1: water for HPLC + acetonitrile 85:15 v/v (for elution of G 28279 and
G 30033)
Mobile phase 2: water for HPLC + acetonitrile 70-60:30-40 v/v (for elution of GS 26379)
Metabolite stock solutions: 1000 ng/ml each of G 30033 and GS 26379, and 100 ng/ml of
G 28279, in absolute ethanol or methanol
Metabolite standard solutions: Mix 1.0 ml of the G 30033 and 10.0 ml of the G 28279 stock
solutions, equivalent to 1000 \xg of each compound. Progressively dilute both, this mixture
and a 1000-jig aliquot of the GS 26379 stock solution, with water for HPLC to yield two series
of standard solutions containing 0.02, 0.01, 0.005 and 0.001 ng/ml each of G 30033 and
G 28279, or GS 26379, respectively
Metabolite solutions for recovery experiments: Mix aliquots of the three stock solutions and
progressively dilute with water for HPLC to obtain two solutions containing 0.05 and
0.01 ng/ml of each compound

6 Procedure
6.1 Sample preparation
Transfer 100 ml of the analytical sample (G) into a tared 500-ml round-bottomed flask and
rotary-evaporate to 2-4 ml with 70-80 C bath temperature; then add water for HPLC to ad-
just the weight of the concentrate to 4.0 g. Using a Pasteur pipet, transfer the solution (or sus-
pension) into a 10-ml centrifuge tube previously washed with 2-3 ml acetonitrile. Rinse the
flask with 4 ml acetonitrile and pipet the rinsings also into the centrifuge tube. Cap with a
polyethylene cap, shake, and centrifuge for 2-4 min at 1000 g.
Method W 13 415
Rinse a tared 100-ml round-bottomed flask with 2-3 ml acetonitrile, discard the rinsings,
and decant the supernatant centrifugate into the flask. Suspend the residue in the centrifuge
tube in 2 ml acetonitrile, cap the tube, and centrifuge again. Combine the clear supernatant
with the first centrifugate and rotary-evaporate with 40-50 C bath temperature to less than
4 g. Then adjust the weight of the solution to 5.0 g by adding water for HPLC. This is equiva-
lent to a volume of 5 ml (VEnd).

Inject an aliquot (Vj) of the solution derived from 6.1 into the sample loop of the high-per-
formance liquid chromatograph.
A scheme of the HPLC column switching system is given in Fig. 1.
Fig. 1. Set-up of apparatus for HPLC determination using column switching.

1, Filling of sample loop; 2, Dosing and elution of column 1; 3, Transfer from column 1 to column 2;
4, Elution of column 2. Positions of the valves: L, load; I, inject.
416 Method W 13
Pumps 1 and 2 Constant volume pumps, model LC 250/1 (Kratos)
Injector Spark Promis programmed automatic injection (Spark
Holland), equipped with a 500-jj.l sample loop in the
injection valve (Rheodyne)
Columns 1 and 2 Stainless steel, 4 mm i.d., 12 cm long; packed with
Nucleosil 100 C18, particle size 5 \\m (Knauer
No. B7-Y76)
Column switching valves MUST (Spark Holland)
Mobile phase 1 Water for HPLC + acetonitrile 85:15 v/v
Mobile phase 2 Water for HPLC + acetonitrile 70-60:30-40 v/v
Flow rates 1 ml/min
Detector UV detector Spectroflow 783 (Kratos), programmable,
equipped for the control of the MUST column switch-
ing valves
Wavelength 220 m
G 28279 G 30033 GS 26379
Mobile phases
(for columns 1 and 2) Mobile phase 1 Mobile phase 1 Mobile phase 2
Retention times 10 min 30 s 20 min 12 s 8 min 30 s
Setting the switching times: The switching times must be set anew before the start of each
analysis series for GS 26379 and for G 28279 plus G 30033. To do this, connect the exit from
column 1 with the detector inlet and inject approx. 500 pi standard solution (0.02 fig/ml) into
the high-performance liquid chromatograph under the conditions given for the appropriate
substance(s). Follow the elution run with the recorder. Determine the switching intervals, i.e.
the times in which the substances are fully eluted.
Typical switching times
for G 28279 and G 30033: Begin switching 5 min
End switching 11 min 12 s
Cycle time 22 min
for GS 26379: Begin switching 4 min 18 s
End switching 5 min 6 s
Cycle time 10 min 30 s
Method W 13 417
Fig. 2. G 30033 (desethyl-atrazine) and

G 28279 (desethyl-simazine) in water.
Chromatogram 1: Standard mixture repre-
senting 0.5 ng of each metabolite.
Chromatogram 2: Water for HPLC, fortified
with 0.1 pig/l of each metabolite. Aliquot in-
jected equivalent to 10 ml water or 1 ng of
each compound added.
Chromatogram 3: Ground water. Aliquot in-
jected equivalent to 10 ml water.
418 Method W 13
Fig. 3. GS 26379 (desethyl-terbuthyl-

azine) in water.
Chromatogram 1: Standard mixture repre-
senting 0.5 ng GS 26379.
Chromatogram 2: Water for HPLC, forti-
fied with 0.1 ng/1 GS 26379. Aliquot in-
jected equivalent to 10 ml
water or 1 ng metabolite.
Chromatogram 3: Ground water. Aliquot
injected equivalent to 10 ml water.
Method W 13 419
7 Evaluation
7.1 Method
Inject 500 ul of each standard solution (equivalent to 0.5, 2.5, 5 and 10 ng of each metabolite)
into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs.
ng of each metabolite. Also inject 500-ul aliquots of the sample solutions. For the heights of
the peaks obtained for these solutions, read the appropriate amounts of the identified metabo-
lite from the corresponding calibration curve.

The recoveries from water for HPLC, fortified with the metabolites at levels of 0.1, 0.5 and
2 M-g/1, ranged from 84 to 119% and averaged 98% with a standard deviation of 6.2% (see
Table). Blanks usually did not occur. The routine limit of determination was 0.05 ng/1 for
each compound.
Table. Percent recoveries from water for HPLC fortified with G 28279, G 30033 and GS 26379.
Added (ng/1) G 28279 G 30033 G S 26379

0.1 89/90/95/99/99/100/ 94/95/98/101/102/ 84/86/96/102/104/
101/103/119 102/103/103/105/105 109
0.5 89/95/96/98/98/100/ 87/89/98/98/99/100/ 93/93/95/98/101/101
100/104/105 102
2 97/97 88/88/98/99/99 94/96

The residue R, expressed in |ng/l, of an identified metabolite is calculated from the following
equation:
p w A -v E n d
where
Vj = portion of volume VEnd injected into high-performance liquid chromatograph (vol-
ume of sample loop) (in ul)
WA = amount of metabolite for V{ read from calibration curve (in ng)
420 Method W 13
8 Important points
Acetonitrile is added to the concentrated analytical sample in 6.1 to precipitate the salts in so-
lution, in order to prevent blockages in the columns and tubes of the high-performance liquid
chromatograph.
9 References
No data
10 Authors
Ciba Geigy AG, Agricultural Division, Basle, Switzerland, G. Formica and C. Giannone
Part 6
Pesticide Residue Analytical Methods for
Water Using the AMD Technique
Thin-Layer Chromatographic Analysis of
Pesticides and Metabolites Using the
Automated Multiple Development (AMD)
Technique
1 Introduction
The technique for the automated multiple development (AMD) of thin-layer chromatograms
has been especially developed for the analysis of pesticide residues in drinking water.
High-performance thin-layer chromatography (HPTLC) silica gel plates are developed auto-
matically in many (e.g. 20 to 30) individual steps, thereby permitting the mobile phase to ad-
vance somewhat farther in each succeeding cycle. Solvent mixtures of different composition
can be used for each cycle, so that a reproducible gradient elution is obtained. The final deter-
mination is usually based on measuring the UV absorbance in a TLC scanner, using up to six
different wavelengths.
Applying the AMD technique results in much better separations than would be obtained
with conventional thin-layer chromatography. Therefore, several pesticides can be simulta-
neously determined in the same extract. A high throughput can also be achieved because up
to 18 spots can be applied onto a single TLC plate. The AMD technique can be used most
successfully if it is preceded by a broad-spectrum cleanup procedure such as described, e.g.,
in Cleanup Methods 7 and 8 (pp. 37 ff and pp. 41 ff, this Volume).
2 Outline of method
An aliquot of the cleaned-up sample solution is applied as a 4 mm long band onto a HPTLC
plate, using a TLC applicator. The plate is developed automatically in the AMD unit and dried
at 120 C with a stream of nitrogen.
The chromatogram is evaluated by measuring the UV absorbance at six different wave-
lengths in a TLC scanner. Using a multi-colour plotter, the absorption curves obtained for the
individual tracks are plotted, displaying the curves for the six wavelengths superimposed in
different colours. Compounds that do not, or only weakly, absorb UV light can be converted
to coloured derivatives by immersing the plate into the solution of a suitable reagent.
The results are confirmed by chromatographing a second aliquot of the sample solution,
using an elution gradient of entirely different selectivity (confirmative gradient) than was used
in the first analysis. A further check can be made by recording and comparing the UV spectra
of the peaks of the standard and sample solutions on the plate developed with the confirma-
tive gradient.
424 AMD Technique
3 Apparatus
Sample vials, 5-ml, with cone-shaped inside, e.g. conic ampoules N 18-5 (Macherey-Nagel
No. 702240)
TLC applicator, suitable for applying sample solutions in the form of narrow bands to
HPTLC plates, e.g. Linomat IV (Camag)
Micro syringes, 100-ul, suitable for TLC applicator
AMD system, consisting of a development unit, controller and accessories (Camag)
Vacuum pump
Vacuum manometer
Computer-controlled measurement and evaluation system for TLC:
Camag TLC scanner II,
Computer HP 9816,
Multi-colour plotter HP 7550 A,
TLC calculation software 88;
all obtainable from Camag
Glass tank, for simultaneously developing 5 TLC plates 20 cm x 20 cm, with glass cover
(Desaga)
Laboratory hot plate, electronically controlled (Desaga), equipped with:
Plate drying chamber, made from two 23 cm x 23 cm glass plates, placed in a U-shaped alu-
minium frame at a distance of 2.5 cm. The fourth side of the chamber is open to allow a TLC
plate to be inserted. The two opposite sides of the frame have a track each extending to half
height to allow the insertion of a second TLC plate. The upper glass plate has a 1-cm dia. hole
cut near to its rear edge to allow nitrogen or compressed air to be introduced
Chromatogram dipping device for HPTLC plates (only for compounds that do not, or only
weakly, absorb UV light)
4 Reagents
Acetonitrile, for chromatography (Baker No. 9392)
Carbon disulphide, p.a. (Baker No. 8021)
Dichloromethane, for chromatography (Baker No. 32222)
Diisopropyl ether (Baker No. 8072)
n-Heptane, p.a. (Riedel-de Haen No. 32287)
Methanol, p.a. (Merck No. 6009)
2-Propanol (isopropanol), for residue analysis (Merck No. 998)
Solvent mixture: acetonitrile + methanol + n-heptane 50:50:5 v/v/v
Internal standard solution: 0.1 mg/ml diphenyl sulphone in methanol
Pesticide standard solutions: 10 |ug/ml of each compound + 10 \xg/m\ diphenyl sulphone in
methanol
Ammonia solution, p.a., approx. 25% (Riedel-de Haen No. 30501)
Formic acid, p.a., 98-100% (Riedel-de Haen No. 33015)
Nitrogen, passed through granular activated charcoal (DeguSorb AS IV, Degussa)
Pre-coated HPTLC silica gel 60 glass plates, 10 cm x 20 cm, without fluorescent indicator
(Merck No. 5641), prepared as follows: Scrape off 5 mm each from the right and left edges
of a HPTLC plate. Place this plate and a second, unlayered glass plate of the same size in the
AMD Technique 425
glass tank, previously filled with 2 1 isopropanol, and allow to stand for at least 1 h, or for
up to several days. Remove the HPTLC plate and put it, silica gel side up, into the plate drying
chamber placed on the hot plate. Insert the second, unlayered glass plate approx. 1 cm above
in the frame track. Dry both plates at approx. 120 C with purified nitrogen or compressed
air for 30 min. Immediately after drying, lay the glass plate onto the silica gel layer of the
HPTLC plate, and seal with adhesive tape. Wrap the plates in aluminium foil and keep wrap-
ped until used
5 Applying the solutions onto the TLC plates

Dissolve the residue obtained, e.g. from Cleanup Method 8, step 6.4 or 6.5, in 200 L| L1 solvent
mixture in a sample vial. Pull 100 ul of this solution into a microsyringe. Using the TLC appli-
cator, apply the solution in the form of a 4 mm long band at a distance of 8 mm from the
bottom edge of the TLC plate. The distance between the individual tracks should be 6 mm;
leaving an empty space of 13 mm from both the left and right edges of the plate, each plate
then has room for 18 tracks. Set the application rate at 6 s per ul. For comparison, apply 5
to 50 JLII of the pesticide standard solution (equivalent to 50 to 500 ng of each compound).
Continue with the thin-layer chromatographic separation, using the screening gradient for
developing the chromatogram (see 6.3).
6 Thin-layer chromatographic determination

6.1 Principle of the AMD technique
In AMD, the plate is automatically cycled through a pre-set number of developments in the
AMD chamber. In each succeeding development, the mobile phase is permitted to advance
somewhat farther by a constant distance. Each time the components of the sample and stan-
dard solutions are separated and, at the same time, focused anew.
First, the chamber is evacuated to remove the last traces of solvent from the starting lines
of the sample or standard solutions. The chamber is flushed with purified nitrogen and, with
the addition of eluting solvent, the first cycle is started. A few seconds later, the cycle is termi-
nated by removing the solvent with suction. Then the next cycle is started including drying,
ventilating, and a somewhat longer elution, followed by removal of the eluting solvent. This
is continued until the whole programme is completed.
For each cycle, new mobile phase is introduced into the tank. If its composition differs from
the preceding one, this will result in a gradient elution. A gradient for silica gel begins strongly
polar (strongly eluting) and ends less polar (weakly eluting). Only when this sequence is fol-
lowed, a reproducible elution on silica gel can be achieved. The course of a gradient elution
with stepwise separation of a mixture is illustrated in the following scheme:
426 AMD Technique
25-[
n:
Fig. 1. Gradient elution by AMD; s, start; n, number of cycles; S, starting line; ch, chromatographable
portion of the extract; a to d, components of this portion; , solvent front.
6.2 Chromatographic separation

A typical elution gradient for HPTLC silica gel plates that extends from a methanol-dichloro-
methane mixture via dichloromethane to carbon disulphide is shown in the following
diagram:
CH 2 CI 2
100-
80-
60-
40-
20- CH3OH
5 10 15 20 25 n
Fig. 2. Typical concentration profile of an elution gradient; n, number of cycles.
This gradient can also be superimposed with a pH gradient from weakly alkaline to moder-
ately acid, in order to cover acid, basic and neutral substances in the same chromatogram. The
determining factor for the direction of the gradient (from alkaline to acid) is the binder of
the silica gel, which consists of an alkali carboxylate. The actual chromatographic separation
is preceded by a three-step cycle for focusing the spots. 0.01 % ammonia is added to the eluant,
which enhances somewhat the alkalinity of the plate. To the following eluants, 0.1% formic
acid is added. Due to the buffer effect of alkali salt + ammonia and formic acid, alkaline
conditions prevail during the early elution cycles. These conditions are gradually shifted to the
acid side in the following cycles.
AMD Technique 427
Six reservoirs are available on the AMD apparatus to produce the gradients. For each cycle,
a mixer is filled completely with solvent from the reservoirs. From this, half of the solvent mix-
ture is used as eluant for the next cycle, and the other half remains in the mixer. The mixer
is then re-filled with new solvent from one of the reservoirs. So when dichloromethane is used
as eluant for one cycle, and the mixer is re-filled with methanol, then the eluant for the next
cycle is dichloromethane-methanol 1:1 v/v.
After the three runs of the first cycle for focusing the spots, often an entirely different sol-
vent mixture is used as eluant. The mixer must therefore be completely emptied ("Empty
mixer after step 3"). A time programme for a 25-cycle gradient elution, the first cycle of three
runs serving to focus the spots, is shown in 6.3, Separation conditions.
After completion of the last cycle, the HPTLC plate is placed into the plate drying chamber
and dried at approx. 120 C for 3 min in a purified stream of nitrogen. It is then left to cool,
and the UV absorption on the individual tracks is measured in the TLC scanner.
6.3 Separation conditions

Separation chamber Camag AMD chamber
Gradient programmes
(Mobile phase components in v/v)
Screening gradient Confirmative gradient
Solvent Reservoir Reservoir
2 3 4 5 6 1 2 3 4 5 6
Carbon disulphide 100 50 100
Dichloromethane 90 90 100 100 100
Diisopropyl ether 90 90 100 100 50
Methanol 10 10 10 10
Ammonia solution 0.01 0.01
Formic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Time programme for the gradient elution of 25 cycles.
Run Cycle Time Run Cycle Time

Reservoir Reservoir
No. No. min No. No.
1 1 0.2 13 11 3.5
2 1 0.2 14 12 4.0
3 1 0.2 15 13 4.5
Empty mixer 16 14 5.0
17 15 5.7
4 2 0.5 18 16 6.5
5 3 0.8 19 17 7.2
6 4 1.0 20 18 8.0
7 5 1.3 21 19 9.0
22 20 10.0
8 6 1.6 23 21 11.0
9 7 1.9 24 22 11.9
10 8 2.3 25 23 12.8
11 9 2.7 26 24 13.7
12 10 3.1 27 25 14.6
600 [mV/10 Abs]
2 4 >
o
CD
O
IT
CD
0 10 20 30 40 50 60 70 80 90 [mm]
A10G A220 Ad'-l." A260 A280 A300 A650
Fig. 3. Screening chromatogram of a standard mixture, using the conditions as described in 6.3 and 6.4.
IS, diphenyl sulphone (internal standard); 1, omethoate; 2, carbendazim; 3, triadimenol; 4, metamitron; 5, triadimefon; 6, methabenzthiazuron;
7, azinphos-methyl; 8, azinphos-ethyl; 9, parathion. 200 ng of each compound, equivalent to 0.4 |LXg/l in a water sample (after Burger 1988). The remis-
sion curve at 650 nm was registered after staining the organophosphate spots by reaction with 4-(4-nitrobenzyl)pyridine and tetraethylenepentamine.
AMD Technique 429
1=
T3
a
1 o
o U
430 AMD Technique
Drying period
before first cycle at least 5 min
after each cycle 3 min
after final cycle 10 min
6.4 Measuring conditions

Apparatus and Controller Camag TLC Scanner II, linked to Computer HP 9816 with
Camag 88 TLC evaluation software
Y position for zeroing 4 mm
Y start position 4 mm
Y track end 90 mm
X start position 15 mm
X track distance 10 mm
Feed 0.1 mm
Pause 0.01 s
No. of values per measuring point 5
Peak optimization 0
Sensitivity Automatic set
Amplification (span) 7
Peak threshold 5
Smoothing 3
Light source Deuterium lamp
Slit 0.1 x 3 mm
Spectral slit width 10 nm
Measuring wavelengths 190 or 200 nm, 220, 240, 260, 280, 300 nm
Additional conditions for recording UV spectra:

Pilot wavelength According to UV spectrum of compound
Measuring range 190-350 nm
Resolution 2 nm
No. of values per measuring point 20
6.5 Plot parameters

To evaluate the sample and standard peaks, for each track the remission curves are drawn, with
the six wavelengths superimposed in different colours. By this means each peak is defined not
only by its distance from the starting line but also by its absorbance at the various wavelengths
(see also Figs. 3 and 4).
Scale Y = 200 mAU
Baseline correction 1
Plot section Only plot the range containing the compounds of in-
terest
6.6 Confirmation of results

Positive results obtained with the screening gradient must be confirmed in any case. For this
purpose, apply the second half of the sample solution derived from Section 5 onto another
HPTLC plate and chromatograph using the confirmative gradient as described in 6.3. Apply
AMD Technique 431
only those compounds for comparison, for which a signal was obtained in the screening chro-
matogram of the sample solution.
7 Evaluation
7.1 Method
Quantitation is performed by measuring the peak heights on the tracks of the sample solutions
and comparing them with the peak heights obtained on the tracks of the standard solutions,
both for the chromatograms developed with the confirmative gradient. The results are cor-
rected with the aid of the peak height of the internal standard in the tracks of the sample and
standard solutions. The peaks of the solutions should exhibit comparable heights. On no ac-
count peak heights obtained at different wavelengths should be compared for quantitation
purposes.
Depending on the UV absorbancy of the individual compounds, in general approx. 25 ng
can be determined from standard solutions.

The residue R, expressed in \ig/l, of an identified compound is calculated from the following
equation:
HA-Wst-HIst-WIA
HSfWISt-HIAG
where
W st = amount of compound applied onto HPTLC plate with standard solution (in ng)
WIA = amount of internal standard applied onto HPTLC plate with sample solution (in ng)
WISt = amount of internal standard applied onto HPTLC plate with standard solution
(in ng)
HA = peak height obtained for the compound in the sample solution (in mm)
H S t = peak height obtained for the compound in the standard solution (in mm)
H I A = peak height obtained for the internal standard in the sample solution (in mm)
H I S t = peak height obtained for the internal standard in the standard solution (in mm)
8 Important points
For a reproducible gradient elution, an optimal vacuum must be achieved in the AMD cham-
ber during the drying steps. Therefore, make sure that the chamber is vacuum-tight before
each gradient run. Proceed as follows: Apply vacuum to the chamber so that the maximum
432 AMD Technique
vacuum (approx. 4 mbar) is reached after 30-60 s. Then turn the three-way valve to a position
that the connection between pump and chamber is interrupted. The pressure in the chamber
should then not rise above 16 mbar within 1 min.
When no sufficient separation of the compounds was achieved, the gradient programme
must be changed. For this purpose, measure the distance from the start position to the solvent
front (max. 90 mm) and divide it by the number of cycles; e.g., for a distance of 90 mm and
25 cycles this results in 3.6 mm per cycle. If the compound in question travels a distance of
36 mm, corresponding to approx. 10 cycles, then the polarity of the following gradients must
be raised to improve the separation.
If the confirmative chromatogram gives a positive result, measure and compare the com-
plete UV spectra of both the sample and standard peaks on the plate in the reflection mode
(see e.g. Figure 5). For additional confirmation, isolate the compound in question from the
adsorbent and identify it by mass spectrometry.
Compounds that do not, or only weakly, absorb UV light can be converted to coloured de-
rivatives by immersing the plate into the solution of a suitable reagent before measuring as
described in 6.4 (see 9. References). In other cases, they can be converted to fluorescent com-
pounds which are determined at their characteristic wavelengths in the TLC scanner.
(Abs-UV]
190 240 290 340 390 440 nm
Fig. 5. UV spectra of methabenzthiazuron peaks, measured on the HPTLC plate in the reflection mode.
from a standard solution,
from a sample solution.
AMD Technique 433
9 References
K. Burger, Multiple method for ultratrace determination: Pesticide active ingredients in
ground and drinking water analyzed by TLC/AMD (Automated Multiple Development),
H. Jork, W. Funk, W. Fischer and H. Wimmer, Dunnschicht-Chromatographie. Reagenzien
und Nachweismethoden. Band 1 a; Physikalische und chemische Nachweismethoden:
Grundlagen, Reagenzien I, VCH Verlagsgesellschaft, Weinheim 1989.
K. Burger, J. Kohler and H. Jork, Application of AMD to the determination of crop protec-
tion agents in drinking water. Part 1: Fundamentals and method, J. Planar Chromatogr. 3,
504-510 (1990).
10 Author
Examples for Applying the AMD Technique
to the Determination of Pesticide Residues in
Ground and Drinking Waters
Example 1
Azinphos-ethyl azinphos-methyl bitertanol, carbofuran, metamitron, methabenzthiazuron,
parathion, triadimefon, and triadimenol (German version, Method W 9, published 1991).
Bromacil, cymoxanil diuron, lenacil, and metribuzin (German version Method W 10, pub-
lished 1991).
1.1 Apparatus and Reagents

See 3. Apparatus and 4. Reagents on pp. 424f, this Volume; for preparing and cleanup of the
analytical sample, see Cleanup Method 8, pp. 41 ff, this Volume. Additionally the following
standard solutions are required:
Compound standard solution 1:10 |ug/ml of each azinphos-ethyl, azinphos-methyl, bitert-
anol, carbofuran, triadimefon and diphenyl sulphone (internal standard) in methanol
Compound standard solution 2:10 pig/ml of each metamitron, methabenzthiazuron, para-
thion, triadimenol and diphenyl sulphone (internal standard) in methanol
Compound standard solution 3:10 jig/ml of each bromacil, cymoxanil, diuron, lenacil,
metribuzin and diphenyl sulphone (internal standard) in methanol
1.2 Cleanup
Proceed with 1000 ml of the analytical sample (G) as described in Cleanup Method 8,
pp. 41 ff, this Volume.
1.3 AMD conditions

Proceed as described in Sections 5 and 6 on pp. 425 ff, this Volume, with the following altera-
tions:
Gradient programmes for the compounds contained in Compound standard solutions 1 and 2 (mobile
phase components in v/v)
1 2 3 4 5 6 1 2 3 4 5 6
Acetonitrile 100 30 100 25
Carbon disulphide 80 100 100
Dichloromethane 70 100 100 20
Diisopropyl ether 75 100 100 100
Formic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1
436 AMD Technique - Examples
Screening Confirmative
gradient gradient
Rf values for
Compound standard solution 1
bitertanol 0.16 0.23
triadimefon 0.37 -*)
carbofuran 0.42 0.63
azinphos-methyl 0.55 0.71
azinphos-ethyl 0.58 0.79
diphenyl sulphone 0.63 0.97
triadimenol 0.15 0.22
metamitron 0.18 0.19
methabenzthiazuron 0.40 0.42
triadimefon 0.58**)
parathion 0.80 0.96
*) Compound standard solution 1 without triadimefon,

**) Compound standard solution 2 with triadimefon added.
Gradient programmes for the compounds contained in Compound standard solution 3 (mobile phase
components in v/v)
1 2 3 4 5 6 1 2 3 4 5 6
Carbon disulphide 100 100
Diisopropyl ether 100 30 100 100 100
Methanol 10 10 10 70
Formic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
gradient gradient
Rf values for
lenacil 0.25 0.34
bromacil 0.33 0.41
diuron 0.39 0.23
cymoxanil 0.43 0.23
metribuzin 0.55 0.60
1.4 Evaluation
For quantitation and calculation of the residues, proceed as described in 7.1 and 7.2, p. 431,
this Volume.
The recoveries from control samples, fortified with the compounds at levels of 0.05 to
0.4 ng/1, are given in Table 1. The limit of detection was approx. 0.03 fig/1, and the limit of
determination was approx. 0.05 ng/1.
AMD Technique - Examples 437
Table 1. Percent recoveries from water, fortified with the compounds contained in Compound standard
solutions 1, 2, and 3.
Added (ng/1)
Compound
0.05 0.1 0.15 0.2 0.25 0.3 0.4
Standard solutions 1 and 2

Azinphos-ethyl 92 85 94 91 90 89 100
Azinphos-methyl 100 96 70 107 100 96 112
Bitertanol 116 90 95 103 90 85 100
Carbofuran 128 94 87 98 100 85 91
Metamitron 98 82 81 79 70 69 94
Methabenzthiazuron 106 85 93 95 93 90 107
Parathion 108 83 98 89 93 87 81
Triadimefon 111 99 97 95 97 93 88
Triadimenol 100 90 92 88 91 88 95
Standard solution 3
Bromacil 114 94 86 95 90 87 95
Cymoxanil 93 93 103 90 103 111 115
Diuron 122 109 96 98 89 93 97
Lenacil 97 101 110 107 118 104 104
Metribuzin 92 78 74 95 88 82 95
1.5 Important points

The recoveries of bitertanol, metribuzin and lenacil can be lower with water samples contain-
ing a high proportion of humic acids.
1.6 Author
Example 2
Benalaxyl bendiocarb, bentazone, dinoseb, dinoterb, DNOQ metalaxyl, nitrothal-isopropyl
pendimethalin, and terbumeton (German version Method W 11, published 1991).
The esters of fluazifop-P, fluroxypyn haloxyfop, and triclopyr can also be analyzed using this
technique.

See 3. Apparatus and 4. Reagents on pp. 424f, this Volume; for preparing and cleanup of the
analytical sample, see Cleanup Method 8, pp. 41 ff, this Volume. Additionally the following
standard solutions are required:
Compound standard solution 4: 10 |j,g/ml of each benalaxyl, dinoterb, DNOC, fluazifop-P-
butyl, fluroxypyr-1-methylheptyl, haloxyfop-ethoxyethyl, and metalaxyl in methanol
Compound standard solution 5: 10 ng/ml of each bendiocarb, bentazone, dinoseb,

nitrothal-isopropyl, pendimethalin, terbumeton, and triclopyr-butoxyethyl in methanol
An internal standard is not required.
2.2 Cleanup
Proceed with 1000 ml of the analytical sample (G) as described in Cleanup Method 8,
pp. 41 ff, this Volume. Do not add internal standard to the analytical sample.
2.3 AMD conditions

Dissolve the residue derived from Cleanup Method 8, step 6.4 or 6.5, in 200 ul solvent mixture
(VEnd) in a sample vial. Apply approx. 100 \i\ of this solution (Vi) onto the HPTLC plate.
Also apply 3 to 30 \x\ of the Compound standard solutions 4 or 5 (equivalent to 30 to 300 ng
of each compound).
Proceed as described in Section 6 on pp. 425 ff, this Volume, with the following alterations:
Gradient programmes for the compounds contained in Compound standard solutions 4 and 5 (mobile
phase components in v/v)
1 2 3 4 5 6 1 2 3 4 5 6
Acetonitrile 100 30 100 25
Carbon disulphide 100 100
Dichloromethane 70 100 100 100
Formic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
gradient gradient
Rf values for
metalaxyl 0.36 0.30
benalaxyl 0.64 0.70
haloxyfop-ethoxyethyl 0.73 0.80
fluazifop-P-butyl 0.79 0.79
fluroxypyr-1-methylheptyl 0.84 0.90
DNOC 0.88 0.92
dinoterb 0.93 0.98
terbumeton 0.31 0.32
bendiocarb 0.73 0.69
nitrothal-isopropyl 0.75 0.92
triclopyr-butoxyethyl 0.84 0.91
bentazone 0.88 0.83
dinoseb 0.91 0.95
pendimethalin 0.97 0.97
2.4 Evaluation
2.4.1 Method
Quantitation is performed by measuring the peak heights on the tracks of the sample solutions
and comparing them with the peak heights obtained on the tracks of the standard solutions.
The peaks from both solutions should exhibit comparable heights.
Quantitation can also be performed by the calibration technique. Prepare calibration curves
by plotting the peak heights obtained for each compound on the standard solution tracks vs.
ng of the respective compound. For the heights of the peaks obtained from the sample solu-
tions, read the appropriate amounts from the corresponding calibration curves.
When a suitable software is available (e.g. Camag 88 TLC evaluation software), quantitation
can also be performed by computer calculation from the raw data using a calibration curve.
2.4.2 Recoveries, limit of detection and limit of determination

The recoveries from control samples, fortified with the compounds at levels of 0.1 to 0.5 |xg/l,
ranged from 70 to 110% and averaged 88%; see Table 2. The limit of detection was approx.
0.05 ng/1, a n d the limit of determination was approx. 0.1 \ig/\. Blank values interfering with
the determination of the polar compounds metalaxyl and terbumeton were frequently ob-
served in the analysis of waters containing high proportions of humic acids. Moreover, recov-
eries were reduced in such cases.
Table 2. Percent recoveries from water, fortified with the compounds contained in Compound standard
solutions 4 and 5 (means of 2-4 experiments).
Added ftig/1)
Compound 0 1 Q2 Q5
Benalaxyl 97 68 82
Bendiocarb 105 78 93
Bentazone *> 89 93 85
Dinoseb 106 98 94
Dinoterb 88 82 87
DNOC 95 76 93
Fluazifop-P-butyl*0 96 76 87
Fluroxypyr-1-methylheptyl *> 77 92 75
Haloxyfop-ethoxyethyl*> 84 91 79
Metalaxyl *} 74 89 77
Nitrothal-isopropyl 93 84 89
Pendimethalin *} 111 78 85
Terbumeton *> 93 69
Triclopyr-butoxyethyl *> 106 88 85
*> The recoveries are not valid for waters with very high humic acid content.
2.4.3 Calculation of residues

The residue R, expressed in jig/1, of an identified compound is calculated from the following
equations:
when comparing peak heights

H A -W St -V End
H st Vi G
when using a calibration curve

R w A -v E n d
where

V E n d = terminal volume of sample solution from 2.3 (in ul)
Vt = portion of volume V E n d applied onto H P T L C plate (in ul)
Wst = a m o u n t of c o m p o u n d applied onto H P T L C plate with standard solution (in ng)
WA = a m o u n t of c o m p o u n d for V{ read from calibration curve (in ng)
HA = peak height obtained from V^ (in m m )
HSt = peak height obtained from W S t (in m m )

Using 10 cm x 20 cm pre-coated HPTLC silica gel 60 glass plates with an extra thin layer of
0.1 mm (Merck No. 11764), the intensity of the peaks was increased. The Rf values for the
compounds, applied to these plates and run under screening conditions, were as follows (see
also Fig. 1 and 2):
Rf values for
Compound standard solution 4 Compound standard solution 5
metalaxyl 0.32 terbumeton 0.24
benalaxyl 0.53 bendiocarb 0.61
haloxyfop-ethoxyethyl 0.64 nitrothal-isopropyl 0.65
fluazifop-P-butyl 0.72 triclopyr-butoxyethyl 0.72
fluroxypyr-1 -methylheptyl 0.78 bentazone 0.76
DNOC 0.87 dinoseb 0.84
dinoterb 0.91 pendimethalin 0.91
2.6 Authors
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, H. Kohle and
H.-G. Nolting
250 [mV/10 Abs]
70 80 90 [mm]
Fig. 1. Screening chromatogram of Compound standard solution 4.
1, metalaxyl; 2, benalaxyl; 3, haloxyfop-ethoxyethyl; 4, fluazifop-P-butyl; 5, fluroxypyr-1-methylheptyl;
6, DNOC; 7, dinoterb. 200 ng of each compound, HPTLC silica gel plate, layer thickness 0.1 mm, meas-
urement wavelengths 200, 220, 240, 260, 280, and 300 nm.
400 [mV/10 Abs]
30 40 50 60 70 80 90 [mm]
Fig. 2. Screening chromatogram of Compound standard solution 5.
8, terbumeton; 9, bendiocarb; 10, nitrothal-isopropyl; 11, triclopyr-butoxyethyl; 12, bentazone;
13, dinoseb; 14, pendimethalin. For conditions, see Fig. 1.
Example 3
Amitrole (German version Method W 12, published 1991).

3.1.1 Apparatus
For cleanup of the analytical sample:
pH meter
Solid phase extraction system, e.g. Visiprep SPE Vacuum Manifold (Supelco No. 5-7030)
Disposable column, volume 3 ml, filled with 500 mg silica gel (Baker No. 7086-03)
Sample vials, 5-ml, with cone-shaped inside, e.g. conic ampoules N 18-5 (Macherey-Nagel
No. 702240)
Device for evaporating solvents in a nitrogen stream, suitable to take the 5-ml sample vials,
e.g. Silli-Therm heating module with Silli-Vap evaporator and Reacti-Bar heating block (Pierce
No. 19793, 19792 and 19785, respectively)
For AMD thin-layer chromatography: See p. 424, this Volume. Additionally the following
items are required:
Double chambered tank, for developing 20 cm x 10 cm TLC plates, with glass cover (Camag
No. 022.5253)
Second dipping tank for HPTLC plates (e.g. Desaga No. 124152), or additional glass insert
suitable for the dipping tank Baron-DC-Tauchfix (Desaga No. 124162)
Hot-air blower
3.1.2 Reagents
Acetonitrile, for chromatography (Merck No. 30)
Dichloromethane, for chromatography (Merck No. 6044)
Diisopropyl ether, p.a. (Baker No. 8072)
Methanol, for chromatography (Merck No. 6007)
Eluting mixture: acetonitrile + methanolic ammonia solution 8:2 v/v
Amitrole standard solution: 1 jug/ml methanol
Chromogenic solution: 0.1 g/100 ml N-(l-naphthyl)ethylenediamine dihydrochloride p.a.
(Merck No. 6237) in dichloromethane + methanol mixture 8:2 v/v
Ammonium amidosulphonate solution: 0.1 g/100 ml ammonium amidosulphonate p.a.
(Merck No. 1220) in dichloromethane + methanol mixture 75:25 v/v
Sulphuric acid, 0.5 mol/1 H 2 SO 4 ; prepared from sulphuric acid, p.a., 95-979/0 (Merck
No. 731)
Hydrochloric acid, p.a., 32% (Merck No. 319)
Formic acid, p.a., 98-100% (Merck No. 264)
Ammonia gas (Merck Lecture Bottle No. 823209 with valve No. 823002)
Methanolic ammonia solution: Saturate 500 ml methanol in a 1-1 two-necked flask, under ice
cooling, with gaseous ammonia. Dilute the solution with a further 500 ml of ice-cold metha-
nol, stopper, and store in a deep freeze
Sodium nitrite, p.a. (Riedel-de Haen No. 31443)
Nitrogen, passed through granular activated charcoal (DeguSorb AS IV, Degussa)
Pre-coated HPTLC silica gel 60 glass plates, 10 cm x 20 cm, without fluorescent indicator
(Merck No. 5641), prepared as described on pp. 424 f, this Volume
3.2 Cleanup
Mount the disposable column onto the vacuum unit of the extraction system and condition
it with 5 ml eluting mixture. Do not allow the column to run dry.
Acidify 20 ml of the analytical sample (G) in a 50-ml round-bottomed flask to pH 2 with
sulphuric acid and rotary-evaporate to dryness. Take up the residue in 2 ml eluting mixture
and transfer the solution quantitatively onto the disposable column, then add a further 3 ml
eluting mixture to the column. Collect the total eluate (approx. 5 ml) in a sample vial and ap-
ply slight suction to the column to drive the remainder of the eluting mixture into the vial.
Transfer the vial to the heating block (35 C) of the evaporation device and evaporate to dry-
ness under a stream of nitrogen.
3.3 Thin-layer chromatographic determination

Proceed as described on pp. 425ff., this Volume, with the following alterations:
3.3.1 Applying the solutions onto the TLC plate

Dissolve the residue derived from 3.2 in 200 ul methanol (VEnd). Using the TLC applicator,
apply approx. 100 ul of this solution (Vj) onto a thin-layer plate, at a distance of 8 mm from
the bottom edge, in the form of a 7 mm long band. The distance between the individual tracks
should be 3 mm; leaving an empty space of 11.5 mm from both the left and right edges of
the plate, each plate then has room for 18 tracks. Set the application rate at 6 s per ul. For
comparison, apply 1, 5, 10, and 20 ul of the amitrole standard solution (equivalent to 1 to
20 ng amitrole).
3.3.2 AMD conditions

Separation chamber Camag AMD chamber
Gradient programmes (Mobile phase components in v/v)
1 2 3 4 5 1 2 3 4 5
Methanol 40 28 14
Methanolic ammonia solution 100 20 10
Formic acid 0.1 0.1 0.1 0.1 0.1
Time programme for the gradient elution of 20 cycles.
Run Cycle Time Run Cycle Time

Reservoir Reservoir
No. No. min No. No. min
1 1 0.2 13 11 3.5
2 1 0.2 14 12 4.0
3 1 0.2 15 13 4.5
Empty mixer 16 14 5.0
17 15 5.7
4 2 0.5 18 16 6.5
5 3 0.8 19 17 7.2
6 4 1.0 20 18 8.0
7 5 1.3 21 19 9.0
22 20 10.0
8 6 1.6
9 7 1.9
10 8 2.3
11 9 2.7
12 10 3.1
Drying period
before first cycle at least 5 min
after each cycle 3 min
after final cycle 10 min
Rf value for amitrole 0.26 0.32
3.3.3 Visualization
Add approx. 10 ml hydrochloric acid dropwise to approx. 12 g sodium nitrite in one of the
chambers of the double chambered tank. Close the tank with the glass cover. When the tank
is full of brown fumes, allow the dried thin-layer plate to stand in the second, empty chamber
of the tank for 10 s. Longer exposure times can lead to oxidations on the plate. Remove the
plate from the tank and dry for about 1 min in a fume hood with a hot-air blower to remove
nitrous gases. Dip the plate into the chromogenic solution for 3 s, using the chromatogram
dipping device, and dry for 20 s with the hot-air blower. Dip the plate into the ammonium
amidosulphonate solution for 3 s, using the chromatogram dipping device, and dry for 20 s
with the hot-air blower. In the course of this, the pink amitrole spots turn yellow.
Then measure the absorbance of the spots immediately!
3.3.4 Measuring conditions and plot parameters

See p. 430, this Volume, with the following alterations:
Light source Tungsten lamp
Measuring wavelength 490 nm
Scale Y = 200 mAU
Baseline correction 1
Plot section Only plot the range containing amitrole
3.4 Evaluation
3.4.1 Method
Quantitation is performed from the chromatograms developed with the confirmative gradient
as described for Example 2 on p. 439, this Volume.
3.4.2 Recoveries and limit of determination

The recoveries from control water samples, fortified with amitrole at levels of 0.05 to 5 ^g/1,
ranged from 77 to 93% and averaged 89%. The limit of determination was approx. 0.05 jug/1.
3.4.3 Calculation of residues

The residue R, expressed in u.g/1 amitrole, is calculated from the equations described for
Example 2 on p. 440.

According to step 3.2, the dry residue is taken up in 2 ml eluting mixture. If greater amounts
of material remain undissolved, recovery rates of amitrole may be reduced. In these cases,
therefore, the analytical sample in 3.2 should be adjusted to pH 2 with hydrochloric acid
(1 mol/1) instead of sulphuric acid.
When many plates are to be subjected to colour development, the following alteration in
the visualization procedure is recommended: Add 20 ml fuming hydrochloric acid p.a. (Merck
No. 317) to one of the chambers of the double chambered tank. Place the dried thin-layer plate
into the second, empty chamber of the tank for 10 min; then remove excess hydrochloric acid
fumes from the plate by exposing it to a hot-air blow for 20 s. Next, transfer the plate into
a thin-layer tank (Desaga No. 120101) and introduce nitric oxide from a gas cylinder (Merck
Lecture Bottle No. 823317, with valve No. 823009) into the tank, until it is filled with brown
fumes. Then remove excess nitrous gases from the plate by exposing it to a hot-air blow for
3 min. Continue as described in 3.3.3, beginning "Dip the plate into the chromogenic solution
The amitrole azo-derivative is stable for approx. 1 to 2 h; any measurements must be termi-
nated within this time span.
3.6 Author
Cumulative Indexes for Volumes 1 and 2
Index of Determinate Pesticides,
Metabolites and Related Compounds
(Index of Compounds)
Numbers in italics refer to Multiple Pesticide Residue Analytical Methods; numbers
in italics marked with an asterisk refer to Individual Pesticide Residue Analytical
Methods.
Acephate 1: 81*; 2: 25, 31, 301, 317 Aziprotryne 1: 221, 283; 2: 31, 313
Acetaldehyde, see Metaldehyde Azocyclotin 1: 222; 2: 343
Acetaldehyde 2,4-dinitrophenylhydrazone 2: 239 Azocyclotin, methyl derivative 2: 343
Acetanilide 1: 252
Acetanilides, substituted 1: 252 Barban 2: 25, 31, 273
3-Acetylamino-l,2,4-triazole, see Monoacetyl- Benalaxyl 2: 301, 437
amitrole Benazolin, methyl ester 2: 25
N-Acetyl-glufosinate, dimethyl ester 2: 218 Bendiocarb 2: 25, 31, 301, 349, 437
Alachlor 2: 25, 31, 301, 317 Benfluralin 2: 31, 301, 317
Aldicarb 1: 87*; 2: 25, 31, 301, 349 Benodanil 2: 31
Aldicarb sulphone 1: 87; 2: 31, 358 Benomyl 2: 69* 111
Aldicarb sulphoxide 1: 87; 2: 358 Bensulide 2: 31
Aldrin 1: 51, 73, 77, 221, 283, 297, 309, 327, Bentazone 2: 302, 437
383; 2: 25, 31, 313, 317, 387 Benzoic acids, chlorinated 1: 171
Allethrin 2: 25 Benzoylprop-ethyl 2: 31, 317
Allidochlor 2: 31 3,4-Benzpyrene (benzo[a]pyrene) 2: 35
Ametryn 1: 221, 265, 283; 2: 31, 313, 317, 403 Benzyl thiocyanate 1: 90
Amidithion 1: 221, 335; 2: 31, 317 Bifenox 2: 31, 302, 317
Aminocarb 2: 301, 349 Bifenthrin 2: 302, 333
5-Amino-4-chloropyridazin-3(2H)-one, see Binapacryl 1: 68, 77, 222, 383; 2: 31, 197* 317
Chloridazon, metabolites Biphenyl 2: 31, 294
2-Amino-3,5-dibromoacetophenone 2: 273 Bis(4-chlorophenyl)methanol (DBH) 2: 31
Aminomethylphosphonic acid, see AMPA Bisdithiocarbamate fungicides 1: 353, 407
Amitraz 2: 31 Bitertanol 1: 78, 222, 383; 2: 31, 77* 87* 317,
Amitrole 2: 49* 301, 442 377, 435
AMPA 2: 229 Bromacil 1: 222, 283; 2: 25, 31, 313, 317, 435
Anilazine 1: 77, 221, 383; 2: 31, 59* 317 Bromacil, N-methyl derivative 2: 25
Anilide herbicides 1: 246, 263 Bromide, inorganic 1: 377
Aniline 1: 247 Bromine-containing fumigants 1: 377
Anilines, substituted 1: 241, 251 4-Bromoacetanilide 1: 252
Anthraquinone 2: 31, 301, 317, 377 4-Bromoaniline 1: 247
Aroclor 1016-1268, see PCB 4-Bromo-3-chloroacetanilide 1: 252
Atraton 1: 221, 265; 2: 31 4-Bromo-3-chloroaniline 1: 247
Atrazine 1: 221, 265, 283, 347; 2: 25, 31, 313, 2-Bromoethanol 1: 377
317, 403 4-Bromo-l-naphthylacetic acid methyl ester 1:167
Azinphos-ethyl 1: 64, 73, 77, 221, 283, 335, Bromophos 1: 64, 77, 222, 283, 297, 309, 335,
371, 383; 2: 31, 313, 317, 428, 429, 435 371, 383; 2: 25, 31, 313, 317
Azinphos-methyl 1: 221, 283; 2: 25, 31, 313, Bromophos-ethyl 1: 64, 77, 222, 283, 335, 371,
317, 428, 429, 435 383; 2: 25, 31, 313, 317
450 Index of Compounds
Bromopropylate 2: 31, 302, 575, 317 Chlorfenvinphos 1: 64, 73, 77, 223, 283, 309,
Bromoxynil 2: 31, 99* 335, 371, 383; 2: 25, 32, 313, 317
Bromoxynil, methyl ether 2: 25, 99 Chlorflurenol 2: 127* 303, 313
Bromoxynil octanoate 2: 31, 99, 302, 317 Chlorflurenol, methyl ester 2: 127
Brompyrazon 2: 31 Chloridazon 1: 246, 263; 2: 25, 32, 755* 273,
Bupirimate 1: 222, 283; 2: 313 303, 577
Butocarboxim 2: 302, 349 Chloridazon, metabolites 2: 135
Butocarboxim, metabolites 2: 358 Chlormephos 2: 32, 303, 577
Buturon 1: 222, 241, 251 Chlormequat 2: 36
Butylhydroxyanisole 2: 35 4-Chloroacetanilide 1: 252
Butylhydroxytoluene 2: 35 4-Chloroaniline 1: 247
2-(2-Chloroanilino)-4,6-dimethoxy-l,3,5-triazine,
Camphechlor 1: 73, 77, 222, 297, 383; 2: 31, see Dimethoxy anilazine
317 Chlorobenzilate 2: 32, 303, 575, 577
Captafol 1: 78, 93* 99* 222, 283, 383, 401; 2-Chlorofluorenone 2: 128
2: 25, 31, 313, 317, 359, 377 2-Chloro-9-hydroxyfluorene 2: 128
Captan 1: 78, 105* 222, 283, 327, 383, 401; 3-Chloro-4-methoxyaniline 1: 247
2: 25, 31, 313, 317, 359, 377 3-Chloro-4-methylacetanilide 1: 252
Carbaryl 2: 25, 31, 302, 349 3-Chloro-4-methylaniline 1: 247; 2: 26
Carbendazim 2: 25, 69* 107* 302, 428, 429 Chloroneb 2: 25, 32, 303, 577
Carbendazim, pentafluorobenzyl derivative 2-Chloro-4-nitroaniline 2: 32
2:107 Chloroparaffins (C12 - C18) 2:35
Carbetamide 2: 25 Chlorophenols 2: 35
Carbofuran 2: 25, 31, 113* 302, 349, 435 4-(4'-Chlorophenoxy)acetanilide 1: 252
Carbofuran, metabolite, see 3-Hydroxy- 4-Chlorophenoxyacetic acid 2: 32
carbofuran Chlorophenoxyalkanoic acids 1: 78, 171;
Carbon disulphide 1: 353, 359, 411 2: 369, 393
Carbophenothion 1: 73, 77, 223, 283, 309, 335, 4-(4'-Chlorophenoxy)aniline 1: 247
361, 383; 2: 31, 313, 317 Chloropropylate 2: 32, 303, 575, 577
Carbophenothion oxygen analogue 2: 31 Chlorothalonil 2: 32, 303, 577, 377
Carbophenothion sulphone 1: 223, 365, 368, Chlorotoluron 1: 223, 241, 251; 2: 26, 32, 577
369 Chlorotriazine herbicides, desalkyl metabolites
Carbophenothion sulphone, oxygen analogue 1: 59, 347; 2: 413
1: 369 Chloroxuron 1: 223, 241, 251; 2: 26, 32, 577
Carbophenothion-methyl 2: 31, 302, 317 Chlorpropham 1: 223, 527; 2: 26, 32, 273, 577
Carbosulfan 2: 775* Chlorpropham, dibromo derivative 1: 321
Chinalphos, see Quinalphos Chlorpyrifos 1: 77, 223, 283, 297, 335, 383;
Chinomethionat (quinomethionate) 2: 31, 302, 2: 26, 32, 575, 577
317, 377 Chlorpyrifos-methyl 1: 223, 283; 2: 32, 575,
Chloranil 2: 31 577
Chlorazine 2: 31 Chlorsulfuron 2: 145*
Chlorbenside 2: 31, 303, 313, 317 Chlorthal 1: 223, 283; 2: 575
Chlorbenside sulphone 2: 31, 303, 317 Chlorthal-dimethyl 2: 26, 32, 303, 577
Chlorbromuron 1: 223, 241, 251; 2: 25, 31 Chlorthiamid 1: 126; 2: 26
Chlorbufam 2: 25, 31 Chlorthion 1: 64, 224, 335, 371
a-Chlordane (cis-chlordane) 1: 77, 223, 297, Chlorthiophos 1: 109* 224, 283; 2: 32, 575, 577
309, 327, 383; 2: 25, 31, 317 Chlorthiophos oxygen analogue 1: 110
y-Chlordane (trans-chlordane) 1: 73, 77, 223, Chlorthiophos sulphone 1: 110
297, 309, 327, 383; 2: 25, 31, 317 Chlorthiophos sulphoxide 1: 110
Chlordecone 1: 295; 2: 32 Cinerins 1: 236; 2: 26, 323
Chlorfenethol 2: 32 Clophen A 3 0 - A 60, T 64, see PCB
Chlorfenprop-methyl 2: 25, 32, 303, 317 CMPP, see Mecoprop
Chlorfenson 1: 73, 77, 223, 383; 2: 32, 313, Copper 2: 153
317 Copper oxychloride 2: 755*
Index of Compounds 451
Coumaphos 2: 32, 303, 317 Demeton-S-methyl sulphoxide (oxydemeton-

Coumarin 2: 35 methyl) 1: 225, 368, 369; 2: 34, 317
Coumithoate 2: 32 Desalkyl triazine metabolites 1: 59, 347; 2: 413
Crotoxyphos 2: 32, 304, 317 Desethyl-atrazine 1: 226, 347; 2: 413
Crufomate 2: 32, 304, 317 N-Desethyl-pirimiphos-methyl 1: 192; 2: 32,
Cyanazine 2: 26, 32, 304, 313, 317 304, 317
Cyanofenphos 1: 224, 283; 2: 32, 313, 317 Desethyl-simazine, see Des-tert-butyl-terbuthyl-
Cyanophos 2: 32, 304, 313, 317 azine
Cycluron 2: 32 bis(Desethyl)-simazine 1: 225, 347
Cyfluthrin 2: 304, 333 Desethyl-terbuthylazine 2: 305, 413
Cyhalothrin 2: 304, 333 Desmethyl-formamido-pirimicarb 1: 183,
Cyhexatin 1: 224; 2: 343 2: 305, 349
Cyhexatin, methyl derivative 2: 343 Desmethyl-norflurazon 2: 32
Cymoxanil 2: 32, 157* 304, 317, 435 Desmethyl-pirimicarb 1: 183, 2: 305, 349
Cypermethrin 1: 224; 2: 26, 32, 317, 333 Desmetryn 1: 226, 265, 283; 2: 26, 32, 313
Des-tert-butyl-desethyl-terbumeton 1: 226, 347
2,4-D 2: 32, 163% 304, 369, 393 Des-tert-butyl-desethyl-terbutryn 1: 226, 347
2,4-D, dinitro derivative of methyl ester 2: 369 Des-tert-butyl-terbumeton 1: 226, 347
2,4-D, isooctyl ester 2: 26, 374 Des-tert-butyl-terbuthylazine 1: 226, 347; 2: 413
2,4-D, methyl ester 2: 26, 32, 374 Des-tert-butyl-terbutryn 1: 226, 347
2,4-D, trichloroethyl ester 2: 393 Dialifos 2: 26, 32, 305, 313, 317, 359
Dalapon 1: 117*; 2: 26 Di-allate 2: 26, 32, 305, 317
Dalapon, methyl ester 1: 117 Diazinon 1: 64, 73, 77, 226, 283, 309, 335, 371,
Dazomet 2: 26 383; 2: 26, 32, 313, 317
2,4-DB 2: 32, 304, 393 Diazoxon 1: 226, 371
2,4-DB, methyl ester 2: 26 Dibrom, see Naled
2,4-DB, trichloroethyl ester 2: 393 Dibutyl phthalate 2: 35
p,p'-DDA 2: 32 Dicamba, methyl ester 2: 26
o,p'-DDD 1: 73, 77, 224, 297, 383; 2: 32, 317 Dichlobenil 1: 123* 226, 283; 2: 26, 32, 169*
p,p'-DDD 1: 51, 73, 77, 224, 283, 297, 309, 313, 317
327, 383; 2: 32, 313, 317 Dichlofenthion 1: 64, 226, 283, 335, 371; 2: 26,
o,p'-DDE 1: 73, 77, 224, 383; 2: 32, 317, 387 32, 313, 317
p,p'-DDE 1: 51, 73, 77, 224, 283, 297, 309, 327, Dichlofluanid 1: 78, 227, 283, 327, 383; 2: 26,
383; 2: 32, 313, 317, 387 32, 777* 191* 313, 317, 377
o,p'-DDT 1: 51, 73, 77, 224, 297, 309, 327, Dichlofluanid, metabolite, see DMSA
383; 2: 26, 32, 317, 387 Dichlone 2: 32
p,p'-DDT 1: 51, 68, 73, 77, 225, 283, 297, 309, 3,4-Dichloroacetanilide 1: 252
327, 383; 2: 26, 32, 313, 317, 387 3,4-Dichloroaniline 1: 247; 2: 273
Decachlorobiphenyl 2: 32 3,5-Dichloroaniline 1: 213
DEF 2: 304, 317 2,6-Dichlorobenzamide 2: 32
Deltamethrin 1: 225; 2: 32, 317, 333 Dichlorobenzenes 2: 35
Demephion-O 2: 32 p,p'-Dichlorobenzophenone 2: 32, 305, 317
Demephion-S 2: 32 2,7-Dichlorofluorenone 2: 133
Demeton-O 1: 225, 361; 2: 32 2,7-Dichloro-9-hydroxyfluorene carboxylic acid,
Demeton-O sulphone 1: 225, 365, 368, 369 methyl ester 2: 133
Demeton-O sulphone, oxygen analogue 1: 369 Dichlorophenols 2: 35
Demeton-S 1: 225, 361; 2: 32 2,4-Dichlorophenoxy-phenoxypropionic acid
Demeton-S sulphone 1: 225, 365, 368, 369; 2: 32
2: 32, 317 3,5-Dichlorophenyl-chloroacetamide 1: 213
Demeton-S sulphoxide 1: 225, 368, 369; 2: 32, Dichlorprop (2,4-DP) 2: 32, 163* 305, 369, 393
317 Dichlorprop, dinitro derivative of methyl ester
Demeton-S-methyl 1: 225,335, 361; 2: 26, 32,317 2: 369
Demeton-S-methyl sulphone 1: 225, 365, 368, Dichlorprop, isooctyl ester 2: 26
369; 2: 32, 317 Dichlorprop, methyl ester 2: 26, 163
Dichlorprop, trichloroethyl ester 2: 393 DMST 2: 32, 177, 195, 377

Dichlorvos 1: 64, 227, 283, 335, 371; 2: 26, 32, DNOC 2: 306, 437
313, 317 DNOC, methyl ether 2: 26
Diclofop 2: 400 Dodine 2: 26, 36
Diclofop, trichloroethyl ester 2: 400 2,4-DP, see Dichlorprop
Diclofop-methyl 1: 727*; 2: 32, 305, 317
Diclofop-methyl, metabolite 1: 127, 133 Edifenphos 2: 33, 306, 317
Dicloran 1: 399; 2: 32, 305, 317 Endosulfan 1: 228, 327; 2: 26
Dicofol 1: 77, 227, 283, 297, 327, 383; 2: 26, a-Endosulfan 1: 73, 77, 283, 383; 2: 33, 313,
32, 313, 317 317, 387
Dicrotophos 1: 77, 227, 383; 2: 32, 317 P-Endosulfan 1: 73, 77, 295, 383; 2: 33, 313,
Dieldrin 1: 51, 73, 77, 227, 283, 297, 309, 327, 317, 387
383; 2: 26, 32, 313, 317, 387 Endosulfan sulphate 1: 73, 77, 228, 295, 383;
Dienochlor 2: 32 2: 33, 313, 317
Dihydrocoumarin 2: 35 Endrin 1: 73, 77, 228, 297, 309, 327, 383;
Dimefox 1: 77, 227, 335, 383; 2: 32, 317 2: 26, 33, 317, 387
Dimethachlor 1: 227, 283; 2: 32, 313, 317 EPN 2: 33, 306, 317
Dimethirimol, methyl ether 2: 26 Ethidimuron 2: 33
Dimethoate 1: 64, 77, 227, 283, 335, 371, 383; Ethiofencarb 2: 26, 306, 349
2: 26, 32, 313, 317 Ethiofencarb, metabolites 2: 358
Dimethoate oxygen analogue, see Omethoate Ethion 1: 64, 73, 77, 228, 283, 297, 335, 371,
Dimethoxy anilazine 2: 32, 59 383; 2: 33, 313, 317
Dimethylaminosulphanilide, see DMSA Ethirimol 2: 26, 36
Dimethylaminosulphotoluidide, see DMST Ethirimol, methyl ether 2: 26
Dimethyl 5-nitroisophthalate, see Nitrothal- Ethoprophos 1: 228, 283; 2: 33, 313, 317
isopropyl, methyl esters of metabolites Ethoxyquin 2: 33
O,O-Dimethyl phosphonate 2: 211 O-Ethyl O-methyl phosphonate, see Fosetyl,
Dinitramine 2: 32, 305, 317 methyl ester
Dinobuton 2: 32, 197* 305, 317 Ethylene bisdithiocarbamate fungicides 1: 353,
Dinocap 1: 78, 227, 383; 2: 32, 317 407
Dinoseb 2: 306, 437 Ethylene thiourea 1: 135* 411
Dinoseb acetate 2: 32 Ethylvanillin 2: 35
Dinoterb 2: 306, 437 Etridiazole 2: 33
Dinoterb, methyl ether 2: 26 Etrimfos 1: 228, 283; 2: 27, 33, 313, 317
Dioxacarb 2: 26, 32, 306, 349 ETU, see Ethylene thiourea
Dioxathion 1: 73, 227, 283, 335; 2: 32, 313, 317
Diphenamid 2: 26, 32 Famophos 2: 33, 306, 317
Diphenylamine 2: 32 Fenamiphos 1: 228, 361; 2: 33, 313, 317
Diphenyl sulphone 2: 42, 404, 428, 429, 435 Fenamiphos sulphone 1: 228, 365, 368, 369
Dipropetryn 2: 32 Fenamiphos sulphoxide 1: 229, 368, 369
Dipropyl isocinchomeronate 2: 32 Fenarimol 2: 27, 33, 306, 313
Diquat 2: 36 Fenazaflor 2: 33
Disulfoton 1: 64, 227, 283, 335, 361, 371; Fenbutatin oxide 1: 229; 2: 343
2: 26, 32, 313, 317 Fenbutatin oxide, methyl derivative 2: 343
Disulfoton sulphone 1: 77, 227, 335, 365, 368, Fenchlorphos 1: 64, 73, 77, 229, 283, 297, 309,
369, 371, 383; 2: 32, 317 335, 371, 383; 2: 33, 313, 317
Disulfoton sulphoxide 1: 77, 228, 368, 369, Fenitrothion 1: 64, 77, 229, 283, 335, 371, 383;
383; 2: 32, 317 2: 27, 33, 313, 317
Ditalimfos 1: 77, 228, 283, 383; 2: 32, 313, Fenoprop 2: 33, 306, 393
317, 359 Fenoprop, isooctyl ester 2: 27
Dithianon 2: 33 Fenoprop, methyl ester 2: 27
Dithiocarbamate fungicides 1: 353, 407 Fenoprop, trichloroethyl ester 2: 393
Diuron 1: 228, 241, 251; 2: 26, 273, 435 Fenpropathrin 1: 229; 2: 333
DMSA 2: 32, 177, 195, 377 Fenson 1: 73, 77, 229, 383; 2: 33, 313, 317
Fensulfothion 1: 64, 229, 283, 335, 361, 371; Genite 2: 33, 307, 317
2: 33, 313, 317 Glufosinate 2: 217*
Fensulfothion sulphone 1: 229, 365, 368, 369; Glufosinate, metabolite, see 3-(Methylphos-
2: 33 phinico)propionic acid
Fensulfothion sulphone, oxygen analogue Glufosinate-ammonium, see Glufosinate
1: 369; 2: 33 Glyphosate 2: 229*
Fensulfothion sulphoxide, oxygen analogue Glyphosate, metabolite, see AMPA
2:33
Fenthion 1: 64, 229, 283, 335, 361, 371; 2 33, Halowax 1000-1051 2: 35
313, 317 Haloxyfop-(2-ethoxyethyl) 2: 307, 437
Fenthion sulphone 1: 229, 365, 368, 369; 2: 33 HCB 1: 51, 73, 77, 231, 295, 297, 309, 327,
Fenthion sulphone, oxygen analogue 1: 369; 383; 2: 33, 317, 387
2: 33 HCH isomers 1: 51, 231
Fenthion sulphoxide 1: 229, 368, 369; 2: 33 a-HCH 1: 51, 73, 77, 283, 297, 309, 327, 383;
Fenthion sulphoxide, oxygen analogue 2: 33 2: 33, 313, 317, 387
Fentin, methyl derivative 2: 343 (3-HCH 1: 51, 73, 77, 283, 297, 309, 327, 383;
Fentin acetate, chloride, hydroxide 1: 230; 2: 33, 313, 317, 387
2:343 y-HCH (lindane) 1: 51, 73, 77, 230, 283, 297,
Fenuron 1: 230, 241, 251; 2: 27, 273 309, 327, 383; 2: 27, 33, 313, 317, 387
Fenvalerate 1: 229; 2: 33, 317, 333 5-HCH 1: 73, 77, 297, 309, 327, 383; 2: 33,
Flamprop-isopropyl 2: 27 317, 387
Flamprop-methyl 2: 27 6-HCH 1: 297; 2: 33, 317, 387
Fluazifop-butyl 2: 33, 306, 437 Heptachlor 1: 51, 73, 77, 231, 283, 297, 309,
Flubenzimine 2: 33, 307, 317 327, 383; 2: 27, 33, 313, 317, 387
Fluchloralin 2: 33, 307, 313, 317 Heptachlor epoxide 1: 51, 73, 77, 231, 283,
Flucythrinate 2: 307, 333 297, 309, 327, 383; 2: 33, 313, 317, 387
Fluometuron 1: 230, 241, 251 Heptenophos 1: 149* 231, 283; 2: 33, 313, 317
Fluorenone 2: 128 Herbicides, substituted phenyl urea 1: 241, 251
Fluorodifen 2: 307, 313 Herbicides, phenoxyalkanoic acid 1: 171;
Fluotrimazole 1: 78, 230, 383; 2: 33, 317, 2:163* 369, 393
377 Herbicides, sulphonylurea 2: 145*
Flurenol 2: 127* Herbicides, triazine 1: 57, 265, 283, 347;
Flurenol, n-butyl ester 2: 127 2: 313, 403, 413
Fluroxypyr 2: 33 Hexabromobiphenyl 2: 35
Fluroxypyr, n-butyl ester 2: 33 Hexachlorobenzene, see HCB
Fluroxypyr-(l-methylheptyl) 2: 33, 307, 437 Hexachlorocyclohexane, see HCH
Fluvalinate 2: 33, 307, 317 Hexazinone 2: 33
Folpet 1: 143* 230, 283, 327, 401; 2: 33, 313, Hostatox (chlorinated indene) 2: 36
317, 359 3-Hydroxy-carbofuran 2: 113, 307, 349
Fonofos 1: 230, 283; 2: 33, 205* 313, 317 9-Hydroxyfluorene 2: 128
Fonofos oxygen analogue 2: 33
Formothion 1: 77, 230, 283, 383; 2: 27, 33, Imazalil 1: 78, 231, 383; 2: 33, 317, 377
313, 317 Insecticides, methyl carbamate 2: 349
Fosetyl 2: 211* Insecticides, organochlorine 1: 51, 57, 71, 77,
Fosetyl, metabolite, see Phosphorous acid 283, 297, 309, 325, 327, 383; 2: 313, 317, 387
Fosetyl, methyl ester 2: 211 Insecticides, organophosphorus 1: 57, 61, 68,
Fosetyl-aluminium, see Fosetyl 71, 77, 283, 297, 309, 325, 335, 361, 371, 383;
Fuberidazole 1: 78, 230, 383; 2: 33, 317, 377 2: 313, 317
Fumigants, bromine-containing 1: 377 Insecticides, pyrethrin 2: 323
Fungicides, bisdithiocarbamate 1: 353, 407 Insecticides, pyrethroid 2: 333
Fungicides, organotin 2: 343 Iodofenphos 1: 77, 231, 297, 383; 2: 27, 33,
Fungicides, phthalimide 1: 93, 105, 143, 401; 313, 317
2: 359, 377 Ioxynil 2: 33, 99*
Fungicides, thiuram disulphide 1: 353 Ioxynil, isooctyl ether 2: 27
Ioxynil, methyl ether 2: 27, 99 Mecoprop, isooctyl ester 2: 27

Ioxynil octanoate 2: 33, 99, 307, 317 Mecoprop, methyl ester 2: 27
Ipazine 2: 33 Mecoprop, trichloroethyl ester 2: 393
Iprodione 1: 231, 283; 2: 33, 313, 317 Mephosfolan 2: 33, 309, 317
Isobenzan 2: 33, 308, 317 Mercaptodimethur 2: 27, 309, 349
Isobumeton, see Secbumeton Mercaptodimethur, metabolites 2: 358
Isocarbamid 2: 33, 308, 317 Merphos 2: 33, 309, 317
Isodrin 1: 77, 231, 383; 2: 33, 317 Metalaxyl 1: 753* 232, 283; 2: 33, 313, 317,
Isofenphos 1: 232, 283; 2: 33, 313 437
Isomethiozin 2: 33 Metaldehyde 2: 239*
Isopropalin 2: 33, 308, 317 Metamitron 2: 27, 33, 309, 428, 429, 435
Isopropyl methyl 5-nitroisophthalate, see Metazachlor 2: 309, 313
Nitrothal-isopropyl, methyl esters of Methabenzthiazuron 2: 27, 33, 309, 577, 428,
metabolites 429, 435
Isopropyl 5-nitroisophthalate, see Nitrothal- Methacrifos 2: 33
isopropyl, metabolites Methamidophos 1: 77, 81% 232, 383; 2: 33, 317
Isoproturon 2: 27 Metham-sodium 2: 36
Methazole 2: 27
Jasmolins 1: 237; 2: 27, 323 Methidathion 1: 77, 232, 283, 335, 383; 2: 27,
33, 313, 317
3-Keto-carbofuran 2: 308, 349 Methiocarb, see Mercaptodimethur
8-Keto-endrin 2: 33, 308, 317 Methomyl 1: 757*; 2: 27, 309, 349
Methomyl oxime 1: 161
Lauronitrile 2: 349 Methomyl sulphone 2: 358
Lenacil 2: 27, 33, 308, 317, 435 Methomyl sulphoxide 2: 358
Lenacil, N-methyl derivative 2: 27 Methoprotryne 1: 232, 265, 283; 2: 34, 313
Leptophos 2: 33, 308, 317 Methoxychlor 1: 73, 77, 232, 283, 297, 309,
Leptophos, desbromo derivative 2: 33 327, 383; 2: 34, 313, 317, 387
Lindane, see y-HCH Methyl bromide 1: 232, 377
Linuron 1: 232, 241, 251; 2; 27, 33, 273, 317 Methyl carbamate insecticides 2: 349
Methyl (4-chloro-2-methyl-5,6-dinitrophenoxy)-
Malaoxon 1: 77, 232, 283, 335, 371, 383; 2: 33, acetate 2: 370
313, 317 Methyl 4-(4-chloro-2-methyl-5,6-dinitrophenoxy)-
Malathion 1: 64, 73, 77, 232, 283, 309, 335, butyrate 2: 370
371, 383; 2: 27, 33, 313, 317 Methyl-2-(4-chloro-2-methyl-5,6-dinitrophenoxy)-
Mancozeb 1: 232, 353, 407; 2: 36 propionate 2: 370
Maneb 1: 232, 353, 407; 2: 36 Methyl (2,4-dichloro-3,6-dinitrophenoxy)acetate
MCPA 1: 78; 2: 33, 308, 369, 393 2: 370
MCPA, dinitro derivative of methyl ester Methyl 2-(2,4-dichloro-3,6-dinitrophenoxy)-
2: 369 propionate 2: 370
MCPA, isooctyl ester 2: 374 Methyl 2,2-dichloropropionate 1: 117
MCPA, pentafluorobenzyl ester 2: 33 Methyl pentachlorophenyl sulphide 1: 232, 297
MCPA, trichloroethyl ester 2: 393 Methyl (2,4,5-trichloro-6-nitrophenoxy)acetate
MCPA-(2-butoxyethyl) 2: 33, 308, 317 2: 370
MCPB 2: 33, 308, 369, 393 3-Methylaniline 2: 269
MCPB, dinitro derivative of methyl ester 2: 369 6-Methylcoumarin 2: 36
MCPB, isooctyl ester 2: 27 Methylmetiram 2: 36
MCPB, methyl ester 2: 27 tris(2-Methyl-2-phenylpropyl)tin compound, see
MCPB, trichloroethyl ester 2: 393 Fenbutatin oxide
MCPP, see Mecoprop 3-(Methylphosphinico)propionic acid 2: 217
Mecarbam 2: 33, 308, 317 3-(Methylphosphinico)propionic acid dimethyl
Mecoprop 2: 33, 308, 369, 393 ester 2: 218
Mecoprop, dinitro derivative of methyl ester Metiram 1: 233, 407; 2: 36
2: 369 Metobromuron 1: 233, 241, 251; 2: 27, 34
Metolachlor 2: 34, 309, 313, 317, 406 Oxamyl 2: 34, 261*

Metoxuron 1: 233, 241; 2: 27 Oxamyl oximino derivative 2: 261
Metribuzin 1: 78, 233, 383; 2: 27, 34, 245* Oxychlordane (octachlor epoxide) 1: 77, 233,
313, 317, 435 297, 309, 383; 2: 34, 317
Metsulfuron 2: 145* Oxydemeton-methyl, see Demeton-S-methyl
Metsulfuron-methyl, see Metsulfuron sulphoxide
Mevinphos 1: 64, 77, 233, 283, 335, 371, 383;
2: 27, 34, 313, 317 Paraoxon 1: 64, 77, 234, 283, 335, 371, 383;
Mirex 2: 34, 309, 317 2: 34, 313, 317
Molinate 2: 34 Paraoxon-methyl 1: 234, 371; 2: 34, 317
Monalide 1: 246, 263; 2: 273 Paraquat 1: 177*; 2: 36
Monoacetyl-amitrole 2: 49 Parathion 1: 64, 68, 73, 77, 234, 283, 309, 335,
Monocrotophos 2: 27, 34, 309, 317 371, 383; 2: 27, 34, 313, 317, 428, 429, 435
Monolinuron 1: 233, 241, 251; 2: 27, 34, 317 Parathion-methyl 1: 64, 73, 77, 234, 283, 335,
Monuron 1: 233, 241, 251; 2: 273 371, 383; 2: 27, 34, 313, 317
Morfamquat 2: 36 PCB 1: 51, 73, 77, 234, 297, 318, 383;
Morphothion 2: 34, 309, 317 2: 35
Pencycuron, methyl derivative 2: 34
Pendimethalin 2: 27, 34, 310, 313, 317, 437
Nabam 1: 233, 407; 2: 36
PCCH, see Pentachlorocyclohex-1-ene
Naled 1: 64, 233, 283, 335, 371; 2: 34, 313, 317
Pentachloroaniline 1: 78, 234, 295, 297, 383;
2-Naphthoxyacetic acid 2: 34
2: 34, 317
1-Naphthylacetic acid 1: 167*
Pentachloroanisole 2: 34, 317
Napropamide 2: 27, 34
Pentachlorobenzene 1: 234, 297; 2: 34, 317
Neburon 1: 233, 241, 251; 2: 34, 273
Pentachlorocyclohex-1-ene 1: 234, 297
Nicotine 2: 27, 34
Pentachloronitrobenzene, see Quintozene
Nitralin 2: 34, 309, 317
Pentachlorophenol 2: 34
Nitrapyrin 2: 34
Permethrin 1: 234; 2: 27, 34, 317, 333
Nitrofen 1: 173*; 2: 27, 34, 310, 313, 317
Perthane 1: 234, 283; 2: 34, 313, 317
5-Nitroisophthalic acid, see Nitrothal-isopropyl,
Phenkapton 1: 64, 234, 283, 297, 335, 371;
metabolites
2: 34, 313, 317
4-Nitrophenol 2: 34
Phenmedipham 2: 27, 34, 269% 310, 317
Nitrothal-isopropyl 2: 34, 253* 310, 317, 437
Phenothrin 2: 341
Nitrothal-isopropyl, metabolites 2: 253
Phenoxyalkanoic acid herbicides 1: 171;
Nitrothal-isopropyl, methyl esters of metabolites
2:163* 369, 393
2:254
Phenthoate 2: 34, 310, 317
Norazin 2: 34
Phenylcarbamate herbicides 1: 246, 263
Norflurazon 2: 34
o-Phenylphenol 2: 294
Nuarimol 2: 27
Phenylurea herbicides 1: 241, 251
Phorate 1: 64, 235, 283, 335, 361, 371; 2: 34,
Octachlorodipropyl ether (S 421) 2: 34, 310, 313, 317
317 Phorate oxygen analogue 2: 34
Octachlorostyrene 2: 36, 310 Phorate sulphone 1: 235, 365, 368, 369
Octadecanonitrile 2: 349 Phorate sulphone, oxygen analogue 1: 369
Omethoate 1: 77, 233, 335, 371, 383; 2: 27, 34, Phorate sulphoxide 2: 34
377, 428, 429 Phosalone 1: 73, 77, 235, 283, 383; 2: 28, 34,
Organochlorine pesticides 1: 51, 57, 71, 77, 313, 317
283, 297, 309, 325, 327, 383; 2: 313, 317, 387 Phosfolan 2: 34
Organophosphorus pesticides 1: 57, 61, 68, 71, Phosmet 2: 34, 310, 359
77, 283, 297, 309, 325, 335, 361, 371, 383; Phosphamidon 1: 235, 295, 335; 2: 34, 317
2:313, 317 Phosphorous acid 2: 211
Organotin compounds 2: 343 Phoxim 2: 34, 310, 317
Organotin compounds, metabolites 2: 348 Phthalimide fungicides 1: 93, 105, 143, 401;
Oxadiazon 2: 27, 34, 310, 317 2: 359, 377
Phthalimides 2: 359 Simazine 1: 237, 265, 283, 347; 2: 28, 34, 313,
Piperonyl butoxide 1: 78, 235, 383; 2: 34, 317, 317, 403
323 Simeton 1: 237, 347; 2: 34
Piperonyl butoxide, tribromo derivative 2: 323 Simetryn 2: 34
Pirimicarb 1: 183*; 2: 28, 34, 310, 317, 349 Sprout suppressants 1: 321
Pirimicarb, metabolites 1: 183 Strobane T 2: 34, 311, 317
Pirimiphos-ethyl 2: 28, 34, 310, 317 Substituted acetanilides 1: 252
Pirimiphos-methyl 1: 191* 235, 283, 399; Substituted anilines 1: 241, 251
2: 28, 34, 313, 317 Substituted phenyl urea herbicides 1: 241, 251
Pirimiphos-methyl, metabolite 1: 192 Sulfallate 2: 34
Plifenate 2: 34 Sulfotep 1: 77, 237, 283, 335, 383; 2: 34, 313, 317
Polychlorinated biphenyls, see PCB Sulphonylurea herbicides 2: 145*
Polychlorinated terphenyl (50% Cl) 2: 36 Sulphur 2: 34, 287*
Potato sprout suppressants 1: 321 Sulprofos 2: 34, 311, 317
Procymidone 1: 235, 283; 2: 34, 313, 317
Profenofos 1: 235, 283; 2: 34, 313, 317 2,4,5-T 1: 78; 2: 34, 311, 369, 393
Profluralin 2: 34, 311, 313, 317 2,4,5-T, amyl ester 2: 34
Promecarb 2: 311, 349 2,4,5-T, hexyl ester 2: 34
Prometon 1: 235, 265; 2: 34 2,4,5-T, methyl ester 2: 374
Prometryn 1: 235, 265, 283; 2: 34, 313, 403 2,4,5-T, nitro derivative of methyl ester 2: 369
Propachlor 2: 28, 34, 275* 311, 317 2,4,5-T, trichloroethyl ester 2: 393
Propanil 2: 28, 34, 273, 311, 317 2,4,5-T-butyl 2: 34
Propargite 2: 34 2,4,5-T-isooctyl 2: 34, 374
Propazine 1: 235, 265, 283; 2: 34, 313, 403 TDE, see DDD
Propetamphos 2: 34 Tecnazene (TCNB) 1: 77, 238, 283, 327, 383;
Propham 1: 235, 246, 263, 321; 2: 28, 273 2: 28, 34, 313, 317
Propham, dibromo derivative 1: 321 Terbacil 2: 28, 35, 311, 313, 317
Propiconazole 2: 281* 311, 317 Terbacil, N-methyl derivative 2: 28
Propineb 1: 236, 353, 407; 2: 36 Terbufos 2: 35, 311, 313, 317
Propoxur 1: 78, 236, 383; 2: 28, 34, 317, 349 Terbumeton 1: 238, 347; 2: 437
Propylene bisdithiocarbamate fungicides 1: 353, Terbuthylazine 1: 238, 265, 347; 2: 35, 403
407 Terbutryn 1: 238, 265, 283, 347; 2: 35, 313,
Propylene thiourea 1: 411 317, 403
Propyzamide 1: 236, 283; 2: 34, 313, 317 2,3,4,6-Tetrachloroanisole 2: 35
Prothiofos 1: 236, 283; 2: 34, 313, 317 Tetrachlorobenzenes 2: 36
Pyrazon, see Chloridazon 1,2,3,4-Tetrachlorodibenzodioxin (1,2,3,4-TCDD)
Pyrazophos 1: 35, 68, 77, 197* 236, 283, 383; 2: 36
2: 34, 313, 317 l,2,4,5-Tetrachloro-3-nitrobenzene, see Tecnazene
Pyrethrins 1: 78, 236, 237, 283, 383; 2: 28, 34, 2,3,4,5-Tetrachloronitrobenzene 2: 35, 317
313, 317, 323, 341 Tetrachlorophenols 2: 36
Pyrethroids 2: 333 Tetrachlorvinphos 1: 203* 238, 283; 2: 28, 35,
313, 317
Quinalphos (chinalphos) 2: 34, 311, 313, 317 Tetradifon 1: 77, 238, 283, 383; 2: 35, 313, 317
Quinomethionate, see Chinomethionat Tetramethrin 2: 35, 311, 377, 341
Quintozene 1: 68, 73, 77, 237, 283, 297, 327, O,O,O',O'-Tetrapropyl dithiopyrophosphate 2: 35
383; 2: 28, 34, 313, 317 Tetrasul 1: 73, 77, 238, 283, 383; 2: 28, 35,
313, 317
Rabenzazole 1: 78, 237, 383; 2: 34, 317, 377 Thiabendazole 2: 28, 35, 291* 295*
Resmethrin 1: 78, 237, 383; 2: 28, 34, 377, 341 Thiofanox 2: 28, 312, 349
Thiofanox sulphone 2: 35, 358
S 421, see Octachlorodipropyl ether Thiofanox sulphoxide 2: 358
Salithion 2: 34, 311, 317 Thiometon 1: 238, 335; 2: 28, 35
Secbumeton 1: 237, 265, 347; 2: 34 Thionazin (zinophos) 1: 64, 77, 238, 283, 335,
Siduron 1: 237, 241, 251 371, 383; 2: 35, 373, 377
Thiophanate-methyl 2: 28, 69* 111 Trichloronat 2: 35, 312, 313, 317

Thiram (TMTD) 1: 238, 353; 2: 28 Trichlorophenols 2: 36
Thiuram disulphide fungicides 1: 353 Tricyclohexyltin compounds 2: 343
Tolclofos-methyl 2: 312, 313 Triclopyr-(2-butoxyethyl) 2: 312, 437
Tolylfluanid 1: 238, 283; 2: 35, 177* 191* 313, Tridemorph 2: 28
317, 377 Trietazine 2: 28, 35
Tolylfluanid, metabolite, see DMST 3-(Trifluormethyl)acetanilide 1: 252
Toxaphene, see Camphechlor 3-(Trifluormethyl)aniline 1: 247
2,4,5-TP, see Fenoprop Trifluralin 1: 78, 238, 383; 2: 28, 35, 313, 317
Triadimefon 1: 78, 238, 283, 383; 2: 35, 87* 2,4,6-Trimethylaniline 2: 270
313, 317, 377, 428, 429, 435 Triphenyltin compounds 2: 343
Triadimenol 1: 78, 238, 383; 2: 35, 87* 317,
377, 428, 429, 435 Urea herbicides 1: 241, 251
Tri-allate 2: 28, 35, 312, 313, 317
Triamiphos 1: 295, 399; 2: 35, 312, 317
Triazine herbicides 1: 57, 265, 283, 347; 2: 313, Vamidothion 1: 64, 238, 371; 2: 28, 35
403, 413 Vamidothion sulphone 1: 238, 371; 2: 35
Triazine herbicides, desalkyl metabolites 1: 57, Vamidothion sulphoxide 2: 35
347; 2: 413 Vinclozolin 1: 78, 213* 238, 283, 383; 2: 28,
Triazophos 1: 35, 68, 77, 209* 238, 283, 383; 35, 313, 317
2: 35, 313, 317 Vinclozolin, metabolites 1: 213
Triazoxide 2: 312, 317, 377 Vondozeb 1: 238, 407
2,4,6-Tribromo-3-methylaniline 2: 269
4,5,7-Tribromo-6-propyl-l,3-benzodioxol, see Zineb 1: 238, 353, 407; 2: 36
Piperonyl butoxide, tribromo derivative Zinochlor, see Anilazine
Trichlorfon 1: 238, 335; 2: 28, 35, 317 Zinophos, see Thionazin
Trichlorobenzenes 2: 36 Ziram 2: 36
Index of Analytical Materials
Alfalfa Apples (contd.)

Triazine herbicides 1: 265 Organophosphorus pesticides 1: 283, 335, 361,
371, 383; 2: 313, 317
Organotin compounds 2: 343
Almonds
Oxamyl 2: 261
Glufosinate 2: 217
Phenyl urea herbicides 1: 241
Phthalimide fungicides 1: 401
Animal fats Phthalimides 2: 359
Chlorthiophos 1: 109 Piperonyl butoxide 2: 323
Glufosinate 2: 217 Pirimicarb 1: 183
Nitrogen-containing pesticides 1: 383; 2: 317 Pirimiphos-methyl 1: 191
Organochlorine pesticides 1: 297, 309, 383; Pyrazophos 1: 197
2: 317 Pyrethrins 2: 323
Organophosphorus pesticides 1: 297, 309, 383; Pyrethroids 2: 333
2: 317 Sulphur 2: 287
Tetrachlorvinphos 1: 203
Thiuram disulphide fungicides 1: 353
Apple juice Tolylfluanid 2: 177, 191
Tolylfluanid 2: 191 Triadimefon 2: 87
Triadimenol 2: 87
Apple peel Triazine herbicides 1: 265, 283; 2: 313
Thiabendazole 2: 291 Triazophos 1: 209
Vinclozolin 1: 213
Apples
Acephate 1: 81 Applesauce
Amitrole 2: 49 Organophosphorus pesticides 1: 371
Binapacryl 2: 197 Tolylfluanid 2: 191
Bitertanol 2: 77, 87
Captafol 1: 93
Captan 1: 105 Apricots
Chlorthiophos 1: 109 Bitertanol 2: 87
Dichlobenil 2: 169 Organochlorine pesticides 1: 283; 2: 313
Dichlofluanid 2: 177 Organophosphorus pesticides 1: 283; 2: 313
Dinobuton 2: 197 Triazine herbicides 1: 265, 283; 2: 313
Dithiocarbamate fungicides 1: 353, 407
Ethylene thiourea 1: 135 Artichokes
Glufosinate 2: 217 Anilazine 2: 59
Heptenophos 1: 149 Bitertanol 2: 87
Metalaxyl 1: 153 Pyrazophos 1: 197
Methamidophos 1: 81
Methomyl 1: 161
Methyl carbamate insecticides 2: 349 Asparagus
1-Naphthylacetic acid 1: 167 Glufosinate 2: 217
Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 327
Nitrothal-isopropyl 2: 253 Organophosphorus pesticides 1: 335
Organochlorine pesticides 1: 283, 327, 383; Phenyl urea herbicides 1: 241
2: 313, 317 Triazine herbicides 1: 265
460 Index of Analytical Materials
Aubergines Barley, green matter (contd.)

Anilazine 2: 59 Dithiocarbamate fungicides 1: 353
Organochlorine pesticides 1: 283; 2: 313 Phthalimide fungicides 1: 401
Organophosphorus pesticides 1: 283; 2: 313 Propiconazole 2: 281
Organotin compounds 2: 343 Thiuram disulphide fungicides 1: 353
Triazine herbicides 1: 283; 2: 313 Triadimefon 2: 87
Triadimenol 2: 87
Banana peel
Thiabendazole 2: 291 Barley, straw
Anilazine 2: 59
Bananas Bitertanol 2: 77, 87
Bitertanol 2: 87 Bromoxynil 2: 99
Glufosinate 2: 217 Captafol 1: 99
Nitrogen-containing pesticides 1: 383; 2: 317 Chlorflurenol 2: 127
Organochlorine pesticides 1: 383; 2: 317 Dithiocarbamate fungicides 1: 353
Organophosphorus pesticides 1: 383; 2: 317 Flurenol 2: 127
Triadimefon 2: 87 Ioxynil 2: 99
Triadimenol 2: 87 Phenyl urea herbicides 1: 241, 251
Triazophos 1: 209 Phthalimide fungicides 1: 401
Propiconazole 2: 281
Barley, ears Pyrazophos 1: 197
Dithiocarbamate fungicides 1: 353 Thiuram disulphide fungicides 1: 353
Thiuram disulphide fungicides 1: 353 Triadimefon 2: 87
Triadimenol 2: 87
Barley, grains Triazine herbicides 1: 265
Anilazine 2: 59
Bitertanol 2: 77, 87 Beans, green
Bromine-containing fumigants 1: 377 Acephate 1: 81
Bromoxynil 2: 99 Anilazine 2: 59
Captafol 1: 99 Bitertanol 2: 87
Chlorflurenol 2: 127 Chlorthiophos 1: 109
Diclofop-methyl 1: 127 Dithiocarbamate fungicides 1: 407
Dithiocarbamate fungicides 1: 353 Ethylene thiourea 1: 135
Flurenol 2: 127 Glufosinate 2: 217
Ioxynil 2: 99 Methamidophos 1: 81
Metalaxyl 1: 153 Methyl carbamate insecticides 2: 349
Metribuzin 2: 245 Nitrogen-containing pesticides 1: 383; 2: 317
Nitrogen-containing pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 383; 2: 313,
Organochlorine pesticides 1: 383; 2: 317 317
Organophosphorus pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 283, 335, 371,
Phenyl urea herbicides 1: 241, 251 383; 2: 313, 317
Phthalimide fungicides 1: 401 Organotin compounds 2: 343
Pirimiphos-methyl 1: 191 Pirimicarb 1: 183
Propiconazole 2: 281 Pirimiphos-methyl 1: 191
Pyrazophos 1: 197 Pyrazophos 1: 197
Thiuram disulphide fungicides 1: 353 Triazine herbicides 1: 283; 2: 313
Triadimefon 2: 87 Vinclozolin 1: 213
Triadimenol 2: 87
Triazine herbicides 1: 265 Beans, pods
Vinclozolin 1: 213
Barley, green matter
Anilazine 2: 59 Beer
Bitertanol 2: 77, 87 Ethylene thiourea 1: 135
Captafol 1: 99 Glyphosate 2: 229
Beer (contd.) Brussels sprouts

Metalaxyl 1: 153 Chlorthiophos 1: 109
Nitrogen-containing pesticides 1: 383; 2: 317 Fonofos 2: 205
Organochlorine pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 371
Organophosphorus pesticides 1: 383; 2: 317 Pirimicarb 1: 183
Vinclozolin 1: 213 Pirimiphos-methyl 1: 191
Triazine herbicides 1: 265
Bilberries Triazophos 1: 209
Dalapon 1: 117
Butter
Organochlorine pesticides 1: 297, 309
Blackberries Organophosphorus pesticides 1: 297, 309
Vinclozolin 1: 213
Caraway
Black currants Glufosinate 2: 217
see Currants, black
Carrots
Acephate 1: 81
Bran
Dithiocarbamate fungicides 1: 353, 407
Pirimiphos-methyl 1: 191
Fonofos 2: 205
Heptenophos 1: 149
Brassicas* Methamidophos 1: 81
Carbofuran 2: 113 Methyl carbamate insecticides 2: 349
Carbosulfan 2: 113 Metribuzin 2: 245
Fonofos 2: 205 Nitrogen-containing pesticides 1: 383; 2: 317
Glufosinate 2: 217 Organochlorine pesticides 1: 283, 383; 2: 313,
Metalaxyl 1: 153 317
Metaldehyde 2: 239 Organophosphorus pesticides 1: 283, 335, 361,
Methyl carbamate insecticides 349 371, 383; 2: 313, 317
Nitrogen-containing pesticides 383; 2: 317 Oxamyl 2: 261
Organochlorine pesticides 1: 283, 383; 2: 313, Phenyl urea herbicides 1: 241
317 Pirimiphos-methyl 1: 191
Organophosphorus pesticides 1: 283, 335, 361, Pyrazophos 1: 197
371, 383; 2: 313, 317 Pyrethroids 2: 333
Phenyl urea herbicides 1: 241 Thiuram disulphide fungicides 1: 353
Pirimicarb 1: 183 Triazine herbicides 1: 265, 283; 2: 313
Propachlor 2: 275 Cauliflower
Pyrethroids 2: 333 Chlorthiophos 1: 109
Tetrachlorvinphos 1: 203 Ethylene thiourea 1: 135
Triazine herbicides 1: 265, 283; 2: 313 Fonofos 2: 205
Triazophos 1: 209 Nitrofen 1: 173
Vinclozolin 1: 213 Nitrogen-containing pesticides 1: 383; 2: 317
Organochlorine pesticides 1: 327, 383; 2: 317
Bread Organophosphorus pesticides 1: 335, 371, 383;
Glyphosate 2: 229 2: 317
Pirimiphos-methyl 1: 191 Propachlor 2: 275
Pyrethroids 2: 333
Triazophos 1: 209
Broad beans
Metribuzin 2: 245 Celeriac, bulbs
Dithiocarbamate fungicides 1: 353
Broccoli Ethylene thiourea 1: 135
Metalaxyl 1: 153 Organochlorine pesticides 1: 283; 2: 313
Celeriac, bulbs (contd.) Cereal grains (contd.)

Organophosphorus pesticides 1: 283, 335; Thiuram disulphide fungicides 1: 353
2:313 Triadimefon 2: 87
Organotin compounds 2: 343 Triadimenol 2: 87
Oxamyl 2: 261 Triazine herbicides 1: 265
Phenyl urea herbicides 1: 241 Triazophos 1: 209
Pyrethroids 2: 333 Cereal green matter*
Thiuram disulphide fungicides 1: 353 Anilazine 2: 59
Triazine herbicides 1: 265, 283; 2: 313 Bitertanol 2: 77, 87
Bromoxynil 2: 99
Celeriac, leaves Captafol 1: 99
Dithiocarbamate fungicides 1: 353 Carbendazim 2: 107
Ethylene thiourea 1: 135 Chlorsulfuron 2: 145
Organotin compounds 2: 343 2,4-D 2: 163
Oxamyl 2: 261 Dalapon 1: 117
Thiuram disulphide fungicides 1: 353 Dichlorprop 2: 163
Cereal grains* Ioxynil 2: 99
Anilazine 2: 59 Metsulfuron 2: 145
Benomyl 2: 69 Phthalimide fungicides 1: 401
Bitertanol 2: 77, 87 Phthalimides 2: 359
Bromine-containing fumigants 1: 377 Propiconazole 2: 281
Bromoxynil 2: 99 Thiuram disulphide fungicides 1: 353
Captafol 1: 93, 99 Triadimefon 2: 87
Carbendazim 2: 69, 107 Triadimenol 2: 87
Chlorflurenol 2: 127
Chlorsulfuron 2: 145 Cereal products, milled
2,4-D 2: 163 Glyphosate 2: 229
Dalapon 1: 117 Bromine-containing fumigants 1: 377
Dichlorprop 2: 163
Diclofop-methyl 1: 127 Cereal straw*
Dithiocarbamate fungicides 1: 353 Anilazine 2: 59
Ethylene thiourea 1: 135 Benomyl 2: 69
Flurenol 2: 127 Bitertanol 2: 77, 87
Glufosinate 2: 217 Bromoxynil 2: 99
Glyphosate 2: 229 Captafol 1: 93, 99
Heptenophos 1: 149 Carbendazim 2: 69, 107
Ioxynil 2: 99 Chlorflurenol 2: 127
Metalaxyl 1: 153 Chlorsulfuron 2: 145
Metribuzin 2: 245 2,4-D 2: 163
Metsulfuron 2: 145 Dalapon 1: 117
Nitrofen 1: 173 Dichlorprop 2: 163
Nitrogen-containing pesticides 1: 383; 2: 317 Diclofop-methyl 1: 127
Organochlorine pesticides 1: 383; 2: 317 Dithiocarbamate fungicides 1: 353
Organophosphorus pesticides 1: 383; 2: 317 Ethylene thiourea 1: 135
Phenyl urea herbicides 1: 241, 251 Flurenol 2: 127
Phthalimide fungicides 1: 401 Glyphosate 2: 229
Phthalimides 2: 359 Ioxynil 2: 99
Pirimiphos-methyl 1: 191 Metribuzin 2: 245
Propiconazole 2: 281 Metsulfuron 2: 145
Pyrazophos 1: 197 Nitrofen 1: 173
Pyrethroids 2: 333 Phenyl urea herbicides 1: 241, 251
Thiophanate-methyl 2: 69 Phthalimide fungicides 1: 401
Cereal straw (contd.) Chives

Propiconazole 2: 281 Pirimicarb 1: 183
Pyrazophos 1: 197
Thiophanate-methyl 2: 69
Chocolate
Organochlorine pesticides 1: 297
Triadimefon 2: 87
Organophosphorus pesticides 1: 297
Triadimenol 2: 87
Citrus fruit*
Cheese, cheese spread Chlorthiophos 1: 109
Organochlorine pesticides 1: 297, 309 Glufosinate 2: 217
Organophosphorus pesticides 1: 297, 309 Heptenophos 1: 149
Metalaxyl 1: 153
Cherries Nitrogen-contanining pesticides 1: 383; 2: 317
Amitrole 2: 49 Organochlorine pesticides 1: 283, 327, 383;
Bitertanol 2: 77, 87 2: 313, 317
Chlorthiophos 1: 109 Organophosphorus pesticides 1: 283, 383;
Dithiocarbamate fungicides 1: 353 2: 313, 317
Ethylene thiourea 1: 135 Organotin compounds 2: 343
Heptenophos 1: 149 Oxamyl 2: 261
Methyl carbamate insecticides 2: 349 Pirimiphos-methyl 1: 191
Nitrogen-containing pesticides 1: 383; 2: 317 Triazine herbicides 1: 265, 283; 2: 313
Organochlorine pesticides 1: 283, 327, 383; Triazophos 1: 209
2: 313, 317
Organophosphorus pesticides 1: 283, 335, 361, Citrus fruit peel*
383; 2: 313, 317 Thiabendazole 2: 291, 295
Pyrethroids 2: 333
Triazine herbicides 1: 283; 2: 313 Clover
Vinclozolin 1: 213 Anilazine 2: 59
Cherries: conserves, juice, press pulp Cocoa

Bitertanol 2: 77 Triazine herbicides 1: 265
Cherries, sour
Glufosinate 2: 217 Cocoa butter
Vinclozolin 1: 213 Organochlorine pesticides 1: 297, 309
Organophosphorus pesticides 1: 297, 309
Chicoree leaves
Metaldehyde 2: 239 Cocoa powder
Chillies Organophosphorus pesticides 1: 297
Organochlorine pesticides 1: 283; 2: 313
Organophosphorus pesticides 1: 283; 2: 313
Cocoa products*
Triazine herbicides 1: 283; 2: 313
Nitrogen-containing pesticides 1: 383; 2: 317
Organochlorine pesticides 1: 297, 309, 383;
Chinese cabbage
2:317
Glufosinate 2: 217
Organophosphorus pesticides 1: 297, 309, 383;
Metaldehyde 2: 239
2: 317
Organophosphorus pesticides 1: 283, 371; 2: 313
Triazine herbicides 1: 283; 2: 313 Coconut oil
Triazophos 1: 209 Organochlorine pesticides 1: 297, 309
Vinclozolin 1: 213 Organophosphorus pesticides 1: 297, 309
Coffee Cucumbers (contd.)

Nitrogen-containing pesticides 1: 383; 2: 317 Triadimefon 2: 87
Organochlorine pesticides 1: 383; 2: 317 Triadimenol 2: 87
Organophosphorus pesticides 1: 383; 2: 317 Triazine herbicides 1: 283; 2: 313
Triazine herbicides 1: 265 Vinclozolin 1: 213
Triazophos 1: 209
Curly kale
Coffee, raw Nitrogen-containing pesticides 1: 383; 2: 317
Anilazine 2: 59 Organochlorine pesticides 1: 383; 2: 317
Oxamyl 2: 261 Organophosphorus pesticides 1: 361, 371, 383;
2: 317
Condensed milk Phenyl urea herbicides 1: 241
Organochlorine pesticides 1: 297, 309 Pirimiphos-methyl 1: 191
Organophosphorus pesticides 1: 297, 309 Tetrachlorvinphos 1: 203
Corn, sweet
Triazophos 1: 209
Currants, black
Corn salad
Heptenophos 1: 149
Metaldehyde 2: 239 Pirimiphos-methyl 1: 191
Organochlorine pesticides 1: 283; 2: 313 Thiuram disulphide fungicides 1: 353
Organophosphorus pesticides 1: 283; 2: 313 Vinclozolin 1: 213
Vinclozolin 1: 213
Currants, red
Cotton leaves Chlorthiophos 1: 109
Phenyl urea herbicides 1: 241 Dichlobenil 2: 169
Triazine herbicides 1: 265 Dithiocarbamate fungicides 1: 353
Heptenophos 1: 149
Cotton seed Nitrogen-containing pesticides 1: 383; 2: 317
Phenyl urea herbicides 1: 241 Organochlorine pesticides 1: 383; 2: 317
Triazine herbicides 1: 265 Organophosphorus pesticides 1: 383; 2: 317
Oxamyl 2: 261 Piperonyl butoxide 2: 323
Pyrethrins 2: 323
Cucumbers
Binapacryl 2: 197
Dinobuton 2: 197 Cut lettuce
Dithiocarbamate fungicides 1: 353, 407 Metaldehyde 2: 239
Flurenol 2: 127
Dairy products*
Organochlorine pesticides 1: 283, 383; 2: 313,
317
Organophosphorus pesticides 1: 283, 335, 361,
2: 317
383; 2: 313, 317
Piperonyl butoxide 2: 323
2: 317
Pyrazophos 1: 197
Pyrethrins 2: 323 Dandelion
Pyrethroids 2: 333 Organochlorine pesticides 1: 283; 2: 313
Sulphur 2: 287 Organophosphorus pesticides 1: 283; 2: 313
Dried egg products* Fats, animal*

Nitrogen-containing pesticides 1: 383; 2: 317 Chlorthiophos 1: 109
Organochlorine pesticides 1: 297, 309, 383; Glufosinate 2: 217
2:317 Nitrogen-containing pesticides 1: 383; 2: 317
Organophosphorus pesticides 1: 297, 309, 383; Organochlorine pesticides 1: 297, 309, 383;
2: 317 2: 317
2: 317
Dried egg yolk
Organophosphorus pesticides 1: 297, 309 Fats, vegetable*
Chlorthiophos 1: 109
Glufosinate 2: 217
Dried fruit
Bromine-containing fumigants 1: 377
2: 317
Dried mushrooms Organophosphorus pesticides 1: 297, 309, 383;
Bromine-containing fumigants 1: 377 2: 317
Dried skim milk
Organophosphorus pesticides 1: 297 Fish
Dried vegetables
Fodder beet, foliage and root
Phenmedipham 2: 269
Dried whole egg
Organophosphorus pesticides 1: 297, 309 Fruit, dried and fresh
Egg products, dried*
see Dried egg products Garlic
Anilazine 2: 59
Vinclozolin 1: 213
Egg yolk, dried*
see Dried egg yolk
Gooseberries
Vinclozolin 1: 213
Eggs, fresh
Organophosphorus pesticides 1: 297, 309 Grapefruit
Heptenophos 1: 149
Oxamyl 2: 261
Endives
Metaldehyde 2: 239
Organochlorine pesticides 1: 283, 327; 2: 313 Grapefruit peel
Organophosphorus pesticides 1: 283; 2: 313 Thiabendazole 2: 291, 295
Pyrethrins 2: 323 Grape juice
Pyrethroids 2: 333 Ethylene thiourea 1: 135
Triazine herbicides 1: 283; 2: 313 Vinclozolin 1: 213
Evening primrose oil Grape must

Glufosinate 2: 217 Chlorthiophos 1: 109
Grapes Greengages
Acephate 1: 81 Organochlorine pesticides 1: 327
Amitrole 2: 49
Captafol 1: 93 Groundnuts
Carbofuran 2: 113 see Peanuts
Carbosulfan 2: 113
Chlorthiophos 1: 109 Hay
Copper oxychloride 2: 153 Dalapon 1: 117
Cymoxanil 2: 157 Glyphosate 2: 229
2,4-D 2: 163
Dichlobenil 2: 169
Head cabbage
Dichlofluanid 2: 177, 191
Carbofuran 2: 113
Dichlorprop 2: 163
Carbosulfan 2: 113
Fonofos 2: 205
Ethylene thiourea 1: 135
Metalaxyl 1: 153
Folpet 1: 143
Methyl carbamate insecticides 2: 349
Fosetyl 2: 211
Glyphosate 2: 229
Metalaxyl 1: 153
317
Methamidophos 1: 81
Organophosphorus pesticides 1: 283, 335, 361,
Methomyl 1: 161
371, 383; 2: 313, 317
1-Naphthylacetic acid 1: 167
Pirimicarb 1: 183
317
Propachlor 2: 275
Triazine herbicides 1: 265, 283; 2: 313
2: 313, 317
Triazophos 1: 209
Oxamyl 2: 261
Phenyl urea herbicides 1: 241 Herbs (spices)
Phthalimide fungicides 1: 401 Nitrogen-containing pesticides 1: 383; 2: 317
Phthalimides 2: 359 Organochlorine pesticides 1: 383; 2: 317
Propiconazole 2: 281 Organophosphorus pesticides 1: 383; 2: 317
Pyrazophos 1: 197
Pyrethroids 2: 333 Honey
Sulphur 2: 287 Organochlorine pesticides 1: 283; 2: 313
Tetrachlorvinphos 1: 203 Organophosphorus pesticides 1: 283; 2: 313
Tolylfluanid 2: 177
Triadimefon 2: 87 Hop cones
Triadimenol 2: 87 Anilazine 2: 59
Triazine herbicides 1: 265, 283; 2: 313 Ethylene thiourea 1: 135
Vinclozolin 1: 213 Fosetyl 2: 211
Heptenophos 1: 149
Grass Metalaxyl 1: 153
2,4-D 2: 163 Methomyl 1: 161
Dichlobenil 2: 169 Nitrogen-containing pesticides 1: 383; 2: 317
Dichlorprop 2: 163 Organochlorine pesticides 1: 383; 2: 317
Glyphosate 2: 229 Organophosphorus pesticides 1: 383; 2: 317
Oxamyl 2: 261 Pyrazophos 1: 197
Triazine herbicides 1: 265 Sulphur 2: 287
Triadimefon 2: 87
Grass silage Triadimenol 2: 87
Glyphosate 2: 229 Vinclozolin 1: 213
Hop foliage Leeks (contd.)

Sulphur 2: 287 Pirimiphos-methyl 1: 191
Propachlor 2: 275
Instant foods based on milk Thiuram disulphide fungicides 1: 353
Organochlorine pesticides 1: 297 Triazine herbicides 1: 265, 283; 2: 313
Jams Lemons
Bitertanol 2: 77 Glufosinate 2: 217
Vinclozolin 1: 213 Organochlorine pesticides 1: 327
Juices* Triazophos 1: 209
Chlorthiophos 1: 109 Lettuce
Ethylene thiourea 1: 135 Acephate 1: 81
Metalaxyl 1: 153 Benomyl 2: 69
Nitrogen-containing pesticides 1: 383; 2: 317 Bromine-containing fumigants 1: 377
Organochlorine pesticides 1: 383; 2: 317 Carbendazim 2: 69
Organophosphorus pesticides 1: 383; 2: 317 Carbofuran 2: 113
Tolylfluanid 2: 191 Carbosulfan 2: 113
Triadimefon 2: 87 Chlorthiophos 1: 109
Triadimenol 2: 87 Dichlofluanid 2: 177
Vinclozolin 1: 213 Dithiocarbamate fungicides 1: 353, 407
Ethylene thiourea 1: 135
Kidneys Folpet 1: 143
Chlorthiophos 1: 109 Fosetyl 2: 211
Glufosinate 2: 217 Heptenophos 1: 149
Metalaxyl 1: 153
Kiwi fruit Metaldehyde 2: 239
Glufosinate 2: 217 Methamidophos 1: 81
Methomyl 1: 161
Kohlrabi Methyl carbamate insecticides 2: 349
Fonofos 2: 205 Nitrogen-containing pesticides 1: 383; 2: 317
Heptenophos 1: 149 Organochlorine pesticides 1: 283, 383; 2: 313,
Methyl carbamate insecticides 2: 349 317
Nitrogen-containing pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 283, 335, 361,
Organochlorine pesticides 1: 283, 327, 383; 371, 383; 2: 313, 317
2: 313, 317 Oxamyl 2: 261
Organophosphorus pesticides 1: 283, 335, 371, Phenyl urea herbicides 1: 241
383; 2: 313, 317 Phthalimide fungicides 1: 401
Triazine herbicides 1: 283; 2: 313 Phthalimides 2: 359
Lard Pirimicarb 1: 183
Organochlorine pesticides 1: 297, 309 Pirimiphos-methyl 1: 191
Organophosphorus pesticides 1: 297, 309 Pyrethrins 2: 323
Thiophanate-methyl 2: 69
Leeks Thiuram disulphide fungicides 1: 353
Dithiocarbamate fungicides 1: 353 Tolylfluanid 2: 177
Ethylene thiourea 1: 135 Triazine herbicides 1: 265, 283; 2: 313
Nitrofen 1: 173
Organochlorine pesticides 1: 283; 2: 313 Liver
Organophosphorus pesticides 1: 283, 361; 2: 313 Chlorthiophos 1: 109
Phenyl urea herbicides 1: 241 Glufosinate 2: 217
Maize cobs Meat

Metalaxyl 1: 153 Chlorthiophos 1: 109
Tetrachlorvinphos 1: 203 Glufosinate 2: 217
Maize, green matter 2: 317
Chlorthiophos 1: 109 Organophosphorus pesticides 1: 297, 309, 383;
Metalaxyl 1: 153 2: 317
Triazophos 1: 209
Meat products*
Maize, kernels Glufosinate 2: 217
Acephate 1: 81 Nitrogen-containing pesticides 1: 383; 2: 317
Carbofuran 2: 113 Organochlorine pesticides 1:297, 309, 383; 2: 317
Carbosulfan 2: 113 Organophosphorus pesticides 1: 297, 309, 383;
Chlorthiophos 1: 109 2: 317
Fonofos 2: 205
Glufosinate 2: 217 Melons
Heptenophos 1: 149 Bitertanol 2: 87
Methamidophos 1: 81 Nitrogen-containing pesticides 1: 383; 2: 317
Phenyl urea herbicides 1: 241 Organochlorine pesticides 1: 327, 383; 2: 317
Propachlor 2: 275 Organophosphorus pesticides 1: 383; 2: 317
Pyrethroids 2: 333 Organotin compounds 2: 343
Triazine herbicides 1: 265 Pyrazophos 1: 197
Triazophos 1: 209 Triadimefon 2: 87
Triadimenol 2: 87
Maize, leaves Milk, condensed

Metalaxyl 1: 153 see Condensed milk
Triazine herbicides 1: 265 Milk, fresh, milk powder
Maize, stalks Organochlorine pesticides 1:297, 309,383; 2: 317
Metalaxyl 1: 153 Organophosphorus pesticides 1: 297, 309, 383;
Phenyl urea herbicides 1: 241 2: 317
Milled cereal products
Mandarine oranges
Chlorthiophos 1: 109 Mirabellas
Organochlorine pesticides 1: 283, 327; 2: 313 Glufosinate 2: 217
Triazine herbicides 1: 283; 2: 313 Mud (sludge)
Diclofop-methyl 1: 127
Mangold
Chloridazon 2: 135 Mushrooms
Diclofop-methyl 1: 127 Bromine-containing fumigants 1: 377
Phenmedipham 2: 269 Dalapon 1: 117
Piperonyl butoxide 2: 323 Organochlorine pesticides 1: 283; 2: 313
Pyrethrins 2: 323 Organophosphorus pesticides 1: 283; 2: 313
Margarine
Organochlorine pesticides 1: 297, 309 Mushrooms, dried
Organophosphorus pesticides 1: 297, 309 Bromine-containing fumigants 1: 377
Must Oils, vegetable*

Amitrole 2: 49 see Fats, vegetable
Cymoxanil 2: 157
Dichlofluanid 2: 177, 191
Olive oil
Tolylfluanid 2: 177
Triadimefon 2: 87
Triadimenol 2: 87 Onions, bulbs
Anilazine 2: 59
Nectarines
Fonofos 2: 205
Metalaxyl 1: 153
Nitrofen 1: 173
Nuts Nitrogen-containing pesticides 1: 383; 2: 317
Bromine-containing fumigants 1: 377 Organochlorine pesticides 1: 383; 2: 317
Nitrogen-containing pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 361, 383;
Organochlorine pesticides 1: 383; 2: 317 2: 317
Organophosphorus pesticides 1: 383; 2: 317 Phenyl urea herbicides 1: 241
Oat, grains Propachlor 2: 275
Bromine-containing fumigants 1: 377 Pyrethroids 2: 333
Heptenophos 1: 149 Triazine herbicides 1: 265
Nitrogen-containing pesticides 1: 383; 2: 317 Vinclozolin 1: 213
Organophosphorus pesticides 1: 383; 2: 317 Onions, stalks
Phenyl urea herbicides 1: 241, 251 Vinclozolin 1: 213
Orange juice
Oat, straw Metalaxyl 1: 153
Phenyl urea herbicides 1: 241, 251
Orange peel
Metalaxyl 1: 153
Oil seeds* Thiabendazole 2: 291, 295
Bitertanol 2: 87
Carbofuran 2: 113
Oranges
Carbosulfan 2: 113
Glufosinate 2: 217
Heptenophos 1: 149
Metalaxyl 1: 153
Glufosinate 2: 217
Glyphosate 2: 229
Metalaxyl 1: 153
Oxamyl 2: 261
Triazophos 1: 209
Oxamyl 2: 261
Pirimiphos-methyl 1: 191 Parsley
Pyrethroids 2: 333 Bromine-containing fumigants 1: 377
Triazine herbicides 1: 265 Organochlorine pesticides 1: 283; 2: 313
Triazophos 1: 209 Organophosphorus pesticides 1: 283; 2: 313
Vinclozolin 1: 213 Triazine herbicides 1: 283; 2: 313
Peaches Pears (contd.)

Bitertanol 2: 87 Organophosphorus pesticides 1: 283, 335, 383;
Captafol 1: 93 2: 313, 317
Chlorthiophos 1: 109 Phenyl urea herbicides 1: 241
Dithiocarbamate fungicides 1: 353 Phthalimide fungicides 1: 401
1-Naphthylacetic acid 1: 167 Pirimiphos-methyl 1: 191
Nitrogen-containing pesticides 1: 383; 2: 317 Thiuram disulphide fungicides 1: 353
Organochlorine pesticides 1: 283, 383; 2: 313, Tolylfluanid 2: 191
317 Triadimefon 2: 87
Organophosphorus pesticides 1: 283, 383; Triadimenol 2: 87
2: 313, 317 Triazine herbicides 1: 265, 283; 2: 313
Organotin compounds 2: 343 Vinclozolin 1: 213
Oxamyl 2: 261
Pyrazophos 1: 197
Tetrachlorvinphos 1: 203 Peas
Thiuram disulphide fungicides 1: 353 Ethylene thiourea 1: 135
Triadimefon 2: 87 Glufosinate 2: 217
Triadimenol 2: 87 Glyphosate 2: 229
Triazine herbicides 1: 265, 283; 2: 313 Metalaxyl 1: 153
Vinclozolin 1: 213 Phenyl urea herbicides 1: 241
Propachlor 2: 275
Peanut foliage
Oxamyl 2: 261
Peanut oil Pepper seeds

Organochlorine pesticides 1: 297, 309 Metalaxyl 1: 153
Organophosphorus pesticides 1: 297, 309 Nitrogen-containing pesticides 1: 383; 2: 317
Peanut shells Organophosphorus pesticides 1: 383; 2: 317
Bitertanol 2: 87
Oxamyl 2: 261
Peppers, sweet
Peanuts Anilazine 2: 59
Bitertanol 2: 87 Heptenophos 1: 149
Nitrogen-containing pesticides 1: 383; 2: 317 Nitrogen-containing pesticides 1: 383; 2: 317
Organochlorine pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 283, 327, 383;
Organophosphorus pesticides 1: 383; 2: 317 2: 313, 317
Oxamyl 2: 261 Organophosphorus pesticides 1: 283, 335, 383;
Pirimiphos-methyl 1: 191 2: 313, 317
Oxamyl 2: 261
Pear juice, pear conserves Triadimefon 2: 87
Tolylfluanid 2: 191 Triadimenol 2: 87
Pears
Amitrole 2: 49
Bitertanol 2: 77, 87 Pineapples
Captan 1: 105 Metalaxyl 1: 153
Dichlobenil 2: 169 Nitrogen-containing pesticides 1: 383; 2: 317
Dithiocarbamate fungicides 1: 353 Organochlorine pesticides 1: 283, 383; 2: 313,
1-Naphthylacetic acid 1: 167 317
Nitrogen-containing pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 283, 335, 383;
Organochlorine pesticides 1: 283, 383; 2: 313, 2: 313, 317
317 Triazine herbicides 1: 265, 283; 2: 313
Plums Potatoes (contd.)

Acephate 1: 81 Piperonyl butoxide 2: 323
Bitertanol 2: 77, 87 Propham 1: 321
Chlorthiophos 1: 109 Pyrazophos 1: 197
Glufosinate 2: 217 Pyrethrins 2: 323
Methamidophos 1: 81 Pyrethroids 2: 333
1-Naphthylacetic acid 1: 167 Thiuram disulphide fungicides 1: 353
Nitrogen-containing pesticides 1: 383; 2: 317 Triazine herbicides 1: 265, 283; 2: 313
Organochlorine pesticides 1: 283, 383; 2: 313, Triazophos 1: 209
317 Vinclozolin 1: 213
Organophosphorus pesticides 1: 283, 383;
2: 313, 317
Organotin compounds 2: 343 Poultry meat
Piperonyl butoxide 2: 323 Organochlorine pesticides 1: 297, 309
Pyrethrins 2: 323 Organophosphorus pesticides 1: 297, 309
Vinclozolin 1: 213
Radicchio
Anilazine 2: 59
Plums: jam, puree
Bitertanol 2: 77
Radishes
Poppy seeds Fonofos 2: 205
Phenyl urea herbicides 1: 251 Metaldehyde 2: 239
Potato peel Organochlorine pesticides 1: 283; 2: 313
Thiabendazole 2: 291 Organophosphorus pesticides 1: 283; 2: 313
Propachlor 2: 275
Potato tops Triazine herbicides 1: 265, 283; 2: 313
Metribuzin 2: 245 Vinclozolin 1: 213
Potatoes Radishes, small

Anilazine 2: 59 Captafol 1: 93
Captafol 1: 93 Dithiocarbamate fungicides 1: 353
Chlorpropham 1: 321 Fonofos 2: 205
Chlorthiophos 1: 109 Heptenophos 1: 149
Cymoxanil 2: 157 Organochlorine pesticides 1: 283; 2: 313
2,4-D 2: 163 Organophosphorus pesticides 1: 283, 335;
Dalapon 1: 117 2:313
Dichlorprop 2: 163 Pirimiphos-methyl 1: 191
Dithiocarbamate fungicides 1: 353, 407 Thiuram disulphide fungicides 1: 353
Ethylene thiourea 1: 135 Triazine herbicides 1: 265, 283; 2: 313
Glufosinate 2: 217 Triazophos 1: 209
Heptenophos 1: 149 Vinclozolin 1: 213
Metalaxyl 1: 153
Metribuzin 2: 245
Nitrogen-containing pesticides 1: 383; 2: 317 Rape, green matter
Organochlorine pesticides 1: 283, 327, 383; Anilazine 2: 59
2: 313, 317 Carbofuran 2: 113
Organophosphorus pesticides 1: 283, 335, 361, Carbosulfan 2: 113
383; 2: 313, 317 Dithiocarbamate fungicides 1: 353
Oxamyl 2: 261 Glufosinate 2: 217
Phenyl urea herbicides 1: 241 Pyrethroids 2: 333
Phthalimides 2: 359 Thiuram disulphide fungicides 1: 353
Rape, seeds Rye, grains

Carbofuran 2: 113 Bromine-containing fumigants 1: 377
Carbosulfan 2: 113 Captafol 1: 99
Chlorthiophos 1: 109 Nitrogen-containing pesticides 1: 383; 2: 317
Diclofop-methyl 1: 127 Organochlorine pesticides 1: 383; 2: 317
Glufosinate 2: 217 Organophosphorus pesticides 1: 383; 2: 317
Glyphosate 2: 229 Phenyl urea herbicides 1: 241
Pyrethroids 2: 333 Propiconazole 2: 281
Triazophos 1: 209 Triadimefon 2: 87
Vinclozolin 1: 213 Triadimenol 2: 87
Rape, straw
Vinclozolin 1: 213 Rye, green matter
Captafol 1: 99
Propiconazole 2: 281
Rape oil Triadimefon 2: 87
Chlorthiophos 1: 109 Triadimenol 2: 87
Raspberries Rye, straw

Dichlofluanid 2: 177 Captafol 1: 99
Pirimiphos-methyl 1: 191 Phenyl urea herbicides 1: 241
Tolylfluanid 2: 177 Propiconazole 2: 281
Triazine herbicides 1: 265 Triadimefon 2: 87
Vinclozolin 1: 213 Triadimenol 2: 87
Red beet, edible root Sage

Chloridazon 2: 135 Bromine-containing fumigants 1: 377
Phenmedipham 2: 269
Red beet, foliage Savoy cabbage

Chloridazon 2: 135 Acephate 1: 81
Diclofop-methyl 1: 127 Chlorthiophos 1: 109
Methamidophos 1: 81
Red cabbage
Fonofos 2: 205
Organochlorine pesticides 1: 283; 2: 313 Skim milk powder
Organophosphorus pesticides 1: 283, 335, 361, Organochlorine pesticides 1: 297
371; 2: 313 Organophosphorus pesticides 1: 297
Pirimiphos-methyl 1: 191 Sludge (mud)
Triazine herbicides 1: 265, 283; 2: 313 Diclofop-methyl 1: 127
Small radishes
Red currants
see Radishes, small
see Currants, red
Soil
Rice, grains Aldicarb 1: 87
Pirimiphos-methyl 1: 191 Amitrole 2: 49
Pyrazophos 1: 197 Anilazine 2: 59
Soil (contd.) Sour cherries

Binapacryl 2: 197 Glufosinate 2: 217
Bitertanol 2: 77, 87 Vinclozolin 1: 213
Bromoxynil 2: 99
Captafol 1: 93 Soybean oil
Carbendazim 2: 107 Chlorthiophos 1: 109
Carbofuran 2: 113 Organochlorine pesticides 1: 297, 309
Carbosulfan 2: 113 Organophosphorus pesticides 1: 297, 309
Chloridazon 2: 135 Soybeans
Chlorsulfuron 2: 145 Chlorthiophos 1: 109
Chlorthiophos 1: 109 Diclofop-methyl 1: 127
Cymoxanil 2: 157 Glufosinate 2: 217
Dalapon 1: 117 Phenyl urea herbicides 1: 241
Dichlobenil 1: 123; 2: 169 Triazine herbicides 1: 265
Dinobuton 2: 197
Spelt, grains
Flurenol 2: 127
Fonofos 2: 205
Glufosinate 2: 217
Glyphosate 2: 229 Spices (herbs)
Heptenophos 1: 149 see Herbs (spices)
Ioxynil 2: 99
Metalaxyl 1: 153 Spinach
Methomyl 1: 161 Anilazine 2: 59
Metribuzin 2: 245 Chlorthiophos 1: 109
Metsulfuron 2: 145 Metalaxyl 1: 153
Nitrothal-isopropyl 2: 253 Metaldehyde 2: 239
Organotin compounds 2: 343 Organochlorine pesticides 1: 283; 2: 313
Oxamyl 2: 261 Organophosphorus pesticides 1: 283, 335, 361,
Paraquat 1: 177 371; 2: 313
Phenyl urea herbicides 1: 241, 251 Piperonyl butoxide 2: 323
Phthalimide fungicides 1: 401 Pyrethrins 2: 323
Phthalimides 2: 359 Triazine herbicides 1: 265, 283; 2: 313
Pirimicarb 1: 183
Pirimiphos-methyl 1: 191 Strawberries
Propachlor 2: 275 Aldicarb 1: 87
Propiconazole 2: 281 Dichlofluanid 2: 177, 191
Pyrazophos 1: 197 Dithiocarbamate fungicides 1: 353
Pyrethroids 2: 333 Fosetyl 2: 211
Sulphur 2: 287 Heptenophos 1: 149
Tetrachlorvinphos 1: 203 Metalaxyl 1: 153
Thiuram disulphide fungicides 1: 353 Metaldehyde 2: 239
Triadimefon 2: 87 Methomyl 1: 161
Triadimenol 2: 87 Methyl carbamate insecticides 2: 349
Triazine herbicides 1: 265, 347 Nitrogen-containing pesticides 1: 383; 2: 317
Triazine herbicides, desalkyl metabolites Organochlorine pesticides 1: 283, 327, 383;
1: 347 2: 313, 317
Triazophos 1: 209 Organophosphorus pesticides 1: 283, 335, 383;
Vinclozolin 1: 213 2: 313, 317
Sorghum Phenmedipham 2: 269
Triazine herbicides 1: 265 Phenyl urea herbicides 1: 241
Strawberries (contd.) Sugar beet, foliage (contd.)

Pirimiphos-methyl 1: 191 Dithiocarbamate fungicides 1: 353
Sulphur 2: 287 Glufosinate 2: 217
Tetrachlorvinphos 1: 203 Glyphosate 2: 229
Thiuram disulphide fungicides 1: 353 Metalaxyl 1: 153
Tolylfluanid 2: 177, 191 Organotin compounds 2: 343
Triazine herbicides 1: 265, 283; 2: 313 Phenmedipham 2: 269
Vinclozolin 1: 213 Thiuram disulphide fungicides 1: 353
Triadimefon 2: 87
Strawberry plants Triadimenol 2: 87
Aldicarb 1: 87
Sugar beet, syrup
Suet (tallow) Vinclozolin 1: 213
Organophosphorus pesticides 1: 297, 309 Sugar cane
Sugar, raw
Vinclozolin 1: 213 Sunflower oil
Glufosinate 2: 217
Sugar beet, dry chips Organochlorine pesticides 1: 297, 309
Vinclozolin 1: 213 Organophosphorus pesticides 1: 297, 309
Sugar beet, edible root
Aldicarb 1: 87 Sunflower seeds
Bitertanol 2: 77, 87 Glufosinate 2: 217
Carbofuran 2: 113 Metalaxyl 1: 153
Carbosulfan 2: 113 Phenyl urea herbicides 1: 241
Chloridazon 2: 135 Triazine herbicides 1: 265
Dithiocarbamate fungicides 1: 353 Sweet corn
Glufosinate 2: 217 Triazophos 1: 209
Glyphosate 2: 229
Heptenophos 1: 149 Sweet peppers
Metalaxyl 1: 153 see Peppers, sweet
Organochlorine pesticides 1: 383; 2: 317 Tallow (suet)
Organophosphorus pesticides 1: 383; 2: 317 see Suet (tallow)
Phenmedipham 2: 269 Tea, tealike products
Piperonyl butoxide 2: 323 Bitertanol 2: 77
Propachlor 2: 275 Nitrogen-containing pesticides 1: 383; 2: 317
Pyrethrins 2: 323 Organochlorine pesticides 1: 383; 2: 317
Pyrethroids 2: 333 Organophosphorus pesticides 1: 383; 2: 317
Triadimefon 2: 87 Tobacco
Triadimenol 2: 87 Ethylene thiourea 1: 135
Triazine herbicides 1: 265 Metalaxyl 1: 153
Vinclozolin 1: 213 Nitrogen-containing pesticides 1: 383; 2: 317
Sugar beet, foliage Organophosphorus pesticides 1: 383; 2: 317
Bitertanol 2: 77, 87 Oxamyl 2: 261
Chloridazon 2: 135 Phenyl urea herbicides 1: 241
Chlorthiophos 1: 109 Pyrazophos 1: 197
Tomato juice Vegetable fats and oils* (contd.)

Ethylene thiourea 1: 135 Organophosphorus pesticides 1: 297, 309, 383;
2:317
Tomato leaves Phenyl urea herbicides 1: 241
Metribuzin 2: 245 Pirimiphos-methyl 1: 191
Vinclozolin 1: 213
Vegetables, dried and fresh
Tomatoes Bromine-containing fumigants 1: 377
Acephate 1: 81
Anilazine 2: 59 Water
Bitertanol 2: 77 Acephate 1: 81
Carbofuran 2: 113 Amitrole 2: 49, 442
Carbosulfan 2: 113 Anilazine 2: 59
Chlorthiophos 1: 109 Azinphos-ethyl 2: 435
Cymoxanil 2: 157 Azinphos-methyl 2: 435
Dichlofluanid 2: 177 Benalaxyl 2: 437
Dithiocarbamate fungicides 1: 407 Bendiocarb 2: 437
Ethylene thiourea 1: 135 Bentazone 2: 437
Folpet 1: 143 Binapacryl 2: 197
Heptenophos 1: 149 Bitertanol 2: 77, 87, 435
Metalaxyl 1: 153 Bromacil 2: 435
Methamidophos 1: 81 Bromoxynil 2: 99
Methomyl 1: 161 Captafol 1: 93
Methyl carbamate insecticides 2: 349 Carbofuran 2: 435
Metribuzin 2: 245 Chlorflurenol 2: 127
Nitrogen-containing pesticides 1: 383; 2: 317 Chloridazon 2: 135
Organochlorine pesticides 1: 283, 327, 383; Chlorotriazine desalkyl metabolites
2: 313, 317 2: 413
Organophosphorus pesticides 1: 283, 335, 361, Chlorsulfuron 2: 145
383; 2: 313, 317 Chlorthiophos 1: 109
Organotin compounds 2: 343 Cymoxanil 2: 157, 435
Oxamyl 2: 261 Dalapon 1: 117
Phenyl urea herbicides 1: 241 Desalkyl metabolites of chlorotriazines
Phthalimide fungicides 1: 401 2:413
Piperonyl butoxide 2: 323 Dichlobenil 1: 123; 2: 169
Pirimiphos-methyl 1: 191 Dinobuton 2: 197
Pyrethrins 2: 323 Dinoseb 2: 437
Pyrethroids 2: 333 Dinoterb 2: 437
Tolylfluanid 2: 177 Diuron 2: 435
Triadimefon 2: 87 DNOC 2: 437
Triadimenol 2: 87 Ethylene thiourea 1: 135
Triazine herbicides 1: 283; 2: 313 Fluazifop-P-butyl 2: 437
Triazophos 1: 209 Flurenol 2: 127
Vinclozolin 1: 213 Fluroxypyr-(l-methylheptyl) 2: 437
Fonofos 2: 205
Turnips, foliage and edible root Fosetyl 2: 211
Anilazine 2: 59 Fungicides 2: 377
Glufosinate 2: 217
Vegetable fats and oils* Glyphosate 2: 229
Chlorthiophos 1: 109 Haloxyfop-(2-ethoxyethyl) 2: 437
Glufosinate 2: 217 Ioxynil 2: 99
Nitrogen-containing pesticides 1: 383; 2: 317 Lenacil 2: 435
Organochlorine pesticides 1: 297, 309, 383; Metalaxyl 1: 153; 2: 437
2: 317 Metamitron 2: 435
Water (contd.) Wheat grains (contd.)

Methabenzthiazuron 2: 435 Pyrazophos 1: 197
Methamidophos 1: 81 Pyrethroids 2: 333
Methomyl 1: 161 Thiophanate-methyl 2: 69
Metribuzin 2: 245, 435 Thiuram disulphide fungicides 1: 353
Metsulfuron 2: 145 Triadimefon 2: 87
Nitrofen 1: 173 Triadimenol 2: 87
Nitrothal-isopropyl 2: 253, 437 Triazine herbicides 1: 265
Organochlorine insecticides 2: 387 Triazophos 1: 209
Oxamyl 2: 261 Wheat, green matter
Paraquat 1: 177 Anilazine 2: 59
Parathion 2: 435 Bromoxynil 2: 99
Pendimethalin 2: 437 Dalapon 1: 117
Phenoxyalkanoic acid herbicides 2: 369, 393 Dithiocarbamate fungicides 1: 353
Phthalimides 2: 359 Ioxynil 2: 99
Pirimicarb 1: 183 Phthalimides 2: 359
Pirimiphos-methyl 1: 191 Propiconazole 2: 281
Propachlor 2: 275 Thiuram disulphide fungicides 1: 353
Propiconazole 2: 281 Triadimefon 2: 87
Pyrethroids 2: 333 Triadimenol 2: 87
Terbumeton 2: 437
Tetrachlorvinphos 1: 203 Wheat, straw
Triadimefon 2: 87, 435 Anilazine 2: 59
Triadimenol 2: 87, 435 Benomyl 2: 69
Triazine herbicides 2: 403 Captafol 1: 93
Triazine herbicides, desalkyl metabolites 2: 413 Carbendazim 2: 69
Triclopyr-(2-butoxyethyl) 2: 437 Chlorflurenol 2: 127
Dalapon 1: 117
Wheat grains Diclofop-methyl 1: 127
Anilazine 2: 59 Dithiocarbamate fungicides 1: 353
Benomyl 2: 69 Ethylene thiourea 1: 135
Bromine-containing fumigants 1: 377 Flurenol 2: 127
Bromoxynil 2: 99 Metribuzin 2: 245
Captafol 1: 93 Nitrofen 1: 173
Carbendazim 2: 69 Phenyl urea herbicides 1: 241, 251
Chlorflurenol 2: 127 Propiconazole 2: 281
Dalapon 1: 117 Pyrazophos 1: 197
Diclofop-methyl 1: 127 Thiophanate-methyl 2: 69
Dithiocarbamate fungicides 1: 353 Thiuram disulphide fungicides 1: 353
Ethylene thiourea 1: 135 Triadimefon 2: 87
Flurenol 2: 127 Triadimenol 2: 87
Glufosinate 2: 217 Triazine herbicides 1: 265
Heptenophos 1: 149
Ioxynil 2: 99 Whole egg, dried
Metribuzin 2: 245 see Dried whole egg
Nitrofen 1: 173
Nitrogen-containing pesticides 1: 383; 2: 317 Whole milk powder
Organochlorine pesticides 1: 383; 2: 317 Organochlorine pesticides 1: 297, 309
Organophosphorus pesticides 1: 383; 2: 317 Organophosphorus pesticides 1: 297, 309
Phthalimides 2: 359 Wine
Pirimiphos-methyl 1: 191 Amitrole 2: 49
Propiconazole 2: 281 Dichlofluanid 2: 177, 191
Wine (contd.) Witloof chicory

Ethylene thiourea 1: 135 Organochlorine pesticides 1: 283; 2: 313
Folpet 1: 143 Organophosphorus pesticides 1: 283; 2: 313
Fosetyl 2: 211 Phenmedipham 2: 269
Metalaxyl 1: 153 Triazine herbicides 1: 283; 2: 313
Nitrogen-containing pesticides 1: 383; 2: 317 Vinclozolin 1: 213
Organochlorine pesticides 1:383; 2:317
Organophosphorus pesticides 1: 383; 2: 317 Youngberries
Propiconazole 2: 281 Metalaxyl 1: 153
Tolylfluanid 2: 177
Triadimefon 2: 87
Triadimenol 2: 87
Vinclozolin 1: 213
* see also special entries

List of Suppliers Referenced in the Text-Matter
of the Manual
Aldrich Chemical Co., 1001 West Saint Paul Ave., Milwaukee, WI 53233, USA; Aldrich Europe, Poststr.
56, Postfach 2004, D-4054 Nettetal 2, FRG.
Alltech Associates, Inc., 2051 Waukegan Road, Deerfield, IL 60015, USA; Alltech Europe, Begonia-
straat 5, B-9731 Eke, Belgium.
Alpine AG, Maschinenfabrik, Peter-D6rfler-Str. 13-25, D-8900 Augsburg, FRG.
Analytical Bio-Chemistry Laboratories, 7200 ABC Lane, P.O. Box 1097, Columbia, MO 65205, USA; in
Europe: N. Foss Electric GmbH, Waidmannstr. 12b, D-2000 Hamburg 50, FRG.
Analytichem International, Inc., 24201 Frampton Ave., Harbor City, CA 90710, USA.
J. T. Baker Inc., 222 Red School Lane, Phillipsburg, NJ 08865, USA; in Europe: J. T. Baker B.V., Rijster-
borgherweg 20, P.O. Box 1, NL-7400 AA Deventer, The Netherlands.
Becker: see Packard.
Beckman Instruments Inc., 2500 Harbor Blvd., Fullerton, CA 92634, USA.
Bender und Hobein GmbH, Lindwurmstr. 71-73, Postfach 150229, D-8000 Miinchen 15, FRG.
J. C. Binzer Papierfabrik, Berleburger Str. 71, D-3559 Hatzfeld 1, FRG.
Bio-Rad Laboratories, Chemical Division, 3300 Regatta Blvd., Richmond, CA 94804, USA; Heide-
mannstr. 164, Postfach 450133, D-8000 Miinchen 45, FRG.
Bischoff Analysentechnik und -gerate GmbH, Boblinger Str. 23, D-7250 Leonberg, FRG.
Boehringer Ingelheim KG, D-6507 Ingelheim, FRG.
Brownlee Labs, 2045 Martin Ave., Santa Clara, CA 95050, USA.
Camag, Sonnenmattstr. 11, CH-4132 Muttenz, Switzerland; Bismarckstr. 27-29, D-1000 Berlin 41, FRG;
Camag Scientific Inc., P.O. Box 563, Wrightsville Beach, NC 28480, USA.
Carlo Erba Strumentazione S.P.A., Strada Rivoltana, 1-20090 Rodano (Milano), Italy.
Chemie-Mineralien KG, Loningstr. 35, D-2800 Bremen, FRG.
Chemie und Filter GmbH Verfahrenstechnik, Im Schumachergewann 7, D-6900 Heidelberg, FRG.
Chromatronix Inc., 2300 Leghorn St., Mt. View, CA 94043, USA.
Chrompack International B.V., Kuipersweg 6, P.O. Box 8033, NL-4330 EA Middelburg, The Netherlands;
Chrompack Inc., 1130 Route 202, Raritan, NJ 08869, USA.
Corning Glass Works, Inc., Science Products Division, Corning, NY 14831, USA; Corning Laboratory
Products, 1 Prince's Street, Richmond, Surrey TW9 1DZ, U.K.
Degussa AG, GB/AC GKA, Postfach 110533, D-6000 Frankfurt 11, FRG.
Desaga GmbH, Postfach 101969, D-6900 Heidelberg 1, FRG.
Dohrmann Div., Xertex Corp., 3240 Scott Blvd., Santa Clara, CA 95042, USA.
Dow Corning Corp., P.O. Box 994, Midland, MI 48686, USA.
Du Pont Co., Inc., 1007 Market St., Wilmington, DE 19898, USA; Du Pont de Nemours (Deutschland),
Du Pont-Str. 1, D-6380 Bad Homburg, FRG.
EGA-Chemie: see Aldrich.
Gunther Ehle, Glasinstrumentenfabrik, Holzheimer Str. 56 a, D-6250 Limburg, FRG.
F & M Scientific: see Hewlett-Packard.
480 List of Suppliers
Finnigan MAT, 355 River Oaks Pkwy., San Jose, CA 95134, USA; Paradise, Hemel Hempstead, Herts.
HP2 4TG, U.K.
Fluka A C , CH-9470 Buchs, Switzerland; Fluka Chem. Corp., 980 South Second St., Ronkonkoma, NY
11779, USA.
Gerstel GmbH, Aktienstr. 232-234, D-4330 Mulheim/Ruhr 1, FRG.
Heraeus-Christ: see Heraeus Sepatech
Heraeus Quarzschmelze GmbH, Quarzstrafie, D-6450 Hanau, FRG.
Heraeus Sepatech GmbH, Postfach 1220, D-3360 Osterode, FRG.
Heraeus-Wittmann GmbH, Englerstr. 11, D-6900 Heidelberg 1, FRG.
Hewlett-Packard Co., Mail Stop 2083, 3000 Hanover St., Palo Alto, CA 94304, USA.
Hormuth-Vetter: Vetter KG, Malscher Strafle, D-6837 St. Leon-Rot 2, FRG.
ICN Biomedicals, Inc., Chromatography Division, 3300 Hyland Ave., Costa Mesa, CA 92626, USA;
Miihlgrabenstr. 10, D-5309 Meckenheim, FRG.
J & W Scientific, 91 Blue Ravine Rd., Folsom, CA, 95630, USA.
Janke & Kunkel GmbH & Co., KG, Neumagenstr. 27, D-7813 Staufen, FRG.
Janssen Pharmaceutica N.V., B-2340 Beerse, Belgium.
Johns-Manville: Manville, Ken-Caryl Ranch, P.O. Box 5108, Denver, CO 80217, USA.
Dr. H. Knauer, Wissenschaftl. Gerate KG, Heuchelheimer Str. 9, D-6380 Bad Homburg, FRG.
Kontes Glass Co., Spruce St., P.O. Box 729, Vineland, NJ 08360, USA.
Kontron Instruments, Inc., 9 Plymouth St., Everett, MA 02149, USA.
Kratos Analytical, Inc., 535 E. Crescent Ave., Ramsey, NJ 07446, USA.
Lehmann & Voss & Co., Alsterufer 19, D-2000 Hamburg 36, FRG.
LKB Products, Pharmacia LKB, Box 305, S-16126 Bromma, Sweden.
Macherey-Nagel, Postfach 101352, D-5160 Diiren, FRG.
Mallinckrodt, Inc., 675 McDonnell Blvd., P.O. Box 5840, St. Louis, MO 63134, USA.
Mega: see Carlo Erba.
Melpar: see Tracor.
E. Merck, Frankfurter Str. 250, Postfach 4119, D-6100 Darmstadt, FRG.
Messer Griesheim GmbH, Industriegase, Homberger Str. 12, D-4000 Dtisseldorf, FRG.
Metrawatt GmbH, Thomas-Mann-Str. 16-20, Postfach 1333, D-8500 Niirnberg, FRG.
Micro Tek Instrument: see Tracor.
Millipore Corporation, 80 Ashby Rd., Bedford, MA 01730, USA.
Nopco Chemical Co., 350 Mt. Kemble Ave., Morristown, NJ 07960, USA.
Orlita GmbH & Co. KG, Dosiertechnik, Max-Eyth-Str. 10, D-6300 GieJkn, FRG.
Packard Instrument Co., Inc., 2200 Warrenville Rd., Downers Grove, IL 60515, USA.
Perkin-Elmer Ltd., Maxwell Rd., Beaconsfield, Bucks. HP9 1QA, U.K.; The Perkin-Elmer Corp., 761
Main Ave., Norwalk, CT 06859, USA.
Pharmacia LKB Biotechnology, Bjorkgatan 30, S-75182 Uppsala, Sweden; 800 Centennial Ave.,
Piscataway, NJ 08854, USA.
Phase Separations, Deeside Industrial Estate, Queensferry, Clwyd CH5 2LR, U.K.; 140 Water St., Nor-
walk CT 06854, USA.
Philips, Scientific and Analytical Equipment Div., Lelyweg 1, NL^7602 EA Almelo, The Netherlands;
Philips Scientific, Analytical Div., York St., Cambridge CB1 2PX, U.K.
Pierce Chemical Co., 3747 N. Meridian Rd., P.O. Box 117, Rockford, IL 61105, USA; Pierce Europe B.V.,
P.O. Box 1512, NI^3260 BA Oud-Beijerland, The Netherlands.
List of Suppliers 481
Promochem GmbH, Postfach 1246, D-4230 Wesel, FRG.

Pye Unicam Ltd., York St., Cambridge CB1 2PX, U.K.
Quickfit: see Corning.
W. Reiss & Co., Krahenweg 5, D-2000 Hamburg 61, FRG.
Restek Corp., 110 Benner Circle, Bellefonte, PA 16823, USA.
Retsch GmbH & Co. KG, Rheinische Str. 36, Postfach 1554, D-5657 Haan 1, FRG.
Rheodyne, Inc., P.O. Box 996, Cotati, CA 94931, USA.
Riedel-de Haen AG, Wunstorfer Str. 40, Postfach 100262, D-3016 Seelze, FRG.
Carl Roth GmbH & Co., Schoemperlenstr. 1-5, Postfach 211162, D-7500 Karlsruhe 21, FRG.
Sartorius GmbH, Weender Landstr. 94-108, Postfach 3243, D-3400 Gottingen, FRG; Sartorius Corp.,
140 Wilbur PL, Bohemia NY 11716, USA.
Schleicher & Schull, Postfach 4, D-3354 Dassel, FRG; 10 Optical Ave., Keene, NH 03431, USA.
Schoeffel Instrument Corp., 24 Booker St., Westwood, NJ 07675, USA.
Schott & Gen., Jenaer Glaswerke, Hattenbergstr. 10, Postfach 2480, D-6500 Mainz, FRG; Schott
America, 3 Odell Plaza, Yonkers, NY 10701, USA.
Serva Feinbiochemica GmbH & Co., Carl-Benz-Str. 7, Postfach 105260, D-6900 Heidelberg 1, FRG;
Serva Biochemicals, 50 A & S Dr., Paramus, NJ 07652, USA.
Shimadzu Scientific Instruments, Inc., 7102 Riverwood Dr., Columbia MD 21046, USA; Shimadzu
Europa, Albert-Hahn-Str. 6-10, D-4100 Duisburg 29, FRG.
Spark Holland B.V., P.O. Box 388, NI^7800 AJ Emmen, The Netherlands.
Spectra-Physics, Autolab Div., 3333 North First St., San Jose, CA 95134, USA.
Supelco, Inc., Supelco Park, Bellefonte, PA 16823, USA.
Tracor Instruments, Inc., 6500 Tracor Lane, Austin, TX 78725, USA.
Valco Instruments Co., Inc., P.O. Box 55603, Houston, TX 77255, USA; Valco Europe, Untertannberg
7, CH-6214 Schenkon, Switzerland.
Varian Instrument Group, 220 Humboldt C t , Sunnyvale, CA 94089, USA.
Waters Chromatography, 34 Maple St., Milford, MA 01757, USA.
Weisser: supplied by Hans Otto, Feldstr. 26-28, D-2000 Hamburg 6, FRG.
Whatman Inc., 9 Bridewell PL, Clifton, NJ 07014, USA; Whatman Ltd., Springfield Mill, Maidstone,
Kent ME14 2LE, U.K.
Woelm: see ICN Biomedicals.
CarlZeiss, Postfach 1369-1380, D-7082 Oberkochen, FRG; One Zeiss Dr., Thornwood, NY 10541, USA.
Author Index
Becker, G. 1: 283; 2: 313 Kirkland, J. J. 1: 161

Bertrand, A. 2: 211 Kohle, H. 2: 37, 113, 145, 333, 359, 437
Beutel, P. 1: 213; 2: 253 Koflmann, A. 1: 87
Blacha-Puller, M. 1: 183; 2: 99, 145, 197, 211, KoBmann, K. 2: 269
275, 359 Krohn, H. 1: 173
Bos, U 1: 37 Kunzler, K. 1: 127
Brauckhoff, S. 2: 349 Meemken, H.-A. 1: 51
Brennecke, R. 2: 59, 77, 87, 177, 377 Mollhoff, E. 2: 49, 343
Bullock, D. J. W. 1: 183, 191 Muller, M. A. 2: 211
Burger, K. 2: 41, 423, 435, 442 Nolting, H.-G. 1: 93, 167, 173, 177, 183, 203;
Btittler, B. 1: 99, 105, 143, 401; 2: 281 2: 37, 99, 113, 145, 197, 211, 275, 323, 333,
Cowell, J. E. 2: 229 359, 437
Dick, J. P. 1: 183 Nord, P.-J. 2: 229
Drescher, N. 1: 21, 135; 2: 369 Otteneder, H. 2: 291, 295
Ebing, W. 1: 57, 61, 87, 335, 371, 407 Otto, S. 1: 117, 135, 213; 2: 135, 163
Eichler, D. 1: 109; 2: 127, 157, 205, 239, 393 Pease, H. L. 1: 161; 2: 261
Eichner, M. 1: 57, 327, 335 Pflugmacher, J. 1: 57, 61, 335, 371, 407
Elzner, J. 2: 287 Ramsteiner, K. 1: 57, 153, 251, 347
Formica, G. 2: 69, 403, 413 Reding, M. A. 2: 229
Frehse, H. 1: 37; 2: 3 Richtarsky, G. W. 1: 167
Gaudernack, E. 1: 65 Schmidt, G. 1: 177
Giannone, C. 2: 69, 403, 413 Schmidt, M. 2: 169
Gorbach, S. 1: 29, 37, 65, 127, 149; 2: 3 Schulte, E. 1: 51
Gottschild, D. 2: 37 Siebers, J. 1: 81, 183, 191; 2: 99, 113, 145, 197,
Habersaat, B. 1: 51 211, 275, 323, 333, 359
Hahn, H. 1: 321 Slates, R. V. 2: 145
Hamann, R. 2: 169 Sochor, H. 2: 217
Hershberger, L. W. 2: 145 Specht, W. 1: 71, 75, 309, 383; 2: 31, 107,
Herzel, F. 1: 123, 177 317
Heupt, W. 2: 127, 157 Stijve, T. 1: 297, 377
Hezel, U. 2: 291 Thier, H.-P. 1: 17, 29, 37, 71, 167, 297, 309,
Hild, J. 1: 361 321, 353, 361, 377, 383; 2: 3, 317, 323, 349
Holt, R. F. 1: 161; 2: 261 Thier, W. 1: 197, 209
Holtz, K.-H. 2: 3 Vogel, W. 2: 239
Hormann, W. D. 1: 99, 105, 143, 153, 265, Vogeler, K. 2: 191, 377
401; 2: 69, 281, 403 Voss, G. 1: 241
Ipach, R. 2: 153 Walter, H.-F. 2: 3
Jarczyk, H. J. 2: 49, 245 Weil, L. 1 : 2 3 ; 2: 387
Karlhuber, B. 1: 57, 347 Weinmann, W. D. 1: 21, 37, 81, 93, 167, 173,
Keller, W. 1: 117, 135; 2: 135, 163, 253 177, 183, 191, 203, 2: 323
Kennedy, S. H. 1: 183 Winkler, S. 1: 65
Kettrup, A. 2: 169 Wolf, A. 1: 93, 203
Kirchhoff, J. 1: 29 Zahnow, E. W. 2: 145

Manual of Pesticide Residue Analysis Volume II PDF

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DFG

ISBN 3-527-27017-5 (VCH, Weinheim) ISBN 0-89573-957-7 (VCH, New York)

Pesticides Commission VCH

Translators: J. Edwards t and Carole Ann Traedgold

Library of Congress Card No. applied for.

Deutsche Bibliothek Cataloguing-in-Publication Data:

VCH Verlagsgesellschaft mbH, D-6940 Weinheim (Federal Republic of Germany), 1992

Printed on acid-free and chlorine-free paper.

Part 1: Introduction and Instructions (contd.)

Part 2: Cleanup Methods (contd.)

Part 3: Individual Pesticide Residue Analytical Methods (contd.)

Glufosinate, 651 217

Part 4: Multiple Pesticide Residue Analytical Methods (contd.)

Part 5: Multiple Pesticide Residue Analytical Methods for Water

Cumulative Indexes for Volumes 1 and 2

Senate Commission for Pesticides, Deutsche Forschungsgemeinschaft

Part 1: Introduction and Instructions

Part 2: Cleanup Methods

Part 3: Individual Pesticide Residue Analytical Methods

Part 4: Multiple Pesticide Residue Analytical Methods

Dr. Claus Klotzsche Bruelweg 36, CH-4147 Aesch/Schweiz

Prof. Dr. Ulrich Mohr Abteilung fur experimentelle Pathologie der

Prof. Dr. Friedrich-Karl Institut fur Toxikologie der Universitat

Prof. Dr. Heinz Schmutterer Institut fiir Phytopathologie und

Prof. Dr. Fidelis Selenka Institut fiir Hygiene der Ruhr-Universitat

Prof. Dr. Hans-Peter Thier Institut fiir Lebensmittelchemie der Universitat

Dr. Ludwig Weil Institut fiir Wasserchemie und Chemische Balneologie

Prof. Dr. Fritz Herzel Bundesgesundheitsamt

Prof. Dr. Alfred-G. Hildebrandt Institut fiir Arzneimittel des Bundesgesundheitsamtes

Secretaries of the Senate Commission for Pesticides

Members and Guests

Dr. Egon Mollhoff Bayer AG, PF-A/CE-RA,

Prof. Dr. Hans Zeumer t

2 Calibration curve concept

2.2 Establishing the calibration curve

for which the corresponding signal values

3 Limit of detection (LDC)

X=0 LDC X = Concentration

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

3.2 Decision rules

X=0 LDC X = Concentration

3.3 Determining the limit of detection

3.3.2 Graphical derivation of LDC

4 Limit of determination (LDM)

4.2 Consequences of Requirement I

4.3 Consequences of Requirement II

4.4 Consequences of Requirement III

4.5 Determining the limit of determination

6 Mathematical formulation of the concept

Formula Auxiliary Term

6.2 Derivation of the calibration straight line

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

Formula Auxiliary '.Term

A = Y- B X Theoretical Blank Value

6.3 Prediction interval

t s f = critical value of the t distribution (two-sided) for a confidence level S,

sR = y - E (Yj - A - BXj)2 = standard error of estimate: "mean"

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

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)

6.6 Derivation of LDC

Y(X) + t SY ^ Y(X) + t Vo Y(X) =| Y(X) (1 + t Vo)