Table Of ContentPesticide Analytical
Methodology
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Pesticide Analytical
Methodology
John Harvey, Jr., EDITOR
E. I. Du Pont de Nemours and Company
Gunter Zweig, EDITOR
U.S. Environmental Protection Agency
ASSOCIAT
Richard Cannizzaro
Henry Dishburger
Joseph Sherma
Based on a symposium
jointly sponsored by the
Divisions of Pesticide Chemistry
and Analytical Chemistry
at the 178th Meeting of the
American Chemical Society
Washington, D.C.,
September 9-14, 1979.
ACS SYMPOSIUM SERIES 136
AMERICAN CHEMICAL SOCIETY
WASHINGTON, D. C. 1980
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Library of Congress CIP Data
Pesticide analytical methodology.
(ACS symposium series; 136 ISSN 0097-6156)
Includes bibliographies and index.
1. Pesticides—Analysis—Congresses. 2. Pesticides—
Environmental aspects—Congresses.
I. Harvey, John, 1925- . II. Zweig, Gunter. III.
American Chemical Society. Division of Pesticide
Chemistry. IV. American Chemical Society. Division
of Analytical Chemistry. V. Series: American Chemis
try Society. ACS symposium series; 136.
SB960.P47 632'.95'028 80-19470
ISBN 0-8412-0581-7 ASCMC 8 136 1-406 1980
Copyright © 1980
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In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
ACS Symposium Series
M. Joan Comstock Series Editor
Advisory Board
David L. Allara W. Jeffrey Howe
Kenneth B. Bischoff James D. Idol, Jr.
Donald G. Crosby James P. Lodge
Donald D. Dollberg Leon Petrakis
Robert E. Feeney F. Sherwood Rowland
Jack Halpern Alan C. Sartorelli
Brian M. Harney Raymond B. Seymour
Robert A. Hofstader Gunter Zweig
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
FOREWORD
The ACS SYMPOSIU
a medium for publishing symposia quickly in book form. The
format of the Series parallels that of the continuing ADVANCES
EST CHEMISTRY SERIES except that in order to save time the
papers are not typeset but are reproduced as they are sub
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and reports of research are acceptable since symposia may
embrace both types of presentation.
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
PREFACE
Analytical methodology for pesticide determinations has greatly advanced
^ during the past 30 years. The emphasis during the first 20 years was
placed on increased sensitivity of detection. That these efforts were suc
cessful is attested by achieving sensitivity both quantitatively and qualita
tively in the femtogram range (10"15 g).
In a recent review of pesticide analytical methodology (Zweig, G.
"The Vanishing Zero—Ten Years Later," JAOAC, 1978 67(2) 229-248)
the author pleaded with
resolving power, and identification capability for sub-microgram quantities
of pesticide residues. The rationale was that the toxicologists lag far
behind the analytical chemists and often are not able to assess the toxico-
logical significance of minute levels of pesticide residues.
We believe that this symposium on Recent Advances in Pesticide
Analytical Methodology fulfills this plea. High-performance liquid chro
matography (HPLC) has made the greatest advances. Chapters on HPLC
cover subjects on metabolism studies; automation of HPLC; evaluation of
LC columns; the effect of the mobile phase on reversed-phase chromatog
raphy; the electrochemical or amperometric detector; and fluorogenic
detection.
Quantitative thin-layer chromatography (TLC) has progressed con
siderably and has been adapted to forensic chemistry of pesticide poisoning
incidents. High-performance TLC (HPTLC) with precoated plates is
discussed separately.
Fourier transform infrared spectroscopy; negative ion spectroscopy;
and immunochemical technology are the subjects of several chapters. A
chapter on cleanup presents a general challenge for all types of analytical
techniques. Chemical derivatization of pesticides and metabolites enhances
the detectability and ability to analyze small quantities of pesticide residues.
Applications of modern analytical techniques also are discussed rela
tive to the analysis of tetrachlorodibenzo-p-dioxin; organotin compounds;
airborne pesticides; computerized data processing for metabolism studies;
and human exposure studies.
The organizers of this symposium wish to thank the contributors for
their excellent talks, written chapters, and cooperation in meeting all
ix
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
deadlines. The Division of Pesticide Chemistry also wishes to acknowledge
the joint sponsorship of this symposium with the Division of Analytical
Chemistry.
Biochemicals Department JOHN HARVEY, JR.
Experimental Station
E. I. Du Pont de Nemours and Company
Wilmington, Delaware 19898
Hazard Evaluation Division GUNTER ZWEIG
Environmental Protection Agency
Washington, D.C. 20460
April 17, 1980
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Modern High-Performance Liquid
Chromatography in Pesticide Metabolism Studies
JOHN HARVEY, JR.
E. I. Du Pont de Nemours & Company, Inc., Biochemicals Department,
Experimental Station, Wilmington, DE 19898
This paper is the onl i th liquid chromatograph
portion of this symposiu
tography specifically from the viewpoint of the pesticide
metabolism chemist. A residue analyst knows what compound he must
analyze for, and develops his method with the properties of that
substance in mind. On the other hand, the pesticide metabolism
chemist has a different problem. At the conclusion of the treat
ment, exposure, and harvest phases of a radiolabeled metabolism
study, he divides his material into appropriate samples, and
extracts each sample with selected solvents to obtain the
radioactive materials in soluble form. Typically these extracts
consist of low levels (ppm) of carbon-14 labeled metabolites in a
complicated mixture of normal natural products from the plant,
animal, or soil source. The identity of each metabolite is
unknown, and each must be isolated from the natural background
and from other labeled metabolites in sufficient quantity and in
adequate purity for identification studies, usually by mass
spectrometry. The situation is rather 1 ike looking for the
proverbial "needle in the haystack" when one does not know the
size, shape, or composition of the needle, or even how many needles
there are in the stack. At this point a separation technique must
be selected with certain important requirements in mind.
First, the technique must be non-destructive. We must have
confidence that the material we isolate and identify is the same
as the metabolite the bio-system produced. Second, the technique
must be powerful enough to effect the desired purification. Third,
we need to achieve the required purity and quantity with an
economical expenditure of time and resources. High performance
liquid chromatography (hereafter referred to as HPLC) is ideally
suited to this challenge.
• The conditions of HPLC are mild - usually ambient
temperatures - with ordinary solvents from hexane to
water. Only in very rare instances have rearrangements
or degradations been reported under LC conditions, and
when suspected, alternative routes are usually available
to provide the separation without the degradation.
0-8412-05 81 -7/80/47-136-001 $05.00/0
© 1980 American Chemical Society
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
2 PESTICIDE ANALYTICAL METHODOLOGY
• HPLC is a powerful separation technique. Commercially
available columns of 4.6mm i.d. χ 25cm length regularly
exhibit 8,000 - 12,000 or more theoretical plates.
• HPLC is fast and economical in terms of labor and
reagents. The effluent fractions come off the column in
small volumes of solvents readily available for liquid
scintillation counting, and/or further purification steps.
• In addition, HPLC is extremely versatile, one chromato-
graph being capable of separating compounds by any of 4-6
distinct modes of chromatography by simple interchanges
of columns. When the capacity for infinite variation of
the composition of the mobile phase is added to the
available choices in the stationary phase a nearly
unlimited versatility is displayed.
It is not my purpos
to discuss the fine point
selection, or new advances in detectors. The papers that follow
deal with recent developments in these areas, and what they
report is as applicable to pesticide metabolism analyses as to
residue analyses. Instead, I shall describe a working radio-
chromatograph for the pesticide research laboratory and discuss
some of the problems associated with this type of instrument.
I shall close by describing some of the possibilities and
procedures open to the analyst in the study of pesticide
metabolism problems.
The Radiochromatograph
A schematic diagram of a radiochromatograph is shown in
Figure 1. In its simplest form any chromatograph consists of a
solvent reservoir, a constant flow high pressure chromatographic
pump, a suitable chromatographic column, and one or more
detectors. An injection valve is the most convenient way of
introducing the sample onto the column, and a recorder produces
the permanent record of detector responses. For the ultimate in
versatility the addition of a device for generating a solvent
gradient is highly desirable. In our own laboratory we have
several radiochromatographs of varying complexity. The one thing
they all have in common is that they are lab-assembled
instruments, with all of the component parts being commercially
available. This approach allows the ultimate in flexibility,
including the capacity to replace individual components at any
time whether because of breakdown, obsolescence, or change in
requirements. In our radio-LC's the general detector is an
ultraviolet absorbance type. This serves, when analyzing crude
mixtures, to indicate where the non-labeled UV absorbing
components may be found, and also enables us to establish the
retention time of model compounds or standards. This type of
detector is sensitive and convenient for many purposes, but any
of the many available LC detectors may be substituted (index of
refraction, infrared, etc.) depending on the user's requirements.
In Pesticide Analytical Methodology; Harvey, J., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
