7.1
7.1
© Springer-Verlag Berlin Heidelberg 2005
II.7.1 Simultaneous analysis
of pesticides by GC/MS
by Shinji Kageyama and Makoto Ueki
Introduction
Agricultual chemicals include not only pesticides for protecting plants, such as insecticides,
germicides, herbicides and rodenticides, but also fertilizers and growth regulating substances
being used in agricultural production and horticulture. In poisoning cases with agricultural
chemicals, the causative poisons are largely the pesticides.  ere are many cases, in which the
poisoning due to exposure to an organophosphorus pesticide is obvious with clinical symp-
toms [1]. However, there are more than 5,000 agricultural chemicals registered and commer-
cially available in Japanese markets [2]; it is essential to identify a causative chemical to make
the  nal clinical diagnosis in a poisoning-suspected case.
Since sensitive and simultaneous analysis of multiple compounds is possible using GC/MS,
the method is being widely used for analysis of pesticides for environmental specimens, such
as water and soil [3–6]. In this chapter, among main pesticides which had been reported caus-
ative for poisoning cases [7], more than 30 kinds of pesticides have been picked up from or-
ganophosphorus, organochlorine, carbamate and triazine pesticides, and a method for simul-
taneous analysis of many pesticides by GC/MS is presented. For alkylpyridinium and amino
acid type herbicides, their analyses are described in other chapters.
Reagents and their preparation
i. Reagents
• Pure n-hexane for organic trace analysis (Merck, Darmstadt, Germany and other manu-
facturers) is directly used or redistilled befor use according to need.
• Many of the authentic standards of pesticides can be purchased from Supelco, Bellefonte,
PA, USA; but fenobucarb, salithion, thiometon, ethylthiomethon, propanil, cyanofenphos
and isoxathion cannot be obtained from the above manufacturer, but can be obtained from
Wako Pure Chemical Industries, Ltd., Osaka, Japan. As internal standard (IS), fenitrothion-
d

ture); septum purge  ow rate: 11.0 mL/min; purge time: 1.0 min; total  ow rate: 14.3 mL/min;
column  ow rate: 1.0 mL/min (constant  ow-rate mode); interface temperature: 150 °C; MS
ionization mode: EI; electron energy: 70 eV; scan range: m/z 40–550; dwell time of SIM mea-
surements: 10 s; SIM ions to be used: listed in
> Table 1.1.
Procedure
i. A 2-mL volume of whole blood or urine
b
is mixed well with 3 mL of puri ed water.
ii. A 100-µL volume of IS (fenitrothion-d
6
) solution is added to the above mixture and mixed
well.
iii.  e pH of the mixture is adjusted to 3.5 by adding 1.2 M hydrochloric acid solution.
iv. A 8-mL volume of n-hexane is mixed with the above mixture and shaken for 10 min for
extraction
c
.
v. It is centrifuged at 1,000 g for 10 min.
vi.  e n-hexane layer is carefully transferred to a glass vial, and evaporated to dryness under
reduced pressure
d
.
vii.  e residue is dissolved in 100 µL n-hexane.
viii. A 2-µL aliquot of it is subjected to GC/MS analysis
e
.
ix. By comparison with the data obtained from a spiked specimen, the identi cation
f
and

diazinon 0.89 304 137 179
ethylthiomethon 0.90 88 186 274
From 10.84 min
propanil 0.94 163 161 217
metribuzin 0.95 198 214 144
carbaryl 0.97 144 115 201
alachlor 0.97 160 188 269
From 11.58 min
fenitrothion-d
6
(IS) 1.00 283 266
pirimiphos-methyl 1.02 290 305 276
fenitrothion 1.00 277 125 260
malathion 1.02 173 125 285
fenthion 1.03 278 109 125
aldrin 1.04 263 66 364
parathion 1.04 291 109 137
From 12.79 min
methidathion 1.14 145 85 302
chlordene 1.14 373 375 377
chlordene-trans 1.15 373 375 377
chlordene-cis 1.17 373 375 410
endosulfan 1.17 339 241 341
From 13.91 min
p, p’-DDE 1.20 246 316 318
dieldrin 1.21 79 380 108
isoxathion 1.24 313 177 208
nitrofen 1.24 283 285 202
endrin 1.25 263 265 81
p, p’-DDD 1.28 235 237 165

upon quantitation, it is desirable to construct a calibration curve using blank specimens of the
same matrix, into which various amounts of a target compound are spiked.
For pesticides with good recoveries, the coe cients of variation were 1.6–9.6 % for urine
and 1.3–11.3 % for blood (n = 10); for those with low recoveries, the values were 3.2–18.5 %.
By liquid-liquid extraction with n-hexane under acidic conditions, the lipid components in
blood are also extracted and cause impurity peaks in TICs. Even in such conditions, clean
peaks of target compounds can be obtained by mass chromatography using monitor ions listed
in
> Table 1.1.
Blood and urine specimens, into which the authentic pesticides had been spiked, were stored
at –20 ± 5 °C and 4 ± 3 °C for 1, 2, 3, 4, 9, 10, 11, 12, 13 and 14 days to test the stability of the pes-
ticides during storage. Most pesticides except DDVP were stable in a frozen state; their coe cients
of inter-day variation were as good as 5–10 %.  e coe cients of inter-day variation for DDVP
were 24 % for urine and 49.3 % for blood; the poor reproducibility found for DDVP is not due to
the analytical method, but due to its unstableness during storage. Such unstableness becomes more
marked during storage under refrigeration; a er storage only for several days, DDVP became
undetectable in some spacimens. Under refrigerated conditions, most pesticides showed their
10–20 % loss. Upon analysis, an adequate storage of specimens (in a refrigerated or frozen state) is
necessary for each compound. For compounds with poor stability, their standard solutions should
be prepared just before use to be spiked to blank specimens for quantitative analysis.
 e detection limits are di erent in di erent pesticides; they were 1–64 ng/mL for blood
specimens and 1–254 ng/mL for urine specimens.  erefore the present method is sensitive
enough to detect and identify causative pesticides in poisoning cases.  e upper limits for
linearity of each pesticide are about several hundred nanograms/mL. In actual cases, very high
concentrations of pesticides are occasionally encountered; in such cases, the amount of a spec-
imen is reduced or diluted to obtain quantitative results.
532 Simultaneous analysis of pesticides by GC/MS
By this method, 2 isomers for permethrin, fenvalerate and chlordene, and 4 isomers for
cypermethrin could be separated. Usually, for qualitative analysis, the scan mode is employed;
for quantitation with high sensitivity, the SIM mode is used. When scan measurements are

thion, 4; pirimiphos-methyl, 4; methidathion, 3; permethrin, 2; fenvalerate, 1; and parathion,
1. Not less than 2 kinds of pesticides were detected from 43 cases. Methomyl was detected in a
case, in which paraquat poisoning had been suspected; a high level of sumithion was detected
from urine in a case, in which chlorfenapyr ingestion had been suspected.  e analytical results
were sometimes di erent from ones, which had been expected on the basis of early informa-
tions. For the  nal identi cation of a causative poison, the present screening method by
GC/MS for a wide range of pesticides is very useful.
Notes
a)  is is a semiquantitative method using one point standard.  e tentative concentration of
a pesticide in a specimen is calculated as follows:
Concentration of a pesticide in a specimen = (calibrator concentration) × (peak area
obtained from a specimen ÷ peak area of IS added to a specimen) × (peak area of IS added
to the calibrator solution ÷ peak area of the calibrator).
b) Stomach contents can be treated in the same way. For accurate quantitation, a calibration
curve is constructed by adding various amounts of a test compound to the blank speci-
mens.  e addition tests can be also used by adding a known amount of a test compound
to a part of a test specimen; the peak area di erence between added and non-added speci-
mens can be used for calculation of the concentration of the test compound in the non-
added specimen.
c) Since emulsion formation easily takes place upon extraction, the shaking should not be
vigorous, but gentle.
d) Upon evaporation to dryness, excessive drying causing sublimation should be avoided.
Especially for DDVP, care should be taken for its evaporation, because DDVP is relatively
volatile, causing unstableness of its recovery rate.
e) To  nd out false-positive results caused by endogenous compounds or contamination from
experimental environments, it is desirable to analyze distilled water and blank specimens
obtained from unexposed and healthy subjects simultaneously. When a high concentration
of a test compound is contained in a specimen, care should be taken against the carry-over
of the compound. In such a case, a blank organic solvent such as n-hexane should be in-
jected at high injection and oven temperatures for washing before the next analysis.

Japan, No. 33. National Police Agency, Tokyo, pp 32–102 (in Japanese)
8) Pharmaceutical Society of Japan (ed) (1982) Standard Methods of Chemical Analysis in Poisoning. With Com-
mentary. 2nd edn. Nanzando, Tokyo, pp 272–307 (in Japanese)
9) Tu AT (1999) Principle of Toxicology – Science of Poisons. Jiho Inc., Tokyo, pp 15–24 (in Japanese)