Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 58) pdf

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

6
was obtained from (Hayashi Pure Chemical Ind., Ltd, Osaka, Japan). Other common
chemicals used were of the highest purity commercially abailable.
ii. Preparation
• Standard solutions for each pesticide are prepared by dissolving each compound in ethanol
(1 mg/mL); each retention times were determined by GC.  e 35 compounds were divided
into 3 groups, where each peak did not overlap or interfere with each other as shown
in
> Figure 1.1. Such 3 mixture standard solutions with known compounds at 1 mg/mL
each are also prepared.
528 Simultaneous analysis of pesticides by GC/MS
• A 10-mL volume of 12 M hydrochloric acid solution is carefully diluted with puri ed water
to prepare 100 mL solution.
• To 2-mL volume each of blank whole blood or urine, which had been obtained from healthy
and unexposed subjects, a 100 µL of each standard solution (1 mg/mL) is added to be used
as a calibrator
a
.
Analytical conditions
Instrument: HP-6890 type GC/HP-5973 type MS equipped with an HP-6890 type autosampler
(all from Agilent Technologies, Palo Alto, CA, USA).
GC column: Ultra-2 (25 m × 0.2 mm i. d.,  lm thickness 0.33 µm, 5 %- phenylmethylsilicone,
Agilent Technologies).
Evaporator: REN-1EN type (Asahi Technoglass, Chiba, Japan).
Vials for sampling are those usable for the autosampler. Before analysis, impurities arising
from the vials and caps should be checked. Other tools are common ones commercially avail-
able.
GC/MS conditions; column (oven) temperature: 50 °C (1 min) → 20 °C/min → 200 °C →
7 °C/min → 290 °C (8 min); analysis time: 29.4 min; injection mode: splitless; injection tem-
perature: 250 °C; injection pressure: about 17 psi (helium pressure at 50 °C of oven tempera-

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

semiquantitation of a pesticide in a test specimen are carried out.
529Similtaneous analysis of pesticides by GC/MS
TICs obtained by GC/MS for extracts of urine, into which pesticides had been spiked. 1: DDVP;
2: fenobucarb; 3: salithion; 4: thiometon; 5: cyanophos; 6: diazinon; 7: ethylthiomethon; 8: propanil;
9: fenitrothion; 10: malathion; 11: fenthion; 12: methidathion; 13: endosulfan; 14: endrin;
15: cyanofenphos; 16: EPN; 17: fenvalerate; 18: simazine; 19: metribuzin; 20: alachlor; 21: aldrin;
22: chlordene; 23: nitrofen; 24: pp’-DDT; 25: permethrin; 26: cypermethrin; 27: pentachlorophenol;
28: γ-BHC; 29: carbaryl; 30: pirimiphos-methyl; 31: parathion; 32: pp’-DDE; 33: dieldrin; 34: isoxathion;
35: pp’-DDD. Multiple peaks appear for chlordene (peak 22), cypermethrin (peak 26) and permethrin
(peak 25), because of the presence of their isomers. Each specimen was spiked urine, to which each
pesticide at a 50 µg/mL concentration had been added.
⊡ Figure 1.1
530 Simultaneous analysis of pesticides by GC/MS
⊡ Table 1.1
SIM ions to be used for simultaneous analysis of pesticides by GC/MS
Target compound Relative retention
time*
Monitor ions (m/z)
From 4.50 min
Methomyl 0.45 58 105 88
DDVP 0.57 185 109 187
From 8.80 min
fenobucarb 0.77 150 121 207
salithion 0.81 216 153 201
thiometon 0.84 125 88 158
simazine 0.85 201 173 186
From 10.12 min
pentachlorophenol 0.87 266 268 165
γ-BHC 0.88 181 219 221
cyanophos 0.88 243 109 125

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

From 15.38 min
cyanofenphos 1.34 157 185 303
p, p’-DDT 1.35 235 237 354
EPN 1.45 157 185 169
permethrin 1.67 183 163 165
cypermethrin 1.85 163 165 209
fenvalerate 2.00 167 419 225
* Relative retention time: those when the retention time of IS was taken as 1.00.
531Similtaneous analysis of pesticides by GC/MS
Assessment and some comments on the method
TICs for the extracts of 2 mL urine, into which 100 µg each of 35 kinds of pesticides had been
spiked, are shown in
> Figure 1.1.  e chromatograms of the 3 groups showed no interfer-
ence of the test peaks by impurities of urine.
 e simultaneous analysis is aimed at screening of compounds in a wide range; the condi-
tions should be set according to an average property of many analytes.  erefore, satisfactory
recoveries cannot be obtained for all compounds. By this method, the recoveries of the pesti-
cides from the spiked urine specimens (41–77 ng/mL) were 68–100 % for organophosphorus
pesticides (except DDVP), benzoepine, fenobucarb, alachlor, cypermethrin and dieldrin; those
from blood specimens were as satisfactory as 68–113 % for all organophosphorus pesticides,
fenobucarb, nitrofen and alchlor.  e recoveries of DDVP, chlordene, aldrin, DDT, DDE,
permethrin, cypermethrin, fenvalerate, metribuzin, simazine and propanil were 42–63 % from
urine and only 8–32 % from blood; for quantitation of these pesticides, the recovery rates
should be improved. Methomyl cannot be detected a er addition of several ten nanograms to
blank specimens; but in actual cases, its high concentrations are frequently detected.  erefore,
at the  rst step, a causative compound is identi ed by screening under the general analytical
conditions. At the second step, the extraction procedure is optimized for each compound to
achieve accurate quantitation. In many cases, a er suitable changes in an extraction solvent
and properties of an aqueous phase, it is not necessary to change instrumental conditions. As
mentioned above, the recovery of each compound is di erent according to a specimen matrix;

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

made in the range of m/z 50–550, the mass spectrum obtained from an unknown peak can be
compared with that included in a public library by computer research; this may enable the
identi cation of a metabolite or a decomposition product of a pesticide, and thus may give a
useful information for analysis of a causative compound in a poisoning case.
Trichlorfon is one of the thermolabile pesticides; under the present GC conditions (injector
temperature 250 °C and the maximal oven temperature 290 °C), trichlorfon can be converted
to DDVP or decomposed to some products. In such a case, discrimination between DDVP and
trichlorfon can be achieved by lowering the injection and maximal oven temperatures down to
about 150 °C (
> Figure 1.2).
Changes in TICs obtained by GC/MS according to different injection and oven temperatures
for detection of trichlorfon. A 1-µL aliquot of the standard trichlorfon solution (10 µg/mL) was
injected. The conditions for the upper panel were: injection temperature, 250 °C; oven tempera-
ture, 50 °C ➞ 290 °C; those for the lower panel: injection temperature, 150 °C; oven temperature,
50 °C ➞ 150 °C. At 250 °C of injection temperature, trichlorfon was decomposed completely;
while at 150 °C trichlorfon appeared as an intense peak. DDVP appeared as a decomposition
product under both conditions.
⊡ Figure 1.2
533Similtaneous analysis of pesticides by GC/MS
When pesticides are incorporated into human bodies, they undergo various metabolisms;
the metabolites are usually very important to estimate a causative poison. Organophosphorus
pesticides are usually oxidized by an oxidase in the liver to convert the P = S group into the
P = O one.  e conversions of malathion into malaoxon and of parathion into paraoxon are
well known. Organochlorine pesticides are also oxidized enzymatically; aldrin and DDT are
converted into dieldrin and DDE, respectively [8]. Carbaryl and many of organophosphorus
pesticides are metabolized into phenolic compounds by acetylcholinesterase; the former com-
pound is known to yield naphthol [9].
In the period from July, 1999 to May, 2000, this method was applied to 304 cases, in which
pesticide poisonings had been suspected.  e numbers of positive results were: sumithion, 78;
malathion, 35; DDVP, 15; propanil, 12; methomyl, 8; carbaryl, 7; EPN, 6; fenthion, 6; isoxa-

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.

f) A guide for identi cation method by GC/MS: in the SIM mode, three main peaks are
selected. Relative ion intensities of the 3 peaks are compared between a test specimen and
534 Simultaneous analysis of pesticides by GC/MS
the authentic compound; their pro le of the test peaks should be similar to that for the
authentic compound and their relative similarity should be in the range of 80–120 %. It is
also essential that there is no overlapping impurity peaks in a blank specimen.  e shi of
retention time of the test peak as compared with that of the authentic peak should be within
± 1 %. All the above conditions are ful lled, the presence of the compound in a test speci-
men can be judged positive. When measurements are made in the scan mode, the identi -
cation is much easier; with almost the same mass spectra and retention times, the com-
pound of the test peak can be judged identical to the authentic one; however, when interfer-
ence by various peaks due to a di erent compound or a peak at a mass number larger than
the molecular weight appears in the mass spectrum, the identity of the compound becomes
questionable.
References
1) Uemura T, Goto R (eds) (1999) Rapid Analytical Methods for Poisons Being Mixed with Foods and Poisoning
Symptoms in Humans. Science Forum, Tokyo, pp 15–53 (in Japanese)
2) National Pesticide Cooperative Society (ed) (1999) Guidebook for Safe and Right Use of Pesticides, 1999 edn.
Ueda Word Process Project, Tokyo, p 21 (in Japanese)
3) Grasso P, Benfenati E (1998) Deuterated internal standards for gas chromatographic-mass spectrometric analy-
sis of polar organophosphorus pesticides in water samples. J Chromatogr A 822:91–99
4) Fukushima M (1999) Analytical methods of pesticides. In: Proceedings of the 24th Meeting of Japanese Society
of Environmental Chemistry. Japanese Society of Analytical Chemistry, Ibaragi, pp 30–45 (in Japanese)
5) Mogami K (1999) Analytical methods of organochlorine compounds. In: Proceedings of the 24th Meeting of
Japanese Society of Environmental Chemistry. Japanese Society of Analytical Chemistry, Ibaragi, pp 58–86 (in
Japanese)
6) Yamaguchi S, Eto S, Eguchi M et al. (1997) Simultaneous analysis of residual pesticides in crops by gas chroma-
tography/mass spectrometry in the scan mode. Bunseki Kagaku 46:905–914 (in Japanese with an English
abstract)
7) National Research Institute of Police Science (ed) (1990) Annual Case Reports of Drug and Toxic Poisoning in

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)