Simultaneous analysis of pesticides by GC/MS

II.7.1 Simultaneous analysis of pesticides by GC/MS by Shinji Kageyama and Makoto Ueki 7.1 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. There are many cases, in which the poisoning due to exposure to an organophosphorus pesticide is obvious with clinical symptoms [1]. However, there are more than 5,000 agricultural chemicals registered and commercially available in Japanese markets [2]; it is essential to identify a causative chemical to make the final 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 causative for poisoning cases [7], more than 30 kinds of pesticides have been picked up from organophosphorus, organochlorine, carbamate and triazine pesticides, and a method for simultaneous 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 manufacturers) 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), fenitrothiond 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. The 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. Springer-Verlag Berlin Heidelberg 2005

528 Simultaneous analysis of pesticides by GC/MS A 10-mL volume of 12 M hydrochloric acid solution is carefully diluted with purified 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., film 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 available. 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 temperature: 250 C; injection pressure: about 17 psi (helium pressure at 50 C of oven temperature); septum purge flow rate: 11.0 ml/min; purge time: 1.0 min; total flow rate: 14.3 ml/min; column flow rate: 1.0 ml/min (constant flow-rate mode); interface temperature: 150 C; MS ionization mode: EI; electron energy: 70 ev; scan range: m/z 40 550; dwell time of SIM measurements: 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 purified water. ii. A 100-µL volume of IS (fenitrothion-d 6 ) solution is added to the above mixture and mixed well. iii. The 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. The n-hexane layer is carefully transferred to a glass vial, and evaporated to dryness under reduced pressure d. vii. The 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 identification f and semiquantitation of a pesticide in a test specimen are carried out.

Similtaneous analysis of pesticides by GC/MS 529 Figure 1.1 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.

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 Monitor ions (m/z) time* 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.

Similtaneous analysis of pesticides by GC/MS 531 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. The chromatograms of the 3 groups showed no interference of the test peaks by impurities of urine. The simultaneous analysis is aimed at screening of compounds in a wide range; the conditions should be set according to an average property of many analytes. Therefore, satisfactory recoveries cannot be obtained for all compounds. By this method, the recoveries of the pesticides 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. The 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 after addition of several ten nanograms to blank specimens; but in actual cases, its high concentrations are frequently detected. Therefore, at the first step, a causative compound is identified 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, after 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 different 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 coefficients 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 pesticides during storage. Most pesticides except DDVP were stable in a frozen state; their coefficients of inter-day variation were as good as 5 10 %. The coefficients 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; after 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. The detection limits are different in different pesticides; they were 1 64 ng/ml for blood specimens and 1 254 ng/ml for urine specimens. Therefore the present method is sensitive enough to detect and identify causative pesticides in poisoning cases. The 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 specimen 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 identification 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). 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 temperature, 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.

Similtaneous analysis of pesticides by GC/MS 533 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. The 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 compound 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. The numbers of positive results were: sumithion, 78; malathion, 35; DDVP, 15; propanil, 12; methomyl, 8; carbaryl, 7; EPN, 6; fenthion, 6; isoxathion, 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. The analytical results were sometimes different from ones, which had been expected on the basis of early informations. For the final identification of a causative poison, the present screening method by GC/MS for a wide range of pesticides is very useful. Notes a) This is a semiquantitative method using one point standard. The 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 specimens. The 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 difference between added and non-added specimens can be used for calculation of the concentration of the test compound in the nonadded 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 find 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 injected at high injection and oven temperatures for washing before the next analysis. f) A guide for identification 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 profile 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. The shift of retention time of the test peak as compared with that of the authentic peak should be within ± 1 %. All the above conditions are fulfilled, the presence of the compound in a test specimen can be judged positive. When measurements are made in the scan mode, the identification is much easier; with almost the same mass spectra and retention times, the compound of the test peak can be judged identical to the authentic one; however, when interference by various peaks due to a different 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 analysis 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 chromatography/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 Commentary. 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)