7. 2 7. 2 © Springer-Verlag Berlin Heidelberg 2005 II.7.2 Organophosphorus pesticides by Masakatsu Sakata Introduction Organophosphorus pesticides ( organophosphate pesticides) are being most widely used as insecticides, and thus cause poisoning cases frequently. e organophosphorus pesticides at the early stage, such as parathion and TEPP, had powerful insecticidal e ects and high toxicity for both humans and beasts, and caused poisoning accidents during spraying. Although many less toxic organophosphorus pesticides were then developed, the resistance to the pesticides was acquired by insects during their repeated use, resulting in less e ectiveness of the pesti- cides. erefore, the development of new pesticides has been being required. Now, about 40 kinds of organophosphorus pesticides are being commercially available in Japan. ey are being controlled by the Poisonous and Deleterious Substances Control Law of Japan. Various types of pesticides with various degrees of toxicity are available; they are class ed into poison- ous, deleterious and common substances. Even with the common substances, such as malathi- on and fenitrothion, the ingestion of their large amounts for suicidal purpose causes fatalities. e fundamental structure of organophosphorus pesticides is: e pesticides are also structurally classi ed according to an element bound with the phos- phorus into the phosphate type, thiono type, thiol type and dithiol type. Many of R 1 and R 2 are dimethoxy or diethoxy groups. To cope with resistant insects, the alkyl moieties of R 1 and R 2 groups were replaced by unsymmetyrical propyl and ethyl groups, respectively, in some pesti- cides. As X structures, alkyl, alkoxy, alkylthio, aryl, heterocyclic, aryloxy and arylthio groups can be mentioned. > Figure 2.1 shows the metabolic pathways of a common thiono type organophosphorus pesticide having dialkoxy groups (R 1 and R 2 ). Organophosphorus pesticides undergo both enhancement of their toxicity and detoxi ca- tion at the same time in mammals. e thiono type pesticide (1) shows almost no inhibitory action on cholinesterase in its unchanged form; it is metabolized by cytochrome P450 (I) into the phosphate type (2), which reveals toxicity. erefore the phosphate type pesticide can inhibit cholinesterase without any metabolic activation. As detoxi cation, dealkylating reaction by enzyme II can be mentioned. e P450 and glutathion are being involved in the enzyme II; the dealkylated form thus produced does not inhibit acetylcholinesterase. e esterases III, which hydrolyze organophosphorus pesticides, as shown in > Fig. 2.1, are phosphorotriester hydrolases. ere are many di erent enzymes responsible for such reac- tions; they are called “ paraoxonase, A-esterase, phosphatase or arylesterase”. Carboxyesterase is 536 Organophosphorus pesticides related with the metabolism of malathion, which has carboxylic acid esters in its leaving groups. e metabolisms of organophosphorus pesticides are usually rapid. It is, therefore, neces- sary to analyze the metabolite(s) together with an unchanged organophosphorus pesticide to assess its poisoning correctly. As analytical instruments for organophosphorus pesticides, GC and HPLC are being used. Especially GC with detectors speci c for phosphorus or sulfur is useful, because of its high sensitivity and speci city. e metabolites are usually highly polar, and thus need derivatiza- tions for GC detection. GC and GC/MS analysis Materials and preparation • Standard compounds: highly pure organophosphorus compounds can be obtained from Supelco (Bellefonte, PA, USA) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). • Standard solutions: each standard compound is dissolved in acetone to prepare 1–10 µg/ mL solutions; these solutions can be further diluted according to needs. As an internal standard (IS), an organophosphorus compound, which does not overlap the peaks of a target compound and impurities, is chosen in preliminary experiments. • Solid-phase extraction cartridges: Sep-Pak C 18 cartridges (Waters, Milford, MA, USA). • Methylating reagent: Diazald ® ( N-methyl-N-nitroso-p-toluenesulfonamide) (Sigma-Aldrich, St. Louis, MO, USA); the distillation device attached with a 25-mL reservoir is constructed Typical metabolic pathways for organophosphorus pesticides in mammals. ⊡ Figure 2.1 537 inside a dra ; the reservoir is immersed in an ice bath. A 0.4-g amount of KOH is dissolved in 10 mL of ethanol/water (9:1, v/v). A 2.14-g amount of N-methyl-N-nitroso-p-toluene- sulfonamide is dissolved in 30 mL diethyl ether (0.01 M) and stored in a 250-mL volume round-bottomed ask under ice-cooling; a er 10 mL of the above KOH-ethanol solution is added to the above solution and mixed, the ask containing the mixture is connected with the distillation device and le at room temperature for several minutes. en, the ask is warmed at 35–40 °C for distillation until the color of the solution in it disappears; this procedure results in collecting about 12 mL diazomethane-diethyl ether solution. If all solution is stored in an airtight container, there is a possibility of its explosion due to generation of N 2 gas. e solution can be stored in a ask with a drying tube at –20 °C. Another method is its storage at –20 °C a er putting 2-mL aliquot each in 10-mL volume vials to be capped airtightly. While the ether solution is yellow, it can be used for methylation. • Organic solvents: they should be of the ultra-pure grade. Analytical conditions GC: an instrument equipped with a ame photometric detector ( FPD, with an interference lter for phosphorus) or a ame thermionic detector ( FTD, the same as the nitrogen-phospho- rus detector, NPD) a GC/MS; qualitatative analysis: total ion chromatograms (TICs) or mass chromatograms; quantitative analysis: selected ion monitoring (SIM) ( > Table 2.1) Capillary columns: DB-1, DB-5 and DB-210 (J&W Scienti c, Folsom, CA, USA); columns with various degrees of polarity should be tested for each pesticide to obtain good separation from other peaks without tailing. ⊡ Table 2.1 Mass spectra of organophosphorus pesticides commercially available in Japan Compound M.W. EI fragment ions Compound M.W. EI fragment ions acephate 183 136, 94, 95, 96, 79 malathion 330 125, 93, 127, 173, 158, 99 chlorofenavinphos 358 267, 269, 323, 81, 325 methidathion 302 145, 85, 93, 125, 146 chlorpyrifos 349 197, 199, 97, 314, 125 methyl parathion 263 109, 125, 263 chlorpyrifos-methyl 322 125, 286, 79, 63 monocrotophos 223 127, 67, 97, 109, 192 cyanophos 243 109, 125, 79, 243, 63 naled 381 109, 145, 79, 185, 147 diazinon 304 179, 137, 152, 304, 93, 153 parathion 291 97, 109, 291, 139, 137 dichlorvos ( DDVP) 220 109, 185, 79, 220, 145 phenthoate 320 274, 121, 93, 125, 79 dimethoate 229 87, 125, 93, 58, 79, 229 phosalone 367 182, 121, 184, 154, 367, 97 disulfoton 174 88, 89, 60, 61, 97 phosmet 317 160, 61, 76, 77, 133 EPN 323 157, 169, 185, 323 pirimiphos-methyl 305 290, 276, 305, 125, 233 ethion 384 231, 153, 97, 125, 121 profenofos 374 97, 208, 139, 338, 295 fenitrothion 277 109, 125, 127, 277, 260 prothiofos 345 113, 267, 309, 262, 63 fenthion 278 278, 125, 109, 153, 168 salithion 216 216, 183, 153, 78 isofenphos 345 58, 213, 255, 185, 96 trichlorfon 256 79, 109, 72, 93, 221 isoxathion 313 105, 177, 313, 159, 77 vamidothion 287 87, 109, 145, 58, 79, 142 GC and GC/MS analysis 538 Organophosphorus pesticides Column temperature: the initial temperature, 60–80 °C; temperature program, 10 °C/min; the nal temperature, the maximum permissible temperature; preliminary experiments are made under these conditions to nd optimal conditions for each compound. Injection temperature: the temperature is optimized in the range of 180–260 °C, because organophosphorus pesticides are relatively thermolabile. Procedures i. Plasma and urine A. Solid-phase extraction i. Each Sep-Pak C 18 cartridge (Waters, Milford, MA, USA) is washed with 10 mL of chloro- form/isopropanol (9:1), 10 mL acetonitrile, 10 mL of acetonitrile/distilled water (1:1) and 10 mL distilled water. ii. A 1-mL volume of plasma or urine is diluted 10-fold with distilled water, and mixed with IS b ; the mixture solution is poured into the above pretreated cartridge at a ow rate of 5 mL/min to trap target compounds including IS in the cartridge. iii. e cartridge is washed with 10 mL distilled water, followed by the elution of the target compounds with 3 mL of chloroform/isopropanol (9:1). e eluate is collected in a glass vial. A small amount of aqueous upper layer is removed with a Pasteur pipette, and the organic layer is evaporated to dryness under a stream of nitrogen. e residue is dissolved in 100 µL acetonitrile, and a 1–2 µL aliquot of it is injected into GC(/MS) [1]. B Liquid-liquid extraction i. A 1-mL volume of plasma or urine, 2 mL distilled water and IS are placed in a 10-mL vol- ume glass centrifuge tube with a ground-in stopper; the pH of the mixture is adjusted to 2 with 1 M HCl solution. ii. A er adding 2 mL chloroform, the tube is vortex-mixed for 2 min and centrifuged at 3,000 rpm for 5 min to separate the chloroform phase; this procedure is repeated twice, and the resulting chloroform phases are combined and evaporated to dryness under a stream of nitrogen c . iii. e residue is dissolved in 50–100 µL acetone; a 1–2 µL aliquot of it is injected into GC/ MS. iv. Methylation of metabolites: a er measurements of the nal acetone solution 2–3 times, the remaining acetone solution is evaporated to dryness; the residue is dissolved in 100 µL of the diazomethane-ether solution and le at room temperature for 5 min d . A er evapora- tion of the solution to dryness under a stream of nitrogen, the residue is again dissolved in 50–100 µL acetone; 1–2 µL of it is injected into GC(/MS). e obtained TIC is carefully compared with that before methylation. When a new peak appears or the amount of an organophosphorus pesticide is increased, there is a possibility of the presence of a metabo- lite e . ii. Organ specimens (acetonitrile extraction) i. A 1–2 g amount of an organ specimen is excised and put in 4 volumes of acetonitrile; the organ tissue is minced into small pieces with surgical scissors and homogenized with a 539 Polytron homogenizer (Kinematica, Luzern, Switzerland) or Ultra-disperser (Yamato, To- kyo, Japan) for 2 min for both extraction and deproteinization. ii. e homogenate is centrifuged at 3,000 rpm for 5 min to separate supernatant solution; to the sediment the same amount of acetonitrile is added, vortex-mixed and centrifuged to obtain the 2nd supernatant solution. iii. Both supernatant solutions are combined and condensed to about 0.5 mL under reduced pressure with warming at not higher than 40 °C. iv. To the condensed solution, 4.5 mL distilled water is added; when insoluble residues are present, they are removed by centrifugation. e clear supernatant solution is subjected to the above liquid-liquid extraction or solid-phase extraction. Assessment and some comments on the method Organophosphorus pesticides show relatively high vapor pressures, and thus suitable for anal- ysis by GC. However, they are generally susceptible to light and heat, and thus unstable. ey are also easily hydrolyzed under alkaline conditions; this should be kept in mind upon extrac- tion procedure. It is possible to achieve sensitive trace analysis of organophosphorus pesticides using an FPD or FTD (NPD), which is speci c for phosphorus. GC/MS, of course, enables sensitive quantitation in the SIM mode. However, when both GC/MS and GC-FPD (FTD) are available, the identi cation by GC/MS, followed by quantitation by GC-FPD (FTD) is most desirable. Unchanged organophosphorus pesticides are lipophilic and thus easily extractable with solid-phase extraction cartridges. However, when various metabolites of the pesticide are simultaneously analyzed, liquid-liquid extraction with chloroform is recommendable. e phosphorus-containing metabolites including dealkylated and hydrolyzed forms are all acidic compounds, which are extractable into chloroform under acidic conditions. When an un- changed organophosphorus pesticide and its metabolite(s) coexist, they can be easily separat- ed; the unchanged form can be extracted into hexane under neutral conditions, while the metabolite(s) can be extracted into chloroform under acidic conditions. e detection limit of EPN, when measured by wide-bore capillary GC-FID, was reported to be 2 ng in an injected volume [1]. When an FPD or FTD is connected with capillary GC, the detection limits of organophosphorus pesticides are as low as in the pg order on-column. Malathion, one of the most popular organophosphorus pesticides, is converted into acid metabolites by the action of carboxylesterase in mammals as shown in > Fig. 2.2 [2]. By methyl-derivatization of these metabolites, compounds with methyl ester(s) in place of ethyl ester(s) can be obtained. A TIC and a gas chromatogram obtained by capillary GC/MS and packed-column GC-FPD, respectively, are shown in > Fig. 2.3. With the packed column, the peaks overlapped, while with the capillary column, they are well separated. GC and GC/MS analysis 540 Organophosphorus pesticides Metabolic pathways for malathion in mammals. ⊡ Figure 2.2 TIC and gas chromatogram for malathion and its metabolites obtained by capillary GC/MS and packed-column GC-FPD, respectively. 1: malaoxon diacid*; 2: malaxon β-monoacid*; 3: malaoxon α-monoacid*; 4: malathion diacid*; 5: malaoxon; 6: malathion β-monoacid*; 7: malathion α-monoacid*; 8: malathion; 9: isomalathion. * methylated derivatives. ⊡ Figure 2.3 541 HPLC and LC/MS analysis Reagents and their preparation ey are almost the same as described in the section of GC and GC/MS analysis. Analytical conditions LC: any instrument equipped with a UV detector or a photodiode array detector can be used, regardless of its manufacturer. Column: an ODS (octadecylsilane-bonded silica gel) column. Mobile phase: a mixture of acetonitrile/distilled water (50:50) is tested for the retention times and peak shapes; the composition ratio is optimized for each analyte. Detection wavelength: most organophosphorus pesticides are detectable at 207 nm. By us- ing a photodiode array detector, identi cation of an unknown compound can be achieved to some extent [3, 4]. LC/MS: the methods using atmospheric pressure chemical ionization (APCI) [5] and ther- mospray (TS) ionization [6] were reported. In the LC/MS analysis of organophosphorus pesti- cides in the APCI mode, there are three methods of detection, viz., the modes of positive ion, negative ion and both ions [5]. Procedure f A 0.5-mL volume of plasma or urine is mixed with 0.5 mL acetonitrile, vortex-mixed for 20 s and centrifuged at 9,500 g for 4 min; 20-µL of the supernatant solution is injected into HPLC (/MS). Assessment and some comments on the method When a photodiode array detector is used for HPLC analysis, it is possible to know that a peak, appearing at the same retention time as that of a target compound, is not due to the target compound by measuring its UV absorption spectrum. It is useful for identi cation of a com- pound to some extent. Especially for the body uid specimens, such as plasma and urine, the addition of acetoni- trile enables both deproteinization and extraction; only a er centrifugation, a part of the su- pernatant solution can be directly injected into HPLC (/MS). is procedure is very simple and rapid, and thus very suitable for clinical tests in poisoning during emergency treatments. e detection limits depend upon the structures of aryl groups bound with the phosphate group. e detection limits using 206 nm wavelength are 14 ng/mL for fenitrothion and 110 ng/mL for the pesticides without chromophores, such as ethylthiomethon. Since at 206 nm of wavelength, the slope of an absorption spectrum is generally very steep, the measurements are unstable; reproducible results can be obtained by using 230 nm for quantitation. However, HPLC and LC/MS analysis 542 Organophosphorus pesticides the sensitivity becomes ten times less at 230 nm. In clinical analysis, there are some pesticides, which cannot be quantitated by HPLC with UV detection. For LC/MS, various interfaces are commercially available. Among them, electrospray ioni- zation (ESI), ion spray ionization and APCI are excellent in view of sensitivity and stability. e sensitivity of LC/MS is lower in a TIC, but comparable in the SIM mode as compared with that of GC/MS. Poisoning case, and toxic and fatal concentrations An 84-year-old female [7] was found dead with vomitus over the pillow at home. A 500-mL bottle of profenofos emulsi able concentrate, the concentration of which was 40 %, was found near the deceased. Approximately 220 mL of the concentrate remained in the bottle. e lapse of time a er death was estimated to be 27 h at the time of autopsy. Autopsy ndings were mio- sis, erosion of the pharynx, larynx and esophagus, and 87 mL of dark brownish uid in the stomach. e concentrations profenofos in blood, urine and gastric contents were 1.2, 0.35 and 3,350 µg/mL, respectively, while the concentration of its metabolite desethylated profenofos in blood was as high as 317 µg/mL. ese results show that the detection of a main metabolite is important together with that of an unchanged form for analysis of an organophosphorus pesticide. > Table 2.2 shows the distribution of malathion, a most widely used organophosphorus pesticide, and its metabolites among blood, bile and some organs in a fatal malathion poison- ing case. e blood concentration of malathion monoacid (21.7 µg/mL) was much higher than that of the unchanged form (0.36 µg/mL). > Table 2.3 summarizes the concentrations of various organophosphorus pesticides in blood, urine and stomach contents so far reported [3, 4, 8–17]. ⊡ Table 2.2 Distribution of malathion and its metabolites in blood, bile and some organs of a human victim in fatal malathion poisoning [8] Concentration (µg/g) Sample Malathion Malaoxon Monoacid Diacid blood 0.36 N.D. 21.7 2.9 bile 0.97 < 0.10 221 11.4 spleen 1.3 N.D. 59.0 18.6 kidney 1.3 N.D. 106 94.4 brain 6.3 < 0.10 2.0 0.16 adipose 80.4 0.29 14.3 2.5 liver N.D. N.D. 13.6 10.6 543 Notes a) Using these detectors, it is possible to detect an organophosphorus pesticide speci cally even in the presence of some impurities in an extract, enabling sensitive analysis. When a target compound is su ciently separated from impurities, GC-FID can be also used. b) As IS, an organophosphorus compound, which does not overlap peaks of a test compound and impurities, is selected in preliminary experiments. e volume of acetone being used as vehicle for IS should be not larger than 100 µL for the Sep-Pak cartridge extraction, because the presence of a non-negligible amount of acetone in a specimen solution may cause the leakage of a target compound upon pouring the solution into the cartridge. When a large amount of acetone has to be mixed with a specimen solution, the acetone solution containing IS is condensed into a small volume under a stream of nitrogen before adding to plasma or urine. ⊡ Table 2.3 Concentrations of organophosphorus pesticides in blood, urine and stomach contents in their poisoning cases Compound Dose (product mL) Blood conc. (µg/mL) (time after ingestion) Urinary conc. (µg/mL) Stomach content conc. (µg/mL) Alive/ dead Reference fenitrothion 50 100 500 17.1 (2 h) 14.5 (2 h) 3.9 (1.5 h) 3.9 (6 h) 1.28 (2 h) 2.4 (27 h) 1.8 (2 h) 0 0 6,100 557 7,500 alive alive alive alive alive dead [3] [3] [4] [9] [9] [10] malathion 1.89 0.36 19.0 (15 h) 2,100 dead dead dead [11] [8] [10] cyanophos 10.9 (8 h) 11.4 (3.5 h) 1,060 24,700 alive alive [10] [10] fenthion 10 0.17 (13 h) 3.8 alive alive [9] [12] prothiofos 370 4.83 (2 h) 0.16 2,610 alive [13] profenofos 1.2 0.35 dead [14] salithion 4.6 (2 h) alive [4] EPN plus 3.9 (5 h) dead [4] edifenphos 1.3 (5 h) dimethoate 21.2 (1.5 h) alive [15] trichlorfon 234 (5 h) 1,990 alive [10] pyridaphenthion 100 10 (6 h) alive [16] methidathion 30 5 (13 h) dead [16] DDVP 29 4.5 dead [17] Poisoning case, and toxic and fatal concentrations 544 Organophosphorus pesticides c) By shaking with chloroform under acidic conditions, both unchanged form of an organo- phosphorus pesticide and its acidic metabolite(s) are extracted into the chloroform layer, but other metabolites are not. d) When the yellow color of diazomethane-ether solution disappears, a 50-µL aliquot of the diazomethane solution should be added again. e) In the case of a mono-dealkylated metabolite of an organophosphorus pesticide having a diethyl phosphate structure, an asymmetrical methyl and ethyl phosphate derivative is produced by methylation; this results in appearance of a new peak. In the case of a metabo- lite of a pesticide having a dimethyl phosphate structure, the desmethylated metabolite goes back to the precursor form by methylation; this reaction results in the increase of the pesticide amount to be detected. e amount of the increase corresponds to that of the dealkylated metabolite. f) e extraction procedures described in the GC and GC/MS analysis section can be also used as pretreatments for HPLC and LC/MS. References 1) Liu J, Suzuki O, Kumazawa T et al. (1989) Rapid isolation with Sep-Pak C 18 cartridges and wide-bore capillary gas chromatography of organoposphate pesticides. Forensic Sci Int 41:67–72 2) Hayasaka M, Kawabata S, Haba A et al. (1994) Analysis of malathion metabolites in biological samples. Jpn J Forensic Toxicol 12:15–25 3) Cho Y, Matsuoka N, Kamiya A (1997) Determination of organophosphorus pesticides in biological samples of acute poisoning by HPLC with diode-array detector. Chem Pharm Bull 45:737–740 4) Mori H, Sato T, Nagase H et al. (1998) A method for rapid analysis of pesticides causing acute poisoning in patients and application of this method to clinical treatment. Jpn J Toxicol Environ Health 44:413–427 5) Kawasaki S, Ueda H (1992) Screening of organophosphorus pesticides using liquid chromatography-atmos- pheric pressure chemical ionization mass spectrometry. J Chromatogr 595:193–202 6) Lacorte S, Barcelo D (1995) Determination of organophosphorus pesticides and their transformation products in river waters by automated on-line solid-phase extraction followed by thermospray liquid chromatography- mass spectrometry. J Chromatogr 712:103–112 7) Gotoh M, Sakata M, Endo T et al. (2001) Profenofos metabolites in human poisoning. Forensic Sci Int 116:221–226 8) Morgade C, Barquet A (1982) Body distribution of malathion and its metabolites in a fatal poisoning by inges- tion. J Toxicol Environ Health 10:321–325 9) Kobayashi R, Kono I, Kawaguchi S et al. (1991) Clinical effects of hemoadsorption therapy in organophosphorus pesticide poisoning. Jpn J Toxicol 4:35–39 (in Japanese) 10) Takahashi A, Taira T, Kanesaka S et al. (1991) Effects of hemoadsorption therapy evaluated by organophosphorus pesticide concentrations in blood. Jpn J Toxicol 4:39–42 (in Japanese) 11) Suzuki O, Hattori H, Asano M (1985) Detection of malathion in victim by gas chromatography/negative ion chemical ionization mass spectrometry. Z Rechtsmed 94:137–143 12) Meyer E, Borry D, Lambert W et al. (1998) Analysis of fenthion in postmortem samples by HPLC with diode-array detection and GC-MS using solid-phase extraction. J Anal Toxicol 22:248–252 13) Sakata M, Goto M, Ubukata K et al. (1999) Prothiofos metabolites in human poisoning. J Toxicol Clin Toxicol 37:327–332 14) Seno H, Hattori H, Kumazawa T et al. (1998) Quantitation of postmortem profenofos levels. J Toxicol Clin Toxicol 36:63–65 15) Kojima T, Yashiki M, Ohtani M et al. (1990) Determination of dimethoate in blood and hemoperfusion cartridge following ingestion of formothion: a case study. Forensic Sci Int 48:79–88 16) Yamashita M (1988) Pharmacodynamics of organophosphorus compounds and their therapeutic drugs in humans. Jpn J Toxicol 1:49–54 (in Japanese with an English abstract) 17) Shimizu K, Shiono H, Fukushima T (1996) Tissue distribution of DDVP after fatal ingestion. Forensic Sci Int 83:61–66 . for malathion in mammals. ⊡ Figure 2.2 TIC and gas chromatogram for malathion and its metabolites obtained by capillary GC/MS and packed-column GC-FPD,. malaoxon diacid*; 2: malaxon β-monoacid*; 3: malaoxon α-monoacid*; 4: malathion diacid*; 5: malaoxon; 6: malathion β-monoacid*; 7: malathion α-monoacid*;