II.7.4 Carbamate Pesticides by Kiyoshi Ameno
Total Page：16
File Type：pdf, Size：1020Kb
7.4 II.7.4 Carbamate pesticides by Kiyoshi Ameno Introduction Among many carbamate pesticides commercially available in Japan, those with relatively high toxicities are shown in > Table 4.1 [1]. Carbamate pesticides are generally classifi ed into N-methylcarbamate insecticides and N -allylcarbamate herbicides in view of their chemical structures and biological actions. Th e number of fatalities due to poisoning by carbamate pes- ticides is 50–100 every year in Japan; many of them are poisoned by methomyl [2]. According to statics reported by National Research Institute of Police Science of Japan, the number of fatalities is highest with paraquat plus diquat, followed by organophosphates and then carba- mates among pesticides. Th e toxicity of carbamate pesticides is due to inhibition of acetylcho- linesterase (AchE) by their binding with the active site of the enzyme; the inhibition of the hydrolysis reaction of acetylcholine (Ach) results in the accumulation of Ach, provoking poisoning symptoms, such as miosis, lacrimation, sweating, hypersalivation and convulsion of extremities. Th e binding of carbamate pesticides to AchE is much weaker than that of organo- phosphorus pesticides, and the former pesticides are easily decomposeda in mammalian bodies. Th erefore, the damages of organs by carbamate pesticides have not been reported. For analysis of carbamate pesticides, methods by GC [3–7], GC/MS [5, 7, 8–10], HPLC [11] and LC/MS [10] were reported. In this chapter, the extraction procedures and analytical methods using the above 4 instruments are described for this group of pesticides. Reagents and their preparation • Th e authentic standards of compounds listed in > Table 4.1 and ethion can be purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. A standard mixture of seven N-methylcarbamate pesticides can be obtained from Kanto Chemicals (Tokyo, Japan)b. Carbofuran, carbaryl and ethion are separately dissolved in acetone to prepare 100 µg/mL solutions as stock solutions for internal standards (ISs)c. Carbendazole (Aldrich, Milwau- kee, WI, USA) is dissolved in distilled water to prepare 100 µg/mL solution. o-Methoxyl- phenol can be also obtained from Aldrich. • Sep-Pak C18 and Oasis MCX cartridges are purchased from Waters (Milford, MA, USA). • Th e solutions to be prepared are: n-hexane/ethyl acetate (1:1, v/v); acetonitrile/distilled water (1:1, v/v); methanol/distilled water (5:59, v/v); 0.1 M HCl solution; and 2 % NaCl solution. • 0.1 M Phosphate buff er solution (pH 7.0): 6.81 g of KH2PO4 is dissolved in 400 mL distilled water, and the pH of the solution is adjusted to 7.0 with 1.0 M NaOH solution; the fi nal volume is adjusted to 500 mL with distilled water. • 5 % Ammonia/methanol solution: 2.5 mL of 3 M NH4OH solution is mixed with 47.5 mL methanol. • HPLC mobile phases: methanol/distilled water (65:35, v/v); acetonitrile/2 mM ammonium formate (30:70 and 80:20, v/v, pH 3.0). © Springer-Verlag Berlin Heidelberg 2005 560 Carbamate pesticides ⊡ Table 4.1 Structures, properties and toxicities of carbamate pesticides Name, MF, MW, property and structure Use Acute oral LD50 (rat, mg/kg) bendiocarb insecticide 40 ~ 156 C11H13NO4 MW: 223.2 MP: 125 ~ 129 benfuracarb insecticide 223 205 C20H30N2O5S MW: 410.5 MP: 110 carbaryl insecticide 850 500 C12H15NO2 MW: 201.2 MP: 142 carbofuran insecticide 8 C12H15NO3 MW: 221.3 MP: 153 ~ 154 carbosulfan insecticide 623 657 C20H32N3O3S MW: 380.5 BP: 124 ~ 128 ethiofencarb insecticide 200 approx. C11H15NO2S MW:: 225.3 MP: 33.4 fenobucarb insecticide 250 185 C12H17NO2 MW: 207.3 MP: 31 ~ 32 furathiocarb insecticide 53 C18H26N2O5S MW: 382.5 BP: 250< Carbamate pesticides 561 ⊡ Table 4.1 (continued) isoprocarb insecticide 450 C11H15NO2 MW: 225.3 MP: 93 ~ 96 metolcarb insecticide 580 498 C9H11NO2 MW: 162.2 MP: 76 ~ 77 methomyl insecticide 17 24 C5H13N2O2S MW: 162.2 MP: 78 ~ 79 oxamyl insecticide 5.4 C7H13N3O3S MW: 219.3 MP: 100 ~ 102 pirimicarb insecticide 147 C11H18N4O2 MW: 238.3 MP: 90.5 propoxur insecticide 50 C11H15NO3 MW: 209.2 MP: 90 thiodicarb insecticide 66 C10H18N4O4S3 MW: 354.5 MP: 173 ~ 174 XMC insecticide 542 C10H13NO2 MW: 179.2 MP: 99 xylylcarb insecticide 375 325 C10H13NO2 MW: 179.2 MP: 79 ~ 80 MF: molecular formula; MW: molecular weight; MP (°C): melting point; BP (°C): boiling point. 562 Carbamate pesticides Extraction methods It is absolutely necessary to extract carbamate pesticides from crude biomedical specimens containing many impurity compounds as pretreatments before instrumental analysis. As spec- imens, body fl uids (whole blood, serum and urine), tissues and stomach contents are objects for analyses. Th e whole blood should be completely hemolyzed before extraction. Homogeni- zation is needed for tissues (organs) specimens; for stomach contents, the supernatant fraction aft er their centrifugation should be used. Liquid-liquid extraction i. A 2-g aliquot of specimens (blood, tissues and stomach contents d) is mixed with 10 mL acetonitrile (containing an appropriate IS), homogenized with a Polytron homogenizer and centrifuged at 3,000 rpm for 5 min to obtain clear supernatant solution. ii. For the sediment, the above i) step of extraction is repeated two times. iii. Th e three acetonitrile supernatant solutions thus obtained are combined and mixed with 80 mL of 2 % NaCl solution and 25 mL of n-hexane/ethyl acetate (1:1, v/v) in a 250-mL volume separating funnele. iv. Th e funnel is shaken for 10 min (with a shaking machine). v. Th e n-hexane/ethyl acetate layer is obtained. vi. Th e layer is evaporated to dryness under reduced pressure in a rotary evaporator at room temperature. vii. Th e residue is dissolved in 100 µL methanol. viii. A 2-µL aliquot of it is injected into GC or GC/MS; a 20-µL aliquot into HPLC. ix. For quantitation, each calibration curve is constructed using peak area ratios of a target compound to IS. A ratio obtained from a specimen is applied to the curve to calculate its concentration. Solid-phase extraction-1 [4] f i. A Sep-Pak C18 cartridge is washed and activated by passing chloroform, acetonitrile, acetonitrile/distilled water (1:1) and distilled water, 10 mL each, successively. ii. A 1-mL volume of a specimen (blood, serum or urine) is mixed with 9 mL distilled water, g stirred well and poured into the Sep-Pak C18 cartridge. iii. It is washed with 10 mL distilled water. iv. A target compound and IS are eluted with 3 mL chloroform. v. A small amount of aqueous layer (upper) of the eluate is carefully removed with a Pasteur pipette h. i vi. Th e above organic extract is dehydrated with anhydrous Na2SO4 . vii. It is evaporated to dryness under a stream of nitrogen at room temperature. viii. Th e residue is dissolved in 100 µL methanol j. ix. A 2-µL aliquot of it is injected into GC or GC/MS; a 20-µL aliquot into HPLC or LC/MS. GC and GC/MS analysis 563 Solid-phase extraction-2 [10] i. An Oasis MCX cartridge k is activated by passing 1 mL methanol and 1 mL of 0.1 M phos- phate buff er solution (pH 7) through it. ii. A 1-mL volume of serum is mixed well with 100 µL of IS (for example carbendazole) so- lution and poured into the cartridge. iii. Th e cartridge is washed with 1 mL distilled water. iv. Th e fi rst elution is made by passing 1 mL methanol through it. v. Th e cartridge is washed with 1 mL of 0.1 M HCl solution. vi. Th e second elution is made by passing 1 mL methanol again. vii. Th e third elution is made by passing 1 mL of 5 % ammonia/methanol. viii. Th e three eluates obtained at the steps iv, vi and vii are combined l, and evaporated to dry- ness under a stream of nitrogen. ix. Th e residue is dissolved in 100 µL methanol for GC or GC/MS analysis; an appropriate amount is injected into GC (/MS). For HPLC (/MS), the residue is dissolved in 100 µL of a mobile phase to be used and injected into HPLC (/MS). GC and GC/MS analysis GC conditions GC columnsm: DB-5 and DB-1 fused silica capillary columns (30 m × 0.25 mm i.d., fi lm thick- ness 0.25 µm, J&W Scientifi c, Folsom, CA, USA). GC conditions; instrument: Shimadzu GC14B (Shimadzu Corp., Kyoto, Japan); detector: fl ame thermionic ionization detector (FTD)n; column temperature: 50 °C → 20 °C/min → 120 °C → 5 °C/min → 260 °C(10 min); injection and detector temperature o: 230 °C; carrier gas: He (13 kPa). GC/MS conditions GC column: a DB-5MS fused silica capillary column (30 m × 0.25 mm i.d., fi lm thickness 0.25 µm, J&W Scientifi c). GC/MS; instrument: Shimadzu GC-MS5000 (Shimadzu Corp.); column temperature: 120 °C (1 min) → 20 °C/min → 240 °C (8 min); injection and separator temperature: 230 °C; carrier gas: He (2.0 mL/min); ionization mode: EI (70 eV). Assessment of the method For GC analysis of carbamate pesticides, an FTD detector is recommendable, because it is specifi c and sensitive, and is not infl uenced by impurities and organic solvents appreciably. Qualitative analysis only with a single column is insuffi cient; multiple columns of diff erent properties should be used for testing an identity of a compound. > Table 4.2 shows retention 564 Carbamate pesticides ⊡ Table 4.2 Retention times of carbamate pesticides obtained by GC* Carbamate Retention time Carbamate Retention time (min) (min) oxamyl 5.9 bendiocarb 15.1 methomyl-oxime 7.1 carbofuran 16.5 propamocarb 9.3 ethiofencarb 18.8 metolcarb (MTMC) 11.2 pirimicarb 18.8 isoprocarb 12.2 carbaryl 19.7 methomyl 12.2 methiocarb 20.7 XMC 12.6 benthiocarb 21.1 xylylcarb (MPMC) 13.3 diethofencarb 21.4 propoxur (PHC) 13.6 furathiocarb 31.5 fenobucarb 13.7 benfuracarb 33.6 * Detection limits were 0.5–1.0 ng on-column.