Determination of Urinary Metabolite of Profenofos after Oral Administration and Dermal Application to Rats

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1 J. Fd Hyg. Safety 17(1), (2002) w profenofos n v s z d *Á Á **Á w œ w w» y k w 5.3 l Determination of Urinary Metabolite of Profenofos after Oral Administration and Dermal Application to Rats Young-Joo Cho*, Kyung-Jin Min, In-Seon Lee** and Chun-Geun Cha %FQBSUNFOUPG1VCMJD)FBMUI,FJNZVOH6OJWFSTJUZ5BFHV,PSFB /BUJPOBM*OTUJUVUFPG"HSJDVMUVSBM4DJFODFBOE5FDIOPMPHZ3%"4VXPO,PSFB 5IF$FOUFSGPS5SBEJUJPOBM.JDSPPSHBOJTN3FTPVSDFT,FJNZVOH6OJWFSTJUZ5BFHV,PSFB ABSTRACT This study was aimed to determine the urinary metabolite of profenofos, one of the organophosphorus pesticides, as the biomarkers of exposure. Urine samples were collected for 24 hours in metabolic cages after oral administration and dermal application of profenofos to rats. Identification of the derivatized urinary metabolite was determined by GC/MS and excretion time courses of the urinary metabolite was analyzed by GC/MS. Urinary metabolite of profenofos, 4-bromo-2-chlorophenol, was detected in rats urine both after oral administration and dermal application of profenofos. Parent compound was not detected in the experiment. In GC/MS, the mass spectral confirmation for 4-bromo-2-chlorophenol ion was identified at m/z bromo-2-chlorophenol was excreted within 48 hours and 72 hours after oral administration and dermal application of profenofos, respectively. In this study, the same urinary metabolite of profenofos was detected both in oral and dermal exposure. Generally, excretion of the urinary metabolite after oral administration was detected faster than after dermal application. It is suggested that urinary 4-bromo-2-chlorophenol could be used as the biomarkers of exposure to profenofos. Key words ý Profenofos, Metabolite, Urine, GC/MS v. ù û w y w w ƒ š. p w y j mw» w. 1) p ³,,,,, ù.», e p,»,», r, v, p. 2)» ü w,,, w acetylcholinesterase ƒ ww acetylcholine g w. 3)» cholinesterase w ƒ w,,, Author to whom correspondence should be addressed. y,, y ùkú. wr x acetylcholinesteraseƒ»,,», š x, y. 4)» ü ƒ ù 24 ü. d 5) w, w w ƒ. y 6) w y š ù, wù w š, ú (<72 hours), w» ¾ d w w l t d w. 7) EPA w» 80% ƒ œm dialkyl phosphatesƒ ù. 8) w dialkyl phosphates w acetylcholinesterase y d ¾ w š. 9)» w v s 20

2 %FUFSNJOBUJPOPG6SJOBSZ.FUBCPMJUFPG1SPGFOPGPTBGUFSPSBM"ENJOJTUSBUJPO_ 21 87% w š., w» w ƒw k v w w. 10,11) z ùkù» s ù w z x ü»ƒ š. 12) v ƒ ƒ ƒ w. tv mw v y, mw n, û mw n. û mw x vd w ƒ, tv s mw t n ƒ v ƒ y» v y w ü w. 13),» w v w» w sƒ w w.» t j, ù 1 profenofos n v s z w y w wš y w w sƒ w» wš w. 150~200 g (5~6 ) Sprague Dawley w œ w g. (normal control group, NC), propylene glycol (propylene glycol treatment control group, PGTC) (treatment group) w ƒƒ 5 x w. x ü» profenofos [O-4-bromo-2-chlorophenyl O-ethyl S- propyl phosphorothioate, 96.3%, ( ) ] w.»» x»» w» w Hewlett Packard (HP) 6890 GC HP 5972A MSD (mass selective detector) w š, z» (Rikakikai, NE-IS),» (Hanil Science, HA ) w»» w. TBAH (tetrabutylammonium hydroxide, 1.0 M in methanol) Aldrich, Wako t w w. Richardson Seiber 14). 1ml w wš, 6N-HCl 1 ml š 1 vortex mixer yww z 5% ethanol/ethyl acetate 1 ml š 2 vortex mixer yww. 2000rpm 15 wš d Áòw ¼ z w d ww 35 C z o ethyl acetate 1ml š. TBAH 40 µl š vortex mixer w yw g š, y 2µl GC/MS w w. w» w GC/MS Table 1. GC/MS» 80 o C, 5 C/min o 150 C¾ o z, 10 C/min o 300 C¾ o. Table 1. GC/MS conditions for the analysis of urinary metabolite. Item GC/MS Instrument Hewlett-packard 6890 GC conditions Column HP-5MS capillary 30 m 0.25 mm (I.D.) Temperature Col. initial temp. 80 initial time 0 min rate 5, 10 o C/min final temp. 150, 300 o C final time 0 min Inj. 280 o C - Carrier gas He, 0.7 ml/min Air - Hydrogen - Type of injection Split (1/10) Injection volume 2 µl Detector - MS conditions MS HP 5972A MSD Ionization mode Electron impact Mass range m/z Electron energy 70 ev

3 22 :PVOH+PP$IPFUBM w Profenofos n œ w (NC), propylene glycol 5 ml/kg n w (PGTC) w, (treatment) profenofos propylene glycol (LD 50 male rat: 358 mg/kg) 1/10 zonde w n w. 15) wr, v s w x 24 l» 5 6cm j» w cm j» profenofos w x s w š š g. v s j 16) (NC), propylene glycol (PGTC) propylene glycol 5ml/kg v s gš, (treat-ment) profenofos propylene glycol v (LD 50 male rat: 3300 mg/kg) 1/10 v sw. w ƒ metabolic cage œ w 24 w wš y g GC/MS w. ¾ -20 C þ o w. wr, profenofos n v s z d w» w w ƒ metabolic cage œ w 6, 12, 24, 48, 72, 96 w w w z GC/MS w. d (peak area) w (peak area) ùkü. š n z d profenofos n z d w. (NC), propylene glycol n w (PGTC) profenofos n w (treatment) ƒƒ 24 w w GC/MS total ion chromatogram Fig. 1 (A), (B), (C). Fig. 1. GC/MS total ion chromatograms of NC urine (A), PGTC urine (B), and treatment urine (C) in oral administration of profenofos to rats. (NC: normal control group, PGTC: propylene glycol treatment group) Fig. 2. GC/MS total ion chromatogram and mass spectrum of urinary metabolite (4-bromo-2-chlorophenol) in oral administration of profenofos to rats.

4 %FUFSNJOBUJPOPG6SJOBSZ.FUBCPMJUFPG1SPGFOPGPTBGUFSPSBM"ENJOJTUSBUJPO_ 23 4-bromo-2-chlorophenol 9 ùkû. GC/MS propylene glycol 4-bromo-2-chlorophenol w e. 4-bromo-2-chlorophenol ƒ m/z=208, 142, 99, 63 š total ion chromatogram rp Fig bromo-2- chlorophenol rp 17) w ƒ w. wr, 4-bromo-2- chlorophenol š y w GC/MS w. v s z d profenofos v s z d w. (NC), propylene glycol n w (PGTC) profeofos n w (treatment) ƒƒ 24 w w GC/MS total ion chromatogram Fig. 3 (A), (B), (C). 4-bromo-2-chlorophenol 9 ùkû. GC/MS propylene glycol 4-bromo-2-chlorophenol w e. 4-bromo-2-chlorophenol ƒ m/ z=208, 142, 99, 63 š total ion chromatogram rp Fig bromo-2-chlorophenol rp 17) w ƒ w. wr, 4-bromo-2-chlorophenol š y w GC/ MS w.» w s sƒ alkyl phosphates phenolic d w,» 6 alkyl phosphates ƒ š» d w alkyl phosphates ù d, phenolic p w s y. 18) profenofos phenolic 4-bromo- 2-chlorophenol profenofos s 4-bromo-2-chlorophenol l t Fig. 3. GC/MS total ion chromatograms of NC urine (A), PGTC urine (B), and treatment urine (C) in dermal application of profenofos to rats. (NC: normal control group, PGTC: propylene glycol treatment group) Fig. 4. GC/MS total ion chromatogram and mass spectrum of urinary metabolite (4-bromo-2-chlorophenol) in dermal application of profenofos to rats.

5 24 :PVOH+PP$IPFUBM Fig. 5. Proposed metabolite of profenofos in rats. Fig. 6. Comparison excretion time courses of urinary 4- bromo-2-chlorophenol in oral and dermal exposure of profenofos to rats. ƒ. wr 4-bromo-2- chlorophenol profenofos ƒ w, Ÿ w, w. x profenofos 17) n v s z 4-bromo-2-chlorophenol y ü w w (Fig. 5). n z d Profenofos n z 6, 12, 24, 48, 72, 96 4-bromo-2-chlorophenol w ùkù Table 2. Profenofos 4-bromo-2-chlorophenol 48 ü 95%ƒ š 12 ƒ, 72 z. v s z d Profenofos v s z 6, 12, 24, 48, 72, 96 4-bromo-2-chlorophenol w ùkù Table 3. Profenofos 4-bromo-2-chlorophenol 48 ü 87%ƒ š 12 ƒ, 96 z. Profenofos n Fig. 6. n 4-bromo-2-chlorophenol w» z x (one-compartment model) d w n z 8.9 š v s z n v s z. Table 2. Excretion time course of urinary 4-bromo-2-chlorophenol in oral administration of profenofos to rats. Percentage of time course (hours) bromo-2-chlorophenol ± ± ± ± 0.24 * ND ND Oral dose level: 35.8 mg/kg (1/10 of LD 50 ). * ND: not detected. Each value represents the mean ± S.E. of 3 experiments and one experiment used five rats. Table 3. Excretion time course of urinary 4-bromo-2-chlorophenol in dermal application of profenofos to rats. Percentage of time course (hours) bromo-2-chlorophenol ± ± ± ± ± 0.29 * ND Dermal dose level: 330 mg/kg (1/10 of LD 50 ) * ND: not detected Each value represents the mean ± S.E. of 3 experiments and one experiment used five rats.

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