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1 Korean Journal of Environmental Agriculture Korean J Environ Agric (2012) Online ISSN: Vol. 31, No. 3, pp Print ISSN: Research Article Open Access 국내미등록유기인계농약의수입농식품에대한다성분잔류분석법 전영환, 1 황정인, 1 안지운, 1 김효영, 1 도정아, 2 오재호, 2 황인균, 2 임무혁, 3 이중근, 4 이영득, 5 김장억 1 * 1 경북대학교응용생명과학부, 2 식품의약품안전평가원, 3 식품의약품안전청, 4 한국보건산업진흥원, 5 대구대학교생명환경학부 Multiresidue Analysis Method for Determination of Unregistered Organophosphorus Pesticides in Korea for Imported Agri-Food Young-Hwan Jeon, 1 Jeong-In Hwang, 1 Ji-Woon Ahn, 1 Hyo-Young Kim, 1 Jung-Ah Do, 2 Jae-Ho Oh, 2 In-Gyun Hwang, 2 Moo-Hyeog Im, 3 Joong-Keun Lee, 4 Young-Deuk Lee 5 and Jang-Eok Kim 1* ( 1 School of Applied Biosciences, Kyungpook National University, Daegu , Korea, 2 National Institute of Food and Drug Safety Evaluation, Osong , Korea, 3 Korea Food and Drug Administration, Osong , Korea, 4 Korea Health Industry Development Institute, Osong , Korea, 5 Division of Life and Environmental Science, Daegu University, Gyeongsan , Korea) Received: 7 September 2012 / Accepted: 21 September 2012 c 2012 The Korean Society of Environmental Agriculture Abstract BACKGROUND: For safety evaluation of imported agri-food in Korea, the multiresidue analysis method was establised for unregistered organophosphorus pesticides, aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos and sulfotep. METHODS AND RESULTS: The used method for multiresidue analysis in brown rice and orange used as representative samples of imported agri-food was the official method of Korean Food and Drug Administration. The results of validation test of 13 organophosphorus pesticides except crotoxyphos for multiresidue analysis method are compared to the criteria such as specificity, linearity, accuracy, precision and limit of quantification. CONCLUSION: The used method for multiresidue analysis of unregistered 13 organophosphorus pesticides except crotoxyphos in Korea can surely be used as an official method for routine analysis of imported agri-food. * 교신저자 (Corresponding author), Phone: ; Fax: ; jekim@knu.ac.kr Key Words: Imported agri-food, Multiresidue analysis, Unregistered organophosphorus pesticides, Validation test 서론 FTA(Free Trade Agreement) 를체결할시우리나라는주로공산품을주요수출품목으로제시하면서상대국의농식품을수입품목으로협상하여왔다. 또한국내의농업여건도농업종사자및경지면적의감소로농업생산성도상대적으로줄어들고있는현실이다. 이러한현실하에서국내시장에는수입농식품의양이계속적으로증가하고있는실정이다 (Park et al., 2004, Jeon et al., 2011). 수입되는농식품에대한우리나라차원에서의농약안전성검사는국민들의건강보호와삶의질향상이란측면에서매우중요한요소이다. 현재우리나라는수입농식품에대한농약안전성검사를식품의약품안전청에서실시하고있는데국내에서 MRL(Maximum Residue Limit) 이설정되어있는농약은국내기준을적용하고있고그렇지않은것은 Codex기준을적용하고있다. 국내나 Codex에기준이설정되지않은농약은 zero tolerance 개념을적용하고있다. 각나라마다농산물의재배환경이다르기때문에발생되는병해충및잡초의종류도달라져각나라마다등록되어사용되고있는농약의종류도다를수있다. 따라서농산물의교역시상호국가간에사용되는농약의종류가다르기때문에여러가지문제가초래되어미국은 zero tolerance기준, 277

2 278 JEON et al. 일본과유럽연합은 PLS(Positive List System) 법을제정하여운영하고있다 (Breen, 2007). 우리나라에서도수입농식품의양이증가되고있는현실에서국내에등록되어있지않은농약의안전성평가를실시하여야하는데일차적으로해야할것이국내미등록농약의잔류분석법의확립이라할수있다. 본연구는국내미등록농약에대한다성분잔류분석법의개발에관한연구의일환으로서국내에미등록된 14종류의유기인계농약을대상으로실시하였다 (Tolosa et al., 1999; Tarbah et al., 2001; Cooke et al., 2004; Lehotay et al., 2004; Fenoll et al., 2007; Qu et al., 2010; Pareja et al., Borras et al., 2011; 2011; Yang et al., 2011; Zhao et al., 2011). 유기인계농약은독성이높은반면에잔류성이길지않기때문에개발된지가상당히오래된농약임에도불구하고전세계적으로널리사용되고있는농약의화학적인계열중의하나이다. 따라서본연구는국내에미등록된 14 종류의유기인계농약 aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos 및 sulfotep 에대한다성분잔류분석법을확립하여국내로수입되는농식품의안전성평가에기여하고자한다. 재료및방법 농약및시약유기인계농약 aspon(91.0%), chlorthion(96.5%), chlorthiophos(96.5%), crotoxyphos(90.2%), demeton-o (90.0%), demeton-s(96.0%), demeton-s-methyl(93.5%), dioxathion(96.0%), heptenophos(92.0%), iodofenphos (98.0%), leptophos(99.0%), methyl-trithion(96.1%), propetamphos(95.0%) 및 sulfotep(94.0%) 등의표준품은 Dr. Ehrenstorfer GmbH (Germany) 사의것을사용하였으며, 이들의화학구조및 log P 값은 Table 1과같다. 정제용컬럼은 Varian Inc.(U.S.A.) 사의 Bond elut-fl(500 mg, 3 ml) cartridge를사용하였다. 잔류농약의분석법을위한유기용매인 acetone, acetonitrile, n-hexane은 Burdick & Jackson(U.S.A.) 사로부터농약잔류분석용을, sodium chloride 는 GR급으로 Junsei Chemical Co.(Japan) 에서구입하여사용하였다. Table 1. Chemical structures and log P of unregistered organophosphorus pesticides in Korea Pesticide Structure Use log P Pesticide Structure Use log P Aspon Insecticide 6.0 Dioxathion Insecticide 5.0 Chlorthion Insecticide 3.5 Heptenophos Insecticide 2.3 Chlorthiophos Insecticide 6.0 Iodofenphos Insecticide 5.5 Crotoxyphos Insecticide 3.3 Leptophos Insecticide 6.3 Demeton-O Insecticide 3.0 Methyl-trithion Insecticide 4.8 Demeton-S Insecticide 2.3 Propetamphos Insecticide 3.8 Demeton-S -methyl Insecticide 1.3 Sulfotep Insecticide 4.0

3 국내미등록농약의잔류분석 279 Table 2. Instrumental conditions of organophosphorus pesticides for multiresidue analysis in brown rice and orange Model Shimadzu GC-2010 gas chromatograph GC/FPD Detector FPD MSD Column Column temperature J&W Scientific DB-17 (30 m 0.25 mm i.d μm ) 80 (2 min.) 10 /min. 250 (21 min.) Shimadzu GC-2010 gas chromatograph GC/MS J&W Scientific DM-5MS (30 m 0.25 mm i.d μm ) 100 (2 min.) 10 /min. 280 (15 min.) Injector port Detector block (Interface) Gas flow rate N₂ 1 ml /min. Makeup N₂ 30.0 ml /min. He 0.9 ml /min. Injection volume 1 μl (Splitless) 1 μl (Splitless) Mass range Ionization - EI, 70 ev 수입농식품다양한수입농식품중대표농산물은지방함량이 2% 이상함유한지방성농산물과비지방성농산물로분류하고, 현미는지방성시료의대표농산물로오렌지는비지방성시료의대표농산물로선정하였다. 기기분석조건 Aspon외 13종의유기인계농약의잔류분석을위하여 GC 는 Shimadzu(Japan) 사의 GC-2010를사용하였고, 컬럼은 DB-5[30 m(length) 0.25 mm(i.d.) 0.25 um(film thickness)] 을사용하였다. 분석된농약의재확인과정을위해사용된 GC/MS는 Shimadzu(Japan) 사의 GC-QP2010 plus MS이었다. 기기분석은 Table 2와같은조건으로분석하였다. 표준검량선작성및정량한계각농약의표준용액은각각의표준품을 1,000 mg/l가되도록 acetone에용해시켜 stock solution으로사용하여이를 0.05, 0.1, 0.2, 0.5, 1.0 및 2.0 mg/l의농도가되도록희석하고 GC/FPD에주입하여크로마토그램의 peak의면적을기준으로표준검량선을작성하였다. 또한각농약의표준용액 mg/l을 1.0 ul씩 GC/FPD에주입하여크로마토그램의 signal과 noise의비를구하여 LOQ(Limit of Quantification) 를설정하였다. 다성분잔류분석법우리나라식품의약품안전청에고시되어있는다종농약다성분분석법 ( 제2법 ) 을이용하였다. 현미와오렌지 50 g에 acetonitrile 100 ml를넣은후혼합추출분쇄기로 3 분간균질화하였다. 이를감압여과한후여액을 sodium chloride g이들어있는 150 ml의분리병에담고마개를막은후 1 분간흔들어섞어주고약 1 시간정치하여 acetonitrile 층과물층을분리시켰다. 상등액인 acetonitrile 층 20 ml를취하여감압농축한후잔류물을 20% acetone 함유 n-hexane 에재용해하였다. 정제는미리활성화시킨 florisil SPE cartridge (3 ml, 500 mg) 의상단에앞서재용해한시료액을가하여시험관에받고이어서 20% acetone 함유 n-hexane 5 ml 로용출하여동일한시험관에받았다. 이용출액을감압농축한후 20% acetone 함유 n-hexane에재용해하여 GC/FPD 로분석하였다. 회수율시험본연구에서확립한농약다성분잔류분석법의효율및신뢰성을검증하기위하여회수율시험을수행하였다. 무처리시료 50 g에 aspon외 13종의표준용액을 LOQ 수준및 10 배수준이되도록첨가한다음상기잔류농약분석법에따라회수율을시험하였다. 결과및고찰 정량한계 (LOQ ; Limit of Quantitation) 의설정 LOQ는시험에사용된시료의양과분석조작중의희석배율그리고분석기기의검출능력등을감안하여산출한수치로, 실험에사용된분석방법으로신뢰성있게정량할수있는한계를의미한다 (Lee et al., 2009). 우리나라의식품공전잔류농약분석법실무해설서에는 0.05 mg/kg 이하또는 MRL 의 1/2 이하까지정량이가능하도록권장하고있다. 본실험에사용된 14종의농약인 aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyltrithion, propetamphos 및 sulfotep의기기의정량한계는 S/N비가 10배이상되는 ng으로설정하였다. 분석정량한계는아래의식에따라 crotoxyphos를제외한 13종

4 280 JEON et al. 농약모두 mg/kg 이하로설정되었고, 이는잔류분석법기준에도적합하였다. 그러나 crotoxyphos는분석정량한계가 0.10 mg/kg으로산출되어잔류분석법기준을초과하는것으로나타났다. LOQ (mg/kg) = [ 기기의정량한계 (ng) 최종시험용액량 (ml)] / [GC 주입량 (μl) 시료량 (g)] 검량선의직선성 Aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos 및 sulfotep의농도별표준희석용액을각 1.0 μl씩을 GC/FPD 에주입하여얻은검량선의회귀방적식은 Table 3과같았으며검량선의직선성을의미하는결정계수 (coefficient of determination, R 2 ) 는 이었다. 회수율시험 현미및오렌지무처리시료에표준용액을 LOQ 수준과 LOQ 수준의 10배의농도가되도록첨가한후앞서확립된분석방법으로회수율을시험한결과는 Table 4와같았으며 LOQ 수준에서는 %, LOQ 수준의 10배에서는 % 로나타나잔류농약분석기준인 % 이내의회수율을만족하였다. 변이계수 (Coefficient of Variation, CV) 역시전체시료에서 5.8% 이하로나타나잔류분석기준인 10% 이내를만족하였다. 또한 GC로분석된크로마토그램은 Fig. 1과같으며전체시료에서간섭피크는확인되지않았다. Crotoxyphos는감도가낮아 mg/kg에서는기기상에서검출되지않았으나 0.4 mg/kg 수준에서는 % 로나타났다. Table 3. Parameters of calibration curves for residue analysis of organophosphorus pesticides Pesticide Brown rice Orange Calibration curve R 2 Calibration curve R 2 Demeton-O y = 973, , y = 397,498-75, Heptenophos y = 4,244, , y = 2,876, , Demeton-S-methyl y = 5,606, , y = 3,971, , Sulfotep y = 16,408, , y = 15,590, , Demeton-S y = 12,002, , y = 10,267, , Propetamphos y = 8,613, , y = 8,235, , Dioxathion y = 2,167, , y = 2,250,932-88, Aspon y = 11,665, , y = 10,584, , Chlorthion y = 6,656, , y = 6,065, , Crotoxyphos y = 501, , y = 243, , Iodofenphos y = 5,556, , y = 4,358, , Chlorthiophos y = 5,541, , y = 4,958, , Methyl-trithion y = 6,014, , y = 5,179, , Leptophos y = 5,700,011-1,174, y = 4,561, , Table 4. Limit of quantitation and recoveries of organophosphorus pesticides in brown rice and orange Pesticide Demeton-O Heptenophos Demeton-S-methyl Sulfotep Fortification level Brown rice Recovery(%) Orange 92.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.2 LOQ* (mg/kg)

5 국내미등록농약의잔류분석 281 Demeton-S Propetamphos Dioxathion Aspon Chlorthion Crotoxyphos Iodofenphos Chlorthiophos Methyl-trithion Leptophos * LOQ : Limit of Quantitation 79.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0 0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fig. 1. Chromatograms of recovery tests for organophosphorus pesticides in brown rice and orange fortified at 0.4 mg/kg by GC-FPD(1: Demeton-O, 2: Heptenophos, 3: Demeton-S-methyl, 4: Sulfotep, 5: Demeton-S, 6: Propetamphos, 7: Dioxathion, 8: Aspon, 9: Chlorthion, 10: Crotoxyphos 11: Iodofenophos, 12: Chlorthiophos, 13: Methyl-trithion, 14: Leptophos).

6 282 JEON et al. GC/MS 를이용한정성확인 GC/FPD로분석된 aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyltrithion, propetamphos 및 sulfotep의정성적확인을위하여 GC/MS 기기분석을실시하였다. 각농약성분들의 total-ion chromatogram(tic) 을분석하고각성분의 EI(Electron ionization) mass spectrum을얻어각구조를확인하였다. 각농약의 TIC와 EI mass spectrum은 Fig. 2와같다. EI mass spectrum으로부터분석된 fragment ion들의화학구 조반영정도와 intensity 를고려하여 selected-ion monitoring(sim) 분석을위한최적 fragment ion을선정하였다. 기기분석성분들의머무름시간및 GC/MS SIM 분석을위한 fragment ion의질량대전하비 (m/z) 값은 Table 5와같다. 선정된 fragment ion을이용하여 SIM mode로분석한결과는 Fig. 3과같다. 전체시료에서간섭피크는확인되지않았으며, crotoxyphos을제외한 aspon 외 12종의농약에대하여정성확인결과 13종의농약모두일치하는것으로나타났다. crotoxyphos의경우 GC/MS의감도및재현성이양호하지못하여정성확인이불가능하였다. Fig. 2. Total-ion chromatograms and electron ionization mass spectra of organophosphorus pesticides by GC/MS(1: Heptenophos, 2: Demeton-O, 3: Demeton-S-methyl, 4: Sulfotep, 5: Demeton-S, 6: Propetamphos, 7: Dioxathion, 8: Aspon, 9: Chlorthion, 10: Iodofenophos, 11: Chlorthiophos, 12: Methyl-trithion, 13: Leptophos).

7 국내미등록농약의잔류분석 283 Fig. 3. Selected-ion monitoring chromatograms of organophosphorus pesticides in brown rice and orange fortified at 0.4 mg/kg by GC/MS(1: Heptenophos, 2: Demeton-O, 3: Demeton-S-methyl, 4: Sulfotep, 5: Demeton-S, 6: Propetamphos, 7: Dioxathion, 8: Aspon, 9: Chlorthion, 10: Iodofenophos, 11: Chlorthiophos, 12: Methyl-trithion, 13: Leptophos). Table 5. Fragment ions of 14 organophosphorus pesticides monitored by GC/MS Pesticide Retention time(min) Molecular Weight Fragment monitored (m/z) SIM 1 SIM 2 Heptenophos Demeton-O Demeton-S-methyl Sulfotep Demeton-S Dioxathion Propetamphos Aspon Chlorthion Iodofenphos Methyl-trithion Chlorthiophos Leptophos Crotoxyphos

8 284 JEON et al. 시험법의밸리데이션 현미와오렌지를대상으로 crotoxyphos을제외한유기인계농약 13종에대한다성분잔류분석법을실시한밸리데이션결과는 Table 6과같다. 특이성, 직선성, 정확성, 정밀성및정량한계가식품의약품안전청에서설정한기준 ( 식품의약품안전청고시제 호 ) 과일치하는것으로나타났다. 따라서현미와오렌지가수입농산물의대표시료임을감안할때, 외국으로부터수입되는농산물로부터 crotoxyphos 을제외한 aspon 외 12종농약의잔류분석은본연구에서확립된농약다성분잔류분석법을적용할수있을것으로사료된다. Table 6. Summaries of validation results for multiresidue analytical method of 13 organophosphorus pesticides in brown rice and orange Requirement for validation (Parameter) Specificity Criterion * No interfering peaks on chromatogram Validation result No interfering peaks on chromatogram Linearity (R 2 ) > 0.99 > Accuracy (Recovery, %) Precision (CV **, %) < 30 < 5.8 LOQ *** 0.05 mg/kg mg/kg * The criteria established by KFDA ** CV : Coefficient of Variation *** LOQ : Limit of Quantitation 요약 외국으로부터수입되는농식품에대하여국내에미등록된유기인계농약 aspon, chlorthion, chlorthiophos, crotoxyphos, demeton-o, demeton-s, demeton-s-methyl, dioxathion, heptenophos, iodofenphos, leptophos, methyl-trithion, propetamphos 및 sulfotep 등 14종에대한안전성평가를위하여다성분잔류분석법을확립하고자하였다. 잔류분석법은식품의약품안전청에서고시한다종농약다성분동시분석법- 제2법에잘적용되었다. 수입농산물의대표시료로선정된현미와오렌지에대한유기인계농약 14종대한잔류분석법의밸리데이션을실시한결과특이성, 직선성, 정확성, 정밀성및정량한계수준을만족시키는것으로나타났다. 단지 crotoxyphos 의경우는기기상의정량한계가낮아서저농도에서의회수율은좋지않은결과를나타내었다. 따라서본연구에서확립된다성분잔류분석법은수입농산물중 crotoxyphos를제외한 aspon 외 12종의유기인계농약에대해적용가능한것으로나타났다. 감사의글 This work was supported in part from a 2011 project of Development of Multiple Analysis Method of Residual Pesticides for Imported Food by National Institute of Food and Drug Safety Evaluation. 참고문헌 Borras, E., Sanchez, P., Munoz, A., Tortajada-Genaro L.A., Development of a gas chromatographymass spectrometry method for the determination of pesticides in gaseous and particulate phases in the atmosphere, Analytica. Chimica. Acta. 699, Breen, J.L., International trade implications of pesticide residues in food, Kor. J. Pesti. Sci Cooke, C.M., Shaw, G., Lester, J.N., Collins, C.D., Determination of solid-liquid partition coefficients (Kd) for diazinon, propetamphos and cis-permethrin, Science of The Total Environment 329(1-3), Fenoll, J., Hellin, P., Martinez, C.M., Miguel, M., Flores, P., Multiresidue method for analysis of pesticides in pepper and tomato by gas chromatography with nitrogen-phosphorus detection, Food Chem. 105(2), Jeon, Y.H., Kim, H.Y.. Hwang, J.I., Kim, J.H., Do, J.A., Im, M.H., Oh, J.H., Kwon, K.S., Lee, J.K., Lee, Y.D., Kim, J.E., Application of multiresidue analysis method of unregistered pesticides in Korea for imported food, Korean J. Environ. Agric. 30(3), Lee, E.M., Lee, H.R., Riu, M.J., Park, H.W., Na, Y.R., Song, H.H., Keum, Y.S., Zhu, Y., Kim, J.H., Establishment of analytical method of prochloraz in cabbage, apple, mandarin, pepper and hulled rice with GC-ECD, Korean J. Environ. Agri. 28(4), Lehotay, S.J., Kok, A., Hiemstra, M., Bodegraven, P., Validation of a fast and easy method for the determination of residues from 229 pesticides in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection, J. AOAC Int. 88(2), Pareja, L., Fernandez-Alba, A.R., Cesio, V., Heinzen, H., Analytical methods for pesticide residues in rice, Trends in Analytical Chemistry 30(2), Park, J.H., Kim, T.K., Oh, C.H., Kim, J.H., Lee, Y.D., Kim, J.E., Analysis of multiple pesticide residues in apples and pears using gas-liquid chromatography, Korean J. Environ. Agri. 23(3),

9 국내미등록농약의잔류분석 Park, S.H., Safety management system of GM food, Food Sci. Indust. 41(2), Qu, L.J., Zhang, H., Zhu, J.H., Yang, G.S., Aboul-Enein, H.Y., Rapid determination of organophosphorous pesticides in leeks by gas chromatography-triple quadrupole mass spectrometry, Food Chem. 122(1), Tarbah, F.A., Mahler, H., Temme, O., Daldrup, T, An analytical method for the rapid screening of organophosphate pesticides in human biological samples and foodstuffs, Forensic Science International 121(1-2), Tolosa, I., Douy, B., Carvalho, F.P., Pesticide residue analysis in foodstuffs applying capillary gas chromatography with mass spectrometric detection, J. Chromatogr. A. 864(1), Tomlin, C.D.S., The Pesticide Manual (14th), BCPC, Hampshire, UK, pp , , , , 1109, Yang, X., Zhang, H., Liu, Y., Wang, J., Zhang, Y.C., Dong, A.J., Zhao, H.T., Sun, C.H., Cui, J., Multiresidue method for determination of 88 pesticides in berry fruits using solid-phase extraction and gas chromatography-mass spectrometry: Determination of 88 pesticides in berries using SPE and GC-MS, Food Chem. 127(2), Zhao, Y.G., Shen, H.Y., Shi, J.W., Chen, X.H., Jin, M.C., Preparation and characterization of amino functionalized nano-composite material and its application for multi-residue analysis of pesticides in cabbage by gas chromatography-.triple quadrupole mass spectrometry, J. Chromatogr. A. 1218(33),

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