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pissn 1229-1153 J. Fd Hyg. Safety Vol. 28, No. 3, pp. 222~226 (2013) http://dx.doi.org/10.13103/jfhs.2013.28.3.222 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr GC-ECD 및 GC-MS 를이용한현미중 chloropicrin 의잔류시험법개발 도정아 1 최정희 1 박혜진 1 박용춘 2 윤혜정 3 최동미 4 오재호 5 * 1 식품의약품안전처식품의약품안전평가원식품위해평가부잔류물질과 2 식품의약품안전처식품의약품안전평가원식품위해평가부신종유해물질팀 3 식품의약품안전처서울지방청유해물질분석과, 4 식품의약품안전처식품영양안전국신소재식품과 5 식품의약품안전처식품의약품안전평가원식품위해평가부식품위해평가과 Development of an Analytical Method for Chloropicrin Determination in Hulled Rice by GC-ECD and GC-MS Jung-Ah Do 1, Jeong-Heui Choi 1, Hyejin Park 1, Yong-Chun Park 2, Hae-Jung Yoon 3, Dongmi Choi 4, and Jae-Ho Oh 5 * 1 Pesticide and Veterinary Drug Residues Division, National Institute of Food and Drug Safety Evaluation, MFDS, 2 New Hazardous Substances Team, National Institute of Food and Drug Safety Evaluation, MFDS, 3 Hazardous Substances Analysis Division, Seoul Reginal Food and Drug Administration, MFDS, 4 Novel Food Division, Food Nutrition and Dietary Safety Bureau, MFDS, 5 Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, MFDS, (Received February 19, 2013/Revised June 10, 2013/Accepted July 29, 2013) ABSTRACT - A simple and sensitive analytical method was developed using gas chromatograph with electron capture detector (GC-ECD) and gas chromatograph-mass spectrometer (GC-MS) for determination and identification of chloropicrin. Because of small molecular weight and high volatile properties of chloropicrin, analytical method was developed utilizing headspace extraction and direct injection to the GC. The developed method was validated using hulled rice sample spiked with chloropicrin at different concentration levels, 0.1 and 0.5 mg/kg. Average recoveries of chloropicrin (using each concentration three replicates) ranged 77.7~79.3% with relative standard deviations less than 10% and calibration solutions concentration in the range 0.005~0.5 µg/ml, and limit of detection (LOD) and limit of quantification (LOQ) were 0.004 and 0.01 mg/kg, respectively. The result showed that developed analytical methods was successfully applied to detect a small amount of chloropicrin in hulled rice. Key words: chloropicrin, headspace extraction, hulled rice, GC-ECD, GC-MS 서론 Chloropicrin은단일탄소유기분자 (single-carbon organic molecule) 로분자량이 164.4로매우작아토양중확산이빠르고강력한자극성최루증기를내는무색액체로일 *Correspondence to: Jae-Ho Oh, Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, MFDS, Tel: 82-43-719-4207, Fax: 82-43-719-4200 E-mail : jado@korea.kr 반적으로뿌리를파괴하는곰팡이에대해선택적인독성을가지고있으며 1-3), 식물정식 14일전토양표면으로부터약 15-25 cm 정도깊이에액체상태로주입되고 48시간이내에대상곰팡이와일부뿌리파괴선충, 토양곤충및기타해충을방제 3) 하는것으로알려져있다. Chloropicrin은 1917년부터살충제로사용되었고, 1920 년에토양훈증제 (preplant soil fumigant) 로처음연구 4-5) 되었으며, 1957년과일과채소재배에있어 chlorpicrin과 methyl bromide (MeBr) 의혼합사용은거대한도약을이끌었다. 많은토양전염병에 chloropicrin이단독으로쓰이기 222

Development of an Analytical Method for Chloropicrin Determination in Hulled Rice by GC-ECD and GC-MS 223 도하지만, MeBr의탁월한제초효과나 1,3-dichloropropene (1,3-D) 의광범위한살선충효과를가지고있지않기때문에대안으로 MeBr (20~33% chloropicrin) 이나 1,3-D 또는광범위한제초특성을가지는 metam sodium, dazomet, pebulate와같은화합물과함께혼합사용이이루어지고있다. Chloropicrin/MeBr 조합은예측가능수확량 (predictable crop yields) 을높임과동시에비료의사용을줄이고또한과일과채소의연작을가능케하였다. 환경적으로 chloropicrin은햇빛에빠르게분해되기때문에중요한오존고갈가능성 (ozone depletion potential) 이없고토양내에서이산화탄소로대사되며, 혐기 / 수중 (anaerobic/aquatic) 상태에서수시간이내 nitromethane으로변환된다. 방사선표지된 (radiolabelled) chloropicrin을처리한토양을이용한식물대사연구에서어떠한식물조직이나수확물에서 chloropicrin이나 nitromethane이발견되지않았다고보고되었다 6). 또한토양중에서 chloropicrin의분해물 ( 이산화탄소, 질산염, 염화물 ) 은식물뿐만아니라토양내서식하는미생물에게기본적인영양소로이용되기도한다. Chloropicrin 은물에낮은용해도 (1.6 g/l, 25 o C) 를갖고있기때문에수중환경에서빠르게움직이지않는다 7-8). 물속에서햇빛에노출시 chloropicrin의반감기는 31.1 시간이었고, 이산화탄소, 중탄산염, 염화물, 질산염및아질산염이최종산물이었으며, 햇빛이없는환경에서 chloropicrin은가수분해를하지않는다고보고 9) 되었다. 대부분의훈증제처럼, chloropicrin은사용이제한된농약으로, 사용량과분포는철저히통제된다. Chloropicrin은중국에서딸기, 땅콩, 생강, 가지, 참외등에훈증제, 살균제, 살선충제및저장곡물의살충제로사용되고있으며, 곡류, 채소류, 콩류에잔류허용기준이 0.1 mg/kg으로설정 10) 되어있다. 국내에서는사용되지않고, 식품의약품안전처공고제2010-294 11) 에따라불검출을원칙으로하는농약이다. 하지만식품중상당부분을수입에의존하는국내식품산업의특성상 chloropicrin과같은국내기준미설정농약이수입식품에잔류하여반입될가능성이있기때문에국내미등록농약에대한시험법확립이필수적이다. 외국에서 chloropicrin 측정을위해대기 12-14), 토양 15), 물 16-18) 등에서 spectrometer와 gas chromatography 등을이용한시험법이다양하게보고되었지만농산물중에잔류하는 chloropicrin 분석방법에대한보고는거의없다. 따라서본연구는국내기준규격미설정농약 chloropicrin에대한식품중잔류농약안전성을확보하기위해서 chloropicrin을신속하고빠르게검출할수있고정확하고신뢰성있는공정시험법을확립하기위해수행되었다. 재료및방법 시약및재료 Chloropicrin 표준품 (95.0%) 은 Accustandard (Connecticut, USA) 에서구입하였고, acetone과 ethyl acetate는 HPLC grade로써 Merck (Darmstadt, Germany) 에서, sodium sulfate anhydrous는 Wako (Osaka, Japan) 에서구매하였다. 멤브레인필터 (0.45 µm, PTFE) 는 Advantec (Tokyo, Japan) 에서 headspace 바이알과마개는 Agilent (Agilent Technologies, USA) 에서구입하였다. 검체인현미는유기농친환경제품을대형유통마트에서구입한후분쇄하여 420 µm 채에통과시켜균질화한후폴리에틸렌지퍼백에담아 50 o C에보관, 실험에사용하였다. 표준원액및표준용액 Chloropicrin 표준물질 0.0526 g을정밀히달아 acetone 100 ml에용해하여 500 µg/ml (500 ppm) 이되도록표준원액을조제하였고, 이를 ethyl acetate로희석하여 0.005, 0.01, 0.02, 0.05, 0.1 및 0.5 µg/ml의표준용액을준비하였다. 표준원액은갈색병에담아 4 o C에보관하였으며, 표준용액은사용시조제하여실험에사용하였다. 추출및정제현미 5g을정밀히달아 sodium sulfate anhydrous 2 g이들어있는 20 ml 헤드스페이스바이알에넣었다. 여기에 ethyl acetate 10 ml를첨가한후헤드스페이스바이알용마개로밀봉하였다. 바이알을 10분간심하게흔들어 5분간정치한다음주사기로상층액약 3mL를취해멤브레인필터로여과하여시험용액으로하였다. 확립된 chloropicrin의시험법을 flow chart로나타내었다 (Fig. 1). Fig. 1. Flow chart for chloropicrin analysis. 기기분석 Chloropicrin 분석을위해현미시료추출을통해얻어진시험용액을 DB-5(30 m 0.25 mm i.d 0.25 µm film thickness) 가장착된 gas chromatograph-electron capture detector (GC-ECD, Agilent 6890, Agilent tedhnologies, USA)

224 Jung-Ah Do, Jeong-Heui Choi, Hyejin Park, Yong-Chun Park, Hae-Jung Yoon, Dongmi Choi, and Jae-Ho Oh Table 1. Analytical conditions for chloropicrin Instrument GC-ECD (6890N, Agilent Technologies, USA) Column DB-5 (30 m 0.25 mm i.d, 0.25 µm film thickness) Oven temperature 45 o C(10 min) 20 o C/min 200 o C(2 min) Injector temperature 260 o C Detector temperature 280 o C Injection volume 1 µl (split mode, 10 : 1) Carrier gas flow 1 ml/min (N 2 ) Table 2. Confirmative conditions for chloropicrin Instrument GC-MS (5973 MSD, Agilent Technologies, USA) Column DB-5MS (30 m 0.25 mm i.d, 0.25 µm) Oven temperature 40 o C(10 min) 20 o C/min 200 o C(2 min) Injector temperature 200 o C Interphase temperature 280 o C Ion source 230 o C Quadruple temperature 150 o C Injection volume 1 µl (splitless mode) Carrier gas flow 1 ml/min (He) 추출과정최적화 Chloropicrin은단일탄소유기분자 (single-carbon organic molecule) 로분자량이 164.4로매우작고증기압이매우높기때문에기존의농약추출법에서같이감압농축이나질소가스농축을수행한다면대부분손실될우려가높다. 예비실험에서시험관에 chloropicrin 표준용액과 2% diethylene glycol 함유 acetone 0.2 ml을함께넣고질소가스로농축후 acetone에재용해하여분석한결과 chloropicrin은전혀검출되지않아농축과정중 chloropicrin이모두휘발됨을확인할수있었다. 그래서헤드스페이스추출법을활용하여검체추출액을직접 GC에주입하는시험법을개발하였다. Headspace 추출법은추출 포집과정에서고유휘발성성분의변화나손실을최소화하는측면에서유리하지만, 추출효율이떨어지는문제점을가지고있다 19). 검체를밀봉된상태에서추출하고추출액중일부를외부와의접촉을최소화하여취하여 GC에주입해야하기때문에헤드스페이스바이알과전용마개를사용하였다. 추출용매로는 ethyl acetate를사용하여 chloropicrin의높은회수율은유지하면서불순물을최소화할수있었다. 에 1µL를주입하였다. 승온조건및자세한기기분석조건은 Table 1에나타내었다. 확립된시험법을이용하여분석된잔류분의신뢰성을확보하기위하여 gas chromatograph-mass spectrometer(gc- MS, 5973 MSD, Agilent Technologies, USA) 를이용하여재확인과정을수행하였다. GC-MS 분석조건은 Table 2 에나타내었다. 결과및고찰 기기선정과 GC 분석조건최적화 Chloropicrin은 Log P ow 값이 2.5인비극성에서중간극성의해리성이없는중성화합물로증기압이 26,660 mpa (25 o CPa) 로높아휘발성이강하여분석을위한기기로 GC 를선정하였다. 또한분자내에 halogen( 염소원자 3개 ) 과 nitro기를보유하고있어 ECD에서높은감도를나타낼것으로판단되어검출기로선택하였다. 확립된기기분석조건 (Table 1) 으로분석한결과 6.5분대의안정적인머무름시간을확인할수있었다. Fig. 2. GC-ECD chromatograms of chloropicrin standard (A), control hulled rice (B), and fortified hulled rice at 0.1 mg/kg (C).

Development of an Analytical Method for Chloropicrin Determination in Hulled Rice by GC-ECD and GC-MS 225 직선성 (Linearity) Chloropicrin 표준원액 500 µg/ml를 ethyl acetate로 0.005, 0.01, 0.02, 0.05, 0.1 및 0.5 µg/ml의농도로희석하여위에서제시한 GC 분석조건으로측정한결과, 표준곡선의상관계수 (r 2 ) 가 0.999로써높은직선성을나타내었다. 선택성 (Selectivity) Fig. 2에서보는바와같이현미무처리검체중 chloropicrin과같은머무름시간을갖는어떤방해물질도검출되지않음으로써현미중 chloropicrin을분석하기위한본시험법이높은분리능과선택성을가짐을확인할수있었다. 정확성과정밀성 (Accuracy and Precision) 현미중시험법의정확성을평가하기위하여처리농도 0.1 및 0.5 mg/kg에서 chloropicrin의회수율실험을 3반복으로수행한결과각농도에서회수율은평균 77.7 ± 1.0% 와 79.3 ± 0.7% 이었다 (Table 3). 코덱스가이드라인 (CAC/GL 40) 에서는회수율의적정법위를 70~120%, 분석오차 10% 이하를적정범위로제시하고있으므로, 본시험법의정확성이양호함을확인할수있었다 21). 또한 3반복실험에대한표준편차가 10% 미만으로개발된시험법이정밀성측면에서도양호함을확인할수있었다 20). 검출한계및정량한계본연구에서확립한시험용액조제및기기시험법을이용하여현미중 chloropicrin의검출한계와정량한계를구하였다. 검출한계는최소검출량이 0.002 ng (S/N = 3) 이었고아래의계산식 21) 에따라 0.004 mg/kg으로나타냈고, 정량한계는최소검출량이 0.005 ng (S/N = 10) 으로아래의계산식 21) 에따라 0.01 mg/kg으로나타났다. Table 4. Selected-ion of GC-MS for chloropicrin Retention time (min) M.W. Exact mass GC-MS를이용한시험법의재확인본연구에서확립된시험법을이용하여분석된잔류분의신뢰성을확보하기위하여재확인과정을수행하였다. GC-MS의분자량범위를 50~180 m/z로스캔하여검체중 chloropicrin을확인하였으며, 분석조건은 Table 4에나타내었다. 확인조건에서얻은 total ion chromatogram (TIC) 과 mass spectra로 selected-ion monitoring (SIM) 분석을위한최적특성이온을선정하였으며 (Fig. 3) 주요특성이온의 m/z 값은 82, 117, 121, 43로나타났다. 요약 Fragment monitored (m/z) 4.5 164.4 162.9 82, 117, 121, 43 Chloropicrin은국내에서는사용되지않으며, 잔류허용기준이불검출로설정된농약이다. 그러나중국에서는훈증제, 살균제, 살선충제및저장곡물의살충제로사용되고있으며, 잔류허용기준이설정되어있다. 따라서본연구에서는중국등에서수입하는식품중국내에서사용하지않는농약에대한안전성을확보하기위하여해당농약을신속하고빠르게검출할수있는시험법을개발하고자하였 1 검출한계 (mg/kg) = 최소검출량 (ng) -------------- 시료량 (g) 최종희석부피 ( ml) -------------------------- 0.002( ng) 1 10( ml) = --------- 시료주입량 ( µl) 5g ( ) ------------------ = 1 ( µl) 0.04 1 정량한계 (mg/kg) = 최소검출량 (ng) -------------- 시료량 (g) 최종희석부피 ( ml) -------------------------- 0.005( ng) 1 10( ml) = --------- 시료주입량 ( µl) 5g ( ) ------------------ = 1 ( µl) 0.01 Table 3. Validation results of analytical method for chloropicrin in hulled rice Sample Fortification (mg/kg) Recovery * (%) Hulled rice 0.1 0.5 * Mean values of triplicates with standard deviation 77.7 ± 1.0 79.3 ± 0.7 Fig. 3. Total ion chromatogram (A) and ms spectrum (B) of chloropicrin.

226 Jung-Ah Do, Jeong-Heui Choi, Hyejin Park, Yong-Chun Park, Hae-Jung Yoon, Dongmi Choi, and Jae-Ho Oh 다. Chloropicrin은분자량이작고휘발성이큰화합물이므로헤드스페이스추출법을활용하여검체추출액을직접 GC에주입하는시험법을개발하였으며, 확립된시험법을검증한결과회수율이 77.7~79.3% 로조사되어코덱스국제가이드라인 (CAC/GL 40) 에적합함을확인하였다. 정량한계또한 0.05 mg/kg 이하로매우우수한감도를가지고있음을확인하였으며, GC-MS를이용한재확인과정을추가로확립하여본시험법의신뢰도를높였다. 확립된시험법은식품에잔류할수있는농약의기준준수여부에대한적합성을판단하기위한공정시험법으로활용되고있다. 감사의글 본연구는 2009년도식품의약품안전처연구개발과제의연구개발비지원 (09071식품안015) 에의해수행되었으며, 이에감사드립니다. 참고문헌 1. Wilhelm S.N. : Chloropicrin as a Soil Fumigant. Niklor Chemical Company, Inc., Chairman, Chloropicrin Manufacturers Task Force, Long Beach, CA 90810-1695. Available at http:// www.ars.usda.gov/is/np/mba/july96/wilhel1.html (1996). 2. O'Neil M.J. : The Merck Index-An Encyclopedia of Chemicals, Drugs, and Biologicals (14th Edition - Version 14.9). Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.. Online version available at: http://www.knovel.com/web/ portal/browse/display?_ext_knovel_display_bookid= 1863&VerticalID=0 (2012). 3. Meister R.T. and Sine C. : Farm Chemicals Handbook. Meister Publishing Company. Willoughby, Ohio, USA (1998). 4. Roark R.C. : USDA Miscellaneous Publication No. 176. A Bibliography of Chloropicrin 1848-1932. United States Department of Agriculture. Washington, DC (1934). 5. Agnihothrudu V. and Mithyantha M.S. : Pesticide Residues - a review of Indian Work. Rallis India Ltd., Bangalore (1978). 6. US Environmental Protection Agency. Pesticide Environmental Fate One Line Summary: Chloropicrin. US EPA Environmental Fate and Effects Division. Washington, DC (1992). 7. Shepler K, Hatton C, and Ruzo L. : Aerobic Soil Metabolism of [ 14 C]Chloropicrin. PTRL West Inc. Richmond, CA. (Unpublished study submitted to USEPA) (1995). 8. Hazardous Substances Data Bank (HSDB) : Accession Number 977. National Library of Medicine, Bethesda, MD. CD ROM version: Micromedix Inc., Denver CO. (1993). 9. Lee H. and Moreno T. : Photohydrolysis of Chloropicrin. Bolsa Research Associates. Hollister, CA. (Unpublished study submitted to USEPA) (1993). 10. 중화인민공화국위생. 식품중농약최대잔류허용량. 중국국가표준화관리. 11. 식품의약품안전청, 식품의약품안전청공고제 2010-294 호, 식품의기준및규격일부개정고시안행정예고 (2010). 12. Muir B., Carrick W.A. and Cooper D.B. : Application of central composite design in the optimisation of thermal desorption parameters for the trace level determination of the chemical warfare agent chloropicrin. Analyst 127(9), 1198-1202 (2002). 13. OSHA. Chloropicrin Method PV2103. Salt Lake City, UT: U.S. Department of Labor, OSHA Salt Lake Technical Center (1991). 14. Carrick WA, Cooper DB and Muir B. : Retrospective identification of chemical warfare agents by high-temperature automatic thermal desorption-gas chromatography-mass spectrometry. J. Chromatogr A 925(1-2), 241-249 (2001). 15. Guo M., Papiernik S.K., Zheng W. and Yates S.R. : Formation and extraction of persistent fumigant residues in soils. Environ Sci Technol ASAP Article 10.1021/es0262535 S0013-936X(02)06253-3 (2003). 16. Castro J.A. and Godoy H. : Spectrophotometric determination of chloropicrin in water. Anal Chim Acta 33, 679-683. (1965). 17. US EPA. Method 551.1 : Determination of chlorinated disinfection byproducts, chlorinated solvents, and halogenated pesticides/herbicides in drinking water by liquid/liquid extraction and gas chromatography with electron capture detection. Cincinnati, OH: U.S. EPA Office of Ground Water and Drinking Water/Technical Support Center (1995). 18. Nikolaou A.D., Lekkas T.D., Golfinopoulos S.K. and Kostopoulou M.N. : Application of different analytical methods for determination of volatile chlorination by-products in drinking water. Talanta, 56(4), 717-726 (2002). 19. Kobayashi A. : Recent progress in flavor research. Nippon Shokuhin Kagaku Kogaku Kaishi, 44, 169-176 (1997). 20. Codex Alimentarius Commission. Guidelines on good laboratory practice in residue analysis, CAC/GL 40, Rome, Italy (2003). 21. Lee Y.D : Practical book of Korea Food Code pesticide residue analysis method. third ed. KFDA, Korea. pp. 79 (2012).