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1 pissn / eissn J. Food Hyg. Saf. Vol. 33, No. 4, pp. 296~305 (2018) Journal of Food Hygiene and Safety Available online at LC-MS/MS 를이용한농산물중살균제 Spiroxamine 의시험법개발및검증 박신민 도정아 * 임승희 윤지혜 박원민 신혜선 국주희 1 정형욱 식품의약품안전처식품의약품안전평가원식품위해평가부잔류물질과, 1 식품의약품안전처식품의약품안전평가원식품위해평가부영양기능연구팀 Development and Validation of Analytical Method for Determination of Fungicide Spiroxamine Residue in Agricultural Commodities Using LC-MS/MS Shin-Min Park, Jung-Ah Do*, Seung-Hee Lim, Ji-Hye Yoon, Won-Min Pak, Hye-Sun Shin, Ju-Hee Kuk 1, and Hyung-Wook Chung Pesticide and Veterinary Drug Residues Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju, Korea 1 Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju, Korea (Received May 9, 2018/Revised May 26, 2018/Accepted July 11, 2018) ABSTRACT - Spiroxamine, one of fungicides, is used to control powdery mildew in various crops and black yellow sigatoka in bananas. The major strength of spiroxamine is to control powdery mildew in various crops and bananas yellow sigatoka in bananas. The compound has shown a high level of activity, good persistence and crop tolerance. Besides powdery mildew, good control of rust, net blotch and Rhynchosporium diseases been indicated in cereals, together with a complementary activity against Septoria diseases. In 2017, the maximum residue limit (MRL) of spiroxamine established in Korea. According to Ministry of ood and rug afety) regulations, spiroxamine residues defined only parent compound. Thus, a analytical method is needed to estimate the residue level of the parent compound. The objective of this study was to develop and validate analytical method for spiroxamine in representative agricultural commodities. Samples were extracted with acetonitrile and partitioned with dichloromethane to remove the interfering substances. The analyte were quantified and confirmed liquid chromatograph-tandem mass spectrometer (LC-MS/MS) in positive-ion mode using multiple reaction monitoring (MRM). Matrix matched calibration curves were linear over the calibration ranges (0.0005~0.1 µg/ml) for the analyte in blank extract with coefficient of determination (r 2 ) > For validation purposes, recovery studies will be carried out at three different concentration levels (LOQ, 10LOQ, and 50LOQ) performing five replicates at each level. The recoveries 70.6~104.6% with relative standard deviations (RSDs) less than 10%. All values were consistent with the criteria ranges in the Codex guidelines (CAC/GL40, 2003) and MFDS guidelines. proposed analytical method be used as an official analytical method in the Republic of Korea. Key words : Spiroxamine, Fungicide, LC-MS/MS, Analytical method, Agricultural commodities Spiroxamine(8-tert-butyl-1,4-dioxaspiro[4.5]decan-2- ylmethyl(ethyl) (propyl)amine) 은전세계 80여개국에등록되어사용되고있는농약으로우리나라에 2017년신규 *Correspondence to: Jung-Ah Do. Pesticide and Veterinary Drug Residues Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety Tel: , Fax: jado@korea.kr 고시된농약이다. Spiroketalamine계살균제로진균활성을나타내며균류의부착기와흡기의형성을막아균류구조에손상을주어사멸시키거나발아관의성장을저해하는기작을갖고있는것으로알려져있으며, 세포벽에물리화학적으로작용하여 uncinula포자를사멸하는기작등을나타낸다. 특히바나나의잎에변색된얼룩점을유발하는 sikatoka병방제에대한활성이있는것으로알려져있으며, 곡류의흰가루병, 녹병, 망반병, Rhynchosporium 병, Septoria병의방제등에이용한다 1-3). 2018년 5월현재, spiroxamine은 EPA, 캐나다에서모화 296

2 Analytical Method for Determination of Spiroxamine 297 합물및 aminodiole를잔류물의정의로설정하고있고, 유럽, 미국, 일본등에서는모화합물만을잔류물로정하여관리하고있다. 국외잔류허용기준은바나나등 300여종의작물에대하여 0.04~50 mg/kg 수준으로설정되어있다 4-7). Spiroxamine의분석법은모화합물을기준으로 GC-MS/MS, LC-MS/MS 등으로분석한사례가보고되었으며, 시험법의경우다성분분석을목적으로농산물, 축산물, 수산물등을분석한문헌이있으나, spiroxamine의물리화학적특성을고려해개발된단성분시험법은없는실정이다 8-12). Spiroxamine은포도와밀의경우체내대사과정에서모화합물의 total radioactive residue (TRR) 가 53.8~76.0% 이며잔류물은 0.2~9.0% 수준을나타낸다고보고되어있다 3-4). 또한안전성에대한평가결과마우스, 랫드의발암성병합시험에서암수모두최고농도군에서발암성이없는것으로평가되었으며, 개대상 1년만성독성시험에서도출된최대무독성용량 (NOAEL, No Observable Adverse Effect Level) 2.47 mg/kg bw/day에종간및개체간차이로안전계수 100을적용하여일일섭취허용량 (ADI, Acceptable Daily Intake) 은 mg/kg bw/day로설정되었다 13). 국내의경우 2016년바나나와포도에대한잔류허용기준이신설요청되었으며, 독성시험및식물체내잔류시험결과에따라잔류물의정의는모화합물로서기준이신설되었다 ( 식품의약품안전처고시제 호, ). 따라서본연구에서는수입농산물중잔류할수있는 spiroxamine 에대하여기준신설식품인바나나와포도뿐만아니라향후기준신설농산물이늘어날것에대비함과동시에, 사용등록이되지않은농산물에오남용하는것을방지하기위하여모든농산물에서 spiroxamine의잔류허용기준적부판정을위한시험법을개발하고자하였다. Materials and Methods 시약및재료 Spiroxamine (97.4%) 표준품은 bayer cropscience사에서제공받아사용하였다. 전처리용시약으로사용된디클로로메탄 (dichloromethane), 아세톤 (acetone), 아세토니트릴 (acetonitrile), 메탄올 (methanol), 헥산 (n-hexane) 및에틸아세테이트 (ethyl acetate) 는 HPLC 등급으로 Merck (Darmstadt, Germany) 에서구입하여사용하였고, 염화나트륨 (sodium chloride, NaCl) 은 Wako (Osaka, Japan), 무수황산나트륨 (anhydrous sodium sulfate) 은 Merck (Darmstadt, Germany), 수산화나트륨 (sodium hydroxide, NaOH) 과암모늄아세테이트 (ammonium acetate) 는 Sigma Aldrich (Buchs, Switzerland), SPE 카트리지 (1 g, 6 cc) 는 Waters (Milford, USA) 제품을구입하여사용하였다. 검체는식품공전농산물분류에서규정하는대표농산물을현미 ( 곡류 ), 감자 ( 서류 ), 대두 ( 두류 ), 감귤 ( 과일류 ), 고추 ( 채소류 ) 로선정하고시중에 서판매하고있는무농약농산물각 1종을구입하여균질화한후밀봉된용기에담아 20 o C이하에서보관하고실험에사용하였다. 표준원액및표준용액의조제 Spiroxamine 1,000 μg/ml의표준원액을제조하여아세토니트릴에희석하여사용하였다. Matrix-matched calibration 을위해각농산물검체의무처리추출물 900 μl에 10 μg/ ml 표준용액 100 μl를넣어 1.0 μg/ml 표준용액을조제한뒤무처리추출액을이용하여단계적으로희석하여 , 0.001, 0.005, 0.01, 0.02, 0.05 및 0.1 μg/ml의 90% 이상의 matrix가첨가된 matrix-matched 표준용액을조제하였다. 표준원액과표준용액은모두갈색병에담아 4 o C 에보관하여실험에사용하였다. 추출및정제균질화된검체 10 g을정밀히달아균질기용기에넣고아세토니트릴 50 ml를가하여진탕기에서 10분간진탕하였다. 진탕후추출물을여과지가깔려있는부흐너깔때기로흡인여과한뒤아세토니트릴 20 ml로잔사및용기를씻어내려앞의여액과합친뒤이를 40 o C이하의수욕상에서감압농축하였다. 농축후잔류물에증류수 100 ml 를가하여녹인후 1 N NaOH 용액을천천히가해 ph 7 로조절하여 500 ml 용량의분액여두에옮기고 10 g의 NaCl을더한뒤디클로로메탄 30 ml를차례로가하고심하게흔들어층이완전히분리될때까지정치시킨후디클로로메탄층을무수황산나트륨에통과시켜감압농축플라스크에받는과정을 2회반복하였다. 이를 40 o C 이하의수욕상에서감압하여용매를모두날려버린후, 잔류물에헥산 10 ml를가하여녹였다. 이를플로리실카트리지에헥산 10 ml를 2~3 방울 / 초의속도로유출하여버리고, 이어서고정상상단이노출되기전에추출과정으로부터얻은추출액중 5mL를카트리지상단에넣어 1~2 방울 / 초의속도로유출시켜버리고고정상상단이노출되기전에헥산 5mL를유출시켜버린후아세톤 / 헥산 (5/95, v/v) 10 ml를유출시켜받은시험액을감압농축플라스크에모았다. 이를 40 o C 이하수욕상에서감압농축후잔류물에아세토니트릴을가하여최종부피 5mL가되게한뒤멤브레인필터 (nylon, 0.2 μm) 로여과한후시험용액으로사용하였다 (Fig. 1). LC-MS/MS 분석조건 Spiroxamine의분석을위하여액체크로마토그래프-질량분석기 (Liquid Chromatograph-Tandem Mass Spectrometer, LC-MS/MS) 를사용하였고, 분석용역상칼럼인 C 18 칼럼을선택하였으며용리방식은 10 mm 아세트산암모늄수용액과 10 mm 아세트산암모늄포함메탄올을이동상으로

3 298 Shin-Min Park et al. Table 1. Analytical conditions for the determination of spiroxamine Instrument LC:Acquity UPLC (Waters, Milford, MA, USA) MS/MS: US/Quattro Primier XE (Waters, Milford, MA, USA) UPLC conditions Column XBridge C 18 (2.1 mm i.d. 100 mm, 3.5 µm) Column temperature 40 o C Flow rate 0.3 ml/min Injection volume 5 µl Mobile phase Gradient table MS/MS conditions A: 10 mm ammonium acetate in methanol B: 10 mm ammonium acetate in distilled water Time (min) Mobile phase A (%) B (%) Ion mode Capillary voltage Source temperature Desolvation temperature Desolvation gas flow Cone gas flow ESI positive mode 1.0 kv 150 o C 500 o C 900 L/h 150 L/h Fig. 1. Flow chart for spiroxamine analysis. 사용하는기울기용리방식을선택하였다. 각대상성분의이온화법으로는 electro-spray ionization (ESI) 법의 positiveion mode를사용하였다. LC-MS/MS 분석조건은 Table 1 과같다. 시험법의검증 Spiroxamine 시험법은잔류물분석에관한 CODEX 가이드라인 21) (CODEX Alimentarius Commission, CAC/GL 40, 2003) 의잔류농약분석기준에근거하여직선성 (linearity), 검출한계 (limit of detection, LOD), 정량한계 (limit of quantification, LOQ), 회수율 (recovery), 재현성 (reproducibility) 에대해유효성을검증하였다. 직선성의확인을위하여 spiroxamine을무처리시료시험용액으로희석하여조제한표준용액 ~0.1 μg/ml의농도범위에대한 각각의피크면적을이용하여검량선을작성하였고, 검량선의상관계수 (coefficient of correlation, r 2 ) 를구하였다. 또한, 검출한계와정량한계는크로마토그램상에서신호대잡음비 (S/N ratio) 각각 3, 10 이상으로하였다. 시험법의정확성및재현성을평가하기위하여무처리시료에 spiroxamine의표준용액을첨가한후분석하여회수율을구하였다. 처리농도는정량한계, 정량한계의 10배, 정량한계의 50배에해당하는농도로수행하였으며각각의농도및시료에대하여 5 반복으로수행하여평균과상대표준편차 (relative standard deviation, RSD) 를계산하여시험법의정확성과정밀성및재현성을평가하였다. Results and Discussion 최적기기분석조건확립 Spiroxamine 은증기압이 Pa (25 o C) 이며 120 o C 이상의온도에서불안정한특성을나타내며, Log P ow 값이

4 Analytical Method for Determination of Spiroxamine 299 산성및중성의 ph에서 1.28~2.79로중간극성을나타내는화합물로 GC분석에비해 LC 분석이유리한것으로판단하였다. 하지만화합물구조에서 conjugation 발색단이나비공유전자쌍을갖는포화화합물의발색단을가지고있지않으므로 HPLC/UVD로의분석이불가할것으로판단되었고, 이를정확하게확인하고자 HPLC의 PDA (Photodiode array) 검출기로 210 nm에서 360 nm까지측정파장을스캔한결과최대흡수파장 (λ max ) 의확인이불가능하였다. 따라서선택성과정확성을확보하고자 Liquid Chromatograph- Tandem Mass Spectrometry (LC-MS/MS) 를분석기기로최종선정하였다. LC-MS/MS는 HPLC에비해낮은정량한계를나타내며, 선정된분자량을기준으로분석되어시료의간섭물질에대해영향을받지않으므로정확성및선택성이높을것으로판단되었다. 분석용칼럼은 spiroxamine의중간극성인 Log P ow 값에근거하여극성도에대해넓은스펙트럼을갖고, 분배의원리로분석물질이분리되는 C 18 역상칼럼을선택하였고, 10 mm 암모늄아세테이트함유메탄올과 10 mm 암모늄아세테이트함유수용액을이동상으로사용하는기울기용리방식을선택하였다. 이동상에사용한암모늄아세테이트는 ion suppression 원리로이동상의 ph를 spiroxamine 의 pka인 6.9와약 1.5 이상의차이를나타내도록조절하며, spiroxamine 분자의이온화억압을유도하고보다높은감도의선명한피크를얻기위해사용하였다. 메탄올은암모늄아세테이트에대한용해도가 100 ml당 7.89 g으로높아, 완충용액으로서사용에용이하여사용하였다 14-15). 대상성분의이온화법으로는 electro-spray ionization (ESI) 법의 positive-ion mode를사용하였고 total ion chromatogram (TIC) 과 mass spectrum을통해분석을위한최적특성이온을선정하였다. 관측질량이 인표준용액 (0.5 μg/ml) 을일정한속도 (10 μl/min) 로질량검출기에직접주입한결과 spiroxamine의관측질량에대해 [M+H] + 형태인 precursor ion 값을 298 mass으로확인하였다. 이때 cone voltage의변화에따른최적화과정을통해 30 V에서최대피크가나타남을확인하였으며, 최적화된 cone voltage 상태에서분석의선택성과검출강도를극대화시키기위하여 MS/MS 분석시 MRM (multiple reaction monitoring) mode로분석하였다. Collision cell에서 collision energy를조절하여최적의 precursor/product ion pair를선정하였고, 가장좋은감도를보이는 product ion을정량이온 (quantification ion) 으로, 다음으로크게검출되는 product ion을정성이온 (qualification ion) 으로설정하여확인하였다. 분석조건에서선정된특성이온은 Table 2에나타내었다. 또한 LC-MS/MS 분석시에는검체중추출성분에의하여대상성분의이온화억압또는증강현상이나타날수있으므로검체별로 matrix-matched calibration 법에준하여정량하였다 16,17). 추출및정제조건의확립 Spiroxamine은 Log P ow 값이 2.8~3.0인화합물로중간극성을띄며, 산성조건에서수용성으로존재하고중성및염기조건에서지용성으로존재한다. Spiroxamine은유기용매에대한용해도가높으며추출시검체내부로의침투성이용이하도록수용성유기용매로추출하고자하였다. 보편적으로사용하고있는수용성유기용매중아세톤과메탄올은아세토니트릴에비해비극성간섭물질에대한추출률이높고, spiroxamine의회수율이아세토니트릴추출시충분히효과적인것으로판단하여아세토니트릴을추출용매로선정하였다. 추출후검체추출액중간섭물질제거를위해디클로로메탄을이용한액-액분배법을적용하였다. Ravelo-Pérez 등 18) 의보고에서는바나나에서 carbaryl 등 8종의잔류농약분석의최적조건을설정하기위해변수로둔조건중액-액분배시염화나트륨의농도를 15, 20, 25% 로두어 salting out을유도한결과염화나트륨의농도가높아질수록최적의회수율을나타내었다고보고하였고, Endo 등 19) 은높은추출효율을얻기위해염을물에첨가하여 salting out을유도한다고보고하였다. 이는액- 액분배시포화수준의염화나트륨첨가를통한이온강도증가로수분층과유기용매층을명확하게분리하여대상물질의분배효율은높이고, 시료내의극성불순물이유기용매층에전이되는것을최소화하기위한원리로판단되었으며이를적용하여분배효율을높였다. 또한비극성유기용매인디클로로메탄에전이된비극성불순물을정제하기위해고상카트리지를이용하여정제하고자하였다. Żwir-Ferenc과 Biziuk 20) 에따르면정제카트리지의선택은분석물질의특성및검체매트릭스등에따라달라지는데, 분석물질이극성인경우순상카트리지를이용한정제가용이하며, 검체를비극성유기용매로추출또 Table 2. Selected-ion of LC-MS/MS for spiroxamine Compound Molecular weight Exact mass (m/z) Spiroxamine a Quantification ion b Collision energy (ev) Precursor ion (m/z) [M+H] + Product ion (m/z) CE b a 20

5 300 Shin-Min Park et al. Table 3. Comparisons of SPE cartridge for spiroxamine analysis Solvent ratio a (v/v) Fraction Florisil elution efficiency (%) Silica elution efficiency (%) (loading 5 ml) /90 2 (10 ml) /80 3 (10 ml) /70 4 (10 ml) /60 5 (10 ml) Total a Solvent ratio of ethyl acetate/dichloromethane Table 4. Comparisons of SPE cartridge for spiroxamine analysis Solvent Solvent ratio (v/v) Fraction Florisil elution efficiency (%) spiroxamine (loading 5 ml) /90 2 (10 ml) 38.4 Ethyl acetate/n-hexane 20/80 3 (10 ml) /70 4 (10 ml) /60 5 (10 ml) 0.1 Total (loading 5 ml) /90 2 (10 ml) 35.1 Acetone/n-Hexane 20/80 3 (10 ml) /70 4 (10 ml) /60 5 (10 ml) - Total (loading 5 ml) /90 2 (10 ml) 25.2 Acetone/Dichloromethane 20/80 3 (10 ml) /70 4 (10 ml) /60 5 (10 ml) 0.1 Total 31.2 는분배했을경우유입된비극성간섭물질의정제를위해극성흡착기를지닌카트리지를사용하여비극성간섭물질은통과시키고상대적으로극성인분석물질을정제한다는보고가있다. 따라서비극성유기용매인디클로로메탄으로액-액분배후유입된비극성간섭물질을정제하고, spiroxamine이중성및염기조건에서비해리상태로존재하며중간극성을띄는특성을고려하여추가적인정제를위해흡착원리를따른순상카트리지로서 hydroxyl기를작용기로갖는플로리실 (florisil), 실리카 (silica) 카트리지를이용하여정제효율을비교하였다. 용매는 spiroxamine에대해비교적용해도가높은비극성유기용매인디클로로메탄 ( 극성지수 : 3.1) 과극성용매를혼합하여분석중극성물질에대한간섭을최소화하기위해에틸아세테이트 ( 극성지수 : 4.4) 용매조합으로정제효율을비교하였다. 플로 리실과실리카카트리지실험결과, 플로리실카트리지의경우에틸아세테이트 / 디클로로메탄의모든분획에서용출되었으나총 50.5% 의회수율을확인하였으며실리카카트리지의경우총 0.4% 의회수율을확인하였다 (Table 3). 따라서다른용매와의정제효율을비교하기위해상대적으로회수율이높았던플로리실카트리지로선정한후용매조성을에틸아세테이트 / 헥산, 아세톤 / 헥산, 아세톤 / 디클로로메탄으로설정하여회수율을확인하였다. 하지만모든용매조성에서최대 40.6% 으로낮은회수율을보였는데, 이는 spiroxamine의 pka에근거하여산성혹은중성의 ph일때이온화되어있는상태이므로일반적으로중성의화합물을분석하는흡착카트리지를사용한회수율이낮은것으로판단되었다 (Table 4). 따라서 spiroxamine의추출효율을높이기위해농산물시료인감귤을대상으로아세토

6 Analytical Method for Determination of Spiroxamine 301 Table 7. Comparisions of solvent ratio on spiroxamine elution efficiency Elution efficiency (%) Fraction Solvent ratio (v/v) a 100 2/98 5/95 10/90 20/80 1 (5 ml) (5 ml) (5 ml) (5 ml) Total a Solvent ratio of acetone/n-hexane Fig. 2. Structure and pka graph of spiroxamine. Table 5. Effects of ph for spiroxamine partition efficiency in mandarin sample ph Mandarin partition efficiency (%) 니트릴로추출후 ph를 6-12로조정하여비교한결과, ph 7인중성조건에서 87.2% 로가장높은회수율을나타내었다. 한편 pka그래프 (Fig. 2) 에서 ph가높아질수록해당화 합물이지용성으로존재하는것을확인할수있었으나중성조건에서강염기조건보다높은회수율을나타내는것을확인하였다. 따라서본시험법에서는앞선결과를바탕으로아세토니트릴로추출한뒤추출액의 ph를 7로조절하여디클로로메탄으로분배하는방법을적용하였다 (Table 5). 농산물검체인감귤을아세토니트릴로추출하여 ph 7로조정한후디클로로메탄으로분배하여정제를진행하였다. 추출액을헥산으로재용해하여플로리실카트리지에용매조성을에틸아세테이트 / 헥산, 아세톤 / 헥산으로설정하고회수율을확인하였다. 실험결과, 에틸아세테이트 / 헥산의경우에틸아세테이트 / 헥산 (20/80, v/v), 에틸아세테이트 / 헥산 (30/70, v/v), 에틸아세테이트 / 헥산 (40/60, v/v) 의분획에서용출되어각각 38.5, 28.8, 1.7% 의회수율로총 69.0% 의회수율을확인하였다. 또한아세톤 / 헥산의경우아세톤 / 헥산 (10/90, v/v), 아세톤 / 헥산 (20/80, v/v) 의분획에서용출되어각각 72.1, 0.9% 의회수율로총 73.0% 의회수율을확인하였다. 따라서아세톤 / 헥산으로정제시로딩에서용출되지않았으며상대적으로높은회수율을확인하였으므로, 아세톤 / 헥산 (10/90, v/v), 아세톤 / 헥산 (20/80, Table 6. Comparisons of elution solvents for spiroxamine analysis Solvent Solvent ratio (v/v) Fraction Florisil elution efficiency (%) Ethyl acetate/n-hexane Acetone/n-Hexane (loading 5 ml) - 10/90 2 (10 ml) - 20/80 3 (10 ml) /70 4 (10 ml) /60 5 (10 ml) 1.7 Total (loading 5 ml) - 10/90 2 (10 ml) /80 3 (10 ml) /70 4 (10 ml) - 40/60 5 (10 ml) - Total 73.0

7 302 Shin-Min Park et al. Fig. 3. Matrix-matched calibration curves of spiroxamine corresponding to: (A) hulled rice, (B) potato, (C) soybean, (D) mandarin and (E) green pepper. Table 8. Validation results of analytical method for the determination of spiroxamine in samples Sample Hulled rice Potato Soybean Mandarin Fortification (mg/kg) MFDS b Recovery ± RSD a (%) Gwangju KFDA c Mean d (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Green pepper ± ± ± ± a Mean values of 5 times repetitions with relative standard deviation b Ministry of Food and Drug Safety c Gwangju Korea Food and Drug Administration d Recovery average of inter-laboratory e Coefficient of variation of inter-laboratory CV e (%)

8 Analytical Method for Determination of Spiroxamine 303 Fig. 4. Representative MRM (quantification ion) chromatograms of spiroxamine corresponding to: 1. hulled rice, 2. mandarin, (A) standard solution at 0.01 mg/kg, (B) control, (C) spiked at mg/kg, (D) spiked at 0.01 mg/kg, and (E) spiked at 0.05 mg/kg. v/v) 의분획을세분화하여정제회수율을확인하여야할것으로판단하였다 (Table 6). 더욱효과적인정제를위해플로리실카트리지를이용하여헥산에재용해한후헥산, 아세톤 / 헥산 (2/98, v/v), 아세톤 / 헥산 (5/95, v/v), 아세톤 / 헥산 (10/90, v/v), 아세톤 / 헥산 (20/80, v/v) 으로세분화하여각용매조성에대해 5mL씩 4개의분획을받아실험을진행한결과, 헥산용매를제외한각기다른분획에서용출되었다. 그중아세톤 / 헥산 (5/95, v/v) 의첫번째와두번째분획 (10 ml) 에서용출되어 78.6% 로가장높은회수율을보였다 (Table 7). 따라서최종정제법으로는헥산으로재용해한추출액 5mL을카트리지에용출시킨후헥산 5mL 로씻어버린뒤아세톤 / 헥산 (5/95, v/v) 10 ml를용출시켜받는것으로확립하였다. 의직선성을확인하기위하여표준원액을 5종의무농약농산물추출물로희석하여 , 0.001, 0.005, 0.01, 0.02, 0.05 그리고 0.1 μg/ml 5 μl를 LC-MS/MS에주입하여분석한결과모두상관계수 (r 2 ) 가 0.99 이상으로높은직선성을보였다 (Fig. 3). 검출한계와정량한계 Spiroxamine의검출한계는기기크로마토그램상에서신호대잡음비 (S/N ratio) 3 이상으로결정하여분석기기의최소검출량 ng에따른검출한계는 mg/ kg이었고, 정량한계는신호대잡음비 (S/N ratio) 10 이상으로결정하여분석기기의최소검출량 ng에따른정량한계는 mg/kg이었다. 시험법의검증 선택성및직선성 Spiroxamine의선택성은표준용액, 무처리시료, 표준용액을첨가한회수율시료의크로마토그램을서로비교하여평가하였다. 5종의무농약농산물시료와표준용액을첨가한시료를확립된시험방법에따라분석한결과, 무농약시료중 spiroxamine의머무름시간과질량대전하비 (m/z) 가같은어떠한간섭물질도검출되지않았으므로검체중 spiroxamine의분석을위해확립된본시험법의높은분리능과선택성을확인할수있었다. Spiroxamine 시험법의회수율 시험법의정확성을평가하기위하여정량한계, 정량한계의 10배, 정량한계의 50배수준인 0.001, 0.01과 0.05 mg/ kg의농도로회수율실험을 5회반복하여수행하였다. 시험결과각농도에서평균회수율은 % 이었고, 이때상대표준편차도 9.9% 미만으로조사되어모든분석물질에대한높은정확성, 재현성및효율성을확인할수있었다. 이결과는잔류물분석에관한 CODEX 가이드라인 21) (CODEX Alimentarius Commission, CAC/GL 40, 2003) 의잔류농약분석기준에서 > mg/kg 0.01 mg/ kg의 30%, > 0.01 mg/kg 0.1 mg/kg의 20%, > 0.1 mg/kg

9 304 Shin-Min Park et al. 1 mg/kg의 15% 보다낮아기준에적합함을확인할수있었다 (Table 8). LC-MS/MS를이용하여분석한농산물중 spiroxamine의회수율크로마토그램은 Fig. 4에제시하였다. Acknowledgement 본연구는식품의약품안전처의연구사업 2017년식품중잔류농약안전관리를위한위해평가및신규시험법확립연구 (17161식위안001) 의지원에의해수행되었으며, 이에감사드립니다. 국문요약 본연구는농산물중 2017년기준신설예정농약 spiroxamine 에대한공정시험법을개발하고자수행하였다. Spiroxamine 은모화합물만이잔류물로정의되므로이를분석하기위한시험법을개발하였고, 확립된시험법은실험실내및실험실간검증을통해공정시험법으로의유효성을확인하였다. Spiroxamine의물리화학적특성을고려하여분석을위해 LC-MS/MS를분석기기로사용하였으며, acetonitrile 으로추출후유기용매를이용하여액액분배조건및 florisil cartridge를이용한 SPE 정제조건을확립하여시료의불순물을효과적으로정제하는시험법을개발하였다. 개발된 Spiroxamine 시험법의직선성은결정계수 (r 2 ) 가 0.99 이상으로우수하였으며, 검출한계및정량한계는각각 , mg/kg으로높은감도를나타내었다. 대표농산물 5 종 ( 현미, 감자, 대두, 감귤, 고추 ) 에대한시험법검증결과평균회수율 (5 반복 ) 은 71.9~98.8% 였고, 분석오차는 10.0% 이하로정확성및재현성이우수함을확인할수있었다. 또한광주지방식약청과의실험실간검증결과에서평균회수율은 70.6~104.6% 를나타내어모두 Codex 가이드라인및식약처가이드라인의기준에부합하는것으로확인되었다. 개발된시험법은낮은검출한계및정량한계, 우수한직선성, 회수율시험을통한양호한정밀성및재현성등이입증되어농산물중 Spiroxamine의분석을위한식품공전공정시험법으로활용되기에적합한것으로판단된다. References 1. Rosales-Conrado, N.: Hydrolysis study and extraction of spiroxamine from soils of different physico-chemical properties. Chemosphere, 77(6), (2009). 2. Gullino, M. L., Leroux, P., Smith, C. M.: Uses and challenges of novel compounds for plant disease control. Crop Prot., 19(1), 1-11 (2000). 3. Bayer CropScience, Application for a Maximum Residue Limit (MRL) or an Import Tolerance for spiroxamine (KWG 4168) on banana and grapes in Korea (2016). 4. European Food Safety Authority, Conclusion on the peer review of the pesticide risk assessment of the active substance spiroxamine (2010). 5. Electronic Code of Federal Regulations. Tolerances and exemptions for pesticide chemical residues in food, 180, (2015). 6. European Commission. EU Pesticides database. europa.eu/food/plant/ pesticides/eu-pesticides-database/public/?event=pesticide.residue.currentmrl&language=en. (2016). 7. The Japan Food Chemical Research Foundation. ne.jp/foundation/agrdtl.php?a_ inq= (2016). 8. Saito-Shida, S., Nemoto, S., Teshima, R., Akiyama, H.: Quantitative analysis of pesticide residues in vegetables and fruits by liquid chromatography quadrupole time-of-flight mass spectrometry. Food Addit. Contam. Part A, 33(1), (2016). 9. Walorczyk, S., Drożdżyński, D., Kierzek, R.: Two-step dispersive-solid phase extraction strategy for pesticide multiresidue analysis in a chlorophyll-containing matrix by gas chromatography-tandem mass spectrometry. J. Chromatogr. A, 1412, (2015). 10. Lozano, A., Rajski, Ł., Uclés, S., Belmonte-Valles, N., Mezcua, M., Fernández-Alba, A. R.: Evaluation of zirconium dioxide-based sorbents to decrease the matrix effect in avocado and almond multiresidue pesticide analysis followed by gas chromatography tandem mass spectrometry. Talanta, 118, (2014). 11. Lozano, A., Kiedrowska, B., Scholten, J., de Kroon, M., de Kok, A., Fernández-Alba, A. R.: Miniaturisation and optimisation of the Dutch mini-luke extraction method for implementation in the routine multi-residue analysis of pesticides in fruits and vegetables. Food Chem., 192, (2016). 12. Ambrus, A., Buczkó, J., Hamow, K. A., Juhász, V., Solymosné Majzik, E., Szemánné Dobrik, H., Szitás, R.: Contribution of sample processing to variability and accuracy of the results of pesticide residue analysis in plant commodities. J. Agric. Food Chem., 64(31), (2016). 13. Ministry of Food and Drug Safety, Spiroxamine Safety Evaluation Report (2016). 14. Mallet, C. R., Lu, Z., Mazzeo, J. R.: A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts. Rapid Commun. Mass Spectrom., 18(1), (2004). 15. McCalley, D. V.: Study of retention and peak shape in hydrophilic interaction chromatography over a wide ph range. J. Chromatogr. A, 1411, (2015). 16. Bauer, A., Luetjohann, J., Rohn, S., Kuballa, J. Jantzen, E.: Ion chromatography tandem mass spectrometry (IC-MS/ MS) multimethod for the determination of highly polar pesticides in plant-derived commodities. Food Control, 86, (2018). 17. Uclés, S., Lozano, A., Sosa, A., Vázquez, P. P., Valverde, A., Fernández-Alba, A. R.: Matrix interference evaluation

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한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과

한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과 - 2 - - 3 - KP 11 Osterici seu Notopterygii Radix et Rhizoma Ostericum koreanum Maximowicz, Notopterygium incisum Ting, Notopterygium