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pissn 1229-1153 / eissn 2465-9223 J. Food Hyg. Saf. Vol. 31, No. 5, pp. 319~326 (2016) https://doi.org/10.13103/jfhs.2016.31.5.319 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr LC-MS/MS 를이용한수산물중콜리스틴분석법개발 신다솜 강희승 이수빈 조윤제 천소영 정지윤 * 이규식 식품의약품안전처식품의약품안전평가원식품위해평가부잔류물질과 Development of Analytical Method for Colistin in Fish and Shrimp using Liquid Chromatography Mass Spectrometry Dasom Shin, Hui-Seung Kang, Soo-Bin Lee, Yoon-Jae Cho, So-Young Cheon, Jiyoon Jeong*, and Gyu-Seek Rhee Pesticide and Veterinary Drug Residues Division, National Institute of Food & Drug Safety Evaluation, Osong, Chungcheongbuk-do 28159, Korea (Received July 11,2016/Revised August 16, 2016/Accepted September 6, 2016) ABSTRACT - Colistin is a last resort antimicrobial agent against multi-drug resistant Gram-negative bacteria. This study was conducted to develop an analytical method to determine colistin in fish and shrimp. The analytes were confirmed and quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM). The sample was extracted with acidified 5% methanol (containing 0.5% formic acid). Then, solid phase extraction (SPE) was used for cleanup. Matrix-matched calibration curves were linear over the calibration ranges (0.05-1.2 mg/kg) for all the analytes into blank sample with r 2 > 0.99. All the values fulfilled the criteria requested by the Codex guidelines. Average recoveries ranged from 85.9% to 107.9%. The repeatability of measurements, expressed as the coefficient of variation (CV, %), was less than 15%. The limit of detection (LOD) was 0.02 mg/kg, and the limit of quantitation (LOQ) was 0.05 mg/kg. This improved method showed higher accuracy and acceptable sensitivity to meet the CAC guideline requirements and is applicable for the analysis of residual colistin (A+B) in fish and shrimp. Key words : colistin, fish, shrimp, analytical method, LC-MS/MS 콜리스틴 (Colistin) 은폴리믹신계열의폴리펩타이드계항균제로 Bacillus colistinus로부터 1950년에분리되어발견되었으며, 콜리스틴 A( 폴리믹신 E1) 와 B( 폴리믹신 E2) 로분류되어있다 1). 콜리스틴 A와 B는구조적으로지방산의사슬일부가다른형태로구성되어있다 (Table 1). 콜리스틴은대표적인부작용으로신장독성이있어주요항생제로사용되지하였으나, Pseudomonas, Klebsiella, and Acinetobacter spp. 등항생제다제내성균이많아지면서사용되기시작하였다 2,3). 최근임상연구에서폐렴및뇌수막염치료에콜리스틴유효성이확인됨에따라다제내성균치료를목적으로사용이늘어나고있는추세이다 4). 콜리스틴은전세계적으로축수산물의양식에연간 12,000톤이 *Correspondence to: Jiyoon Jeong, Pesticide and Veterinary Drug Residues Division, National Institute of Food and Drug Safety Evaluation, Osong, Chungcheongbuk-do 28159, Korea Tel: 82-43-719-4203, Fax: 82-43-719-4200 E-mail: stopyoon@korea.kr 상이사용되는것으로추정되고있으며, 중국, 유럽등에서널리사용되고있다. 하지만돼지에서분리된 E. coli에서의콜리스틴내성유전자 (mcr-1) 가최근발견되면서콜리스틴내성과관련된우려가증가하고있다 5). 일본의후생노동성에서는허용물질목록관리제도 (positive list system, PLS) 를통하여축산물중콜리스틴의잔류허용기준 (maximum residue level, MRL) 을 0.15~0.2 mg/kg, 계란 0.3 mg/kg으로설정하여관리하고있다 6). 국제식품규격위원회 (CODEX) 및유럽연합 (EU) 에서도축산물 (0.15~ 0.2 mg/kg) 및계란 (0.3 mg/kg) 중콜리스틴에대한잔류허용기준을동일하게관리하고있다 7). 우리나라에서도식품의약품안전처는축산물에대해국외기관과유사한수준인 0.15~0.3 mg/kg, 어류및갑각류에대해 0.15 mg/kg로잔류허용기준 (MRL) 을설정하여관리하고있다 11). 콜리스틴을분석하기위한액체크로마토그래피 (liquid chromatography) 와형광검출법 (fluorescence detection), 효소면역분석법 (ELISA) 등의시험법이개발되어왔다 12-16). 319

320 Dasom Shin, Hui-Seung Kang, Soo-Bin Lee, Yoon-Jae Cho, So-Young Cheon, Jiyoon Jeong, and Gyu-Seek Rhee Table 1. Molecular structure and physico-chemical properties of colistin Property IUPAC Name Classification Content N-(4-amino-1-(1-(4-amino-1-oxo-1-(3,12,23-tris(2-aminoethyl)-20-(1-hydroxyethyl)-6,9-diisobutyl- 2,5,8,11,14,19,22-heptaoxo-1,4,7,10,13,18-hexaazacyclotricosan-15-Ylamino)butan-2-ylamino)-3- hydroxybutan-2-ylamino)-1-oxobutan-2-yl)-n,5-dimethyl-heptanamide Polymyxin antibiotic Molecular formula Molecular weight Colistin A : C 53 H 100 N 16 O 13 Colistin B : C 52 H 98 N 16 O 13 Colistin A : 1169 g/mol Colistin B : 1155 g/mol pka 11.6 pk ow 2.4 Solubility Freely soluble (in water) Structure Li 등 (2003) 은액체크로마토그래프 (HPLC) UV 및형광검출기를이용하여인체혈장중콜리스틴 A와 B, 콜리스틴메탄설포네이트를분석하였으며, 최적온도 (37 o C) 와 ph 7.4를확립하였다. Wan 등 (2006) 은 LC-MS/MS와고상추출법 (SPE, solid phase extraction) 을활용하여콜리스틴 A와 B에대한시험법을개발하였으며, 폴리믹신 B를내부표준물질로활용하여, 콜리스틴 A와 B에대한정량한계 (LOQ) 를 1-16 μg/kg로확보하였다. Ma 등 (2008) LC- MS/MS와 HLB 카트리지를활용하여인체시료에서콜리스틴을분석한결과소변및혈장중콜리스틴 A와 B에대해서정량한계를각각 0.016-0.056 μg/ml 수준으로확 보하였다. Xu 등 (2012) 는수산물 8종에대한콜리스틴 A 와 B에대해 LC-MS/MS와고상추출법을활용한시험법을개발하였으며, 회수율은 73~83% 수준이었으며정량한계는 40 μg/kg로확보하였다. Mercier 등 (2014) 등은콜리스틴과콜리스티메테이트소디움 (colistimethate sodium, CMS) 에대한액체크로마토그래프-질량분석기 (LC-MS/MS) 와 HLB 카트리지를활용하여인체시료에서고감도분석법을확보하였다 ( 정량한계, 0.006-0.014 μg/ml). 하지만, 우리나라식품공전 (5.3.35) 에서는 LC-MS를이용하여콜리스틴 A와아프라마이신에대한동시분석으로제시되어있다. 현재의시험법을적용하였을경우 40% 이하의낮은회수율을보여주었다. 본연구를통해회수율개선뿐만아 니라콜리스틴 A와 B를모두포함하여잔류허용기준 (MRL) 에따라수산물중콜리스틴에대해고감도로분석이가능한공인된분석법을개발하고자하였다. Materials and Methods 시약및재료콜리스틴설페이트 (colistin sulfate, 62.9%) 의표준품은 U.S.Pharmacopeial (MD, USA) 로부터구입하여사용하였다 (Table 1). 아세토니트릴 (acetonitrile), 메탄올 (methanol) 등은 Merck (Darmstadt, Germany) 에서 HPLC 등급으로구입하여사용하였다. 또한, 고상추출카트리지로사용한 hydrophilic-lipophilic balance (HLB, 6cc, 200 mg) 는 Waters (Oasis, MA, USA) 에서구입하여사용하였다. 개미산 (formic acid) 등그이외의분석용시약및용매는특급또는분석용을사용하였다. 시험용시료는수산물섭취량과소비량을고려하였으며 8), 수산물의지방함량및조직특성을고려하여갑각류 ( 새우 ), 해수어 ( 넙치 ), 담수어 ( 장어 ) 를선정하였다. 시중에서유통되고있는시료를껍질, 내장을제거한부위 ( 근육 ) 만을분쇄하고균질화한후, 분석전까지냉동고 ( 20 o C) 에보관하였다. 공시료 (blank) 는동일한시험법을거쳐콜리스틴이잔류되지않음을확인한후시험용시료로사용하였다.

Development of Analytical Method for Colistin in Fish and Shrimp using Liquid Chromatography Mass Spectrometry 321 Table 2. LC-MS/MS parameter for the analysis of colistin LC system Waters, UPLC Fig. 1. Flow chart of analysis procedure for colistin. 표준원액및표준용액의조제콜리스틴표준품을 15.89 mg을정밀히달아 100 ml 폴리프로필렌 (polypropylene) 재질의볼륨플라스크에 50% 메탄올로정용하여, 100 mg/l 표준원액을조제하였다. 이를 1% 개미산을함유한메탄올로 0.5, 0.75, 1.5, 3, 6, 12 mg/ L 희석하여시료에각각 200 μl 첨가하여시료전처리과정과동일하게처리한뒤표준용액으로준비하였다. 표준원액과표준용액은폴리프로필렌재질용기에담아냉동보관 ( 20 o C) 하였으며, 표준용액은사용직전에희석하여실험에사용하였다. 실험에사용한모든시험용기는폴리프로필렌재질을사용하였다. 추출및정제넙치, 장어, 새우각각의시료를균질화하여 2g을 50 ml 폴리프로필렌재질의튜브에취하고추출용액 0.5% 개미산을함유한 5% 메탄올 5mL를넣고가볍게진탕한후 12,000 g, 4 o C에서약 10분간원심분리하였다. 상층액을다른 50 ml 폴리프로필렌재질의튜브에취한후남은잔사에위와동일하게처리하여상층액을합하였다. 미리메탄올 5mL와물 5mL로 HLB 카트리지를활성화시킨후추출액을 1~3 ml/min의속도로충진제에흡착시키고물 5mL를넣어같은유속으로세척하였다. 이때, 감압하여물을완전히제거한후공기를통과시켜건조하였다. 1% 개미산을함유한메탄올 4mL를 15 ml 폴리프로필렌재질의튜브에용출하였다. 이용액을균질화한후 polytetrafluoroethylene (PTFE) 0.2 μm 멤브레인소수성필터로여과하여폴리프로필렌재질의바이알에담아시험용액으로사용하였다 (Fig. 1). Column Column temp. Injection vol. Flow rate Mobile phase Gradient Mass spectrometry Ionization mode Capillary temp. Spray voltage Collision gas Waters X-SELECT C 18 (2.1 mm i.d. 150 mm, 3.5 μm) 40 o C 5 μl 0.3 ml/min. A = ammonium formate : formic acid : water (1:2.5:497.5 = v/v/v) B = 1% formic acid in acetonitrile Time (min) Mobile phase A(%) B(%) 0 100 0 1 100 0 8 10 90 9 10 90 9.5 100 0 12 100 0 Waters, Xevo TQ-S ESI positive 500 o C 3.5 kv Ar 기기분석조건콜리스틴분석을위해사용한 LC-MS/MS와컬럼은 Waters 사의 US/Xevo TQ-S (Milford, MA, USA), X-SELECT C 18 (2.1 150 mm, 3.5 μm, Dublin, Ireland) 을각각사용하였다. 이동상 A는암모늄포메이트 : 개미산 : 물 (1 : 2.5 : 497.5 = v/v/v), 이동상 B는 1% 개미산을함유한아세토니트릴을선택하여기울기용리방식을선택하였다. 콜리스틴의이온화법은 electro-spray ionization (ESI) 법의양이온모드 (positive ion mode) 로 MS조건을최적화하였으며, multiple reaction monitoring (MRM) 조건으로분석하였다 (Table 2). 분석법검증개선된분석법은 CAC/GL 16번과 71번 9,10) 및최근식품의약품안전처에서발간한식품등시험법마련표준절차에관한가이드라인 11) 에따라서직선성 (linearity), 정확성 (accuracy), 정밀성 (precision), 정량한계 (limit of quantification, LOQ) 를측정하여유효성을검증하였다. 직선성은혼합표준용액을잔류허용기준농도의 1/3, 1/2, 1, 2, 4, 8배가되도록시료전처리과정과동일하게거친시료액을이용하여 LC-MS/MS에주입하여얻어진피크면적으로검량곡선을작성하고직선성을구하였다. 정확성과정밀성은공시료에잔류허용기준의 0.5, 1, 2배농도로표준용액을첨가

322 Dasom Shin, Hui-Seung Kang, Soo-Bin Lee, Yoon-Jae Cho, So-Young Cheon, Jiyoon Jeong, and Gyu-Seek Rhee 한후, 회수율과상대표준편차 (relative standard deviation, RSD, %) 를측정하였다. 검출한계 (Limit of detection, LOD) 는각신호대잡음비 (signal to noise ratio, S/N ratio) 3배이상인농도로계산하였으며, 정량한계는각각신호대비잡음비가 10배이상인농도로계산하였다. 또한, 신뢰성재고를위하여실험실간시험법검증 (inter-laboratory verification) 을수행하였다. 시험법을검증하기위해식품등시험검사기관 A와식품등시험검사기관 B를선정하여수행하였고, 대상시료는시험법을확립한곳과동일하게넙치, 장어및새우에서회수율검증을수행하였다. 수행농도는분석법확립에대한 CODEX의식품중동물용의약품잔류분석법에대한가이드라인에근거하여진행하였으며, 재현성검증을위하여 5회반복이상, 모든기기분석조건은동일하게진행하였다. Results and Discussion 분석법조건선정현식품공전 (5.3.35) 에서는 LC-MS 를이용하여분석하도 록고시되어있지만, 수산물시료의복잡한매트릭스 (matrix) 에서콜리스틴대상물질의존재여부를정확하고낮은농도의정량한계확보와고감도분석을위해 LC-MS/MS 를선택하였다. 이온화법으로는전기분무이온화 (electro spray ionization, ESI) 방법으로 positive mode 조건으로분석되었다. 콜리스틴은평균분자량이 1162 g/mol의고분자물질이지만, amine기 (-NH 2 ) 가여러개붙어있고 Log P ow 값이 2.4로극성물질이기때문에전기분무이온화를통해되어전하를띠게되어다가이온형태로확인할수있었다. 콜리스틴 (A[M 1 ]+B[M 2 ]) 의최적 MS조건및기기조건을 Table 2에제시하였다. 이동상에사용된암모늄포메이트는 protonation enhancer로서분자내 amine기에서 protonation 을용이하게하여콜리스틴 A의 [M 1 +2H] 2+, B의 [M 2 +2H] 2+ 를생성하도록하였다. 콜리스틴표준용액 (50 μg/l) 을질량검출기에직접주입하여 cone voltage (10-50 V) 를조절하여 35 V에서최대강도를나타내는것을확인하였다. 선구이온 (precursor ion) 은 full scan mode 에서질량스펙트럼을확인하여, 콜리스틴 A의 [M 1 +2H] 2+ (m/z 585), B의 [M 2 +2H] 2+ (m/z 578) Fig. 2. Precursor-products ions full scan mass spectrum of colistin A and B. Table 3. Selected-ion of LC-MS/MS for colistin Compound Retention time (min) Exact mass (m/z) Precursor ion (m/z) Product ion (m/z) Collision Energy (ev) Colistin A 4.65 1169.5 585 Colistin B 4.45 1155.4 578 *Quantification ion 101* 202 569 101* 227 579 30 13 16 30 20 16

Development of Analytical Method for Colistin in Fish and Shrimp using Liquid Chromatography Mass Spectrometry 323 형태의이온의 peak를확인하였다. MS/MS 분석시 collision cell에서의 energy를조절하여콜리스틴 A에서의가장높은감도를보이는생성이온 m/z 101 ([α,γ-diaminobutyric acid-γ-nh 2 ] + ) 을정량이온 (quantification ion) 으로선택하고, 다음으로크게검출되는두개의생성이온 m/z 202 ([α,γdiaminobutyric acid-threonine-h] + ), m/z 569 ([M 1 +2H-H 2 O] 2+ ) 을정성이온 (qualification ion) 으로설정하였다. 콜리스틴 B에서의가장높은감도를보이는생성이온 m/z 101 ([α,γdiaminobutyric acid-γ-nh 2 ] + ) 을정량이온으로선택하고, 다음으로크게검출되는두개의생성이온, m/z 227 ([6- methylheptanoic acid- α,γ-diaminobutyric acid-γ-nh 2 +H] + ), m/z 576 ([M 2 +2H-H 2 O] 2+ ) 을정성이온으로설정하였다 (Fig. 2). 선정된이온과머무름시간은 Table 3에나타내었다. 추출및정제조건선정식품공전 (5.3.35) 축 수산물에대한콜리스틴시험법은아프라마이신과동시분석법으로적용하게되어있으나수산물시료에적용하였을경우, 전처리과정에서극성인콜리스틴의물질특성을만족하지못한채아세토니트릴로추출하여농축후비극성인용매로재용해하는과정으로 인하여회수율이낮은결과를보여주었다. 또한, 식품공전의정제과정에서사용된 florisil 카트리지는약한염기성을갖는흡착제로유기용매로부터낮은극성부터중간극성물질의분석에사용되기때문에분석물질인콜리스틴과는맞지않는시험법이라고판단하였다. 본연구를통하여시험법을개선하고자폴리펩타이드계인콜리스틴의특성 17) 을고려하여추출용매를극성용매로선택하였다. 콜리스틴이수산물에존재하는경우중 cell membrane 내인지질에단단히결합되어있는조직적특성을고려하여단백질의가수분해하여침전시키는역할을하는개미산을선택후비율을조절하여 0.5% 의개미산을추출용매에포함시켰다. 하지만개미산수용액을사용할경우에는전처리과정중시료에유화액 (emulsion) 이생길수있기때문에전처리과정이어려워진다. 이유화액속의모든간섭물질을카트리지로정제하였을때 matrix effect가크게나타나지않았기때문에 0.5% 개미산용매에서극성도는최대한높이고비극성간섭물질의추출률을비교적낮추기위해서 5% 의메탄올을첨가하였다. 0.5% 개미산을포함한 5% 메탄올로추출후모든화합물을위한균형-역상흡착제인 HLB 카트리지를사용하여정제과정을거쳤다. 시험용액을기기분석전불순물을한번더제거하기위한멤브레인필터를선택하기위하여분석에가장많이쓰이는 4가지를선정하여회수율실험을수행하여산 / 염기가함유된유기용매정제에가장적합하고감도가가장뛰어난 0.2 μm PTFE 멤브레인필터를선택하였다 (Fig. 3). Fig. 3. Comparison of peak intensity by membrane disc filter type (0.15 mg/l). 분석법검증콜리스틴분석법의선택성, 특이성, 직선성을검증하기위해넙치, 장어, 새우의무처리시료, 표준용액, 표준용액을첨가한회수율시료의크로마토그램을서로비교하였다 (Fig. 4). 그결과, 무처리검체중콜리스틴 (A+B) 과같은머무름시간을갖는어떤방해물질도검출되지않았다. 따라서, 콜리스틴 (A+B) 을분석하기위한본시험법이높은분리능과선택성을가짐을확인할수있었다콜리스틴표준원액을물로희석하여시료에잔류허용기준의농도별로 spiking 하여시료전처리과정과동일하게거친시료액을위에서제시한기기분석조건으로측정하여 matrix-matched 검량곡선을작성한결과를 Table 4에제시하였다. 상관계수가 0.99 이상으로 Codex에서권장하는 r 2 >0.98와비교해도매우만족할만한수준이었으며, 검출한계와정량한계는각각 0.02 mg/kg, 0.05 mg/kg이었다. 본시험법의정확성을평가하기위하여넙치, 장어, 새우에처리농도를잔류허용기준 (MRL) 의 0.5, 1, 2배농도로표준용액을 spiking하여회수율실험을 5회반복으로수행하여정확성과정밀성을평가하였다. 그결과넙치, 장어및새우시료에대한콜리스틴의회수율과정밀성은 92.1~107%, 4.64~11.8% 로조사되어정확성과정밀성모

324 Dasom Shin, Hui-Seung Kang, Soo-Bin Lee, Yoon-Jae Cho, So-Young Cheon, Jiyoon Jeong, and Gyu-Seek Rhee Fig. 4. Chromatograms of colistin A (m/z 585 m/z 101) and B (m/z 578 m/z 101) : (a) blank in flatfish, (a-1) matrix matched standards at 0.15 mg/kg in flatfish, (a-2) colistin MRL (0.15 mg/kg) recovery test in flatfish, (b) blank in eel, (b-1) matrix matched standards at 0.15 mg/kg in eel, (b-2) colistin MRL (0.15 mg/kg) recovery test in eel, (c) blank in shrimp, (c-1) matrix matched standards at 0.15 mg/kg in shrimp and (c-2) colistin MRL (0.15 mg/kg) recovery test in shrimp. Table 4. Standard curve range, linearity, correlation coefficients, LOD and LOQ of colistin Compound Colistin Matrices Standard curve range (mg/kg) Linearity r 2 Flatfish y = 44,901.1135x + 4,343.3532 0.9990 Eel 0.05-1.2 y = 58,373.5229x + 3,018.2587 0.9906 Shrimp y = 28,294.1862x + 2,005.1642 0.9986 Table 5. Validation results of the analytical method of colistin (A + B) in flatfish, eel and shrimp (n = 5) Concentration (mg/kg) flatfish eel shrimp Recovery (%) CV (%) Recovery (%) CV (%) Recovery (%) CV (%) 0.075 (1/2MRL) 102 4.64 85.9 7.06 95.5 9.38 0.15 (MRL) 102 8.61 105 7.51 87.6 11.8 0.30 (2MRL) 95.7 10.8 108 8.71 92.7 7.21 두 CAC 권장기준을충족하며, 잔류동물용의약품시험법의적합성을확인할수있었다 (Table 5). 신뢰성과재현성확보를위해실험실간검증을실시하였으며직선성, 회수율과정밀성을평가한결과직선성은모든분석기관에서의상관계수가 0.99이상으로확인되었고, 전체회수율평균은 82.3~111% 이었으며, CV값은 0.18~9.91% 로모두적합한수준이었다 (Table 6). 현재까지수산물중잔류하는동물용의약품콜리스틴분석법에대한연구자료는저조한편이다 12-16). 하지만가장유사한 Xu 등 (2012) 의연구결과와비교하였을때, 우수한감도와 10% 이상높은회수율및상대적으로낮은상대표준편차를얻을수있었고, 정량한계또한 2배적은 injection volume 으로도유사한수준의정량성을확보할수있었다. 현식품공전의시험법은수산물에적용하였을때, 분석대상물질과시험법이맞지않아회수율이낮았다. 따라서, 확립된시험법을통해높은회수율과정확성그리고정량성을확보하였고콜리스틴 A, B 성분모두를관리하게되면서수산물중잔류할수있는콜리스틴에대한안전관리에활용할수있을것으로사료되는바이다. 검출한계및정량한계콜리스틴의검출한계는기기상에서 S/N ratio 3 이상으로결정하여수산물의경우분석기기에따른검출한계는

Development of Analytical Method for Colistin in Fish and Shrimp using Liquid Chromatography Mass Spectrometry 325 Table 6. Recovery and CV of colistin by inter-laboratory verification (n = 5) Flatfish Eel Shrimp Fortified Conc. (mg/kg) Inter-laboratory verification (colistin) Laboratory A Laboratory B Laboratory C Total Recovery (%) CV (%) Recovery (%) CV (%) Recovery (%) CV (%) Recovery (%) CV (%) 0.075 102 4.64 96.7 0.86 96.7 8.17 98.6 4.56 0.150 102 8.61 112 0.61 91.8 3.26 102 4.16 0.300 95.7 10.8 95.7 0.19 85.8 14.3 92.4 8.45 0.075 85.9 7.06 98.4 0.18 110 5.16 98.0 4.13 0.150 105 7.51 94.2 0.81 84.7 4.64 94.7 4.32 0.300 108 8.71 92.4 0.38 83.3 10.2 94.6 6.44 0.075 95.5 9.38 79.5 8.16 103 0.78 92.8 6.11 0.150 87.6 11.8 76.2 2.76 103 15.1 88.9 9.91 0.300 92.7 7.21 82.3 8.96 83.9 6.74 86.3 7.64 0.02 mg/kg이었고, 정량한계는 S/N ratio 10 이상으로결정하여 0.05 mg/kg이었다. 우리나라의수산물에대한콜리스틴잔류허용기준인 0.15 mg/kg의 1/2 미만으로본분석법으로잔류허용기준준수여부를판별하기에문제가없을것으로판단된다. Acknowledgement 본연구는식품의약품안전처연구개발과제 (15161MFDS665 and 16161MFDS602) 에의해수행되었으며, 이에감사드립니다. 국문요약 LC-MS/MS를이용하여폴리펩타이드계동물용의약품인콜리스틴에대한시험법을확립하여정량성및정밀성을확보하였으며, 확립된시험법의적용성검증을위해국제식품규격위원회기준에따라특이성, 정확성, 직선성, 정밀성, 검출한계, 정량한계등을검증하였다. 콜리스틴표준용액을잔류허용기준의농도에따라검량선을작성한결과 0.99 이상의직선성을확인할수있었으며, 본실험에서의평균회수율은 85.9~107% 이었다. 또한, 분석오차는 11.8% 이하로정확성및재현성이우수하였으며, 검출한계는 0.02 mg/kg, 정량한계는 0.05 mg/kg이었다. 또한, 실험실간교차검증을통하여신뢰성을확보하였다. 확립된분석법은양식수산물중잔류할수있는동물용의약품인콜리스틴에대한안전관리에활용할수있을것으로판단된다. References 1. J. A. Orwa, A. V. Gerven, E. Roets, J. Hoogmartens: Validation of a Liquid Chromatography Method for Analysis of Colistin Sulphate. Chromatographia, 51, No. 7/8 (2000). 2. V. Pintado, Lucía García San Miguel, G. Fabio, M. Blanca, C. Javier, F. Jesu s, M. D. Pilar, M. Santiago: Intravenous colistin sulphomethate sodium for therapy of infections due to multidrug-resistant gram-negative bacteria. J. of Infection, 56, 185-190 (2008). 3. C. Boudewijn, C. Marco, B. Keith, G. Kari, G. Kornelia, H. Anja, J. Helen, L. Ernesto, L. N. Antonio, et al.: Use of colistin-containing products within the European Union and European Economic Area (EU/EEA): development of resistance in animals and possible impact on human and animal health. Int. J. of Antimicrob. Agents, 46, 297-306 (2015). 4. Y. H. Choi, MD.: Treatment of drug resistant bacteria: new bugs, old drugs, and new therapeutic approaches. J. Korean Med. Assoc., 57(10), 837-844 (2014). 5. M. Joseph B and L. Shawn: From Pigs to Patients: Transmissible, Single Gene-mediated Resistandce to Colistin. J. of Med. Microbiol. & Diagnosis, 5:1 (2016). 6. Ministry of Health, Labour and Welfare (MHLW), Available from: http://www.mhlw.go.jp/english/. Accessed (2002). 7. European Medicines Evaluation Agency (EMEA), Available from: http://www.ema.europa.eu/ema/. Accessed (2006). 8. Ministry of Helth and Welfare: Korea National Health and Nutrition Examination Survey, Available from: http://knhanes. cdc.go.kr/. Accessed (2014). 9. Codex guidelines for the establishment of a regulatory program for control of veterinary drug residues in foods. CAC/ GL 16, 1-46 (1993). 10. Codex Alimentarius Committee. Guidelines for the design and implementation of national regulatory food safty assurance programme associated with the use of veterinary drugs in food producing animals. CAC/GL 71 (2012). 11. Korean Food Standards Codex: Ministry of Food and Drug Safety (2016). 12. Jian Li, W. M. Robert, L. N. Roger, J. D. Turnidge and Kingsley Coulthard: Stability of Colistin and Colistin Methanesulfonate in Aqueous Media and Plasma as Determined

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