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1 Korean Journal of Environmental Agriculture Korean J Environ Agric (2013) Online ISSN: Vol. 32, No. 1, pp Print ISSN: Research Article Open Access HPLC-PDA 를이용한닭고기중 Nitroxoline 분석법개발 조윤제, 1 채영식, 1 김재은, 1 김재영, 1 강일현, 1 이상목, 1 도정아, 1 오재호, 1 장문익, 1* 홍진환 1 1 식품의약품안전처식품의약품안전평가원식품위해평가부잔류물질과 Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA Yoon-Jae Cho, 1 Young-Sik Chae, 1 Jae-Eun Kim, 1 Jae-Young Kim, 1 Ilhyun Kang, 1 Sang-Mok Lee, 1 Jung-Ah Do, 1 Jae-Ho Oh, 1 Moon-Ik Chang 1* and Jin-Hwan Hong 1 ( 1 Residues of Pesticide and Veterinary Drugs in Foods Division, Department of Food safety Evaluation, National Institute of Food & Drug Safety Evaluation, Ministry of Food and Drug Safety Chungwon , Korea) Received: 2 January 2013 / Revised: 14 March 2013 / Accepted: 25 March 2013 c 2013 The Korean Society of Environmental Agriculture This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract BACKGROUND: Nitroxoline is an antibiotic agent. It is used for the treatment of the second bacterial infection by the colibacillosis, salmonellosis and viral disease of the poultry. When the nitroxoline is indiscreetly used, the problem about the abuse of the antibiotics can occur. Therefore, this study presented the residue analytical method of nitroxoline in food for the safety management of animal farming products. METHODS AND RESULTS: A simple, sensitive and specific method for nitroxoline in chicken muscle by high performance liquid chromatograph with PDA was developed. Sample extraction with acetonitrile, purification with SPE cartridge (MCX) were applied, then quantitation by HPLC with C18 column under the gradient condition with 0.1 % tetrabutylammonium hydroxide-phosphoric acid and methanol was performed. Standard calibration curve presented linearity with the correlation coefficient (r 2 ) > 0.999, analysed from 0.02 to 0.5 mg/l concentration. * 교신저자 (Corresponding author), Phone: ; Fax: ; @korea.kr Limit of quantitation in chicken muscle showed 0.02 mg/kg, and average recoveries ranged from 72.9 to 88.1 % in chicken muscle. The repeatability of measurements expressed as coefficient of variation (CV %) was less than 12 % in 0.02 and 0.04 mg/kg. CONCLUSION(S): Newly developed method for nitroxoline in chicken muscle was applicable to food inspection with the acceptable level of sensitivity, repeatability and reproducibility. Key Words: Analytical method, Chicken, HPLC-PDA, Nitroxoline, Residue 서론 Nitroxoline(5-nitro-8-hydroxyquinoline) 은항생제로서항혈관형성, 항암및항진균효과를광범위하게나타낸다 (Karpińska et al., 2010; Pelletier et al., 1995). 의학적으로는요로감염증의치료에이용되는데, 요로감염증을일으키는그람양성및음성균에대하여활성을나타내며, 특히 Candida albicans 균에큰효과를나타낸다 (Ghoneim et al., 2011). 또한, 동물에게는주로가금류의대장균증, 살모넬라증및바이러스성질병에기인한 2차세균감염증을치료하는항생제로이용된다 ( 잔류동물용의약품기준선진화를위 70

Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA 71 한기획연구 (KFDA), 2010). 국내축 수산용동물용의약품의최근 5 년간사용량은약 13,000여톤정도로비교적많은양이사용되고있다 (Shin et al., 2011).

항생제중하나인 nitroxoline의경우, 국내에서의사용량이 2009년에는 260 kg, 2010년에는 129 kg정도로다른항생제에비해적은양이지만 (Shin et al., 2012), 인체에미치는영향이현재까지보고된바없으며, 국내는물론제외국에서도기준및규격이설정되어있지않은상태이다 ( 잔류동물용의약품기준선진화를위한기획연구 (KFDA), 2010).

2 Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA 71 한기획연구 (KFDA), 2010). 국내축 수산용동물용의약품의최근 5 년간사용량은약 13,000여톤정도로비교적많은양이사용되고있다 (Shin et al., 2011). 현대의축 수산업에서항생제사용은불가피하나사용된항생제가축산물로의이행및잔류로이어지면, 식품위생등의문제로나타날수있으며, 최근문제시되고있는축 수산식품내동물용항생제의잔류와내성등의부작용으로인해심장기능및임신율의저하, 체내간장 신장등장기의기능장애, 발육저하, 독성발현및내성균의발생과같은유해작용을나타낼수있다 ( 잔류동물용의약품기준선진화를위한기획연구 (KFDA), 2010). 항생제중하나인 nitroxoline의경우, 국내에서의사용량이 2009년에는 260 kg, 2010년에는 129 kg정도로다른항생제에비해적은양이지만 (Shin et al., 2012), 인체에미치는영향이현재까지보고된바없으며, 국내는물론제외국에서도기준및규격이설정되어있지않은상태이다 ( 잔류동물용의약품기준선진화를위한기획연구 (KFDA), 2010). 따라서항생제의무분별한사용을야기시킬수있으며, 더나아가항생제의오 남용의문제가나타날수도있다. 현재까지임상등에관련된몇몇문헌 (Karpińska et al., 2010; Szabó et al., 1995; Wang et al., 2001; Ghoneim et al., 2011; Štefane et al., 2012; Kang et al., 2003) 을제외하고는식품중잔류분석을수행한연구는거의보고된바없을뿐만아니라, 특히가금류에대한잔류분석연구는전무한상황이다. 최근우리나라에서는사용이허가된동물용의약품에대한기준을설정, 강화하고있는추세에따라독성및잔류자료들이요구되는상황에서가금류에사용하기위하여등록된 nitroxoline의안전관리를위한잔류분석법이필요함에 따라 nitroxoline 의효율적이고신속한분석법을확립하고자하였다. 재료및방법 시료시중에서유통되고있는가금류 ( 닭고기 ) 를구입하여공시험을거쳐 nitroxoline이잔류되지않은시료를대상으로회수율시험을하였다. 대상시료의근육을믹서기로마쇄하여균질화한후 20 g 단위로소분하여분석전까지냉동실 (-20 ) 에보관하면서사용하였다. 시약및재료 Nitroxoline(8-Hydroxy-5-nitroquinoline, 96.0 %) 표준품은 Sigma-Aldrich(St. Louis, MO, USA) 로부터구입하여사용하였다. 표준용액은메탄올 (Merck, Darmstadt, Germany) 을사용하여조제하였으며, nitroxoline의물리화학적특성은 Table 1에나타내었다. 액체크로마토그래피이동상및전처리용시약으로는아세토니트릴및메탄올의경우 HPLC급시약 (Merck, Darmstadt, Germany) 을사용하였다. 또한, 수산화암모늄, 개미산및인산은특급시약 (Wako, Osaka, Japan) 을구입하여사용하였고, TBAOH(tetrabutylammonium hydroxide solution(assay % in water)) 는 Sigma-Aldrich(St. Louis, MO, USA) 에서구입하여사용하였다. 분석에사용한고상추출카트리지 (SPE) 는 MCX 카트리지 (mixed-mode cation exchange (500 mg, 6 ml), Waters, Bellefonte, PA, USA) 를이용하여활성화과정을거친후사용하였다. Table 1. Physicochemical properties and structure of nitroxoline IUPAC name 5-Nitroquinolin-8-ol CAS No Melting point Molecular weight (C 9H 6N 2O 3) Boiling point at 760 mmhg Density g/ml Log Pow 1) Vapor pressure (25, mpa) mmhg at 25, mpa at 25 pka - Solubility Soluble in Water Toxicity ORL-RAT : LD 50 > 104 mg/kg 1) n-octanol/water partition coefficient.

3 72 CHO et al. 표준용액의조제 Nitroxoline 표준물질약 10 mg을정밀히달아메탄올에녹여 100 μg/ml 표준원액으로조제하였으며, 표준용액은표준원액을실험전에메탄올로희석하여사용하였다. 표준원액과표준용액은모두갈색병에담아 -20 냉동실에보관하여사용하였다. 시료전처리균질화한닭고기근육시료 5 g을 50 ml 원심분리관에취하고 20 ml 아세토니트릴을가한후진탕하여추출과정을거쳤다. 이를 9,000 G, 4 에서 10분간원심분리하고얻어진상징액을농축플라스크에취하였다. 이잔류물에아세토니트릴 20 ml를가하고위의원심분리과정을재반복한후얻어진상등액을상기상등액과합하여, 40 이하의수욕상에서감압농축하였다. 농축에의해얻어진잔류물은메탄올 2 ml로재용해하여, 미리메탄올 2 ml 및물 2 ml로활성화시킨 MCX 카트리지에흡착시켰다. 흡착된카트리지에 5 % 개미산수용액 2 ml 및 5 % 수산화암모늄함유메탄올 2 ml로세척한후, 5 % 수산화암모늄함유메탄올 2 ml로용출하였다. 용출액은 40 이하의수욕상에서질소농축하였 고잔류물은메탄올 2 ml 로재용해하여 0.20 μm 멤브레인필터로여과하고, 이를시험용액으로사용하였다 (Fig. 1). 기기분석조건 Nitroxoline의잔류량은 PDA 검출기 (photodiode-array detector) 가장착된액체크로마토그래프 (Shiseido JP/Nanospace SI-2, Tokyo, Japan) 를사용하였다. 분석용컬럼은 XBridge C 18(4.6 mm 250 mm, 5 μm: Waters, Dublin, Ireland), 컬럼온도는 40, 이동상으로는용매 A: 0.1 % TBAOH- 인산와용매 B: 메탄올을이용하여용매기울기조건으로분석하였으며, 유속은 1 ml/min, 주입량은 20 μl로하여 360 nm에서측정하였다 (Table 2). Nitroxoline의확인시험은먼저질량분석기조건을확립하기위해서표준용액을사용하여컬럼을통과하지않고직접탠덤질량분석기 (tandem mass spectrometry) 로도입하였다. 텐덤질량분석기를통해 nitroxoline의 precursor ion을선택하고최적의충돌에너지 (collision energy) 를선택한다음 product ion을탐색하여표준물질에대한 fragmentation pattern을확인하였다 (Table 3). 5 g sample + 20 ml acetonitrile Centrifugation at 4, 9,000 G for 10 min-1 Purification (MCX cartridge, 500 mg, 6 cc) Take supernatant-1 Conditioning with 2 ml MeOH, 2 ml Water 1 2 Sample loading Evaporate at 40 Washing with 2 ml 5 % formic acid(in water) Reconstitution with 2 ml methanol Elution with 2 ml 5 % NH 4OH in methanol Concentration Evaporate at 40 Reconstitution with 2 ml methanol Fig. 1. Analytical procedure for nitroxoline residues in chicken sample. HPLC-PDA analysis

4 Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA 73 Table 2. HPLC-PDA parameter for the analysis of nitroxoline Parameter Conditions HPLC Shideido JP/Nanospace SI-2 Column C 18 (4.6 mm 250 mm, 5 μm) Flow rate 1.0 ml/min Injection volume 20 μl Column temperature 40 Mobile phase A : 0.1 % TBAOH(tetrabutylammonium hydroxide)-phosphoric acid B : Methanol min A (%) B (%) Wavelength 360 nm Table 3. LC-MS/MS parameter for the analysis of nitroxoline Parameter Conditions LC Thermo Accela High Speed LC Column C 18 (2.1 mm 150 mm, 3 μm) Flow rate 0.2 ml/min Injection volume 10 μl Column temperature 40 Mobile phase A : 0.1 % trifluoroacetic acid in water B : 0.1 % trifluoroacetic acid in methanol min A (%) B (%) Mass Spectrometry Thermo TSQ Vantage Ionization mode Positive ion electrospray Spray voltage 5,000 V Capillary temperature 350 Collision gas Ar Collision energy Precursor Ion (m/z) Collision energy (ev)

5 74 CHO et al. 분석법검증 최종적으로닭근육시료를이용하여개발된 nitroxoline 의분석법은 CAC Guide line(codex Alimentarius Commission guidelines for the establishment of a regulatory programme for control of veterinary drug residues in foods; CAC/GL 16, 1993) 에준하여직선성 (Linearity), 회수율, 정량한계 (LOQ), 재현성 (Reproducibility) 등으로유효성검증을실시하였다. 닭근육시료로부터 nitroxoline mg/kg의농도에대한각각의피크면적을이용하여검량선을작성하였고, 각검량선의상관계수 (coefficient of correlation, r 2 ) 를구하였다. 정량한계는분석결과를수치화할수있는최저한계를의미하는데, 각국및기관에서는보통 S/N 6 10 범위를상용하고있으며, 국내에설정된기준은국제기준을수용하고있다 (Lee, 2012). 따라서이를근거로하여 matrix blank에첨가한 nitroxoline의크로마토그램에서신호대잡음비 (S/N ratio) 가 10 이상이되는농도값을이용해다음과같은계산식에의해정량한계를산출하였다. Limit of quantitation (mg/kg) = a b w a: nitroxoline의검출량 (mg/l) b: 최종희석부피 (L) w: 검체량 (kg) 이에대한분석법의정확성을평가하기위하여최대잔류허용기준 (MRL) 이설정되어있지않은 nitroxoline은 1 및 2 배 LOQ(0.02 및 0.04 mg/kg) 농도로회수율을측정하였다. 분석크로마토그램으로부터얻은피크의면적을검량선에대입하여시료에대한 nitroxoline의회수율을구하였고, 6반복실험을통해정확성과정밀성을평가하였다. 또한, HPLC/PDA 및 LC-MS/MS를보유한특정실험실 1곳을선정하여동일한전처리과정과분석조건으로 5회반복실험을통하여분석법을검증 (verification) 하였다. 된기기분석조건으로분석한 nitroxoline 에대한크로마토그램과 UV 스펙트럼은 Fig. 2 와같다. Fig. 2. Chromatogram and spectrum of nitroxoline standard (2 ng). 특이성및검량곡선본연구의분석조건에대한특이성 (specificity) 을검증하기위해, blank test를한결과, Fig. 3에서보는바와같이 nitroxoline의머무름시간대에어떠한방해물질또는 matrix 가검출되지않은결과를나타내어본분석법의높은분리능과선택성을가짐을확인하였다. Nitroxoline 표준용액의직선성 (linearity) 시험결과, nitroxoline에대한상관계수 (r 2 ) 가 0.999의직선성을나타내었으며 (Table 4), 이는 CAC에서권장하는 r 2 >0.95에적합하여매우만족할만한수준의결과를확인하였다. (A) 결과및고찰 분석법최적조건확립 Nitroxoline 을분석하기위한기기조건으로 0.1 % TBAOH- 인산 (A) 와메탄올 (B) 두가지용매로용매기울기조건을설정하여머무름시간과감도를확인한결과, 높은감도와적당한머무름시간을나타내었다. 분석파장은 UV 스펙트럼확인결과, 217 nm에서가장높은흡광도를보였으나기본선 (baseline) 이불안정할뿐만아니라표준물질의피크 (peak) 또한늘어짐현상이나타나분석에어려움이있어다음으로높은흡광도를보이는 360 nm를선택하여위의문제점들을해결하였다. 분석에사용된칼럼은보편적으로사용되는분석용칼럼인 C 18(4.6 mm 250 mm, 5 μm) 을선택하였으며, 분석유속은 1.0 ml/ 분, 주입량은 20 μl 을적용하였다. 확립 (B)

6 Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA 75 (C) 수는 %, %(Table 5) 로나타나, CAC에서권장하는회수율및변동계수에충족하는결과를나타내어잔류동물용의약품분석법으로서적합함을확인하였다. Table 5. Recovery, CV and LOQ of nitroxoline in chicken muscle Fig. 3. Chromatograms of nitroxoline standard at 2 ng (A), blank chicken sample (B), and fortified chicken sample at 2 ng (C). Table 4. Standard calibration curve range, linearity and r 2 of nitroxoline Standard curve Compound Sample Linearity r 2 range (mg/l) Nitroxoline Chicken y=21176x muscle 정량한계및회수율 Nitroxoline의정량한계는크로마토그램상에서신호대잡음비 (S/N ratio) 를 10 이상으로하였다. 분석법의정확성을평가하기위하여닭고기시료 5 g에 nitroxoline을각각 LOQ 및 2 LOQ(0.02 및 0.04 mg/kg) 농도가되도록표준물질을첨가하고시험용액조제과정을거쳐회수율실험을수행하였다. 분석크로마토그램으로부터얻은피크의머무름시간을비교하고 (Fig. 3), 피크에대한면적을구한뒤, 검량선을통해시험용액중 nitroxoline의함량을확인함으로써회수율을구하였고, 6반복실험을통해정확성과정밀성을평가하였다. 회수율시험결과, 닭고기시료에서의회수율과변동계 Sample Chicken muscle Nitroxoline Fortified Conc. Recovery 1) CV (mg/kg) (%) (%) ± ± LOQ (mg/kg) ) Mean values of six replicate with standard deviations. 확인시험 Nitroxoline의 HPLC-PDA를통한분석조건은설정되었지만, 분석물질에대한정성적확인과차후 LC-MS/MS 를통한분석조건설정을위해 nitroxoline에대한확인이온 (confirmation ion) 을설정하였다. Full scan mode에서 precursor ion을질량스펙트럼을통해확인한다음충돌에너지 (collision energy) 를설정하여 product ion을생성한후높은감도와양호한스펙트럼을보이는이온을선택하여최적의 MRM(multiple reaction monitoring) 조건을설정하였다. Nitroxoline은 ESI(electro-spray ionization) 이온화방법으로 positive mode에서높은감도를보였으며, 모분자인 m/z 191.0에충돌에너지를각각 46, 39 및 21 ev를가하여확인이온 m/z 89.1, 그리고 145.0을얻어 nitroxoline 에대한확인시험을확립하였다 (Fig. 4 및 5). [M+H] + Fig. 4. Precursor ion spectrum and product ion spectrum of nitroxoline standard solution in MeOH.

7 76 CHO et al. m/z m/z m/z Fig. 5. LC-MS/MS Chromatograms of nitroxoline standard at 20 ng. 실험실간분석법검증 개발된 nitroxoline 의분석법에대한실험실간검증을 HPLC-PDA 및 LC-MS/MS를보유한특정실험실 1곳을선정하여동일한전처리과정및분석조건으로 5회반복실험을수행하여이를통해분석법의신뢰성을재고하고자검증 (verification) 을수행하였다. 실험실간분석법검증결과, 확립된분석법의회수율은 %, 변동계수가 % 의결과를나타내었다 (Table 6). Table 6. Comparison of analytical methods for nitroxoline in inter-laboratory Sample (Laboratory) Chicken muscle (A) Spiked Conc. (mg/kg) Nitroxoline Mean recovery (%) RSD (%) 따라서가금류에사용되고있는 nitroxoline의잔류분석에대하여효율적인시료전처리방법과최적의정제과정뿐만아니라높은수준의정밀성, 정확성및재현성을나타내는효과적인분석법으로확인되었다. 따라서본 nitroxoline의분석법은축산식품의안전관리에기여할것으로판단된다. 요약 우리나라는 2007년부터원료축산물에대한동물용의약품잔류허용기준설정을강화하면서관련시험법을식품공전에등재하여활용하고있다. 더불어국외에서는국제식품규격위원회와유럽연합의규제강화추이에따라불검출기준물질에대한 MRL 설정등낮은농도의정량한계를가지는검증된분석법개발등이활발히진행되고있다. 따라서본연구는가금류중잔류허용기준은아직설정되어있지않으나, 식품안전성조사및연구등을위해서는시험법의확립이필요 한 nitroxoline의닭고기근육중잔류시험법을개발하고자하였다. 분석에사용된검체는닭고기의근육을이용하였다. 검체에아세트로니트릴을가하여추출한후, 이를감압농축하여메탄올로녹인다음 MCX 카트리지로정제한후 HPLC-PDA에주입하였다. 기기분석은 C18 컬럼을사용하였고, 0.1 % TBAOH- 인산용액과메탄올을기울기조건으로하여 360 nm에서측정하였다. 또한, 액체크로마토그래프-질량분석기를통해확인시험을수행하였으며, 모든검증은 CAC 가이드라인 (CAC/GL 16, 1993; CAC/GL 71, 2009) 규정에따라실시하였다. 그결과, nitroxoline의 LOQ는 0.02 mg/kg 수준이었고, 회수율은 % 로나타났다. 또한, 변동계수는 % 로 CAC 가이드라인 (CAC/GL 16, 1993; CAC/GL 71, 2009) 규정에만족하는수준이었다. 따라서개발된분석법은잔류동물용의약품의분석에있어보다신속하고경제적인분석및모니터링에적용가능할것으로기대된다. 감사의글 This study was supported by a grant (12161KFDA023) from Korea Food and Drug Administration in 참고문헌 Codex Alimentarius Commission, Codex Guidelines for the Establishment of a Regulatory Programme for Control of Veterinary Drug Residues in Foods, Codex Committee on Residues of Veterinary Drugs in Foods; CAC/GL 16, CODEX, Italy. Codex Alimentarius Commission, Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals; CAC/GL 71, Codex, Italy.

8 Development and Validation of Analytical Method for Nitroxoline in Chicken Using HPLC-PDA 77 Ghoneim, M.M., El-Desoky, H.S., Electrochemistry of the antibacterial and antifungal drug nitroxoline and its determination in bulk form, pharmaceutical formulation and human blood, Bioelectroch. 80, Kang, X., Wang, Z., Quantitative high-performance liquid chromatographic analysis of nitroxoline and structurally related compounds, Chromatographia 57, Karpińska, G., Mazurek, A.P., Dobrowolski, J.C., On tautomerism and substituent effect in 8-hydroxyquinoline-derived medicine molecules, J. Mol. Struc. Theochem. 961, Lee, Y.D., Practical book of Korea Food Code pesticide residue analysis method, pp. 79, Korea Food & Drug Administration, Korea. Pelletier, C., Prognon, P., Bourlioux, P., Roles of divalent cations and ph in mechanism of action of nitroxoline against Escherichia coli strains, Antimicrob, Agents Chemother. 39, Shin, H.C., Kim, T.S., Development of management system and standardization for veterinary drug residue, pp. 7-8, Korea Food & Drug Administration, Korea. Štefane, B., Požgan, F., Sosič, I, Gobec, S., A microwave-assisted nucelophilic substitution reaction on a quinoline system: the syntehsis of amino analogues of nitroxoline, Tetrahedron Lett. 53, Szabó, G., Guczi, J., Examination of silica-salicylic acid and silica-8-hydroxyquinoline HPLC stationary phases for estimation of the adsorption coefficient of soil for some aromatic hydrocarbons, Chemosphere 30, Wang, H., Wang, S.W., Zhang, H.S., Spectrofluorimetic determination of cysteine based on the fluorescence inhibition of Cd(Ⅱ)-8-hydroxyquinoline-5-sulphonic acid complex by cysteine, Talanta. 53,

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