Microsoft Word 张燕-二校 1483

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1 6 4 Vol. 6 No Journal of Food Safety and Quality Apr., 张燕 *, 舒平, 阚海勋, 赵浩军, 杨卫花, 王坤, 张蕾 (, ) 摘要 : 目的 10 - 方法,, 0.22 μm, - ( 0.1% 5 mmol/l ), C 18, 20 min 10 ; (ESI + ), (MRM), 结果 0.1~20 μg/l, 10, 0.999; 0.2 μg/kg~1.0 μg/kg, 1.0 μg/kg~5.0 μg/kg; 70.2%~108.2%, 15% 结论 10, 关键词 : ; ; ; - Determination of 10 residual penicillin in dairy products by ultra high performance liquid chromatography-tandem mass spectrometry ZHANG Yan *, SHU Ping, KAN Hai-Xun, ZHAO Hao-Jun, YANG Wei-Hua, WANG Kun, ZHAGN Lei (Dali State Comprehensive Technical Inspection Center, Dali , China) ABSTRACT: Objective To establish a method for simultaneous determination of 10 residual penicillin in dairy products by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The sample was extracted with acetonitrile and degreased with saturated acetonitrile n-hexane and filtered through 0.22 μm millipore membrane. The filtrate was detected by ultra high performance liquid chromatography-tandem mass spectrometry, identified by electrospray ionization (ESI) in positive mode using multiple reactions monitoring mode, and quantified with external standard method. The sample extract was separated on a C 18 column by gradient elution in 20 min with acetronitrile-water (containing 0.1% formic acid and 5 mmol/l ammonium acetate) as mobile phase. Results The calibration curve of the 10 penicillins residues showed a good linearity in the range of 0.1 μg/l~20.0 μg/l in various dairy products in the matrix with the correlation coefficient r 2 > The detection limit of the method was 0.2 μg/kg~1.0 μg/kg for liquid milk and 1.0 μg/kg~5.0 μg/kg for milk powder. The recoveries of spiking levels ranged from 70.2% to 108.2%, and the relative standard deviation (RSD) was less than 15%. Conclusion The method for determination of residues of 10 penicillin antibiotics in dairy products is simple, rapid and accurate, and could satisfy the * 通讯作者 :,, [email protected] *Corresponding author: ZHANG Yan, Engineer, Dali State Comprehensive Technical Inspection Center, Economic Development Zone 1 box, Dali 67100, China. [email protected]

2 requirements for monitoring of penicillin residues KEY WORDS: penicillin; dairy product; antibiotic residues; ultra performance liquid chromatography-tandem mass spectrometry 1 引言, [1],,, [2],,,, [3,4],, 10~50 μg/kg, 4~30 μg/kg [5],,, [2] [2] [2,7] [7] [3,9,14] [5-8,10-13,15-20], , 2013, GB/T 材料与方法 2.1 仪器与试剂 Ekspert ultralc 100-XL ( AB ); SCIEX QTRAP4500 ( AB ); (0.01 mg ); TG16-WS ( ); Diret-Q5 ( Milipore); AS20500BDT ( ); ( IKA); 0.22 μm( ); Research plus 10~1000 μl ( Eppendorf) ( Merck); (() ); (Acros Organics); ( ); (); ( ); ( 92.0%) ( 91.0%) ( 98.0%, Dr. Ehrenstorfer )( 93.7%, ) 2.2 仪器条件色谱条件 : DiKMA Endeavorsil TM C μm mm; : 0.2 ml/min; : 40 ; : 5 μl; : 0.1% 5 mmol/l +, 质谱条件 : (ESI + ); : (MRM); (IS): 5500 V; (GS1): 50.0 psi; (CUR): 30 psi; (CAD): Medium; (GS2): 55 psi; (TEM): 500 ; ; (DP); (EP); (CE) (CXP) 2 (min) 表 1 梯度洗脱比例 Table 1 Gradient elution ratio (ml/min) 0.1% 5 mmol/l A (%) B (%)

3 4, : Table 2 表 2 10 种青霉素类抗生素的质谱定性定量离子对及其参考条件 Mass spectrometry qualitative and quantitative ion of 10 penicillin antibiotics and reference conditions (m/z) DP EP CE CXP 366.0/208.0* / / /105.9 * / / /218.0* / /165.0* / /160.2* / /159.9* / /160* / /277.0* / /199.0* / / / / /160.0* / * 2.3 样品前处理液体乳 : 5.0 g 50 ml, 10 ml, 1 min, 10 min, 8000 r/min 5 min, 2 ml, 2 ml 2,, 乳粉 : 1.0 g 50 ml, 4 ml, 10 ml, 混合标准溶液的制备 g( 0.01 mg)10 10 ml,

4 (30:70=v:v) 100 µg/ml 1.0 µg/ml : μg/l ; μg/l 3 结果与讨论 3.1 样品前处理方法的优化,,,, [5,11-20] - GB/T GB/T 22975,, ph, HLB, -,, HLB ProElut PLS,, ph, HLB,,,,,, HLB,, 3.2 色谱条件的选择 0.1%+ +0.1% 5 mmol/l, C 18 1, 0.1% 5 mmol/l,, 95% 3 min, 3 min 70% 6 min, 6~12 min 70% 45%, DiKMA Endeavorsil TM C 18,,, 质谱条件的选择 Ekspert ultralc 100-XL+AB SCIEX QTRAP4500,, 5 μl/min mg/l, 10 (Q1 ), m/z (470.0), (MS2),,,, 方法的线性范围及相关性,, μg/l, μg/l,, 3 3,, μg/l ; μg/l (MRM)TIC 1, μg/l, 1.0 μg/l 3.5 准确度和精密度实验 5 g 1 g 6, μg/l, 4 MRM ( 3, 4) ~10.0 μg/l,

5 4, : %~108.2%, RSD 15%(n=6) 3.5 检出限, μg/l, S/N μg/kg, S/N 3, Table 3 表 3 10 种青霉素线性方程线性相关系数及检出限 Linear regression equations with correlation coefficients and limits of detection of 10 penicillins RT(min) r 0.5~ Y=1662.7X ~ Y=6541.6X ~ Y=37699X ~ Y=126886X ~ Y=10493X ~ Y=7965.8X ~ Y=6014.5X ~ Y=7416.3X ~ Y=34544X ~ Y=4707.8X (MRM) TIC Fig. 1 TIC of MRM chromatogram of standard solution

6 1488 食品安全质量检测学报第6卷查出结果作为检出限(LOD), 结合取样量计算检出甲氧苯青霉素为液体乳 0.2 μg/kg, 乳粉 1.0 μg/kg; 其限得出苯咪青霉素邻氯青霉素乙氧萘青霉素余 6 种为液体乳 1.0 μg/kg, 乳粉为 5.0 μg/kg Fig. 2 Fig. 3 图 2 标准溶液的多反应监测(MRM)图谱 Multiple reaction monitoring (MRM) of standard solution 图 3 乳制品中空白样品多反应监测(MRM) Multiple reaction monitoring (MRM) of the blank sample in dairy products

7 4, : Fig. 4 4 (MRM) Multiple reaction monitoring (MRM) of adding standard material in dairy products Table 4 表 4 10 种青霉素类药物的回收率及精密度 Spike recoveries and RSD for 10 penicillins in dairy products /(μg/kg) /% /% /(μg/kg) /% /%

8 续表 4 /(μg/kg) /% /% /(μg/kg) /% /% 实际样品测定, , 10, 40 4 结论 10,,,,,,,,, ; 参考文献 [1] Dayan AD. Allergy to antimicrobial residues in food: assessment of the risk to man [J]. Vet Microbiol, 1993, 35(3-4): [2]. [M]. 2009: Peng GF. Selected standard amount detection method commonly used in veterinary drug residue [M]. 2009: [3] NY/T HPLC[S]. NY/T Determination of ampicillin residue in milk: HPLC [S]. [4],,. [M]. 2007: Wu YN, Shao B, Shen JZ. Detecting and monitoring techniques of veterinary drug residues[m]. 2007: [5],,,. - 9 [J]., 2011, 18(32): Chen RC, Jia HT, Ai LF, et al. Determination of nine penicillin residues in milk and milk powder by HPLC-MS/MS [J]. Food Sci, 2011, 18(32): [6] Verzegnassi L, Royer D, Mottier P, et al. Analysis of chloramphenicol in honeys of different geographical origin by liquid chromatography coupled to electrospray ionization tandem mass spectrometry [J]. Food Addit Contam, 2003, 20(4): [7],,. [J]., 2009, 37(2): Wei D, Wang CL, Liang SZ. Study on detection method of amoxicillin residue in milk [J]. Jiangsu Agri Sci, 2009, 37(2): [8] Takada W, Kihara AT, Kitamura N, et al. Quantitative determination method for trace amount of penicillin contaminants in commercially available drug product by HPLC coupled with tandem mass spectrometry [J]. Chem Pharm Bull,

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Guo J, Zhang CF, Gao GQ, et al. Research progress on determination of residues of antibiotic in milk by high performance liquid chromatography-tandem mass spectrometry [J].

9 4, : , 53(2): [9],,,. 2 [J]., 2010, 29(4): Zhang Y, Yang YL, Zhang DY. et al. A high performance liquid milk residue 2Penicillin chromatography with pre column derivatization detection [J]. J Instrum anal, 2010, 29(4): [10],,,. - [J]., 2014, 1(41): Guo J, Zhang CF, Gao GQ, et al. Research progress on determination of residues of antibiotic in milk by high performance liquid chromatography-tandem mass spectrometry [J]. China Anim Husb Vet Med, 2014, 1(41): [11] GB/T G V -[S]. GB/T Determination of amoxicillin, ampicillin, piperacillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin, dicloxacillin residues in fugu and eel-lc-ms-ms method [S]. [12] GB/T [S]. GB/T Method for the determination of nine penicillins residues in livestork and poultry muscles-lc -MS-MS method [S]. [13] GB/T G V -[S]. GB/T Method for the determination of penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin, dicloxacillin residues in honey-lc-ms-ms method [S]. [14] [S]. The agriculture ministry announcement No High performance liquid chromatographic method for determination of penicillins residues in milk [S]. [15] GB/T G V -[S]. GB/T Determination of amoxicillin, ampicillin, piperacillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin, dicloxacillin residues in milk and milk powder LC-MS-MS method [S]. [16] GB/T / [S]. GB/T Determination of penicillins residues in foodstuffs of animal origin-lc-ms/ms method [S]. [17],,,. - 6 [J]., 2009, 34(6): Qin F, Zheng WJ, Cheng GL, et al. Determination of residues of 6 type of penicillin in milk by high performance liquid chromatography tandem mass spectrometry [J]. Chin J Antib, 2009, 34(6): [18],,. - 9 [J]., 2012, 41(2): Zhao W, Du G, Li XR. Determination of residues of 9 type of penicillin in milk by high performance liquid chromatography tandem mass spectrometry [J]. J Zhejiang Univ, 2012, 41(2): [19],,,. - 7 [J]., 2014, 24(11): Xiao HZ, Liu HH, Zhen Z, et al. Determination of seven penicilins residues in milk products by high performance liquid chromatography-tandem mass spectrometry [J]. Chin J Health Lab Technol, 2014, 24(11): [20],. - [J]., 2014, 42(10): Liu XL, Shan Y. Determination of penicillins antibioctics in milk by using HPLC MS-MS [J]. China Dairy Ind, 2014, 42(10): 作者简介 ( 责任编辑 : 白洪健 ) 张燕, 工程师, 主要研究方向为食品安全检测 [email protected]

材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ

材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ [ 应用纪要 ] Rapid identification of 5 illegal additives in slimming health food by Xevo TQ-S 郝刚苏州市食品药品检验所, 苏州 15104 关键词超高效液相色谱 - 质谱, 多反应监测, 减肥类保健食品非法添加, 快速定性定量