6 4 Vol. 6 No. 4 2015 4 Journal of Food Safety and Quality Apr., 2015 10 张燕 *, 舒平, 阚海勋, 赵浩军, 杨卫花, 王坤, 张蕾 (, 671000) 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 671000, 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 >0.999. 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 :,, E-mail: shiyan1230@163.com *Corresponding author: ZHANG Yan, Engineer, Dali State Comprehensive Technical Inspection Center, Economic Development Zone 1 box, Dali 67100, China. E-mail: shiyan1230@163.com
1484 6 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], 2011 10, 2013, GB/T 21315 10 2 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 2.2.1 色谱条件 : DiKMA Endeavorsil TM C 18 1.8 μm 100 2.1 mm; : 0.2 ml/min; : 40 ; : 5 μl; : 0.1% 5 mmol/l +, 1. 2.2.2 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 (%) 0.00 0.2 95 5 3.00 0.2 95 5 6.00 0.2 70 30 12.0 0.2 45 55 13.0 0.2 95 5 20.0 0.2 95 5
4, : 10 1485 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* 50.0 10.0 17.0 5.8 366.0/160.0 45.0 10.0 20.0 12.0 366.0/349.0 50.0 10.0 17.0 13.0 350.1/105.9 * 60.0 10.0 21.0 10.0 350.1/159.9 60.0 10.0 19.3 17.0 350.1/192.1 60.0 10.0 18.7 10.0 462.0/218.0* 60.0 13.0 26.0 10.0 462.0/246.0 60.0 13.0 19.0 10.0 381.0/165.0* 60.0 10.0 25.0 13.0 381.0/221.9 60.0 10.0 21.0 13.0 335.0/160.2* 62.0 3.0 16.7 9.0 335.0/175.0 62.0 3.0 18.8 10.0 351.0/159.9* 62.0 3.0 16.0 12.0 351.0/114.0 62.0 3.0 21.0 10.0 402.0/160* 70.0 10.0 16.0 10.0 402.0/243 60.0 10.0 20.0 10.0 436.0/277.0* 60.0 10.0 21.0 10.0 436.0/160.0 60.0 10.0 22.0 10.0 415.0/199.0* 67.0 10.0 19.0 10.0 415.0/171.0 63.0 10.0 24.0 10.0 492.0/182.2 80.0 10.0 24.0 15.0 492.0/333.0 70.0 10.0 21.9 16.0 492.0/447.9 80.0 10.0 19.5 17.0 470.0/160.0* 71.0 10.0 24.0 15.0 470.0/310.0 72.0 10.0 20.0 15.0 * 2.3 样品前处理 2.3.1 液体乳 : 5.0 g 50 ml, 10 ml, 1 min, 10 min, 8000 r/min 5 min, 2 ml, 2 ml 2,, 0.22 2.3.2 乳粉 : 1.0 g 50 ml, 4 ml, 10 ml, 2.3.1 2.4 混合标准溶液的制备 0.0010 g( 0.01 mg)10 10 ml,
1486 6 (30:70=v:v) 100 µg/ml 1.0 µg/ml : 0.5 1.0 2.5 5.0 10.0 20.0 μg/l ; 0.1 0.25 0.5 1.0 2.5 5.0 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 21315 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,,, 10 3.3 质谱条件的选择 Ekspert ultralc 100-XL+AB SCIEX QTRAP4500,, 5 μl/min 10 0.5 mg/l, 10 (Q1 ), m/z 366.0 350.1 462.0 381.0 335.0 351.0 402.0 436.0 415.0 492.0(470.0), (MS2),,,, 10 3.4 方法的线性范围及相关性,, 0.1 0.25 0.5 1.0 2.5 5.0 μg/l, 6 0.5 1.0 2.5 5.0 10.0 20.0 μg/l,, 3 3,, 0.1 5.0 μg/l ; 6 0.5 20.0 μg/l (MRM)TIC 1, 2 0.1 μg/l, 1.0 μg/l 3.5 准确度和精密度实验 5 g 1 g 6, 0.25 0.5 2.5 5.0 10.0 μg/l, 4 MRM ( 3, 4) 4 0.25~10.0 μg/l,
4, : 10 1487 70.2%~108.2%, RSD 15%(n=6) 3.5 检出限, 10 0.1 0.5 μg/l, S/N 3 0.5 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 0.5~20.0 2.76 Y=1662.7X 149.49 0.9991 0.5~20.0 7.15 Y=6541.6X+376.18 0.9992 0.1~5.0 8.56 Y=37699X+677.44 0.9992 0.1~5.0 8.91 Y=126886X 776.2 0.9996 0.5~20.0 9.30 Y=10493X 1786.7 0.9993 0.5~20.0 9.65 Y=7965.8X+1464.2 0.9997 0.5~20.0 9.96 Y=6014.5X+1700.5 0.9993 0.1~5.0 10.1 Y=7416.3X+627.02 0.9993 0.1~5.0 10.62 Y=34544X+1811.8 0.9994 0.5~20.0 11.08 Y=4707.8X+142.78 0.9997 1 (MRM) TIC Fig. 1 TIC of MRM chromatogram of standard solution
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
4, : 10 1489 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) /% /% 0.5 70.3-102.1 12.1 2.5 79.3-102.6 9.8 2.5 72.5-99.8 9.8 5.0 74.2-106.7 12.3 10.0 73.1-102.9 11.3 10.0 71.6-98.3 11.9 0.5 74.2-93.5 14.3 2.5 82.3-95.7 9.5 2.5 70.6-104.3 12.5 5.0 79.3-102.6 10.8 10.0 71.3-97.6 8.9 10.0 82.6-105.6 11.6 0.25 76.2-102.6 12.3 0.5 76.3-96.3 12.3 0.5 71.9-97.5 9.8 1.0 81.2-106.8 6.3 2.5 73.6-102.6 12.9 2.5 87.9-105.3 9.4 0.25 80.3-101.1 6.8 0.5 75.2-95.3 8.9 0.5 85.4-103.4 9.7 1.0 89.2-106.7 10.7 2.5 86.9-106.7 7.6 2.5 82.3-102.9 5.9 0.5 74.5-98.4 11.3 2.5 76.4-95.8 10.1 2.5 77.9-101.6 9.8 5.0 82.6-100.9 12.0 10.0 85.9-105.2 12.6 10.0 83.9-107.4 5.7 0.5 79.6-103.6 8.6 2.5 79.2-94.8 6.9 2.5 82.1-102.9 7.6 5.0 70.2-102.5 11.3 10.0 86.2-104.8 12.6 10.0 76.1-105.3 13.8
1490 6 续表 4 /(μg/kg) /% /% /(μg/kg) /% /% 0.5 79.3-98.5 11.6 2.5 82.3-103.9 5.7 2.5 82.6-99.5 8.7 5.0 73.8-102.5 10.3 10.0 87.9-103.9 10.6 10.0 83.1-97.2 13.4 0.25 79.2-99.6 8.3 0.5 72.5-95.3 9.6 0.5 82.3-105.3 7.9 1.0 76.5-104.3 11.3 2.5 89.7-99.3 6.5 2.5 81.2-106.5 8.5 0.25 86.2-108.2 12.3 0.5 82.3-104.7 6.9 0.5 81.9-103.6 10.5 1.0 85.9-102.6 7.8 2.5 85.9-106.7 12.9 2.5 81.6-103.7 10.3 0.5 73.4-97.6 9.5 2.5 72.3-95.3 9.2 2.5 72.3-95.2 10.9 5.0 75.3-103.8 10.6 10.0 80.9-104.3 7.2 10.0 89.5-106.9 12.3 3.6 实际样品测定, 35 5 40, 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): 223 226. [2]. [M]. 2009: 109 117. Peng GF. Selected standard amount detection method commonly used in veterinary drug residue [M]. 2009: 109 117. [3] NY/T 829-2004 -HPLC[S]. NY/T 829-2004 Determination of ampicillin residue in milk: HPLC [S]. [4],,. [M]. 2007: 314 351. Wu YN, Shao B, Shen JZ. Detecting and monitoring techniques of veterinary drug residues[m]. 2007: 314 351. [5],,,. - 9 [J]., 2011, 18(32): 249 252. 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): 249 252. [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): 335 342. [7],,. [J]., 2009, 37(2): 335 342. Wei D, Wang CL, Liang SZ. Study on detection method of amoxicillin residue in milk [J]. Jiangsu Agri Sci, 2009, 37(2): 335 342. [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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