4 6 Vol. 4 No. 6 2013 12 Journal of Food Safety and Quality Dec., 2013 - β- 张 立 1, 李娜思 2, 冯峰 1, 王文枝 1, 杨丙成 2, 储晓刚 1* (1.,, 100123; 2., 200237) 摘要 : 目的 β- ( ) ( ) - 方法,, Oasis HLB, Waters UPLC BEH C 18 ( 50 mm 2.1 mm, 1.7 μm ), (ESI - ), (MRM), MRM, 结果 4 1 100 µg/l, r>0. 997, 4 85.5%~95.5%, 0.1~0.2 µg/l 结论,, β- 关键词 : - ; ; β- ; Simultaneous determination of β-lactam antibiotics and β-lactamase inhibitors in milk by ultra pressure liquid chromatography tandem mass spectrometry ZHANG Li 1, LI Na-Si 2, FENG Feng 1, WANG Wen-Zhi 1, YANG Bing-Cheng 2, CHU Xiao-Gang 1* (1. Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China; 2. School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China) ABSTRACT: Objective An ultra pressure liquid chromatography (UPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of two β-lactam antibiotics (cefotaxime and cefoperazone) and two β-lactamase inhibitors (tazobactam and sulbactam) in bovine milk and infant formula. Methods The analysts were extracted from milk sample with acetonitrile, defatted with liquid-liquid extraction by hexane, and further purified by Oasis HLB solid phase extraction cartridges. The extracted analysts were separated by UPLC BEH C 18 column (50 mm 2.1 mm, 1.7 μm), and determined by UPLC-MS/MS under negative ionization mode. Results The method showed a good linearity over the range of 1 100 µg/l, with the linear correlation coefficient r>0.997. The mean recoveries for four analysts in bovine milk ranged from 85.5% to 95.5%. The limit of detection was 0.1 0.2 µg/l. Conclusion This method is rapid, sensitive and repeatable, and it could be performed for the determination of β-lactam antibiotics and β-lactamase inhibitors in milk-marix. KEY WORDS: ultra-high pressure liquid chromatography-tandem mass spectrometry; β-lactamase inhibitors; β-lactam antibiotics; milk-matrix 基金项目 : (2012104002) (2011BAK10B04) Fund: Supported by Public Science and Technology Research Funds Project (2012104002) and the National Key Technology R&D Program (2011BAK10B04) * 通讯作者 :,, E-mail: xgchu@vip.163.com *Corresponding author: CHU Xiao-Gang, Researcher, Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Jia 3 Gaobeidianbei Road, Beijing 100123, China. E-mail: xgchu@vip.163.com
1822 4 1 引言 β-, [1-3],, β-, β- [4-7], β-,, [8], β-,, β-, β- [9], β-, β- β- [10], β-,, β- β- β- [11,12], β- β-, 2 β- 2 β- ( 1),, -, β- β- - 2 材料与方法 2.1 试剂与仪器 (cefoperazone, CFP) (cefotaxime, CFT) (sulbactam, SUL) (tazobactam, TAZ)( 99%), Dr. Ehrenstorfer, 1 Fig. 1 Structures of four chemicals
6, : - β- 1823 J. T. Baker ;, Millipore 20 ( Waters ) API 5500 ( Applied Biosystem ), Waters UPLC,, Analyst 1.5.1 ; Avanti J-26 XPI ( Beckman ); XP 105( Mettler ); SA-31( Yamato ); Milli-Q Advantage A10 ( Millipore ); 0.22 μm ( PALL ) 2.2 实验方法 2.2.1 样品前处理 2.0 g 50 ml, 2 ml 60 ( 2.0 ml), 6 ml, 1 min, 5000 r/min 10 min, 5 ml 50 ml, 8 ml, 1 min, 5000 r/min 5 min,, 5 ml 0.05 mol/l (ph=8.5), Oasis HLB ( 5 ml 5 ml 0.05 mol/l (ph=8.5) ) 3 ml 0.05 mol/l 2 ml, 3 ml, ; 2 ml,, 2.2.2 仪器分析条件 : Waters Acquity UPLC BEH C 18 ( 50 mm 2.1 mm, 1.7 μm); : 40 ; : 4 ; : 5 μl; A 0.1% (v/v), B 0.1% (v/v) : 0~3.0 min, 5%~50% B; 3.1~4.0 min, 95% B; 4.1~5.0 min, 5% B, 0.25 ml/min,, (MRM);, (CAD) 41 kpa, (CUR) 138 kpa, (GS1) 413 kpa, (GS2) 345 kpa (IS) -4.5 kv, (TEM) 400, Q0 (EP) -10 V, (CXP) -11 V, 4 /, (DP) (CE) 1 3 结果与讨论 3.1 样品前处理条件的优化,, [7] β- Table 1 表 1 四种化合物的质谱参数 Mass spectrometer parameters of the four analytes (V) (V) 1 (TAZ) 299.0 138.1* 80 18 207.0 17 2 (SUL) 232.0 140.0* 100 24 64.2 26 3 (CFT) 455.0 239.0* 100 16 124.1 36 4 (CFP) 644.0 115.2* 80 20 *Quantitative daughter ion 188.0 17
1824 4 [6],,,,, Waters C 18 MAX Oasis HLB ( 500 mg, 6 ml), Waters 2 2, C 18 ; MAX SUL ; HLB 4 ( 90% 98%), Oasis HLB Oasis HLB, 10%,,, SPE ( 2.2.1) SPE, ph ph 8.5,, ph 8.5 3.2 质谱条件的优化 ESI + ESI -,, SUL TAZ CFT ESI -, CFP ESI + ESI -,, ESI - 4 3 3.3 线性关系与检出限 0.1 1 5 10 25 100 µg/l,,, 3 10 (S/N), ( 2) 4, (20 µg/l) 4 2 SPE Fig. 2 Comparison of different SPE sorbents Table 2 表 2 四种目标化合物线性关系, 检出限以及定量限 (n=6) Linearity, LOD and LOQ of the four chemicals in milk samples (n=6) (µg/l) LOD(µg/L) LOQ(µg/L) TAZ Y=718X+156 0.9986 1~100 0.1 0.35 SUL Y=620X+213 0.9989 1~100 0.1 0.35 CFT Y=480X+301 0.9987 2~100 0.2 0.6 CFP Y=3040X-2060 0.9993 1~100 0.1 0.35
6, : - β- 1825 3 4 Fig. 3 Extracted ion chromatographs of four standards 3.4 方法回收率与精密度 3 (2 10 100 µg/l), 6, 3, 85.5% 95.5%, (RSD) 9%, 3.5 样品分析 20, β-, 4, β- 4 结论 - β-, β-
1826 4 4 4 ( ) ( ) ( 下, 1) Fig. 4 Total ion chromatographies of 4 standard chemicals(up); spiked sample(middle); blank sample(down) 表 3 四种目标化合物在样品中的回收率和精密度 (n=6) Table 3 Recoveries and precision of the four chemicals in milk samples(n=6) / (µg/l) /% RSD/% TAZ 2 90.6 8.7 10 94.1 7.2 100 95.4 5.6 SUL 2 86.7 7.9 10 88.8 5.6 100 93.2 5.6 CFT 2 92.6 6.5 10 94.3 5.8 100 95.4 4.2 CFP 2 92.5 6.7 10 93.0 6.5 100 94.2 4.3, β- 参考文献 [1],,,. β- [J]., 2009, 72(5): 394 400. Zhang Q, Ye NS, Gu XX, et al. Development and Application of Analytical Methods for Analyses of β Lactam Antibiotics Residues [J]. Chem, 2009, 72(5): 394 400. [2],,. [J]., 2010, 38(9): 42 44. Meng J, Wang R, Zhu JH. New problem in the detection of antibiotics in raw milk [J]. Chin Dairy Ind, 2010, 38(9): 42 44. [3] Holthoon F, Mulder PPJ, Bennekom EO. Quantitative analysis of
6, : - β- 1827 penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry [J]. Anal Bioanal Chem, 2010, 396: 3027 3040. [4] Riediker S, Stadler RH, et al. Simultaneous determination of five beta lactam antibiotics in bovine milk using liquid chromatography coupled with electrospray ionization tandem mass spectrometry [J]. Anal Chem, 2001, 73: 1614 1621. [5] Lara FJ, Garcia-Capana AM, Alés-Barrero F, et al. Multiresidue method for the determination of quinolone antibiotics in bovine raw milk by capillary electrophoresis tandem mass spectrometry [J]. Anal Chem, 2006, 78: 7665 7673. [6],,,. 4 β [J]., 2008, 26(6): 682 686. Zhang Q, Ye NS, Gu XX, et al. Simultaneous determination of four β lactam antibiotics in m ilk by solid phase extraction m icellar electro kinetic chromatography [J]. Chin J Chromatogr, 2008, 26(6): 682 686. [7] Li H, Xia X, Xue YN, et al. Simultaneous determination of amoxicillin and prednisolone in bovine milk using ultra high performance liquid chromatography tandem mass spectrometry [J]. J Chromatogr B, 2012, 900: 59 63. [8],,,. [J]., 2007, 19: 113 116. Cui SH, Li JY, Ma Y, et al. Characterization and detection of antibiotic destroyer in raw milk [J]. Chin J Food Hyg, 2007, 19: 113 116. [9],,. β [J]., 2011, 27: 1020 1024. Li JR, Hu KF, Zhang YP. Development of a detection method for β lactamase as antibiotic destroyer in milk [J]. Mod Food Sci Technol, 2011, 27: 1020 1024. [10],,,. [J]., 2012, 33 (16): 64 66. Liu SS, Shan Y, Man CX, et al. Determination of sulbactam in raw milk by the high performance liquid chromatography photodiode array detection [J]. Sci Technol Food Ind, 2012, 33 (16): 64 66. [11],,,. β [J]., 2013, 32 (7): 47 51. Zheng XY, Lin Q, You FM, et al. Simultaneous determination of β lactamase inhibitors such as clavulanate potassium, tazobactam and salbactam residues in dairy products by UPLC MS/MS [J]. Chin J Anal Lab, 2013, 32 (7): 47 51. [12],,,. [J]., 2012, 31 (6): 700 704. Xue X, Wang J, Liu GL, et al. Determination of sulbactam in milk by hydrophilic interaction chromatography tandem mass spectrometry( HILIC MS /MS) [J]. J Instrum Anal, 2012, 31 (6): 700 704. 作者简介 ( 责任编辑 : 赵静 ) 张立, 学士, 副研究员, 主要研究方向为食品检验检疫技术 E-mail: zhangli@caiq.gov.cn, fengf_2005@163.com 储晓刚, 研究员, 主要研究方向为食品安全 E-mail: xgchu@vip.163.com