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1 6 9 Vol. 6 No Journal of Food Safety and Quality Sep., 王建凤 1, 刘艳 1*, 杨一帆 2, 贾丽 1, 黄华 3, 杜振霞 2, 范筱京 1, 张经华 (1.,, ; 2., ; 3., ) 1 摘要 : 目的 (SPE-UPLC-MS/MS) 6 方法 0.05 mol/l, HLB,, 结果 6(S/N=3) (S/N=10) 0.05~0.4 μg/kg 0.1~1.6 μg/kg 66.3%~110.0% 结论, 关键词 : ; ; ; Determination of 6 kinds of cephalosporins in milk by solid phase extraction coupled with ultra high performance liquid chromatography-tandem mass spectrometry WANG Jian-Feng 1, LIU Yan 1*, YANG Yi-Fan 2, JIA Li 1, HUANG Hua 3, DU Zhen-Xia 2, FAN Xiao-Jing 1, ZHANG Jing-Hua 1 (1. Beijing Engineering Research Center of Food Safety Analysis, Beijing Centre for Physical and Chemical Analysis, Beijing , China; 2. College of Science, Beijing University of Chemical Technology, Beijing , China; 3. Beijing Municipal Center for Food Safety Monitoring and Risk Assessment, Beijing , China) ABSTRACT: Objective To developed a method for the detection of 6 kinds of cephalosporins in milk by solid phase extraction coupled with ultra high performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). Methods The samples were extracted with 0.05 mol/l phosphate buffer solution, cleaned up by HLB solid phase extraction column, and then analyzed by UPLC-MS/MS with multiple reaction monitoring (MRM) mode. Results The LODs and LOQs of 6 kinds of cephalosporins were in the ranged of 0.05~0.4 μg/kg and 0.1~1.6 μg/kg, respectively, with the mean recoveries varied from 66.25% to %. Conclusion The established method is simple, rapid and effective, and suitable for the determination of 6 kinds of cephalosporins in milk samples. KEY WORDS: solid phase extraction; ultra performance liquid chromatography-tandem mass spectrometry; cephalosporin; milk 基金项目 : (Z ) Fund: Supported by Beijing Municipal Natural Science Foundation (Z ) * 通讯作者 :,, xgly36@163.com *Corresponding author: LIU Yan, Associate Researcher, Beijing Center for Physical and Chemical Analysis, No. 27, West Third Ring Road, Haidian District, Beijing , China. xgly36@163.com

2 9, : 引言,, [1,2],,,, [3-5],,, [6,7], -(UPLC-MS/MS) [8-14] Li [9,10] 2 UPLC-MS/MS,,, 82.5%~98.3%, RSD 5.5% [11] 3 HPLC-MS/MS,,, 81%, RSD 12% Hou [12] UPLC-MS/MS,, 94.6%~117.1%, RSD 14%, 6 SPE-UPLC-MS/MS, 2 材料与方法 2.1 试剂与仪器 (, ); (, Optima ); (, Alfa Aesar ); (, ); (, ); (, ) UPLC XEVO-TQ MS ( Waters ); Acquity UPLC BEH C 18 (2.1 mm 50 mm, 1.7 μm, Waters ); CR22G ( Hitachi ); TGL-16G ( ); Vortex-Genie 2 ( Scientific Industries ); KQ-500DE ( ); N-EVAP112 ( Organomation ); OASIS HLB (6 cc, 500 mg, Waters ) 2.2 色谱质谱条件 : BEH C 18 (2.1 mm 50 mm, 1.7 μm); : 0.5 mmol/l (A, ph=5) (B); : 0.2 ml/min; : 25 ; : 5 μl; : 0~2 min, 5%~10%B; 2~5 min, 10%~25%B; 5~7 min, 25%~35%B; 7~9 min, 35%~95%B; 9~11.5 min, 95%~5%B : (MRM), ESI(+) MRM 样品处理溶液配制 0.05 mol/l (PBS): 11.4 g 1 L, 1 mol/l NaOH 6 mol/l HCl ph ; 0.5 mmol/l (A): g 0.5 L, 0.45 μm, ph 5 6, 1000 mg/l, 4 - (5:95, V:V), 1 mg/l 样品提取 (2.0±0.2) g, 6 ml PBS, 20 s, 样品净化 6 ml, 3 ml PBS 3 ml/min 3 ml PBS 3 ml,, 12 ml, 6 ml,, 40 - (5:95, V:V) 1 ml, 0.22 μm

3 Table 1 表 1 6 种头孢菌素类抗生素的 MRM 质谱参数 MS parameters in MRM mode of 6 kinds of cephalosporins (m/z) (m/z) (ev) (V) 114.0* C 16 H 17 N 3 O 5 S cefadroxil * C 16 H 17 N 3 O 4 S cefalexin monohydrate * C 23 H 26 N 6 O 9 S cefquinome sulfate * 8 20 C 16 H 19 N 3 O 4 S cefradine * C 20 H 25 N 5 O 7 S cefetametpivoxyl * C 16 H 15 N 2 NaO 6 S cefalotin sodium *: (Quantitative ion) 3 结果与讨论 3.1 色谱条件和质谱条件质谱条件的优化, - (5:95, V:V) 6 1 mg/l,, 6 ESI+ ;, 2,, 色谱条件的优化 C 18, - - 2, 6, 1, -, 6 -,, -,, ph 3 4 5, 6, 2, ph 3 ; ph 4 ; ph 5 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (efetametpivoxyl) Fig. 1 Influence of different mobile phase on the responsive intensity of 6 kinds of cephalosporin compounds in milk

4 9, : (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxy) 6. 2 ph 6 Fig. 2 Influence of different ph of mobile phase on the responsive intensity of 6 kinds of cephalosporin compounds in milk 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) 6. 3 ph 6 Fig. 3 Influence of different ph of selected solvent on the recoveries of 6 cephalosporins compounds in milk ph 5 ph %, ph 4 ph 5, ph 样品前处理的优化提取液 ph 值的优化,,, ph, 1 mol/l NaOH 6 mol/l HCl PBS ph, ph , ph 6.5 PBS, ph 10 PBS 提取液体积的优化, ml PBS, 4,,,, 6 ml 1. (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) Fig. 4 Influence of the volume of selected solvent on the recoveries of 6 kinds of cephalosporin compounds in milk 淋洗液体积的优化,, HLB,,

5 ml 12 ml, ph (cefadroxil) 2. (cefalexin monohydrate) 3. (cefquinome sulfate) 4. (cefradine) 5. (cefetametpivoxyl) Fig. 5 Influence of the volume of selected solvent on the recoveries of 6 cephalosporin compounds in milk 3.3 方法学考察检出限和定量限,, 3(S/N=3) ; 10(S/N=10) 基质标准曲线及线性范围,,, 1~100 ng/ml, ; 2~200 ng/ml, ; 4~400 ng/ml, 6 (r 2 ) 回收率和精密度 , 2, 66.2%~110.0%, (RSD) 15.3% 表 2 6 种头孢菌素类抗生素在牛奶基质中的检出限定量限添加回收率和相对标准偏差及基质添加曲线线性相关系数的变化范围 (n=3) Table 2 LOD, LOQ, range of recovery, relative standard deviation and linear correlation coefficient of 6 kinds of cephalosporin compounds in milk (n=3) LOD(μg/kg) LOQ(μg/kg) r 2 (μg/kg) (%) % 0.89% cefadroxil % 3.73% % 7.16% % 3.01% cefalexin monohydrate % 6.46% % 2.18% % 8.30% cefquinome sulfate % 15.22% % 4.37% % 3.49% cefradine % 7.06% % 8.35% % 12.1% cefetametpivoxyl % 3.82% % 3.20% % 8.27% cefalotin sodium % 0.22% % 6.85% RSD (%)

6 9, : 实际样品检测 10,, 3, 6 4 结论 SPE-UPLC-MS/MS 6 ph mol/l PBS, ph 10 PBS HLB,, EC 2002/657/EC [15] 100 μg/kg,, 参考文献 [1],,,. - [J]., 2006, 6(6): Cai YE, Cai YQ, Mou SF, et al. High performance liquid chromatography-ultraviolet photometric detection for the simultaneous determination of cephalosporins in human urineand bovine milk [J]. Chin J Anal Chem, 2006, 6(6): [2],,,. - 5 [J]., 2010, 38(5): Li XM, Cao YZ, Zhang JJ, et al. Determination of 5 cephalothins in honey by high performance liquid chromatography coupled with electrospray tandem mass spectrometry [J]. Chin J Anal Chem, 2010, 38(5): [3] Beconi-Barker MG. Determination of ceftiofur and its desfuroylceftiofurrelated metabolites in swine tissues by high performance liquid chromatography [J]. J Chromatogr, 1995, 673(2): [4],. 7-ACA [J]., 2006, 19(5): Wang JF, Qu HT. Quantify and identify the assay of 7-ACA in cefazolin sodium [J]. Heilongjiang Med J, 2006, 19(5): [5],,. [J]., 2006, 30(6): Fu DC, Di R, Li B. Review on the new development of cephalosporin antibiotics [J]. J Hebei Normal Univ, 2006, 30(6): [6],,,. [J]., 2007, 6: Fan YY, Qi L, Yang SM, et al. Determination of cephalosporins residues in pork by high performance liquid chromatographymass spectrometry [J]. Mod Sci Instrum, 2007, 6: [7],,,. 19 β- [J]., 2010, 29(7): Zhu WX, Liu YF, Yuan P, et al. Fast determination of 19 β-lactams and its metabolites in milk by ultra-performance liquid chromatography tandem mass spectrometry and dispersion solid-phase extraction [J]. Chin J Anal Lab, 2010, 29(7): [8]. [J]., 2010, 38(3): Sun YX. Review on determination of antibiotics residues in dairy products by high performance liquid chromatography [J]. J Shanxi Agric Sci, 2010, 38(3): [9] Li NS, Feng F, Yang B, et al. Simultaneous determination of β-lactam antibiotics and β-lactamase inhibitors in bovine milk by ultra performance liquid chromatography-tandem mass spectrometry [J]. J Chromatogr B, 2014, : [10]. - [D]. :, Li NS. Determination of antibiotics residues and dicyandiamide in bovine milk by UPLC-MS/MS [D]. Shanghai: East China University of Science and Technology, [11]. [D]. :, Zhang AB. Studies on determin-ation of cephapirinbenzathine and cephalonium residues in milk and withdrawal time for milk of their intrammary infusion [D]. Yangzhou: Yangzhou University, [12] Hou XL, Wu YL, Lv Y, et al. Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for determination of 10 cephalosporins and desacetylcefapirin in milk [J]. J Chromatogr B, 2013, 931: [13]. [D]. :, Zhang Q. Approach for determination of antibiotics residues in

7 foodstuffs of animal origin [D]. Beijing: Capital Normal University, 作者简介 [14],,,. - / 76 [J]., 2010, 38(3): 王建凤, 硕士, 助理研究员, 主要研究方向为分析化学 wangjf871218@163.com Guo DH, Deng XJ, Zhao SZ, et al. Simultaneous determination of 76 veterinary drug residues in foodstuffs of animal origin by solid phase extraction liquid chromatography tandem mass spectrometry [J]. Chin J Anal Chem, 2010, 38(3): [15] European Commission Decision 2002/657/ EC [S]. 刘艳, 博士, 副研究员, 主要研究方向为食品安全检测技术 xgly36@163.com ( 责任编辑 : 李振飞 )

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI