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1 6 10 Vol. 6 No Journal of Food Safety and Quality Oct., β- 1, 2, 刘名扬 3*, 郭祯 1, 赵景红 2, 朱程云 1, 刘月 (1., ; 2., ; 3.,, ) 1 摘要 : 目的 / (C 18 /WCX) 10 β- ( ) 方法 C 18 /WCX, : :, 1 ml/min; 30 ; 254 nm, Unitary C 18 结果,, 10 β- C 18 /WCX ph, C 18 /WCX 2 结论, β- 关键词 : ; / ; β- Simultaneous determination of 10 kinds of β-lactam antibiotics by reverse-phase exchange mixed-mode chromatography LIU Ming-Yang 1, 2, 3*, GUO Zhen 1, ZHAO Jing-Hong 2, ZHU Cheng-Yun 1, LIU Yue 1 (1. College of Environment and Chemical Engineering, Dalian Jiaotong University, Dalian , China; 2. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian , China; 3. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian , China) ABSTRACT: Objective To establish a method for simultaneous determination of 10 kinds of β-lactam antibiotics (cefadroxil, ceftezole sodium, cefuroxime sodium, cefazolin, cephradine, cefazedone, cemandil sodium, cephalothin, pipracil and cloxacillin sodium) by reversed-phase liquid chromatography/weak cation exchange (RPLC/WCX, C 18 /WCX). Methods Chromatographic separation was achieved on a C 18 /WCX, using a mixture of acetonitrile, ammonium formate and water as mobile phase with a flow rate at 1 ml/min and 30, and the detector was set at 254 nm. Results Compared with the conventional reversed-phase chromatography Unitary C 18, the selectivity of C 18 /WCX could be improved. It was found that the mechanism of samples separated on C 18 /WCX were hydrophobic interaction and ion exchange interaction by regulated by the salt concentration and ph value. Conclusion It showed that the mixed-mode chromatography would be a potential approach for the analysis and separation of β-lactam antibiotics. 基金项目 : ( ) Fund: Supported by the National Natural Science Foundation of China ( ) * 通讯作者 :,,, liumy@mail.tsinghua.edu.cn *Corresponding author: LIU Ming-Yang, Researcher, Professor, College of Environment and Chemical Engineering, Dalian Jiaotong University, Dalian , China. liumy@mail.tsinghua.edu.cn

2 KEY WORDS: mixed-mode chromatography; reversed-phase liquid chromatography/ weak cation exchange; β-lactam antibiotics 1 引言 2 β-, 1, [1-4] [5-8],, [9,10] 3-, 2, β-,,, C 18 /WCX, 10 β- (8 2 ), 3 C 18 /WCX, Unitary C 18 1 Fig. 1 Structure of penicillins and cephalosporins / (C 18 /WCX), 2 C 18 /WCX Fig. 2 C 18 /WCX mixed-mode stationary phase

3 10, : 10 β Fig β- The structure of ten kinds of beta lactam antibiotics 2 材料与方法 2.1 材料与试剂 (NH 4 FA, 99%) (98%) (98%) (ACN, 99.9%, HPLC/PREP ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ) (CAS ),, 90% 2.2 仪器与设备 Waters Alliance 2695, Waters 2489 ( Waters ); Milli-Q ( Millipore ); DHG-9140A ( ); AB135S ( Mettler Toledo ) 2.3 实验方法 β- 内酰胺类抗生素混合溶液的配制 3.3 mg 3.5 mg 3.4 mg 3.5 mg 2.1 mg 3.5 mg 3.0 mg 5.6 mg 9.8 mg 8.1 mg, 1 ml ( 100 mmol/l, ph 6.5, 4:1, ), μm, 4 60 μl 40 μl 40 μl 40 μl 110 μl 35 μl 85 μl 50 μl 40 μl 200 μl, 0.6 ml,

4 色谱分离条件选择 C 18/ WCX(150 mm 4.6 mm, 5 μm) Unitary C 18 (150 mm 4.6 mm, 5 μm); 100 mmol/l ph 6.5 ; 1 ml/min; 30 ; 254 nm C 18 /WCX, 5 mmol/l, 0~15 min, 5%~15% ; 15~20 min, 15%~60% ; Unitary C 18, 5 mmol/l, 0~5 min, 5%~15% ; 5~10 min, 15% ; 10~20 min, 15%~40% 3 结果与讨论 C 18 /WCX,, β-, 10 β- C 18 /WCX,, 3.1 中性条件下 β- 内酰胺类抗生素在 C 18 /WCX 和 Unitary C 18 上分离效果 : (5 mmol/l, ph 6.5), C 18 /WCX, 1, 10 β-,, 4(b) Unitary C 18, 1, ;, 2,, 4(a), 10 β- C 18 /WCX Unitary C 18 2, Unitary C 18, C 18 /WCX,, Unitary C 18, C 18 /WCX Unitary C 18, 2, C 18 /WCX, 1,,, 2 β- : (i), (ii) ph, (Unitary C 18 ) 3.2 离子强度对保留时间的影响 ph 6.5, 1 2, mmol/l, C 18 /WCX( 5a) Unitary C 18 ( 5b) 2 5a, 10 β- C 18 /WCX, C 18 /WCX,,,,,, Unitary C 18, Unitary C 18,,, ( 5b) 表 1 以 C 18 /WCX 为固定相时流动相的梯度条件 Table 1 Gadient conditions of mobile phase with C 18 /WCX as stationary phase /min 100 mmol/l (ph 6.5) 表 2 以 Unitary C 18 为固定相时流动相的梯度条件 Table 2 Gradient conditions of mobile phase with Unitary C 18 as stationary phase /min 100 mmol/l (ph 6.5)

5 10, : 10 β Unitary C 18 (a) C 18 /WCX(b) Fig. 4 Separation of samples in the stationary phase of Unitary C 18 (a)and C 18 /WCX(b) after gradient optimization 1: ; 2: ; 3: ; 4: ; 5: ; 6: ; 7: ; 8: ; 9: ; 10: 1: cefadroxil; 2: ceftezole sodium; 3: cefuroxime sodium; 4: cefazolin; 5: cephradine; 6: cefazedone; 7: cemandil sodium 8: cephalothin; 9: pipracil; 10: cloxacillin sodium 5 C 18 /WCX (a) Unitary C 18 (b) Fig. 5 Effect of the ammonium formate concentration on the retention time of samples in stationary phase of C 18 WCX (a) and Unitary C 18 (b) ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ) cefadroxil ( ); ceftezole sodium ( ); cefuroxime sodium ( ); cefazolin ( ); cephradine ( ); cefazedone ( ); cemandil sodium ( ); cephalothin ( ); pipracil ( ); cloxacillin sodium ( )

6 C 18 /WCX (a) Unitary C 18 (b) ph Fig. 6 Effect of the ammonium formate ph on the retention time of samples in stationary phase of C 18 /WCX (a) and Unitary C 18 (b) ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ); ( ) cefadroxil ( ); ceftezole sodium ( ); cefuroxime sodium ( ); cefazolin ( ); cephradine ( ); cefazedone ( ); cemandil sodium ( ); cephalothin ( ); pipracil ( ); cloxacillin sodium ( ) 3.3 ph 值对保留时间的影响 C 18 /WCX, 1 5 mmol/l ph , 10 β- Unitary C 18, 2 C 18 /WCX ( 6a), ph, 10 β- Unitary C 18 ( 6b), ph,,, ph3.5~6.5,, C 18 /WCX, ph2.7~3.5, ph,, ph,,,, C 18 /WCX,,, Unitary C 18, ph,,, C 18 /WCX Unitary C 18, ph, C 18 /WCX β-, Unitary C 18 β-, ph, Unitary C 18,, ph,, β-, C 18 /WCX β- 4 结论, 10 β- C 18 /WCX ph, C 18 /WCX 2,, ; Unitary C 18 C 18 /WCX β- 参考文献 [1] Marina VP, Bienvenida GL, Juan FGR, et al. Multiclass detection and quantitation of antibiotics and veterinary drugs in shrimps by

10, : 10 β- 4141 fast liquid chromatography time-of-flight mass spectrometry [J]. Talanta, 2011, 85: 1419 1427. [2] Camara M, Gallego-Pico A, Garcinuno RM, et al.

7 10, : 10 β fast liquid chromatography time-of-flight mass spectrometry [J]. Talanta, 2011, 85: [2] Camara M, Gallego-Pico A, Garcinuno RM, et al. An HPLC-DAD method for the simultaneous determination of nine β-lactam antibiotics in ewe milk [J]. Food Chem, 2013, 141: [3] Juhel MG, Delepine B, Gautier S, et al. Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach [J]. Food Addit Contam, 2009, 26(11): [4] Lina K, Marinella F, Martin S, et al. Fully automatedanalysis of Lactams in bovine milk by online solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry [J]. Anal Chem, 2009, 81(11): [5] Carolina QM, Berengere C, Ana MGC, et al. Convenient solid phase extraction of cephalosporins in milk using a molecularly imprinted polymer [J]. Food Chem, 2012, 135: [6] Didier O, Emmanuelle C, Philippe J, et al. Comprehensive fast multiresidue screening of 150 veterinary drugs in milk by ultra-performance liquid chromatography coupled to time of flight mass spectrometry [J]. J Chromatogr B, 2009, 877: [7] Junza A, Amatya R, Barron D, et al. Comparative study of the LC MS/MS and UPLC MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC [J]. J Chromatogr B, 2011, 879: [8] Karageorgou EG, Samanidou VF. Application of ultrasound-assisted matrix solid-phase dispersion extraction to the HPLC confirmatory determination of cephalosporin residues in milk [J]. J Sepa Sci, 2010, (33): [9] Wang CR, Guo ZM, Long Z, et al. Overloading study of basic compounds with a positively charged C 18 column in liquid chromatography [J]. J Chromatogr A, 2013, 1281: [10] Snyder LR, Dolan JW, Carr PW, et al. The hydrophobic-subtraction model of reversed-phase column selectivity [J]. J Chromatogr A, 2004, 1060: [11] Cai X, Guo Z, Xue X, et al. Two-dimensional liquid chromatography separation of peptides using reversed-phase/weak cation-exchange mixed-mode column in first dimension [J]. J Chromatogr A, 2012, 1228: [12],,,. [J]., 2013, 31(4): Dong XF, Cai XM, Shen AJ, et al. Development and application of separation materials for mixed-mode chromatography [J]. Chin J Chromatogr, 2013, 31(4): 作者简介 ( 责任编辑 : 李振飞 ) 刘名扬, 博士, 研究员, 教授, 主要研究方向为光谱色谱仪器分析 liumy@mail.tsinghua.edu.cn

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