6 10 Vol. 6 No. 10 2015 10 Journal of Food Safety and Quality Oct., 2015 10 β- 1, 2, 刘名扬 3*, 郭祯 1, 赵景红 2, 朱程云 1, 刘月 (1., 116028; 2., 116001; 3.,, 116023) 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 116028, China; 2. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China; 3. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, 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 : (21177040) Fund: Supported by the National Natural Science Foundation of China (21177040) * 通讯作者 :,,, E-mail: liumy@mail.tsinghua.edu.cn *Corresponding author: LIU Ming-Yang, Researcher, Professor, College of Environment and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China. E-mail: liumy@mail.tsinghua.edu.cn
4136 6 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
10, : 10 β- 4137 Fig. 3 3 10 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 66592-87-8) (CAS 26973-24-0) (CAS 56238-63-2) (CAS 25953-19-9) (CAS 58456-86-3) (CAS 56187-47-4) (CAS 42540-40-9) (CAS 58-71-9) (CAS 66258-76-2) (CAS 7081-44-9),, 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 2.3.1 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, ), 10 0.22 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,
4138 6 2.3.2 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, 5 10 20 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) 0 5 5 90 15 5 15 80 20 5 60 35 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) 0 5 5 90 5 5 15 80 10 5 15 80 20 5 40 55
10, : 10 β- 4139 4 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 ( )
4140 6 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 6.5 5.5 4.5 3.5 2.7, 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
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