6 12 Vol. 6 No. 12 2015 12 Journal of Food Safety and Quality Dec., 2015 王凤美 *, 何京橙, 张鸿伟, 王妍婷, 张晓梅 (, 266002) 摘要 : 目的 - 方法,, - (ultra performance liquid chromatography tandem mass spectrometry), 结果, 0.99, 0.5 μg/kg 结论 关键词 : ; ; ; ; - Determination of clenbuterol, ractopamine and salbutamol in animal plasma by ultra high performance liquid chromatography-tandem mass spectrometry WANG Feng-Mei *, HE Jing-Cheng, ZHANG Hong-Wei, WANG Yan-Ting, ZHANG Xiao-Mei (Technical Center for Inspection and Quarantine of Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266002, China) ABSTRACT: Objective A method based on ultra performance liquid chromatography-tandem mass spectrometry(uplc-ms/ms) has been developed for the determination of beta-agonists residues including clenbuterol, ractopamine, and salbutamol in animal plasma. Method The analytes were extracted with NH 4 AC solution from samples and hydrolysised with beta-glucuronidase overnight at 37. The extract was filtered and the filtrate was cleaned by Oasis MCX solid phase extraction cartridge. Quantification of the 3 analytes was achieved by UPLC-MS/MS with multiple reaction monitoring (MRM) using internal standard method. Results The method had a good recovery for the clenbuterol, ractopamine, and salbutamol, and the correlation coefficient was more than 0.99 with the limit of quantitation (LOQ) 0.5 μg/kg. Conclusion The method is suitable for the determination of clenbuterol, ractopamine, and salbutamol residues in animal plasma. KEY WORDS: plasma; clenbuterol; ractopamine; salbutamol; ultra performance liquid chromatography tandem mass spectrometry 基金项目 : (2013B324) Fund: Supported by the Inspection and Quarantine Standards (2013B324) * 通讯作者 :,,, E-mail: fengmeiw@126.com *Corresponding author: WANG Feng-Mei, Senior Engineer, Technical Center of Shandong Entry-Exit Inspection & Quarantine Bureau, No.70, Qutangxia Road, Shinan District, Qingdao 266002, China. E-mail: fengmeiw@126.com
5024 6 1 引言 β-,,,,, β-,,, β- -,, [1-3],,, [4-5] 3 β- [6-9], 3, -, 2 材料与方法 2.1 仪器与试剂 Acquility Quattro Premier XE - ( Waters ); Himac CR22GII ( HITACHI ); MS1Minishaker ( IKA ); FA1104 ( ); Milli-Q(18.2 MΩ cm) ( Millipore ) (, Fluka ); (, Merck ); (, Mallinckrodt Bake ) : 77.0 g, 500 ml,, ph 5.2 (clenbuterol)( Dr., 98.5%); (ractopamine)( Dr., 98.0%); (salbutamol)( Dr., 98.5%) (clenbuterol-d9) (ractopamine-d3)( CDN, 99.0%); (salbutamol -D3)( Dr., 99.0%) β- / ( Sigma ) 2.2 对照品溶液的配制 ( 10.0 mg),, 10 ml, -18, 2.3 样品溶液的制备 2.3.1 样品的制备与保存 10 ml,, -20 2.3.2 样品提取 1.0 g ( 0.01 g), 50 ml,, 15 ml, β- / 20 µl,, 37 4 10000 r/min 5 min,,, 2 ml,, 2.3.3 样品净化 MCX, 3 ml 3 ml 3 ml, 1.0 ml/min MCX, 3 ml 3 ml 3 ml 50% 3 ml, ; MCX 2 min, 3 ml - - (50:45:5,V:V:V), 45, 1 ml 0.1%, 0.22 µm, - 2.4 色谱 - 质谱条件 2.4.1 色谱条件 : : Waters ACQUITY UPLC BEH C 18 (2.1 mm 100 mm, 1.7 μm, Waters ); : 300 μl /min; : 10 μl; : 35 ; A ( 10 mmol/l 0.1% ), B ;
12, : - 5025 1 1 表 1 β- 受体激动剂测定的梯度洗脱程序 Table 1 Elution condition for the determination of beta-agonists Min 10 mmol/l ( 0.1% )/% % 0.0 95 5 6 0.5 95 5 6 5.0 40 60 6 6.0 10 90 6 6.1 95 5 6 8.0 95 5 6 2.4.2 质谱条件 : : (electrospray ionization, EI) ; : (multiple reaction monitoring, MRM), ; : ; (V): 3300 ; ( ): 350; ( ): 120; ( ): 380; (L/h): 800; (L/h): 50; 3 UPLC-MS/ MS 2 3 方法学考察与讨论 3.1 样品的选择, [10-14],, ;,, 1 (MRM) Fig. 1 MRM chromatography of salbutamol ractopamine and clenbuterol ( : 1.57 min, : 2.54 min, : 2.86 min)
5026 6 Lether [10], -, Lei [11],,, 3.2 前处理过程优化 β-,,,, ; [15],, N- [16],, 95%, 40% 2.0 mol/l (ph 5.2), β- /, 37 3.3 方法学评价 3.3.1 线性关系及测定低限 0.1% 2.2,, : 0.5 1.0 5.0 10.0 50.0 μg/kg, -D9 -D3 -D5 1.0 μg/kg, 3 3 3 β- (r) 0.99, 3 β- 0.5~50 μg/kg 10, 0.5 μg/kg 3.3.2 回收率 3, LOQ 2LOQ 10LOQ 3, 6, (RSD), 3 β- 95.57%~101.73%, RSD 3.26%~7.96%( 4) 表 2 3 种化合物的质谱分析参数 Table 2 Analysis parameters of the 3 compound (m/z) (m/z) (s) (V) (V) 277.0 240.0 203* 0.05 25 15 259.1 0.05 25 10 147.8* 0.05 22 18 221.8 0.05 22 10 163.9* 0.05 25 15 302.0 106.9 0.05 25 28 -D9 286.2 204.0 0.08 30 15 -D3 243.3 151.1 0.08 30 18 -D3 305.2 166.9 0.08 30 15 表 3 3 种 β- 受体激动剂线性范围 相关系数和测定低限 Table 3 Linear ranges, correlation coefficients of 3 β-agonists (μg/kg) (r) 0.5~50 Y=0.1438X-0.0006 0.9974 0.5~50 Y=0.3666X+0.0491 0.9989 0.5~50 Y=0.3528X-0.0031 0.9978
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