Microsoft Word 王凤美-二校 出5023

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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 王凤美 *, 何京橙, 张鸿伟, 王妍婷, 张晓梅 (, ) 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 , 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 :,,, [email protected] *Corresponding author: WANG Feng-Mei, Senior Engineer, Technical Center of Shandong Entry-Exit Inspection & Quarantine Bureau, No.70, Qutangxia Road, Shinan District, Qingdao , China. [email protected]

2 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 样品的制备与保存 10 ml,, 样品提取 1.0 g ( 0.01 g), 50 ml,, 15 ml, β- / 20 µl,, r/min 5 min,,, 2 ml,, 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, 色谱 - 质谱条件 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 ;

3 12, : 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% )/% % 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)

4 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- /, 方法学评价 线性关系及测定低限 0.1% 2.2,, : μg/kg, -D9 -D3 -D5 1.0 μg/kg, β- (r) 0.99, 3 β- 0.5~50 μg/kg 10, 0.5 μg/kg 回收率 3, LOQ 2LOQ 10LOQ 3, 6, (RSD), 3 β %~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) * * * D D D 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 ~50 Y=0.3666X ~50 Y=0.3528X

5 12, : Table 4 表 4 空白血浆中 3 种 β- 受体激动剂的添加回收 (n=18) Recoveries of the 3 β-agonists spiked in plasma samples (n=18) (%) RSD(%) (%) RSD(%) (%) RSD(%) 结论 -,, 3 β- 参考文献 [1],, β2- [J]., 2014, 42(10): Liu J, Xie YF, Ren DD, et al. Determination of residues of 26β2-agonists in pork liver by reversed phase high performance liquid chromatography-tandem mass spectrometry [J]. Chin J Anal Chem, 2014, 42(10): [2],,,. HPLC-q/LTQ MS 10 β- [J]., 2014, 34(20): Zhang Q, Zhang SZ, Qu L, et al. Determination of 10 kinds of β-agonists in meat and meat products by matrix solid-phase dispersion-high performance liquid chromatography tandem quadrupole linear ion trap mass spectrometry [J]. Food Sci, 2014, 34(20): [3],,. β- [J]., 2015, 6(2): Xiong L, Li WH, Gao YQ, et al. A review of inspection technology of β-agonist drug residues in meat [J]. J Food Saf & Qual, 2015, 6(2): [4] Jelka P, Tihomira G, Igor B, et al. Clenbuterol residues in plasma and urine samples of food-producing pigs during and after subchronic exposure to a growth-promoting dose [J]. Food Technol Biotechnol. 2009, 47(1): [5] Guan FY, Uboh CE, Lawrence RS, et al. Quantification of clenbuterol in equine plasma, urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry [J]. Rapid Commun Mass Spectrom, 2002, 16: [6],, β- [J],, 2014, 42(12): Li XM, Gao Y, Su XO, et al. Direct analysis of combined β-agonist in sheep urine using on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry [J]. Chin J Anal Chem. 2014, 42(12): [7],, β- [J]., 2010, 28(8): Nie JR, Zhu ML, Lian J, et al. Determination of fifteen β-agonists in animal urine by high performance liquid chromatography-tandem mass spectrometry [J]. Chin J Chromatogr, 2010, 28(8): [8],,. - 4 β- [J]., 2008, 36(12): Wang FM, Zhang HW, Pang SP, et al. Determination of four beta-agonists residues in products of animal origin and urine by ultra chromatography coupled with tandem mass spectrometry [J]. Chin J Anal Chem, 2008, 36(12): [9],,. [J]., 2009, 4: 2 5. Yang T, Sun ZX, Bao WH. Effects of sulfated glucomannan on the antiviral activity of broiler infected with IBDV [J]. Ningbo Agric Sci Technol, 2009, 4:2 5. [10] Lether AF, Harkins JD, Karpiesiuk W, et al. Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS/MS after oral and intratracheal administration [J]. J Anal Toxicol, 2001, 25: [11] Lei YC, Tai YF, Tai YT, et al. Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assey in Taiwan [J]. J Agnic Food Chem, 2008,

6 : [12] Jlhansson MA, Hellenas KE. Matrix effects in immunobiosensor determination of clenbuterol in urine and serum [J]. Analyst, 2004, 129: [13] Schmeer K, Sauter T, Schmid J. Rapid pharmacokinetic screening of salbutamol in plasma samples by column-switching high-performance liquid chromatography-electrospray mass spectrometry [J]. J Chromatogr A, 1997, 777: [14] Wu JW, Ding CG, Ge QH, et al. Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study [J]. J Chromatogr B, 2011, 879: [15],,. - [J]., 2014, 42(11): Gu X, Liu YM, Yao T, et al. Identification of major metabolites of salbutamol in swine urine and plasma using-high performance liquid chromatography-electrospray-time of flight-mass spectrometry [J]. Chin J Anal Chem, 2014, 42(11): [16],,. β2- [J]., 2014, 42(5): Li Y, Su XO, Zhang W, et al. Study on influences of enzynolysis and organic solvent extraction on determination of 2 kinds ofβ2-agonist residues in sheep plasma and urine [J]. Chin J Anal Chem, 2014, 42(5): 作者简介 ( 责任编辑 : 金延秋 ) 王凤美, 硕士, 高级工程师, 主要研究方向为食品安全检测 [email protected]