Microsoft Word 张罡-校对-3778

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1 5 12 Vol. 5 No Journal of Food Safety and Quality Dec., 2014 张罡 1, 王凤美 1*, 汤志旭 1, 王树峰 2, 管恩平 2, 颜显辉 3 (1., ; 2., ; 3., ) 摘要 : 目的 - (high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS) 方法 ( 50 kg),, 35 d, 32 d d , 3, l.0 mol/l, : (3:7, v:v), (mixed-mode cationic exchange, MCX) LC-MS/MS d , 3, HPLC-MS/MS 结果 ( d µg/kg);, d μg/kg 结论,, 关键词 : ; ; - Determination of clenbuterol residues in pig hairs by high performance liquid chromatography coupled with tandem mass spectrometry and investigation of the residue and elimination regularity of clenbuterol ZHANG Gang 1, WANG Feng-Mei 1*, TANG Zhi-Xu 1, WANG Shu-Feng 2, GUAN En-Ping 2, YAN Xian-Hui 3 (1. Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao , China; 2. Huangdao Entry-Exit Inspection and Quarantine Bureau, Huangdao , China; 3. Technical Center for Inspection and Quarantine of Rongcheng Entry-Exit Inspection and Quarantine Bureau, Rongcheng , China) ABSTRACT: Objective To develop a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the residues of clenbuterol in pig hair and investigate the residue and elimination regularity of clenbuterol. Methods Growing white pigs (the average weight of 50 kg) were fed with the diet that contained clenbuterol for 35 d, withdrawal 32 d. Three hair samples were collected at each time point (d 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 32, and d 35 after the first drug administration). The 基金项目 : (2011IK189) (2013B324) Fund: Supported by the Scientific and Technological Project of the General Administration of Quality Supervision, Inspection and Quarantine of the People s Republic of China (2011IK189) and the Inspection and Quarantine Standards (2013B324) * 通讯作者 :,, fengmeiw@126.com *Corresponding author: WANG Feng-Mei, Senior Engineer, Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No.70, Qutangxia Road, Shinan District, Qingdao , China. fengmeiw@126.com

0 mol/l, : (3:7, v:v), (mixed-mode cationic exchange, MCX) LC-MS/MS d 0 3 5 7 9 11 13 15 17 19 21 23 25 26 27 30 32, 3, HPLC-MS/MS 结果 ( d 35 866.

2 12, : hairs were dissolved by sodium hydroxide, extracted by tert-butyl alcohol: ethyl acetate (3:7, v:v), after purified with MCX solid phase extraction cartridge, then the residual amount of clenbuterol in these samples was determined by HPLC-MS/MS. Three pigs were killed at each time point (d 0, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 30, and d 32 after the last drug administration), their hair samples were taken and the residues of clenbuterol were determined by LC-MS/MS. Results The residual amount of clenbuterol in the hair samples presented a rising trend while the drug accumulate in the pig ( µg/kg at 35 d). The residual amount of clenbuterol rose at first, and then reduced slowly after withdrawal. The residual amount of clenbuterol was still to 87 μg/kg after withdrawal 32 days. Conclusion This is a simple operation, with a good repeatability and high recovery rate, which is suitable to detect the residue of clenbuterol in animal s hair. This study can help to offer the reference and basis for supervision of edible animal feeding process. KEY WORDS: β-agonists; pig hairs; high performance liquid chromatography-tandem mass spectrometry 1 引言 (clenbuterol),,,, [1-3] /22/EC [4],, [5,6], β-, β-, β- [7,8], [9,10], - [11,12] - [13,14], β-, ( ),, ;, β-, β-, - (high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS),, β- 2 材料与方法 2.1 动物实验与样品采集 3 ( 50 kg), 1.5 mg/kg, 2, 35 d, 32 d : 2, , 3, 8:00~10:00 3, : 仪器与试剂 Agilent1100 ( Agilent ); API 4000 ( AB ): ; Himac CR 22GⅡ ( Hitachi ); Pierce Model 1878 ; PHS-3C PH ( ); Oasis MCX (3 ml, 60 mg)( Waters ) ( Dr. Ehrenstorfer ); ( C/D/N ISOTopes ); ( Burdick & Jackson ); ( Burdick & Jackson ); ( ), ( J.T.Baker ); Milli-Q 2.3 标准溶液的配制 ( 10 mg) 4 10 ml,,

Three pigs were killed at each time point (d 0, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 30, and d 32 after the last drug administration), their hair samples were taken and the residues of

3 3780 5,, 1 g/l, µl, 10 ml, 10 mg/l, 样品处理毛发的清洗 3,, 5 mm 提取 1 g, 50 ml,, l mol/l 10 ml,, 80 l h,, ph 9.5~10.0, 10 ml : (3:7),, 2 40, 2 10 ml 0.1 mol/l, 净化 0.1 mol/l 5 ml MCX, 1.0 ml/min, 0.1 mol/l - (1:1, v:v) 5 ml, - - (50:45:5, v:v) ( 1.0 ml/min),, 45, 1.0 ml0.1%, 0.22 μm, 2.5 测定色谱条件 CAPCELL PAK C8 (4.6 mm 250 mm, 5 μm); A: 10 mmol/l ( 0.15% ) B:, T: 0~7~13~13.1~18 min, 10%~25%~30%~10%~10% B; :0.5 ml/min; : 35 ; : 10 L 质谱条件 (electrospray ionization, ESI) ; : (multiple reaction monitoring, MRM); : 5500 V; : 550 ; : 11 L/min; : 35 psi; 1 3 结果与讨论 3.1 毛发的预处理, 3, 3, β-, 3.2 提取条件的优化 β-, NaOH KOH [15,16],,, ml 1 mol/l NaOH 1 g, 2 β- [17,18], 0.1 mol/l HCl,, β- [14], SN/T ,,, [19] K β-,,, β- 表 1 克伦特罗主要参考质谱参数 Table 1 Optimized spectrometric parameters of clenbuterol (m/z) (m/z) (ms) (V) (ev) 203.0* D *

15% ) B:, T: 0~7~13~13.1~18 min, 10%~25%~30%~10%~10% B; :0.5 ml/min; : 35 ; : 10 L 2.5.2 质谱条件 (electrospray ionization, ESI) ; : (multiple reaction monitoring, MRM); : 5500 V; : 550 ; : 11 L/min; : 35 psi; 1 3 结果与讨论 3.

4 12, : 表 2 毛发在不同温度下需要的水解时间 Table 2 Hydrolysis time of animal hair under different temperature ( ) (min) 方法检测限与线性范围 1 g, 0.1 µg/kg, 0.5 µg/kg, 0.1~100.0 ng/ml, 0.99,, 100~1000 ng/ml,, 0.99( 3) 3.4 回收率及精密度 3, , 蓄积代谢规律研究毛发中克仑特罗的蓄积规律, ( 2a) 2a,, μg/kg,,, [16], 2 [17] (10 mg/kg), 5 d,,, 2a, 19~29, 19~21, μg/kg μg/kg, μg/kg, 29~35 d, 2 d 80 μg/kg 197 μg/kg μg/kg 毛发中克仑特罗的代谢规律, ( 2b) 2b,,,, 0 d μg/kg, μg/kg,,, 7, 420 μg/kg, μg/kg 13,, μg/kg, μg/kg, 32 d 87 μg/kg Table 3 表 3 克伦特罗的线性方程及相关系数 Regression equation and correlation coefficients of clenbuterol (ng/ml) (r 2 ) 0.1~100 Y=1.62X ~1000 Y=1.24X (µg/kg) 表 4 克伦特罗的添加回收率试验数据 (n=6) Table 4 Recoveries of clenbuterol (n=6) (%) (%) (%) (%)

33 μg/kg,,, [16], 2 [17] (10 mg/kg), 5 d,,, 2a, 19~29, 19~21, 186.83 μg/kg 384.47 μg/kg, 197.64 μg/kg, 29~35 d, 2 d 80 μg/kg 197 μg/kg 35 866.75 μg/kg 3.5.2 毛发中克仑特罗的代谢规律, ( 2b) 2b,,,, 0 d 866.

5 Fig. 2 2 Metabolism regularity of accumulation clenbuterol in pig hair 4 结论 HPLC-MS/MS,,, 35 d μg/kg;,, 32 d 87 μg/kg,,, 参考文献 [1]. 24 [J]., 2009, 47(30): Jiang L. Clinical analysis of 24 cases of clenbuterol poisoning rescue [J]. China Mod Doct, 2009, 47(30): [2]. [J]., 2002, 9(6): 706. Zhou SF. Investigation with the consumption of the pig liver caused by clenbuterol poisoning [J]. Pract Prev Med, 2002, 9(6): 706. [3] Martine Z, Navaari JE. Food poisoning related to consumption of illcit B-agonist in liver [J]. Lancet, 1990, 336(8726): [4] Directive EC. Council directive 96/22EC of 29 April 1996 concerning the prohibition on the use in stock farming of certain substances having a hormonal other hypostatic action and of beta-agonist, and repealing directives 81/602 [J]. EEC, 1996, 88(146): 3 9. [5] 176. [R]., Number 176 of People's Republic of China Ministry of Agriculture Bulletin. Against drug directory varieties used in animal feed and drinking water [R]. People's Republic of China Ministry of Agriculture, [6] 193. [R]., Number 193 of People's Republic of China Ministry of Agriculture Bulletin. List of veterinary drugs and other compounds in food animals disabled [R]. People's Republic of China Ministry of Agriculture, [7],,,. [J]., 2003, 22(3): Dai H, Yuan ZN, Huang ZQ, et al. Determination of clenbuterol hydrochloride and salbutamol in feed by HPLC [J]. J Instrum Anal, 2003, 22(3): [8] Gleixner A, Sauerwein H, Meyer HD. Detection of the anabolic-adrenoceptor agonist clenbuterol in human scalp hair by HPLC/EIA [J]. Clin Chem, 1996, 42: [9],. [J]., 2006, 16(5): Liu WW, Niu WM, Jiang LF, et al. ELISA and GC-MS determination of clenbuterol in pig urine [J]. Chin J Health Lab Technol, 2006, 16(5): [10],. [J]., 2005, 36(4): Wang JP, Shen JZ. Detection of salbutamol and clenbuterol in swine liver and swine urine by enzyme linked immunosorbent assays [J]. Acta Vet Zootech Sin, 2005, 36(4): [11],. - [J]., 2006, 40(11): 5 8. Zhu YL, Shao DJ. Simultaneous determination of clenbuterol

Investigation with the consumption of the pig liver caused by clenbuterol poisoning [J]. Pract Prev Med, 2002, 9(6): 706. [3] Martine Z, Navaari JE.

6 12, : hydrochloride and ractopamine residues in swine liver by GC/MS [J]. Chin J Vet Drug, 2006, 40(11): 5 8. [12],,. - [J]., 2006, 34(8) : Wu YL, Li XW, Liu SY, et al. Determination of residual clenbuterol and ractopamine in animal liver tissues by solid phase extraction with gas chromatography-mass spectrometry [J]. Chin J Anal Chem, 2006, 34(8): [13],,,. HPLC-MS/MS [J]., 2008, 42(8): Nie JR, Hong ZT, Lian J, et al. Rapid determination of ractopamine and clenbuterol in animal urine with HPLC-MS/MS [J]. Chin J Vet Drug, 2008, 42(8): [14],,,. - 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 performance liquid chromatography coupled with tandem mass spectrometry [J]. Chin J Anal Chem, 2008, 36(12): [15] Fene CA, Vaquez BI. Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography-mass spectrometry [J]. J Chromat B, 726(1999): [16] Nielen MWF, Lasaroms JJP. Multiresidue analysis of beta-agonists in bovine and porcine urine,feed and hair using liquid chromatography electrospray ionization tandem mass spectrometry [J]. Anal Bioanal Chem, 2008, 391: [17],. HPLC-MS/MS [J]., 2008, 44(11): Su XO, Shen JZ. Studies on the Determination of clenbutrol in swine hairs using LC-MS/MS and its residue elimination [J]. Anim Product, 2008, 44(11): [18] Hernandez-Carrasquilla M. External contamination of bovine hair with β2-agonist compounds:evaluation of decontamination strategies [J]. J Chromat B, 767(2002): [19] Carrasquilla HM. Analysis of β-agonist compounds in bovine hair. Evaluation of procedures for extraction of hair [J]. J Chromat B, 2000, 51: 作者简介 ( 责任编辑 : 张宏梁 ) 张罡, 助理工程师, 主要研究方向为食品中兽药残留分析检测 zhanggang2062@163.com 王凤美, 高级工程师, 主要研究方向为药物残留分析 fengmeiw@126.com

[13],,,. HPLC-MS/MS [J]., 2008, 42(8): 17 20. Nie JR, Hong ZT, Lian J, et al. Rapid determination of ractopamine and clenbuterol in animal urine with HPLC-MS/MS [J].

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

Microsoft Word 张 罡-校对-3778

Microsoft Word 张罡-校对-3778