色谱/质谱法检测β2-兴奋剂残留研究

Study on of Simultaneous Determination of β2-agonist Residues in Swine Urine by Liquid Chromatography/Mass Spectrometry

作者: 专业:养殖 导师:陈斌 年度:2013 学位:硕士  院校: 重庆师范大学

关键词
β2-兴奋剂 猪尿 液质联机 残留方法

Keywords
β2-Adrenergic Agonist, Swine Urine, LC-MS, residue Analytical
        β兴奋剂曾用于人类哮喘和早产的治疗,也曾用于家畜的哮喘和早产的治疗。由于其具有促进动物生长、提高瘦肉率的作用曾被作为饲料添加剂应用于动物养殖业。这些药物作为动物生长促进剂的非法使用对食品安全和消费者存在风险等因素,如盐酸克伦特罗导致了几例中毒爆发在欧盟和中国人。目前我国已严格禁止β兴奋剂在中国饲料和饮用水使用使用,是开发一个简单的和准确的动物尿液中这些药物的检测方法是非常必要的,并对生产商进行有效的监控。本文建立了尿中硫酸特布他林、马布特罗、西马特罗、西布特罗、氯丙那林、沙丁胺醇、莱克多巴胺、盐酸克仑特罗、班布特罗、溴布特罗10种β2-兴奋剂同时检测的高效液相色谱/质谱方法,并将方法应用于猪尿中实际样品的测定。猪尿用氢氧化钠水溶液调节pH至9.8后用叔丁基甲醚和叔丁醇混合液进行萃取分离,上清液用氮气吹干后用甲酸水溶液溶解,溶液经混合阳离子固相萃取柱净化,氨水甲醇溶液洗脱,氮气吹干,最后用流动相溶解,进行检测。在0.5、1.0、5.0、10.0、20.0和50.0ng/ml六个添加水平,尿中硫酸特布他林、马布特罗、西马特罗、西布特罗、氯丙那林、沙丁胺醇、莱克多巴胺、盐酸克仑特罗、班布特罗、溴布特罗的平均回收率范围为86.4%~113.0%,变异系数范围为7.6%~14.6%,检测限为0.1ng/ml。该技术应用于猪尿中实际样品的测定,结果准确可靠。其平均回收率≥86.4%,相对标准偏差低于14.6%。用该方法检测猪尿液中10种β2-受体激动剂残留结果显示,精密度高,准确可靠,该方法在指导残留监控和裁定贸易争端等领域具有广阔的应用空间。
    β2-adrenergic agonists was developed for the therapeutic treatment of asthma andpreterm labor in humans and has been used in domestic livestock for the same purposes.Because they can promote animal growth, improve the lean meat rate so they were usedas feed additives in animal husbandry. The illicit use of these drugs as growth promotersin animals can pose a risk for food safety and consumer protection. For instance,clenbuterol had led to several cases of intoxication outbreaks in humans in the EuropeanUnion and China. On the basis of such epidemiological evidence, The entire class ofβ2-adrenergic agonists is also strictly prohibited for use in feeds and drinking water inChina. Therefore, it is very imperative to develop a simple and accurate method for thedetection of these drugs in animal feed, so producer compliance with the regulationscould be effectively monitored.In this paper, a high performance liquid chromatography/mass spectrometry(LC-MS) method was developed for simultaneous determination of ClenbuterolHydrochloride, Clorprenaline, Mabuterol In swine urine. And the method is applied todetermine incurred swine urine samples. Urines adjusted pH to9.8with NaOH solutionwere extracted with tert-butyl alcohol and tert-butyl alcohol-methyl ether mixturesolution(50:50, v/v). The extracts were evaporated, the residues were soluted with0.1%formic acid solution, and cleaned up by cation solid phase extraction columns.The columns were eluted with ammonia methanol. After evaporated, the residue wassoluted with, the analytes were determined. At the fortified levels of0.5,1,5,10,20and50ng/ml, the mean recoveries of β2-adrenergic agonists ranged from86.4%113.0%,the coefficents of variation (CV) ranged from7.6%14.6%,the limits of detection(LOD) were0.1ng/ml in swine urine, the quantitations of detection(LOQ) were0.5μg/Lin swine urine.In this study, the average recovery rates were more than86.4%, the CVs werelower than14.6%. We present a sample pretreatment method based on cation solidphase extraction columns to purify urines samples of swine, the sample eluates were determined by LC/MS. The results showed the developed LC-MS was simple, practical,and reliable method for swine urine samples.
        

色谱/质谱法检测β_2-兴奋剂残留研究

摘要5-6
ABSTRACT6-7
1 引言10-13
    1.1 食品安全以及隐患10
    1.2 国内食品安全现状10-11
    1.3 食品中的污染物11
    1.4 食品污染物检测的困难11-12
    1.5 食品污染物中禁用物质和兽药残留研究12-13
2 综述13-27
    2.1 β_2-兴奋剂概述13-16
        2.1.1 理化性质13-14
        2.1.2 药理毒理性质14-16
    2.2 β_2-兴奋剂残留限量要求16-19
        2.2.1 最高残留限量与最低执法限量16-17
        2.2.2 β_2-兴奋剂残留限量规定17-19
    2.3 β_2-兴奋剂残留研究现状19-21
        2.3.1 盐酸克仑特罗残留研究19-20
        2.3.2 莱克多巴胺残留研究20-21
    2.4 β_2-兴奋剂检测方法21-24
        2.4.1 酶联免疫吸附分析方法21-22
        2.4.2 β_2-兴奋剂的确证性检测方法22-23
        2.4.3 高效液相色谱法23-24
        2.4.4 气相色谱/质谱联用法24
    2.5 目前研究中存在的主要问题24-25
    2.6 选题价值及目的意义25-26
    2.7 研究内容26-27
3 材料及方法27-32
    3.1 试验材料27-29
        3.1.1 药品与试剂27-29
        3.1.2 仪器设备29
    3.2 试验方法29-32
        3.2.1 试验设计29
        3.2.2 色谱条件及质谱条件29-30
        3.2.3 溶液配制30
        3.2.4 标准曲线制备30-31
        3.2.5 提取31
        3.2.6 净化31
        3.2.7 样品测定31-32
4 结果与讨论32-39
    4.1 仪器条件的选择32-35
        4.1.1 色谱条件的选择32
        4.1.2 质谱条件的优化32-35
    4.2 样品的处理35
        4.2.1 提取35
        4.2.2 净化35
    4.3 方法性能35-38
    4.4 应用38-39
5 结论与设想39-40
    5.1 结论39
    5.2 进一步研究设想39-40
参考文献40-44
附录:作者攻读硕士学位期间发表论文及科研情况44-45
致谢45


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