【作者】 刘晓云；

【导师】 杨连生；

【作者基本信息】 华南理工大学 ， 制糖工程， 2010， 博士

【摘要】 盐酸莱克多巴胺(Ractopamine hydrochloride)又称激动剂,是一种苯酚胺类β-肾上腺素激动剂,具有促进动物生长、提高瘦肉率的作用,但大量或长期超量使用易在动物可食组织中残留蓄积,引发一系列的中毒事件,欧盟等已立法禁止在畜禽生产上使用该类药物,我国也明确禁止使用,但目前检测技术灵敏度低,同时检测标准也不统一,筛查方法与确证方法的符合性仅为30～50%,难以通过监测控制莱克多巴胺的使用。因此有必要通过科学的研究建立适合莱克多巴胺控制需求的检测方案。本文研究目的是建立适合尿液以及毛发检测的方便快捷的筛查检测方法以及高灵敏度定量确证检测方法,通过动物药代动力研究模型,分析不同时期莱克多巴胺在尿液以及毛发中的药物消除规律,确定筛查方法以及确证方法的检测标准,为动物莱克多巴胺检测方案提供理论指导。文章分别建立了尿液定量分析的GC/MS以及LC/MS/MS的定量分析方法,经验证,方法可以实现准确定量分析,符合尿液中莱克多巴胺定量检测的需求。文章还探讨毛发检测的可行性,证实所建立的方法可以实现在10～10000 pg/mg范围内准确定量分析,符合毛发中莱克多巴胺定量检测的需求。文章通过对动物的喂药实验建立非房室药动力学模型,通过动物喂药模型,采用GC/MS, LC/MS/MS等方法定量分析收集到的尿液以及毛发样本,建立浓度-时间曲线图,分析不同时期的消除规律,结果表明,在喂食莱克多巴胺第二天尿液和毛发中均会出现莱克多巴胺成分,尿液中莱克多巴胺及其代谢物含量呈现逐步减少的趋势,消除期为3.5天,而毛发中莱克多巴胺成分,在喂药初期也出现减少趋势,但经过一段时间后即达到稳定,维持一定的药物含量,停药后含量减少,消除期为22.9天;研究中还同时发现,尿液以及毛发中药物的浓度与动物喂药以及停药时间存在一定的关系,可以通过药物浓度对动物用药进行溯源探讨。从动物药代消除规律发现,药物在动物体内代谢极快,停药后样本中莱克多巴胺含量极低,因此建立了适合大批样本筛查测试的ELISA检测方法以及适合现场筛查测试的荧光免疫层析方法,结果证实两种方法均能用于尿液中莱克多巴胺的测试;同时也对毛发的免疫检测进行了探讨,发现了毛发的免疫筛查检测的可行性,但还不足以用于毛发的筛查检测工作。为确保筛查方法与确证方法的一致性,文章比较了不同筛查及确证方法在动物用药期以及停药后检测情况,在用药期,用免疫筛查方法以及色谱确证方法均能测出样本中的莱克多巴胺,且筛查方法与确证方法检出率一致。停药后用1ng/mL灵敏度的筛查方法结合5ng/mL的色谱确正方法可以将莱克多巴胺检测的窗口期控制在用药开始第1天到停药后3～6天内。最后,文章探讨了毛发用于莱克多巴胺监控的意义,结果发现,在用药全程以及停药后30天内均可检测出莱克多巴胺药物。毛发检测方法可以有效弥补尿液检测窗口期短的缺陷,可将莱克多巴胺监控的窗口期由原有的用药开始至停药后3～5天延长到停药后30天。总的来说,本文所建立的方法可以实现优势互补,满足现场到实验室整个环节的应用。更多还原

【Abstract】 Ractopamine belongs to Beta-agonist compounds and has been often used as growth-promoting agent to improving the feed efficiency and enhance lean meat to fat ratio in many animal species. It is a forbidden molecule as feed additive in most countries including China because of the many reported collective intoxication outbreaks in humans. For most labs, It was hard to get a high sensitivity method to control ractopamine, the cutoff is different for different method, so the Compliance rate for screening method and confirmation method was just 30～50%, this is a big comfuse for ractopamine controlling. So it is necessary to research different detection methods to control the using of ractopamine in animals.The objective of this study was to establish a practical method for Screening and confirmation of Ractopamine residues in urine and hair. The pharmacokinetics and the elimination regularity of ractopamine in urine and hair were firstly studied. This research will be a scientific guidance for ractopamine control.An efficient and sensitive Gas chromatography-mass spectrometry(GC/MS) and a higher sensitive Liquid Chromatography with Tandem Mass Spectrometric (LC/MS/MS) analytical method for detection of ractopamine in urine was developed and validated. The result showed these two methods all can used in quantitative of ractopamine in urine samples. A quantitative analytical procedure for the determination of ractopamine in pig hair has been developed; result showed that this method can be used for ractopamine detection in hair samples.To research the requirement for detection, this article set up a pharmacokinetics mode to study the elimination regularity of ractopamine in urine and hair. The concentration of ractopamine in urine and hair samples durine feeding and withdrawal period was anaylized by GC/MS and LC/MS/MS methods. Compare the changes of concentration with time, we found that the concentration of ractopamine in urine decline with time, the elimilation time was 3.5 days. The concentration in hair can get a stable and keep the same level after some decline time and the elimilation time was 22.9 days. Ractopamine of urine eliminated very fast after withdrawal drugs. In this study, we also found there are relationship between feeding time and withdrawal time with concentration, we can tracing the history of feeding drug from them.From elimination regularity of ractopamine in urine, we found concentration of ractopamine was very low and is hard to trace after withdrawal drug. To screening test, a higher sensitive immunoassay is necessary. We set up an ELISA method to screening samples for using in labs and a Fluorescence Chromatography immunoassay to screening samples on site. Data improved this two immunoassay were sensitive with 1ng/mL LOQ, and can be used in urine screening. We also try to test hair samples by this two method but we fail, but we still can sure it is possible to test ractopamine in hair.To use screening and confirmation method, we compared different method. By test different urine samples during feeding and withdrawal period, we found all method can test ractopamine from samples during feeding time. But we can test ractopamine up to 3～6 days by screening with 1ng/mL cutoff, and all positive samples can be confirmed by GC/MS and LC/MS/MS with 5ng/mL cutoff.At last part in this article, we discussed the meanings of hair test in ractopamine control. Hair test can detect ractopamine up to 30 days after withdrawal drugs, so it can be used to trace feeding history and confirm the false negative and positive result by urine.Overall, all method can used in ractopamine control for different purpose.更多还原