猪体内甲硝唑残留靶标筛选研究

Marker residue is the direct target to monitor the drug residues in animal products. Whether the designation of marker residue is scientific and reliable determines directly the safety of the animal product. Therefore, How to findout and identify the various unknown residues in the complex biologic matrix has been the key technique bottleneck to designate the marker residue. Metronidazole is a veterinary drug which has been banned or limitted to use in food anmals in many countries including China. It could be metabolized into several substances in animals to make residues in animal products which harms the health of humanbeings. Because of the scarcity of scientific research, the marker residue of metronidazole keeps unclear till to now, and the parent drug has been detected to monitor the residue of metronidazole in food animals, which can not gurantee the food safety. This program hopes to study the procedures and methods for the seperation, purification,qualification and quantification of metronidazole related substances in blood, bile, excreta and tissues using isotopic tracing method coupled with advanced techniques of sample pretreatment and chemical analysis. Based on the methods, metabolism, distribution and residue depletion of metronidazole in pigs using isotopic tracing method would be investigated to demonstrate the composition, structures and distribution of residues related to metronidazole in pigs. Then, the dynamic characteristics of the residues would be described qualitatively and quantitatively using liquid scintillation counter and high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry. Finally, the target residue including marker residue and target tissue of metronidazole in pigs would be designated. This research would identify the scientific and accurate target residue for metronidazole in pigs which would improve the food safety of metronidazole. The procedures and techniques in this research would provide valuable thoughts and methods for the screening and identification of target residue of all the other veterinary drugs.

残留标志物是判定动物性产品中兽药残留安全性的直接监控靶标，其科学性与准确性直接决定动物性产品的安全性。如何发现并鉴定存在于复杂生物基质中形态各异的未知残留物是制约兽药残留靶标确定的关键技术瓶颈。甲硝唑是国际公认的禁限用兽药品种，在体内存在多种残留形态。因缺乏科学研究，其残留监控仅检测原形，无法确保食品安全性。本项目采用国际前沿的同位素示踪方法，结合先进的样品前处理技术与理化分析技术，研究猪体液、排泄物及主要器官组织中甲硝唑未知残留物的分离、纯化及定性定量分析方法，探索复杂生物基质中未知代谢物发现与鉴定与策略。在此基础上，开展体内代谢和残留消除研究，揭示甲硝唑在猪体内残留物的组成、形态、分布及其量变与质变规律，确定甲硝唑在猪体的残留靶标。研究成果将为甲硝唑的残留控制提供科学准确的监控靶标与技术手段，为其他兽药残留靶标的筛选与确定提供可借鉴的研究思路与方法。

甲硝唑是国际公认的禁限用药，具有潜在的致癌性与致突变作用。实际生产中仍存在违规使用和滥用现象，严重危害动物性产品安全性。由于缺乏代谢与残留消除研究等食品安全性评价核心资料，甲硝唑在动物性产品中的残留监控靶标迄今不明。本项目采用LC/MS-IT-TOF与LC-MS/MS联用技术，在建立体液、排泄物及组织中甲硝唑及其代谢物定性定量分析方法的基础上，开展了甲硝唑在猪体内的代谢及组织残留消除规律研究，确定残留靶组织和残留标示物。. 猪经灌服推荐剂量甲硝唑(25mg/kg bw)后，收集尿液、粪、血浆、胆汁和肌肉、肝脏、肾脏、脂肪等可食性组织样品。尿液、血浆和胆汁中甲硝唑及其代谢物采用固相萃取柱Cleanert PEP-2提取净化，可提取6种代谢物，药物回收率达85%-92%。粪和组织中甲硝唑及其代谢物采用液液萃取法提取，证明单一溶剂乙腈的提取效率最高（92.8%），可提出6种代谢物。对各样品中不同时段中甲硝唑及其代谢物的结构与组成进行鉴定。结果显示，甲硝唑在猪体内主要发生三种代谢方式：侧链羟基化、羟基化侧链氧化和葡萄糖醛酸（或硫酸）结合。猪体内除甲硝唑原型(MNZ)外，形成4种新的代谢产物，分别为羟基甲硝唑(HM)、羧基甲硝唑(MAA)、甲硝唑葡萄糖醛酸结合物(Glu-MNZ)和甲硝唑硫酸结合物(Sul-MNZ)。其中MNZ、HM和MAA均可在肝脏、肾脏、肌肉、胃、肺、心脏等9种组织中检出。MAA在所有组织中消除最快，3d后即不可检出；停药5d后，所有组织中均只能检测到MNZ，肝脏和肾脏甲硝唑残留时间最长，可持续到14d。甲硝唑在猪肾脏中消除最慢，消除半衰期达4.33d。由此确定甲硝唑在猪体内的残留标示物为甲硝唑原型，残留靶组织为肾脏。. 本研究结果首次揭示了甲硝唑在猪体内残留物的组成、形态、分布及其量变与质变规律，确定甲硝唑在猪体的残留靶标，为甲硝唑的残留控制提供科学准确的监控靶标与技术手段，为其他兽药残留靶标的筛选与确定提供可借鉴的研究思路与方法。