【作者】 陶燕飞；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2011， 博士

【摘要】 在世界人口急剧膨胀的今天,合理的使用兽药可以提高畜禽产量,但是兽药的不合理使用无疑会导致动物体内药物的滞留或蓄积,并以残留的方式进入人体及生态系统。兽药残留对人类及环境的危害主要是慢性、远期和累积性的,如致癌、发育毒性、体内蓄积、免疫抑制、致敏和诱导耐药菌株等。动物性食品中的兽药残留已成为公认的农业和环境问题,因此,在食品安全这个全球关注的热点问题中,如何快速、准确地检测畜禽产品中残留的兽药问题就成为了重中之重的问题。与药品分析不同,残留分析的特殊性和复杂性在于痕量、动态的待测物存在于复杂的生物样品中,在于将分析手段与兽药的理化性质、体内过程、存在状态以及药理毒理相结合,在于样品基质和待测组分的不确定性,所以分离和检测是残留分析的两个基本方面,高分辨率和高灵敏度是其发展的两大精髓,人们在努力改进残留分析效能的同时更注重提高分析效率、降低分析成本和减少环境污染。食品中兽药残留分析存在的困难包括：样品基质背景复杂、前处理过程繁琐,需要耗费较多的时间、被测成分浓度较低、分析仪器的定性能力受到限制、仪器检测灵敏度不够等一系列问题。如何解决这些问题,满足目前越来越严格的法规的要求,是许多科技工作者研究的方向。选择简洁、有效的样品处理方法,可以得到事半功倍的效果。液相色谱-串联质谱技术的发展,为分析鉴定难挥发、热不稳定化合物的结构提供了非常有用的数据。本课题深入探索研究简便、快速、低廉和有效的快速溶剂萃取和超声提取的样品前处理技术及快速、灵敏的多组分同时分析的液相色谱串联质谱联用技术,建立了动物性食品中15种氨基苷类、6种呋喃类及水产品中孔雀石绿其相关化合物残留定量确证方法,本研究可为此三大类药物的残留监控提供技术支持和理论依据,对动物源食品安全评价具有指导意义和参考价值。1.动物性食品中氨基苷类药物多残留分析方法研究氨基苷类药物(Aminoglycosides, AGs)在临床主要用于对革兰氏阴性菌、绿脓杆菌等感染的治疗,其不合理使用易在动物组织中造成残留。已报道文献主要采用了传统的液液提取方法,新的样品前处理方法报道较少,且对于氨基苷类多残留的定量确证方法主要采用七氟丁酸酐作为离子对使之在反相色谱柱上有保留,样品前处理过程比较繁琐,如何建立一种能同时检测动物性食品中多组份氨基苷类药物残留的高效、快捷、灵敏的分析方法就显得尤为重要。本研究建立了快速溶剂萃取及高效液相色谱串联质谱法检测动物性食品包括猪、鸡、牛、羊的肌肉和肝脏,鸡蛋和牛奶中15种氨基苷类包括安普霉素、链霉素、双氢链霉素、大观霉素、庆大霉素C1、妥布霉素、巴龙霉素、潮霉素B、核糖霉素、春雷霉素、阿米卡星、新霉素B、奈替米星、卡那霉素A、西索米星。样品采用乙腈/三氟乙酸(TCA)溶液快速溶剂法(ASE)提取,ASE的最佳条件是萃取温度为70℃,萃取压力为1500psi,两次循环静态提取,每次提取时间为10min,合并萃取液,减压蒸馏除去有机层后,用氨水调pH值为7.0,CBA弱阳离子交换柱净化,利用一种亲水作用色谱柱即链霉素专用质谱柱(ST柱),流动相只需加少量的更易挥发的三氟乙酸离子对试剂,15种氨基苷类药物得到了很好的保留,成功解决了极性强的化合物色谱保留差的问题,根据添加回收实验获得本方法的灵敏度为15种目标化合物在不同样品中最低检测限为10μg/kg,最低定量限为20μg/kg。对比已有的文献报道,本方法涉及的药物范围更宽(包括了氨基苷类抗菌和抗虫药物),采用的前处理方法提取效率更高、重现性好、可操作性强,改进了分析检测手段,为难挥发、热不稳定、极性强的化合物分析方法提供了有价值的技术参考。2.动物性组织中呋喃苯烯酸钠和硝呋烯腙药物分析方法研究呋喃苯烯酸钠和硝呋烯腙属于呋喃类药物,禁止用于饲料添加剂,但是这两种化合物的残留检测方法报道极少,定量确证方法同时检测呋喃苯烯酸钠和硝呋烯未见报道,本研究建立了超声提取动物性食品包括猪、牛、羊的肌肉、肝脏、肾脏和脂肪组织,鸡肌肉和肝脏,鱼肌肉和皮及鸡蛋和牛奶中呋喃苯烯酸钠和硝呋烯腙残留的LC-MS/MS定量确证方法。优化了溶剂及用量的选择、超声提取的时问、温度的选择,提取完毕后氮气吹干溶剂,50%乙腈/水复溶,正己烷去脂,HLB柱净化,质谱条件优化的结果是采用正离子扫描硝呋烯腙(m/z 361.1>222.1)和负离子扫描呋喃苯烯酸钠(m/z 257.8>213.9),本方法在猪、牛、羊、鸡肌肉和鸡蛋、牛奶中的CCα为NIT 0.07-0.24μg/kg, NFS 0.08-0.28μg/kg,在肝脏和肾脏样品中的CCa为NIT0.21-0.66μg/kg, NFS为0.31-0.61μg/kg; NIT的CCβ在猪、牛、羊、鸡肌肉和鸡蛋、牛奶中为0.18-0.48μg/kg,在肝脏和肾脏样品中为0.41-0.84μg/kg, NFS在各组织中为0.21-0.81μg/kg。对比已有的文献报道,本研究首次报道了动物性食品中呋喃苯烯酸钠和硝呋烯腙残留检测的定量确证方法,采用超声提取,方法简便可行,利于推广使用。3.动物可食性组织中硝基呋喃类药物分析方法研究硝基呋喃类药物代谢物的残留检测方法报道虽然很多,但大都是需要过夜衍生,前处理繁琐耗时,本研究通过前处理方法技术的摸索,建立了动物性食品包括猪、牛、羊的肌肉、肝脏、肾脏和脂肪组织,鸡肌肉和肝脏,鱼肌肉和皮以及鸡蛋和牛奶中硝基呋喃类药物代谢物3-氨基-2-恶唑酮(3-amino-2-oxalidinone, AOZ)、5-吗啉甲基-3-氨基-2-嗯唑烷基酮(5-morpholinomethyl-3-amino-2-oxalidinone, AMOZ)、1氨基-乙内酰脲(1-amino-hydantoin, AHD)和氨基脲(semicarbazide, SEM)的LC-MS/MS定量确证方法。组织样品采用ASE提取,溶剂为甲醇／三氯乙酸,再用邻硝基苯甲醛超声衍生1h,调pH值7.4后,用乙酸乙酯提取,HLB净化,分析物采用高效液相色谱／串联质谱定性检测。结果表明,4种硝基呋喃类药物代谢物在不同样品中最低检测限为0.1μg/kg,最低定量限为0.5μg/kg。对比已有的文献报道,虽然关于硝基呋喃类药物代谢物在各动物性食品中的残留检测方法报道很多,但本研究突破传统的液液提取及过夜衍生的方法,采用快速溶剂提取游离的和结合态的代谢物后进行超声衍生,大大缩短了分析时间且提高了萃取效率,为硝基呋喃类药物残留分析提供另一个技术支撑。4.水产品中孔雀石绿、无色孔雀石绿、结晶紫和无色结晶紫分析方法研究孔雀石绿和无色孔雀石绿禁止用于水产品,如何建立一种能同时检测水产品中孔雀石绿、无色孔雀石绿、结晶紫和无色结晶紫残留的高效、快捷、灵敏的分析方法就显得尤为重要。本研究建立了快速溶剂萃取鲫、鳗鱼、三文鱼、蟹、虾及贝样中孔雀石绿、无色孔雀石绿、结晶紫和无色结晶紫多残留LC-MS-MS方法。各组织样品采用McIlvaine buffer (pH 3)/乙腈作为提取溶剂,采用快速溶剂萃取法,并优化萃取参数,结果表明,目标化合物在不同样品中最低检测限为0.007-0.05μg/kg,最低定量限为0.02-0.11μg/kg。对比已有的文献报道,虽然关于孔雀石绿的残留检测方法报道非常多,但由于这类化合物稳定性差,前处理上仍存在回收率偏低的问题,本研究通过添加稳定性试剂,采用快速溶剂萃取,缩短了分析提取的时间,大大提高了回收率,重现性更好,为实现自动化操作提供科学依据。本课题通过对样品前处理及色谱质谱条件优化或建立新的提取方法,建立了动物源食品中极性大的常用药物和三大类禁用药物多残留定量确证方法,分离分析技术的改进和创新大大提高了这几种药物残留分析的灵敏度,且前处理可操作性更强,对于分析人员和仪器更绿色环保。最终更有利于方法的推广,为保障人类和动物健康提供有力技术支撑。更多还原

【Abstract】 At today of world population growing rapidly, using rationally of veterinary can improve the production of livestock and poultry, but using irrationally of veterinary in animals will undoubtedly result in the residues or accumulation of drugs, and lead to the drugs going into the human body and ecological systems. Veterinary residues are harmful to humans and the environment which are mainly chronic, long-term and cumulative, such as cancer, developmental toxicity, accumulating in the body, immune suppression, such as sensitization and induction of drug-resistant strains. Veterinary residues in food have become recognized as agricultural and environmental issues, therefore, in food safety it is a hot issue of global concern on how to detect veterinary residues in livestock products quickly and accurately. And analysis of different drugs, residue analysis is that the particularity and complexity of the trace, dynamic analytes present in complex biological samples, analytical tools and veterinary drugs lies in the physical and chemical properties, in vivo process, existing state and toxicology combination is that the sample matrix and the uncertainty component to be tested. So the separation and detection are two basic aspects of residue analysis, high resolution and high sensitivity is the essence of its development. People devote to the methods which were improved the effectiveness of residue analysis at the same time, the analysis efficiency, at the same time to reduce costs and environmental pollution.The difficulties in the analysis of veterinary residues include:the complex background of the sample matrix, the cumbersome pretreatment sample and the trace concentration of measured component, the limited qualitative ability of the instruments, and a series of sensitivity problem. Many scientists take up the researches how to solve these problems in order to meet the current requirements of increasingly stringent regulations. Choosing simple, effective method of sample preparation can be a multiplier effect. Liquid chromatography-tandem mass spectrometry technology for the analysis and identification the structures of nonvolatile, thermally unstable compounds, provides very useful data.Based on this aspect, this study aims to explore analysis methods for determination of aminoglycosides, nitrofurans, malachite green and gentian violet and its related metabolites and amitraz in foods of animal origin, and investigate various kinds of parameters deeply. Accelerated solvent extraction will be firstly combined with LC-MS/MS. The optimized ASE method reduces the use of solvents and extraction time compared to traditional liquid-liquid extractions. It generates less hazardous waste and was more benign to the environment. Our study will provide technical support for monitoring, and it also has a great value for evaluation on animal food safety.1. Development of a method for simultaneous quantification and confirmation of aminoglycosides in the foods of animal originA simple and especially rapid method-using accelerated solvent extraction (ASE) has been developed for the quantitative determination of fifteen aminoglycosides in muscle and liver of porcine, chicken and bovine, egg and milk. Using accelerated solvent extraction (ASE) instrument, parameters such as extraction temperature (40-90℃) and pressure (500-2500psi) were investigated and the selected extraction (70℃,1500psi for 10min in two cycles) was most effective.finally for liquid chromatography tandem mass spectrometry analysis. The analytes were separated by a specialized column for aminoglycosides, and eluted with 0.01% trifluoroacetic acid and acetonitrile. High correlation coefficients (r>0.999) of calibration curves for the analytes were obtained within linear from 10 to 1000μg/kg. Meanwhile, using sisomicin as internal standard, reasonable recoveries (71.4-93.9%) of the 15 aminoglycosides spiked in meat were demonstrated with excellent relative standard deviation (RSD). This is a quantitative method with simple pretreatment, rapid determination and high sensitivity, and it can be applied in the determination and quantification of multi-aminoglycosides. Contrasting with the reported literatures, this method involves a wider range of drugs (including the amino-glycosides and insect antimicrobial drugs), using the pre-treatment to extract efficient, reproducible, easy to operate, improve analysis tests, difficult for volatile, thermally unstable, polar compounds analysis methods provide a valuable technical reference. 2. Development of a method for simultaneous quantification and confirmation of nitrovin and sodium nifurstyrenate in the foods of animal originA specific and sensitive method based on liquid chromatography-tandem mass spectrometry using an electro-spray ionization source has been developed for the determination of nitrovin and sodium nifurstyrenate residues in the muscle and liver of porcine and chicken and in the muscle of fish, egg and milk.. This method includes the procedures as following:extraction using acetonitrile as extraction solvent by ultrasound-assisted extraction, defattening with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, in multiple reaction monitoring (MRM) mode, under negative scan mode acquiring two diagnostic product ions for confirmation of sodium nifurstyrenate, and positive mode for nitrovin. The sensitivity results are that the averaged decision limits (CCa; a 1%) ranged from 0.09μg/kg to 0.26μg/kg while the detection capability (CCβ;β5%) ranged from 0.33μg/kg to 0.97μg/kg. Meanwhile, reasonable recoveries (71%-110%) spiked in the tissues such as muscles and livers showed excellent relative standard deviation (RSD). This is a quantitative method with simple pretreatment, rapid determination and high sensitivity, and it can be applied in the determination and quantification of nitrovin and sodium nifurstyrenate residues in complex foods from animals. Contrasting with the reported literatures, this is the first report about the quantitative and confirmation method for nitrovin and sodium nifurstyrenate residues in animal food using ultrasonic extraction, which is simple and feasible, and easy to make more widespread.3. Development of a method for simultaneous quantification and confirmation of metabolites of nitrofurans in the foods of animal originA rapid method-using accelerated solvent extraction (ASE) and ultra-sound derivatised has been developed for the quantitative determination of 4 nitrofurans (3-amino-2-oxalidinone, AOZ; 5-morpholinomethyl-3-amino-2-oxalidinone, AMOZ; 1-amino-hydantoin, AHD; semicarbazide, SEM)in muscle and liver of porcine, chicken and bovine, egg and milk. The procedure consisted of an methanol/TCA extraction conducted at elevated temperature (90℃) and pressure (1500psi), after further clean-up, the extraction solution was concentrated and finally for LC-MS/MS analysis. The LOD was 0.1μg/kg and LOQ was 0.5μg/kg. Recoveries were in the range of 73.7%-111.2%, with RSD less than 15%. The simple method reduced the time for sample pretreatment, and met the requirement for nitrofurans residue analysis. Contrasting with the reported literatures, there are a lot of literatures on the determination of metabolites of nitrofuran drugs residues in various animal food. This research break through the traditional methods of liquid-liquid extraction and overnight derivatization, using accelerated solvent extraction of free and the conjugated metabolites and derivatization with the ultrasound method, greatly reducing the analysis time and improving the extraction efficiency. The method can provide another technical support for the analysis of nitrofuran drug residues in intricate matrix.4. Development of a method for simultaneous quantification and confirmation of malachite green, gentian violet and their leuco-metabolites in aquatic productsAn automated method had been developed for the determination of Malachite green and Gentian violet as well as their leuco-metabolites in aquatic products by liquid chromatography-tandem mass spectrometry with accelerated solvent extraction and auto solid-phase cleanup. The target analytes were extracted using accelerated solvent extraction (ASE) and then purified using auto solid-phase clean-up. The ASE conditions as:solvent, temperature, pressure, static time, and cell size were optimized. The optimum extraction conditions were set as the following aspects:using 22mL ASE cell, McIlvaine buffer (pH 3)/acetonitrile/ hexane (2/10/2, v/v) as the extraction solvent; pressure at 1500 psi; temperature at 60℃; static time 5 min(static time); one cycles. The extracts were purified on OASIS MCX SPE column. Detection and quantification of Malachite green; gentian violet; leucomalachite green; leucogentian violet were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). An averaged decision limits (CCa) and detection capability (CCβ) of the method were in the range of 0.005μg/kg-0.012μg/kg and 0.08μg/kg-0.13μg/kg in shrimp and salmon. The recoveries of Malachite green; Gentian violet; Leucomalachite green; Leucogentian violet at levels of 0.1-1.0μg/kg averaged from 82.1 to 102.9% with the relative standard derivation less than 14.6%. This method is precise, sensitive and highly efficient in extraction. What’s more, after routine applications, it’s turned out that this method is suitable for the determination of Malachite green, Gentian violet and their leuco-metabolites in aquatic products. Contrasting with the reported literatures, although there are many methods about the analysis the residues of malachite green. But because of poor stability of these compounds, it’s still exist the problem of low recoveries of these compounds. In this study adding to the stability reagents, using the accelerated solvent extraction (ASE), which shorten the analysis time and greatly improve the recovery rate, and gain better reproducibility for the realization of automation to provide a scientific basis.From above, analysis methods for determination of four classes of veterinary drugs in the foods of animal origin were studied in this dissertation. Accerated solvent extraction was used, and various parameters were optimized according to characters of drugs and matrix. All results from the study can provide advanced technologies and reasonable evidence for monitoring or surveillance for these kinds of veterinary drugs residue. And all of them have great values for food safety and drug safety re-evaluation.更多还原