【作者】 李娜；

【导师】 刘永明；

【作者基本信息】 烟台大学 ， 分析化学， 2016， 硕士

【摘要】 动物源性食品中农兽药残留分析分为前处理与检测两步,目前,仪器水平已经能够满足测定要求,然而,样品前处理方法存在步骤多、有机溶剂消耗大、回收率低等缺陷。本文对现有的检测方法及样品前处理技术进行了综述,利用平衡透析法与HPLC法、GC-MS法分别探讨了氨基甲酸酯类、盐酸克伦特罗、丁香酚和吡喹酮等药物与蛋白质之间的结合作用,并深入调查了药物在实际样品中的存在状态,为复杂样品中样品前处理技术发展提供了理论指导。研究了CH3CN–K2HPO4–H2O双水相体系提取法和有机水溶液提取法等样品前处理方法,建立了快速高效的动物源性食品中氨基甲酸酯类、盐酸克伦特罗、丁香酚和吡喹酮等药物残留的分析方法,并用于实际样品的测定。论文主要分为五个部分:第一部分:阐述动物性食品安全现状,总结了农兽药残留检测技术及样品前处理方法,并说明了本文的研究内容和研究意义。第二部分:以BSA作为模拟蛋白,利用高效液相色谱法来研究鱼肉和虾肉样品中蛋白质对提取四种氨基甲酸酯类药物的影响,结果表明蛋白质与四种氨基甲酸酯类药物强结合。利用CH3CN-K2HPO4-H2O双水相技术,可破坏这种强结合作用有效提取出药物。在上相萃取液中加入PSA、C18去除极性和脂类杂质,直接进行液相色谱检测。在最优实验条件下,应用此前处理方法与HPLC法结合测定鱼和虾样品中的四种氨基甲酸酯类药物,加标回收率、RSD与检出限范围分别为80.1%~96.7%,1.37%~5.19%和0.26~8.8ng/g。本方法是有效的前处理方法且为测定复杂动物源性样品中氨基甲酸酯类药物残留提供了一个理论基础。第三部分:直接以生物亲和的甲醇-盐酸水溶液提取结合液相色谱法,建立了一种快速提取与测定肉和血清样品中盐酸克伦特罗药物残留的方法。实验研究了盐酸克伦特罗药物在猪肉与猪血清样品中的存在状态。研究表明:盐酸克伦特罗与猪肉、猪血清样品中的蛋白质存在较强结合作用,用50%的甲醇盐酸水溶液作为提取溶剂时,药物残留的提取效率最好且提取液不需净化。试验测定了猪肉、猪血清、牛肉和牛血清样品中的盐酸克伦特罗药物残留量,盐酸克伦特罗的质量浓度在0.01~5.00μg/mL范围内与峰面积呈良好的线性关系,方法的检出限为1.8ng/g,相对标准偏差在2.1%~5.1%之间,回收率在87.4%~99.2%之间。第四部分:以乙腈-磷酸氢二钾-水双水相体系高效提取富含蛋白的鱼肉和虾肉中残留的丁香酚药物,结合气相色谱-质谱联用(GC-MS)技术实现了对鱼肉和虾肉中丁香酚药物残留的快速准确检测。利用平衡透析法结合气相色谱-质谱联用(GC-MS)技术研究了丁香酚药物与蛋白质的结合情况及在真实鱼肉和虾肉中的存在形式。实验表明丁香酚药物在富含蛋白的鱼和虾样品中以结合态形式存在。实验利用双水相基质去除法与QuEChERS法两种方法同时处理富含蛋白的鱼肉、虾肉样品,结果显示双水相基质去除法的提取回收率高。将此前处理方法应用于测定真实鱼肉、虾肉样品中的丁香酚残留量,检出限为2.2 ng/g,回收率范围为80.6%~95.9%。第五部分:利用荧光光谱法研究了吡喹酮药物对蛋白质的猝灭方式,平衡常数及结合点位数,结果表明蛋白质分子与吡喹酮药物牢固结合。为了破坏结合作用提高萃取效率,实验优化了多种萃取体系以及萃取体系浓度,80%的CH3CN-K2HPO4-H2O萃取体系较好。本文对实验的各参数进行了研究,在最优实验条件和仪器参数下将此方法应用在检测实际鱼肉和虾肉样品中的吡喹酮药物残留,检出限为6.0 ng/g,回收率范围是80.4%~91.6%,相对标准偏差为2.46%~3.42%之间。更多还原

【Abstract】 The most commonly analysis of pesticide and veterinary drug residues in foodstuffs of animal origin can be divided into two steps: the sample pre-treatment and the determination of using instrument. Recently, testing equipment can meet the requirements for analyzing of pesticide and veterinary drug residues. However, the reported sample pre-treatment methods were complex, solvent-consuming,low-recovery and so on. This thesis has reviewed the literatures of sample preparation techniques. Equilibrium dialysis method with HPLC and GC-MS method were combined to investigate the binding interaction of drugs and protein, as well as drugs and real samples, further to explain the state of drugs in real sample. The studied drugs are including carbamates, clenbuterol, eugenol and praziquantel. Above all, the investigation provided a theoretical guidance for the pre-treatment technology in complex samples. CH3CN-K2HPO4-H2 O aqueous two-phase extraction method and organic solution extraction method were studied. And fast and efficient methods were established for analysis of residues in foodstuffs of animal origin.Part one: This section described the situation of security in foodstuffs of animal origin, and reviewed the detection of technologies and the sample pretreatment methods for analyzing of pesticide and veterinary drug residues.Part two: Using bovine serum albumin(BSA) as the model protein,high-performance liquid chromatography(HPLC) was applied to study the effect of protein on the extraction of four carbamate pesticides in fish and shrimp meat samples,the results showed that the strong interaction between protein and four carbamate pesticides. Based on CH3CN-K2HPO4-H2 O aqueous two-phase extraction system, a high-efficient extraction was obtained, then add PSA、C18 to eliminate the fat and other polar impurities, last analyze directly with HPLC. Under the optimal conditions, this method was applied to determination quickly of four carbamate pesticides in fish and shrimp sample with High-Performance Liquid Chromatography(HPLC), satisfactory spiked recoveries between 80.1% and 96.7% with the relative standard deviation valuesfrom 1.37% to 3.76%, the limits of detection ranging from 0.26 ng/g to 8.8 ng/g. It was verified that this method was an efficient pretreatment and could provide a basis of how to determine carbamate pesticides in complex animal origin samples.Part three: Direct using the methanol-hydrochloric acid aqueous solution as extracting solution, a rapid method was established in this experiment for the determination of clenbuterol in meat and serum by HPLC. The existence of the state of the clenbuterol in pork and porcine serum sample was investigated. The research showed that there is a strong interaction between clenbuterol and protein in pork as well as porcine serum. In the 50%(volume fraction) methanol-water extracting solution, obtained a high-efficient extraction of clenbuterol and analysed directly by HPLC. This proposed method was available to determination of clenbuterol in pork,porcine serum beef and bovine serum by high performance liquid chromatography, the clenbuterol had good linear response within the range of 0.01~5.00 μg/m L. Overall limits of detection were 1.8ng/g, Relative standard deviation values were at2.1%~5.1%, and recoveries were 87.4%~99.2%.Part four:Based on CH3CN-K2HPO4-H2 O aquatic two phase system, a fast and high-effecient sample pretreatment method was developed for analyzing eugenol in fish and shrimp sample by Gas Chromatography- Mass Spectroscopy(GC–MS). Using equilibrium dialysis method and GC–MS were applied to study the binding interaction between eugenol and protein. Meanwhile, the effects of protein matrix on the extraction of eugenol from fish and shrimp meat were detailedly researched. The research indicated that there is a strong interaction between eugenol and fish as well as shrimp which were wealthy in protein. The aquatic two phase system and Qu ECh ERS method were applied to sample in same time, the aquatic two phase system obtained high-efficient extraction. This advised method was applied to analysis of eugenol in fish and shrimp samples. The detection limit was 2.2 ng/g and the spiked recoveries were 80.6%~95.9%.Part five: Fluorescence spectrometry was made use of study the interaction between drugs and protein, including the quenching, the equilibrium constants and the number of binding. The results showed that the protein has a strong interaction withpraziquantel, To improve the extraction efficiency, some extraction systems and the extraction systems concentrations were investigated. However, in the 80%(volume fraction) CH3CN-K2HPO4-H2 O aquatic two phase system, obtained a high-efficient extraction of praziquantel. Under the optimal conditions, this method was applied to the quick determination of praziquantel residues in fish and shrimp samples, the detection limit was 6.0 ng/g, satisfactory spiked recoveries between 80.4% and 91.6%with the relative standard deviation values from 2.46% to 3.42%.更多还原