The Research Of Key Technologies In Quantitative And Confirmative Methods For The Determination Of Veterinary Drugs Multi-Residues In Animal Derived Food

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.