| To ensure confidence and to limit the residues of these drugs in animal products, accurate detection and quantitative determination of these molecules in food of animal origin are of paramount importance. However, prior studies mostly focused on the improvement of detection, the main method for sample pretreatment is liquid to liquid extraction, only a very few paid attention to sample preparation techniques, additionally, most of the methods in the previous literatures only allow the determination of one or two analytes, or only focus on one matrix.Accelerated solvent extraction (ASE) is a recent advance in sample preparation for trace analyte and this technique uses conventional solvents at elevated pressures and temperatures to extract solid samples quickly. The process takes advantage of the increasing solubility of analyte at temperatures and pressures well above the common, raising the diffusion rate and decreasing the viscosity and surface tension, so the kinetic processes for analytes desorbing from the matrix are accelerated.Based on this aspect, this study aims to explore analysis methods for determination of tetracycline. sulfanilamide, fluoquinolones, cyromazine and its related metabolites and amitraz in foods of animal origin, and investigate various kinds of parameters deeply. Pressurized liquid extraction will be firstly combined with HPLC and LC-MS/MS. The optimized PLE 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 tetracyclines in the foods of animal originA simple and especially rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of seven tetracyclines in muscle and liver of porcine, chicken and bovine. The procedure consisted of a TCA/methanol extraction conducted at elevated temperature (60℃) and pressure (65 bar), after further clean-up, the extraction solution was concentrated and finally for liquid chromatography tandem mass spectrometry analysis. For HPLC, the limits of quantitation (LOQ) was lower than 20μg/kg, recoveries of 7 tetracyclines in spiked tissues were from 75.6-103.5%. For LC-MS/MS, LOQs were 0.5μg/kg for 7 tetracyclines in muscle, liver and kidney. The limits of detection (LOD) were 0.2μg/kg. Recoveries of 7 tetracyclines in tissues spiked from 0.5 to 2.0μg/kg were in the range of 78.8%～105.2%. Coefficients of variation were in the range of 5.7%～10.8%. The simple method reduced the time for sample pretreatment, and met the requirement for tetracycline residue analysis.2. Development of a method for simultaneous quantification and confirmation of sulfonamides in the foods of animal originA rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of 18 sulfonamides in muscle and liver of porcine, chicken and bovine. The procedure consisted of an acetonitrile extraction conducted at elevated temperature (60℃) and pressure (65 bar), after further clean-up, the extraction solution was concentrated and finally for HPLC and LC-MS/MS analysis. For HPLC, the LOD was 5μg/kg and LOQ were 10 [ig/kg for 18 sulfonamides in foods of animal origin, recoveries of 20 drugs in spiked tissues were from 71.1%～118.3%. For LC-MS/MS, the LOD was 0.5μg/kg and LOQ was 1μg/kg, respectively. Recoveries of 20 drugs in spiked tissues were in the range of 70.5%-117.5%. Coefficients of variation were less than 13%. The simple method reduced the time for sample pretreatment, and met the requirement for sulfonamides residue analysis.3. Development of a method for simultaneous quantification and confirmation of fluoroquinolones in the foods of animal originA rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of 15 fluoroquinolones in muscle and liver of porcine, chicken and bovine. The procedure consisted of an acetonitrile extraction conducted at elevated temperature (65℃) and pressure (85 bar), after further clean-up, the extraction solution was concentrated and finally for HPLC and LC-MS/MS analysis. For HPLC, the LOD was 5μg/kg and LOQ were 10μg/kg for 15 fluoroquinolones in foods of animal origin, recoveries of 15 drugs in spiked tissues were in the range of 70.6%～111.1%, with RSD less than 15%. For LC-MS/MS, the LOD was 0.5μg/kg and LOQ was 1μg/kg. recoveries of 20 drugs in spiked tissues 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 fluoquinolones residue analysis.4. Development of a method for simultaneous quantification and confirmation of cyromazine, melamine and its related metabolites in the foods of animal originSimple and sensitive methods have been developed for simultaneous detection of cyromazine, melamine and their metabolites (ammeline, ammelide and cyanuric acid) in samples of animal origins. These include a high performance liquid chromatography (HPLC) method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and are useful in regular monitoring and in toxicity studies of these molecules. Representative samples used in this study include muscles and livers of swine, bovine, sheep and chicken, kidneys of swine, bovine and sheep, and milk powder. A new sample preparation procedure with pressurized liquid extraction (PLE) at 90 bar and 70℃was investigated. Quantification of these five compounds by HPLC was achieved using an APS-2 column with UV detection at 230 nm. Limit of detection (LOD) was at 20μg/kg, and limit of quantification (LOQ) was at 40 p.g/kg. Recoveries of the five analytes in spiked samples ranged from 72.2% to 115.4% with RSD less than 12%. Confirmatory analysis of the analytes was performed using LC-MS/MS in Selected Reaction Monitoring (SRM) mode. The LOD and LOQ were 5μg/kg and 10μg/kg, respectively.5. Development of a method for simultaneous quantification and confirmation of amitraz and 2,4- dimethylaniline in the foods of animal originA method has been developed for determination and confirmation of amitraz and its main metabolite,2,4-dimethylaniline, in food animal tissues by using gas chromatography electron capture detector (GC-ECD) and gas chromatography mass spectrometry detector (GC-MS). This method is based on a new extraction procedure using accelerated solvent extraction (ASE). It consists of an n-hexane/methanol extraction step, a cleaning-up step by BakerBond octadecyl C18 silica bonded cartridge, hydrolysis and derivatization to 2,4- dimethyl-7-F-butyramide for GC-ECD analysis. For confirmation using GC-MS, hydrolysis and derivatization were not needed. Parameters for extraction pressure, temperature and cycle of ASE, clean-up, derivatization and analysis procedure have been optimized. Spike recoveries from 50 to 300μg/kg levels were found to be between 72.4% and 101.3% with relative standard deviation less than 11.5% in GC-ECD, from 5 to 20μg/kg levels were found to be between 77.4% and 107.1 % with relative standard deviation less than 11.6% in GC-MS. The LOD and LOQ are 5p.g/kg and 10μg/kg, respectively, for these two analytes using GC-ECD. For GC-MS, LOD and LOQ were 2 and 5μg/kg, respectively.From above, analysis methods for determination of five 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. |
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