Research On The Methodology For Rapidscreening And Multi-Residue Quantification Of Antibiotics And Their Metabolites In Animal-Derived Food

Antibiotics have been widely utilized in medical and veterinary practice to treat and prevent diseases and to enhance feed efficiency. If they are not used correctly, the practice could lead to the presence of antibiotics residues in foods of animal origin. Antibiotics were degredated, and metabolites cannot be ignored, because some of them have still bioactive and potentially toxic, stable and mobile in the animal derived food or the environment. Their control is highly important for the agricultural environment, food industry and human health. To the people foodstuff is all-important, and to the foodstuff safe is all-important. The food safety risk monitoring is very essential. Therefore, it is of important theoretical significance and practical value to study the methodology for rapid screening and multi-residue quantification of antibiotics and their metabolites in animal-derived food.A thorough review of the concerned aspects was conducted in the first chapter. The review showed there are three main problems related to the determination of antibiotics and antibiotic metabolites multi-residues in foods of animal origin, as follows. First, there were the residues of multiple antibiotics with difference in chemical polarity and the low concentration levels extremely in animal-derived food. Second, the maximum residue levels(MRLs) may differ considerably in different countries and the information of antibiotic metabolites is extremely deficient. Finally, the study on the database for antibiotics and relative metabolites is little by in vivo and in vitro. Therefore, the research target of the present dissertation was proposed. The first aim was to develop the accurate-mass database including antibiotics and relative metabolites by in vivo and in vitro using ultra performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry(UPLC-ESI-Q-TOF-MS). Based on the use of an accurate-mass database, the analytical method of rapid screening and quantification was established. The main conclusions and original aspects of the dissertation are summarized as follow: 1. The development of a method for rapid screening, identification and quantification of fluoroquinolones, sulfonamides and relative metabolites in dairy productsA novel UPLC–Q-TOF/MS method for quantification and confirmation of eight fluoroquinolones, five sulphonamides and four acetyled metabolites in milk is developed. The simultaneous extraction and effective cleanup for seventeen target analytes in dairy products samples were achieved with acetonitrile-20% trichloroacetic acid(v:v,100:1) as solvent and Oasis HLB SPE cartridges as sorbent. Qualitatively screening and quantitative analysis were carried out by full scan/ MSE mode, based on an accurate-mass database. The matrix-matched standard calibration curves showed a good linearity(R2>0.983) for all antibiotics and their metabolites. The limits of quantification were in the range of 0.5-0.8 μg/kg for FQs and 0.5-13.0 μg/kg for SAs and related metabolites. The average recoveries at three spiked levels were in the range 69%-92% with relative standard deviation was less than 15%. This novel approach has high speed and sensitivity, and was successfully applied for the screening, confirmation and quantification analysis of fluoroquinolones, sulfonamides and relative metabolites in dairy products. 2. The development of a platform of antibiotic metabolism by in vitro and in vivoThe in vitro metabolism of phenacetinum was investigated by high performance extraction coupled with UPLC-Q-TOF/MS using ICR mice liver homogenate. In vivo metabolism of phenacetinum was also investigated. The paracetamol was identified as the major metabolites, which was same as the in vitro study. A platform of the antibiotic metabolism in vitro and in vivo was developed. 3. The screening and determination of antibiotic metabolites by in vitro and in vivoUsing UPLC-Q-TOF-MSE with Metabolynx software coupled with high performance extraction, an accurate-mass database of antibiotics and antibiotic metabolites was established by in vitro and in vivo. Based on the accurate-mass database, a rapid screening and multi-residue quantification of antibiotics and their metabolites in animal-derived food was developed employing UPLC-Q-TOF/MSE. It can be successfully applied for the screening, confirmation and quantification analysis of fluoroquinolones and sulfonamides and relative metabolites in dairy products. 4. The screening and determination of cyromazine and relative metabolites by in vitro and in vivoUsing UPLC-Q-TOF-MSE with Metabolynx software coupled with high performance extraction, an accurate-mass database of cyromazine and relative metabolites melamine, ammelide, ammeline and cyanuric acid, as well as dicyandiamide was estabilished by in vitro and in vivo. Based on the accurate- mass database, the matrix-matched internal standard calibration curves was developed for the determination of cyromazine and metabolites melamine, ammelide, ammeline and cyanuric acid, as well as dicyandiamide in dairy products. The limits of quantification were in the range of 1.6-66.4 μg/kg for all target analytes. The average recoveries at three spiked levels were in the range 66.2-104% with relative standard deviation was less than 20%. CYRO and MM residues in some infant milk powder samples were confirmed. Quantitation of positive samples was performed using the internal standard method.