Determination Of Beta-Lactam Antibiotics And Their Metabolites In Food Of Animal Origin

The unique structure ofβ-lactam antibiotics is the four-memberβ-lactam ring; it is one of the most widespread antibiotics.β-lactam antibiotics are widely used in veterinary medicine for preventing and treating animal diseases because of their strong antibacterial function, and trace amounts of antibiotics or their metabolites may remain in the foodstuff of animal origin due to improper use of antibiotics. Antibiotics residues in food may cause allergic reactions in some individuals. In addition, the wide use ofβ-lactam antibiotics may reduce their efficacy to treat diseases, due to the occurrence of new strains of bacterial. At present, various agencies usually use the parent compounds as the marker residues but not the metabolites, but high amounts of those metabolites may pose a human health hazard. Therefore not only the parent compounds ofβ-lactam antibiotics but also their metabolites should be detected to ensure the safety of food for people.The thesis was majored in determiningβ-lactam antibiotics and their major metabolites in bovine milk and muscle with ultra performance liquid chromatography combined with triple quadrupole mass spectrometer (UPLC-MS/MS), the established methods were validated with a set of parameters (linearity, limit of detection, limit of quantification, recovery, precision and specificity). This thesis included the following work and conclusions:1. The method for determination of 9β-lactam antibiotics (amoxicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, cefazolin, cefalexin, ceftiofur, cefotaxime) in bovine milk with UPLC-MS/MS was established. Waters Acquity UPLC BEH Shield RP18 was used as the analytical column, acetonitrile and water with 0.02% formic acid as the mobile phase. All the targets were completely separated within 6min using the optimized chromatographic conditions. Penicillin V was used as the internal standard, the linear correlation coefficients were all more than 0.9980. At the spiked concentrations of 0.5MRL,1MRL and 2MRL, the recoveries were 91%-104%, RSD<10%, LODs (S/N>3) were 0.04-0.2ng·mL-1,LOQs (S/N>10) were set as half of MRLs. The developed method was applied to detect 20 bovine milk samples available from the supermarkets in Beijing, and no sample was contaminated withβ-lactam antibiotics at level of MRLs.2. The method for determination of amoxicillin, penicillin G and their five major metabolites in bovine milk with UPLC-MS/MS was developed. Waters Acquity UPLC HSS T3 was used as the analytical column, water with 0.02% formic acid and 5mmol·L-1 ammonium acetate combined with acetonitrile as the mobile phase. All the targets were completely separated within 6min using the optimized chromatographic conditions. Penicillin V was used as the internal standard, the linear correlation coefficients were all more than 0.9985. LODs were 0.02-1.0ng·mL-1, LOQs were 2.5-5.0ng·mL-1.At the spiked concentrations of LOQ,2LOQ and 4LOQ, the recoveries were 85%-108%, RSD<13%. The developed method was applied to detect 40 bovine milk samples available from the supermarkets in Beijing, and high amounts of penicillin G metabolites were observed in some bovine milk samples. These provided evidence for adding metabolites in current methods.3. The method for determination of amoxicillin, penicillin G and their five major metabolites in bovine muscle with UPLC-MS/MS was developed. Waters Acquity UPLC HSS T3 was used as the analytical column, water with 0.02% formic acid and 5mmol·L-1 ammonium acetate combined with acetonitrile as the mobile phase. All the targets were completely separated within 6min using the optimized chromatographic conditions. Penicillin V was used as the internal standard, the linear correlation coefficients were all more than 0.9952. LODs were 0.05-3.0μg·kg-1, LOQs were all 25μg·kg-1. At the spiked concentrations of LOQ,2LOQ and 4LOQ, the recoveries were 91%-106%, RSD<9%. The established method was applied to detect 20 bovine muscle samples available from Pollution-free Food Action Plan, and no sample was contaminated with any of the targets.