| With the development of antibody preparation, fluorescent tracer marker, chromatography and mass spectrometry technologies, there are many novel and rapid methods emerging for detection of small molecule compounds. Aminoglycosides (AGs) are a class of antibiotic drugs containing amino sugars and amino alcohols ring structure, which are widely used to treat mastitis in dairy and to promote growth in our country. At the same time, the monitoring for AGs antibiotics residue in animal foods is an important part in China’s food safety system. Therefore, it is very important significance to explore and develop new rapid screening methods and validation methods for detection of AGs antibiotics residue, based on the combination of existing residue detection method with new analytical technologies.In this study, Antigen-enzyme tracers were prepared, and visual gel-ELISA methods were established. For detection of apramycin, the sensitivity of the gel-ELISA method was0.5μg/L, and the Cut-off values for raw milk, muscles and livers was3μg/L,3μg/kg, and10μg/kg, respectively.It was a two-steps method with the detection time of20min. For detection of gentamicin and kanamycin simultaneously, the sensitivity of the gel-ELISA method was2.0μg/L, and the Cut-off values for raw milk was5μg/L. It was a one-step method with the detection time of15min.Antibody-fluorescent microspheres tracers were prepared, and Fluorescent immunochromatographic methods were established. For detection of gentamicin, it was a yes/no method, with the Cut-off value of100μg/L in solution and milk. Vertical chromatography mode in wells was applied, and the detection time was within20min. For detection of kanamycin, it was a qualitative method, with the LOD of5μg/L. The dynamic range IC20～IC80was calculated as9.5-100μg/L, and the IC5o was24.8μg/L. When the blank samples were spiked at25,50and100μg/L, the mean recovery ranged from78.4～92.7%, with intra-assay CVs ranging from10.8～12.4%.In the study, low-density bonded C18column was selected for determination of AGs based on hydrophilic interaction chromatography (HILIC) mode, and a UPLC-MS/MS method for determination of12AGs antibiotics residue in milk was developed successfully. The common reagents (such as acetonitrile, formic acid and water) were used as mobile phase, and gradient elution mode was used. The results showed sharp symmetrical peaks and good retention for12AGs antibiotics, with the retention time of more than1min. There is no ion-pair reagents used in the method.The result showed good linear relationships for analytes ranged from20-5000μg/L, and the LOD and LOQ for the method were10～50μg/L and20～100μg/L, respectively. Fortification at the range of20～1000μg/L, mean recoveries were60.9%～109.2%with intra-and inter-assay CVs of4.6%-14.8%and6.0%～16.2%in milk.In summary, the methods established in the study were rapid and effective, which could provide effective techniques and enrich the detection system for AGs antibiotics residue monitoring. |
