Abstract:A method for determining the residues of benzylpenicillin，cloxacillin，and ampicillin in meat was established based on solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry （SPE-UPLC-MS/MS）. The main factors affecting the efficiency of solid phase extraction，such as the type and volume of eluent and sample loading rate，were optimized. At the same time，the extraction and purification conditions of samples were investigated. The optimal purification effect was achieved at the sample flow rate of 3 mL/min with 6 mL acetonitrile. The HPLC separation was carried out in a UPLC C18 column with 0.1% formic acid aqueous solution-acetonitrile as the mobile phase for gradient elution. The data were collected by dynamic multiple reaction monitoring （MRM） in positive ion mode （electrospray ionization） and quantified by the external standard method. The correlation coefficients of the linear regression equations of the three target analytes were all greater than 0.999，and the limits of detection （S/N=3） and quantification （S/N=10） ranged from 0.05 to 0.10 μg/kg and from 0.1 to 0.4 μg/kg，respectively. The average recovery of the 3 analytes varied within 82.7%-96.8% and the relative standard deviations were within 1.92%-4.61%. With high precision，high reproducibility，high resolution，and short analysis time，the established SPE-UPLC-MS/MS method is suitable for qualitative and quantitative determination of penicillins in meat.