Abstract:A ultra performance liquid chromatography tandem quadrupole mass spectrometry method was developed for the analysis of nine penicillin residues（amoxicillin，ampicillin，cloxacillin，dicloxacillin，nafcillin，oxacillin，gpenicillin，penicillin，methicillin）in milk，simutaneously.Protein and fat in the samples were removed by acetonitrile and n-hexane respectively，then the samples were purified on HLB column and concentrated under N2.The analytes were separated on an Agilent Eclipse plus C18column（2.1×100 mm，dp1.8 μm）with gradient elution consisted of 0.01 mol/L ammonium acetate（pH4.5）and acetonitrile，and analyzed under the mode of MRM.The results showed good linearity in the ranges of 0.1 μg/L-100 μg/L.The recoveries were between 67.7%and 92.8%，and the values of the RSD（n=6）were lower than 15%for all the analytes.The limits of quantitatitation（LOQ）for the analytes range from 0.1 μg/kg to 4 μg/kg.The method was suitable to detect penicillins in milk simultaneously with good accuracy and reputability.