Objective The study was aimed to establish a HPLC method for the determination of amoxicillin, ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, and dicloxicillin in feeds.

Method Firstly, the samples of formula feed, concentrated feed and concentrate supplement were extracted with 20% acetonitrile solution, and the premixed feed samples were extracted by EDTA-Mcllvaine buffer solution and purified by HLB solid-phase extraction column, and then derivated by the reagents. Eventually, the samples were separated on a C₁₈(150 mm×4.6 mm, i.d 5 μm)column by gradient elution with acetonitrile -phosphate buffer solution as mobile phase, detected by UV detecor at 325 nm, calibrated by matrix matching standard working solution and quantified by external standard method.

Result There were good linear relationships among the seven kinds of penicillins of formula feed, concentrated feed and concentrate supplement in the concentration range of 0.25-10 μg/mL, and there were good relationships among the seven kinds of penicillins of premixed additive feed in the concentration range of 0.10-10μg/mL. The average recovery rates were in the range of 69.6%-103.7% with RSD value less than 15%. The limit of detection(LOD)of compound feed, concentrated feed and concentrate supplement was 2.5 mg/kg, and the limit of quantification(LOQ)was 5 mg/kg. The LOD of premixed additive feed was 1.0 mg/kg and the LOQ was 2.0 mg/kg.

Conclusion The method was proved to be specific, rapid, accurate and reproducible for determination of seven penicillin antibiotics in daily feed.