HPLC法测定饲料中7种青霉素类药物含量

Determination of seven Penicillins in Feed by High Performance Liquid Chromatograph

【目的】建立饲料中7种青霉素药物(阿莫西林、氨苄西林、青霉素G、青霉素V、苯唑西林、氯唑西林和双氯西林)含量的高效液相色谱测定方法。【方法】配合饲料、浓缩饲料、精料补充料样品采用20%乙腈溶液提取,添加剂预混合饲料样品经EDTA-Mcllvaine缓冲液提取,HLB小柱净化,衍生化后,用C18(250 mm×4.6 mm,i.d 5μm)色谱柱,以磷酸盐缓冲溶液-乙腈二元流动相梯度洗脱,325 nm紫外波长检测,基质匹配标准工作液校准,外标法定量。【结果】配合饲料、浓缩饲料和精料补充料中7种青霉素类药物在0.25~10μg/mL浓度范围内线性关系良好,添加剂预混和饲料中7种青霉素类药物在0.10~10μg/mL浓度范围内线性关系良好;加标回收率为69.6%~103.7%,RSD值<15%。配合饲料、浓缩饲料、精料补充料的检测限为2.5 mg/kg,定量限为5 mg/kg;添加剂预混合饲料的检测限为1.0 mg/kg,定量限为2.0 mg/kg。【结论】该方法具有快速、准确、专属性和重现性好的特点,适合日常饲料样品中7种青霉素类药物含量测定。

【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 5μm)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 0.25-10μg/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.