title: A rapid HPLC method for indirect quantification of β-lactamase activity in milk.
abstract: To circumvent the strictly regulated limits of antibiotics in milk, illegal addition of β-lactamase to lower the antibiotic levels in milk has been reported recently in China. Herein, we describe a fast, sensitive, and robust HPLC-UV method for the determination of β-lactamase activity in milk, based on an indirect quantification strategy. The test milk sample was mixed with a known amount of penicillin G, a specific substrate of β-lactamase. After incubation, an aliquot of the mixture was injected into the HPLC-UV system to quantify the remaining penicillin G in less than 10 min. Comparative analysis of the amount of penicillin G before and after incubation was used to indirectly deduce the activity of β-lactamase in the test sample. This method was successfully applied to milk products with different fat percentages, resulting in a detection limit of 0.6 U/mL. Good recoveries, ranging from 94 to 105%, were obtained from blank milk samples spiked with β-lactamase at levels of 2 to 50 U/mL, with relative standard deviations <6%. A good correlation was demonstrated between the HPLC method and the conventional culture-based assay. Using this method, the activity changes in β-lactamase during milk pasteurization, sterilization, and storage were investigated. The advantages of low-cost, accurate quantification and easily accessible instrumentation make the proposed method an ideal alternative for high-throughput routine analysis in the dairy industry.
authors: China National Center for Food Safety Risk Assessment, Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, P. R. China.
authors: Shuang
authors: S
authors: Zhou
authors: China National Center for Food Safety Risk Assessment, Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, P. R. China.
authors: Dan
authors: D
authors: Wang
authors: China National Center for Food Safety Risk Assessment, Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, P. R. China. Electronic address: zhaoyf@cfsa.net.cn.
authors: Yunfeng
authors: Y
authors: Zhao
authors: China National Center for Food Safety Risk Assessment, Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, P. R. China. Electronic address: wuyongning@cfsa.net.cn.
authors: Yongning
authors: Y
authors: Wu
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D061353:Food Storage; D057230:Limit of Detection; D008892:Milk / Q000201:enzymology*; D061352:Pasteurization; D010400:Penicillin G / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013242:Sterilization; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D001618:beta-Lactamases; D010400:Penicillin G
keywords: antibiotics; high-performance liquid chromatography (HPLC); milk; β-lactamase