LC-Receptorgram: A Method for Identification and Quantitation of β-Lactams in Milk by Liquid Chromatography with Microbial Receptor Assay

Abstract

Abstract A rapid determinative method was developed for identification and quantitation of common β-lactam drugs (e.g., amoxicillin, ampicillin, cephapirin, penicillin G, ceftiofur, and cloxacillin) in milk. This method combines the advantages of liquid chromatography (LC) with the selectivity and sensitivity of a microbial receptor assay. This LC-receptorgram procedure identifies and quantitates β-lactams at 10 ppb or lower within 3 h. A simple purification scheme, using solid-phase extraction, gave recoveries ranging from 50% for amoxicillin to 80-90% for other β-lactams. Individual drugs were separated by LC using a reversed-phase C8 column and an isocratic buffer system containing methanol (35%) in phosphate buffer (50 mM, pH 6.0). The Charm II quantitative procedure was used to detect (5-lactams in the LC fractions. In a blind study of raw milk fortified with β-lactams, the coefficients of variance for quantitation of 6 β-lactams at the safe levels ranged from 4.1 to 13%. The confidence level for selectivity was greater than 99%, and for detection of positives at safe levels, it was 95% or greater. Analysis of 6 β-lactams in incurred samples showed that all 6 drugs are depleted to below 10 ppb in less than 48 h. Incurred samples with cephapirin and ceftiofur contained active metabolites, in addition to parent drug, that were detected by the LC-receptorgram. In market milk samples that screened positive in routine testing programs, penicillin G, cephapirin, and ceftiofur were identified by the LC-receptorgram as the most common contaminants. In several samples containing cephapirin or ceftiofur, an active metabolite was detected in addition to the parent drug. Only the parent drug was detected for other β-lactams.