HPLC Receptorgram: A Method for Confirmation and Identification of Antimicrobial Drugs by Using Liquid Chromatography with Microbial Receptor Assay. I. Sulfonamides in Milk

Abstract

Abstract A determinative method was developed for confirmation, identification, and quantitation of 12 sulfonamide residues and p-aminobenzoic acid (PABA) in milk. This method, termed "HPLC receptorgram," uses liquid chromatography in conjunction with the microbial receptor assay (MRA) to monitor sulfonamides. The MRA for sulfonamides (commercialized as the Charm II test) uses a microcrobial receptor and [3H]sulfamethazine tracer. Sulfonamides in milk bind to the receptor and inhibit the binding of the tracer to the receptor. Milk samples spiked at 10 ppb for each sulfonamide result in at least a 50% decrease in binding on the HPLC receptorgram, with the exception of sulfanilamide and sulfacetamide, which give approximately a 25% decrease. Additionally, milk samples spiked at the minimum detection level of the MRA assay can be confirmed, identified, and quantitated with coefficients of variation (%) of 7.6-24. Analysis of market milk samples found positive by the MRA showed 9 samples containing sulfamethazine, 4 samples containing sulfadimethoxine, 2 samples containing sulfadoxine, 2 samples containing sulfadiazine or sulfathiazole, and 1 sample each containing sulfisoxazole or sulfapyridine. Three samples contained multiple sulfonamide residues. Ten sulfonamides are determined with an isocratic buffer elution system containing 22% acetonitrile mobile phase. p-Aminobenzoic acid (PABA), an interfering sulfonamide analog, sulfanilamide, and sulfacetamide are eluted by using a 10% acetonitrile mobile phase