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

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

Analysis and Occurrence of 14 Sulfonamide Antibacterials and Chloramphenicol in Honey by Solid-Phase Extraction Followed by LC/MS/MS Analysis

“…Sulfonamide and CAP residue detection has evolved over recent years. Enzyme-linked immunoassays have been proven to be effective in detecting entire classes of compounds in a variety of foods including honey – . This technique cannot, however, differentiate between compounds within a given class.…”

Development and Evaluation of an Enzyme Immunoassay for Sulfamerazine in Milk

“…Food and Drug Administration's (FDA) establishing a safe level for all sulfonamides at 10 ppb (Zomer et al, 1992), there is a need for EIAs that detect sulfonamides other than SMT. An FDA report has indicated that one of the major sulfonamide contaminants in milk is sulfamerazine (National Survey of Shelf Milk for Sulfonamides and Tetracyclines, April 3,1990).…”

Direct ELlSAs for Sulfathiazole in Milk and Honey with Special Emphasis on Enzyme Conjugate Preparation

“…Legal tolerance levels for sulfonamides are continuously reevaluated as new toxicological information becomes available (Long et al, 1990). In the U.S., the FDA has established 10 ppb as the safe level for all sulfonamides (Zomer et al, 1992). Current Canadian regulations stipulate maximum residue levels (of 10 ppb) of the SA sulfadimethoxine in milk (Canadian Gazette, 1991).…”

“…Address inquiries to Dr. Peter Sporns. Eachem, 1983;Horie et al, 1992;Jiirgens, 1982;Larocque et al, 1990;Parks, 1982;Roudaut and Moretain, 1990;Akiyama, 1991, 1992;Van Poucke et al, 1991;Weber and Smedley, 1983;Zomer et al, 1992), but often have low recoveries and reproducibility (Horwitz,198 l), and require extraction and extensive clean-up procedures (Agarwal, 1992;Horwitz, 198 1). Furthermore, detection limits of reverse-phase chromatographic methods are usually in the order of 1000 ppb (Belliardo, 1981;Diaz et al, 1990;Jiirgens, 1982), which is outside the range required.…”