Assay for cephapirin and ampicillin in raw milk by high-performance liquid chromatography--integrated pulsed amperometric detection

The FDA has issued guidelines governing the use of antibiotics in cattle and routinely tests for the presence of antibiotics in milk. Unfortunately, these compounds are often difficult to detect by direct methods because they often lack a chromophore or fluorophore. Integrated pulsed amperometric detection (IPAD) following reversed-phase liquid chromatography is well-suited for this analysis because it is selective, sensitive, and direct; i.e., derivatization is not required. This work involves the development of a simple, rapid assay for the determination of beta-lactam antibiotic residues in milk using HPLC-IPAD, specifically, ampicillin and cephapirin. Since the analyst studied here are detectable by UV detection, a comparison between IPAD and UV detection will be made. Sample preparation schemes that involve the extraction of antibiotics of interest from the milk matrix and subsequent cleanup are an important aspect of this project. These procedures will be discussed in detail. In addition, analytical figures of merit and IPAD wave form optimization will be addressed.