Simultaneous Determination of Amoxicillin and Ampicillin in Bovine Milk by HPLC with Fluorescence Detection

Abstract

A sensitive liquid chromatographic analytical method using fluorescence detection was developed for the simultaneous determination of amoxicillin and ampicillin residues in raw and processed bovine milk. Aliquots of raw or processed milk (5 mL) were diluted to 40 mL with 0.01 M KH2PO4 (pH 4.5) buffer, and the soluble proteins were precipitated with the addition of sodium tungstate and sulfuric acid followed by centrifugation. The drug residues were concentrated by passing the supernatant through a C18 solid phase extraction cartridge. Amoxicillin and ampicillin were eluted from the cartridge and reacted with salicylaldehyde to form fluorescent derivatives, which were then analyzed with liquid chromatography and fluorescence detection. Average recoveries of spiked amoxicillin and ampicillin at 5, 10, and 20 ng/mL were >80%, with coefficients of variation (CV) <5%. The limit of detection (LOD) and limit of quantitation (LOQ) for amoxicillin were 1.1 and 2.4 ng/mL, respectively. The LOD and LOQ for ampicillin were 1.0 and 1.7 ng/mL, respectively. Keywords: Amoxicillin; ampicillin; antibiotics; milk; HPLC