Analysis of Amoxicillin residues in raw milk by high performance liquid chromatography with fluorescence detection

Abstract

High performance liquid chromatography (HPLC) coupled with fluorescent detection was demonstrated for the analysis of amoxicillin residues in raw milk. Factors affection the fluorescent derivative formation (e.g. trichloroacetic acid (TCA), formaldehyde concentrations, reaction time and temperature) and the HPLC separation (e.g. buffer concentrations and pH and concentrations of organic solvent) were investigated. The derivative with the highest fluorescent intensity was obtained when amoxicillin was reacted with 20% w/v TCA, 7% v/v formaldehyde at 100oC for 30 min. Separation of the derivative was achieved in phosphate buffer (50 mM, pH 5.6) containing 24% v/v acetonitrile. Percent relative standard deviations (%RSDs) for injection, intra-day and inter-day precision were within 3.1% for the retention time and 2.3% for peak area. The percent recovery was 100.6% with the %RSD of 1.8%. The limit detection (LOD) and limit of quantitation (LOQ) were 20 and 50 ppb, respectively. The developed method was rapid, simple and sensitive for the analysis of amoxicillin residues in raw milk in trace amounts.