Rapid method for the quantification of amoxicillin and its major metabolites in pig tissues by liquid chromatography-tandem mass spectrometry with emphasis on stability issues

A fast method for the quantitative determination of amoxicillin (AMO), amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione (DIKETO) in pig edible tissues (kidney, liver, fat and muscle) with liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method uses a simple liquid-liquid extraction of the tissue matrix with a 10 mM potassium dihydrogen phosphate buffer (pH 4.5) as extraction solvent. After deproteinisation by ultrafiltration, the tissue extract was directly injected onto the LC column. Chromatographic separation of the components was performed on a PLRP-S polymeric column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray MS/MS mode. The method was fully validated according to EU requirements (linearity, precision, trueness, quantification limit, detection limit and specificity). The stability of the components was evaluated over the pH range from 1.2 to 8.0. Biological samples of pigs medicated with AMO and AMO/clavulanic acid were analyzed using the developed method.