Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

Abstract

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C8 reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min−1. There is good linearity in the range of 0.5–500 ng mL−1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL−1 and 0.08 ng mL−1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL−1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL−1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.