Simple and Rapid LC Method for the Determination of Amoxicillin in Plasma

Abstract

A rapid, simple, accurate, sensitive and reproducible high performance liquid chromatographic (HPLC) method for the quantitation of amoxicillin in human plasma using cefadroxil as an internal standard (IS) has been developed and validated. The procedure involves an ultrafiltration step prior to a reversed-phase liquid chromatography. The drug and the IS were eluted from Symmetry® C18 stainless steel column (5 μm, 150 × 4.6 mm I.D.) at room temperature with a mobile phase consisting of methanol: 75 mM potassium dihydrogen phosphate buffer solution (10:90,v/v) (pH adjusted to 3.0 with phosphoric acid), at a flow rate of 1.5 mL min−1. The effluent was monitored using a UV detector set at 228 nm. Each analysis required no longer than 10 min. Quantitation was achieved by measurement of the peak area ratio of the drug to the internal standard, and the limit of quantification of amoxicillin in plasma was 0.5 μg mL−1 (RSD% and DEVs% were <15%). The method showed good precision: the intraday RSD% values for amoxicillin were in the range of 1.41–6.86% whereas the values for interday were in the range of 1.13–8.16% at four different concentrations. The method is rapid (total run time <12 min) and accurate (DEVs,% <10%). The mean relative recovery was 99.67% and the mean absolute recovery was 86.68%. Stability testing shows that amoxicillin is stable in plasma for at least 4 weeks when stored at −70 °C. The method was successfully applied in a pharmacokinetic study involving healthy rabbits.