Quantitative Determination of Clavulanic Acid in Plasma by HPLC: Application to a Pharmacokinetic Study

Abstract

A rapid, simple, accurate, sensitive, and reproducible high performance liquid chromatographic (HPLC) method for quantification of clavulanic acid (CA) in human plasma using metronidazole as an internal standard has been developed and validated. Following derivatization with imidazole, plasma protein was precipitated using acetonitrile. The drug and the internal standard were eluted from a 4‐µm stainless steel Novapak® C18 column (150 × 3.9 mm2 I.D.) at room temperature, with a mobile phase consisting of 4% methanol in 0.01 M potassium dihydrogen phosphate buffer (pH adjusted to 3.2), at a flow rate of 1.8 mL/min. The effluent was monitored using a UV detector set at 311 nm. Each analysis required no longer than 8 min. Quantitation was achieved by measurement of the peak height ratio of the drug to the internal standard, and the limit of quantification of CA in plasma was 100 ng/mL. The intraday coefficient of variation (C.V., %) ranged from 3.30% to 3.93% and interday C.V. ranged from 1.74% to 2.74% at three different concentrations. The mean relative recovery was 100.35% and the mean absolute recovery was 97.63%. A stability test shows that CA is stable in plasma for at least 4 weeks when stored at −70°C. The method was successfully applied in a bioavailability/bioequivalence study involving amoxicillin/CA combination (250/62.5 mg) administered orally to 24 healthy volunteers.