SIMULTANEOUS DETERMINATION OF AMFENAC SODIUM AND ITS METABOLITE (7-BENZOYL-2-OXINDOLE) IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A simple, selective, sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of amfenac sodium (AMF) and its metabolite (7-benzoyl-2-oxindole, S1) in plasma was established. To 100 mul of plasma, purified water (100 mul), ammonium sulphate (0.2 g) and ethanol (100 mul) containing fenbufen (100 mug/ml, internal standard) were added. After centrifugation the ethanol layer was directly injected into a reversed-phase ODS column. AMF and S1 were eluted using a gradient buffer system of 20-80% acetonitrile-phosphate buffer (pH 7.0), and detected at 245 nm. The analytical recoveries of AMF at concentrations of 0.5, 2 and 10 mug/ml in plasma were ca. 92.1, 95.4 and 94.6%, respectively. The coefficients of variation of the AMF recoveries were below 4.6%. The S1 recoveries at the concentrations of 40, 200 and 400 ng/m) were ca. 93.4, 98.7 and 95.3%, and the coefficients of variation were below 6.0%. The coefficients of variation for intra- and inter-day variation of AMF (5 mug/ml) and S1 (100 ng/ml) were 4.5 and 3.6% and 4.2 and 4.2%, respectively. The detection limits of AMF and S1 in plasma were as low as 0.1 mug/ml and 20 ng/ml, and the coefficients of variation were 4.0 and 4.4%, respectively. The method was applied to determine the plasma concentrations of AMF and S1 after oral administration of AMF capsules (100 mg of AMF) to human volunteers.