Quantitation of acetazolamide in plasma by high-performance liquid chromatography

A method for estimating acetazolamide concentrations in human plasma is described. Buffered plasma (pH 4.8) containing chlorothiazide as an internal standard is extracted twice with ethyl acetate. The extract is evaporated, redissolved, and chromatographed on silica gel with hexane-chloroform-methanol-acetic acid (65:25:10:0.25) as the mobile phase. The extraction efficiencies were greater than 90%, the coefficients of variation at 1 and 30 micrograms/ml of plasma were 3.5 and 2.0%, respectively, and the calibration curves were linear and had an intercept of essentially zero. The suitability of the method for pharmacokinetic studies was verified in a normal volunteer dosed with 250-mg (solution) and 500-mg (sustained-release tablet) acetazolamide formulations.