DETERMINATION OF METHAZOLAMIDE CONCENTRATIONS IN HUMAN BIOLOGICAL-FLUIDS USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma . In vivo, methazolamide readily distributes into red blood cells. The refore, both blood and plasma concentration data are needed in order t o characterize the pharmacokinetics of methazolamide. In the present s tudy, an analytical method using high performance liquid chromatograph y was validated for determination of methazolamide concentrations in s everal biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibito r, methazolamide was readily quantitated in whole blood, plasma and ur ine. Sample preparation involved liquid-liquid extraction with ethyl a cetate followed by a washing step using phosphate buffer (pH 8.0). Aft er back extraction into glycine buffer (pH 10.0), samples were then wa shed with ether and injected onto the chromatograph. Chromatography wa s performed using a C-18, 5 mu m reverse-phase column with UV detectio n at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium a cetate (pH 4.0) and acetonitrile (20%). The assay was validated over t wo standard concentration ranges from 1 to 100 mu g ml(-1), concentrat ions reflective of those expected in vivo. Calibration curves were lin ear for all biological fluids and coefficients of variation for interd ay and intraday reproducibility studies were less than 8% (range 3.1-7 .9%). The method was used to measure methazolamide concentrations in b lood, plasma and urine following oral administration to five human sub jects. (C) 1998 Elsevier Science B.V. All rights reserved.