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. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid-liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 microns reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 micrograms ml-1, concentrations reflective of those expected in vivo, Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1-7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.