Determination of acetazolamide in biological fluids by reverse-phase high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of acetazolamide in whole blood, plasma, and urine was developed. Samples of biological fluids containing various concentrations of acetazolamide were spiked with the internal standard, sulfadiazine. Samples were then mixed with a 50% ammonium sulfamate solution. Whole blood samples were heated for 25 s in boiling water. All samples were extracted with ethyl acetate; a phosphate buffer (pH 8.0) was used to wash the extracts. Acetazolamide was back-extracted into a glycine buffer (pH 10.0), which was then washed with ether. Separation of acetazolamide and internal standard from other biological constituents was achieved on a 10-micron C18 reverse-phase column using an acetonitrile-methanol-acetate buffer (pH 4.0). The eluant was monitored at 254 nm. All calibration curves were linear, and the results from reproducibility studies were excellent. Application of the method to human pharmacokinetic studies was demonstrated.