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

A high-performance liquid chromatographic (HPLC) assay for acetazolamide is presented. A 100-microliter sample is mixed with an aliquot of the internal standard solution and the mixture, buffered at pH 4.5, is extracted with ethyl acetate. The extract is evaporated to dryness and the residue is analyzed by HPLC, using a reverse-phase octadecylsilane column. The wavelength of the detection is 254 nm. The coefficient of variation (CV) in the within-day analysis of replicate 10-microgram/ml acetazolamide samples in human blood plasma was 6.5%, while the between-day CV was 7.1%. The procedures was developed for the 1-25 microgram/ml acetazolamide concentration range. The internal standard used is similar in chemical structure to acetazolamide and can be readily prepared in one step from a commercially available precursor. In addition to blood serum or plasma, the assay can also use aqueous and vitreous humor samples. Theophylline and acetaminophen interfere in the assay. The technique was used to determine the concentration of acetazolamide in the blood serum of human volunteers after an oral dose of the drug, and in the aqueous and vitreous humors of rabbits after an intravenous dose of acetazolamide.