Solid-phase extraction of acetazolamide from biological fluids and subsequent analysis by high-performance liquid chromatography

Abstract

A sensitive, relatively fast and simple to operate high-performance liquid chromatographic method for the determination of acetazolamide in plasma and saliva is described. Quantitative extraction of the drug from both plasma and saliva was achieved using commercially available reversed-phase octadecylsilane-bonded silica column (Bond-Elut C 18, 2.8 ml capacity). Acetazolamide and the internal standard are retained on the Bond-Elut C 18 column and reproducibly recovered by elution with methanol. Liquid—liquid partition chromatography, carried out on a 30-cm μPorasil column (10-μm porous silica) using a mobile phase consisting of dichloromethane—ethanol—water—glacial acetic acid (500:65:65:1), provided adequate separation with acceptable retention times. Acetazolamide levels in the region 50–100 ng/ml can be determined in 100 μl of plasma or 200 μl of saliva employing ultraviolet detection at 254 nm with a sensitivity of 0.005 absorbance units full scale. Although the method is primarily used to determine steady-state drug levels in paediatric patients, its general applicability is illustrated by the 24-h plasma and saliva concentration profiles obtained from a male volunteer following oral administration of acetazolamide.