Determination of Fluconazole in Human Serum by Solid-Phase Extraction and Reversed-Phase High-Performance Liquid Chromatography

Abstract

A simple, rapid, and accurate reversed-phase octadecylsilyl high-performance liquid chromatographic method using solid-phase column extraction is described for measuring fluconazole in human serum. The column eluent was monitored by ultraviolet absorption at 210 nm. Fluconazole was extracted from diluted serum by adsorption on a small Bond-Elut C18 cartridge after the addition of UK48,134 as the internal standard and recovered by elution with methanol. The methanol was then evaporated to dryness and the residue reconstituted in 200 microliters of mobile phase and filtered prior to injecting an aliquot (50 microliters) onto an Adsorbosphere C18 column (4.6 x 250 mm, 5 microns particle size), using a mobile phase of 25 mM tris(hydroxymethyl)aminomethane-phosphate buffer (pH 7.0):acetonitrile (75:25, vol/vol). The retention times were 6.6 min for fluconazole and 9.0 min for the internal standard. The assay was precise, with inter- and intraassay coefficients of variation of less than or equal to 2.9% and less than or equal to 2.1%, respectively, and with good linearity (r = 1.000) in the range of 0.1 to 25 micrograms/ml. The duration of each analysis was 15 min and the minimum detectable serum concentration was 0.1 microgram/ml.