Liquid chromatographic method for the determination of plasma itraconazole and its hydroxy metabolite in pharmacokinetic/bioavailability studies

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of itraconazole and its active metabolite, hydroxyitraconazole, in human plasma. Prior to analysis, both compounds together with the internal standard were extracted from alkalinized plasma samples using a 3:2 (v/v) mixture of 2,2,4-trimethylpentane and dichloromethane. The mobile phase comprised 0.02 M potassium dihydrogen phosphate-acetonitrile (1:1, v/v) adjusted to pH 3.0. Analysis was run at flow-rate of 0.9 ml/min with excitation and emission wavelengths set at 260 and 365 nm, respectively. Itraconazole was found to adsorb on glass or plastic tubes, but could be circumvented by prior treating the tubes using 10% dichlorodimethylsilane in toluene. Moreover, rinsing the injector port with acetonitrile helped to overcome any carry-over effect. This problem was not encountered with hydroxyitraconazole. The method was sensitive with limit of quantification of 3 ng/ml for itraconazole and 6 ng/ml for hydroxyitraconazole. The calibration curve was linear over a concentration range of 2.8–720 ng/ml for itraconazole and 5.6–720 ng/ml for the hydroxy metabolite. Mean recovery value of the extraction procedure for both compounds was about 85%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 15%. Hence, the method is suitable for use in pharmacokinetic and bioavailability studies of itraconazole.