Bioassay for Serum Itraconazole Concentrations Using Hydroxyitraconazole Standards

Low concentrations of itraconazole in serum have been associated with therapeutic failure. Variable interpatient bioavailability and detrimental drug interactions with p450 enzyme-inducing agents are well documented. Thus, routine monitoring of serum itraconazole concentrations in patients with life-threatening mycoses is essential for patient care. Present high-performance liquid chromatography (HPLC) methods measure only concentrations of itraconazole and not its active metabolite hydroxyitraconazole. Bioassay methods using itraconazole standards overestimate concentrations in serum as measured by HPLC. We have developed a bioassay for total serum itraconazole and hydroxyitraconazole concentrations using hydroxyitraconazole standards. Itraconazole and hydroxyitraconazole concentrations in 40 clinical samples were assayed by HPLC. Total drug concentrations were measured in the same samples by bioassay with itraconazole or hydroxyitraconazole standards. The correlation of concentrations measured by the last bioassay method with HPLC determinations of both compounds was excellent (r = 0.98, slope = 0.5), with acceptable reproducibility. Small errors were seen at extremes of concentrations. The ratio of hydroxyitraconazole to itraconazole in serum varied from 0.76 to 3.2. The use of hydroxyitraconazole standards rather than itraconazole standards for determination of total itraconazole and hydroxyitraconazole concentrations in serum by bioassay gives accurate and reproducible results that correlate well with total itraconazole and hydroxyitraconazole concentrations as measured by HPLC. Our data show that although hydroxyitraconazole gives larger inhibition zones than itraconazole in bioassay standards, this is not true of patient samples, in which the two compounds make equivalent contributions.