Sensitive bioassay for ketoconazole in serum and cerebrospinal fluid

Ketoconazole is a broad-spectrum antifungal agent which appears promising for treatment of a variety of systemic mycoses. Pharmacokinetic studies are limited due to a lack of readily available methods for quantitation of ketoconazole in serum or cerebrospinal fluid. We developed a rapid, simple bioassay for measurement of ketoconazole alone or in the presence of therapeutic levels of amphotericin B, using an agar diffusion assay incorporating Candida pseudotropicalis. Pairs of 8-mm wells cut in the seeded assay medium were filled with four duplicate ketoconazole standards and duplicate patient specimens. Zones of inhibition were visible after 7 to 8 h of incubation, but were most easily measured after overnight growth. The assay allowed determinations of serum ketoconazole levels as low as 0.3 microgram/ml with a 4.4% coefficient of variation. Thirty-five serum samples from patients receiving the drug were assayed by this method, and the results were compared with the Coccidioides immitis endospore assay. The correlation coefficient between the assays was 0.90. This assay allows any microbiology laboratory to easily and safely determine ketoconazole levels in serum or cerebrospinal fluid.