Determination of ketoconazole in micro-samples of human plasma by HPLC with fluorescence detection. Application in a pharmacokinetic study

Ketoconazole has demonstrated usefulness in different therapeutic applications, therefore a simple, rapid and sensitive high performance liquid chromatographic assay with fluorescence detection was developed and validated for the quantification of ketoconazole in micro-samples (100 μL) of human plasma. After extraction with a rapid process by acidic protein precipitation, the chromatographic separation was achieved using a XBridge C18 column (150 × 4.6 mm) and a mixture of 0.1% triethylamine adjusted to pH 3 with orthofosforic acid and acetonitrile (62:38 v/v) as mobile phase. The wavelengths were set at 237 nm (excitation) and 370 nm (emission). All validation tests were obtained with accuracy and precision over the concentration range of 0.05-20 μg/mL. This method was successfully applied in a small pharmacokinetic study of ketoconazole in healthy adult volunteers, concluding is suitable for the determination of ketoconazole in micro-samples of human plasma with low time consuming, using economical reagents and less laborious procedures.