A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma.

A stereospecific high-performance liquid chromatographic assay was developed for the quantitation of ketoconazole enantiomers (KTZ) in rat plasma. After protein precipitation of 100 microL plasma using acetonitrile, a wash step was performed using hexane. The supernatant was removed and KTZ enantiomers and amiodarone, the internal standard, were extracted using liquid-liquid extraction with tert-butyl methyl ether. After transfer and evaporation of the organic layer, the residue was reconstituted in mobile phase and injected into the HPLC through a chiral column. The mobile phase consisted of hexane:ethanol:2-propanol with diethyl amine, pumped at 1.5 mL/min. All components eluted within 18 min. KTZ enantiomers were baseline resolved and peaks were symmetrical in appearance with no interferences. Calibration curves were linear over the range 62.5-5000 ng/mL of enantiomer. The intraday and interday CV% assessments were <or=19 and <13%, respectively, and mean error was <4% for both enantiomers. The lower limit of quantitation was 62.5 ng/mL for each enantiomer based on 100 microL rat plasma. In rats, plasma concentrations of (+)-KTZ were higher than those of antipode after single oral doses. The assay was shown to be sensitive and appropriate for use in pharmacokinetics study of KTZ in rat. Copyright (c) 2007 John Wiley & Sons, Ltd.