High-performance liquid chromatography of theneuroactive steroids alphaxalone and pregnanolone in plasma using dansyl hydrazine as fluorescent label: application to a pharmacokinetic-pharmacodynamic study in rats

摘要

This report describes a rapid and sensitive analytical method for the quantification of the neuroactive steroids alphaxalone~ and pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extraction of the compounds and internal standard pregnenolone with dichiorome-thane, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-p.m Microsphere C,, column with fluorescence detection at wavelengths 332 nm and 516 nin for excitation and emission, respectively The mobile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)—acetonitrile (45:55, vfv for alphaxalone and 40:60, v/v for pregnanolone) with a flow-rate of 1 ml/min. The total run time was --35 nun In the concentration range of 0.010-10 p.g mi’, the intra- and inter-assay coefficients of variation were less than 17% for both methods In 50 j~l plasma samples the corresponding limits of detection were 10 ng mi-I (signal-to-noise ratio3) The utility of the analytical method was established by analyzing plasma samples from rats, which had received an intravenous admimatration of 5 mg kg’ alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min’ kg’, 814 mg kg’ and 13.5 mm for alphaxalone and 69.2 ml min’ kg’, 1638 ml kg~’ and 27.8 mm for pregnanolone, respectively. Due to its simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids.