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

This report describes a rapid and sensitive analytical method for the quant ification of the neuroactive steroids alphaxalone and pregnanolone in rat p lasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extracti on of the compounds and internal standard pregnenolone with dichloromethane , followed by isocratic reversed-phase high-performance liquid chromatograp hy on a 3-mu m Microsphere C-18 column with fluorescence detection at wavel engths 332 nm and 516 mm for excitation and emission, respectively. The mob ile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)-acetonitri le (45:55, v/v for alphaxalone and 40:60, v/v for pregnanolone) with a flow -rate of 1 ml/min. The total run time was similar to 35 min. In the concent ration range of 0.010-10 mu g ml(-1), the intra- and inter-assay coefficien ts of variation were less than 17% for both methods. In 50 mu l plasma samp les the corresponding limits of detection were 10 ng ml(-1) (signal-to-nois e ratio=3). The utility of the analytical method was established by analyzi ng plasma samples from rats, which had received an intravenous administrati on of 5 mg kg(-1) alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min(-1 ) kg(-1), 814 mg kg(-1) and 13.5 min for alphaxalone and 69.2 ml min(-1) kg (-1), 1638 ml kg(-1) and 27.8 min for pregnanolone, respectively. Due to it s simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids. (C) 2000 Elsevier Scienc e B.V. All rights reserved.