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

摘要: This report describes a rapid and sensitive analytical method for the quantification of neuroactive steroids alphaxalone pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The involves protein precipitation, alkaline extraction compounds internal standard pregnenolone dichloromethane, followed by isocratic reversed-phase high-performance liquid chromatography on 3-microm Microsphere C18 column fluorescence detection at wavelengths 332 nm 516 excitation emission, respectively. mobile phase consists mixture 25 mM acetate buffer (pH 3.7)-acetonitrile (45:55, v/v 40:60, pregnanolone) flow-rate 1 ml/min. total run time was approximately 35 min. In concentration range 0.010-10 microg ml(-1), intra- inter-assay coefficients variation were less than 17% both methods. 50 microl samples corresponding limits 10 ng ml(-1) (signal-to-noise ratio=3). utility established analyzing from rats, which had received an intravenous administration 5 mg kg(-1) or pregnanolone. Values clearance, volume distribution steady state terminal half life 71.9 ml min(-1) kg(-1), 814 13.5 min 69.2 1,638 27.8 pregnanolone, Due to its simplicity sensitivity this can be used routine basis pharmacokinetic analysis steroids.