A sensitive and selective HPLC/ESI-MS/MS assay for the simultaneous quantification of 16-dehydropregnenolone and its major metabolites in rabbit plasma

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M1), 5-pregnene-3β-ol-20-one (M2), 5-pregnene-3β, 20-diol (M3), 5-pregnene-3β-ol-16, 17-epoxi-20-one (M4) and 5,16-pregnadien-3β, 11-diol-20-one (M5) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 μm, 100 mm × 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS–MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56–400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M3 and M5 where linearity was over the 3.13–400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at −60 °C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.