LC-MS/MS simultaneous analysis of allopregnanolone, epiallopregnanolone, pregnanolone, dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate in human plasma

Aim: Several neuropsychopharmacological properties have been attributed to the 3 alpha-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate. gamma-aminobutyric acid (GABA(A)) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC-MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min(-1) resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml(-1) (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then applied to monitor these neurosteroids in plasma samples from ten volunteers. Conclusion: For the first time, a straightforward and reliable method for the chromatographic separation of allopregnanolone, epiallopregnanolone and pregnanolone, as well as of dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate was carried out, with optimal accuracy, sensitivity and specificity.