A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3α-androstanediol (5α-androstane-3α,17β-diol) in plasma

The testosterone metabolite 3α-androstanediol (5α-androstane-3α,17-diol) is a potential GABAA receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3α-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3α-androstanediol in rat plasma. Standard 3α-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3α-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3α-androstanediol, which was almost completely prevented by pretreatment with the 5α-reductase inhibitor finasteride, indicating that 3α-androstanediol is synthesized from testosterone via a 5α-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3α-androstanediol in small amounts (200 μl) of plasma and possibly other biological samples.