Studies on neurosteroids XVIII: LC–MS analysis of changes in rat brain and serum testosterone levels induced by immobilization stress and ethanol administration

The liquid chromatography–mass spectrometry (LC–MS) methods were developed and validated for the determination of testosterone (T) in the brain and serum of rats and of 5α-androstane-3α,17β-diol (ADIOL), a metabolite of T, in the brain of rats. After derivatization of T with 2-hydrazino-1-methylpyridine and of ADIOL with p-nitrobenzoyl chloride, the detection sensitivities of T and ADIOL using LC–MS were increased 70- and 400-times superior to those of intact T and intact ADIOL, respectively. Those LC–MS methods are specific and reliable for the analysis of trace amounts of T and ADIOL in small amounts of samples. The animal studies using the developed methods showed that the brain and serum levels of T and the brain levels of ADIOL were not changed by stress or ethanol administration but the concentration ratio of the brain T to serum T in the stressed rats was higher than that in untreated rats. The low levels of endogenous AIDOL in brain of stressed and unrestrained rats found in this study demonstrated that the contribution to anesthetic and anxiolytic effects of ADIOL via γ-aminobutyric acid type A receptors may be negligible.