ABSTRACT
Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.
Subject(s)
17-Ketosteroids/analysis , Androstanols/analysis , Brain Chemistry , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Testosterone/analysis , 17-Ketosteroids/blood , Androstanols/blood , Animals , Male , Rats , Rats, Wistar , Reproducibility of Results , Restraint, Physical , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , Testosterone/bloodABSTRACT
A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of the rat brain 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol) has been developed and validated. The brain extract was purified using solid-phase extraction cartridges, derivatized with isonicotinoyl azide, and subjected to LC-MS/MS. The method was accurate and reproducible, and the limit of quantitation was 0.1 ng/g tissue when a 100-mg tissue sample was used. The change in the brain 3alpha,5alpha-Adiol level by immobilization stress was also analyzed using the developed method.
Subject(s)
Androstane-3,17-diol/analysis , Brain/metabolism , Androstane-3,17-diol/metabolism , Animals , Calibration , Chromatography, Liquid , Male , Rats , Rats, Wistar , Reproducibility of Results , Restraint, Physical , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization , Stress, Psychological/metabolism , Tandem Mass SpectrometryABSTRACT
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 5alpha-androstane-3alpha,17beta-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 gamma-aminobutyric acid type A receptors may be negligible.
