title: A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma.
abstract: The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) 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 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-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 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.
authors.affiliation: Department of Molecular Biomedical Sciences, North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, USA. samba_reddy@ncsu.edu
authors.forename: Doodipala S
authors.initials: DS
authors.lastname: Reddy
authors.forename: Lata
authors.initials: L
authors.lastname: Venkatarangan
authors.forename: Benjamin
authors.initials: B
authors.lastname: Chien
authors.forename: Kumar
authors.initials: K
authors.lastname: Ramu
mesh_terms: D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism; D058891:5-alpha Reductase Inhibitors; D015113:Androstane-3,17-diol / Q000096:biosynthesis / Q000097:blood* / Q000592:standards; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D018120:Finasteride / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D013739:Testosterone / Q000494:pharmacology
chemical_list: D058891:5-alpha Reductase Inhibitors; D015113:Androstane-3,17-diol; D013739:Testosterone; D018120:Finasteride; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase