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Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5 α ‐androstane‐3 α ,17 β ‐diol, in rat brain and serum using liquid chromatography–tandem mass spectrometry
Author(s) -
Higashi Tatsuya,
Yokoi Hiroyuki,
Nagura Yukiko,
Nishio Tadashi,
Shimada Kazutake
Publication year - 2008
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1078
Subject(s) - chemistry , chromatography , pregnenolone , electrospray ionization , androsterone , androstane , neuroactive steroid , solid phase extraction , tandem mass spectrometry , mass spectrometry , liquid chromatography–mass spectrometry , testosterone (patch) , steroid , endocrinology , biochemistry , stereochemistry , receptor , hormone , gabaa receptor , medicine
The development and validation of liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI‐MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5 α ‐androstan‐3 α ‐ol‐17‐one, 3 α ,5 α ‐A) and 5 α ‐androstane‐3 α ,17 β ‐diol (3 α ,5 α ‐Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol–acetic acid, purified using solid‐phase extraction cartridges, derivatized with an ESI‐active reagent, isonicotinoyl azide (INA), and then subjected to LC–ESI‐MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 µL sample; the intra‐ and inter‐assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1–103.7% for both androgens. The animal study using the methods suggests that most of 3 α ,5 α ‐Adiol found in the brain is derived from the periphery, while 3 α ,5 α ‐A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3 α ,5 α ‐Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5 α ‐reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3 α ,5 α ‐A and 3 α ,5 α ‐Adiol levels and increased only the brain level of androstenedione, the precursor of 3 α ,5 α ‐A. Copyright © 2008 John Wiley & Sons, Ltd.