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Studies on neurosteroids XXII. Liquid chromatography–tandem mass spectrometric method for profiling rat brain 3‐oxo‐4‐ene‐neuroactive steroids
Author(s) -
Higashi Tatsuya,
Nagahama Akihiro,
Mukai Yoshiyuki,
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.891
Subject(s) - chemistry , chromatography , neuroactive steroid , electrospray ionization , derivatization , tandem mass spectrometry , solid phase extraction , steroid , liquid chromatography–mass spectrometry , androstenedione , selected reaction monitoring , pregnenolone , acetic acid , mass spectrometry , biochemistry , hormone , androgen , receptor , gabaa receptor
A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the simultaneous determination of five 3‐oxo‐4‐ene‐neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20 α ‐dihydroprogesterone and 20 β ‐dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol‐acetic acid, purified using solid‐phase extraction cartridges and subjected to LC–ESI–MS/MS. The method does not require derivatization. Deuterium‐labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra‐ and inter‐assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6–103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented. Copyright © 2007 John Wiley & Sons, Ltd.