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Updated: Jul 13, 2026
Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts
Published on: September 21, 2014
Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain
Tatsuya Higashi1, Akihiro Nagahama, Yoshiyuki Mukai
1Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. higashi@p.kanazawa-u.ac.jp
Abstract:
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), 20alpha-dihydroprogesterone and 20beta-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.
