Neurosteroids in rat brain: extraction, isolation and analysis by nanoscale liquid chromatography-electrospray mass spectrometry

Liu, S., Sjovall, J. and Griffiths, W.J. (2003) Neurosteroids in rat brain: extraction, isolation and analysis by nanoscale liquid chromatography-electrospray mass spectrometry. Analytical Chemistry, 75 . pp. 5835-5846. 10.1021/ac0346297.

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DOI: 10.1021/ac0346297

Abstract

Neurosteroids in Rat Brain: Extraction, Isolation, and Analysis by Nanoscale Liquid Chromatography-Electrospray Mass Spectrometry Suya Liu, Jan Sjövall, and William J. Griffiths* Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden, and Department of Pharmaceutical & Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, U.K. Received for review June 9, 2003. Accepted August 14, 2003. Abstract: A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 g/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-13C2]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.

Item Type:Article
Additional Information:Full text available in print and electronically at the School of Pharmacy Library.
Departments, units and centres:Department of Pharmaceutical and Biological Chemistry > Department of Pharmaceutical and Biological Chemistry
ID Code:629
Journal or Publication Title:Analytical Chemistry
Deposited By:Library Staff
Deposited On:18 Jan 2007
Last Modified:19 Sep 2007 11:15

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