Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation.

Karu, K., Turton, J.A., Wang, Y. and Griffiths, W.J. (2011) Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation. Chemistry and Physics of Lipids . 10.1016/j.chemphyslip.2011.04.011.

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DOI: 10.1016/j.chemphyslip.2011.04.011

Abstract

Oxysterols are present in mammalian brain at ng/g-μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MS(n)). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC-MS(n) provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3±3.4μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [(2)H(7)]-labelled cholesterol and cholesterol autoxidation products identified.

Item Type:Article
Departments, units and centres:Department of Pharmacology > Centre for Toxicology
ID Code:2150
Journal or Publication Title:Chemistry and Physics of Lipids
Deposited By:Library Staff
Deposited On:09 Jun 2011 12:56
Last Modified:09 Jun 2011 12:56

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