A Highly Sensitive Method For The Quantification Of Key Oxysterols In Plasma Using Liquid Chromatography With High Resolution Mass Spectrometry


Oxysterols are oxygenated derivatives of cholesterol, which have been demonstrated to be present at increased levels in the plasma of patients with cardiovascular disease and are considered as possible biomarkers for disease onset. High resolving power and mass accuracy afforded by mass spectrometers based on Orbitrap™ technology provides unique advantages for the screening and simultaneous quantitation of oxysterols in complex biological matrices, which are normally present in trace amounts. However, the chromatographic separation remains crucial since many oxysterols are structural isomers. A sensitive method for the quantification of nine key oxysterols, namely 7-α-hydroxycholesterol, 7-β-hydroxycholesterol, 7-ketocholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 5α, 6α- epoxycholestanol, 5β, 6β-epoxycholestanol and 6α-hydroxy-5α-cholestane, using high resolution mass spectrometry is described. The method is applicable to both human and mouse plasma. Liquid/liquid extraction with dichloromethane and methanol was applied to mouse plasma samples without any additional derivatization step, and an alkaline hydrolysis was employed to cleave sterol-fatty acid conjugates. The oxysterols were separated by reversed phase liquid chromatography and detected using atmospheric pressure chemical ionization in positive ion mode on a Gexactive™ mass spectrometer operated in full scan mode with a resolving power of 70.000 FWHM. Target analytes were confirmed using accurate mass and were quantified using peak area ratios, with individual deuterium-labeled internal standards present for each target compound. The method showed analytical accuracy based on a standard addition recovery test within +/-10% and excellent linearity across the calibration range from 5 to 5000 ng/ml (r2>0.9948). Precision within 15% was achieved for the whole range of analytes. Compared to traditional LC-MS/MS approaches, this method demonstrated accuracy and sensitivity without any additional need for analyte derivatization, and the acquisition of full scan data maintained the opportunity for retrospective data evaluation to investigate related analyte species, which were not considered at the point of the analysis.