Fast LC–MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque.

Background: A rapid liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. Method: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid–liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. Results: The detection limit was 0.1ng/ml and the linearity ranged from 0.5 to 0.75 to 2000ng/ml for the oxysterols and from 50 to 1000μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5). Conclusion: This rapid LC–MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions. [ABSTRACT FROM AUTHOR]