Preanalytical standardization for reactive oxygen species derived oxysterol analysis in human plasma by liquid chromatography–tandem mass spectrometry

• Published in: Biochemical and biophysical research communications 2014-04, Vol.446 (3), p.726-730
• Main Authors: Helmschrodt, C., Becker, S., Thiery, J., Ceglarek, U.
• Format: Article
• Language: English
• Subjects: Biobanking condition; Blood Specimen Collection - methods; Butylated hydroxytoluene; Cholesterol - analogs & derivatives; Cholesterol - blood; Chromatography, Liquid - methods; Chromatography, Liquid - standards; Cryopreservation - methods; Edetic Acid - chemistry; Free oxysterol; Humans; Hydroxycholesterols - blood; Long-term storage; Oxidation-Reduction; Plasma - chemistry; Preanalytical protocol; Reactive oxygen species; Reactive Oxygen Species - analysis; Reactive Oxygen Species - chemistry; Reproducibility of Results; Sitosterols - chemistry; Tandem Mass Spectrometry - methods; Tandem Mass Spectrometry - standards; Temperature; Time Factors
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.12.087

•Validation of a preanalytical protocol for oxysterol analysis.•Comparison of cryobanking conditions for oxysterol analysis.•Data for long-term storage up to 2years. The analysis of the oxysterols 7-keto-, 7-α/β-hydroxy-, 5α,6α-epoxy-, 5β,6β-epoxycholesterol and cholestane-3β,5α,6β-triol derived from reactive oxygen species (ROS) is of interest as biomarkers in the field of atherosclerosis. Preanalytical validation is a crucial point to minimize the susceptibility of oxysterols to in vitro autoxidation. The aim of this study was to standardize a preanalytical protocol for ROS-derived oxysterol analysis by liquid chromatography–tandem mass spectrometry in human plasma. Sample matrices were compared and stability of free oxysterols in whole blood and EDTA-plasma was investigated with regard to short-term storage until sample preparation, freeze–thaw cycles, addition of butylated hydroxytoluene and long-term storage up to 1year at different temperatures (−20°C, −80°C and −130°C) as well as different storage containers (safe-lock tubes, cryo tubes and straws). Sample preparation prior LC–MS/MS analysis was reduced to a simple concentration and protein precipitation step. Storing EDTA-whole blood for 30min at room temperature resulted in