The analysis of the oxysterols 7-keto-, 7-alpha/beta-hydroxy-, 5 alpha,6 alpha-epoxy-, 5 beta,6 beta-epoxycholesterol and cholestane-3 beta,5 alpha,6 beta-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 1 year at different temperatures (-20 degrees C, 80 degrees C and -130 degrees 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 30 min at room temperature resulted in <25% concentration changes, within acceptable change limits (ACL). In freshly prepared plasma samples, free oxysterols were stable for 90 min stored at 4 degrees C with concentration changes <23.5% (within ACL). Up to nine freeze-thaw cycles did not affect analyte concentrations (concentration-change -8.5% to +5.0%). 7-Ketocholesterol was stable for 2 years stored <-80 degrees C; concentration changes below 20.5% (within ACL). The remaining oxysterols were stored for a maximum of 2-4 weeks without exceeding ACL. The addition of BHT did not reveal improvement in analyte stability for storage at -80 or -130 degrees C. We developed a standardized preanalytical protocol for oxysterol analysis based on LC-MS/MS, compared cryobanking conditions for each oxysterol and present data for long-term storage up to 2 years. (C) 2013 Elsevier Inc. All rights reserved.