title: Preanalytical standardization for reactive oxygen species derived oxysterol analysis in human plasma by liquid chromatography-tandem mass spectrometry.
abstract: 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 1 year 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 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 °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 °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 °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.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany. Electronic address: christin.helmschrodt@medizin.uni-leipzig.de.
authors.forename: C
authors.initials: C
authors.lastname: Helmschrodt
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Becker
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Thiery
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Ceglarek
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods; D002784:Cholesterol / Q000031:analogs & derivatives / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D015925:Cryopreservation / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood*; D010084:Oxidation-Reduction; D010949:Plasma / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012855:Sitosterols / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards; D013696:Temperature; D013997:Time Factors
chemical_list: C119784:5,6-epoxycholesterol; C116880:7-ketositosterol; D006888:Hydroxycholesterols; D017382:Reactive Oxygen Species; D012855:Sitosterols; C011724:cholest-5-en-3 beta,7 alpha-diol; D002784:Cholesterol; D004492:Edetic Acid
keywords: Biobanking condition; Butylated hydroxytoluene; Free oxysterol; Long-term storage; Preanalytical protocol; Reactive oxygen species