title: Quantification of oxysterols in human plasma and red blood cells by liquid chromatography high-resolution tandem mass spectrometry.
abstract: Oxysterols are important intermediates in numerous metabolic and catabolic pathways and their biological significance is also proven. The present paper describes a reliable and short liquid chromatography-high-resolution mass spectrometry method (LC-MS/HR-MS) for the quantification of 8 different oxysterols (24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol and cholestan-3β,5α,6β-triol) in human plasma and red blood cells. Oxysterols were extracted with iso-octane after saponification of esterified sterols. Due to the poor ionization efficiency of the target compounds in electrospray ionization (ESI) derivatization of the molecules has been performed with N,N-dimethylglycine (DMG). Within less than 8 min we were able to achieve baseline separation of the isobaric 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol and 7β-hydroxycholesterol. Moreover, high mass resolution was advantageously applied to resolve quasi-isobaric interferences. The method was validated based on the recommendations of US Food and Drug Administration and the European Medicines Agency guidelines. Oxysterol concentrations were determined in human plasma and red blood cells from healthy volunteers. Furthermore, the applicability for clinical use has been proven by the analysis of oxysterols as biomarkers in Niemann-Pick type C or cerebrotendinous xanthomatosis patients.
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauÿ-Allee 11, D-93053 Regensburg, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Pataj
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauÿ-Allee 11, D-93053 Regensburg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Liebisch
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauÿ-Allee 11, D-93053 Regensburg, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Schmitz
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauÿ-Allee 11, D-93053 Regensburg, Germany. Electronic address: Silke.Matysik@klinik.uni-regensburg.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Matysik
mesh_terms: D015415:Biomarkers / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood*; D007653:Ketocholesterols / Q000097:blood; D052556:Niemann-Pick Disease, Type C / Q000097:blood; D010949:Plasma; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods; D019294:Xanthomatosis, Cerebrotendinous / Q000097:blood
chemical_list: D015415:Biomarkers; D006888:Hydroxycholesterols; D007653:Ketocholesterols; C099828:cholest-5-ene-3,4-diol; C011724:cholest-5-en-3 beta,7 alpha-diol; C076996:27-hydroxycholesterol; C007997:25-hydroxycholesterol; C003001:7-ketocholesterol
keywords: HR-MS (high-resolution mass spectrometry); Human plasma; Niemann–Pick type C; Orbitrap; Oxysterols; Red blood cells