title: High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.
abstract: Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.
authors.affiliation: Department of Chemistry, University of Oslo, PO Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Hanne
authors.initials: H
authors.lastname: Roberg-Larsen
authors.forename: Martin Frank
authors.initials: MF
authors.lastname: Strand
authors.forename: Anders
authors.initials: A
authors.lastname: Grimsmo
authors.forename: Petter Angell
authors.initials: PA
authors.lastname: Olsen
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Dembinski
authors.forename: Frode
authors.initials: F
authors.lastname: Rise
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
authors.forename: Stefan
authors.initials: S
authors.lastname: Krauss
authors.forename: Steven Ray
authors.initials: SR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D005374:Filtration; D053823:Hedgehog Proteins; D006801:Humans; D006888:Hydroxycholesterols / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: C562171:2-bromo-N-(4-(8-methylimidazo(1,2-a)pyridin-2-yl)phenyl)benzamide; D001549:Benzamides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D053823:Hedgehog Proteins; D006888:Hydroxycholesterols