Title of article :
High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography–mass spectrometry
Author/Authors :
Roberg-Larsen، نويسنده , , Hanne and Strand، نويسنده , , Martin Frank and Grimsmo، نويسنده , , Anders and Olsen، نويسنده , , Petter Angell and Dembinski، نويسنده , , Jennifer L. and Rise، نويسنده , , Frode and Lundanes، نويسنده , , Elsa and Greibrokk، نويسنده , , Tyge and Krauss، نويسنده , , Stefan and Wilson، نويسنده , , Steven Ray، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2012
Pages :
7
From page :
291
To page :
297
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 1 mm 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.
Keywords :
oxysterols , LC–MS , AFFL-SPE-LC , Hedgehog , On-line filtration
Journal title :
Journal of Chromatography A
Serial Year :
2012
Journal title :
Journal of Chromatography A
Record number :
1516438
Link To Document :
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