High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography–mass spectrometry

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.

► Oxysterols play roles in regulation of cancer related signal pathways.
► Sample preparation must deal with complex cell matrix, excess derivatization reagent and unwanted autoxidation.
► AFFL-SPE-LC allows for simple and highly automated sample clean-up and separation.
► The method was used to determine oxysterol levels in several cell types, cell sub-populations.
► Significant alterations were observed upon delivery of a Hh antagonist.