title: High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).
abstract: Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.
authors.affiliation: Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Liebisch
authors.forename: Marion
authors.initials: M
authors.lastname: Binder
authors.forename: Rainer
authors.initials: R
authors.lastname: Schifferer
authors.forename: Thomas
authors.initials: T
authors.lastname: Langmann
authors.forename: Berta
authors.initials: B
authors.lastname: Schulz
authors.forename: Gerd
authors.initials: G
authors.lastname: Schmitz
mesh_terms: D002784:Cholesterol / Q000032:analysis*; D002788:Cholesterol Esters / Q000032:analysis*; D002855:Chromatography, Thin Layer; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D002788:Cholesterol Esters; D002784:Cholesterol