title: Detection of dihydroxycholesterols in human plasma using HPLC-ESI-MS/MS.
abstract: We report a straightforward sample preparation procedure and a direct liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of 7alpha,25-dihydroxycholesterol (7α25-OHC) and 7alpha,27-dihydroxycholesterol (7α27-OHC). By applying a slow protein precipitation approach using cold ethanol, we were able to detect and quantify 7α25-OHC and 7α27-OHC in a fast and reliable manner. The average concentrations from 20 healthy individuals were determined to be 0.21±0.05nM for 7α25-OHC and 3.4±0.1nM for 7α27-OHC. In addition, we are the first to report the average degrees of esterification (n=8) to be 73.8% and 82% for 7α25-OHC and 7α27-OHC, respectively. Using the established method, we achieved the sensitivity sufficient for detecting low abundant dihydroxylated oxysterols in healthy individuals. This result should enable extension of these studies towards a comprehensive analysis of oxysterol levels under disease conditions.
authors: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address: ratnaningrum.karuna@novartis.com.
authors: Ratna
authors: R
authors: Karuna
authors: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.
authors: Isabelle
authors: I
authors: Christen
authors: Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.
authors: Andreas W
authors: AW
authors: Sailer
authors: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.
authors: Francis
authors: F
authors: Bitsch
authors: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address: juan.zhang@novartis.com.
authors: Juan
authors: J
authors: Zhang
mesh_terms: D053898:Biosynthetic Pathways; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004951:Esterification; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood* / Q000737:chemistry; D007536:Isomerism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006888:Hydroxycholesterols
keywords: 7alpha,25-dihydroxycholesterol; 7alpha,27-dihydroxycholesterol or 7alpha,(25R)26-dihydroxycholesterol; G protein-coupled receptor; Hydroxycholesterol; Mass spectrometry; Oxysterols