title: Development and validation of an HPLC-MS method for the simultaneous quantification of key oxysterols, endocannabinoids, and ceramides: variations in metabolic syndrome.
abstract: Oxysterols, ceramides, and endocannabinoids are three families of bioactive lipids suggested to be involved in obesity and metabolic syndrome. To facilitate the quantification of these potentially interconnected lipids, we have developed and validated a liquid chromatography coupled to mass spectrometry method allowing for their simultaneous quantification from tissues. Sample purification is of great importance when quantifying oxysterols due to the potential artifactual conversion of cholesterol into oxysterols. Therefore, we developed a novel solid-phase extraction procedure and demonstrated that it allowed for good recoveries of the three families of analytes without artifactual oxidation of cholesterol. The oxysterols, ceramides, and endocannabinoids and their respective internal standards were chromatographically separated by HPLC and ionized using the atmospheric pressure chemical ionization (APCI) source of an LTQ-orbitrap mass spectrometer. The repeatability and bias were within the acceptance limits for all 23 lipids of interest. The sensitivity (limit of detection (LOD) and limit of quantification (LOQ)) and specificity of the method allowed us to quantify all the analytes in the liver and adipose tissue of control and high-fat diet-fed C57BL/6 mice. We found that 16 weeks of high-fat diet strongly impacted the hepatic levels of several oxysterols, ceramides, and endocannabinoids. A partial least-squares discriminant analysis (PLS-DA) based on the variations of the hepatic levels of these 23 bioactive lipids allowed differentiating the lean mice from the obese mice.
authors.affiliation: Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Av. E. Mounier, 72 (B1.72.01), 1200, Brussels, Belgium.
authors.forename: Valentin
authors.initials: V
authors.lastname: Mutemberezi
authors.affiliation: Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Av. E. Mounier, 72 (B1.72.01), 1200, Brussels, Belgium.
authors.forename: Julien
authors.initials: J
authors.lastname: Masquelier
authors.affiliation: Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Av. E. Mounier, 72 (B1.72.01), 1200, Brussels, Belgium.
authors.forename: Owein
authors.initials: O
authors.lastname: Guillemot-Legris
authors.affiliation: Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Av. E. Mounier, 72 (B1.72.01), 1200, Brussels, Belgium. giulio.muccioli@uclouvain.be.
authors.forename: Giulio G
authors.initials: GG
authors.lastname: Muccioli
mesh_terms: D000818:Animals; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D063388:Endocannabinoids / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D024821:Metabolic Syndrome / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D013261:Sterols / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002518:Ceramides; D063388:Endocannabinoids; D013261:Sterols
keywords: Ceramides; Endocannabinoids; HPLC-MS; Metabolic syndrome; Oxysterols; Validation