Development and validation of an HPLC-MS method for the simultaneous quantification of key oxysterols, endocannabinoids, and ceramides: variations in metabolic syndrome
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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.
KeywordsCeramides Endocannabinoids Oxysterols HPLC-MS Validation Metabolic syndrome
Extracted ion chromatogram
Reactive oxygen species
The authors thank Dr. Alhouayek for her helpful suggestions. V.M. and O.G.-L. are research fellows of the “Fonds pour la recherche dans l’industrie et l’agriculture” (FRIA, Belgium). G.G.M. is the recipient of subsidies from the Fonds Spéciaux de Recherches (FSR, Université catholique de Louvain) and from the FRS-FNRS, Belgium (grants CC 1.5.034.10, FRFC 2.4555.08, J.0160.13). The MASSMET platform is acknowledged for the use of the mass spectrometer.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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