A cutting-edge approach based on UHPLC-MS to simultaneously investigate oxysterols and cholesterol precursors in biological samples: Validation in Huntington's disease mouse model

• Published in: Talanta open 2024-08, Vol.9, p.100278, Article 100278
• Main Authors: Passoni, Alice, Favagrossa, Monica, Valenza, Marta, Birolini, Giulia, Lanno, Alessia, Mariotti, Caterina, Cattaneo, Elena, Salmona, Mario, Colombo, Laura, Bagnati, Renzo
• Format: Article
• Language: English
• Subjects: Cholesterol metabolism; Huntington's disease; Liquid chromatography-mass spectrometry; Mouse brain; Mouse plasma
• ISSN: 2666-8319; 2666-8319
• DOI: 10.1016/j.talo.2023.100278

•Reduced cholesterol biosynthesis in the brain is an early dysfunction in Huntington's disease.•Liquid chromatography-mass spectrometry is a powerful technique for cholesterol metabolites quantitation in biological samples.•Validation of analytical methods regarding sample preparation and instrumental analysis is critical for metabolic studies.•Identifying reproducible and reliable changes in cholesterol metabolism in Huntington's disease has important implications for developing new biomarkers and new therapies. Brain is most cholesterol-rich organ in the body. Since cholesterol does not cross the blood brain barrier, its metabolism is provided in situ by astrocytes and neurons, and it is crucial for maintaining sterol levels and neuronal integrity and function. Recent studies have shown that the levels of cholesterol precursors and metabolites are lower in the brains of animal models of Huntington's disease (HD) while reduced levels of its catabolite are detected in the plasma of patients. In this study, we introduce a novel analytical method designed to fulfill the complex analytical requirements associated with cholesterol metabolites detection in neurodegenerative disorders. The method allows for the simultaneous quantification of a specific set of oxysterols along with cholesterol precursors in biological samples. The proposed method uses an Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) system operating in multiple reaction monitoring (MRM). Since sterols can be found in biological matrices in either free form or esterified to various fatty acids, a three-step extraction procedure was devised, consisting of alkaline hydrolysis, liquid-liquid extraction and final concentration omitting the need for a solid-phase extraction (SPE) step. The validated method achieved a detection limit of 10 ng/mL in plasma and 1 ng/mg in brain tissue, reaching a comparable sensitivity to previously published LC-MS and GC–MS methods. All target analytes were separated on a reverse-phase column employing a segmented gradient and a temperature ramp. This strategy enabled the elution and separation of all selected metabolites within a 30-minutes timeframe. This innovative approach was employed to quantify cholesterol metabolites in both plasma and brain samples from wild-type (WT) and R6/2 mice, a mouse model of HD. The results obtained from the sample analysis highlighted a significant reduction in desmosterol levels in the R6/2 brain at 12 weeks