Enhanced lipidomics workflows for plasma and extracellular vesicles through advanced liquid chromatography-tandem...

Lipidomics, a subfield of metabolomics, provides comprehensive analysis of lipids in biological systems and is essential for biomedical research, driven by advances in analytical technologies. Lipids are crucial biomolecules in cellular functions and have been increasingly recognized for their role in physiological and pathological processes. This study focuses on advanced strategies for the development, validation, and implementation of untargeted lipidomics methods in human plasma and extracellular vesicles (EVs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validation demonstrated excellent accuracy (precision and trueness) (81-120 % of nominal value), precision with inter-day repeatability below 20 %, limits of quantification ranging from 0.25 to 25 μM, and recovery rates exceeding 80 % for most lipid classes, as well as matrix effects. Plasma samples were used as a proof-of-concept study, and the method was ultimately applied to human macrophage-derived EVs. Lipid extraction utilized four liquid-liquid extraction methods to ensure broad lipid class coverage, high recovery, and repeatability. Additionally, we demonstrated that a sonication-assisted homogenization step effectively facilitates lipid extraction from EVs. Through untargeted lipidomics, our study identifies and quantifies a diverse range of lipid species in human plasma (225 lipids analytes) and macrophage-derived EVs (124 lipids analytes) within different classes. Overall, we present sophisticated approaches that combine pre-analytical lipid extraction techniques with high-resolution LC-MS/MS to enhance lipidomics research. This approach enhances the characterization of lipid profiles and their biological implications, paving the way for applications in personalized medicine and the discovery of novel lipid biomarkers associated with EVs biogenesis.