Evaluating Novel Direct Injection Liquid Chromatography-Mass Spectrometry Method and Extraction-Based Workflows for...

Lipids of extracellular vesicles (EVs) are attracting attention due to their crucial biological functions and potential roles in processes such as carcinogenesis. This study compares three commonly used lipid extraction techniques, i.e., liquid-liquid extraction, single-phase extraction, and solid-phase extraction, with a novel direct injection liquid chromatography-mass spectrometry (DI-LC-MS) workflow tailored to EV lipidomics. In the DI-LC-MS approach, EVs are disrupted and released directly in the chromatographic system, enabling the analysis of lipids without a prior extraction step. The applicability of the DI-LC-MS workflow was demonstrated by profiling lipids in mammalian and bacterial EVs. The lipidome coverage and high precision of the DI-LC-MS method (coefficient of variation of peak area lower than 20% for all the identified lipids) enabled identification of differences in lipid profiles of EV samples. The column used in the DI-LC-MS method exhibited a sufficient lifespan and stability for comparative lipidomic studies. Lipidome coverage, lipid species distribution, and precision varied across the studied workflows; our findings highlight the strengths and limitations of these methods. The DI-LC-MS emerges as a sustainable alternative for EV lipidomic studies by eliminating the need for sample preparation and reducing analysis time, solvent use, and chemical noise while requiring less than 1 μL of sample.