On-Valve μSFE-SFC-MS Enables Fast Analysis of Single Oocyte Lipidome

Lipid regulation and remodeling are pivotal in follicle development and oocyte maturation. Performing lipidome analysis on single oocytes is essential for optimizing assisted reproductive techniques and improving pregnancy outcomes. Conventional organic solvent extraction faces challenges in single-cell analysis, including excessive sample dilution and time-consuming processing. In this study, we developed an on-valve microflow supercritical fluid extraction and chromatography-mass spectrometry (μSFE-SFC-MS) method. Lipids in intact single oocytes are extracted online by supercritical carbon dioxide fluid, separated by a C18 capillary column, ionized, and annotated. Notably, this method significantly reduces sample pretreatment and chromatographic separation time to just 15 min per sample, compared to about 2 h of the conventional method. We analyzed of 276 lipid species from a single oocyte. Lipidomic differences in oocyte maturation stages clarify distinct metabolic remodeling of phospholipids into sphingomyelins and glycerides. The differential analysis suggests that several lipid species can be used as criteria for determining oocyte maturation at the single-cell level. The present work offers a fast and high-coverage lipidomic analysis for single oocytes, also providing a workflow for other single-cell and trace samples.