Supercritical fluid chromatography/Orbitrap mass spectrometry based lipidomics platform coupled with automated lipid identification software for accurate lipid profiling

•The combination of SFC/MS and Lipid Search software enabled high-throughput lipid profiling.•This lipidomics method enabled the separation based on polar head groups and fatty acyl moieties.•Improvement of the separation enabled more accurate lipid profiling.

We developed a practical analytical system for high-throughput and comprehensive lipid profiling using a supercritical fluid chromatography (SFC) system coupled to an Orbitrap Fourier transform mass spectrometer (Orbitrap FT-MS). Using our SFC method, polar lipid molecular species were separated based on not only their fatty acyl moieties but also their polar head groups, using a single octadecylsilyl (ODS) column. In addition, because automatic data processing software was used for the identification of lipid molecular species, the analysis time including data processing was about a half an hour per sample. A variety of lipid molecular species were detected in mouse plasma, and isomers which often co-elute in reverse phase separation were identified accurately and quantified individually. To the best of our knowledge, this is the first report describing the chromatographic separation of lipids based on both fatty acyl moieties and polar head groups, using a single ODS column. Our results demonstrate that SFC/MS is a powerful tool for the simultaneous analysis of diverse lipid molecular species.