Evaluation of separation performance and quantification accuracy in lipidomics methods

In mass-spectrometry-based lipidomics, diverse analytical methods are employed in different institutions, resulting in variations in isomer identification, the number of detected compounds, and quantitation accuracy. The primary analytical methods include flow injection (FI), reversed-phase liquid chromatography (RP-LC), and hydrophilic interaction liquid chromatography (HILIC). Recently, supercritical fluid chromatography (SFC) has gained attention for its enhanced separation of hydrophobic and structural isomers. However, the most suitable analytical method for lipidomics remains in debate because of the scarcity of comparative performance studies. In this study, we evaluated the quantitative performance of four methods (FI, RP-LC, HILIC, and SFC) each connected to a triple quadrupole mass spectrometer (MS/MS) using the NIST SRM1950 plasma reference under identical conditions, focusing purely on methodological differences. Quantification was performed for 355 lipid species across 14 lipid classes using one deuterated standard per class as an internal standard. The results revealed no significant differences in quantification across six lipid classes, whereas other classes showed notable method-specific variations. Additionally, chromatographic performance, including analysis time, pressure drop, theoretical plate height, and isomer separation, was compared between HILIC-MS/MS and SFC-MS/MS. SFC-MS/MS outperformed HILIC-MS/MS in all parameters, including height equivalent to a theoretical plate, resolution, peak height, and structural isomer separation performance. We conclude that although all methods are applicable in lipidomics, method selection and optimization should be consistent with specific analytical requirements, including target lipid class, sample consumption, and analysis time.