Accurate Determination of Circulatory Lipids Using a Combination of HILIC-MRM and RPLC-PRM

Circulatory lipids are important markers for characterizing disease phenotypes; however, accurately determining lipid species remains a significant challenge in lipidomic analysis. Here, we present a novel analytical workflow for accurate lipidome characterization in human plasma using mass spectrometry (MS) through the integration of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC). This workflow enables rapid screening of 1,966 lipid species across 18 lipid classes using HILIC-multiple reaction monitoring (MRM), which enables facile identification of lipid species by lipid class-based separations. In the NIST Standard Reference Material for Human Plasma (SRM 1950), 489 lipid species were identified using HILIC-MRM and subsequently analyzed with RPLC-parallel reaction monitoring (PRM) to resolve potential lipid isobars within the same lipid class. Notably, RPLC-PRM identified 70 additional lipidomic features in SRM 1950 that were not detectable with HILIC-MRM. Furthermore, a high correlation (Pearson correlation coefficient = 0.81) was observed regarding the concentrations of lipid species not carrying isobaric interferences in between HILIC-MRM and RPLC-PRM, indicating that the individual lipid concentrations measured by each platform can be integrated. The workflow was further applied to a cohort of 284 human plasma samples from chronic kidney disease (CKD) patients, successfully profiling lipidomic phenotypes across CKD subtypes. These findings demonstrate that combining HILIC-MRM and RPLC-PRM as complementary platforms enhances the accuracy and comprehensiveness of lipidomic analysis.