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.