Anal Chim Acta. 2026 Oct 1;1417:345759. doi: 10.1016/j.aca.2026.345759. Epub 2026 Jun 2.
ABSTRACT
BACKGROUND: Lipidomes are highly complex and variable, and high-throughput analytical strategies are essential for comprehensive lipidome profiling in large-scale analysis. The use of hydrophilic interaction liquid chromatography (HILIC) coupled with conventional untargeted acquisition strategies remains unpopular due to the challenges in interpreting complex spectral data. To address this gap, we present a detailed lipidomic workflow based on a fast 6.7 min-long HILIC separation method coupled with data-dependent acquisition/sequential window acquisition of all theoretical fragment ions analysis (DDA/SWATH) and improve its applicability with a novel framework for untargeted data processing. In parallel, we also established a targeted high-resolution multiple reaction monitoring (MRMHR) method for comprehensive multi-class lipidomic profiling. To evaluate the applicability of these two approaches in large-scale clinical lipidomics, we used both workflows for the analysis of 240 case-control matched plasma samples from a population-based prospective cohort.
RESULTS: After method development and optimization, application of the two complementary workflows to 240 plasma samples generated lipid profiles of over 400 and 500 features for targeted and untargeted analysis, respectively. As expected, the coverage of lipids by the untargeted workflow was notably different from that of the targeted workflow at the lipid species level. In terms of quantitation of lipids detected in both approaches, we observed differences in the quantitation of lipids in individual samples, but statistical comparisons of sample groups remained largely concordant. Furthermore, we demonstrated that the choice of internal standards for normalization can influence downstream statistical outcomes.
SIGNIFICANCE: Two fast HILIC-based workflows were demonstrated to be applicable for large-scale clinical lipidomics and represent reliable high-throughput alternatives to existing longer separation methods for both untargeted and targeted analyses using high-resolution mass spectrometry. Furthermore, the influence of targeted and untargeted approaches, together with the assessment of internal standard selection on downstream statistical outcomes, provides important methodological considerations for large-scale lipidomic studies.
PMID:42508890 | DOI:10.1016/j.aca.2026.345759