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Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This new analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is based on lipid class separation using 1.7 μm particle bridged ethylene hybrid silica columns and a gradient of methanol-water-ammonium acetate mixture as a modifier. The method enables a fast separation of 30 nonpolar and polar lipid classes within 6-min analysis time covering six main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive- and negative-ion full scan and tandem mass spectra measured with high mass accuracy and high resolving power. The method is used for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive, and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in clinical research.