In-depth phospholipid profiling of lymphoma cells by HPLC coupled with trapped ion mobility spectrometry and high-resolution tandem mass spectrometry

), exact mass-to-charge ratio (m/z), collision cross section (CCS) and mobility-resolved MS/MS data. Applied to human lymphoma cell lipid extracts, the workflow enabled confident identification of 263 individual lipid species spanning 10 phospholipid and 2 sphingolipid subclasses, including the resolution of 63 isomeric species at the fatty-acyl compositional level. The multidimensional approach allowed partial discrimination of fatty-acyl compositional, sn- and double bond positional isomers. Notably, characteristic deviations in both retention time and ion mobility were observed for plasmalogens relative to alkyl-ether linked phospholipids, reflecting the unique physicochemical properties of the vinyl-ether linkage. These systematic offsets enabled confident plasmalogen assignment in representative cases, supported by authentic standards, co-chromatograms and mobility-resolved fragmentation data. Collectively, this streamlined analytical platform markedly expands phospholipidome coverage and provides enhanced structural resolution of complex lipid mixtures.