Profiling of lipid species by normal-phase liquid chromatography, nanoelectrospray ionization, and ion trap–orbitrap mass spectrometry

Detailed analysis of lipid species can be challenging due to their structural diversity and wide concentration range in cells, tissues, and biofluids. To address these analytical challenges, we devised a reproducible, sensitive, and integrated lipidomics workflow based on normal-phase liquid chromatography–Fourier transform mass spectrometry (LC–FTMS) and LC–ITMS2 (ion trap tandem mass spectrometry) for profiling and structural analysis of lipid species. The workflow uses a normal-phase LC system for efficient separation of apolar and polar lipid species combined with sensitive and specific analysis powered by a chip-based nanoelectrospray ion source and a hybrid ion trap–orbitrap mass spectrometer. The workflow was executed using a primary LC–FTMS survey routine for identification and profiling of lipid species based on high-mass accuracy and retention time followed by a targeted LC–ITMS2 routine for characterizing the fatty acid moieties of identified lipid species. We benchmarked the performance of the workflow by characterizing the chromatographic properties of the LC–MS system for general lipid analysis. In addition, we demonstrate the efficacy of the workflow by reporting a study of low-abundant triacylglycerol and ceramide species in mouse brain cerebellum and 3T3-L1 adipocytes, respectively. The workflow described here is generic and can be extended for detailed lipid analysis of sample matrices having a wide range of lipid compositions.