Continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry of complex lipidomic samples

Abstract

A new continuous comprehensive two-dimensional liquid chromatography-electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 μm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 μm, core-shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes.

Quantitative analysis of N‐acylphosphatidylethanolamine molecular species in rat brain using solid‐phase extraction combined with reversed‐phase chromatography and tandem mass spectrometry

“…With the RP chromatography used, retention increases with the total number of carbon atoms, while double bonds lead to a decrease in retention, as described by the equivalent carbon number model for lipids 43 and demonstrated in Supporting Information Fig. S2.…”

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

“…Holčapek et al. applied RPLC × HILIC–MS for the analysis of complex lipidomic samples . Because the authors focused on phospholipids, the gradient span in the HILIC dimension could be narrow, allowing relatively short re‐equilibration times.…”

Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis

“…For example, two-dimensional LC-MS/MS was used to enhance the profiling of ceramide and phosphatidylcholine species in mouse liver [32]. For lipidomic profiling of biological samples, 143 lipid species from 10 lipid classes were identified in human plasma and porcine brain samples using a continuous two dimensional LC-MS/MS method, with RPLC as the first dimension and HILIC as the second dimension [17]. Using an online NPLC/RPLC-MS/MS method, Nie et al identified 721 endogenous lipid species from 12 lipid classes in rat peritoneal surface, but only 415 of them were structurally confirmed using MS/MS [13].…”

“…Both modes have advantages and disadvantages. Although RPLC in the first dimension and HILIC in the second dimension have been demonstrated [17], most of the on-line mode uses NPLC/HILIC in the first dimension and RPLC in the second dimension for maximum separation power. Due to the incompatibility of the effluents from the first dimension with the second dimension column, stop-flow in the first NPLC dimension coupled with a solvent evaporation interface [13], dual loop trapping coupled with solvent evaporation for continuous separation in the first NPLC dimension [18], stop-flow in the first HILIC dimension [5], and stop-flow in the first HILIC dimension coupled with trap column [19] have been developed over the years.…”