Highly repeatable and selective ultrahigh-performance supercritical fluid chromatography – Mass spectrometry interclass separation in lipidomic studies

Abstract

Extensive research has recently established ultrahigh-performance supercritical fluid chromatography – mass spectrometry (UHPSFC/MS) coupling as an excellent tool for the fast and comprehensive characterization of lipids. However, the long-term instability of retention times using UHPSFC needs to be addressed, which is especially undesirable in lipidomic studies involving large clinical cohorts that are based on the comparison of lipid profiles between disease and control samples. In this work, we focused on conducting a detailed evaluation of the UHPSFC interclass separation of total lipid extracts for selective and repeatable lipidomic analysis. The selectivity of 9 chromatographic columns with different chemistry and the effect of particular chromatographic conditions on the lipid class resolution were carefully assessed. The most pronounced influence was found for the solvent type that was used as a modifier, which enabled the lipid class resolution to be finely adjusted. When propan-2-ol was added to methanol, a positive effect was observed for the resolution of mainly polar lipid classes and the intraclass resolution of lipid species was mildly suppressed; these were both favorable for the interclass separation used in this work. The optimized UHPSFC/MS method is based on a diol column and a gradient of methanol – propan-2-ol – water – ammonium formate modifier that provides the resolution of all lipid classes in brain samples within 7 min of analysis. This method has shown excellent retention times repeatability for the 555 injections in lipidomic study with the standard deviations below 0.012 min for all lipids. This demonstrates the ability of the optimized UHPSFC/MS method for highly selective and repeatable lipidomic analysis of biological samples.