Chiral supercritical fluid chromatography of monoacylglycerol and diacylglycerol enantiomers in biological samples: Adjusting selectivity via column coupling

The distinction of lipid isomers is gaining more attention in lipidomics due to their different biochemical properties in the organism. Herein, we aimed to develop a method for the analysis of monoacylglycerol (MG) and diacylglycerol (DG) enantiomers in biological samples using chiral supercritical fluid chromatography and mass spectrometry (SFC-MS). Amylose-based chiral columns showed a certain degree of separation of MG and DG isomers, but low selectivity for the acylglycerol classes in total lipid extracts, which could not be improved by modifier composition or other chromatographic conditions. The coelution of MG and DG enantiomers with highly concentrated triacylglycerols (TGs) negatively affected their MS determination based on the peak area ratio, therefore the interclass selectivity of chiral SFC was adjusted by coupling with an achiral column. The connection of the amylose tris-(3,5-dimethylphenylcarbamate) chiral column with octadecyl achiral column using a methanol as a modifier provided an excellent interclass separation of acylglycerols with the resolution of 5.53 and 15.17 for oleic acid-based MG/DG and DG/TG classes, respectively. The developed method enabled the determination of MG and DG enantiomers in complex total lipid extracts of biological samples in a 15 min gradient without time-consuming sample prefractionation. Chiral SFC-MS analysis of egg yolk, human plasma, and porcine brain samples showed different ratios of enantiomers, suggesting their unique roles within each sample type.