A novel online two-dimensional supercritical fluid chromatography/reversed phase liquid chromatography-mass spectrometry method for lipid profiling.

A novel online two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography-triple-quadrupole mass spectrometry (2D SFC/RPLC-QQQ MS) method based on a vacuum solvent evaporation interface was developed for lipid profiling in human plasma, in which lipid classes were separated by the first-dimension SFC and different lipid molecular species were further separated by the second-dimension RPLC. All separation condition parameters were carefully optimized, and their influence on the chromatographic behavior of lipids is discussed. Finally, the recoveries of 11 lipid standards were all more than 88% for the interface. Besides, the limit of detection for these lipid standards was on the order of nanograms per milliliter, and the relative standard deviations of the peak area and retention time ranged from 1.54% to 19.85% and from 0.00% to 0.10%, respectively. The final 2D SFC/RPLC-QQQ MS method allowed the identification of 370 endogenous lipid species from ten lipid classes, including diacylglycerol, triacylglycerol, ceramide, glucosylceramide, galactosylceramide, lactosylceramide, sphingomyelin, acylcarnitine, phosphatidylcholine, and lysophosphatidylethanolamine, in human plasma within 38 min, which was used for screening potential lipid biomarkers in breast cancer. The 2D SFC/RPLC-QQQ MS method is a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery. Graphical Abstract.