Comprehensive Quantitative Profiling of Less Polar Lipids in Human Plasma Using Validated Reversed-Phase UHPSFC/MS/MS

The quantitative analysis of lipids is challenging due to their structural diversity and the coexistence of numerous isomers in biological samples. Here, we present a robust and validated method based on reversed-phase ultrahigh-performance supercritical fluid chromatography-tandem mass spectrometry (RP-UHPSFC/MS/MS) for the high-throughput profiling of less polar lipids in human plasma. The sample preparation workflow involves an initial Folch-based extraction, followed by chemical derivatization with benzoyl chloride, and the final liquid–liquid extraction into hexane to isolate less polar acylated analytes. This strategy enables the sensitive profiling of six major lipid classes (TG, DG, MG, SE, ST, and FA) in positive ion mode in less than 18 min. The use of two C18 columns in series provides high chromatographic resolution, sufficient to separate isomeric species, including cis/trans and positional isomers of double bonds in fatty acids. The method was rigorously validated for five lipid classes, except free FA, and the implementation of response factors was shown to be essential for the accurate quantification of sterol esters. A total of 147 lipid species were quantified in NIST SRM 1950 human plasma, demonstrating its suitability for large-scale lipidomic studies focused on less polar lipids.