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.