Comprehensive Lipidomic Characterization of Human Blood by High-Throughput UHPSFC/MS using Bioinert Column and Modified MTBE Extraction

Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Here, we introduce a novel ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method using a bioinert column, enabling the separation of lipids with a broad polarity range from nonpolar to ionic species. The UHPSFC/MS method was optimized using 79 lipid species across 41 lipid subclasses, achieving a total run time of 7.5 min, including the column equilibration. The comparison of the separation with conventional and bioinert columns revealed a substantial improvement in peak shapes for ionic lipid classes, such as PS, LPS, PA, LPA, CerP, and SPBP. Additionally, we introduce a combination of the modified chloroform-free extraction followed by a hexane elimination step, which reduces concentrations of nonpolar lipids allowing the injection of more concentrated extracts and minimize ion source contamination. The optimized methodology was applied for the untargeted analysis of human plasma and erythrocyte-rich fraction to achieve highly confident identification of 657 lipid species across 37 lipid subclasses in human blood. The method followed the recommendations for validation of (bio)analytical methods, and its accuracy was confirmed by quantitative analysis of the reference material NIST SRM 1950, with the determined concentrations in agreement with the consensus values from ring trials. The current methodology represents a novel high-throughput and comprehensive quantitative lipidomic method for biological samples. Moreover, the findings provide the potential for the development of bioinert components for SFC platforms.