Four-Dimensional\nLipidomic Analysis Using Comprehensive\nOnline UHPLC × UHPSFC/Tandem Mass Spectrometry

Multidimensional chromatography offers enhanced chromatographic\nresolution and peak capacity, which are crucial for analyzing complex\nsamples. This study presents a novel comprehensive online multidimensional\nchromatography method for the lipidomic analysis of biological samples,\ncombining lipid class and lipid species separation approaches. The\nmethod combines optimized reversed-phase ultrahigh-performance liquid\nchromatography (RP-UHPLC) in the first dimension, utilizing a 150\nmm long C18 column, with ultrahigh-performance supercritical fluid\nchromatography (UHPSFC) in the second dimension, using a 10 mm long\nsilica column, both with sub-2 μm particles. A key advantage\nof employing UHPSFC in the second dimension is its ability to perform\nultrafast analysis using gradient elution with a sampling time of\n0.55 min. This approach offers a significant increase in the peak\ncapacity. Compared to our routinely used 1D methods, the peak capacity\nof the 4D system is 10 times higher than RP-UHPLC and 18 times higher\nthan UHPSFC. The entire chromatographic system is coupled with a high-resolution\nquadrupole-time-of-flight (QTOF) mass analyzer using electrospray\nionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS)\nand with positive- and negative-ion polarities, enabling the detailed\ncharacterization of the lipidome. The confident identification of\nlipid species is achieved through characteristic ions in both polarity\nmodes, information from MS elevated energy (MS<sup>E</sup>) and fast\ndata-dependent analysis scans, and mass accuracy below 5 ppm. This\nanalytical method has been used to characterize the lipidomic profile\nof the total lipid extract from human plasma, which has led to the\nidentification of 298 lipid species from 16 lipid subclasses.