A Procedure\nfor Solid-Phase Extractions Using Metal-Oxide-Coated\nSilica Column in Lipidomics

Lipid\nenrichment is indispensable for enhancing the coverage\nof\ntargeted molecules in mass spectrometry (MS)-based lipidomics studies.\nIn this study, we developed a simple stepwise fractionation method\nusing a titanium- and zirconium-dioxide-coated solid-phase extraction\n(SPE) silica column that separates neutral lipids, phospholipids,\nand other lipids, including fatty acids (FAs) and glycolipids. Chloroform\nwas used to dissolve the lipids, and neutral lipids, including steryl\nesters,\ndiacylglycerols, and triacylglycerols, were collected in the loading\nfraction. Second, methanol with formic acid (99:1, v/v) was used to\nretrieve FAs, ceramides, and glycolipids, including glycosylated ceramides\nand glycosylated diacylglycerols, by competing for affinity with the\nLewis acid sites on the metal oxide surface. Finally, phospholipids\nstrongly retained via chemoaffinity interactions were eluted using\na solution containing 5% ammonia and high water content (45:50 v/v,\n2-propanol:water), which canceled the electrostatic and chelating\ninteractions with the SPE column. High average reproducibility of\n<10% and coverage of ∼100% compared to those of the non-SPE\nsamples were demonstrated by untargeted lipidomics of human plasma\nand mouse brain, testis, and feces. The advantage of our procedure\nwas showcased by characterizing minor lipid subclasses, including\ndihexosylceramides containing very long-chain polyunsaturated FA in\nthe testis, monogalactosyl and digalactosyl monoacylglycerols in feces,\nand acetylated and glycolylated derivatives of gangliosides in the\nbrain that were not detected using conventional solvent extraction\nmethods. Likewise, the value of our method in biology is maximized\nduring glycolipidome profiling in the absence of neutral lipids and\nphospholipids that cover more than 80% of the chromatographic peaks.