A Procedure for Solid-Phase Extractions Using Metal-Oxide-Coated Silica Column in Lipidomics

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