A procedure for solid phase extractions using metal oxide coated silica column in lipidomics

Abstract

Lipid enrichment is often indispensable to enhance the coverage of targeted molecules in mass spectrometry (MS)-based lip-idomics studies. This study developed a simple stepwise fractionation method using titanium and zirconium dioxide coated solid phase (SP) silica column that separates neutral lipids, phospholipids, and other lipids including glycolipids and several polar lipids such as sulfatides. Our procedure used chloroform to dissolve lipids and the neutral lipids including cholesteryl ester, di- and triacyl glycerols were collected in the loading fraction. Secondly, methanol with formic acid (99:1, v/v) retrieved sphingolipids, glycolipids, and fatty acids by competing for affinity with Lewis acid sites on the metal oxide surface. Finally, phospholipids strongly retained via chemoaffinity interaction were eluted using a solution containing 5% ammonia and high water content (45:50 v/v, 2-propanol:water), which cancels electrostatic- and chelating interactions with the SP column. The high average reproducibility of <10% and the coverage of ~100% compared to non-SP extracts were demonstrated by untarget-ed lipidomics of human plasma, and mouse brain and feces. The advantage of our procedure was showcased by characterizing minor lipid subclasses mono- and digalactosyl monoacylglycerols in feces and acetylated- and glycolylated derivatives of gan-gliosides in brain that were not detected in conventional monophase or liquid-liquid extraction methods. Likewise, the value of our method in biology is maximized during glycolipidome profiling by the absence of phospholipids and glycolipids that cover >80% of the chromatographic peaks.