Sample preparation and analysis of gangliosides and phospholipids from plasma by liquid chromatography coupled to mass spectrometry

Gangliosides (GG) and phospholipids (PL) are two classes of complex and polar lipids exhibiting a wide structural heterogeneity. LC/MS offers an access to this huge diversity but also faces some challenges. Efficient extraction and purification sample methods are thus required to perform accurate and reliable analyses. To this end, GG and PL were extracted from human plasma with three solvent mixtures (CHCl3/MeOH/H2O) of increasing polarity, in comparison with the reference Folch method. GG were further purified with a Phree Phospholipid Removal column, whose sorbent is designed to selectively remove PL. GG and PL classes were then separated under HILIC conditions before analysis using a QqQ mass spectrometer.Our method enabled a fast, straightforward and efficient extraction of both complex lipids, PL and GG. The extraction efficiency was much higher than the Folch method for the especially polar lipids that are GG and Lyso-PC, and similar for the various PL classes (PG, PI, PE, PS, PC, Sphingomyelin). The relative quantitative analysis of GG, performed by single reaction monitoring thanks to their characteristic sialic acid fragment (m/z 290), revealed a massive ion-suppression effect (up to 90%) due to co-elution of PC with the less polar GG. Removal of PL is thus essential for an accurate GG analysis by LC/MS and the Phree Phospholipid Removal column appeared as a powerful tool to achieve this purification step.Our sample preparation method allows the accurate analysis of both GG and PL classes from a single biological extract