Targeted Lipidomics of Sphingolipids

Lipids were extracted using a 1:1 mixture of methanol and butanol containing a panel of internal lipid standards (Avanti^® Polar Lipids). Sphingolipids were quantified using targeted lipidomics on a TSQ Altis triple quadrupole mass spectrometer, following lipid separation using an Agilent Eclipse Plus C8 column on a Vanquish^™ ultra high-performance liquid chromatography system [35 (link)]. Peaks were integrated using Xcalibur [35 (link)]. Absolute concentrations were determined by normalization against corresponding internal standards and tissue weight. Hierarchical clustering was employed to group similar patterns of lipid changes using Euclidean distance and Ward’s algorithm, where Z-scores were calculated to measure the relative deviation of individual lipid concentrations from the mean values (Metaboanalyst 5.0).