Quantification of Ceramides and Diacylglycerols

Ceramides and diacylglycerols (DAG) were measured according to the methods as previously described [43 (link),44 (link)]. Briefly, lipids were extracted from ~20mg of tissue using an extraction mixture consisting of isopropanol:water:ethyl acetate (35:5:60; v:v:v). The ceramides and DAG were measured using an Agilent 6460 triple quadrupole mass spectrometer. Both sphingolipids and DAG were analyzed using a positive ion electrospray ionization source with multiple reaction monitoring. Chromatographic separation was performed using an Agilent 1290 Infinity Ultra Performance Liquid Chromatograph. The analytical column was a reverse-phase Zorbax SB-C8, 2.1x150 mm, 1.8 μm (Agilent, USA). The separation was conducted in a binary gradient using 2 mM ammonium formate, 0.15% formic acid in methanol as Solvent A and 1.5 mM ammonium formate, 0.1% formic acid in water as Solvent B at a flow rate of 0.4 ml/min. C17:0-ceramide and 1,3-dipentadecanoyl-rac-glycerol (Avanti Polar Lipids, USA) were used as the internal standards.