1 3 dipentadecanoyl rac glycerol

Ceramides and diacylglycerols were measured according to the methods described by Blachnio-Zabielska et al. [35 (link), 36 (link)]. Briefly, lipids were extracted from ~20 mg of tissue by the use of the extraction mixture composed of isopropanol : water : ethyl acetate (35 : 5 : 60; v : v : v). Quantitative measurement of ceramides and diacylglycerols was made using an Agilent 6460 triple quadrupole mass spectrometer. Both ceramides and diacylglycerols were analyzed using positive ion electrospray ionization (ESI) source with multiple reaction monitoring (MRM). The chromatographic separation was performed using an Agilent 1290 Infinity Ultra Performance Liquid Chromatography (UPLC). The analytical column was a reverse-phase Zorbax SB-C8, 2.1 × 150 mm, 1.8 μm (Agilent, Santa Clara, CA). Chromatographic separation was conducted in 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 the flow rate of 0.4 mL/min. C17:0-ceramide and 1,3-dipentadecanoyl-rac-glycerol (Avanti Polar Lipids, Alabaster, AL) were used as internal standards for ceramides and diacylglycerols, respectively. The HPLC grade methanol and water as well as formic acid and ammonium formate were obtained from Sigma-Aldrich (St. Louis, MO).

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.