Determination of ceramides by LC-MS/MS

The sphingolipids were analyzed using previously reported method²⁵ with modification. Briefly, cell pellet (7.5 × 10⁶ cells) added with 50 µl of internal standard (C17:0-ceramide, 1 pM) was extracted twice with ethyl acetate/2-propanol/water (60/28/12; v/v/v). Sphingolipids were separated using gradient elution with mobile phase A (2 mM ammonium formate and 0.2% formic acid (v/v) in water) and B (1 mM ammonium formate and 0.2% formic acid (v/v) in methanol) on an Agilent 1200 series high-performance liquid chromatography (HPLC) system with Spectra C8SR column (3 µm, 150 × 3.0 mm, Peeke Scientific, Redwood, CA). Mass spectrometric detection was performed by multiple reaction monitoring (MRM) mode on a Sciex 4000 QTRAP mass spectrometer (AB Sciex, Framingham, MA) operating in positive ion mode. Analyst software 1.6.3 (AB Sciex) was used for the data acquisition as well as processing. Sphingolipid data generated from liquid chromatography-tandem mass spectrometry (LC-MS/MS) were normalized to lipid phosphate as previously described²⁶.