C₁₈ ceramide analysis in mammalian cells employing reversed-phase high-performance liquid chromatography tandem mass spectrometry

Ceramides play an important role in diverse cellular functions such as differentiation, cell cycle progression, cell-cell adhesion, senescence, and apoptosis. Here we report a method of extracting lipids from mammalian cells and quantifying ceramide, where the assay conditions were optimized for reproducibility, linearity, recovery, and sensitivity. Simultaneous chromatographic separations were carried out by reversed-phase high-performance liquid chromatography coupled to electrospray ionization using a Pursuit 3 Diphenyl column (50 × 2.0 mm) and supported by a mobile phase consisting of acetonitrile plus 0.1% formic acid and 25 mM ammonium acetate. Ceramides were detected in the multiple reaction mode by tandem mass spectrometry in the positive ion mode, and all extracted ion peaks were integrated for quantitative analysis. The limits of detection and quantification achieved were 0.2 and 1.0 pg on column, respectively. Using this method, we successfully quantified and compared differences in C₁₈ ceramide levels induced by two DNA-damaging agents, mitomycin C and daunorubicin, and two apoptosis-inducing ligands, tumor necrosis factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL). This work, therefore, describes a method that will be helpful for investigating how ceramide is regulated by different chemotherapeutic agents and will help us to better understand the mechanisms of signal transduction involving ceramide.