Ceramide mass analysis by normal-phase high-performance liquid chromatography

Ceramide (Cer) and its generation from sphingomyelin by sphingomyelinase has received a lot of attention during the past few years, mainly because its formation in cells is associated with stress responses and apoptosis induction. Assays employed to quantitate cellular Cer levels are often indirect, depending on enzyme activities or based on metabolic labeling using radioactive precursors, which do not provide real mass data. This chapter describes a nonenzymatic, nonradioactive mass assay to quantitate Cer by normal-phase high-performance liquid chromatography (HPLC) after its conversion to nonpolar, chromophoric benzoate derivatives. Ceramide derivatives can be separated from other (benzoylated) lipids by isocratic normal-phase HPLC with a ChromSpher silica analytical column (4.6 × 250 mm, 5-pm pore size) and a mobile phase consisting of cyclohexane/0.45% (v/v) 2-propanol at a flow rate of 1.5 ml/min. The solvent mixture should be degassed (either by sonication or under vacuum) before use.