Simultaneous Quantitative Determination Method for Sphingolipid Metabolites by Liquid Chromatography/Ionspray Ionization Tandem Mass Spectrometry

Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simultaneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/MS). The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid–liquid extraction procedure with a mixture of chloroform and methanol. After separation on a short C18silica column eluted in a gradient mode, the metabolites were detected by MS/MS. This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/106cells. The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day. We also detected endogenous sphingosine 1-phosphate in HL-60 cells. The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase. It was found that sphingomyelinase fromBacillus cereusmay have selectivity for acyl chain length.