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 simulataneously 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 C18 silica 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/ 10(6) cells. 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 from Bacillus cereus may have selectivity for acyl chain length.
Mano
N
N
Section of Analytical Chemistry, Tsukuba Research Laboratories, Eisar Company Ltd., Ibaraki, Japan.
Oda
Y
Y
Yamada
K
K
Asakawa
N
N
Katayama
K
K
D002518:Ceramides / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D008246:Lysophospholipids; D013058:Mass Spectrometry / Q000379:methods*; D013107:Sphingolipids / Q000032:analysis*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis
D002518:Ceramides; D008246:Lysophospholipids; D013107:Sphingolipids; C060506:sphingosine 1-phosphate; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine