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

✍ Scribed by Nariyasu Mano; Yoshiya Oda; Koji Yamada; Naoki Asakawa; Kouichi Katayama

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 170 KB
• Volume: 244
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.9891

✦ Synopsis

added to cells, sphingolipid-derived molecules, sphin-Sphingolipid metabolites ceramide, sphingomyelin, gosine and ceramide, elicit various pharmacological resphingosine, psycosine, sphingosylphosphorylcholine, sponses, such as platelet and neutrophil activation, inand dimethylsphingosine were separated and simultahibition of growth factor action, modulation of receptor neously quantitated by liquid chromatography/ionfunction, and inhibition of phorbol ester-induced respray ionization tandem mass spectrometry (LC/MS/ sponses (3-10). Sphingolipids and lysosphingolipids MS). The use of glassware throughout minimized also affect significant cellular responses and exhibit losses due to adsorption and the pretreatment of this antitumor promoter activities in various mammalian method consisted of simple liquid-liquid extraction cells. At least some of these molecules may function as procedure with a mixture of chloroform and methanol. endogenous modulators of cell function and possibly as After separation on a short C 18 silica column eluted in second messengers. Nonetheless, their mechanisms of a gradient mode, the metabolites were detected by MS/ action remain largely enigmatic. In view of the increas-MS. This assay allows simultaneously quantification of ing interest in sphingolipid metabolites sensitive and these metabolites over a range of at least 0.1 to 100 ng/ specific methods are needed to measure their endoge-10 6 cells. The LC/MS/MS analyses took 10 to 15 min per nous levels. sample and we could examine up to 50 samples per Several methods are available for the quantitation day. We also detected endogenous sphingosine 1-phosof sphingolipid metabolites by chromatography after phate in HL-60 cells. The utility of the method was fluorescent (11-14) or radioisotope derivatization (15demonstrated by examining changes in metabolites