Development of an LC-MS/MS method for the analysis of free sphingoid bases using 4-fluoro-7-nitrobenzofurazan (NBD-F)

The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis-8 and trans-8 isomers of sphingoid bases by reversed phase HPLC on a C18 column. The extraction and derivatization procedures and LC-MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.