In-depth structural characterization of sphingoid bases via derivatization and electron-activated dissociation tandem mass spectrometry

Sphingoid bases (SPHs) serve as the core structural backbone of all sphingolipid classes, with their diversity arising from intrachain modifications such as carbon–carbon double bonds (CC), hydroxyl groups, and methyl branching. Traditional tandem mass spectrometry (MS/MS) relying on collision-induced dissociation (CID) often fails to yield diagnostic fragmentation patterns for precise localization of these modifications, underscoring the need for advanced dissociation techniques. In this work, we present a novel analytical strategy combining carnitine derivatization of sphingoid amines with electron-activated dissociation (EAD) in MS² to enable in-depth structural characterization. This technique uniquely suppresses intrachain fragmentation while generating diagnostic ions at modification sites, resulting in simplified mass spectra that facilitate unambiguous identification of subtle structural variations. This method was integrated into a reversed-phase liquid chromatography-mass spectrometry workflow and further applied for in-depth profiling of total SPHs in Astragalus and Escherichia coli (E. coli). Our analysis uncovered two pairs of regioisomeric SPHs: CC positional isomers of SPH d18:1 in Astragalus and hydroxylation positional isomers of SPH t18:1 in E. coli, demonstrating the method's utility in resolving structurally complex sphingolipid bases.