Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization

Negative-ion electrospray ionization tandem quadrupole mass spectrometry provides a useful method for the structural characterization of ceramides. Fragment ions referring to the identities of the fatty acid substituent and of the long chain base of the molecules a-re readily available and the structure of ceramides can be easily determined. A unique fragmentation pathway Which leads to formation of the fatty acid carboxylate anions (RCO2-) Was observed. This fragmentation is initiated by cleavage of the C2-C3 bond of the LCB to yield a N-acylaminoethanol anion ([RCONHCH2CH2O](-)), followed by rearrangement to a carboxyethylamine ([RCO2CH2CH2NH](-)) intermediate, which further dissociates to a RCO2- ion. This pathway is confirmed by the CAD tandem mass spectrum of the synthetic N-acylaminoethanol standard and of the deuterated analogs of ceramides obtained by H-D exchange. The observation of RCO2- ion species permits an unambiguous identification of the fatty acyl moiety of ceramides. Tandem mass spectrometry methods for characterization of structural isomers of ceramides using product-ion scanning and for identification of specific ceramide subclasses in biological mixtures using neutral loss scanning are also demonstrated. (J Am Soc Mass Spectrom 2002, 13, 558-570) (C) 2002 American Society for Mass Spectrometry.