Electrospray ionization with higher-energy collision dissociation tandem mass spectrometry toward characterization of ceramides as [M + Li]⁺ ions: Mechanisms of fragmentation and structural identification

Ceramide is a huge lipid family consisting of diversified structures in which various modifications are seen in the fatty acyl chain and the long chain base (LCB). In this contribution, a higher collision energy (HCD) linear ion-trap mass spectrometric method (LIT MSⁿ) was applied to study the mechanisms underlying the fragmentation processes of ceramide molecules in 12 subclasses, which were desorbed by ESI as the [M + Li]⁺ ions. Multiple sets of fragment ions reflecting the fatty acyl chain and LCB were observed in the HCD MS² spectra for all the ceramide classes, resulting in unambiguous definition of the ceramide structures, including the chain length and the modification (α-hydroxy-, β-hydroxy-, ω-hydroxy-FA) of the fatty acyl moiety, and the types of LCB (sphingosine, phytosphigosine, 6-hydroxy-sphingosine). Thereby, this approach permits differentiation of isomeric structures and ceramide species in the biological specimen can be unveiled in detail. By application of sequential MS³, the double bond position along the fatty acyl chain of the molecule can be located, and a complete structural characterization of ceramides can be achieved.