1-O-acylceramide is a new class of epidermal ceramide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H − H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H − H2O − (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H − H2O − (1-O-FA)]+ ions that yielded multiple sets of specific ions.
