Characterization of sphingomyelins in lipid extracts using a HPLC-MS-Offline-NMR method.

Sphingomyelins were characterized using a combination of a novel isocratic reversed-phase HPLC method with electrospray time-of-flight mass spectrometric detection and optional online MS/MS. The constitution of the sphingomyelins is determined by MS/MS experiments. Baseline separation of 17 compounds of a bovine brain extract (2 main compounds and 15 minor or trace compounds) was achieved with a mobile phase consisting of methanol, 2-propanol, THF, and water on a RP-18-phenyl column. In parallel, the HPLC fraction were sampled to a 600-MHz NMR spectrometer to acquire 1D and 2D NMR spectra and to elucidate the molecular structure of individual sphingomyelin components.