Analysis of native neutral glycosphingolipids by combined high performance liquid chromatography/mass spectrometry

Conditions for normal phase high performance liquid chromatography-chemical ionization mass spectrometry of underivatized glycosphingolipids with the use of a moving belt transport interface were investigated. Chromatography was performed on a 2 mm ID X 3 cm column packed with 5 micron spherical silica. A gradient of increasing methanol and water in methylene chloride was used to resolve mono-, di-, tri-, and tetraglycosylceramides and sulfatides in less than 15 min. A polyimide belt was used to transport the sample residues to the mass spectrometer where ammonia chemical ionization mass spectrometry was used to obtain spectra. One to 5 micrograms of each component was sufficient to obtain full spectral scans. Mono- and dihexosylceramide spectra showed [M + H]+ ions of good abundance, while the higher glycosphingolipids have molecular weights that exceed the range of the mass spectrometer utilized. All glycosphingolipids gave ions characteristic of their ceramide, fatty acid, long chain base and carbohydrate components. Sequence information which reflected the relative position of hexose and N-acetylhexosamine residues was also obtained.