High-Performance Liquid Chromatography-Mass Spectrometry of Glycosphingolipids: I. Structural Characterization of Molecular Species of GlcCer and IV3βGal-Gb4Cer1

A method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of glycosphingolipids was developed, which involves a frit interface between the HPLC and the MS. The molecular species of glucosylceramide (GlcCer) purified from the spleen of a patient with Gaucher's disease and galactosylglobotetraosylceramide (IV3 beta Gal-Gb4Cer) from mouse kidney were analyzed using this system on a reversed-phase column, with methanol containing 1% glycerol as the elution solvent. The injection of 1 microgram of GlcCer gave the mass spectra of seven major molecular species, the pseudo-molecular ion for each of the seven molecular species being observed at m/z 698, 726, 754, 782, 808, 796, and 810, respectively. The injection of 200 pg of synthetic N-stearoyl glucosylsphingosine (d18:1) gave a clear peak with the single ion monitoring method detecting the pseudo-molecular ion at m/z 726. The injection of 5 micrograms of IV3 beta Gal-Gb4Cer gave the mass spectra of six major molecular species, the pseudo-molecular ions being observed at m/z 1,489, 1,471, 1,515, 1,497, 1,517, and 1,499. This report deals with a new HPLC/FAB/MS system, which was successfully applied to the structural characterization of the molecular species of neutral glycosphingolipids, and the system is a quite promising for development into a quantitative method for glycosphingolipids with high sensitivity and specificity.