Analysis of derivatized ceramides and neutral glycosphingolipids by high-performance tandem mass spectrometry

Microscale reduction of ceramides and neutral glycosphingolipids has been evaluated as a means of improving their analysis by fast atom bombardment mass spectrometry, alone and in combination with tandem mass spectrometry. Reduction (conversion of the amide to an amine) of native ceramides and glycosphingolipids containing one to three sugars yields derivatives that show significant signal enhancement. This sensitivity increase allows the acquisition of normal and tandem fast atom bombardment mass spectra from a submicrogram amount of sample. Concomitant permethylation is required for glycosphingolipids that contain more than three sugars. Collision induced dissociation mass spectra of protonated molecular ions, recorded on a four sector instrument, show improved fragmentation allowing the simultaneous characterization of both the ceramide and carbohydrate portions of glycosphingolipids. The reductions are carried out at the nanogram to microgram level with borane, reacting the solid sample with condensed reagent vapor. The borane reduction method has been adapted for this class of substances by adding an oxidation step in order to convert unsaturated lipids to hydroxylated derivatives by oxidation of the resulting organoborane. This approach, used in conjunction with tandem mass spectrometry, allows the determination of olefinic bond location. Labeled derivatives have been prepared by reacting the substrates with trideuterioborane and were used to ascertain the fragmentations and localize olefinic bonds. The collision induced fragmentation of reduced ceramides and neutral glycosphingolipids is only weakly affected by the presence of additional functionalities, such as methoxyl (after permethylation) and hydroxyl groups (resulting from hydroboration and oxidation), a characteristic which facilitates interpretation of the spectra of unknown compounds.