High-performance liquid chromatographic resolution of p-nitrobenzyloxyamine derivatives of brain gangliosides

A new quantitative procedure for the high-performance liquid chromatographic (HPLC) resolution of human brain gangliosides employing reversed-phase chromatography is described. To provide a derivative which can be determined by UV absorption techniques, p-nitrobenzyloxyamine was coupled to the gangliosides. Derivatization involves ozonation and cleavage of the ceramide double bond followed by oxime formation to the nascent aldehyde. Individual gangliosides, as they were resolved by HPLC, were collected. These fractions were then identified by thin-layer chromatography (TLC) and by gas chromatography of their monosaccharides. Quantitative results were obtained along with a marked increase in sensitivity over conventional resorcinol-hydrochloric acid quantitation of TLC-resolved gangliosides.