Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography

A new anion-exchange HPLC method for separation of gangliosides has been developed using TMAE (trimethylaminoethyl)-Fractogel as the stationary phase and a gradient system of ammonium acetate in methanol for elution. Chromatography of mouse brain gangliosides resulted in separation according not only to their degree of sialylation into mono-, di-, tri- and tetrasialogangliosides, but also in separation of positional isomers within the four elution domains, e.g. GD1a (IV3Neu5Ac,II3Neu5Ac-GgOse4Cer) and GD1b (II3(Neu5Ac)2-GgOse4Cer) of the disialoganglioside fraction. Furthermore, base-line separation of alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides e.g. IV3Neu5Ac-nLcOse4Cer and IV6Neu5Ac-nLcOse4Cer, isolated from human granulocytes, was achieved and pure fractions of each ganglioside were obtained. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the isolation of pure gangliosides on a preparative scale by one-step column chromatography.