Analytical and preparative procedures are described for high-performance liquid chromatography (HPLC) fractionation of gangliosides without previous derivatization. These procedures make use of a reversed-phase Lichrosorb RB-8 or pBondapak RP-18 column, and of a mixture of acetonitrile and 5 niM phosphate buffer, at fixed or varying volume ratios, as solvent system. Peak elution from the column is monitored by flow through reading of absorbance at 195 nm. Under all the described conditions HPLC is capable of resolving all common gangliosides and of separating each of them into four molecular species containing C18sphingosine, C 18-sphinganine, C20-sphingosine, or C20-sphinganine.
The analytical method has been successfully applied to fractionation of ganglioside mixtures from calf brain and to verification of homogeneity of singleganglioside preparations. It is suitable for quantitative purposes, with high sensitivity (detection limit, 0.1 nmole) and precision (SD less than 10% of mean values in the concentration range 0.1-50 nmoles). The semipreparative method, which provides successive cycles of analysis in a fully automated way, enables the preparation in 2-4 days of 100-mg amounts of each molecular species starting from single gangliosides, like GMl and GDla. The preparative method makes use of acetonitrile-phosphate buffer-tetrahydrofuran as eluting solvent, and requires the addition to the starting ganglioside of the corresponding radioactive compound as tracer. This procedure, applied to GM1 ganglioside, is devised for processing up to 50 mg of ganglioside per analysis.