Analysis and quantitation of gangliosides as p-bromophenacyl derivatives by high-performance liquid chromatography

A highly sensitive method for the analysis and quantitation of gangliosides by high-performance liquid chromatography is described. The method involves simple, quantitative and specific derivatization of gangliosides, involving their carboxylic acid groups, to UV-absorbing p-bromophenacyl derivatives. The molar extinction coefficient of the derivatives at maximum absorption at 261 nm was about 23,000. The acidic fraction separated by DEAE-Sephadex column chromatography was directly derivatized and the reaction mixture was directly injected into a high-performance liquid chromatograph without any purification of the derivatives. Monosialogangliosides, GM4, GM3, GM2, and GM1, were well separated by normal phase high-performance liquid chromatography. The standard curve for quantitation of gangliosides was linear up to 100 micrograms of ganglioside-sialic acid and the lower limit of detection was about 10 ng. Also, by reverse phase high performance liquid chromatography, the molecular species of gangliosides were successfully analyzed. The method was shown to be applicable to the analysis of gangliosides in biological materials.