Critical study of the alditol acetate method for quantitating small quantities of hexoses and hexosamines in gangliosides.

An extensive investigation of and improvement in the method for quantitating the carbohydrates of glycolipids by the gas--liquid chromatography of their alditol acetate derivatives is described. The effects of duration and temperature of hydrolysis, neutralization after hydrolysis, and acetylation time and temperature on the relative detector responses of mixtures of free as well as ganglioside hexoses and hexosamines were extensively studied. It is concluded that optimum results are obtained with the following conditions: hydrolysis at 100 degree C for 8 to 12 h, a 40--60 min reduction, acetylation at 100 degree C for 30 min. When quantitating the carbohydrate components of gangliosides, the most reliable results will be obtained using as an external standard a glycolipid whose chemical structure is similar to that of the sample. Using this procedure, reliable results can be obtained with the initial glycolipid sample containing as little as 1 microgram of each sugar.