Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard

A highly sensitive method for quantification of sialic acids in ganglioside s was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedi oxy)benzene (DMB) and separated on a reversed-phase C-18 column with an iso cratic elution, As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived fro m the reagents. When gangliosides were subjected to acid hydrolysis, the ve locity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore ga ngliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was sy nthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation, Fo lch partition was used to decrease lipophilic materials included in the sam ple, and the sialic acids released were recovered from the upper phase, The present method has a satisfactory sensitivity in the simultaneous quantifi cation of NeuAc and NeuGc in purified gangliosides as well as in crude lipi d fractions containing a variety of gangliosides. (C) 2000 Academic Press.