Analysis of reducing sugars as their chromophoric hydrazones by high-performance liquid chromatography

A chromophoric hydrazide, 4'-N,N-dimethylamino-4-azobenzene sulfonyl hydrazide (DABS-hydrazide), was prepared from 4'-N,N-dimethylamino-4-azobenzene sulfonyl chloride by reaction with hydrazine. Reducing sugars were derivatized with DABS-hydrazide at 50 degrees C for 120 min. The chromophoric hydrazones were separated by reversed-phase HPLC isocratically using a short column (4.6 X 50 mm) and 0.08 M acetic acid-acetone as an eluant with no sample pretreatment and were quantitated at the picomole level. This method was applied to the sugar analysis of 5 micrograms of glycoproteins. Dansyl hydrazide derivatives of sugars were also separated by this HPLC system.