High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives

We found that 1-phenyl-3-methyl-5-pyrazolone reacts with reducing carbohydrates almost quantitatively to yield 2:1 compounds having no stereoisomers, which strongly absorb the uv light at 245 nm and are easily oxidizable on a glassy carbon electrode. Reversephase partition chromatography on a column of Capcell Pak C18 with uv or electrochemical detection allowed rapid analysis of aldoses and N-acetylhexosamines with the detection limit of 1 pmol or 100 fmol, respectively. This method proved especially useful for analysis of component monosaccharides of glycoproteins. It was also shown to be valid for separation of reducing oligosaccharides; maltodextrins with a degree of polymerization up to 19 were similarly derivatized and separated on this stationary phase.