ANALYSIS OF THE MORGAN-ELSON CHROMOGENS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The Morgan-Elson method for quantitative N-acetylhexosamine analysis i s a two-step procedure comprising alkali treatment of the sugar and su bsequent condensation of the resulting chromogens with p-dimethylamino benzaldehyde (Ehrlich's reagent) to yield a colored product. In the pr esent investigation, the products formed in the first step of the proc edure were analyzed by high-performance liquid chromatography (HPLC) o n a reversed-phase (C-18) column, which was eluted with a water-methan ol gradient; the absorbance of the effluent was monitored at 229 nm. T he profile generated fi om alkali-treated N-acetylglucosamine exhibite d two major peaks, in a ratio of similar to 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the pe ak area, was eluted in an intermediate position, and several smaller p eaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's re agent, indicating that they were Morgan-Elson chromogens. The HPLC pro file of alkali-treated N-acetylmannosamine was identical to that of th e products generated from N-acetylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the ch romogens. N-Acetylgalactosamine yielded two major peaks, which were el uted in the same positions as the two major products formed from N-ace tylglucosamine, but the intermediate peak seen in the N-acetylglucosam ine pattern was absent. The HPLC procedure allowed detection of as lit tle as similar to 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when o nly small amounts of sample are available for quantitative analysis. ( C) 1997 Academic Press.