Abstract
The Morgan–Elson method for quantitativeN-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens withp-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water–methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treatedN-acetylglucosamine exhibited two major peaks, in a ratio of ∼2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan–Elson chromogens. The HPLC profile of alkali-treatedN-acetylmannosamine was identical to that of the products generated fromN-acetylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens.N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed fromN-acetylglucosamine, but the intermediate peak seen in theN-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as ∼25 ng ofN-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan–Elson procedure when only small amounts of sample are available for quantitative analysis.
Published Version
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