Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography.
The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-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-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 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-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, 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 from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-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.
Rodén
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Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham 35294, USA.
Yu
H
H
Jin
J
J
Ekborg
G
G
Estock
A
A
Krishna
N R
NR
Livant
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P
D000116:Acetylgalactosamine / Q000032:analysis / Q000378:metabolism; D000117:Acetylglucosamine / Q000032:analysis / Q000378:metabolism; D001881:Borates; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002863:Chromogenic Compounds / Q000032:analysis* / Q000737:chemistry; D003116:Color; D006595:Hexosamines / Q000032:analysis / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D001881:Borates; D002863:Chromogenic Compounds; D006595:Hexosamines; D000116:Acetylgalactosamine; D000117:Acetylglucosamine; C002022:N-acetylmannosamine