Simultaneous determination of neutral sugars and uronic acid constituents in a novel bacterial polysaccharide using gas chromatography-mass spectrometry

Abstract

The purified novel bacterial polysaccharide was acid-hydrolyzed, followed by the subsequent derivatization using ethanethiol-trifluoroacetic acid and acetic anhydride-pyridine systems sequentially. Our findings differ from the previous reports in that the glucuronic acid was obtained through effective derivatization. The neutral sugars and glucuronic acid were analyzed using gas chromatography-mass spectrometry (GC-MS) with xyloses as an internal standard. The polysaccharide was found to be composed of fucose, glucose, glucuronic acid and galactose, with the relative molar ratio of 1.50: 1.0: 0.79: 2.06. The neutral sugars ratio was similar to the relative molar ratio for fucose, glucose and galactose of 1.76: 1.0: 1.98 through alditol acetates determined by GC. The percentages of glucuronic acid analyzed using either the carbazole and sulfuric acid method or the above method were 16.19% and 14.85%, respectively. These results indicate that it is practicable to use the derivatization method and GC-MS to quantitatively analyze neutral sugars and glucuronic acid simultaneously in polysaccharide. For GC-MS analysis, the procedure was developed for the simultaneous determination of the derivatives in 25 min, and was performed using an HP-5MS column. Molecular ion peaks were observed in the electron ionization (EI) mass spectra. The fragmentation mechanism for glucuronic acid derivative is discussed in detail.