Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Carbohydrate-Containing Proteins
Determination of neutral sugars and aminosugars as alditol acetates by GLC

The method is rapid and experimentally straightforward. Amino sugars are first deaminated by Treatment with nitrous acid, yielding 2,5-anhydromannose (from glucosamine), 2,5-anhydrotalose (from galactosamine), and glucose (from mannosamine) as the major products. Alditol acetates prepared directly from amino sugars exhibit significantly longer retention times compared to those of neutral sugar derivatives on commonly used columns.

Deamination. To an aqueous solution of the sample (0.1 mL) containing up to 2 mg of amino sugar, 100 mg of solid sodium nitrite is added. The mixture is cooled in ice and treated with 0.1 mL of 9 M sulfuric acid, kept for 5 min, and then allowed to warm gradually to room Temperature. After 25 min, the solution is made alkaline with 0.2 mL of 15 M aqueous ammonia, followed by reduction andalkylation According to the Procedure described in [29].

Reduction. To 0.1 mL of the sugar solution in 1 M ammonia, 1 mL of a sodium borohydride solution (2% w/v) in anhydrous dimethyl sulfoxide is added. The mixture is kept for 90 min at 40 °C, and excess borohydride is decomposed by The addition of 0.1 mL of glacial acetic acid.

Acetylation. 1-Methylimidazole (0.2 mL) as a catalyst and then acetic anhydride (2 mL) are added to the reduced monosaccharide. The mixture is stirred, left at room temperature for 10 min, and then treated with 5 mL of Water to decompose excess acetic anhydride. After cooling, 1 mL of dichloroethane is added, and the mixture is shaken. Following phase Separation, the lower layer is removed for GLC analysis. A 2 µL aliquot is injected onto the Column, and the remainder is stored at —20 °C.

Gas-Liquid Chromatography. A SCOT Glass capillary column (28.5 m×0.5 mm) with a Silar 10C liquid phase is used. Temperature program: heating to 190 °C in 4 min, and then up to 250 °C at a rate of 4 deg/min.

Fifteen alditol acetates of deaminated amino sugars were separated in less than 20 min. The method has been successfully applied to the analysis of Chitin and Glycoproteins.