Sugar analysis using alditol acetates

The hydrolysis of glycosides and polysaccharides to reducing sugars and the concomitant conversion to alditol acetates (borohydride reduction and acetylation) is a standard method to analyse polysaccharides containing aldoses, ketoses, deoxyaldoses, and acetamidohexoses and other related sugars. Sugars that cannot be observed with this analysis are uronic acids, ulosonic acids (e.g. Kdo), 4-aminosugars, or charged species like phosphorylated sugars which may arise in a hydrolysate. The acids are not observed because the sodium salt of the acid that is formed on addition of NaBH₄ is not volatile. Of special concern is the difference in rate of hydrolysis between sugars. Thus, uronic acids are seldom completely hydrolysed and the sugar that the uronic acid is linked to is underrepresented. 2-Acetamidohexoses undergo partial N-deacetylation and the resulting 2-aminosugars are not hydrolysed at all and both the 2-aminosugar and the sugar to which it is linked are underrepresented. Normally, yields of 60-80% are obtained. 4-acetamido sugars are hydrolysed to pyrrole derivatives which are polymerised and such sugars are therefore not observed. Methanolysis is however possible with 4-acetamido sugars.

Note that all ketoses on reduction are expected to yield approximately equal amounts of both the R- and the S-isomer, which may or may not separate on GLC analysis. Thus, fructose gives an equimolar mixture of glucitol and mannitol.


Fructose		Glucitol (Sorbit)		Mannitol

A number of acids may be used for the hydrolysis, the most common are trifluoroacetic acid, (TFA) sulfuric acid, and hydrochloric acid. Trifluoroacetic acid is somewhat weaker than the others but is fairly easy to evaporate.

Flow scheme

Hydrolysis

Reduction

Acetylation

Reagents

Trifluoroacetic acid (TFA), 0.5 or 2 M
Methanol
Sodium borohydride, 0.25 M in 1 M NH₄OH
Ammonia 1 M
Acetic acid 10% in methanol
Acetic anhydride
Pyridine

Procedure

Transfer sample (ca 0.2 mg, ~1 µmol) to a 13 x 100 mm screw cap tube.
Option: Add internal standard, 50 µg of xylose or suitable sugar.
Hydrolyse in ~0.3 mL 2M TFA at 120° for 2h or in 0.5M TFA at 100° 12-16 h.
Evaporate the solution to dryness by a stream of compressed air, add 0.5mL MeOH, and evaporate. Repeat once.
Reduce with 0.3 mL fresh solution of NaBH₄ in NH₃ for 30 min at 20°
Quench with 0.5 mL 10% HOAc in MeOH, evaporate to dryness. Add 0.5 mL 10% HOAc in MeOH and evaporate to dryness. Repeat once or twice. Add 0.5mL MeOH and evaporate to dryness. Repeat once or twice.
Acetylate with 0.1 mL Ac₂O and 0.1 mL pyridine 100° 20 min. Add 50 µL of water if problems.
Evaporate the solution and add 0.5 mL toluene, evaporate to dryness. Repeat once.
Partition between 0.5 mL H₂O and 0.5 mL EtOAc by stirring fast but not violently using a triangular magnetic rod in a conical vial (Reacti-vial type) for a couple of minutes. Transfer the upper EtOAc phase to the old rinsed tube. Add another 0.5 mL EtOAc and extract. Repeat a third time. Concentrate to dryness dissolve in ca 0.2 mL EtOAc, transfer into sample tube and concentrate to 25-50 µL.

Comments

For an exact weight of a sample of ca 0.2 mg take 1-2 mg and dissolve in 1 mL of water and take out appropriate volume.
Too little liquid will give only drops on the wall and little in the bottom, so use >0.3 mL.
Use blow down equipment throughout, normally compressed air is dry enough to be used, if not use N₂ .
Trace amounts of acid are almost impossible to get rid of but will not harm this step. Bubbles of hydrogen on the bottom normally indicate excess NaBH₄ but in ammonia they may be scarce. The solution of NaBH₄ in ammonia easily lasts a week. If not enough BH₄ ^- is present cyclic acetates will be formed which are fast-moving on the GLC column and with m/z 115 and 157 as prominent peaks in the mass spectrum.
NaBD₄ -reduction, which is performed as described above except that NaBD₄ is used instead of NaBH₄ , and will give CHDOAc as the top fragment.
Acetic acid will convert the borohydride to boric acid which must be removed by acidic methanol as its methyl ester i.e. methyl borate. The procedure must be repeated. If boric acid is left it may form complexes with the alditols and cause underacetylation.
Be careful so that no water is removed with the organic phase. Use holder for the Reacti-vial. Alternatively partition may be effected with the pasteur pipette.

Test substances, Standards