Glucosamine Rapid Assay

1. Place sample (containing 0.5 – 10 µg GlcN) in a Pyrex screw capped tube.
2. Add HCl to a final concentration of 2N and a final volume of 0.6 ml. Flush with nitrogen.
3. Stopper tightly and hydrolyze 16h at 100^oC.
4. Prepare standards containing 0 – 20 µg GlcN from a stock solution of 1.5 mg/ml. (Use 0, 3, 5, 8, 10, 12 ml of stock solution and add water to make up to 300 µl. Then add 300 µl 4N HCl to a final volume of 0.6 ml and final concentration of 2N). Standards need not be heated.
5. Neutralize samples and standards with 0.4 ml 2M Na₂CO₃ (10.6 g in 50 ml). (pH – 7 with the slight excess of Na₂CO₃)
6. Shake gently and add 0.5 ml (freshly prepared) 2% acetyl acetone in 1.5M Na₂CO₃. (15.9 g Sodium Carbonate + 2 ml acetyl acetone made up to 100 ml).
7. Stopper tightly and heat in boiling water bath for 20 min.
8. Cool. Add 1 ml EtOH.
9. Add 0.5 ml Ehrlichs reagent. (1 g p-dimethylaminobenzaldehyde in 15 ml EtOH and 15 ml c/HCl)
10. Shake tubes vigorously to expel excess CO₂.
11. Maximum colour development is reached in 5 min. Chromophore stable for 1 to 2 hours.
12. Read absorbance at 530 nm.

NOTE: NaCl affects colour. Therefore standards and sample should contain the same amount of salt to avoid colour depression and erroneous results. Dialysis helps to remove NaCl.