Glucosamine Rapid AssayMRTHOD:
- Place sample (containing 0.5 - 10 µg GlcN) in a Pyrex screw capped tube.
- Add HCl to a final concentration of 2N and a final volume of 0.6 ml. Flush with nitrogen.
- Stopper tightly and hydrolyze 16h at 100oC.
- 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.
- Neutralize samples and standards with 0.4 ml 2M Na2CO3 (10.6 g in 50 ml). (pH - 7 with the slight excess of Na2CO3)
- 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).
- Stopper tightly and heat in boiling water bath for 20 min.
- Cool. Add 1 ml EtOH.
- Add 0.5 ml Ehrlichs reagent. (1 g p dimethylaminobenzaldehyde in 15 ml EtOH and 15 ml c/HCl)
- Shake tubes vigorously to expel excess CO2.
- Maximum colour development is reached in 5 min. Chromophore stable for 1 to 2 hours.
- Read absorbance at 530 nm.
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.