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R.E.W. Hancock Laboratory: Methods 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 100oC.
  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.