GB/T 20365-2006Determination method for chondroitin sulfate and glucosamine hydrochloride - Liquid chromatography method (English PDF)

1 Scope

This Standard specifies the method for determining the content of chondroitin sulfate and glucosamine hydrochloride by liquid chromatography method.

This Standard applies to the determination of chondroitin sulfate and glucosamine hydrochloride extracted from cartilage tissues of cattle, pigs and sharks.

2 Normative References

The provisions in following documents become the provisions of this Standard through reference in this Standard. For dated references, the subsequent amendments (excluding corrigendum) or revisions do not apply to this Standard, however, parties who reach an agreement based on this Standard are encouraged to study if the latest versions of these documents are applicable. For undated references, the latest edition of the referenced document applies.

GB/T 6682 Water for analytical laboratory use - Specification and test methods (GB/T 6682-1992, neq ISO 3696:1987)

3 Principle

The chondroitin sulfate sample is uniformly dispersed with acetonitrile; dissolve in water; separate by liquid chromatography; measure at a wavelength of 192 nm; and quantify by the external standard method.

4 Reagents and Materials

4.1 Water: Grade-1 water specified in GB/T 6682.

4.2 Acetonitrile: Liquid chromatography grade.

4.3 Sodium pentane sulfonate: Purity >=99.5%.

4.4 Standard product of chondroitin sulfate A: Purity is about 70%; and the rest is chondroitin sulfate C. See Appendix A for the structural formula.

4.5 Standard product of chondroitin sulfate B: Purity is about 90%; and the rest are chondroitin sulfate A and C. See Appendix A for the structural formula.

4.6 Standard product of chondroitin sulfate C: Purity is about 90%; and the rest is chondroitin sulfate A. See Appendix A for the structural formula.

4.7 Glucosamine hydrochloride: Purity>=99.0%.

4.8 Standard solution: Weigh 25.0 mg of chondroitin sulfate standard A (or B, or C) which has been dried at 105°C for 4 h, and 25.0 mg of glucosamine hydrochloride in a beaker; add 5.0 mL of acetonitrile; shake to disperse evenly. Then add water; dissolve it with ultrasonic waves; transfer to a 50 mL volumetric flask; and dilute to the mark with water. The concentration of chondroitin sulfate is about 0.5 mg/mL; and the concentration of glucosamine hydrochloride is about 0.5 mg/mL.

4.9 Sodium pentane sulfonate solution: Weigh 0.87g of sodium pentane sulfonate solution and dissolve it in 500 mL of water. The concentration of this solution is 10 mmol/L.

5 Instruments and apparatuses

5.1 Ultrasonic generator.

5.2 Analytical balance: Sensitivity is 0.1mg.

5.3 High-performance liquid chromatography: Equipped with a diode array detector or an ultraviolet detector.

5.4 Filter membrane: polytetrafluoroethylene (PTFE) membrane, 0.2µm.

6 Test Procedures

7 Calculation of Results

The content of the tested substance is calculated according to Formula (1):

Where:

Wi - the percentage of the tested substance in the sample, in %;

Ai - the chromatographic peak area of the tested substance in the sample solution; mis - the mass of the standard product of tested substance in the standard solution, in mg;

Ais - the chromatographic peak area of the standard product of tested substance in the standard solution; m - the mass of the sample, in mg. […]