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Method for quantitatively detecting hyaluronic acid fragment structure change

A technology of hyaluronic acid and structural change, which is applied in material analysis by observing the effect on chemical indicators, measuring devices, and analysis by chemical reaction of materials, etc. , It is difficult to detect structural changes and the degree of changes

Active Publication Date: 2012-01-18
SHANDONG BLOOMAGE HYINC BIOPHARM CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the hyaluronic acid fragments produced by the degradation of hyaluronic acid are a mixture of small hyaluronic acid molecular fragments of different lengths, and it is difficult to detect changes in its structure and the degree of change using general techniques
Nuclear magnetic resonance technology (NMR) can be used to determine the structure of substances, but this technology requires high sample purity, and the equipment is expensive, and the detection cost is high, so it cannot be widely used

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1.1 Sample: Hyaluronic acid fragments produced by hyaluronidase degradation of animal tissues, with an average molecular weight of 5800.

[0022] 1.2 Method:

[0023] a) Sample pretreatment

[0024] Precisely weigh the appropriate amount of hyaluronic acid reference substance and hyaluronic acid fragment sample and place them in a 50ml volumetric flask, dissolve them with sodium acetate buffer solution and constant volume, add hyaluronidase, and bathe in water at 37°C for 2h;

[0025] b) Chromatographic conditions

[0026] The chromatographic conditions of high performance liquid chromatography are amino column (4.6mm×250mm), the mobile phase is 0.5 mol / L sodium phosphate buffer; the flow rate is 0.5ml / min; the column temperature is 30°C; the detection wavelength is 232nm; The volume is 20 μL. The carbazole method refers to the content determination method of sodium hyaluronate in the European Pharmacopoeia.

[0027] c) Calculation of results

[0028] Use high perf...

Embodiment 2

[0032] 2.1 Sample: hyaluronic acid fragments produced by hydrochloric acid degradation, with an average molecular weight of 11600.

[0033] 2.2 Method: The chromatographic condition of high performance liquid chromatography is amino column (4.6mm×250mm), the mobile phase is 0.5 mol / L sodium phosphate buffer solution; the flow rate is 0.5ml / min; the column temperature is 30°C; the detection wavelength is 232nm ; The injection volume was 20 μl. The carbazole method refers to the content determination method of sodium hyaluronate in the European Pharmacopoeia.

[0034] 2.3 Results: The result of carbazole method was 95.99%, the result of high performance liquid chromatography was 77.64%, the difference between the two was 18.35%, indicating that the structure of hyaluronic acid fragments has changed, that is, the degradation of hydrochloric acid has a negative effect on the structure of hyaluronic acid. Has a destructive effect.

Embodiment 3

[0036] 3.1 Sample: hyaluronic acid fragments produced by hydrochloric acid degradation, with an average molecular weight of 7200.

[0037] 3.2 Method: The chromatographic conditions of high performance liquid chromatography are amino column (4.6mm×250mm), the mobile phase is 0.5 mol / L sodium phosphate buffer solution; the flow rate is 0.5ml / min; the column temperature is 30°C; the detection wavelength is 232nm ; The injection volume was 20 μl. The carbazole method refers to the content determination method of sodium hyaluronate in the European Pharmacopoeia.

[0038] 3.3 Results: The result of the carbazole method was 95.48%, the result of the high performance liquid chromatography was 62.38%, and the difference between the two was 33.10%. Combined with the experimental data of Example 2, it shows that under the same degradation conditions, the lower the average molecular weight of the hyaluronic acid fragment, the more serious its structural damage.

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Abstract

The invention relates to a method for quantitatively detecting the structure change of a hyaluronic acid fragment produced by degrading a polymer sodium hyaluronate. A high performance liquid chromatography and a carbazole method are used for simultaneously measuring the content of the hyaluronic acid fragment, and a difference between the contents obtained by the two methods is used for quantifying the structure change of the hyaluronic acid fragment prepared by different methods. The method provided by the invention is suitable for the hyaluronic acid fragment prepared by different methods,can evaluate the damage degree to the hyaluronic acid structure caused by the degradation method and preliminarily judge the production method of the hyaluronic acid fragment according to the measurement result, and is an important measure for evaluating the quality of the hyaluronic acid fragment.

Description

technical field [0001] The invention relates to a method for measuring hyaluronic acid, in particular to a method for quantitatively detecting structural changes of hyaluronic acid fragments. Background technique [0002] Hyaluronic acid is a straight-chain polymer mucopolysaccharide formed by the repeated connection of disaccharide units composed of glucuronic acid and acetylglucosamine. Hyaluronic acid fragments are generally degraded by the macromolecule sodium hyaluronate, and its degradation methods can be divided into three categories: physical degradation (such as heating, microwave, ultrasonic, radiation, etc.), chemical degradation (such as acid, alkali, oxidation, etc. ) and biodegradation (e.g. hyaluronidase). Using hyaluronic acid extracted from animal tissue to degrade hyaluronic acid, only the glycosidic bond at the enzyme cleavage site is broken, and its disaccharide structural unit remains unchanged. However, in the process of physical degradation and chem...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N21/78
Inventor 王海英郭学平栾贻宏刘爱华
Owner SHANDONG BLOOMAGE HYINC BIOPHARM CORP LTD
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