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A method for measuring the degree of cross-linking of cross-linked hyaluronic acid or its salt

A technology of cross-linked hyaluronic acid and its determination method, which is applied in the field of determination of the degree of cross-linking, can solve problems such as the inability to measure the degree of cross-linking, and achieve stable, reliable and highly repeatable results

Active Publication Date: 2020-12-04
SHANGHAI JINGFENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods can only detect the content of the residual cross-linking agent in the gel, or qualitatively analyze the degree of cross-linking, but cannot be used for the determination of the degree of cross-linking

Method used

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  • A method for measuring the degree of cross-linking of cross-linked hyaluronic acid or its salt
  • A method for measuring the degree of cross-linking of cross-linked hyaluronic acid or its salt
  • A method for measuring the degree of cross-linking of cross-linked hyaluronic acid or its salt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] A method for measuring the degree of cross-linking of cross-linked hyaluronic acid or a salt thereof, comprising the steps of:

[0054] (1) Take 0.7 g of sodium hyaluronate cross-linked with dimercapto polyethylene glycol as the sample to be tested, dissolve it in 3 mL of phosphate buffer (5 mmol / L, pH=6), add 1×10 3 IU hyaluronidase, react in a water bath at 37°C for 24h, shake well, take 1mL of the reaction solution, 3 Centrifuge at r / min for 15 minutes, take the supernatant and pass through a 0.22 μm filter membrane to obtain a filtrate containing oligosaccharides and disaccharides;

[0055] (2) Get 10 μ L of the above-mentioned filtrate, separate and detect it with size exclusion chromatography and ultraviolet absorption, obtain the absorbance-retention time chromatogram, and measure the chromatographic peak areas of the two respectively;

[0056] (3) Calculate the degree of crosslinking, where A 1 For the chromatographic peak area of ​​the oligosaccharide, A 2...

Embodiment 2

[0067] The difference from Example 1 is that the concentration of sodium hyaluronate cross-linked with dimercapto polyethylene glycol is 0.35g / 3mL, and the amount of hyaluronidase added is 0.5×10 3 IU.

Embodiment 3

[0069] The difference from Example 1 is that the concentration of sodium hyaluronate cross-linked with dimercapto polyethylene glycol is 1.05g / 3mL, and the amount of hyaluronidase added is 1.5×10 3 IU.

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Abstract

The invention provides a determining method of the crosslinking degree of crosslinked HA (hyaluronic acid) or salt thereof. The determining method comprises the steps as follows: (1) crosslinked HA orsalt thereof is subjected to an enzymatic hydrolysis reaction under the action of hyaluronidase to produce oligosaccharide and disaccharide; (2) oligosaccharide and disaccharide are separated and detected through liquid chromatography and ultraviolet absorption, an absorbance-retention time chromatogram is obtained, and chromatographic peak areas of oligosaccharide and disaccharide are determinedrespectively; (3) the crosslinking degree is calculated according to the formula shown in the description, wherein A1 is the chromatographic peak area of oligosaccharide while A2 is the chromatographic peak area of disaccharide. The determining method can directly determine the crosslinking degree of crosslinked HA or salt thereof and has higher repeatability, and determination results are stableand reliable.

Description

technical field [0001] The invention belongs to the technical field of chemical analysis, in particular to a method for measuring the crosslinking degree of crosslinked hyaluronic acid or a salt thereof. Background technique [0002] Hyaluronic acid (HA) is a chain-like polyanionic mucopolysaccharide composed of repeated connections of glucuronic acid and acetylglucosamine disaccharide units. It is an important component of vitreous body, skin, joint synovial fluid and cartilage tissue. Various cellular activities and physiological processes. HA has been widely used clinically because of its good moisturizing properties, lubricity, viscoelasticity, non-immunogenicity and promotion of drug absorption. However, natural HA has shortcomings such as strong water solubility, easy diffusion in tissues and degradation by enzymes, and short retention time in the body, while modified and cross-linked HA derivatives can make up for these shortcomings. Some cross-linked HA products ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 张兆利郑琳松刘珊程庆有
Owner SHANGHAI JINGFENG PHARMA