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Method for determining content of quaternary ammonium salt in hyaluronic acid-quaternary ammonium salt polymer

A technology of hyaluronic acid and quaternary ammonium salt, which is applied in the detection field, can solve the problems of interference detection accuracy, tailing, and alcohol precipitation method is not applicable, and achieve high detection accuracy, separation, and accurate determination.

Pending Publication Date: 2022-04-26
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, no direct method for detecting GTA or DTA has been found. Quaternary ammonium salts are used as cations, and ion chromatography is commonly used for detection. Using conventional quaternary ammonium salt detection methods to detect GTA or DTA will cause tailing and retention time Phenomena such as drift, interference detection accuracy
In addition, the product contains hyaluronic acid, which needs to be pre-treated. Due to the strong hydrophilicity of hyaluronic acid, the solution after hydrolysis contains a large amount of organic sugars, which are adsorbed by the chromatographic column and damage the life of the chromatographic column. Sedimentation of hyaluronic acid in liquid
At present, the commonly used sedimentation method is alcohol precipitation, but because the polymer reaction changes the properties of hyaluronic acid, the alcohol precipitation method is no longer applicable, and a new detection method needs to be developed

Method used

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  • Method for determining content of quaternary ammonium salt in hyaluronic acid-quaternary ammonium salt polymer
  • Method for determining content of quaternary ammonium salt in hyaluronic acid-quaternary ammonium salt polymer
  • Method for determining content of quaternary ammonium salt in hyaluronic acid-quaternary ammonium salt polymer

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preparation example Construction

[0035] Preparation of the test solution: hydrolyze the hyaluronic acid-quaternary ammonium salt polymer to obtain a mixed solution of hyaluronic acid and quaternary ammonium salt, then add salt solution and acetonitrile to settle the hyaluronic acid, and obtain the supernatant as the Test solution;

[0036] Use ion chromatography to measure the content of free quaternary ammonium salt in the test solution.

[0037] The present invention does not impose any limitation on the above hydrolysis method, which can be hydrolyzed by conventional methods in the art, for example, using sodium hydroxide solution to hydrolyze the hyaluronic acid-quaternary ammonium salt polymer.

[0038] The present invention adopts the above-mentioned salt solution and acetonitrile to settle the hyaluronic acid, which can reduce the solubility of the hyaluronic acid in the aqueous solution, and finally precipitate the hyaluronic acid, so that the content of the quaternary ammonium salt can be accurately ...

Embodiment 1

[0085] 1. Reagents and materials

[0086] Sodium chloride (Sinopharm Chemical Reagent Co., Ltd.), methanesulfonic acid (Thermo Fisher Scientific Co., Ltd.), acetonitrile (Sinopharm Chemical Reagent Co., Ltd.)

[0087] 2,3-Epoxypropyltrimethylammonium Chloride (GTA) (Aladdin), 2,3-Dihydroxypropyl-trimethylammonium Chloride (DTA) (Jiangsu Aikang Biomedical Research and Development Co., Ltd.) , hyaluronic acid-quaternary ammonium salt polymer (Bloomage Biotechnology Co., Ltd.)

[0088] 2. Chromatographic conditions

[0089] Chromatographic column: CS12A (Thermo Fisher Scientific Co., Ltd., 4×250mm, 5μm);

[0090] Mobile phase: 10mmol / L methanesulfonic acid-30% acetonitrile solution;

[0091] Flow rate: 1ml / min;

[0092] Injection volume: 25μL; column temperature: 30℃

[0093] Suppressor current: 35mA.

[0094] Description of chromatographic conditions:

[0095] Among them, the mobile phase, flow rate, and column temperature are all recommended parameters in the instruction...

Embodiment 2

[0126] The methanesulfonic acid concentration of the mobile phase of the chromatography in embodiment 1 is changed to 15mmol / L, and other conditions are identical with embodiment 1, measure quaternary ammonium salt content in need testing solution.

[0127] The detection range and detection limit of this method are shown in Table 4, and the detection limit is calculated as three times the signal-to-noise ratio.

[0128] The detection range of test product in table 4 embodiment 2

[0129] sample Linear rangemg / L linear equation R 2

The detection limit peak time Separation GTA 5-100 Y=0.0238X-0.0021 0.9999 0.5mg / g 5.3min 3.41 DTA 5-100 Y=0.0197X-0.0098 0.9995 1mg / g 4.2min 1.98

[0130] As can be seen from Table 1, the linear range of GTA is 5-100 mg / L, and the detection limit is 0.5 mg / g, and the linear range of DTA is 5-100 mg / L, and the detection limit is 1 mg / g.

[0131] Then configure standard substances with different ...

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Abstract

The invention discloses a method for measuring the content of quaternary ammonium salt in a hyaluronic acid-quaternary ammonium salt polymer, which comprises the following steps: preparation of a test solution: hydrolyzing the hyaluronic acid-quaternary ammonium salt polymer to obtain a mixed solution of hyaluronic acid and quaternary ammonium salt, then adding a salt solution and acetonitrile to settle the hyaluronic acid, and filtering to obtain the test solution; the obtained supernate is used as a test solution; and determining the content of the free quaternary ammonium salt in the test solution by using an ion chromatography. According to the method, the solubility of the hyaluronic acid in the aqueous solution is reduced under the combined action of the high-concentration salt solution and the acetonitrile, so that the content of the quaternary ammonium salt can be accurately determined, and the method has relatively high detection accuracy and separation degree.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a method for measuring the content of quaternary ammonium salts in hyaluronic acid-quaternary ammonium salt polymers. Background technique [0002] Hyaluronic acid contains multiple active groups that can be modified. Quaternary ammonium salts can be linked to active groups of hyaluronic acid to form hyaluronic acid grafted polymers. 2,3-Epoxypropyltrimethylammonium chloride (Glycidyl trimethylammonium chloride, GTA) is a commonly used quaternary ammonium salt, which can be grafted with hyaluronic acid to form HA-GTA polymer. After the reaction, some GTA will remain in the product, and the purity of the HA-GTA polymer can be obtained by detecting the content of the residual GTA in the product. At the same time, in order to verify the grafting rate of GTA, the HA-GTA polymer needs to be decomposed to obtain 2,3-dihydroxypropyl-trimethyl ammonium chloride ((2,3-Dihydroxypropyl)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/96
CPCG01N30/96
Inventor 万丽佳魏健康静
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD