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Identification method and application of heparin and heparan sulfate

A technology of heparan sulfate and heparin, which is applied in the field of medical detection and achieves the effects of high sensitivity, low detection limit and quantification limit, and good repeatability

Active Publication Date: 2020-09-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, since the quality standards of new glycosaminoglycan innovative drugs require definite composition and structure, strict quantitative analysis of their components is required. Few studies have been reported on similar glycosaminoglycan drugs

Method used

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  • Identification method and application of heparin and heparan sulfate
  • Identification method and application of heparin and heparan sulfate
  • Identification method and application of heparin and heparan sulfate

Examples

Experimental program
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preparation example Construction

[0042] (2) Preparation of the test solution: Take 10.0 mg of the test powder, accurately weigh it, place it in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, and prepare a sample stock solution with a concentration of 1.00 mg / mL. Accurately measure 100 μL of the sample stock solution, add 900 μL of enzyme reaction buffer and mix well, take 50 μL and add the mixed heparinase I, II, III (10 μL, 40 μL, 5 μL respectively), and put it in a 37°C incubator for 24 hours. After the reaction, centrifuge at 14000rpm for 10min, and take the supernatant to obtain the test solution.

[0043] (3) Determination: Accurately measure the test solution and the reference solution respectively, inject them into a high-performance liquid chromatography-mass spectrometer for separation, and adopt gradient elution, mobile phase A phase: 10mM NH 4 Ac-water, mobile phase B phase: 10mM NH 4 Ac-90% acetonitrile.

[0044] The present invention selects and optimizes to chromatogra...

Embodiment 1

[0063] A method for liquid-mass quantitative determination of a heparin-like drug disaccharide mixed standard product, comprising the steps of:

[0064] first step:

[0065] Accurately measure an appropriate amount of the mixed standard solution, add enzyme reaction buffer and mix evenly, add the mixed heparinase I, II, III (10 μL, 40 μL, 5 μL, respectively), and react in a temperature-controlled box at 37°C for 24 hours. After the reaction, centrifuge at 14000rpm for 10min, and take the supernatant to obtain the reference solution.

[0066] Step two:

[0067] Take 10.0 mg of the test product powder, accurately weigh it, place it in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, and prepare a sample stock solution with a concentration of 1.00 mg / mL. Accurately measure 100 μL of the sample stock solution, add 900 μL of enzyme reaction buffer and mix well, take 50 μL and add the mixed heparinase I, II, III (10 μL, 40 μL, 5 μL respectively), and put it ...

Embodiment 2

[0081] A kind of identification method of heparin and heparan sulfate, as follows:

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Abstract

The invention provides an identification method and application of heparin and heparan sulfate, and belongs to the technical field of medicine detection. Research finds that eight disaccharide components of 0S, NS, 6S, 2S, 2SNS, 6SNS, 2S6S and TriS are rich in heparin drugs, and can be well used for identifying heparin and heparan sulfate as a mixed standard substance. Therefore, a set of quantitative detection method based on hydrophilic interaction chromatography-mass spectrometry is established for the eight components, and the method has the advantages of being easy and convenient to operate, short in consumed time, high in selectivity, high in sensitivity, good in specificity, good in repeatability and the like. The method can be used for simply, conveniently and accurately identifying heparin and heparan sulfate, a method and data reference are provided for constructing a more complete quality control standard system of glycosaminoglycan drugs, so that the method has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for identifying heparin and heparan sulfate and an application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Heparin and heparan sulfate are endogenous, complex and polydisperse linear sulfated glycosaminoglycans, which are formed by linking glucuronic acid or idonic acid and glucosamine through 1→4 glycosidic bonds. Repeated composition of disaccharide units, each unit has variable sulfation and isomerization, and the length of sugar chains is different, making it a multi-component mixture with uneven molecular weight and complex stru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72
CPCG01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N2030/324
Inventor 王凤山刘冬科
Owner SHANDONG UNIV
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