CN113607834A - Thrombin and determination method for molecular weight distribution of active ingredients in preparation thereof - Google Patents
Thrombin and determination method for molecular weight distribution of active ingredients in preparation thereof Download PDFInfo
- Publication number
- CN113607834A CN113607834A CN202110811484.8A CN202110811484A CN113607834A CN 113607834 A CN113607834 A CN 113607834A CN 202110811484 A CN202110811484 A CN 202110811484A CN 113607834 A CN113607834 A CN 113607834A
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- Prior art keywords
- molecular weight
- thrombin
- weight distribution
- preparation
- determination
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Links
- 108090000190 Thrombin Proteins 0.000 title claims abstract description 30
- 229960004072 thrombin Drugs 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000004480 active ingredient Substances 0.000 title claims abstract description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims abstract description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims abstract description 5
- 150000004676 glycans Chemical class 0.000 claims abstract description 4
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 4
- 239000005017 polysaccharide Substances 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims 3
- 229920001503 Glucan Polymers 0.000 claims 1
- 229920002307 Dextran Polymers 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 239000000543 intermediate Substances 0.000 abstract description 3
- 239000000843 powder Substances 0.000 description 7
- 230000000740 bleeding effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention discloses a thrombin and a method for measuring the molecular weight distribution of active ingredients in a preparation thereof, which uses a gel column special for polysaccharide and takes 0.2-1.0mol/L sodium sulfate solution with pH of 4.5-5.5 (preferably pH of 5.0) as a mobile phase; the column temperature is 30-40 ℃; the flow rate is 0.5-1.0ml per minute; a differential refractive detector. The standard sample is composed of a series of components with known molecular weight of dextran in the middle, and the weight average molecular weight of the dextran is 1000-50000 respectively; the method realizes the determination of the molecular weight distribution of the thrombin, is suitable for the determination of the molecular weight distribution of thrombin raw materials, intermediates and finished products, and fills the blank of no thrombin molecular weight determination method in the current Chinese pharmacopoeia.
Description
Technical Field
The invention relates to a thrombin and a method for measuring the molecular weight distribution of active ingredients in the preparation thereof
Background
Thrombin (Thrombin), a white to off-white amorphous substance, is generally a lyophilized powder. Thrombin is a blood stopping agent, and is mainly suitable for stopping bleeding of small blood vessels, capillaries and parenchymal viscera which are difficult to stop bleeding clinically; can be used for stopping bleeding of trauma, operation, oral cavity, ear, nose, throat, urinary system, burn, orthopedics, neurosurgery, ophthalmology, gynecology and obstetrics, and digestive tract. The thrombin directly acts on the last step of the blood coagulation process to promote the conversion of soluble fibrinogen in the plasma into insoluble fibrin, thereby achieving the purpose of quick-acting hemostasis. The preparation is thrombin freeze-dried powder.
The existing thrombin freeze-dried powder determination mainly has the following problems:
1) the reference substance is complicated to use and has a non-uniform structure, which may affect the measurement result.
2) The thrombin freeze-dried powder is directly measured, although simple peak assignment is carried out on the thrombin freeze-dried powder, the thrombin freeze-dried powder only belongs to the molecular weight, and the influence of preparation auxiliary materials and the like on the thrombin freeze-dried powder is not determined through comparison research with raw materials.
3) With an ultraviolet detector, it may result in some substances that do not absorb ultraviolet light being undetectable.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect that the molecular weight of thrombin freeze-dried powder in the prior art is not accurately measured, and provides a method for measuring the molecular weight distribution of active ingredients in thrombin and a preparation thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
a thrombin and its preparation effective component molecular weight distribution determination method comprises using polysaccharide special gel column, using 0.2-1.0mol/L sodium sulfate solution (preferably pH5.0) as mobile phase; the column temperature is 30-40 ℃; the flow rate is 0.5-1.0ml per minute; a differential refractive detector. The standard sample is composed of a series of components with known molecular weight dextran (dextran) in the middle, and the weight average molecular weight is 1000-50000 respectively.
Dissolving the product with mobile phase to obtain solution containing 10mg per 1 ml. And (4) injecting 25 mu l of the mixture into a liquid chromatograph, and recording the chromatogram. And processing by using GPC special software.
Has the advantages that: the invention adopts HPLC method to realize the determination of thrombin molecular weight distribution, is suitable for the determination of thrombin raw material, intermediate and finished product molecular weight distribution, and fills the blank of no thrombin molecular weight determination method in the current Chinese pharmacopoeia.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a molecular weight distribution measurement chart of the thrombin raw material in example.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Examples
A method for measuring molecular weight distribution of thrombin and effective components in its preparation comprises using gel column special for measuring polysaccharide, and 0.2mol/L sodium sulfate solution (pH5.0) as mobile phase; the column temperature is 35 ℃; flow rate 0.5ml per minute; a differential refractive detector. The number of theoretical plates should be not less than 5000, calculated as glucose peaks. The standard sample is composed of a series of components with known molecular weight of dextran and weight average molecular weight of 1000-50000 respectively. Taking a proper amount of sample, adding mobile phase for dissolving to prepare a solution containing about 10mg of the solution in each 1ml, filtering, and standing overnight. And (4) injecting 25 mu l of the mixture into a liquid chromatograph, and recording the chromatogram. And processing by using GPC special software.
Thrombin starting materials, intermediates and formulations were each assayed according to the methods described above and the results are as follows:
the accuracy of the method is verified, and the result is as follows:
finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A thrombin and its preparation effective component molecular weight distribution determination method, characterized by, use the specialized gel column of polysaccharide, regard 0.2-1.0mol/L sodium sulfate solution as the mobile phase; the column temperature is 30-40 ℃, and the flow rate is 0.5-1.0ml per minute; measuring by a differential refraction detector;
the standard sample is composed of a series of components with known molecular weight of glucan and weight average molecular weights of 1000-50000 respectively;
and (3) taking a proper amount of samples, adding a mobile phase for dissolving to prepare a sample solution, injecting the sample solution into a liquid chromatograph, and recording a chromatogram.
2. The method for measuring the molecular weight distribution of the active ingredient in thrombin and a preparation thereof according to claim 1, wherein the pH of the sodium sulfate solution is 4.5 to 5.5.
3. The method of claim 1, wherein the thrombin comprises about 8-12 mg of thrombin per 1ml of the sample solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110811484.8A CN113607834A (en) | 2021-07-19 | 2021-07-19 | Thrombin and determination method for molecular weight distribution of active ingredients in preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110811484.8A CN113607834A (en) | 2021-07-19 | 2021-07-19 | Thrombin and determination method for molecular weight distribution of active ingredients in preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113607834A true CN113607834A (en) | 2021-11-05 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110811484.8A Pending CN113607834A (en) | 2021-07-19 | 2021-07-19 | Thrombin and determination method for molecular weight distribution of active ingredients in preparation thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173506A (en) * | 2011-10-09 | 2013-06-26 | 清华大学 | Method for controlling production of low-molecular-weight heparin |
| US20160244739A1 (en) * | 2015-02-25 | 2016-08-25 | Omrix Biopharmaceuticals Ltd. | Method for purifying and quantifying thrombin and its degradation polypeptides |
| CN109682903A (en) * | 2019-02-19 | 2019-04-26 | 天津赛诺制药有限公司 | A kind of detection method of astragalus root polysaccharide molecular weight |
-
2021
- 2021-07-19 CN CN202110811484.8A patent/CN113607834A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173506A (en) * | 2011-10-09 | 2013-06-26 | 清华大学 | Method for controlling production of low-molecular-weight heparin |
| US20160244739A1 (en) * | 2015-02-25 | 2016-08-25 | Omrix Biopharmaceuticals Ltd. | Method for purifying and quantifying thrombin and its degradation polypeptides |
| CN109682903A (en) * | 2019-02-19 | 2019-04-26 | 天津赛诺制药有限公司 | A kind of detection method of astragalus root polysaccharide molecular weight |
Non-Patent Citations (3)
| Title |
|---|
| YILAN OUYANG等: "Qualitative and quantitative analysis of heparin and low molecular weight heparins using size exclusion chromatography with multiple angle laser scattering/refractive index and inductively coupled plasma/mass spectrometry detectors", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 王赛贞等: "高效凝胶渗透色谱法测定双灵固本散中多糖肽的分子量及其分布", 《海峡药学》 * |
| 陈英红等: "高效凝胶渗透色谱法测定凝血酶冻干粉的分子量及其分布", 《特产研究》 * |
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Application publication date: 20211105 |