Detection method of nadroparin calcium disaccharide spectrum

A technique for detecting nadroparin calcium and its detection method, which is applied in the field of raw drug detection, can solve the problems of inability to obtain the detection result of nadroparin calcium, low sensitivity, and high operation requirements, and achieve low performance requirements, high sensitivity, and detection low limit effect

Pending Publication Date: 2020-06-05
BEIJING SAISHENG PHARMA
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  • Claims
  • Application Information

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Problems solved by technology

[0006] Although enoxaparin and nadroparin calcium belong to low-molecular-weight heparins, their preparation process is completely different from that of nadroparin calcium, and there are large differences in their characteristic structures. Simply applying the detection method of enoxaparin cannot obtain more accurate results. Nadroparin Calcium Test Results
[0007] However, other existing research methods with complete enzymatic hydrolysis, such as the use of thin-layer chromatography, have low sensitivity; HPLC analysis requires pre-column derivatization treatment, which consumes a lot of analysis time and introduces complex matrices. And some derivatization reactions require a very small amount of reaction reagents, which have high requirements for operation, and the results are accidental

Method used

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  • Detection method of nadroparin calcium disaccharide spectrum
  • Detection method of nadroparin calcium disaccharide spectrum
  • Detection method of nadroparin calcium disaccharide spectrum

Examples

Experimental program
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Embodiment 1

[0054] This embodiment provides a method for detecting nadroparin calcium, comprising:

[0055] S1, the preparation of the solution:

[0056] (1) Preparation of enzymolysis buffer: Weigh 10 mg of bovine serum albumin and 32 mg of calcium acetate and dissolve them in 60 ml of water, then add 580 μl of glacial acetic acid to the solution, mix well and adjust the pH to 7.0 with 2M sodium oxide solution. Then make up to 100ml with water. Pass the prepared enzymatic hydrolysis buffer through a 0.22 μm microporous membrane for later use.

[0057] (2) Preparation of heparinase solution: mix equal amounts of heparinase I, heparinase II, and heparinase Ш, dilute to 0.4 IU / ml with enzymatic hydrolysis buffer, and set aside;

[0058] (3) Preparation of Nadroparin Calcium sample solution: take Nadroparin Calcium sample (label: 20181127), dilute with water and prepare a solution with a concentration of 20 μg / μl;

[0059] S2, complete enzymatic hydrolysis:

[0060] Take 10 μl of nadropa...

Embodiment 2

[0081] According to the method for detecting nadroparin calcium in Example 1, the difference is that: the nadroparin calcium sample is replaced by a nadroparin calcium intermediate sample (label: intermediate 1129-3). Chromatographic results such as Figure 5 .

[0082] Investigation test on the influence of enzymatic hydrolysis conditions on the test results:

[0083] 1. Condition investigation and verification of complete enzymatic hydrolysis:

[0084] According to the method for detecting nadroparin calcium in Example 1, the difference is: adjust the amount of heparanase added (20 μl, 40 μl, 60 μl, 80 μl, 100 μl, 120 μl) and adjust the enzymatic hydrolysis time (36h, 48h, 60h, 72h, 84h ).

[0085] The specific adjustment method is shown in Table 1.

[0086] Table 1

[0087] time Enzyme dosage 36h 20μl, 40μl, 60μl, 80μl, 100μl, 120μl 48h 20μl, 40μl, 60μl, 80μl, 100μl, 120μl 60h 20μl, 40μl, 60μl, 80μl, 100μl, 120μl 72h 20μl, 40μl, 60μl...

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Abstract

The invention belongs to the technical field of raw material medicine detection, and particularly relates to a detection method of nadroparin calcium disaccharide spectrum. The detection method of thenadroparin calcium disaccharide spectrum comprises the following steps: firstly, adding mixed heparinase into a sample solution for complete enzymolysis, and then detecting an enzymolysis product byadopting an anion exchange chromatography method. According to the method, aiming at the characteristics of nadroparin calcium, the enzymolysis conditions of nadroparin calcium are deeply researched,and the disaccharide component of nadroparin calcium is detected by selecting the matched anion exchange chromatography, so that the detection result is more accurate, the sensitivity is higher, precolumn derivatization is not needed, and the operation is simpler and more convenient. The method can provide structural information for exploration of a raw material synthesis process, and can also effectively control the quality of nadroparin calcium, so that the quality of nadroparin calcium is stable, controllable, efficient and safe.

Description

technical field [0001] The invention belongs to the technical field of crude drug detection, and in particular relates to a method for detecting the calcidisaccharide spectrum of nadroparin. Background technique [0002] Heparin is a highly sulfated glycosaminoglycan extracted from mammalian tissues. Low molecular weight heparin (LMWH) is produced by the decomposition and degradation of heparin. It has anti-Xa activity, but its anti-IIa factor activity is small. Therefore, it can inhibit the formation of thrombus and arteriovenous thrombosis in vivo and in vitro, but it does not affect platelet aggregation and the combination of fibrinogen and platelets. Existing graded LMWH preparations include Nadroparin Calcium Injection, Enoxaparin Sodium Injection, Dalteparin Sodium Injection, Bemiparin Sodium Injection, etc. [0003] It is necessary to clarify its structure when conducting imitation research of this type of drug. At present, the structural confirmation of low-molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/96
CPCG01N30/96
Inventor 白淑敏吕晓利任立新宋梦薇滕宁宁齐硕
Owner BEIJING SAISHENG PHARMA
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