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Agkistrodon blomhoffii ussurensis defibrase HPLC detection method and application

A detection method, the technology of the white-browed pit viper, is applied in the field of HPLC detection of the white-browed pit viper defibrase, and can solve the problems that the A/B components cannot be accurately quantified, the in vitro activities are different, and the A/B components cannot be completely separated, and the like. To achieve the effect of effective quality control and ensure safety

Pending Publication Date: 2021-08-10
BEIJING SAISHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Even if the present invention adjusts its chromatographic column to a C8 or C18 chromatographic column during research, the result A / B component still cannot achieve complete separation, indicating that the chromatographic system recorded in this document cannot accurately quantify a single A / B component
Although the structure of defibrase A / B components of Agkistrodon halys is very similar, the enzymatic characterization test proves that there are significant differences in the in vitro activities of the two. Based on the consideration of clinical safety and drug accuracy, the A / B components should be accurately quantified especially important

Method used

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  • Agkistrodon blomhoffii ussurensis defibrase HPLC detection method and application
  • Agkistrodon blomhoffii ussurensis defibrase HPLC detection method and application
  • Agkistrodon blomhoffii ussurensis defibrase HPLC detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0046] This embodiment provides a reversed-phase HPLC chromatographic analysis and detection method for defibrase from Agkistrodon halys, and the specific steps are as follows:

[0047] 1. Purity determination:

[0048] The defibrase sample to be tested was taken from Agkistrodon halys, and the preparation process was referred to "Research on the preparation process and quality standard of defibrase" published by Xu Feng. The solution prepared by molecular sieve chromatography multi-step purification separation and extraction was diluted with pure water to a solution containing 1 mg of protein per 1 ml as the test solution.

[0049] Get need testing solution 20 μ L and inject liquid chromatograph to detect, detection condition is as follows:

[0050] C18 reverse-phase analytical column: Agilent ZORBAX 300SB-C18, 4.6mm×250mm, 5μm;

[0051] Mobile phase A: Weigh about 2.4g of sodium dihydrogen phosphate, dissolve it in 950mL of water, then adjust the pH to 6.50 with sodium hyd...

Embodiment 2

[0067] This example verifies the precision, repeatability, and stability of the detection method in Example 1.

[0068] 1. Precision test

[0069] Adopt the defibrase test sample of Agkistrodon halys that is identical with embodiment 1, dilute with pure water to the solution that contains 1mg protein in every 1ml, as need testing solution. According to the purity measurement method of Example 1, the above-mentioned need testing solution is accurately drawn, and 6 needles are continuously injected, the RSD value of the retention time of the two main peaks of component A and component B should be no more than 1.0%, and the RSD value of the peak area should be None greater than 2.0%. The detection results are shown in Table 3. Calculated by retention time, the RSDs of A component and B component were 0.04% and 0.04% respectively; calculated by peak area, the RSDs of A component and B component were 0.54% and 0.78% respectively. %. Show that the method precision of embodiment 1...

Embodiment 3

[0082] This example provides a method for reverse-phase HPLC chromatographic analysis and detection of defibrase from Agkistrodon halys, the purity determination method of which is the same as that of Example 1, the only difference being that the concentration of phosphate buffer salt in mobile phase A is 25 mM.

[0083] For test results, see figure 2 , it can be seen that the separation between the two main peaks and the separation of the adjacent impurity peaks also meet the requirements. Considering that the mixture of salt and organic solvent may precipitate crystals and block the instrument pipeline, the salt concentration should be as small as possible , so preferably 20mM.

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Abstract

The invention relates to the technical field of biochemical detection, and particularly discloses an agkistrodon blomhoffii ussurensis defibrase HPLC detection method and application thereof. According to the HPLC detection method, a mobile phase A, a mobile phase B and a mobile phase C are adopted for elution; the mobile phase A is a 20-25 mM aqueous solution of sodium dihydrogen phosphate, and the pH value of the aqueous solution of sodium dihydrogen phosphate is 5.8-7.0; the mobile phase B is acetonitrile; and the mobile phase C is an aqueous solution of trifluoroacetic acid. Octadecylsilane chemically bonded silica is used as a filling agent in a reversed-phase column, and gradient elution is carried out. By establishing a purity determination method which is simple, convenient and sensitive and has good specificity, accuracy and durability, the quality of the agkistrodon blomhoffii ussurensis defibrase can be effectively controlled, and the quality of the agkistrodon blomhoffii ussurensis defibrase is stable, controllable, efficient and safe. Meanwhile, the method can also be used for identifying defibrases from different snake venom sources, and lays a foundation for improving quality standards of defibrase raw materials and preparations thereof.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to an HPLC detection method and application of defibrase from Agkistrodon halys. Background technique [0002] Defibrase is one of the snake venom thrombin preparations. It has significant anticoagulant effects such as defibrillation, viscosity reduction, depolymerization, and thrombolysis. It is widely used in clinical treatment and prevention of occlusive cardiovascular and cerebrovascular thrombotic diseases. The thrombin-like product extracted from the venom of Agkistrodon sharp kissed Agkistros and Changbaishan Baimei Agkistrodon vipers was named defibrase. In recent years, studies have shown that the defibrase from the two snake venom sources are different in structure, enzymatic characterization, mechanism of action, and clinical manifestations. They belong to isoenzymes, but they cannot be equated with the same substance, and necessary differentiating quality c...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 白淑敏栾美丽齐硕马胜楠梁丽娜
Owner BEIJING SAISHENG PHARMA
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