Method for detecting purity of defibrase of agkistrodon acutus and application of defibrase
By using ChromCore Phenyl type chromatography column and specific mobile phase gradient elution program in HPLC chromatography detection, the problem of separating and identification of subfibrase by white-eyed chromatography is solved, and the accurate purity detection and quality control of subfibrase is achieved, which improves the Optimization of production process and product safety.
Patent Information
- Application Number
- CN202411355394.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2025-01-14
AI Technical Summary
The prior art cannot effectively isolate and identify the two active components of the white-eyed viper sub-fibrase, resulting in high purity detection and inaccurately reflecting the quality of the white-eyed viper sub-fibrase, especially because of the white-eyed viper and white-eyed viper sub-fibrase. The missing identification items affect quality control and production process optimization.
The Phenyl chromatography column was analyzed by ChromCore Phenyl nanospectrum, combined with specific mobile phase gradient elution procedures and elution conditions, including 0.1% aqueous trifluoroacetic acid solution and acetonitrile solution, and accurately separated the white-eye viper fiber by HPLC chromatography detection method. Two active components of the enzyme and identify defibrase enzymes from different snake venom sources.
The accurate purity detection of the purified fibrosis of the white-eyed viper viper and the identification of different sources of snake venom was achieved, the perfection of the quality standards for fibrosis was improved, data support was provided for the optimization of the production process, and the purity and safety of the product were ensured.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of biochemical detection, and in particular to a method for detecting the purity of Agkistrodon acutus defibrase and its application. Background Art
[0002] Defibrase is a type of snake venom thrombin preparation. It has significant anticoagulant effects such as defibrination, viscosity reduction, depolymerization, and thrombolysis. It is widely used in the clinical treatment and prevention of occlusive cardiovascular and cerebrovascular thrombotic diseases. The thrombin-like products extracted from the venom of Agkistrodon acutus and Agkistrodon halys-browed snake in Changbai Mountain are named defibrase. The current quality standard system for defibrase is not perfect, and the project setting is not comprehensive enough, making it difficult to effectively control the quality of defibrase. The focus of defibrase quality control is process control, purity testing of purified samples at different stages, and monitoring of impurity types. However, there is a lack of identification items for defibrase from Agkistrodon acutus and Agkistrodon halys-browed snake in the raw material standards, so the current detection method cannot truly reflect the purity of defibrase.
[0003] There are documents on the HPLC chromatographic detection method for the purity of defibrase. For example, using a TSK-GELG 2000SWXL (7.8mm×300mm) chromatographic column, a mobile phase of 0.2mol / L phosphate buffer (pH6.8), a flow rate of 1.0mL / min, a column temperature of 25°C, a detection wavelength of 280nm, and an injection volume of 300μL, the test results show that the main peak is tailing, impurities are included in the main peak, and the purity test is high, which is greatly affected by the excipients of the preparation.
[0004] For another example, the quality of defibrase from different sources was studied by gel chromatography (SEC), reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase ultra high performance liquid chromatography (RP-UPLC). The test results showed that reversed phase high performance liquid chromatography (HPLC and UPLC) can well identify the defibrase from Agkistrodon acutus and Agkistrodon halys-browed snake. The main component of the defibrase from Agkistrodon acutus has only one peak, while the defibrase from Agkistrodon halys-browed snake has two peaks. However, the specific operation and chromatogram of the detection method are not mentioned in this document.
[0005] For example, using a Vydac214TP54 C4 column, mobile phase A is 0.1% trifluoroacetic acid, mobile phase B is 90% acetonitrile containing 0.1% trifluoroacetic acid, and the flow rate is 0.8 mL min -1 , column temperature 45 ℃, detection wavelength 280nm, injection volume 300μL, detection of Agkistrodon acutus defibrase, able to achieve a main peak of Agkistrodon acutus defibrase and impurities baseline separation, the main peak is one, subsequent experiments proved that this method can not separate the two components of Agkistrodon acutus defibrase.
[0006] For another example, the Hanbang Lichrospher C4 column was used, with mobile phase A being 0.1% trifluoroacetic acid and mobile phase B being acetonitrile-water (90:10) containing 0.1% trifluoroacetic acid, to detect the defibrase from Agkistrodon acutus, and it was verified that the defibrase from Agkistrodon acutus was a cross peak, but baseline separation could not be achieved.
[0007] Some researchers have also developed a reverse phase analysis method for the defibrase of the white-browed pit viper, which can achieve baseline separation of the two components. However, the mobile phase used is complex, containing three phases, and the mobile phase contains a buffer, which has poor durability for the chromatographic column.
[0008] Therefore, the analytical method for defibrase in Agkistrodon acutus needs to be further developed. Summary of the invention
[0009] In view of the problems in the related art, the present application provides a method for detecting the purity of Agkistrodon acutus defibrase and its application. The method is a reversed-phase HPLC chromatographic detection method, which can accurately separate the two active components of Agkistrodon acutus defibrase, lay a foundation for improving and perfecting the quality standard of Agkistrodon acutus defibrase, and provide data support for improving and optimizing the production process. In addition, the method can also be used to identify defibrase from different snake venom sources.
[0010] The chromatographic columns used in the related art are basically C4, C8 or C18 chromatographic columns, which are difficult to effectively separate the main components. This application uses another type of chromatographic column, ChromCore Phenyl, 4.6mm×250mm, 5μm, a phenyl chromatographic column for nanospectral analysis, high-purity monodisperse silica microspheres, surface bonded phenylpropyl, completely capped, with strong retention capacity for aromatic compounds, and widely used in the separation of aromatic and heterocyclic drug molecules and tea polyphenol compounds. It is selectively complementary to the C18 stationary phase. It is suitable for separating various hydrophobic compounds and is widely used in analytical detection in many fields such as pharmaceuticals, food, environment, and chemical industry. Defibrase is a highly hydrophobic enzyme with two components, A and B, both of which have molecular weights of 36KDa~38KDa, which are very close. The protein structures of the two are the same, only the sugar modification is different, and the tertiary structure formed is different, similar to the heterocyclic group formed. Therefore, the use of this chromatographic column can achieve effective separation.
[0011] In the first aspect, the present application provides a method for detecting the purity of Agkistrodon acutus defibrase, which adopts the following technical scheme:
[0012] A method for detecting the purity of Agkistrodon acutus defibrase, wherein the method uses mobile phase A and mobile phase B for elution;
[0013] The mobile phase A is a 0.1% trifluoroacetic acid aqueous solution;
[0014] The mobile phase B is 0.1% trifluoroacetic acid in acetonitrile solution;
[0015] According to volume percentage, the elution procedure is as follows;
[0016] time Mobile phase A Mobile phase B 0 75％ 25％ 35min 35％～45％ 45％～55％ 40min 25％～35％ 65％～75％ 40.1min~45min 75％ 25％
[0017] The chromatographic column is filled with silica gel with neutral functional groups bonded on the surface.
[0018] Optionally, the chromatographic column is a phenyl chromatographic column.
[0019] Optionally, the length of the chromatographic column is 250 mm.
[0020] Optionally, the pore size of the chromatographic column is 5 μm.
[0021] Optionally, the flow rate of the mobile phase is 0.6-0.8 ml / min; the elution temperature is 40-50° C.; and the detection wavelength is 280 nm.
[0022] Optionally, the flow rate of the mobile phase is 0.7 ml / min; and the elution temperature is 45°C.
[0023] In a second aspect, the present application provides the application of the above detection method in the quality control of Agkistrodon acutus defibrase.
[0024] In a third aspect, the present application provides the application of the above detection method in the identification of the defibrase from Agkistrodon acutus and Agkistrodon acutus.
[0025] In a fourth aspect, the present application provides the application of the above detection method in the identification of defibrase from different snake venom sources.
[0026] In summary, the present application includes at least one of the following beneficial technical effects:
[0027] The present application provides a method for detecting the purity of Agkistrodon acutus defibrase and its application. The method is a reversed-phase HPLC chromatographic detection method, which can accurately separate the two active components of Agkistrodon acutus defibrase, lay a foundation for improving and perfecting the quality standard of Agkistrodon acutus defibrase, and provide data support for improving and optimizing the production process. In addition, the method can also be used to identify defibrase from different snake venom sources.
[0028] The chromatographic columns used in the related art are basically C4, C8 or C18 chromatographic columns, which are difficult to effectively separate the main components. This application uses another type of chromatographic column, ChromCore Phenyl, 4.6mm×250mm, 5μm, a phenyl chromatographic column for nanospectral analysis, high-purity monodisperse silica microspheres, surface bonded phenylpropyl, completely capped, with strong retention capacity for aromatic compounds, and widely used in the separation of aromatic and heterocyclic drug molecules and tea polyphenol compounds. It is selectively complementary to the C18 stationary phase. It is suitable for separating various hydrophobic compounds and is widely used in analytical detection in many fields such as pharmaceuticals, food, environment, and chemical industry. Defibrase is a highly hydrophobic enzyme with two components, A and B, both of which have molecular weights of 36KDa~38KDa, which are very close. The protein structures of the two are the same, only the sugar modification is different, and the tertiary structure formed is different, similar to the heterocyclic group formed. Therefore, the use of this chromatographic column can achieve effective separation. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is the chromatogram of the small sample of Agkistrodon acutus defibrase to be tested in Example 1.
[0030] Figure 2 This is the chromatogram of the small sample of Agkistrodon acutus defibrase with the lowest purity in Example 1.
[0031] Figure 3 This is the SDS-PAGE picture of the small sample of Agkistrodon acutus defibrase to be tested in Example 1.
[0032] Figure 4 This is a comparative graph for precision verification in Example 2 (six parallel defibrase test samples).
[0033] Figure 5 This is the chromatogram of the small sample of Agkistrodon acutus defibrase to be tested in Comparative Example 1.
[0034] Figure 6 This is the chromatogram of the sample of Agkistrodon acutus defibrase to be tested in Comparative Example 2.
[0035] Figure 7 This is the chromatogram of the sample of Agkistrodon acutus defibrase to be tested in Comparative Example 3.
[0036] Figure 8 This is a comparative chart of the defibrase from Agkistrodon acutus and Agkistrodon acutus.
[0037] In each chromatogram, the horizontal axis is time (min) and the vertical axis is response value (mAU). DETAILED DESCRIPTION
[0038] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.
[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.
[0040] The endpoints and any values of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0041] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.
[0042] The present application provides a method for detecting the purity of the defibrase from Agkistrodon halys. The detection method uses mobile phase A and mobile phase B for elution; the mobile phase A is a 0.1% trifluoroacetic acid aqueous solution; the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution; according to the volume percentage, the elution procedure is as follows:
[0043] time Mobile phase A Mobile phase B 0 75％ 25％ 35min 35％～45％ 45％～55％ 40min 25％～35％ 65％～75％ 40.1min~45min 75％ 25％
[0044] The chromatographic column is filled with silica gel with neutral functional groups bonded on the surface.
[0045] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.
[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0047] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.
[0048] Example 1
[0049] This embodiment provides a method for detecting the purity of Agkistrodon acutus defibrase.
[0050] The detection method is as follows:
[0051] The protein content of the sample to be tested was detected, and according to the results of the protein content determination, the sample to be tested was prepared into a solution containing 1 mg of protein per 1 ml as the test solution. 10 μL of the test solution was injected into an HPLC liquid chromatograph (Thermo Fisher-Dionex UltiMate U3000) for detection. The detection conditions are as follows:
[0052] Chromatographic column: Phenyl column, ChromCore Phenyl, 4.6mm×250mm, 5μm.
[0053] Mobile phase A: 0.1% trifluoroacetic acid aqueous solution. Preparation method: Measure 1000 ml of filtered purified water, add 1 ml of trifluoroacetic acid, mix well, and ultrasonicate for 15 minutes.
[0054] Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Preparation method: Measure 1000 ml of chromatographically pure acetonitrile, add 1 ml of trifluoroacetic acid, mix well, and ultrasonicate for 15 minutes.
[0055] The elution program is shown in Table 1.
[0056] Table 1 Elution procedure of Example 1
[0057] time Mobile phase A Mobile phase B 0 75％ 25％ 35min 35％～45％ 45％～55％ 40min 25％～35％ 65％～75％ 40.1min~45min 75％ 25％
[0058] Elution time: 45min. Detection wavelength: 280nm. Flow rate: 0.7ml / min. Elution temperature: 45℃.
[0059] Record the chromatogram and the test results are as follows: Figure 1 Calculated by area normalization method.
[0060] Depend on Figure 1 It can be seen that the retention time is 21.900min for the defibrase component A of Agkistrodon acutus, and the retention time is 22.933min for the defibrase component B of Agkistrodon acutus. The peak shapes of the two components are good, the separation degree is 1.83, and the theoretical plate number is above 3000 according to the two main peaks. The sum of the relative peak area ratio is 99.52%. This shows that the detection method can accurately separate the two active components of the defibrase of Agkistrodon acutus.
[0061] At the same time, in order to further reflect the ability of the detection method of this embodiment, the detection method of this embodiment is used to detect the sample with the lowest purity in the process. The test results are as follows: Figure 2 As shown. Figure 2 It can be seen that the separation degree of the defibrase A component of Agkistrodon acutus and other impurities is much greater than 1.5.
[0062] and Figure 3 The above test results are compared with the SDS-PAGE diagram of the sample of Agkistrodon acutus defibrase shown in the figure, which shows that the above test results are consistent with the test results of the SDS-PAGE diagram. It further shows that the detection method can accurately separate the two active components of Agkistrodon acutus defibrase, and can provide a basis for monitoring the generation and removal of impurities in the process.
[0063] Example 2
[0064] This example verifies the precision of the detection method provided in Example 1.
[0065] Dilute the sample of Agkistrodon acutus defibrase to be tested with pure water to a solution containing 1 mg protein per 1 ml, as the test solution, prepare 6 copies in parallel, inject the sample, and record the chromatogram. The RSD values of the retention time of the two main peaks of components A and B should not be greater than 1.0%, and the RSD values of the peak areas should not be greater than 2.0%.
[0066] The test results are shown in Table 2.
[0067] Table 2 Precision verification results
[0068]
[0069] As shown in Table 2, the RSDs of component A and component B are 0.06% and 0.28% respectively according to retention time, and 1.43% and 0.45% respectively according to peak area, indicating that the method of Example 1 has good precision.
[0070] Comparative Example
[0071] Comparative Example 1
[0072] This comparative example provides a method for detecting the purity of Agkistrodon acutus defibrase. The detection object of this comparative example is the same as that of Example 1.
[0073] 20 μL of the test solution was injected into a liquid chromatograph (Thermo Fisher Dionex UltiMate U3000) for detection. The detection method is as follows:
[0074] Chromatographic column: Vydac 214TP54-C4 (5 mm, 250 mm × 4.6 mm) chromatographic column.
[0075] Mobile phase A: 0.1% trifluoroacetic acid in water.
[0076] Mobile phase B: 90% acetonitrile solution (containing 0.1% trifluoroacetic acid).
[0077] The elution procedure was as follows: gradient elution (expressed in terms of the proportion of mobile phase B: 0→5 min, maintained at 40%; 5→30 min, 40%→100%; 30→40 min, maintained at 100%; 40→41 min, 100%→40%).
[0078] Detection wavelength: 280nm. Flow rate: 0.8mL / min. Elution column temperature: 45℃.
[0079] Record the chromatogram and the test results are as follows: Figure 5 shown.
[0080] Depend on Figure 5 It can be seen that although the detection method of Comparative Example 1 can detect two components of the defibrase of Agkistrodon halys, the separation degree is 0.43, which is far less than 1.5, and therefore it is impossible to effectively monitor the purity of the defibrase.
[0081] Comparative Example 2
[0082] This comparative example provides a method for detecting the purity of Agkistrodon acutus defibrase. The detection object of this comparative example is the same as that of Example 1.
[0083] The detection method is as follows:
[0084] Gel analysis column: Biosep S2000, 7.8 mm × 300 mm, 5 μm.
[0085] Mobile phase: 0.2 mol / L sodium phosphate buffer (pH 6.8). Preparation method: Take 175.4 g of disodium hydrogen phosphate and 79.56 g of sodium dihydrogen phosphate, dissolve in 900 ml of water, adjust the pH value to 6.8, add water to 5000 ml, mix, filter, and ultrasonicate for 15 minutes.
[0086] Detection wavelength: 280nm. Flow rate: 1mL / min.
[0087] Record the chromatogram and the test results are as follows: Figure 6 shown.
[0088] Depend on Figure 6 It can be seen that only one main peak was detected in the test results of Comparative Example 2, with a retention time of 8.202 min, and the two components could not be separated. Obviously, using a gel chromatography column to control sample purity cannot accurately reflect the impurity situation, cannot achieve the purpose of quality control, and may pose clinical risks.
[0089] Comparative Example 3
[0090] This comparative example provides a method for detecting the purity of Agkistrodon acutus defibrase. The detection object of this comparative example is the same as that of Example 1.
[0091] Inject 20 μL of 1 mg / ml test solution into a liquid chromatograph (Thermo Fisher Dionex UltiMate U3000) for detection. The detection method is as follows:
[0092] C18 reverse phase analytical column: Agilent ZORBAX 300SB-C18, 4.6 mm × 250 mm, 5 μm;
[0093] Mobile phase A: 20 mM sodium dihydrogen phosphate buffer. The preparation method is as follows: weigh about 2.4 g of sodium dihydrogen phosphate, dissolve it in 950 mL of water, then adjust the pH to 6.50 with sodium hydroxide solution, add water to make up to 1000 mL, mix well, filter, and sonicate for 15 minutes.
[0094] Mobile phase B: pure acetonitrile.
[0095] Mobile phase C: 1% TFA. The preparation method is: add 2 mL TFA to 200 mL water, mix well, and ultrasonicate for 15 minutes.
[0096] The elution procedure was as follows: gradient elution (expressed in terms of the proportion of mobile phase B: 0→5 min, maintaining at 32%; 5→25 min, 32%→42%; 20→20.1 min, 42%→32%; 25.1→30 min, maintaining at 32%, and mobile phase C was 15%).
[0097] Elution time: 30min. Detection wavelength: 280nm. Flow rate: 0.8ml / min. Elution temperature: 40℃.
[0098] Record the chromatogram and the test results are as follows: Figure 7 shown.
[0099] Depend on Figure 7 It can be seen that the detection method of Comparative Example 3 can achieve baseline separation of the two components of the defibrase of Agkistrodon acutus, but the peak shape is not good and the peak is wide. Therefore, the method needs to be improved and optimized. In addition, the mobile phase contains three phases, and the mixing process is prone to poor stability. The mobile phase contains buffer salts, which easily block the column.
[0100] By comparing the test results of Example 1 with those of Comparative Examples 1-3, it can be seen that the detection method of the present application can accurately separate the two active components of Agkistrodon agkistrodon defibrase, laying a foundation for improving and perfecting the quality standard of Agkistrodon agkistrodon defibrase and providing data support for improving and optimizing the production process.
[0101] Example 4
[0102] This example uses the detection method of Example 1 to detect the defibrase of Agkistrodon acutus.
[0103] According to the mobile phase system of Example 1, the retention time of about 25.303 min is Agkistrodon acutus defibrase, and its active ingredient is a single component, which is consistent with the results described in the literature.
[0104] The reversed-phase HPLC test results of Example 1 were compared with the reversed-phase HPLC test results of this example. Figure 8 shown.
[0105] Depend on Figure 8 It can be seen that the retention times of the main peaks of the two snake venom defibrases are significantly different, indicating that the detection method provided in the present application can quickly and accurately identify defibrases from different snake venom sources.
[0106] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A method for detecting the purity of Agkistrodon acutus defibrase, characterized in that: The detection method uses mobile phase A and mobile phase B for elution; The mobile phase A is a 0.1% trifluoroacetic acid aqueous solution; The mobile phase B is 0.1% trifluoroacetic acid in acetonitrile solution; According to volume percentage, the elution procedure is as follows; The chromatographic column is filled with silica gel with neutral functional groups bonded on the surface.
2. The detection method according to claim 1, characterized in that: The chromatographic column is a phenyl chromatographic column.
3. The detection method according to claim 1, characterized in that: The length of the chromatographic column is 250 mm.
4. The detection method according to claim 1, characterized in that: The pore size of the chromatographic column is 5 μm.
5. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.6-0.8 ml / min; the elution temperature is 40-50° C.; and the detection wavelength is 280 nm.
6. The detection method according to claim 5, characterized in that: The flow rate of the mobile phase was 0.7 ml / min; the elution temperature was 45°C.
7. Use of the detection method according to any one of claims 1 to 6 in the quality control of defibrase from Agkistrodon halys.
8. Use of the detection method according to any one of claims 1 to 6 in distinguishing the defibrase from Agkistrodon acutus.
9. Use of the detection method according to any one of claims 1 to 6 in identifying defibrase from different snake venom sources.