A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography

Abstract

The present invention relates to a method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography. The chromatographic conditions of the method are: the chromatographic column adopts an octadecylsilane bonded silica gel chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixed solution of triethylamine buffer and methanol, and the mobile phase B is acetonitrile. The volume ratio of the mobile phase A and the mobile phase B is 4:6. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography provided by the present invention can achieve a separation degree of 1.8 between 7-bromo-1-H-indazole and its impurities, and can effectively separate 7-bromo-1-H-indazole from its impurities. And the method of the present invention adopts a specific isocratic elution mode, and the sample is quickly dissolved in 1min, and has the advantages of good specificity, fast analysis speed, high reproducibility, and high stability, which is convenient for the quality detection and monitoring of 7-bromo-1-H-indazole, and at the same time improves the detection efficiency, which is conducive to the safe promotion and application of 7-bromo-1-H-indazole.

Description

Technical Field

[0001] The invention relates to a method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography, belonging to the technical field of chemical analysis and detection. Background Art

[0002] Indazole compounds or their physiologically acceptable derivatives respectively have the effects of inhibiting Rho kinase activity; dilating blood vessels and protecting vascular function; protecting myocardial cells from hypoxia-reperfusion injury; promoting cellular glucose consumption; and protecting vascular endothelial cells from oxidative damage. They are a class of new compounds that are of great use in the preparation of drugs for the prevention and treatment of cardiovascular and cerebrovascular diseases, diabetes, and the like, such as hypertension, atherosclerosis, cerebral vasospasm, coronary artery spasm, myocardial infarction, heart failure, and diabetic complications.

[0003] 7-bromo-1-H-indazole, as a derivative of indazole, is an important chemical intermediate for human treatment, drug development, etc. High performance liquid chromatography is a commonly used detection method for quantitative determination of substances, but 7-bromo-1-H-indazole contains impurities with polarity similar to that of 7-bromo-1-H-indazole, so it is difficult to directly separate them by existing high performance liquid chromatography. In addition, there is no public detection method for 7-bromo-1-H-indazole at present, and it is urgent to establish a detection method for 7-bromo-1-H-indazole that is simple to operate, highly sensitive, and reproducible. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography.

[0005] The technical solution of the present invention is as follows:

[0006] A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography, wherein the chromatographic conditions are:

[0007] The chromatographic column adopts an octadecylsilane bonded silica gel chromatographic column;

[0008] The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixed solution of triethylamine buffer and methanol, and the mobile phase B is acetonitrile;

[0009] The structural formula of the 7-bromo-1-H-indazole is as follows:

[0010]

[0011] Preferably according to the present invention, the chromatographic column is a Shim-pack GIS C18 chromatographic column with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter).

[0012] Preferably according to the present invention, the volume ratio of the triethylamine buffer to methanol is 9:1.

[0013] Further preferably, the triethylamine buffer is prepared as follows: add 1 ml of triethylamine to 1000 ml of water, and add phosphoric acid to adjust the pH to 6.0.

[0014] Preferably, according to the present invention, the volume ratio of the mobile phase A to the mobile phase B is 4:6. The inventors of the present application found that when 7-bromo-1-H-indazole is detected by high performance liquid chromatography, the ratio of the mobile phase has a great influence on the separation of impurities and the main component. When the mobile phase A: mobile phase B = 4:6 isocratic elution is used, 7-bromo-1-H-indazole and impurities can be well distinguished.

[0015] Preferably according to the present invention, the chromatographic conditions are specifically:

[0016] Chromatographic column: Shim-pack GIS C18 column;

[0017] Mobile phase A: triethylamine buffer: methanol = 9:1;

[0018] Mobile phase B: acetonitrile;

[0019] Column temperature: 30-40°C;

[0020] Detection wavelength: 205~215nm;

[0021] Flow rate: 0.8~1.2mL / min;

[0022] Injection volume: 8-12 μL;

[0023] Analysis time: 20-26 minutes;

[0024] The volume ratio of mobile phase A:mobile phase B was 4:6.

[0025] Preferably according to the present invention, the chromatographic conditions are specifically:

[0026] Chromatographic column: Shim-pack GIS C18 column;

[0027] Mobile phase A: triethylamine buffer: methanol = 9:1;

[0028] Mobile phase B: acetonitrile;

[0029] Column temperature: 35°C;

[0030] Detection wavelength: 210nm;

[0031] Flow rate: 1 mL / min;

[0032] Injection volume: 10.0 μL;

[0033] Analysis time: 23min;

[0034] The volume ratio of mobile phase A:mobile phase B was 4:6.

[0035] Preferably according to the present invention, the method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography specifically comprises the following steps:

[0036] (1) Dilute the sample to be tested with acetonitrile aqueous solution to 0.25-0.75 mg / mL as the test solution; dilute the 7-bromo-1-H-indazole reference substance with acetonitrile aqueous solution to 0.25-0.75 mg / mL as the reference substance solution;

[0037] (2) eluting the test solution under the above-mentioned chromatographic conditions;

[0038] (3) Detect and analyze the characteristic peak area of ​​the reference solution and calculate the content of 7-bromo-1-H-indazole by the external standard method.

[0039] Preferably according to the present invention, in step (1), the concentration of the acetonitrile aqueous solution is 80%.

[0040] The linear equation of the above method is y=31871.1x+5329.5, R 2 =0.9997, where x represents the concentration of 7-bromo-1-H-indazole, in μg / mL; y represents the peak area of ​​7-bromo-1-H-indazole in the chromatogram; R 2 Represents the correlation coefficient.

[0041] In the present invention, "%" means mass percentage.

[0042] The beneficial effects of the present invention are as follows:

[0043] The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography provided by the present invention can effectively determine the content of 7-bromo-1-H-indazole, realize the separation degree of 7-bromo-1-H-indazole and its impurities up to 1.8, and separate 7-bromo-1-H-indazole from its impurities while measuring. At the same time, by optimizing the conditions, the sensitivity of 7-bromo-1-H-indazole detection and the accuracy of its content are further improved. And the method of the present invention adopts a specific isocratic elution mode, and the sample is quickly dissolved in 1min, with the advantages of good specificity, fast analysis speed, high reproducibility and high stability, which is convenient for the quality detection and monitoring of 7-bromo-1-H-indazole, and the detection efficiency is improved at the same time, which is conducive to the safe promotion and application of 7-bromo-1-H-indazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a chromatogram of 7-bromo-1-H-indazole detected by the method of Example 1 of the present invention.

[0045] Figure 2 This is the standard curve for detecting 7-bromo-1-H-indazole using the method in Example 1 of the present invention.

[0046] Figure 3 This is a chromatogram of 7-bromo-1-H-indazole detected by the method of Comparative Example 2 of the present invention.

[0047] Figure 4 This is a chromatogram of 7-bromo-1-H-indazole detected by the method of Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solution of the present application. Unless otherwise specified, the reagents and raw materials used can be purchased through commercial channels. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications.

[0049] Add 1 ml of triethylamine to 1000 ml of water, and add phosphoric acid to adjust the pH to 6.0 to obtain triethylamine buffer.

[0050] Acetonitrile and water were uniformly mixed in a volume ratio of 8:2 to obtain an acetonitrile aqueous solution.

[0051] Example 1

[0052] A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography is as follows:

[0053] An octadecylsilane bonded silica gel chromatographic column was used, with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter), and model number was Shim-pack GIS C18 chromatographic column.

[0054] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is triethylamine buffer: methanol = 9:1 (volume ratio), and mobile phase B is acetonitrile; mobile phase A: mobile phase B = 4:6 (volume ratio).

[0055] Chromatographic parameters: column temperature 35°C, detection wavelength 210 nm, flow rate 1.0 mL / min, injection volume: 10.0 μL, analysis time: 23 min.

[0056] The 7-bromo-1-H-indazole standard was diluted with acetonitrile aqueous solution to obtain standard solutions with gradient concentrations of 10, 100, 250, 500 and 1000 μg / mL as test solutions.

[0057] The solution to be tested was filtered through an organic filter membrane, and the sequence was set. After the instrument was stable, the injection was started. The HPLC test results of 7-bromo-1-H-indazole with a concentration of 500 μg / mL were as follows: Figure 1 shown.

[0058] Depend on Figure 1 It can be seen that the elution position of 7-bromo-1-H-indazole is 4.78 min, and the separation degree between the main peak and the adjacent peak is 1.8.

[0059] The peak area results corresponding to the test solutions of different concentrations are shown in Table 1 below.

[0060] Table 1

[0061] x: 7-bromo-1-H-indazole concentration / μg / mL y: represents the peak area 10 324027 100 3092455 250 7778124 500 17220862 1000 31294850

[0062] The standard curve was drawn according to the data in Table 1. The linear equation of the standard curve was y=31871.1x+5329.5, R 2 =0.9997, where x represents the concentration of 7-bromo-1-H-indazole, in μg / mL; y represents the peak area of ​​7-bromo-1-H-indazole in the chromatogram; R 2 Represents the correlation coefficient. The specific standard curve is shown in Figure 2 shown.

[0063] Comparative Example 1

[0064] A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography is as follows:

[0065] An octadecylsilane bonded silica gel chromatographic column was used, with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter), and model number was Shim-pack GIS C18 chromatographic column.

[0066] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is acetonitrile; mobile phase A:mobile phase B=4:6 (volume ratio).

[0067] Chromatographic parameters: column temperature 35°C, detection wavelength 210 nm, flow rate 1.0 mL / min, injection volume: 10.0 μL, analysis time: 23 min.

[0068] The detection method was the same as that in Example 1. The results showed that the peak position of 7-bromo-1-H-indazole was 5.01 min, the separation degree between the main peak and the adjacent peak was 1.3, and the peak shape was poor.

[0069] Comparative Example 2

[0070] A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography is as follows:

[0071] An octadecylsilane bonded silica gel chromatographic column was used, with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter), and model number was Shim-pack GIS C18 chromatographic column.

[0072] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is triethylamine buffer: methanol = 9:1 (volume ratio), and mobile phase B is acetonitrile; mobile phase A: mobile phase B = 2:8 (volume ratio).

[0073] Chromatographic parameters: column temperature 35°C, detection wavelength 210 nm, flow rate 1.0 mL / min, injection volume: 10.0 μL, analysis time: 23 min.

[0074] The detection method is the same as in Example 1, and the results are as follows Figure 3 Display. Figure 3 It can be seen that the elution position of 7-bromo-1-H-indazole is 2.98 min, the separation degree between the main peak and the adjacent peak is 0.9, and there is blank interference.

[0075] Comparative Example 3

[0076] A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography is as follows:

[0077] An octadecylsilane bonded silica gel chromatographic column was used, with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter), and model number was Shim-pack GIS C18 chromatographic column.

[0078] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is triethylamine buffer: methanol = 9:1 (volume ratio), and mobile phase B is acetonitrile; mobile phase A: mobile phase B = 6:4 (volume ratio).

[0079] Chromatographic parameters: column temperature 35°C, detection wavelength 210 nm, flow rate 1.0 mL / min, injection volume: 10.0 μL, analysis time: 23 min.

[0080] The detection method is the same as in Example 1, and the results are as follows Figure 4 Display. Figure 4 It can be seen that the elution position of 7-bromo-1-H-indazole is 10.29 min, the separation degree between the main peak and the adjacent peak is 2.5, and the peak tailing is serious.

[0081] By comparing Example 1 with Comparative Examples 1 to 3, it can be seen that when the mobile phase and isocratic elution procedure in Example 1 are used, the separation is high, the impurities and 7-bromo-1-H-indazole can be completely separated, the sensitivity and stability of the detection can be guaranteed, and the time used for isocratic elution is short, and the analysis efficiency is higher. However, after changing to water as mobile phase A in Comparative Example 1, there are problems of low separation and poor peak shape. After changing to mobile phase A: mobile phase B = 2:8 (volume ratio) in Comparative Example 2, the separation is low and there is blank interference. After changing to mobile phase A: mobile phase B = 6:4 (volume ratio) in Comparative Example 3, the peak tailing is serious and the analysis time is long, and the analysis efficiency is too low.

[0082] In summary, the method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography provided by the present invention can achieve a separation degree of 1.8 between 7-bromo-1-H-indazole and its impurities, and can effectively separate 7-bromo-1-H-indazole from its impurities. And by optimizing the conditions, the sensitivity of 7-bromo-1-H-indazole detection and the accuracy of its content are further improved. 7-bromo-1-H-indazole can be monitored and analyzed in real time, can be used for the screening of reaction conditions in the synthesis of 7-bromo-1-H-indazole process, and provides an analytical basis for the quality standard of 7-bromo-1-H-indazole.

Claims

1. A method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography, characterized in that: The chromatographic conditions are: The chromatographic column adopts octadecylsilane bonded silica gel chromatographic column; The mobile phase includes mobile phase A and mobile phase B; The mobile phase A is a mixed solution of triethylamine buffer and methanol, and the mobile phase B is acetonitrile; The volume ratio of the triethylamine buffer to methanol is 9:1; the triethylamine buffer is prepared as follows: 1 ml of triethylamine is added with water to 1000 ml, and phosphoric acid is added to adjust the pH to 6.0; the volume ratio of the mobile phase A to the mobile phase B is 4:6; The structural formula of the 7-bromo-1-H-indazole is as follows: 。 2. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, characterized in that: The chromatographic column is a Shim-pack GIS C18 chromatographic column with specifications of 4.6 mm×25 cm×5 μm (inner diameter×column length×particle diameter).

3. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, characterized in that: The chromatographic conditions are specifically: Chromatographic column: Shim-pack GIS C18 column; Mobile phase A: triethylamine buffer: methanol = 9:1; Mobile phase B: acetonitrile; Column temperature: 30~40℃; Detection wavelength: 205~215nm; Flow rate: 0.8~1.2mL / min; Injection volume: 8~12μL; Analysis time: 20~26min; The volume ratio of mobile phase A:mobile phase B was 4:

6.

4. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 3, characterized in that: The chromatographic conditions are specifically: Chromatographic column: Shim-pack GIS C18 column; Mobile phase A: triethylamine buffer: methanol = 9:1; Mobile phase B: acetonitrile; Column temperature: 35°C; Detection wavelength: 210nm; Flow rate: 1 mL / min; Injection volume: 10.0 μL; Analysis time: 23min; The volume ratio of mobile phase A:mobile phase B was 4:

6.

5. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, characterized in that: The specific steps include: (1) Dilute the sample to be tested with acetonitrile aqueous solution to 0.25~0.75 mg / mL as the test solution; dilute the 7-bromo-1-H-indazole reference substance with acetonitrile aqueous solution to 0.25~0.75 mg / mL as the reference substance solution; (2) eluting the test sample solution under the chromatographic conditions; (3) Detect and analyze the characteristic peak area of ​​the reference solution and calculate the content of 7-bromo-1-H-indazole by the external standard method.

6. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 5, characterized in that: In step (1), the concentration of the acetonitrile aqueous solution is 80%.

7. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, characterized in that: The linear equation of this method is y=31871.1x+5329.5, R 2 =0.9997, where x represents the concentration of 7-bromo-1-H-indazole, in μg / mL; y represents the peak area of ​​7-bromo-1-H-indazole in the chromatogram; R 2 Represents the correlation coefficient.

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