CN116046922A - Method for measuring content of 7-bromo-1-H-indazole by high performance liquid chromatography - Google Patents

Method for measuring content of 7-bromo-1-H-indazole by high performance liquid chromatography Download PDF

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CN116046922A
CN116046922A CN202211383158.2A CN202211383158A CN116046922A CN 116046922 A CN116046922 A CN 116046922A CN 202211383158 A CN202211383158 A CN 202211383158A CN 116046922 A CN116046922 A CN 116046922A
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indazole
bromo
mobile phase
content
performance liquid
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杜仪静
宋泽选
董玉灿
张雪
马松
王才朋
刘海东
吴彩英
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Shandong Yanggu Huatai Chemical Co Ltd
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Abstract

The invention relates to a method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography. The chromatographic conditions of the method are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of triethylamine buffer solution and methanol, and the mobile phase B is acetonitrile. The volume ratio of the mobile phase A to the mobile phase B is 4:6. the method for measuring the content of the 7-bromo-1-H-indazole by using the high performance liquid chromatography can realize that the separation degree of the 7-bromo-1-H-indazole and impurities thereof reaches 1.8, and can effectively separate the 7-bromo-1-H-indazole and the impurities thereof. The method adopts a specific isocratic elution mode, the sample is rapidly dissolved in 1min, and the method has the advantages of good specificity, high analysis speed, high reproducibility and high stability, is convenient for quality detection and monitoring of the 7-bromo-1-H-indazole, improves detection efficiency, and is beneficial to safe popularization and application of the 7-bromo-1-H-indazole.

Description

Method for measuring content of 7-bromo-1-H-indazole by high performance liquid chromatography
Technical Field
The invention relates to a method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography, belonging to the technical field of chemical analysis and detection.
Background
Indazole compounds or physiologically acceptable derivatives thereof have the effect of inhibiting Rho kinase activity, respectively; has the functions of dilating blood vessel and protecting blood vessel; has effects in protecting myocardial cells against anoxia reperfusion injury; has effects in promoting cell glucose consumption; has the function of protecting vascular endothelial cells against oxidative damage, and is a novel compound with important application in preparing medicaments for preventing and treating cardiovascular and cerebrovascular diseases, diabetes and the like such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetic complications and the like.
7-bromo-1-H-indazole is an important chemical intermediate for human body treatment, drug development and the like as an indazole derivative. High performance liquid chromatography is a common detection means for quantifying substances, but 7-bromo-1-H-indazole contains impurities with polarity which is not much different from that of 7-bromo-1-H-indazole, so that direct separation by the existing high performance liquid chromatography is difficult. And at present, no detection method for 7-bromo-1-H-indazole is disclosed, and a detection method for 7-bromo-1-H-indazole which is simple to operate, high in sensitivity and good in reproducibility is urgently needed to be established.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for measuring the content of 7-bromo-1-H-indazole by using a high performance liquid chromatography.
The technical scheme of the invention is as follows:
a method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography, wherein the chromatographic conditions are as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of triethylamine buffer solution and methanol, and the mobile phase B is acetonitrile;
the structural formula of the 7-bromo-1-H-indazole is as follows:
Figure BDA0003928808450000021
according to the present invention, the column is preferably a Shim-pack GIS C18 column having a size of 4.6mm×25cm×5 μm (inner diameter×column length×particle diameter).
According to the invention, the volume ratio of the triethylamine buffer to the methanol is preferably 9:1.
further preferably, the triethylamine buffer is formulated as follows: 1ml of triethylamine was added to 1000ml of water, and phosphoric acid was added to adjust the pH to 6.0.
According to the invention, the volume ratio of the mobile phase A to the mobile phase B is preferably 4:6. the inventor of the application finds that when the high performance liquid chromatography is adopted to detect the 7-bromo-1-H-indazole, the proportion of the mobile phase has great influence on the separation of impurities and main components, and the mobile phase A is adopted: mobile phase b=4: when the solution is eluted at 6 isocratic, the 7-bromo-1-H-indazole can be well distinguished from impurities.
According to a preferred embodiment of the invention, the chromatographic conditions are in particular:
chromatographic column: a Shim-pack GIS C18 column;
mobile phase a: triethylamine buffer: methanol=9: 1, a step of;
mobile phase B: acetonitrile;
column temperature: 30-40 ℃;
detection wavelength: 205-215 nm;
flow rate: 0.8-1.2 mL/min;
sample injection volume: 8-12 mu L;
analysis time: 20-26 min;
mobile phase a: the volume ratio of the mobile phase B is 4:6.
according to a preferred embodiment of the invention, the chromatographic conditions are in particular:
chromatographic column: a Shim-pack GIS C18 column;
mobile phase a: triethylamine buffer: methanol=9: 1, a step of;
mobile phase B: acetonitrile;
column temperature: 35 ℃;
detection wavelength: 210nm;
flow rate: 1mL/min;
sample injection volume: 10.0. Mu.L;
analysis time: 23min;
mobile phase a: the volume ratio of the mobile phase B is 4:6.
according to the invention, the method for measuring the content of the 7-bromo-1-H-indazole by using the high performance liquid chromatography specifically comprises the following steps of:
(1) Diluting a sample to be measured to 0.25-0.75 mg/mL by using an acetonitrile aqueous solution to serve as a sample solution; diluting the 7-bromo-1-H-indazole reference substance to 0.25-0.75 mg/mL with acetonitrile aqueous solution to obtain reference substance solution;
(2) Eluting the sample solution under the chromatographic conditions;
(3) And detecting and analyzing the characteristic peak area of the reference substance solution, and calculating the content of the 7-bromo-1-H-indazole by an external standard method.
According to a preferred embodiment of the present invention, in the step (1), the concentration of the acetonitrile aqueous solution is 80%.
The linear equation of the 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 the 7-bromo-1-H-indazole in the chromatogram; r is R 2 Representing the correlation coefficient.
In the present invention, "%" represents mass percent.
The invention has the following beneficial effects:
the method for measuring the content of the 7-bromo-1-H-indazole by using the high performance liquid chromatography can effectively measure the content of the 7-bromo-1-H-indazole, realize that the separation degree of the 7-bromo-1-H-indazole and impurities thereof reaches 1.8, and separate the 7-bromo-1-H-indazole and the impurities thereof during measurement. Meanwhile, the sensitivity of the detection of the 7-bromo-1-H-indazole and the accuracy of the content thereof are further improved by optimizing the conditions. The method adopts a specific isocratic elution mode, the sample is rapidly dissolved in 1min, and the method has the advantages of good specificity, high analysis speed, high reproducibility and high stability, is convenient for quality detection and monitoring of the 7-bromo-1-H-indazole, improves detection efficiency, and is beneficial to safe popularization and application of the 7-bromo-1-H-indazole.
Drawings
FIG. 1 is a chromatogram of the detection of 7-bromo-1-H-indazole according to example 1 of the present invention.
FIG. 2 is a standard curve for detecting 7-bromo-1-H-indazole according to the method of example 1 of the present invention.
FIG. 3 is a chromatogram of the detection of 7-bromo-1-H-indazole according to comparative example 2 of the present invention.
FIG. 4 is a chromatogram of the detection of 7-bromo-1-H-indazole according to comparative example 3 of the present invention.
Detailed Description
The technical solutions of the present application will be clearly and completely described below in connection with the embodiments of the present application. The reagents and starting materials used were purchased commercially, unless otherwise indicated. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
1ml of triethylamine was added to 1000ml of water, and pH was adjusted to 6.0 by adding phosphoric acid to obtain a triethylamine buffer.
Acetonitrile and water are uniformly mixed according to the volume ratio of 8:2, and an acetonitrile aqueous solution is obtained.
Example 1
A method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography comprises the following steps:
octadecylsilane chemically bonded silica gel column (size: 4.6 mm. Times.25 cm. Times.5 μm (inner diameter. Times.column length. Times.particle size)) was used, and the model was a Shim-pack GIS C18 column.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is triethylamine buffer solution: methanol=9: 1 (volume ratio), mobile phase B is acetonitrile; mobile phase a: mobile phase b=4: 6 (volume ratio).
Chromatographic parameters: column temperature 35 ℃, detection wavelength 210nm, flow rate 1.0mL/min, sample injection volume: 10.0 μl, analysis time: 23min.
7-bromo-1-H-indazole standard was diluted with acetonitrile aqueous solution to standard solutions of gradient concentrations 10, 100, 250, 500 and 1000 μg/mL as test solutions.
The solution to be tested is filtered by an organic filter membrane, a sequence is set, and sample injection is started after the instrument is stable, wherein the high performance liquid chromatography detection result of the 7-bromo-1-H-indazole with the concentration of 500 mug/mL is shown in figure 1.
As can be seen from FIG. 1, the peak position of the 7-bromo-1-H-indazole was 4.78min, and the degree of separation between the main peak and the neighboring peak was 1.8.
The peak area results corresponding to the solutions to be measured with different concentrations are shown in table 1 below.
TABLE 1
x: 7-bromo-1-H-indazole concentration/. Mu.g/mL y: representing peak area
10 324027
100 3092455
250 7778124
500 17220862
1000 31294850
A standard curve was plotted according to the data in table 1, with a linear equation of 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 the 7-bromo-1-H-indazole in the chromatogram; r is R 2 A specific standard graph representing the correlation coefficient is shown in fig. 2.
Comparative example 1
A method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography comprises the following steps:
octadecylsilane chemically bonded silica gel column (size: 4.6 mm. Times.25 cm. Times.5 μm (inner diameter. Times.column length. Times.particle size)) was used, and the model was a Shim-pack GIS C18 column.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is water, and the mobile phase B is acetonitrile; mobile phase a: mobile phase b=4: 6 (volume ratio).
Chromatographic parameters: column temperature 35 ℃, detection wavelength 210nm, flow rate 1.0mL/min, sample injection volume: 10.0 μl, analysis time: 23min.
The detection method is the same as that of example 1, and the result shows that the peak position of the 7-bromo-1-H-indazole is 5.01min, the separation degree of a main peak and an adjacent peak is 1.3, and the peak type is poor.
Comparative example 2
A method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography comprises the following steps:
octadecylsilane chemically bonded silica gel column (size: 4.6 mm. Times.25 cm. Times.5 μm (inner diameter. Times.column length. Times.particle size)) was used, and the model was a Shim-pack GIS C18 column.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is triethylamine buffer solution: methanol=9: 1 (volume ratio), mobile phase B is acetonitrile; mobile phase a: mobile phase b=2: 8 (volume ratio).
Chromatographic parameters: column temperature 35 ℃, detection wavelength 210nm, flow rate 1.0mL/min, sample injection volume: 10.0 μl, analysis time: 23min.
The detection method was the same as in example 1, and the results are shown in FIG. 3. As can be seen from FIG. 3, the peak position of the 7-bromo-1-H-indazole is 2.98min, the separation degree of the main peak and the adjacent peak is 0.9, and the interference is left.
Comparative example 3
A method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography comprises the following steps:
octadecylsilane chemically bonded silica gel column (size: 4.6 mm. Times.25 cm. Times.5 μm (inner diameter. Times.column length. Times.particle size)) was used, and the model was a Shim-pack GIS C18 column.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is triethylamine buffer solution: methanol=9: 1 (volume ratio), mobile phase B is acetonitrile; mobile phase a: mobile phase b=6: 4 (volume ratio).
Chromatographic parameters: column temperature 35 ℃, detection wavelength 210nm, flow rate 1.0mL/min, sample injection volume: 10.0 μl, analysis time: 23min.
The detection method was the same as in example 1, and the results are shown in FIG. 4. As can be seen from FIG. 4, the peak position of the 7-bromo-1-H-indazole was 10.29min, the separation degree of the main peak from the neighboring peak was 2.5, and the peak tailing was severe.
As can be seen from the comparison of example 1 and comparative examples 1 to 3, the mobile phase and isocratic elution procedure of example 1 are adopted, the separation degree is high, the impurities and the 7-bromo-1-H-indazole can be completely separated, the sensitivity and stability of detection can be ensured, the time for isocratic elution is short, and the analysis efficiency is high. However, the water of comparative example 1 had problems of low degree of separation and poor peak pattern after being used as mobile phase A. Mobile phase a of comparative example 2 was used instead: mobile phase b=2: after 8 (volume ratio), the degree of separation is low and there is blank interference. Mobile phase a of comparative example 3 was used instead: mobile phase b=6: after 4 (volume ratio), peak tailing was severe and the analysis time was long, and the analysis efficiency was too low.
In conclusion, the method for measuring the content of the 7-bromo-1-H-indazole by using the high performance liquid chromatography can realize that the separation degree of the 7-bromo-1-H-indazole and impurities thereof reaches 1.8, and can effectively separate the 7-bromo-1-H-indazole and the impurities thereof. And the sensitivity of the detection of the 7-bromo-1-H-indazole and the accuracy of the content thereof are further improved by optimizing the conditions. The method can monitor and analyze 7-bromo-1-H-indazole in real time, can be used for screening reaction conditions in the process of synthesizing 7-bromo-1-H-indazole, and simultaneously provides an analysis basis for the quality standard of 7-bromo-1-H-indazole.

Claims (10)

1. A method for measuring the content of 7-bromo-1-H-indazole by high performance liquid chromatography, characterized in that the chromatographic conditions are as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica chromatographic column;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of triethylamine buffer solution and methanol, and the mobile phase B is acetonitrile;
the structural formula of the 7-bromo-1-H-indazole is as follows:
Figure FDA0003928808440000011
2. the method for determining the content of 7-bromo-1-H-indazole according to claim 1, wherein said chromatography column is a Shim-pack GIS C18 chromatography column having a specification of 4.6mm x 25cm x 5 μm (inner diameter x column length x particle size).
3. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, wherein said volume ratio of triethylamine buffer to methanol is 9:1.
4. the method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 3, wherein said triethylamine buffer is formulated according to the following method: 1ml of triethylamine was added to 1000ml of water, and phosphoric acid was added to adjust the pH to 6.0.
5. The method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, wherein said volume ratio of mobile phase a to mobile phase B is 4:6.
6. the method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 1, wherein said chromatographic conditions are in particular:
chromatographic column: a Shim-pack GIS C18 column;
mobile phase a: triethylamine buffer: methanol=9: 1, a step of;
mobile phase B: acetonitrile;
column temperature: 30-40 ℃;
detection wavelength: 205-215 nm;
flow rate: 0.8-1.2 mL/min;
sample injection volume: 8-12 mu L;
analysis time: 20-26 min;
mobile phase a: the volume ratio of the mobile phase B is 4:6.
7. the method for determining the content of 7-bromo-1-H-indazole by high performance liquid chromatography according to claim 6, wherein said chromatographic conditions are in particular:
chromatographic column: a Shim-pack GIS C18 column;
mobile phase a: triethylamine buffer: methanol=9: 1, a step of;
mobile phase B: acetonitrile;
column temperature: 35 ℃;
detection wavelength: 210nm;
flow rate: 1mL/min;
sample injection volume: 10.0. Mu.L;
analysis time: 23min;
mobile phase a: the volume ratio of the mobile phase B is 4:6.
8. the method for measuring the content of 7-bromo-1-H-indazole by using high performance liquid chromatography according to claim 1, comprising the following steps:
(1) Diluting a sample to be measured to 0.25-0.75 mg/mL by using an acetonitrile aqueous solution to serve as a sample solution; diluting the 7-bromo-1-H-indazole reference substance to 0.25-0.75 mg/mL with acetonitrile aqueous solution to obtain reference substance solution;
(2) Eluting the sample solution under the chromatographic conditions;
(3) And detecting and analyzing the characteristic peak area of the reference substance solution, and calculating the content of the 7-bromo-1-H-indazole by an external standard method.
9. The method for determining the content of 7-bromo-1-H-indazole according to claim 8, wherein in step (1), said aqueous acetonitrile solution has a concentration of 80%.
10. The method for determining the content of 7-bromo-1-H-indazole according to claim 1, wherein said method has a linear equation of 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 the 7-bromo-1-H-indazole in the chromatogram; r is R 2 Representing the correlation coefficient.
CN202211383158.2A 2022-11-07 2022-11-07 Method for measuring content of 7-bromo-1-H-indazole by high performance liquid chromatography Pending CN116046922A (en)

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