CN107490630B - Analytical detection method of 7-fluoro-4-hydroxyquinazoline - Google Patents

Analytical detection method of 7-fluoro-4-hydroxyquinazoline Download PDF

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CN107490630B
CN107490630B CN201610408865.0A CN201610408865A CN107490630B CN 107490630 B CN107490630 B CN 107490630B CN 201610408865 A CN201610408865 A CN 201610408865A CN 107490630 B CN107490630 B CN 107490630B
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hydroxyquinazoline
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张贵民
王芳
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Shandong New Time Pharmaceutical Co Ltd
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Abstract

The invention relates to an analytical detection method of 7-fluoro-4-hydroxyquinazoline, which is used for controlling the quality of 7-fluoro-4-hydroxyquinazoline and comprises the steps of using a chromatographic column (C18, 4.6 multiplied by 250mm and 3 mu m) with octadecylsilane chemically bonded silica as a filler, using 0.05-0.15% phosphoric acid water solution and acetonitrile as mobile phases, carrying out gradient elution, and carrying out high performance liquid chromatography analytical detection with the detection wavelength of 247nm and the column temperature of 25-35 ℃. The analysis and detection method can effectively separate the 7-fluoro-4-hydroxyquinazoline and impurities thereof, and has the advantages of high resolution and sensitivity, good repeatability and durability, short analysis time, simple operation, and stable and reliable result.

Description

Analytical detection method of 7-fluoro-4-hydroxyquinazoline
Technical Field
The invention relates to a high performance liquid chromatography analysis method, in particular to an analysis and detection method of 7-fluoro-4-hydroxyquinazoline.
Background
Afatinib is an aniline quinazoline compound, is an irreversible EGFR-HER2 dual tyrosine kinase receptor inhibitor, can irreversibly combine with EGFR-HER2 tyrosine kinase to inhibit the tyrosine kinase activity thereof, further blocks EGFR-HER 2-guided tumor cell signal conduction, inhibits the metastasis and proliferation of tumor cells, and promotes the apoptosis of the tumor cells.
The 7-fluoro-4-hydroxyquinazoline is one of important intermediates for synthesizing afatinib, and the chemical formula of the intermediate is C8H5FN2O, structural formula is:
Figure BDA0001013707320000011
the literature does not describe an analysis and detection method of the intermediate, but the analysis and detection of the intermediate plays an important role in reaction control and yield improvement, and also directly influences the quality of the final product afatinib, so that the establishment of a stable and effective analysis and detection method is necessary for quality control of the intermediate.
Disclosure of Invention
The invention aims to provide an analytical detection method of 7-fluoro-4-hydroxyquinazoline, which is used for controlling the quality of 7-fluoro-4-hydroxyquinazoline.
In order to achieve the purpose of the invention, the inventor finally obtains the following technical scheme through a large number of experiments:
an analytical detection method of 7-fluoro-4-hydroxyquinazoline, which is to perform gradient elution by using a chromatographic column with octadecylsilane chemically bonded silica as a filler, 0.05-0.15% phosphoric acid aqueous solution (volume ratio) as a mobile phase A and acetonitrile as a mobile phase B, and comprises the following steps:
a. taking a proper amount of 7-fluoro-4-hydroxyquinazoline, adding a solvent for dissolving, and preparing a sample solution containing 0.041-0.311 mg of 7-fluoro-4-hydroxyquinazoline per 1 ml;
b. setting the flow rate of a mobile phase to be 0.7-1.1 ml/min, the detection wavelength to be 247nm and the column temperature to be 25-35 ℃;
c. injecting 10 mu l of the sample solution of a into a liquid chromatograph, executing a gradient elution program, and completing the analysis and detection of the 7-fluoro-4-hydroxyquinazoline;
the specification of the chromatographic column is C18, 4.6X 250mm, 3 μm.
The gradient elution was set as follows, in volume ratio:
Figure BDA0001013707320000021
further, the gradient elution is preferably set as follows:
Figure BDA0001013707320000022
the mobile phase A is preferably 0.1% phosphoric acid aqueous solution.
The solvent is prepared from 0.1% phosphoric acid aqueous solution: and the acetonitrile is prepared according to a volume ratio of 90: 10.
The concentration of the sample solution is preferably 0.2 mg/ml.
The flow rate of the mobile phase is preferably 1.0ml/min and the column temperature is preferably 30 ℃.
The method of the present invention is explained in detail in the examples, and the method of the present invention is verified, and the results prove that: the analysis and detection method can effectively separate the 7-fluoro-4-hydroxyquinazoline and impurities thereof, and has the advantages of high separation degree and sensitivity, good repeatability and durability, short analysis time, simple operation, and stable and reliable result, thereby being used for quality control of the 7-fluoro-4-hydroxyquinazoline and providing effective guarantee for the quality of a final finished product.
Drawings
FIG. 1 HPLC chromatogram of 7-fluoro-4-hydroxyquinazoline from example 1.
FIG. 2 HPLC chromatogram of 7-fluoro-4-hydroxyquinazoline from example 2.
FIG. 3 HPLC chromatogram of 7-fluoro-4-hydroxyquinazoline from example 3.
FIG. 4 Linear working curve of 7-fluoro-4-hydroxyquinazoline of example 8.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1
Instruments and conditions: agilent 1260 liquid chromatography system, column: welch Ultimate XB-C18 (4.6X 250mm, 3 μm), detection wavelength 247nm, column temperature 30 ℃, flow rate 1.0ml/min, mobile phase A as 0.1% phosphoric acid aqueous solution (by volume), mobile phase B as acetonitrile, by volume, gradient elution set as:
Figure BDA0001013707320000031
the experimental steps are as follows: dissolving 7-fluoro-4-hydroxyquinazoline with 0.1% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 90:10, quantitatively diluting to prepare solution containing 0.2mg of 7-fluoro-4-hydroxyquinazoline in each 1ml, using the solution as test solution, precisely measuring 10 mu l of the test solution, injecting the solution into a liquid chromatograph, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 1.
FIG. 1 shows that under the chromatographic conditions, the 7-fluoro-4-hydroxyquinazoline peak and the impurity peak can be completely separated, the peak shape is good, the separation degree is high, the retention time of the 7-fluoro-4-hydroxyquinazoline peak is 13.839min, and the separation degree is 3.55.
Example 2
Instruments and conditions: agilent 1260 liquid chromatography system, column: welch Ultimate XB-C18 (4.6X 250mm, 3 μm), detection wavelength 247nm, column temperature 30 ℃, flow rate 0.7ml/min, mobile phase A of 0.05% phosphoric acid in water (by volume), mobile phase B of acetonitrile, gradient elution set up as:
Figure BDA0001013707320000032
Figure BDA0001013707320000041
the experimental steps are as follows: dissolving 7-fluoro-4-hydroxyquinazoline with 0.1% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 90:10, quantitatively diluting to prepare solution containing 0.2mg of 7-fluoro-4-hydroxyquinazoline in each 1ml, using the solution as test solution, precisely measuring 10 mu l of the test solution, injecting the solution into a liquid chromatograph, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 2.
FIG. 2 shows that under the chromatographic conditions, the 7-fluoro-4-hydroxyquinazoline peak and the impurity peak can be completely separated, the peak shape is good, the separation degree is high, the retention time of the 7-fluoro-4-hydroxyquinazoline peak is 14.389min, and the separation degree is 2.87.
Example 3
Instruments and conditions: agilent 1260 liquid chromatography system, column: welch Ultimate XB-C18 (4.6X 250mm, 3 μm), detection wavelength 247nm, column temperature 30 ℃, flow rate 1.1ml/min, mobile phase A of 0.15% phosphoric acid in water (by volume), mobile phase B of acetonitrile, gradient elution set up as:
Figure BDA0001013707320000042
the experimental steps are as follows: dissolving 7-fluoro-4-hydroxyquinazoline with 0.1% phosphoric acid aqueous solution and acetonitrile at a volume ratio of 90:10, quantitatively diluting to prepare solution containing 0.2mg of 7-fluoro-4-hydroxyquinazoline in each 1ml, using the solution as test solution, precisely measuring 10 mu l of the test solution, injecting the solution into a liquid chromatograph, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 3.
FIG. 3 shows that under the chromatographic conditions, the 7-fluoro-4-hydroxyquinazoline peak and the impurity peak can be completely separated, the peak shape is good, the separation degree is high, the retention time of the 7-fluoro-4-hydroxyquinazoline peak is 12.995min, and the separation degree is 3.53.
Example 4
Experiment of system applicability
Instruments and conditions: the same as in example 1.
The experimental steps are as follows: taking a proper amount of the product, precisely weighing, dissolving with 0.1% phosphoric acid aqueous solution and acetonitrile at a volume ratio of 90:10, and diluting to obtain a solution containing 0.2mg per 1ml, wherein the solution is used as a test solution. Taking a sample solution, continuously injecting samples for six times, respectively calculating the peak area of the 7-fluoro-4-hydroxyquinazoline peak and the relative standard deviation of the retention time, and the experimental result is shown in table 1.
TABLE 17 applicability test results for fluoro-4-hydroxyquinazoline system
Figure BDA0001013707320000051
As can be seen from Table 1, the symmetry factors of the peaks of 7-fluoro-4-hydroxyquinazoline are all less than 1.5, the number of theoretical plates is all higher than 3000, the relative standard deviation of the peak areas is 0.073% (limit 2.0%), and the relative standard deviation of the retention time is 0.142% (limit 1.0%). Therefore, under the chromatographic condition, the peak shape of the 7-fluoro-4-hydroxyquinazoline is good, the relative standard deviation is small, and the obtained result is stable and reliable.
Example 5
Repeatability test
Instruments and conditions: the same as in example 1.
The experimental steps are as follows: taking a proper amount of the product, precisely weighing, dissolving and diluting with 0.1% phosphoric acid aqueous solution and acetonitrile with the volume ratio of 90:10 to prepare 0.2mg solution in each 1ml as a test solution, and preparing 6 parts of the test solution by the same method. Taking a test sample solution, determining according to the method, calculating the content of the 7-fluoro-4-hydroxyquinazoline according to an area normalization method, and calculating the relative standard deviation, wherein the experimental result is shown in table 2.
TABLE 27 repeatability of fluoro-4-hydroxyquinazoline
Figure BDA0001013707320000052
Figure BDA0001013707320000061
As can be seen from Table 2, the content of 7-fluoro-4-hydroxyquinazoline in each sample solution is not significantly different, and the relative standard deviation is 0.001%, so that the repeatability of the analysis and detection method is good.
Example 6
Durability test
Instruments and conditions: agilent 1260 liquid chromatography system, column: welch Ultimate XB-C18 (4.6X 250mm, 3 μm) with a detection wavelength of 247nm was flowed as in example 1.
The experimental steps are as follows: taking a proper amount of the product, precisely weighing, dissolving with 0.1% phosphoric acid aqueous solution and acetonitrile at a volume ratio of 90:10, and diluting to obtain a solution containing 0.2mg per 1ml, wherein the solution is used as a test solution. The change of the content of 7-fluoro-4-hydroxyquinazoline (calculated by area normalization) was recorded by varying the column temperature, flow rate and column batch, respectively, and the results are shown in table 3.
TABLE 37 durability test results for fluoro-4-hydroxyquinazoline
Figure BDA0001013707320000062
As can be seen from Table 3, the results of the measurement of the 7-fluoro-4-hydroxyquinazoline content were not significantly different after the column temperature, flow rate and column batch were changed, and it can be seen that the durability of the analytical detection method of the present invention was good.
Example 7
Detection limit
Instruments and conditions: the same as in example 1.
About 10mg of 7-fluoro-4-hydroxyquinazoline was precisely weighed and placed in a 50ml measuring flask as stock solution. Adding 0.1 percent phosphoric acid aqueous solution and acetonitrile with the volume ratio of 90:10 for dissolution, and adopting a gradual dilution method, wherein the concentration when S/N is approximately equal to 3 is taken as the detection limit concentration, the concentration of the 7-fluoro-4-hydroxyquinazoline is 0.01773 mu g/ml, and the detection limit is 0.1773 ng. Therefore, the method and the instrument have higher sensitivity.
Example 8
Linearity and range
Instruments and conditions: the same as in example 1.
The experimental steps are as follows: and (3) precisely weighing 41.4mg of 7-fluoro-4-hydroxyquinazoline, placing the weighed material into a 100ml measuring flask, adding 0.1% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 90:10 to dissolve, and diluting to a scale to obtain a linear stock solution. Precisely measuring linear stock solutions 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, 6.0ml and 7.5ml respectively in 10ml measuring bottles, adding a solvent to dilute to a scale, shaking uniformly, and measuring by the method. Taking the concentration of the test solution as an abscissa and the peak area of the 7-fluoro-4-hydroxyquinazoline as an ordinate to perform linear regression, obtaining a linear regression equation of y 16673x +19.747, and the results are shown in table 4 and attached figure 4.
TABLE 47 Linear test results for fluoro-4-hydroxyquinazoline
Figure BDA0001013707320000071
As can be seen from Table 4 and FIG. 4, the correlation coefficient R of the linear regression equation2Under the chromatographic conditions, the 7-fluoro-4-hydroxyquinazoline has a good linear relation in the concentration range of 0.041-0.311 mg/ml.

Claims (5)

  1. The analytical detection method of the 7-fluoro-4-hydroxyquinazoline adopts a high performance liquid chromatography for analytical detection, and is characterized by comprising the following steps:
    a. taking a proper amount of 7-fluoro-4-hydroxyquinazoline, adding a solvent for dissolving, and preparing a sample solution containing 0.041-0.311 mg of 7-fluoro-4-hydroxyquinazoline per 1 ml;
    b. setting the flow rate of a mobile phase to be 0.7-1.1 ml/min, the detection wavelength to be 247nm and the column temperature to be 25-35 ℃;
    c. injecting 10 mu l of the sample solution A into a liquid chromatograph, and executing a gradient elution program to finish the analysis and detection of the 7-fluoro-4-hydroxyquinazoline;
    wherein, the chromatographic column: c18, 4.6X 250mm, 3 μm;
    the mobile phase is characterized in that 0.05-0.15% phosphoric acid water solution (volume ratio) is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is performed according to the following table in volume ratio:
    Figure DEST_PATH_IMAGE001
    the solvent is prepared from 0.1% phosphoric acid water solution: and the acetonitrile is prepared according to a volume ratio of 90: 10.
  2. 2. The analytical detection method according to claim 1, wherein the concentration of the sample solution is 0.2 mg/ml.
  3. 3. The analytical detection method according to claim 1, wherein the mobile phase flow rate is 1.0ml/min and the column temperature is 30 ℃.
  4. 4. The analytical detection method according to claim 1, wherein the mobile phase a is a 0.1% phosphoric acid aqueous solution.
  5. 5. The analytical detection method according to claim 1, characterized in that the mobile phase gradient elution is arranged to:
    Figure 926571DEST_PATH_IMAGE002
    。
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