CN113933424A - Method for detecting impurities in apixaban starting material - Google Patents
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Abstract
The invention relates to the technical field of drug detection, in particular to a method for detecting impurities in an apixaban starting material. The method for detecting impurities in the apixaban starting material comprises the following steps: detecting the initial material sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: gradient elution is carried out by adopting a mobile phase A and fluidity B; the mobile phase A is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 95: 5; the concentration of the ammonium acetate aqueous solution is 20-40 mmol/L; octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the detection wavelength is 225-235 nm. The method for detecting the impurities in the apixaban starting material can accurately and sensitively detect the impurities in the starting material and can better control the quality of the apixaban.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a method for detecting impurities in an apixaban starting material.
Background
Apixaban is a novel oral XM factor inhibitor which is jointly developed by Beshimasuibao and pfeiri, and is a novel oral anticoagulant drug. By inhibiting an important coagulation factor XM, apixaban prevents thrombin generation and thrombosis. Moreover, apixaban has the advantages of high safety, no need of monitoring to adjust dosage, oral administration, no cross-drug resistance, less adverse reaction and the like.
The apixaban has a corresponding synthesis method according to different reaction raw materials, wherein [ (4-methoxyphenyl) hydrazino ] ethyl chloroacetate is one of starting materials for synthesizing the apixaban, and impurities in the starting materials are inevitably brought into the apixaban product. Therefore, the method has important significance for detecting or controlling the quality of the apixaban starting material, preparing the apixaban and improving the quality stability of the apixaban.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting impurities in apixaban starting materials, so as to control the content of the impurities brought in from the starting materials and improve the quality stability of the apixaban and the like.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the method for detecting impurities in the apixaban starting material comprises the following steps:
detecting the initial material sample solution by adopting high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
gradient elution is carried out by adopting a mobile phase A and fluidity B; the mobile phase A is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 95: 5; the concentration of the ammonium acetate aqueous solution is 20-40 mmol/L;
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
the detection wavelength is 225-235 nm.
The method for detecting the impurities in the apixaban starting material can accurately and sensitively detect the impurities in the starting material and can better control the quality of the apixaban.
In a specific embodiment of the invention, the concentration of the aqueous ammonium acetate solution is 30 mmol/L.
In a specific embodiment of the present invention, the flow rate is 0.8 to 1.2 mL/min.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-35 ℃. Further, the specification of the chromatographic column is Waters
C18，250mm×4.6mm，5μm。
In a specific embodiment of the invention, the gradient elution comprises: in 0-10 min, the volume fraction of the mobile phase A is 88-92%; the volume fraction of the mobile phase A is changed from 88 to 92 percent to 8 to 12 percent in 10 to 10.1 min; in 10.1-20 min, the volume fraction of the mobile phase A is 8-12%; changing the volume fraction of the mobile phase A from 8-12% to 88-92% in 20-20.1 min; and in 20.1-30 min, the volume fraction of the mobile phase A is 88-92%.
In a specific embodiment of the invention, the procedure of the gradient elution is:
| time-min | Mobile phase A-% | Mobile phase B-% |
| 0 | 90 | 10 |
| 10 | 90 | 10 |
| 10.1 | 10 | 90 |
| 20 | 10 | 90 |
| 20.1 | 90 | 10 |
| 30 | 90 | 10 |
。
In a specific embodiment of the present invention, the structural formula of the apixaban starting material is:
the structural formula of the impurities is as follows:
in a specific embodiment of the present invention, the solvent of the test solution is acetonitrile.
In a specific embodiment of the invention, the concentration of the starting material in the sample solution is 15-25 mg/mL.
In a specific embodiment of the invention, the method further comprises performing the high performance liquid chromatography detection on a reference solution. Further, the solvent of the reference solution is acetonitrile; in the reference solution, the concentration of the impurities is 2-4 mug/mL.
In a specific embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
the mobile phase A is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 95: 5;
the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 30 ℃;
the detection wavelength is 230nm, and the flow rate is 1 mL/min.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting the impurities in the apixaban starting material can accurately and sensitively detect the impurities in the starting material and can better control the quality of the apixaban.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a chromatogram of a test solution obtained by the detection method provided in example 1 of the present invention;
FIG. 2 is a chromatogram of a labeled test solution obtained by the detection method provided in embodiment 1 of the present invention;
FIG. 3 is a chromatogram of a control solution obtained by the detection method provided in example 1 of the present invention;
FIG. 4 is a chromatogram of a reference solution of impurity M obtained by the detection method provided in comparative example 1;
FIG. 5 is a chromatogram of a reference solution of impurity M obtained by the detection method provided in comparative example 2;
FIG. 6 is a sensitivity chromatogram of impurities provided in Experimental example 1 of the present invention;
FIG. 7 is a line graph of impurities provided in Experimental example 3 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The method for detecting impurities in the apixaban starting material comprises the following steps:
detecting the initial material sample solution by adopting high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
gradient elution is carried out by adopting a mobile phase A and fluidity B; the mobile phase A is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 95: 5; the concentration of the ammonium acetate aqueous solution is 20-40 mmol/L;
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
the detection wavelength is 225-235 nm.
The method for detecting the impurities in the apixaban starting material can accurately and sensitively detect the impurities in the starting material and can better control the quality of the apixaban.
As in the different embodiments, the concentration of the aqueous ammonium acetate solution in the mobile phase A and the mobile phase B can be 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, and so forth.
In a specific embodiment of the invention, the concentration of the aqueous ammonium acetate solution is 30 mmol/L.
As in the different embodiments, the detection wavelength may be 225nm, 226nm, 227nm, 228nm, 229nm, 230nm, 231nm, 232nm, 233nm, 234nm, 235nm, etc., preferably 228 to 232nm, more preferably 230 nm.
In a specific embodiment of the present invention, the flow rate is 0.8 to 1.2 mL/min.
As in the different embodiments, the flow rate may be such that the flow rate may be 0.8mL/min, 0.9mL/min, 1mL/min, 1.1mL/min, 1.2mL/min, etc., preferably 1 mL/min.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-35 ℃. Further, the specification of the chromatographic column is Waters
C18, 250mm × 4.6mm, 5 μm or a column of comparable performance.
As in the different embodiments, the column temperature of the chromatographic column can be 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, etc., preferably 30 ℃.
In a specific embodiment of the invention, the gradient elution comprises: in 0-10 min, the volume fraction of the mobile phase A is 88-92%; the volume fraction of the mobile phase A is changed from 88 to 92 percent to 8 to 12 percent in 10 to 10.1 min; in 10.1-20 min, the volume fraction of the mobile phase A is 8-12%; changing the volume fraction of the mobile phase A from 8-12% to 88-92% in 20-20.1 min; and in 20.1-30 min, the volume fraction of the mobile phase A is 88-92%.
In a specific embodiment of the invention, the procedure of the gradient elution is:
in a specific embodiment of the present invention, the structural formula of the apixaban starting material is:
the structural formula of the impurity M is as follows:
in a specific embodiment of the present invention, the solvent of the test solution is acetonitrile.
In a specific embodiment of the invention, the concentration of the starting material in the sample solution is 15-25 mg/mL.
In a specific embodiment of the invention, the method further comprises performing the high performance liquid chromatography detection on a reference solution. Further, the solvent of the reference solution is acetonitrile; in the reference solution, the concentration of the impurities is 2-4 mug/mL.
In a specific embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
the mobile phase A is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 95: 5;
the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 30 ℃;
the detection wavelength is 230nm, and the flow rate is 1 mL/min.
In a specific embodiment of the present invention, the method further comprises: and calculating the content of impurities in the test solution by an external standard method. Further, the method for calculating the content of the impurities comprises the following steps: respectively injecting the impurity standard series working solutions into a high performance liquid chromatograph, measuring corresponding peak areas under the detection condition of the high performance liquid chromatograph, and drawing a standard curve of the impurity by taking the concentration of the standard series working solutions as a horizontal coordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of the impurities in the chromatographic detection result of the test solution into the standard curve of the impurities, and calculating to obtain the content of the impurities in the test solution.
Example 1
The embodiment provides a method for detecting impurities in an apixaban starting material, which comprises the following steps:
(1) preparation of reference solution and test solution
Preparing a reference substance solution: precisely weighing a proper amount of an impurity M reference substance, dissolving and diluting the impurity M reference substance by using acetonitrile to prepare a solution containing about 3 micrograms of the impurity M per 1mL, and taking the solution as a reference substance solution;
preparing a test solution: accurately weighing 200mg of apixaban starting material (batch 2M14220601), adding acetonitrile to dissolve and quantitatively diluting the solution into a solution containing about 20mg of apixaban starting material per 1mL, and taking the solution as a test solution;
preparing a standard sample solution: accurately weighing 200mg of apixaban starting material (batch 2M14220601), adding an appropriate amount of impurity M reference substance, adding acetonitrile to dissolve, and quantitatively diluting to obtain a solution containing 20mg of apixaban starting material and 3 μ g of impurity M per 1mL as a standard sample solution.
(2) High performance liquid chromatography detection conditions
The instrument comprises the following steps: an Agilent 1260 type high performance liquid chromatograph, an ultraviolet detector, detection wavelength: 230 nm;
a chromatographic column:
c18, 250mm × 4.6mm, 5 μm; column temperature: 30 ℃;
mobile phase: mobile phase a and mobile phase B; wherein the mobile phase A is a mixed solution of acetonitrile and 30mmol/L ammonium acetate aqueous solution at a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and 30mmol/L ammonium acetate aqueous solution at a volume ratio of 95: 5;
gradient elution was performed with mobile phase a and mobile phase B according to table 1;
flow rate: 1.0 mL/min.
TABLE 1 gradient elution schedule (vol.)
(3) High performance liquid chromatography detection
And (3) precisely measuring the reference solution, the test solution and the added standard test solution by 20 mu L respectively, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording the chromatogram. The chromatogram of the control solution is shown in FIG. 3, wherein the retention time of impurity M is 7.673min, and the peak-off time is moderate. The chromatogram of the test solution is shown in FIG. 1, and no impurity M chromatogram peak is found in the test solution. The chromatogram of the labeled sample solution is shown in FIG. 2, and it is clear from FIG. 2 that the degree of separation between impurity M and the back peak is 3.03, and that the degree of separation is good.
Example 2
This example refers to the detection method of example 1, with the only difference that: in the step (2), the detection wavelength is 225 nm.
Example 3
This example refers to the detection method of example 1, with the only difference that: in the step (2), the detection wavelength is 235 nm.
Comparative example 1
Comparative example 1 provides a method for detecting impurities in an apixaban starting material, comprising the steps of:
(1) preparation of reference substance solution and standard sample solution
Preparing a reference substance solution: precisely weighing a proper amount of an impurity M reference substance, dissolving and diluting the impurity M reference substance by using acetonitrile to prepare a solution containing about 3 mu g of the impurity M in every 1mL of the solution as a reference substance solution;
preparing a standard sample solution: accurately weighing 200mg of apixaban starting material (batch 2M14220601), adding an appropriate amount of impurity M reference substance, adding acetonitrile to dissolve, and quantitatively diluting to obtain a solution containing 20mg of apixaban starting material and 3 μ g of impurity M per 1mL as a standard sample solution.
(2) High performance liquid chromatography detection conditions
The instrument comprises the following steps: an Agilent 1200 type high performance liquid chromatograph, an ultraviolet detector, detection wavelength: 270 nm;
a chromatographic column: waters
C18, 250mm × 4.6mm, 5 μm; column temperature: 30 ℃;
mobile phase: isocratic elution at 90: 10 for mobile phase A and B; wherein, the mobile phase A is trifluoroacetic acid aqueous solution with volume fraction of 0.03%, and the mobile phase B is acetonitrile;
flow rate: 1.0 mL/min.
(3) High performance liquid chromatography detection
And (3) precisely measuring 20 mu L of each of the reference solution and the standard sample solution, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording a chromatogram. The chromatogram of the impurity M control solution is shown in fig. 4, and it is clear from fig. 4 that the impurity M peak is branched.
Comparative example 2
Comparative example 2 provides a method for detecting impurities in an apixaban starting material, comprising the steps of:
(1) preparation of reference substance solution and standard sample solution
Preparing a reference substance solution: precisely weighing a proper amount of an impurity M reference substance, dissolving and diluting the impurity M reference substance by using acetonitrile to prepare a solution containing about 3 mu g of the impurity M in every 1mL of the solution as a reference substance solution;
preparing a standard sample solution: precisely weighing apixaban starting material (batch 2M14220601)200mg, adding an appropriate amount of impurity M reference substance, adding acetonitrile to dissolve, and quantitatively diluting to obtain a solution containing about 20mg of apixaban starting material and 3 μ g of impurity M per 1mL as a standard sample solution.
(2) High performance liquid chromatography detection conditions
The instrument comprises the following steps: an Agilent 1200 type high performance liquid chromatograph, an ultraviolet detector, detection wavelength: 270 nm;
a chromatographic column: waters
C18, 250mm × 4.6mm, 5 μm; column temperature: 30 ℃;
mobile phase: isocratic elution at 90: 10 for mobile phase A and B; wherein the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.01 percent, and the mobile phase B is a mixed solution of methanol and acetonitrile with the volume ratio of 60: 40;
flow rate: 1.0 mL/min.
(3) High performance liquid chromatography detection
And (3) precisely measuring 20 mu L of each of the reference solution and the standard sample solution, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording a chromatogram. The chromatogram of the impurity M control solution is shown in fig. 5, and it is clear from fig. 5 that the impurity M peak is branched.
Experimental example 1
Sensitivity test
(1) A control solution was prepared according to the method for preparing a control solution described in example 1;
preparation of a sensitivity solution: precisely measuring 0.5mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding acetonitrile to dilute to a scale, and shaking up to obtain a sensitivity solution.
(2) With reference to the detection conditions of example 1, 20. mu.L of the sensitive solution was measured with precision, injected into a high performance liquid chromatograph, and the chromatogram was recorded as shown in FIG. 6. As can be seen from FIG. 6, the signal-to-noise ratio of the chromatographic peak corresponding to impurity M of the apixaban starting material was 212.4, > 10.
Experimental example 2
Limit of detection and limit of quantification test
A sensitivity solution was prepared according to the method for preparing a sensitivity solution described in experimental example 1; precisely measuring 1mL of reference solution, placing the reference solution in a 10mL measuring flask, adding acetonitrile to dilute to a scale, and shaking up to obtain a limit solution; precisely measuring 3mL of the quantitative limiting solution, placing the quantitative limiting solution in a 10mL measuring flask, adding acetonitrile to dilute to a scale, and shaking up to be used as the detection limiting solution. Sample injection detection is carried out according to the chromatographic detection conditions in the embodiment 1, the signal-to-noise ratio is more than or equal to 3 as a detection limit, and the signal-to-noise ratio is more than or equal to 10 as a quantification limit.
The specific quantitation limit test results and detection limit test results are shown in tables 2 and 3, respectively.
TABLE 2 quantitative limit results
TABLE 3 detection Limit results
Experimental example 3
Linear test
Preparation of a control stock solution: precisely weighing a proper amount of impurity M reference substance, adding acetonitrile to dissolve and quantitatively diluting to prepare a solution containing about 30 mu g of impurity M in each 1mL of impurity M reference substance as a reference substance stock solution;
preparation of a linear solution: precisely measuring a proper amount of a reference substance stock solution, adding acetonitrile to dilute the reference substance stock solution into a solution with a corresponding concentration, and shaking the solution uniformly to obtain a linear solution.
20. mu.L of each linear solution was precisely measured, and each solution was injected into a high performance liquid chromatograph under the detection conditions of example 1, and the chromatogram was recorded. The test results are shown in Table 4, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 7.
TABLE 4 Linear test results for impurity M
From the above results, it is found that the linear relationship between the concentration and the peak area is good when the impurity M of the apixaban starting material is in the range of 0.01594-6.2035 μ g/mL.
Experimental example 4
Repeatability test
Preparation of a control stock solution: taking a proper amount of an impurity M reference substance, precisely weighing, adding acetonitrile to dissolve, and quantitatively diluting to prepare a solution containing about 30 mu g of the impurity M in every 1mL to obtain a reference substance stock solution;
preparing a reference substance solution: taking a proper amount of impurity M reference substance stock solution, precisely weighing, adding acetonitrile for dissolving, and quantitatively diluting to prepare a solution containing about 3 mu g of impurity M in each 1mL of impurity M reference substance stock solution to obtain a reference substance solution;
preparing a test solution: accurately weighing the apixaban starting material about 200mg, placing the obtained product in a 10mL volumetric flask, adding acetonitrile for dissolution, adding 1mL of a reference substance stock solution, adding acetonitrile for dilution to a scale, and shaking up to obtain a test solution; preparing 6 parts in parallel;
preparation of a sensitivity solution: precisely measuring 0.5mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding acetonitrile to dilute to a scale, and shaking up to obtain a sensitivity solution.
20. mu.L of each solution was precisely measured, and each solution was injected into a high performance liquid chromatograph under the detection conditions in example 1, and the chromatogram was recorded. The test results are shown in Table 5.
TABLE 5 results of the repeatability tests
Remarking: the "peak area" in Table 5 means the peak area of the impurity A
From the above test results, it can be seen that the content of the impurity M in the apixaban starting material was highly reproducible in the prepared 6 test sample solutions.
Experimental example 5
Accuracy test
Preparation of a control stock solution: precisely weighing a proper amount of impurity M reference substance, adding acetonitrile to dissolve and quantitatively diluting to prepare a solution containing about 30 mu g of impurity M in each 1mL of impurity M reference substance as a reference substance stock solution;
preparation of an accuracy solution:
50% accuracy solution: accurately weighing the apixaban starting material (batch 2M14220601) about 200mg, placing the obtained product into a 10mL measuring flask, adding an appropriate amount of acetonitrile, performing ultrasonic treatment to dissolve the product, accurately adding 0.5mL of a reference substance stock solution, diluting the solution to a scale with acetonitrile, and shaking up to obtain a 50% accuracy solution; preparing 3 parts in parallel;
100% accuracy solution: accurately weighing the apixaban starting material (batch 2M14220601) about 200mg, placing the obtained product into a 10mL measuring flask, adding an appropriate amount of acetonitrile, performing ultrasonic treatment to dissolve the product, accurately adding 1mL of a reference substance stock solution, diluting the solution with acetonitrile to a scale, and shaking up the solution to obtain a 100% accuracy solution; preparing 3 parts in parallel;
200% accuracy solution: accurately weighing the apixaban starting material (batch 2M14220601) about 200mg, placing the obtained product into a 10mL measuring flask, adding an appropriate amount of acetonitrile, performing ultrasonic treatment to dissolve the product, accurately adding 2mL of a reference substance stock solution, diluting the solution with acetonitrile to a scale, and shaking up the solution to obtain a 200% accuracy solution; 3 parts are prepared in parallel.
Preparing a blank test solution: accurately weighing the apixaban starting material (batch 2M14220601) about 200mg, placing the starting material into a 10mL measuring flask, adding an appropriate amount of acetonitrile, performing ultrasonic treatment to dissolve and dilute the starting material to a scale, and shaking up the starting material to serve as a blank test solution.
Preparing a reference substance solution: an appropriate amount of the impurity M as a control was precisely weighed, and dissolved and diluted with acetonitrile to prepare a solution containing about 3. mu.g of the impurity M per 1mL as a control solution.
Preparation of a sensitivity solution: precisely measuring 0.5mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding acetonitrile to dilute to a scale, and shaking up to obtain a sensitivity solution.
Each 20. mu.L of each solution was precisely measured, and the solution was injected into a high performance liquid chromatograph under the detection conditions of example 1, and the chromatogram was recorded. The test results are shown in Table 6.
TABLE 6 test results of the accuracy test
From the experimental results of the recovery rate of the impurity M of the apixaban starting material, the recovery rates of 9 accurate samples are between 93% and 97%, which shows that the method has good accuracy.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for detecting impurities in the apixaban starting material is characterized by comprising the following steps of:
detecting the initial material sample solution by adopting high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
gradient elution is carried out by adopting a mobile phase A and fluidity B; the mobile phase A is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and an ammonium acetate aqueous solution in a volume ratio of 95: 5; the concentration of the ammonium acetate aqueous solution is 20-40 mmol/L;
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
the detection wavelength is 225-235 nm.
2. The assay of claim 1, wherein the apixaban starting material has the formula:
the structural formula of the impurities is as follows:
3. the detection method according to claim 1, wherein the flow rate is 0.8 to 1.2 mL/min.
4. The detection method according to claim 1, wherein the column temperature of the chromatographic column is 25 to 35 ℃;
preferably, the specification of the chromatographic column is Waters
C18，250mm×4.6mm，5μm。
5. The detection method according to claim 1, wherein the concentration of the aqueous ammonium acetate solution is 30 mmol/L.
6. The detection method according to claim 1, wherein the gradient elution comprises: in 0-10 min, the volume fraction of the mobile phase A is 88-92%; the volume fraction of the mobile phase A is changed from 88 to 92 percent to 8 to 12 percent in 10 to 10.1 min; in 10.1-20 min, the volume fraction of the mobile phase A is 8-12%; changing the volume fraction of the mobile phase A from 8-12% to 88-92% in 20-20.1 min; and in 20.1-30 min, the volume fraction of the mobile phase A is 88-92%.
7. The detection method according to any one of claims 1 to 6, wherein the gradient elution is performed by:
。
8. The detection method according to claim 1, wherein the solvent of the test solution is acetonitrile;
preferably, in the test solution, the concentration of the starting material is 15-25 mg/mL.
9. The detection method according to claim 1, further comprising performing the high performance liquid chromatography detection on a control solution;
preferably, the solvent of the control solution is acetonitrile; in the reference solution, the concentration of the impurities is 2-4 mug/mL.
10. The detection method according to claim 7, wherein the detection conditions of the high performance liquid chromatography comprise:
the mobile phase A is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 10: 90, and the mobile phase B is a mixed solution of acetonitrile and 30mmol/L ammonium acetate at a volume ratio of 95: 5;
the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 30 ℃;
the detection wavelength is 230nm, and the flow rate is 1 mL/min.
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