CN113504317A - Detection method and application of genotoxic impurities in apixaban - Google Patents
Detection method and application of genotoxic impurities in apixaban Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 71
- 239000012535 impurity Substances 0.000 title claims abstract description 57
- QNZCBYKSOIHPEH-UHFFFAOYSA-N Apixaban Chemical compound C1=CC(OC)=CC=C1N1C(C(=O)N(CC2)C=3C=CC(=CC=3)N3C(CCCC3)=O)=C2C(C(N)=O)=N1 QNZCBYKSOIHPEH-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 229960003886 apixaban Drugs 0.000 title claims abstract description 54
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 51
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 51
- 239000012085 test solution Substances 0.000 claims abstract description 29
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 14
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- 239000000945 filler Substances 0.000 claims abstract description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
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Abstract
The invention relates to the technical field of drug detection, in particular to a detection method and application of genotoxic impurities in apixaban. The detection method of the genotoxic impurities in the apixaban comprises the following steps: detecting the test solution by high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; gradient elution is carried out by adopting a mobile phase A and a mobile phase B; the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3; the detector is an ultraviolet detector, and the detection wavelength is 335 nm-345 nm. The detection method has the advantages of good specificity, high analysis speed, high reproducibility and the like, and can be used for accurately and sensitively detecting the genotoxic impurities in the apixaban.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a detection method and application of genotoxic impurities in apixaban.
Background
The Apixaban is a novel oral Xa factor inhibitor which is jointly developed by Beshimex baishibae and pfeiri, and is a novel oral anticoagulant drug. By inhibiting an important coagulation factor Xa, apixaban prevents thrombin generation and thrombosis. Apixaban has the following structural formula:
in china, apixaban is currently approved for adult patients with hip or knee joint elective replacement to prevent Venous Thromboembolism (VTE). 9 months 2014, FDA approved new indications: (1) prevention of stroke and systemic circulatory embolism in adult patients with non-valvular atrial fibrillation (NVAF); (2) prevention of venous thrombosis in adult patients after hip joint or knee joint phase-selective replacement surgery; (3) treatment of deep vein thrombosis; (4) treatment of pulmonary embolism; (5) reducing the risk of recurrence of DVT and PE.
Genotoxic impurities are impurities that react with DNA, causing DNA damage, induce gene mutations at very low levels, and may be carcinogenic. Thus, control of genotoxic impurities is a major concern for drug quality. However, no detailed report is found for the detection of genotoxic impurities possibly existing in apixaban at present.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for detecting genotoxic impurities in apixaban, so as to better control the quality of apixaban and improve the medication stability and safety.
The second purpose of the invention is to provide the application of the detection method of genotoxic impurities in apixaban in the quality control of apixaban preparations.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the detection method of the genotoxic impurities in the apixaban comprises the following steps:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
gradient elution is carried out by adopting a mobile phase A and a mobile phase B;
the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3;
the detector is an ultraviolet detector, and the detection wavelength is 335 nm-345 nm.
The detection method has the advantages of good specificity, high analysis speed, high reproducibility and the like, and can be used for accurately and sensitively detecting the genotoxic impurities in the apixaban.
In a specific embodiment of the invention, the genotoxic impurity comprises genotoxic impurity A having the formula
In an embodiment of the present invention, the flow rate is 0.8 to 1.2mL/min, preferably 0.9 to 1.1mL/min, and more preferably 1 mL/min.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-35 ℃, and preferably 30 ℃.
In a specific embodiment of the invention, the mobile phase a is a phosphoric acid aqueous solution with a volume fraction of 0.08% to 0.12%, preferably a phosphoric acid aqueous solution with a volume fraction of 0.1%.
In a specific embodiment of the invention, the gradient elution comprises: and changing the volume fraction of the mobile phase A from 58-62% to 38-42% in 0-35 min. Further, the volume fraction of the mobile phase A is changed from 60% to 40% in 0-35 min.
In a specific embodiment of the invention, the procedure of the gradient elution is:
| time-min | Mobile phase A% | Mobile phase B% |
| 0 | 60 | 40 |
| 35 | 40 | 60 |
| 35.1 | 10 | 90 |
| 40 | 10 | 90 |
| 40.1 | 60 | 40 |
| 50 | 60 | 40 |
。
In the inventionIn a specific embodiment, the chromatographic column is Waters in specification
C18，250mm×4.6mm，5μm。
In an embodiment of the present invention, the sample amount of the sample solution may be 5 to 20 μ L, such as 10 μ L.
In a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
octadecylsilane chemically bonded silica is used as a filler for a chromatographic column, and the column temperature is 30 ℃;
the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.1 percent; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3;
the detection wavelength is 340 nm;
the flow rate is 1 mL/min;
the amount of sample was 10. mu.L.
In a specific embodiment of the present invention, the preparation of the test solution comprises: and dissolving the test sample by adopting methanol.
In a specific embodiment of the present invention, the method further comprises: and calculating the content of genotoxic impurities in the test solution by an external standard method.
The invention also provides the application of any one of the detection methods in the quality control of the apixaban preparation.
The detection method of the invention ensures that the detection of genotoxic impurities has good specificity, repeatability, accuracy, sensitivity and the like on the basis of ensuring the high-efficiency separation of the genotoxic impurities and the apixaban, and can better realize the quality control of the apixaban preparation.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection method has the advantages of good specificity, high analysis speed, high reproducibility and the like, and can be used for accurately and sensitively detecting genotoxic impurities in apixaban;
(2) the detection method can be used for the synthesis research and the quality control of the production process of the apixaban, provides a basis for the quality control of the apixaban preparation and ensures the product quality.
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 test solution obtained by the detection method provided in comparative example 1;
FIG. 3 is a sensitivity chromatogram of genotoxic impurity A provided in Experimental example 1 of the present invention;
FIG. 4 is a line graph of genotoxic impurity A 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 detection method of the genotoxic impurities in the apixaban comprises the following steps:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
gradient elution is carried out by adopting a mobile phase A and a mobile phase B;
the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3;
the detector is an ultraviolet detector, and the detection wavelength is 335 nm-345 nm.
The detection method has the advantages of good specificity, high analysis speed, high reproducibility and the like, and can be used for accurately and sensitively detecting the genotoxic impurities in the apixaban.
As in the different embodiments, the detection wavelength may be 335nm, 336nm, 337nm, 338nm, 339nm, 340nm, 341nm, 342nm, 343nm, 344nm, 345nm, etc., preferably 338 to 342nm, more preferably 340 nm.
In a specific embodiment of the invention, the genotoxic impurity comprises genotoxic impurity A having the formula
In an embodiment of the present invention, the flow rate is 0.8 to 1.2mL/min, preferably 0.9 to 1.1mL/min, and more preferably 1 mL/min.
As in various embodiments, the flow rate can be 0.8mL/min, 0.9mL/min, 1mL/min, 1.1mL/min, 1.2mL/min, and the like.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-35 ℃, and preferably 30 ℃.
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 ℃ and so on.
In a specific embodiment of the present invention, the volume fraction of the phosphoric acid aqueous solution of the mobile phase a may be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, etc., preferably 0.08% to 0.12%, more preferably 0.1%.
In a specific embodiment of the invention, the gradient elution comprises: and changing the volume fraction of the mobile phase A from 58-62% to 38-42% in 0-35 min. Further, the volume fraction of the mobile phase A is changed from 60% to 40% in 0-35 min.
In a specific embodiment of the invention, the gradient elution procedure is:
| time-min | Mobile phase A% | Mobile |
| 0 | 60 | 40 |
| 35 | 40 | 60 |
| 35.1 | 10 | 90 |
| 40 | 10 | 90 |
| 40.1 | 60 | 40 |
| 50 | 60 | 40 |
。
In a specific embodiment of the invention, the chromatography column is of the Waters specification
C18, 250mm × 4.6mm, 5 μm or a column of comparable performance.
In a specific embodiment of the present invention, the sample amount of the test solution may be 5 to 20 μ L, and in various embodiments, the sample amount of the test solution may be 5 μ L, 10 μ L, 15 μ L, 20 μ L, or the like, and preferably 10 μ L.
In a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
octadecylsilane chemically bonded silica is used as a filler for a chromatographic column, and the column temperature is 30 ℃;
the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.1 percent; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3;
the detection wavelength is 340 nm;
the flow rate is 1 mL/min;
the amount of sample was 10. mu.L.
In a specific embodiment of the present invention, the preparation of the test solution comprises: and dissolving the test sample by adopting methanol.
In actual operation, methanol is adopted to dissolve the test solution, so that the stability and accuracy of detection can be ensured. When acetonitrile is used as a solvent to dissolve the test sample, the detection stability is poor, and the accuracy is not in a qualified range.
In a specific embodiment of the present invention, the sample may be a crude apixaban.
In a specific embodiment of the present invention, the concentration of apixaban in the test solution is 3-5 mg/mL, such as 4 mg/mL. In actual practice, the concentration of apixaban in the test solution is not limited thereto.
In a specific embodiment of the present invention, the method further comprises: and calculating the content of genotoxic impurities in the test solution by an external standard method.
In a specific embodiment, the method for calculating the content of the genotoxic impurities comprises: respectively injecting the genotoxic 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 genotoxic impurity by taking the concentration of the standard series working solutions as a horizontal coordinate and the peak area as a vertical coordinate; substituting the chromatographic peak area of the genotoxic impurity in the chromatographic detection result of the test solution into the standard curve of the genotoxic impurity, and calculating to obtain the content of the genotoxic impurity in the test solution.
In the specific embodiment of the invention, the method also comprises the step of carrying out high performance liquid chromatography detection on the reference substance solution. Furthermore, the concentration of the control substance gene toxic impurities in the control solution is 0.4-0.8 mug/mL, such as 0.6 mug/mL.
The invention also provides the application of any one of the detection methods in the quality control of the apixaban preparation.
The detection method of the invention ensures that the detection of genotoxic impurities has good specificity, repeatability, accuracy, sensitivity and the like on the basis of ensuring the high-efficiency separation of the genotoxic impurities and the apixaban, and can better realize the quality control of the apixaban preparation.
Example 1
The embodiment provides a method for detecting genotoxic impurities in apixaban, which comprises the following steps:
(1) reference solution and test solution
Preparing a reference substance solution: precisely weighing a proper amount of a reference substance of the genotoxic impurity A, dissolving the reference substance with methanol and diluting the reference substance to prepare a solution containing about 0.6 mu g of the genotoxic impurity A per 1mL, wherein the solution is used as a reference substance solution;
preparing a test solution: the crude apixaban (batch 202011002A) was precisely weighed as 40mg, dissolved in methanol and quantitatively diluted to a solution containing about 4mg of apixaban per 1mL as a test 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: 340 nm;
a chromatographic column: waters
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 phosphoric acid aqueous solution with the volume fraction of 0.1%, and the mobile phase B is an acetonitrile-methanol mixed solution with the volume ratio of 2: 3;
gradient elution was performed with mobile phase a and mobile phase B according to table 1;
flow rate: 1.0 mL/min;
sample introduction volume: 10 μ L.
TABLE 1 gradient elution schedule (vol.)
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 35 | 40 | 60 |
| 35.1 | 10 | 90 |
| 40 | 10 | 90 |
| 40.1 | 60 | 40 |
| 50 | 60 | 40 |
(3) High performance liquid chromatography detection conditions
And (3) precisely measuring 10 mu L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording a chromatogram. In the chromatogram of the control solution, the retention time of the apixaban genotoxic impurity A is 27.972min, and the peak appearance time is moderate. The chromatogram of the sample solution is shown in FIG. 1, and it can be seen from FIG. 1 that the retention time of apixaban is about 10min, the retention time of apixaban and genotoxic impurity A is greatly different, the degree of separation is good, and the detection is not interfered.
Example 2
This example refers to the detection method of example 1, with the only difference that: in step (2), the flow rate was 0.8 mL/min.
Example 3
This example refers to the detection method of example 1, with the only difference that: in step (2), the flow rate was 1.2 mL/min.
Example 4
This example refers to the detection method of example 1, with the only difference that: in the step (2), the column temperature was 25 ℃.
Example 5
This example refers to the detection method of example 1, with the only difference that: in the step (2), the column temperature was 35 ℃.
Comparative example 1
Comparative example 1 the test method of example 1 was referenced, differing only in that: in step (2), the detection wavelength was 240nm, and the chromatogram was recorded as shown in FIG. 2.
As is clear from FIG. 2, when the detection wavelength is 240nm, many interference peaks are present in the chromatogram.
Comparative example 2
Comparative example 2 the test method of example 1 was referenced, differing only in that: in the step (1), the methanol adopted for preparing the reference solution and the test solution is replaced by acetonitrile.
When the solvent is acetonitrile, a standard addition recovery rate test is carried out, three samples with 100% standard addition are preliminarily examined (the sample preparation method refers to the preparation of a solution with 100% accuracy), and the recovery rate of the impurity A in the sample is only about 30%.
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 1mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding methanol to dilute to the scale, and shaking up to obtain a sensitive solution.
(2) With reference to the detection conditions of example 1, 10. 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. 3. As can be seen from FIG. 3, the signal-to-noise ratio of the chromatographic peak corresponding to the apixaban genotoxic impurity A was 15.4 > 10.
Experimental example 2
Limit of detection and limit of quantification test
A control solution was prepared according to the method for preparing a control solution described in example 1; precisely measuring 1mL of reference solution, placing in a 10mL measuring flask, adding methanol to dilute to scale, and shaking up to obtain a limit solution; precisely measuring 2.5mL of the limiting solution, placing the limiting solution in a 10mL measuring flask, adding methanol 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 example 1, and the peak height which is three times of the baseline noise is taken as the detection limit, and the peak height which is ten times of the baseline noise is taken as the quantification limit.
The specific detection limit test results and the quantitative limit test results are shown in tables 2 and 3, respectively.
TABLE 2 detection Limit results
TABLE 3 quantitative limit results
Experimental example 3
Linear test
Preparation of a control stock solution: precisely weighing a proper amount of a reference substance of the gene toxic impurity A, adding methanol for dissolving, and quantitatively diluting to prepare a solution containing about 2 mu g of the reference substance in each 1mL of the reference substance as a reference substance stock solution;
preparation of a linear solution: precisely measuring a proper amount of the reference substance stock solution, adding methanol to dilute the reference substance stock solution into a solution with a corresponding concentration, and shaking the solution uniformly to obtain a linear solution.
10. 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. 4.
TABLE 4 Linear test results for genotoxic impurity A
And (4) conclusion: the genotoxic impurity A in apixaban is in the range of 0.06391-0.95867 mu g/mL, and the linear relation between the concentration and the peak area is good.
Experimental example 4
Repeatability test
Preparing a test solution: precisely weighing about 40mg of the apixaban crude product, adding methanol for dissolving, and quantitatively diluting to prepare a solution containing about 4mg of apixaban in every 1mL as a test solution; preparing 6 parts in parallel;
preparing a reference substance solution: precisely weighing a proper amount of reference substance of the gene toxic impurity A, adding methanol for dissolving, and quantitatively diluting to prepare a solution containing 0.6 mu g of the reference substance per 1 mL;
preparation of a sensitivity solution: precisely measuring 1mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding methanol to dilute to the scale, and shaking up to obtain a sensitive solution.
Each of the solutions was measured precisely at 10. mu.L, 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 5.
TABLE 5 repeatability test results
Remarking: the "peak area" and "content" refer to the peak area and content of the genotoxic impurity a in the apixaban crude product.
And (4) conclusion: according to the test results of the prepared 6 parts of test solution, the content reproducibility of the genotoxic impurity A in the apixaban crude product is good.
Experimental example 5
Accuracy test
Preparation of a control stock solution: precisely weighing a proper amount of a reference substance of the gene toxic impurity A, adding methanol for dissolving, and quantitatively diluting to prepare a solution containing about 2 mu g of the reference substance in each 1mL of the reference substance as a reference substance stock solution;
preparation of an accuracy solution:
50% accuracy solution: precisely weighing about 40mg of apixaban crude product (batch 202011002A), placing the apixaban crude product into a 10mL measuring flask, adding a proper amount of methanol for ultrasonic dissolution, precisely adding 0.12mL of reference substance stock solution, diluting with methanol to a scale, and shaking up to obtain a 50% accuracy solution; preparing 3 parts in parallel;
100% accuracy solution: precisely weighing about 40mg of apixaban crude product, placing the apixaban crude product into a 10mL measuring flask, adding a proper amount of methanol for ultrasonic dissolution, precisely adding 0.24mL of a reference substance stock solution, diluting the solution to a scale with methanol, and shaking up to obtain a 100% accuracy solution; preparing 3 parts in parallel;
150% accuracy solution: precisely weighing about 40mg of apixaban crude product, placing the apixaban crude product into a 10mL measuring flask, adding a proper amount of methanol for ultrasonic dissolution, precisely adding 0.42mL of a reference substance stock solution, diluting the solution to a scale with methanol, and shaking up to obtain a 150% accuracy solution; 3 parts are prepared in parallel.
Preparing a blank test solution: accurately weighing about 40mg of apixaban crude product, placing the apixaban crude product into a 10mL measuring flask, adding a proper amount of methanol for ultrasonic dissolution, diluting to a scale, and shaking up to be used as a blank test solution.
Preparing a reference substance solution: a proper amount of the genotoxic impurity A reference substance is precisely weighed, and dissolved and diluted by methanol to prepare a solution containing about 0.6 mu g of the genotoxic impurity A per 1mL as the reference substance solution.
Preparation of a sensitivity solution: precisely measuring 1mL of the reference solution, placing the reference solution in a 10mL measuring flask, adding methanol to dilute to the scale, and shaking up to obtain a sensitive solution.
Each 10. 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
And (4) conclusion: the recovery rate test result of the genotoxic impurity A in the apixaban shows that the recovery rates of 9 accurate samples are all between 86% and 106%, and the test result data show 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 detection method of the genotoxic impurities in the apixaban is characterized by comprising the following steps:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
gradient elution is carried out by adopting a mobile phase A and a mobile phase B;
the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.15%; the mobile phase B is acetonitrile-methanol mixed liquid with the volume ratio of 2: 3;
the detector is an ultraviolet detector, and the detection wavelength is 335 nm-345 nm.
2. The assay of claim 1 wherein the genotoxic impurity comprises genotoxic impurity a having the formula
Preferably, the detection wavelength is 340 nm;
preferably, the sample volume of the test solution is 5-20 mu L;
more preferably, the sample amount of the sample solution is 10. mu.L.
3. The detection method according to claim 1, wherein the flow rate is 0.8 to 1.2 mL/min;
preferably, the flow rate is 0.9-1.1 mL/min.
4. The detection method according to claim 1, wherein the column temperature of the chromatographic column is 25 to 35 ℃;
the specification of the chromatographic column is Waters
250mm×4.6mm，5μm。
5. The detection method according to claim 1, wherein the mobile phase A is an aqueous phosphoric acid solution with a volume fraction of 0.08-0.12%;
preferably, the mobile phase A is 0.1% by volume of phosphoric acid aqueous solution.
6. The detection method according to any one of claims 1 to 5, wherein the gradient elution comprises: changing the volume fraction of the mobile phase A from 58-62% to 38-42% in 0-35 min;
preferably, the volume fraction of the mobile phase A is changed from 60% to 40% in 0-35 min.
7. The detection method according to any one of claims 1 to 5, wherein the gradient elution is performed by:
。
8. The detection method according to claim 1, wherein the preparation of the test solution comprises: and dissolving the test sample by adopting methanol.
9. The detection method according to claim 1, further comprising: and calculating the content of genotoxic impurities in the test solution by an external standard method.
10. Use of the assay of any one of claims 1 to 9 for quality control of an apixaban formulation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116298027A (en) * | 2022-12-28 | 2023-06-23 | 江苏诺泰澳赛诺生物制药股份有限公司 | Liquid chromatography detection method and application of genotoxic impurities |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104387383A (en) * | 2014-10-24 | 2015-03-04 | 江南大学 | Synthetic method of novel apixaban precursor compound |
| CN105044269A (en) * | 2015-06-30 | 2015-11-11 | 成都百裕科技制药有限公司 | Method for detecting initial material II in apixaban through reversed-phase high performance liquid chromatography |
| US20170008886A1 (en) * | 2014-01-21 | 2017-01-12 | Wanbury Ltd. | A process for the preparation of apixaban and its intermediates |
| CN109627242A (en) * | 2018-12-29 | 2019-04-16 | 江苏豪森药业集团有限公司 | The preparation method of Eliquis |
| CN110467614A (en) * | 2019-09-12 | 2019-11-19 | 江西国药有限责任公司 | A kind of preparation method of Eliquis acid impurity |
-
2021
- 2021-06-22 CN CN202110691647.3A patent/CN113504317A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170008886A1 (en) * | 2014-01-21 | 2017-01-12 | Wanbury Ltd. | A process for the preparation of apixaban and its intermediates |
| CN104387383A (en) * | 2014-10-24 | 2015-03-04 | 江南大学 | Synthetic method of novel apixaban precursor compound |
| CN105044269A (en) * | 2015-06-30 | 2015-11-11 | 成都百裕科技制药有限公司 | Method for detecting initial material II in apixaban through reversed-phase high performance liquid chromatography |
| CN109627242A (en) * | 2018-12-29 | 2019-04-16 | 江苏豪森药业集团有限公司 | The preparation method of Eliquis |
| CN110467614A (en) * | 2019-09-12 | 2019-11-19 | 江西国药有限责任公司 | A kind of preparation method of Eliquis acid impurity |
Non-Patent Citations (6)
| Title |
|---|
| JESSICA B. ELLWANGER 等: "Analytical Quality by Design Approach for a Stability-Indicating Method to Determine Apixaban and Its Related Impurities", 《CHROMATOGRAPHIA》, vol. 83, 8 November 2019 (2019-11-08), pages 65 - 75, XP036977750, DOI: 10.1007/s10337-019-03815-9 * |
| ROBERT KORMANY 等: "Development of a Fast and Robust UHPLC Method for Apixaban In-Process Control Analysis", 《MOLECULES》 * |
| ROBERT KORMANY 等: "Development of a Fast and Robust UHPLC Method for Apixaban In-Process Control Analysis", 《MOLECULES》, vol. 26, 8 June 2021 (2021-06-08), pages 1 - 7 * |
| 严衍禄 等: "分析化学 第4版", 北京农业大学出版社, pages: 207 - 208 * |
| 陈思 等: "阿哌沙班杂质分析与控制", 《中国药学杂志》 * |
| 陈思 等: "阿哌沙班杂质分析与控制", 《中国药学杂志》, vol. 55, no. 22, 30 November 2020 (2020-11-30), pages 1906 - 1910 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116298027A (en) * | 2022-12-28 | 2023-06-23 | 江苏诺泰澳赛诺生物制药股份有限公司 | Liquid chromatography detection method and application of genotoxic impurities |
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