CN114965783B - Method for measuring apixaban initial raw material p-methoxyhydrazone by high performance liquid chromatography - Google Patents

Abstract

The invention provides a method for measuring apixaban initial raw material p-methoxyhydrazone by high performance liquid chromatography, which comprises the steps of firstly preparing system applicability solution of hydroxyhydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone, and preparing a sample solution and a control solution by taking apixaban Sha Banben to be measured; taking the system applicability solution, injecting the system applicability solution into a liquid chromatograph, recording a chromatogram, sequentially taking out peaks of hydroxyhydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone in the system applicability solution, precisely measuring a control solution and a sample solution, injecting the control solution and the sample solution into the liquid chromatograph, and recording the chromatogram. The invention can effectively separate the para-hydroxy hydrazone, the methyl ester hydrazone, the para-methoxyhydrazone and the chloro hydrazone, can effectively control the impurity content in the para-methoxyhydrazone, and has indispensable significance for quality control of the final product apixaban.

Description

Method for measuring apixaban initial raw material p-methoxyhydrazone by high performance liquid chromatography

Technical Field

The invention relates to a method for measuring apixaban initial raw material p-methoxyhydrazone by high performance liquid chromatography, belonging to the technical field of chemical detection and analysis.

Background

Apixaban is a novel oral direct factor Xa inhibitor developed by the combination of the company bai-me-si precious and the company dujie, approved for marketing in the european union at month 3 in 2011, under the trade name Eliquis, and clinically used for preventing Venous Thromboembolism (VTE) in adult patients undergoing a selective hip or knee replacement procedure. On 12 months 28 2012, the FDA approved apixaban for the prevention of stroke and systemic embolism in non-valvular Atrial Fibrillation (AF) patients. On month 21 2014, the FDA approved apixaban for the treatment of Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) and reduced the risk of recurrent DVT and PE following initial treatment.

The metahydrazone is an important starting material for the synthesis of raw materials of the apine Sha Banben product, and is also a genotoxic impurity to be controlled in the final product, and the quality control of the metahydrazone is related to the quality of the final product. Related impurities derived therefrom, particularly paraanisole containing aniline structures, chlorohydrazones containing halogenated benzene structures, and the like, can be regarded as genotoxic impurities. In order to ensure the quality of apixaban final products, reduce the risk of genotoxic impurities in the final products, simultaneously reduce the workload of quality control of the final products, and improve the efficiency of production quality inspection links, the comprehensive impurity control on the methoxyhydrazone is required. However, no literature reports about a method for analyzing substances of the product at present, no accurate determination or separation control process aiming at the metaflumizone and related impurities thereof exists in the existing preparation process of apixaban, the related impurities of the metaflumizone are strictly controlled in a starting material, and the work of controlling gene impurities in a finished product can be effectively reduced according to ICH M7 guiding principle and an impurity control strategy tree, so that the workload of a quality control link is lightened.

Disclosure of Invention

The invention aims to solve the problems in the existing apixaban drug preparation and quality detection processes, and determines and puts forward a method for determining the apixaban starting material to the methoxyhydrazone by a high performance liquid chromatography through proper fumbling experiments, and strictly performs method verification, ensures the scientific and strict of the method, meets the requirements of research and development and production, and plays a key role in controlling the quality of the methoxyhydrazone.

The technical solution of the invention is as follows: the method for measuring the apixaban initial raw material p-methoxyhydrazone by high performance liquid chromatography specifically comprises the following steps:

1) Preparing a system applicability solution: respectively taking about 5mg of hydroxyhydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone, respectively placing into 10ml measuring flask, adding mixed solvent (acetonitrile-water=50:50) to make ultrasonic treatment to dissolve and dilute to scale, shaking uniformly, and using as stock solution; 1ml of each stock solution was precisely measured, placed in the same 200ml measuring flask, diluted to the scale with a mixed solvent (acetonitrile-water=50:50), and shaken well to obtain a system applicability solution.

2) Preparing a test solution: the preparation is carried out in dark, a proper amount of the product is prepared for fresh preparation, the product is precisely weighed, and the mixture is dissolved and diluted into a solution of about 0.5mg in 1ml by adding a mixed solvent (acetonitrile-water=50:50) to be used as a test sample solution.

3) Control solution preparation: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding a mixed solvent (acetonitrile-water=50:50) to dilute to a scale, and shaking uniformly; precisely 5ml was measured and placed in a 10ml measuring flask and shaken well as a control solution.

4) Liquid chromatography detection of the methoxyhydrazone: taking 20 mu l of system applicability solution, injecting the solution into a liquid chromatograph, recording a chromatogram, sequentially taking out peaks of hydroxyhydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone in the system applicability solution, enabling the mutual separation degree of each impurity and main peak to be greater than 1.5, precisely measuring 20 mu l of control solution and sample solution, injecting the solution into the liquid chromatograph, and recording the chromatogram.

Chromatographic conditions and system applicability: octadecylsilane chemically bonded silica as filler (Waters Xbridge Shield RP, 150X 4.6mm,3.5 μm); gradient elution is carried out at the column temperature of 40 ℃; the detector is an ultraviolet detector; buffer-acetonitrile (90:10) was used as mobile phase A, buffer-acetonitrile (5:95) was used as mobile phase B, and linear gradient elution was performed according to the following table at a flow rate of 1.5ml/min, a detection wavelength of 295nm, and a column temperature of 40 ℃. Buffer salts used in the buffer solution include, but are not limited to, sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, and the like, and the concentration thereof is 20-50 mmol/L.

In the chromatogram of the sample solution, if impurity peaks are displayed, the hydroxyhydrazone is not larger than the main peak area (0.5%) of the control solution according to the corrected peak area (multiplied by a correction factor of 0.73), and the methyl ester hydrazone and the chloro hydrazone are not larger than 0.6 times (0.3%) of the main peak area of the control solution; the other impurities should not be greater than 0.2 times (0.1%) the main peak area of the control solution, and the sum of the impurity peak areas should not be greater than 2 times (1.0%) the main peak area of the control solution calculated as corrected peak area.

The specific calculation formula is as follows:

Sum of impurities% = known impurity% + other individual impurities%

Wherein: a _{Related substances} -peak area of the sample solution-related substance; a _{Control of major peak} -peak area of the main peak of the control solution.

Compared with the prior art, the invention has the advantages that: can effectively separate the para-hydroxy hydrazone, the methyl ester hydrazone, the para-methoxy hydrazone and the chloro hydrazone, can effectively control the impurity content in the para-methoxy hydrazone, and has indispensable significance for the quality control of the final product apixaban.

Drawings

FIG. 1 is a liquid chromatogram showing the effect of separating all impurities in an embodiment of the present invention.

FIG. 2 is a typical system applicability liquid chromatogram in an embodiment of the invention.

FIG. 3 is a graph of a diode array detection contour plot in an embodiment of the invention.

Fig. 4 is a 3D scan map of diode array detection in an embodiment of the invention.

FIG. 5 is a graph showing the spectrum of p-anisidine in the examples of the present invention.

FIG. 6 is a spectrum of hydroxyhydrazone in an example of the present invention.

FIG. 7 is a spectrum of methyl ester hydrazone in the examples of the present invention.

FIG. 8 is a spectrum of the p-methoxyhydrazone in the example of the present invention.

FIG. 9 is a spectrum of chlorohydrazone in the examples of the present invention.

Detailed Description

The technical scheme of the invention is further described below according to examples. In the description of the present specification, the contents of each example means that a specific technical feature described in connection therewith is included in at least one embodiment of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features described may be combined in any suitable manner in any one or more embodiments or examples.

For the synthetic process and degradation route of the p-methoxyhydrazone, the possible impurities in the p-methoxyhydrazone are summarized as shown in the following table 1:

TABLE 1 Methoxyhydrazone common impurity name information Table

In order to separate the impurities, inquire the literature and combine the inquireable standard of the preparation, creatively grope a mobile phase system, adopt buffer salt solution to enhance the separation effect of the impurities, adjust the proportion of the mobile phase and the temperature programming process, optimize the sample processing method, and finally establish the high performance liquid chromatography system, the detection spectrum of which is shown in the accompanying figures 1 and 2; meanwhile, the wavelength attribution is carried out by adopting a diode array detector, and the detection results are shown in fig. 3 and fig. 4 in detail. The spectrograms of the para-anisole, the hydroxyhydrazone, the methyl ester hydrazone, the para-methoxyhydrazone and the chloro hydrazone are shown in the accompanying figures 5-9.

The method provided by the invention is validated according to the requirements of the 2020 edition of Chinese pharmacopoeia. The relevant substance methodology verification results are specifically as follows:

1) Wavelength selection

Acceptable standards: the impurities are maximally absorbed around the selected wavelength.

Verification result: the scanning result of the PDA diode array detector on each impurity and the main peak of the metaflumizone shows that the maximum absorption wavelength distribution of the impurity on the anisole, the hydroxyhydrazone, the methyl ester hydrazone, the chlorohydrazone and the metaflumizone is 232-340 nm, the distribution is wider, in order to detect each impurity at the same wavelength, the detection wavelength is confirmed to be 295nm through the confirmation of the absorption diagram of the mixed impurity solution with the same concentration and the like, and each impurity at the wavelength has larger absorption and is similar to the response value of the main peak. The destructive test supports this wavelength as the detection wavelength of the present article.

2) Specialization of

Acceptable standards: the degree of separation must not be less than 2.0, and the purity factor of the main peak should be greater than 980.

Verification result: the blank solvent has no interference, the separation degree of the main component peak and each impurity peak is good, the impurity peak generated under the destruction condition can be separated from the main peak, and the peak purity meets the requirement.

3) System applicability

Acceptable standards: the degree of separation must not be less than 2.0.

Verification result: the mutual separation degree of all known impurities and main peaks is more than 2.0, the theoretical plate number meets the requirement, the retention time of separate sample injection is similar to that of mixed sample injection, and the system applicability is good.

4) Detection limit amount limit

Acceptable standards: the S/N limit is about 10; the S/N of the detection limit is about 3.

Verification result: the minimum detection limit for the anisole is 1.02ng (100 ppm) and the quantitative limit is 1.59ng (150 ppm);

the lowest detection limit of the hydroxyhydrazone is 1.07ng (100 ppm), and the quantitative limit is 1.64ng (12.58 ppm);

the lowest detection limit of the methyl ester hydrazone is 1.13ng (100 ppm), and the quantitative limit is 1.71ng (12.70 ppm);

The minimum detection limit of the metaflumizone is 1.15ng (100 ppm), and the quantitative limit is 1.68ng (12.80 ppm);

The minimum detection limit of the chlorinated hydrazone is 1.11ng (100 ppm), and the quantitative limit is 1.73ng (12.80 ppm);

The detection sensitivity is good.

5) Linearity of

Acceptable standards: the regression coefficient is not less than 0.999.

Verification result: the peak area and the concentration of the para-anisole in the concentration range of 0.077-0.6378 mug/ml are in good linear relation (r=0.9998, n=5).

The peak area and the concentration of the hydroxyhydrazone are in good linear relation (r=0.9994 and n=8) in the concentration range of 0.08847-7.848 mug/ml.

The peak area and the concentration of the methyl ester hydrazone are in good linear relation (r=0.9991, n=6) in the concentration range of 0.08012-2.280 mug/ml.

The peak area and the concentration of the metaflumizone in the concentration range of 0.07072-3.203 mug/ml are in good linear relation (r=0.9995 and n=8).

The peak area and the concentration of the chloro hydrazone are in good linear relation (r=0.9991, n=6) in the concentration range of 0.08172-2.190 mug/ml.

6) Correction factor

Acceptable standards: the values of the correction factors are all within the range of 0.9-1.1, and the correction factors can be not added for calculation.

Verification result: through calculation, the correction factor of the anisole is 1.82, the relative correction factor of the hydroxyhydrazone is 3.51, and the correction factors of other impurities are all in the range of 0.9-1.1.

7) Precision of sample injection

Acceptable standards: RSD <2.0%.

Verification result: the system applicability solution (containing no paraanisole), the paraanisole limit concentration solution, the self control solution and the sample injection precision of the test solution in the standard of the product are all good.

8) Repeatability of

Acceptable standards: RSD <4.0%.

Verification result: the results of repeated detection of 6 samples and samples with the same impurity addition amount show that the impurity contents are calculated according to the self-control method of adding correction factors, and the addition repeatability of the anisole is rsd% =2.44 (n=6); sample reproducibility RSD% = 6.93 (n=6) for 6 parts of hydroxyhydrazone; sample reproducibility RSD% = 7.45 (n=6), sample addition reproducibility RSD% = 3.13 (n=6) for 6 parts of methyl ester hydrazone; sample reproducibility RSD% =9.86% for 6 parts of chlorohydrazone, sample addition reproducibility RSD% =0.94 (n=6). Besides the fact that the hydroxyhydrazone cannot be verified due to sample degradation, the method is good in repeatability on various impurities. Meanwhile, the recovery rate of the low-concentration sample addition of each impurity of 6 samples is 80% -120%, and the measurement results of the external standard method and the self-contrast method are basically consistent.

9) Recovery rate

Acceptable standards: the recovery rate is between 85.0% and 110.0%, and the RSD is not more than 4%.

Verification result: the recovery rate test results of three different levels ranging from low, medium and high ranging from quantification limit to 120% show that: recovery of 9 samples of anisole was 95.26%, RSD% = 3.17 (n=9); recovery of 9 samples of methyl ester hydrazone was 90.38%, RSD% = 2.13 (n=9); recovery of 9 samples of chlorohydrazone was 100.3%, RSD% = 3.15 (n=9); besides the fact that the hydroxyhydrazone cannot be verified due to sample degradation, the recovery rate of each impurity detected by the method is good, the recovery rate is 80.0-120.0%, and the measurement accuracy is good.

10 Stability of solution

Acceptable standards: during the detection time, the detection result is not affected.

Verification result: the solution stability of each impurity test solution is good within 24 hours under the storage condition of 4 ℃; the self-control solution has good solution stability within 12 hours under the storage condition of 4 ℃; the test solution has good solution stability within 8 hours under the storage condition of 4 ℃; the test solution is not placed in a dark place, and the hydroxy hydrazone and the chloro hydrazone are greatly amplified within 24 hours, so that the preparation of the test solution is prompted to be operated in a dark place according to the conditions of other tests.

11 Durability(s)

Acceptable standards: when the detection conditions slightly change, the detection results are not affected.

Verification result: the durability of the analysis method under the conditions of wavelength + -2 nm, relative value change + -0.1 ml/min of flow velocity, relative change + -4% of initial proportion of mobile phase, column temperature change + -5 ℃ and different chromatographic columns is examined, and as a result, the tailing factor of each substance peak under each condition is smaller than 2.0, the separation degree of impurity peaks and other component peaks is larger than 1.5, and the method has good durability.

The verification result shows that each index of the method meets the requirements of the 2020 edition of Chinese pharmacopoeia and is suitable for detecting related substances of the metaflumizone.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (3)

1. The method for measuring the apixaban starting material p-methoxyhydrazone by high performance liquid chromatography is characterized by comprising the following steps of:

1) Preparing a system applicability solution: respectively taking 5mg of hydroxyhydrazone, 5mg of methyl ester hydrazone, 5mg of metaflumizone and 5mg of chlorohydrazone, respectively placing in a 10ml measuring flask, adding a mixed solvent, carrying out ultrasonic treatment to dissolve and dilute to scale, and shaking uniformly to obtain a stock solution; precisely measuring 1ml of each stock solution, placing the stock solutions into a same 200ml measuring flask, diluting to a scale with a mixed solvent, and shaking uniformly to obtain a system applicability solution;

2) Preparing a test solution: performing light-shielding operation, taking a sample to be measured, precisely weighing, adding a mixed solvent for dissolving and diluting to prepare a solution of 0.5mg in each 1ml, and using the solution as a sample solution for temporary preparation;

3) Preparation of control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding mixed solvent to dilute to scale, and shaking; precisely measuring 5ml, placing in a 10ml measuring flask, and shaking uniformly to obtain a control solution;

4) Liquid chromatography detection of the methoxyhydrazone: taking 20 μl of the system applicability solution, injecting into a liquid chromatograph, recording a chromatogram, and sequentially peaking hydroxyhydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone in the system applicability solution; when the mutual separation degree of each impurity and the main peak is more than 1.5, precisely measuring and injecting 20 mu l of each control solution and each sample solution into a liquid chromatograph, and recording a chromatogram;

wherein the chemical structural formula of the metahydrazone, the hydroxyhydrazone, the methyl ester hydrazone and the chlorohydrazone is specifically as follows:

；

The chromatographic conditions of the liquid chromatograph in the step 4) are as follows: a Waters Xbridge Shield RP chromatographic column with the specification of 150 multiplied by 4.6mm is adopted, and the particle size of the filling material is 3.5 mu m; the column temperature is 40 ℃, and the linear gradient elution is carried out according to the following table, and the flow rate is 1.5ml/min; the detector is an ultraviolet detector, and the detection wavelength is 295nm;

；

the mobile phase A and the mobile phase B adopted by the gradient elution are buffer salt-acetonitrile, wherein the mass ratio of the buffer salt to the acetonitrile in the mobile phase A is 90:10, and the mass ratio of the buffer salt to the acetonitrile in the mobile phase B is 5:95;

The buffer salt comprises one or more of sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and ammonium dihydrogen phosphate, and the concentration of the buffer salt is 20-50 mmol/L.

2. The method for determining the starting material p-methoxyhydrazone of apixaban by high performance liquid chromatography according to claim 1, wherein: the mixed solvent is a solution of acetonitrile and water in a mass ratio of 50:50.

3. The method for determining the starting material p-methoxyhydrazone of apixaban by high performance liquid chromatography according to claim 1, wherein: after the chromatograms are recorded in the step 4), if impurity peaks are displayed in the chromatograms of the test sample solutions, the corrected peak areas are multiplied by impurity correction factors of 0.73, the hydroxyl hydrazone is not larger than the main peak areas of the control solutions, and the methyl ester hydrazone and the chloro hydrazone are not larger than 0.6 times of the main peak areas of the control solutions; other impurities are not more than 0.2 times of the main peak area of the control solution, and the sum of the peak areas of the impurities is not more than 2 times of the main peak area of the control solution according to the corrected peak area; the specific calculation formula is as follows:

；

Total%of each impurity = known%of impurity + other individual impurities;

Wherein: a _{Related substances} -peak area of the sample solution-related substance; a _{Control of major peak} -peak area of the main peak of the control solution.