CN114965783A - Method for determining apixaban starting material p-methoxyhydrazone by high performance liquid chromatography - Google Patents

Abstract

The invention provides a method for determining the metaflumizone of an apixaban starting material by high performance liquid chromatography, which comprises the steps of firstly preparing a system applicability solution of hydroxyhydrazone, methylmethylester hydrazone, metaflumizone and chlorinated hydrazone, and preparing a test solution and a reference solution of an apixaban product to be detected; injecting the system adaptability solution into a liquid chromatograph, recording a chromatogram, sequentially peaking hydroxyl hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chlorinated hydrazone in the system adaptability solution, precisely measuring a reference solution and a test solution, injecting the reference solution and the test solution into the liquid chromatograph, and recording the chromatogram. The method can effectively separate the p-hydroxyhydrazone, the methyl ester hydrazone, the p-methoxyhydrazone and the chlorinated hydrazone, can effectively control the impurity content of the p-methoxyhydrazone, and has indispensable significance for the quality control of the final product apixaban.

Description

Method for determining apixaban starting material p-methoxyhydrazone by high performance liquid chromatography

Technical Field

The invention relates to a method for determining the metaflumizone pair of apixaban starting materials by high performance liquid chromatography, belonging to the technical field of chemical detection and analysis.

Background

Apixaban, a novel oral direct factor Xa inhibitor developed by the combination of behmean, precious corporation and fevered corporation, was approved for marketing in the european union 3 months 2011 under the trade name Eliquis, and was used clinically to prevent Venous Thromboembolism (VTE) in adult patients undergoing elective hip or knee replacement. On day 28/12/2012, apixaban was approved by the FDA for the prevention of stroke and systemic embolism in non-valvular Atrial Fibrillation (AF) patients. 8/21 days 2014, apixaban was approved by the FDA for the treatment of Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) and to reduce the risk of recurrent DVT and PE after initiation of treatment.

The metaxazone is an important starting material for synthesizing the raw material of the apixaban product and is genotoxic impurities needing to be controlled in a final product, and the quality control of the metaxazone is related to the quality of the final product. Related impurities derived therefrom, particularly p-anisidine containing an aniline structure, chlorinated hydrazone containing a halogenated benzene structure, and the like, can be considered as genotoxic impurities. In order to ensure the quality of the apixaban final product, reduce the risk of genotoxic impurities in the final product, reduce the workload of quality control of the final product and improve the efficiency of a production quality inspection link, the comprehensive impurity control of the methoxyhydrazone is required. However, no literature reports a related substance analysis method of the product at present, and the existing preparation process of apixaban does not have any precise measurement or separation control process for the methoxyhydrazone and related impurities thereof, and the related impurities of the methoxyhydrazone are strictly controlled in the starting material, so that the work of controlling gene impurities in a finished product can be effectively reduced according to the ICH M7 guiding principle and an impurity control strategy tree, and the work load of a quality control link is reduced.

Disclosure of Invention

The invention aims to solve the problems in the existing apixaban medicine preparation and quality detection processes, determines and provides a method for determining the starting material of apixaban on methoxyhydrazone by using a high performance liquid chromatography through a proper exploratory experiment, strictly performs method verification, ensures the scientific and rigorous method, meets the requirements of research and development and production, and plays a key role in controlling the quality of methoxyhydrazone.

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

1) preparing a system applicability solution: taking 5mg of hydroxy hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chlorinated hydrazone respectively, placing the hydroxy hydrazone, the methyl ester hydrazone, the p-methoxyhydrazone and the chlorinated hydrazone respectively into a 10ml measuring flask, adding a mixed solvent (acetonitrile-water =50: 50) for ultrasonic dissolution, diluting the mixed solvent to a scale, and shaking up to obtain a stock solution; precisely measuring 1ml of each stock solution, placing the stock solutions into the same 200ml measuring flask, diluting the stock solutions to a scale with a mixed solvent (acetonitrile-water =50: 50), and shaking the stock solutions uniformly to obtain a system applicability solution.

2) Preparing a test solution: the preparation method comprises processing in dark, preparing, weighing, dissolving with mixed solvent (acetonitrile-water =50: 50), and diluting to obtain solution of about 0.5mg per 1 ml.

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

4) Liquid chromatography detection of methoxyhydrazone: and (3) injecting 20 mu l of the system adaptability solution into a liquid chromatograph, recording a chromatogram, sequentially outputting peaks of hydroxyl hydrazone, methyl hydrazone, p-methoxyhydrazone and chloro-hydrazone in the system adaptability solution, accurately measuring 20 mu l of each of the reference solution and the sample solution, injecting the reference solution and the sample solution into the liquid chromatograph, and recording the chromatogram.

Chromatographic conditions and system applicability: octadecylsilane chemically bonded silica was used as a filler (Waters Xbridge Shield RP18, 150X 4.6mm, 3.5 μm); the column temperature is 40 ℃, and gradient elution is carried out; the detector is an ultraviolet detector; the following table was followed for linear gradient elution with buffer-acetonitrile (90: 10) as mobile phase A and buffer-acetonitrile (5: 95) as mobile phase B at a flow rate of 1.5ml/min, a detection wavelength of 295nm and a column temperature of 40 ℃. The buffer salt adopted by the buffer solution comprises but is 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 of the buffer salt is 20-50 mmol/L.

If an impurity peak appears in the chromatogram of the test solution, according to the corrected peak area (multiplied by a correction factor of 0.73), the hydroxy hydrazone is not more than the main peak area (0.5%) of the control solution, and the methyl ester hydrazone and the chloro hydrazone are not more than 0.6 times (0.3%) of the main peak area of the control solution; the other impurities should not be more than 0.2 times (0.1%) of the main peak area of the control solution, and the sum of the impurity peak areas should not be more than 2 times (1.0%) of the main peak area of the control solution as calculated from the corrected peak areas.

The specific calculation formula is as follows:

the sum of the impurities% = known impurity% + other individual impurities%

In the formula:A _{related substances} -peak areas of substances of interest of the test solution;A _{control 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 p-hydroxy hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro-hydrazone, can effectively control the impurity content in the p-methoxyhydrazone, and has indispensable significance for the quality control of the final product apixaban.

Drawings

FIG. 1 is a liquid chromatogram of the total impurity separation effect in the example of the present invention.

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

Fig. 3 is a diode array detection contour map in an embodiment of the invention.

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

FIG. 5 is a spectrum of p-anisidine in an example of the present invention.

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

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

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

FIG. 9 is a chart of the spectrum of chlorinated hydrazone in the example of the present invention.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples. In the description of the present specification, the contents of each embodiment means that a specific technical feature described in connection with it 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 embodiment or example. Furthermore, the particular features described may be combined in any suitable manner in any one or more of the embodiments or examples.

Aiming at the synthesis process and the degradation path of the p-methoxyhydrazone, the impurities possibly occurring in the p-methoxyhydrazone are summarized as shown in the following table 1:

TABLE 1 name information table for common methoxyhydrazone impurities

In order to separate the impurities, documents are inquired and the inquired standards of the preparation are combined, a mobile phase system is creatively searched, the separation effect of the impurities is enhanced by adopting a buffer salt solution, the proportion of the mobile phase and the temperature programming process are adjusted, the sample processing method is optimized, the high performance liquid chromatography system is finally established, and the detection spectrum is detailed in attached figures 1 and 2; meanwhile, the wavelength is attributed by adopting a diode array detector, and the detection result is shown in the attached figures 3 and 4 in detail. The spectra of p-anisidine, hydroxy hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro hydrazone are shown in figures 5-9.

The methodology of the method provided by the invention is verified according to the requirements of the China pharmacopoeia 2020 edition. The verification result of the related substance methodology is as follows:

1) wavelength selection

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

And (4) verification result: the scanning result of the PDA diode array detector on each impurity and the main peak of the methoxyhydrazone shows that the maximum absorption wavelength distribution of the impurities on the anisidine, the hydroxyhydrazone, the methyl ester hydrazone, the chlorinated hydrazone and the methoxyhydrazone is from 232nm to 340nm and is wide, and in order to detect each impurity at the same wavelength at the same time, the detection wavelength is determined to be 295nm finally through the confirmation of absorption graphs of mixed impurity solutions and the like with the same concentration, and each impurity at the wavelength has larger absorption and is close to the response value of the main peak. The damage experiment supports this wavelength as the detection wavelength for this product.

2) Specificity

Acceptable standards: the separation degree is not less than 2.0, and the purity factor of the main peak is more than 980.

And (4) verification result: the blank solvent has no interference, the separation degree of the main component peak and each impurity peak is good, the impurity peaks generated under the damage condition can be separated from the main peak, and the peak purity meets the requirement.

3) System applicability

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

And (4) verification result: the mutual separation degree of known impurities and main peaks is more than 2.0, the theoretical plate number meets the requirement, the retention time of single sample injection and mixed sample injection is close, and the system applicability is good.

4) Detection limit amount limit

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

And (4) verification result: the minimum detection limit of p-anisidine is 1.02ng (100 ppm), the quantification limit is 1.59ng (150 ppm);

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

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

the minimum detection limit of the methoxyhydrazone is 1.15ng (100 ppm), and the quantification limit is 1.68ng (12.80 ppm);

the lowest 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

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

And (4) verification result: the peak area of p-anisidine has a good linear relation with the concentration in the concentration range of 0.077-0.6378 [ mu ] g/ml (r =0.9998, n = 5).

The peak area of hydroxyhydrazone in the concentration range of 0.08847-7.848 mu g/ml is in good linear relation with the concentration (r =0.9994, n = 8).

The peak area of methyl ester hydrazone in the concentration range of 0.08012-2.280 mu g/ml is in a good linear relation with the concentration (r =0.9991, n = 6).

The peak area of the p-methoxyhydrazone in the concentration range of 0.07072-3.203 mug/ml is in a good linear relation with the concentration (r =0.9995, n = 8).

The peak area of the chlorinated hydrazone is in a good linear relation with the concentration in the concentration range of 0.08172-2.190 mu g/ml (r =0.9991, n = 6).

6) Correction factor

Acceptable standards: the numerical values of the correction factors are all in the range of 0.9-1.1, and calculation can be carried out without adding the correction factors.

And (4) verification result: through calculation, the correction factor of the anisidine 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 introduction

Acceptable standards: RSD is less than 2.0%.

And (4) verification result: the system applicability solution (without p-anisidine), p-anisidine limit concentration solution, self control solution and test solution in the product standard have good sample injection precision.

8) Repeatability

Acceptable standards: RSD is < 4.0%.

And (4) verification result: the results of repeated detection of 6 samples and samples with the same impurity loading amount show that the content of each impurity is calculated according to a self-control method of adding a correction factor, and RSD% =2.44 (n = 6) for the loading repeatability of anisidine; 6 samples of hydroxyhydrazone had a reproducibility RSD% =6.93 (n = 6); 6 samples of methyl ester hydrazone had a repetitive RSD% =7.45 (n = 6), loading repetitive RSD% =3.13 (n = 6); 6 samples of chlorohydrazone had a% RSD reproducibility of 9.86% and a% RSD reproducibility of loading of 0.94 (n = 6). The method has good repeatability for each impurity except that hydroxyhydrazone degradation of the sample cannot be verified. Meanwhile, the recovery rate of low-concentration sample addition of each impurity of 6 samples is 80-120%, and the measurement results of an external standard method and a self-contrast method are basically consistent.

9) Recovery rate

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

And (4) verification result: the results of the recovery tests at three different levels, low and medium and high, ranging from quantitative limits to 120%, show that: the recovery of 9 samples of anisidine was 95.26%, RSD% =3.17 (n = 9); the recovery of 9 samples of methyl ester hydrazone was 90.38%, RSD% =2.13 (n = 9); the recovery of 9 parts of the chlorinated hydrazone was 100.3%, RSD% =3.15 (n = 9); except that the hydroxyhydrazone can not be verified due to sample degradation, the method has the advantages that the recovery rate of each impurity detected by the method is better and is between 80.0 and 120.0 percent, and the detection accuracy is good.

10) Stability of solution

Acceptable standards: in the detection time, the detection result is not influenced.

And (4) verification result: the stability of the solution is good when the impurity test solutions are stored at 4 ℃ for 24 hours; the self-contrast solution has good stability within 12 hours under the storage condition of 4 ℃; the test solution has good stability within 8 hours under the storage condition of 4 ℃; the test solution is not placed in the dark, the hydroxy hydrazone and the chloro hydrazone have larger amplification within 24 hours, and the preparation of the test solution should be carried out in the dark and prepared just before use according to the conditions of other tests.

11) Durability

Acceptable standards: when the detection condition is slightly changed, the detection result is not influenced.

And (4) verification result: the wavelength is +/-2 nm, the change of the flow rate relative value is +/-0.1 ml/min, the initial proportion of the mobile phase is +/-4%, the change of the column temperature is +/-5 ℃, and the durability of the analysis method under different chromatographic column conditions is examined, so that the tailing factor of each substance peak under each condition is less than 2.0, the separation degree of impurity peaks and other component peaks is more than 1.5, and the durability of the method is good.

The verification result shows that all indexes of the method meet the requirements of the 2020 edition of Chinese pharmacopoeia and are suitable for detecting related substances to the methoxyhydrazone.

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 attributes 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 specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.

Claims (6)

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

1) preparation of system applicability solution: taking 5mg of hydroxy hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chloro-hydrazone respectively, placing the hydroxy hydrazone, the methyl ester hydrazone, the p-methoxyhydrazone and the chloro-hydrazone respectively in a 10ml measuring flask, adding the mixed solvent for ultrasonic dissolution, diluting the mixed solvent to a scale, and shaking up to obtain a stock solution; precisely measuring 1ml of each stock solution, placing the stock solutions into the same 200ml measuring flask, diluting the stock solutions to a scale by using a mixed solvent, and shaking the stock solutions uniformly to obtain a system applicability solution;

2) preparing a test solution: the operation is carried out in a dark place, the apixaban product to be detected is precisely weighed, and is dissolved and diluted by adding a mixed solvent to prepare a solution of about 0.5mg in each 1ml, and the solution is used as a test solution and is prepared for new clinical use;

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

4) liquid chromatography detection of methoxyhydrazone: injecting 20 mu l of the system adaptability solution into a liquid chromatograph, recording a chromatogram, and sequentially peaking hydroxyl hydrazone, methyl ester hydrazone, p-methoxyhydrazone and chlorinated hydrazone in the system adaptability solution; and when the mutual separation degree of each impurity and the main peak is more than 1.5, precisely measuring 20 mu l of each of the reference solution and the test solution, injecting the reference solution and the test solution into a liquid chromatograph, and recording a chromatogram.

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

3. The method for determining the p-methoxyhydrazone of apixaban starting material by high performance liquid chromatography as claimed in claim 1, wherein: the chromatographic conditions of the liquid chromatograph in the step 4) are as follows: a Waters Xbridge Shield RP18 chromatographic column with specification of 150X 4.6mm and a filler particle size of 3.5 μm is adopted; the column temperature is 40 ℃, gradient elution is carried out, and the flow rate is 1.5 ml/min; the detector is an ultraviolet detector, and the detection wavelength is 295 nm.

4. The method for determining the p-methoxyhydrazone of apixaban starting material by high performance liquid chromatography as claimed in claim 3, wherein: the mobile phase A and the mobile phase B adopted in the gradient elution are both buffer salt-acetonitrile, wherein the mass ratio of the buffer salt to the acetonitrile in the mobile phase A to the mobile phase B is 95: 5-5: 95.

5. The method for determining the p-methoxyhydrazone of the apixaban starting material by the high performance liquid chromatography method according to claim 4, wherein the method comprises the following steps: 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.

6. The method for determining the p-methoxyhydrazone of apixaban starting material by high performance liquid chromatography as claimed in claim 1, wherein: after the chromatogram is recorded in the step 4), if an impurity peak appears in the chromatogram of the test solution, multiplying the corrected peak area by an impurity correction factor of 0.73, wherein the hydroxyl hydrazone is not larger than the main peak area of the contrast solution, and the methyl ester hydrazone and the chlorinated hydrazone are not larger than 0.6 times of the main peak area of the contrast solution; the other impurities are not more than 0.2 times of the main peak area of the control solution, and the sum of the impurity peak areas is not more than 2 times of the main peak area of the control solution according to the calculation of the corrected peak area; the specific calculation formula is as follows:

the sum of the impurities% = known impurity% + other individual impurities%

In the formula:A _{related substances} -peak areas of substances of interest of the test solution;A _{control major peak} Peak area of the main peak of the control solution.

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