CN113917043B - Detection method of afatinib maleate genotoxic impurities - Google Patents
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Abstract
The application discloses a detection method of afatinib maleate genotoxic impurities. And (3) using a high performance liquid chromatography, taking 0.01mol/L potassium dihydrogen phosphate solution as a mobile phase A, acetonitrile as a mobile phase B, setting the pH, the flow speed, the column temperature, the detection wavelength and the sample injection amount of the mobile phase A, and performing gradient elution through a C18 chromatographic column. The high performance liquid chromatography adopted by the application can not only effectively separate the afatinib maleate genetic toxic impurity intermediate V, afatinib maleate and other known impurities, but also rapidly detect the genetic toxic impurity intermediate V which is lower than the quality control limit of 1/10, and has the advantages of shorter detection time, higher precision and accuracy, good repeatability, good system applicability and the like, thereby providing an important method detection reference basis for quality control of afatinib medicines.
Description
Technical Field
The application relates to the field of medicine analysis, in particular to a method for detecting genetic toxicity impurities of afatinib maleate.
Background
Afatinib maleate (afatinib dimaleate), chemical name (2E) -N- [4- [ (3-chloro-4-fluorophenyl) amino ] -7- [ [ (3S) -tetrahydrofuran-3-yl ] oxy ] quinazolin-6-yl ] -4- (dimethylamino) -2-butenamide dimaleate, developed by bringeglin, germany, 7 months of the FDA approval in the united states, trade name Gilotrif, dosage form film coated tablets, having 20, 30 and 40 mg 3 gauge. Afatinib is an irreversible Epidermal Growth Factor Receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2) dual tyrosine kinase inhibitor for the first line treatment of patients with metastatic non-small cell lung cancer (NSCLC) in which exon 19 is deleted and exon 21 (L858R) is substituted for mutation. The main impurities currently known for afatinib maleate are impurity a, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I and intermediate V, from starting materials, intermediates and degradation products, of the formula:
the intermediate V contains alkyl phosphate and is a genotoxicity warning structure. According to ICH M7 and the rule of control and guidance of the genetic toxicity impurity of 9306 in the fourth edition of Chinese pharmacopoeia 2020, calculated according to TTC 1.5 mug/day, the limit of the intermediate V is 30ppm. The intermediate V is detected in the afatinib maleate product so as to meet the requirement of the drug quality standard.
Patent CN105588893B discloses a detection method of 0.75ug/mL (30 ppm) of intermediate V system applicability solution, but the detection method has low sensitivity, long detection time reaching 100min, and uncontrollable detection cost, and cannot be widely applied.
The limit of genotoxic impurities is low, and most detection methods also use LC-MS or GC-MS for detection; because the LC-MS or GC-MS instrument is more expensive, most of the QC departments of pharmaceutical enterprises do not have the detection conditions of LC-MS or GC-MS, so the development of the conventional liquid chromatography method for detecting the genotoxic impurities has more practicability.
Disclosure of Invention
The application aims to provide a method for detecting genetic toxic impurities in afatinib maleate products, which has higher retrieval precision, shorter detection time and better system adaptability, so that the aspects of specificity, quantitative limit, detection limit, linear range, repeatability, accuracy and the like completely meet the standards.
In order to achieve the above purpose, the present application provides the following technical solutions:
preparing a sample solution from an afatinib maleate to be detected, preparing a reference substance solution from an afatinib maleate intermediate V reference substance, and then respectively performing HPLC detection by taking a monopotassium phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, and calculating the formula according to an external standard method: content (%) of afatinib maleate intermediate V in test sample=Wherein: ai is the peak area of each impurity in the sample solution; as is the peak area of each impurity of the reference solution; v is the dilution multiple of the sample solution; vs is the dilution of the control solution; wi is the sample weighing amount of the test sample; ws is the sample weighing amount of the reference substance.
Based on the research, the application provides a method for detecting afatinib maleate genotoxic impurities, which adopts high performance liquid chromatography, takes 0.01mol/L potassium dihydrogen phosphate solution as a mobile phase A, takes acetonitrile as a mobile phase B, sets detection wavelength and sample injection amount, and adopts the following steps of gradient elution through a C18 chromatographic column according to the volume ratio: starting to elute with 75% of mobile phase A and 25% of mobile phase B, gradually reducing the proportion of mobile phase A, gradually increasing the proportion of mobile phase B until the mobile phase A reaches 35% in 25 minutes, and continuing to elute for 10 minutes to 35 minutes, increasing the proportion of mobile phase A, reducing the proportion of mobile phase B, adjusting the mobile phase A to 75% and the mobile phase B to 25% in 36 minutes, and continuing to elute for 9 minutes; the detection method can detect 8.0ppm of afatinib maleate toxic impurities, wherein the impurities are intermediates V, and the structural formula is as follows:
according to the embodiment of the application, the quality control limit of the V limit of the intermediate is 30ppm according to the rule of guidelines 9306 for genetic toxicity impurity control of ICH M7 and the fourth edition of Chinese pharmacopoeia 2020. In the quantitative limit test, the quantitative limit concentration of the intermediate V is lower than the quality control limit of the intermediate V, the detection limit concentration is lower than 1/10 of the quality control limit of the intermediate V, and when the high performance liquid chromatography is adopted for gradient washing, the concentration of the intermediate V can be effectively detected at 2.6-8.0 ppm, which indicates that the intermediate V can be effectively detected at more than 1/10 of the quality control limit.
According to the embodiment of the application, when the high performance liquid chromatography of the application is used for gradient washing, the detection wavelength is preferably 230nm, the sample injection amount is preferably 50 mu L, the C18 chromatographic column is preferably YMC Triart C18 column, and the specification is 4.6mm×250mm and 3 mu m.
According to an embodiment of the application, when the high performance liquid chromatography of the application is used for gradient washing, the volume ratio of the initial mobile phase A to the initial mobile phase B is 75:25, 77:23 or 73:27; the pH value of the mobile phase potassium dihydrogen phosphate solution is regulated to 2.8-3.2 by phosphoric acid; the flow rate is 0.8-1.2 mL/min; when the column temperature is 25-35 ℃, the detection method is good in durability, the detection amount of the impurity V is smaller than the detection limit, and the tailing factor of the intermediate V peak in the reference substance solution is 0.8-1.5.
Preferably, the pH of the mobile phase A potassium dihydrogen phosphate solution is adjusted to 3.0 with phosphoric acid at a flow rate of 1.0mL/min and a column temperature of 30 ℃.
According to the embodiment of the application, when the high performance liquid chromatography is adopted for gradient washing, in the specificity test of the afatinib product containing the intermediate V and each known impurity, the blank solvent and each known impurity do not interfere with the detection of the intermediate V, and the separation degree between the intermediate V and the adjacent impurity peaks is more than 1.5;
in the test of the linear range, the intermediate V has good linear relation in the range of 8ppm to 45ppm relative to the concentration of the sample, and the correlation coefficient r is not lower than 0.999;
in an accuracy test, the recovery rate of the intermediate V is 80% -115%, and RSD is less than or equal to 5.0%;
in the repeatability test, the detection amount RSD of 6 samples of the intermediate V is less than or equal to 10 percent;
in the test of intermediate precision, different testers detect the same sample according to repeated test operation on different instruments on different dates, and the detection amount RSD of the intermediate V in 12 samples is less than or equal to 15 percent.
The beneficial effects of the application are as follows: compared with the prior art, the application adopts an acetonitrile-potassium dihydrogen phosphate solution gradient elution system, can effectively separate genetic toxic impurity intermediate V from afatinib maleate and other known impurities, has better peak symmetry and quick peak emergence, can rapidly detect genetic toxic impurity intermediate V which is lower than a quality control limit of 1/10, simultaneously has obvious advantages in specificity, quantitative limit, detection limit, linear range and repeatability, has the advantages of shorter detection time, higher precision and accuracy, good repeatability, good system applicability and the like, and provides an important method detection reference basis for quality control of afatinib medicines.
Drawings
FIG. 1 is an inspection blank solvent liquid chromatogram;
FIG. 2 is a liquid chromatogram of an inspection maleic acid positioning solution;
FIG. 3 is a liquid chromatogram of an inspection intermediate V solution;
FIG. 4 is a diagram of an inspection impurity interference liquid chromatogram;
FIG. 5 is a liquid chromatogram of an inspection sample solution;
FIG. 6 is a liquid chromatogram of inspection of 8.0ppm intermediate V;
FIG. 7 is a linear graph showing the concentration of the sample in the range of 8ppm to 45 ppm.
Detailed description of the preferred embodiments
The application discloses a method for detecting low-limit genetic toxicity impurities in an afatinib maleate product. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is specifically noted that all similar substitutions and modifications will be apparent to those skilled in the art, and are intended to be included in the present application. While the detection method of the present application has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and suitable modifications and combinations of the method applications described herein can be made to practice and use the techniques of the present application without departing from the spirit or scope of the application.
According to the detection method disclosed by the application, the external standard method is used for calculating mainly aiming at the detection of the intermediate V.
The chromatograph used in the embodiment of the detection method of the application is a Siemens high performance liquid chromatograph, and the chromatographic column is a YMC Triart C18 column (4.6 mm multiplied by 250mm,3 μm).
The application is further illustrated by the following examples.
Example 1: chromatographic conditions and chromatographic system of the detection method
Instrument: siemens high performance liquid chromatograph
Chromatographic column: YMC Triart C18 column (4.6 mm. Times.250 mm,3 μm)
Mobile phase a:0.01mol/L potassium dihydrogen phosphate solution, and adjusting pH to 3.0 with phosphoric acid
Mobile phase B: acetonitrile
The gradient procedure was as follows:
and (3) starting to elute with 75% of mobile phase A and 25% of mobile phase B according to the volume ratio, gradually reducing the proportion of mobile phase A, gradually increasing the proportion of mobile phase B until the mobile phase A reaches 35% in 25 minutes and the mobile phase B reaches 65%, continuing to elute for 10 to 35 minutes, increasing the proportion of mobile phase A, reducing the proportion of mobile phase B, adjusting the mobile phase A to 75% in 36 minutes, adjusting the mobile phase B to 25% in 36 minutes, and continuing to elute for 9 minutes.
Flow rate: 1.0mL/min
Column temperature: 30 DEG C
Detection wavelength: 230nm
Sample injection amount: 50 mu L
Test solution: and (5) operating in a dark place. Taking a proper amount of the test sample, precisely weighing, adding acetonitrile-water (30:70) for dissolving and diluting to prepare a solution containing about 0.5mg of the test sample per 1 mL.
Control solution: taking a proper amount of intermediate V reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution containing about 0.5mg of intermediate V stock solution in each 1 mL; a suitable amount of intermediate V stock solution was diluted with acetonitrile-water (30:70) to prepare a solution containing about 0.015. Mu.g per 1mL as a control solution.
The calculation mode is as follows:
content (%) of afatinib maleate intermediate v=
Wherein: ai is the peak area of each impurity in the sample solution;
as is the peak area of each impurity of the reference solution;
v is the dilution multiple of the sample solution;
vs is the dilution of the control solution;
wi is the sample weighing amount of the test sample;
ws is the sample weighing amount of the reference substance.
Example 2: the detection method of the application is a special test
A diluent: acetonitrile-water (30:70)
Intermediate V solution: a proper amount of intermediate V is taken, precisely weighed, and a diluent is added to prepare a solution of about 5 mug in each 1 mL.
Maleic acid positioning solution: proper amount of maleic acid is taken, precisely weighed, and a diluent is added to prepare a solution containing about 200 mug of maleic acid in each 1mL of solution, and the solution is taken as a maleic acid positioning solution.
Impurity interfering solution: and adding a diluent into proper amounts of impurities A, B, C, D, E, F, G, H, I to prepare a mixed solution with about 0.5 mug of each impurity in each 1mL serving as an impurity interference solution.
Afatinib maleate sample solution: and then taking a proper amount of the product, precisely weighing, and adding a diluent to prepare a solution containing about 0.5mg of the product per 1mL, wherein the solution is used as an afatinib maleate sample solution.
Blank solvent, intermediate V solution, maleic acid positioning solution, impurity interference solution and afatinib maleate sample solution are respectively injected according to the chromatographic conditions of the embodiment 1, and chromatograms are recorded, and are shown in figures 1-5 and table 1.
Table 1: results of the proprietary test
Conclusion: the blank solvent and all known impurities do not interfere with the detection of the intermediate V, and the separation degree between the intermediate V and the adjacent impurity peaks is more than 1.5.
Example 3: quantitative limit and detection limit test of the detection method
The detection Limit (LOD) and the quantification Limit (LOQ) are determined according to the signal-to-noise ratio method. The intermediate V solution was diluted stepwise, the measured signal was compared with the baseline noise, and the lowest concentration that could be reliably detected was calculated, and the results are shown in table 2.
Table 2: quantitative limit and detection limit results
The quantitative limit concentration of the intermediate V is lower than the quality control limit of the intermediate V, the detection limit concentration is lower than 1/10 of the quality control limit of the intermediate V, the intermediate V can be effectively detected above the quality control limit of 1/10, and as can be seen from Table 2 and FIG. 6, the intermediate V of 2.6-8.0 ppm can be detected by adopting the high performance liquid chromatography disclosed by the application, and the sensitivity of the application is proved to be good.
Example 4: the linearity and range detection of the detection method of the application
The intermediate V reference substance is weighed in proper amount, dissolved and diluted with methanol to prepare a solution containing about 1.5 mug per 1mL as a stock solution, and 0.5mL, 0.6mL, 0.8mL, 1mL, 1.5 mL-200 mL, 100mL and 100mL measuring bottles are respectively weighed in precise amounts, acetonitrile-water (30:70) is added to dilute to scale, and shaking is carried out to prepare linear 1-linear 6 solutions. The linear relationship was plotted as a function of measured peak area versus analyte concentration, and linear regression was performed using the least squares method, requiring that the value of the linear regression coefficient r be not less than 0.990, and the results are shown in Table 3.
Table 3: results of the linear measurement
As can be seen from table 3 and fig. 7, the detection method of the present application shows a good linear relationship with respect to the intermediate V, in which the linear correlation coefficient r is 0.9996 in the range of 8ppm to 45ppm relative to the concentration of the sample.
Example 5: detection of repeatability of the detection method of the application
An afatinib maleate sample was taken and the test was repeated 6 times according to the test method of example 1 of the present application to verify the good precision of the method, and the results are shown in table 4.
Table 4: repeatability test results
As shown in Table 4, the intermediate V was detected below the limit for 6 times, which proves that the method has good precision.
Example 6: detection of accuracy of the detection method of the application
The ratio (recovery rate) between the measured and theoretical amounts of the reagent for the intermediate V in the labeled sample was determined by the sample-adding recovery method and expressed as a percentage% with the recovery rate required to be 80% -115% to confirm that the method has good accuracy, and the results are shown in Table 5.
Table 5: accuracy test results
As shown in Table 5, the recovery rate of intermediate V is 90.57% -104.08%, which meets the verification requirements (80% -115%). The method was confirmed to have good accuracy.
Example 7: the detection method of the application can detect the stability of the reference substance solution
Taking intermediate V control solution, standing at room temperature, injecting 50 mu L of the intermediate V control solution after 0h, 3h, 10h, 14h, 27h and 4 days respectively, recording a chromatogram, and calculating the relative standard deviation of the peak areas of the intermediate V. The results are shown in Table 6.
Table 6: stability test results of control solution
As is clear from Table 6, the intermediate V control solution was stable when left at room temperature for 4 days.
Example 8: detection of stability of sample solution by the detection method
Sample solutions of afatinib maleate were taken and 50 μl were injected after 0h, 5.5h, 10.5h, 13.5h, 24h and 3 days, respectively, chromatograms were recorded, and the relative standard deviation of peak areas of intermediate V and afatinib was calculated, and the test results are shown in table 7.
Table 7: test results of stability test of sample solution
As is clear from Table 7, the test sample solutions were stable when left at room temperature for 3 days.
Example 9: detection of durability test of the detection method of the present application
The degree of tolerance of the measurement results was not affected when the measurement conditions were slightly changed by changing the pH value of mobile phase a, the ratio of mobile phase B, the column temperature, and the flow rate. The durability results are shown in the following table.
Table 8: chromatographic condition variation parameter
Table 9: adjusting the pH of mobile phase A with phosphoric acid, the influence of different pH flows on the measurement of intermediate V
Table 10: effect of different flow phase ratios on intermediate V determination
Table 11: influence of different column temperatures on the measurement of intermediate V
Table 12: influence of different flow rates on the measurement of intermediate V
As can be seen from tables 8-12, the pH value of mobile phase A is adjusted to 2.8-3.2 by phosphoric acid, and the volume ratio of initial mobile phase A to B is 75:25, 77:23 or 73:27; the column temperature is 25-35 ℃; the flow rate is 0.8-1.2 mL/min, the detection amount of the intermediate V is smaller than the detection limit, and the tailing factor of the intermediate V peak in the reference substance solution is 0.8-1.5. The detection method of the present application, in which the pH of mobile phase a, the initial mobile phase a: B volume ratio, the column temperature, and the flow rate were set within the above ranges, was shown to be excellent in durability.
The foregoing is merely a preferred embodiment of the application, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are also intended to be considered as protective scope of the application.
Claims (6)
1. A method for detecting afatinib maleate genotoxic impurities is characterized in that a high performance liquid chromatography is adopted, 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, the pH value of the mobile phase A is regulated to 2.8-3.2 by phosphoric acid, acetonitrile is used as a mobile phase B, the detection wavelength is set to be 230nm, the sample injection amount is 50 mu L, the sample is subjected to gradient elution through a C18 chromatographic column according to the volume ratio, the C18 chromatographic column is YMC Triart C18, the specification is 4.6mm multiplied by 250mm and 3 mu m, and the elution procedure is as follows: starting to elute with 75% of mobile phase A and 25% of mobile phase B, gradually reducing the proportion of mobile phase A, gradually increasing the proportion of mobile phase B until the mobile phase A reaches 35% in 25 minutes, and continuing to elute for 10 minutes to 35 minutes, increasing the proportion of mobile phase A, reducing the proportion of mobile phase B, adjusting the mobile phase A to 75% and the mobile phase B to 25% in 36 minutes, and continuing to elute for 9 minutes; the detection method can detect 8.0ppm of afatinib maleate genotoxic impurities; wherein the genotoxic impurity is an intermediate V, and the structure of other impurities of the afatinib maleate is as follows:
。
2. the method for detecting afatinib maleate genotoxic impurities according to claim 1, wherein the 0.01mol/L potassium dihydrogen phosphate solution is adjusted to pH 3.0 with phosphoric acid.
3. The method for detecting afatinib maleate genotoxic impurities according to claim 1, wherein the flow rate of the high performance liquid chromatography is 0.8-1.2 ml/min.
4. A method for detecting afatinib maleate genotoxic impurities as in claim 3 wherein said high performance liquid chromatography is performed at a flow rate of 1.0 mL/min.
5. The method for detecting afatinib maleate genotoxic impurities according to claim 1, wherein the column temperature of the high performance liquid chromatography is 25-35 ℃.
6. The method for detecting afatinib maleate genotoxic impurities according to claim 1, wherein the column temperature of the high performance liquid chromatography is 30 ℃.
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