CN113917043A - Detection method of afatinib maleate genotoxic impurities - Google Patents
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Abstract
The invention discloses a method for detecting genetic toxic impurities of afatinib maleate. The high performance liquid chromatography is adopted, 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the pH, the flow rate, the column temperature, the detection wavelength and the sample amount of the mobile phase A are set, and the gradient elution is carried out through a C18 chromatographic column. The high performance liquid chromatography adopted by the invention can effectively separate the afatinib maleate genotoxic impurity intermediate V, afatinib maleate and other known impurities, has good peak type symmetry and fast peak appearance, can quickly detect the genotoxic impurity intermediate V which is lower than the quality control limit 1/10, 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 the quality control of afatinib medicaments.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, and in particular relates to a method for detecting genetic toxicity impurities of afatinib maleate.
Background
Afatinib dimaleate maleate, chemically (2E) -N- [4- [ (3-chloro-4-fluorophenyl) amino ] -7- [ [ (3S) -tetrahydrofuran-3-yl ] oxy ] quinazolin-6-yl ] -4- (dimethylamino) -2-butenamide dimaleate, developed by brigling invar, germany, approved by the FDA in the us at 7 months 2013 for marketing, under the trade name Gilotrif, in the form of film-coated tablets, having 3 specifications of 20, 30 and 40 mg. Afatinib is an irreversible dual tyrosine kinase inhibitor of Epidermal Growth Factor Receptor (EGFR) and human epidermal growth factor receptor 2(HER-2) for first-line treatment of patients with EGFR exon 19 deletion and exon 21(L858R) substitution mutations in metastatic non-small cell lung cancer (NSCLC). The currently known main impurities of afatinib maleate are impurity a, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I and intermediate V, which are derived from starting materials, intermediates and degradation products, and have the following structural formula:
the intermediate V contains alkyl phosphate and is a genetic toxicity warning structure. The limit of the intermediate V is 30ppm calculated according to TTC 1.5 mu g/day according to the guide principle of controlling genotoxic impurities of ICH M7 and the national drug code 2020 edition rules of the four ministry of communications 9306. The intermediate V needs to be detected in an afatinib maleate product so as to meet the requirement of a medicine quality standard.
Patent CN105588893B discloses a detection method of 0.75ug/mL (30 ppm) of intermediate V system applicability solution, but the detection method is not deeply studied, has low sensitivity, long detection time up to 100min, long time consumption and uncontrollable detection cost, and cannot be widely applied.
The limit of genotoxic impurities is low, and most detection methods can also use LC-MS or GC-MS for detection; because LC-MS or GC-MS instruments are expensive, and the large and multiple medicine enterprise QC department does not have the detection conditions of LC-MS or GC-MS, the development of a conventional liquid chromatography method for detecting genotoxic impurities has higher practicability.
Disclosure of Invention
The invention aims to provide the method for detecting the genotoxic impurities in the afatinib maleate product, which has higher retrieval precision, shorter detection time and better system adaptability, so that the specificity, the quantitative limit, the detection limit, the linear range, the repeatability, the accuracy and other aspects of the method completely meet the standard.
In order to achieve the purpose, the invention provides the following technical scheme:
preparing a test sample solution from an afatinib maleate to-be-detected product, preparing a reference product solution from an afatinib maleate intermediate V, performing HPLC (high performance liquid chromatography) detection by using a potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, and calculating a 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 of the test solution; as is the peak area of each impurity of the reference solution; v is the dilution multiple of the test solution; vs is the dilution factor of the control solution; wi is the sample weighing of the sample; ws is the weight of the reference substance.
Based on the research, the invention provides a method for detecting genetic toxic impurities in afatinib maleate, which adopts high performance liquid chromatography, takes 0.01mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, sets the detection wavelength and the sample amount, and adopts a C18 chromatographic column for gradient elution according to the volume ratio, wherein the elution procedure is as follows: starting elution by using 75% of a mobile phase A and 25% of a mobile phase B, gradually reducing the proportion of the mobile phase A, gradually increasing the proportion of the mobile phase B until the proportion of the mobile phase A reaches 35% in 25 minutes and the proportion of the mobile phase B reaches 65%, continuing elution for 10 minutes to 35 minutes, increasing the proportion of the mobile phase A, reducing the proportion of the mobile phase B, adjusting the proportion of the mobile phase A to 75% in 36 minutes, adjusting the proportion of the mobile phase B to 25%, and continuing elution for 9 minutes; the detection method can detect 8.0ppm of afatinib maleate genotoxic impurities, the impurities are intermediate V, and the structural formula is as follows:
according to the embodiment of the invention, the quality control limit of the intermediate V is 30ppm according to the guide principle of ICH M7 and the four ministry of general rules of China, 2020 edition 9306 genotoxic impurity control. In a 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 within the range of 2.6-8.0 ppm, which indicates that the intermediate V can be effectively detected within the quality control limit of 1/10.
According to the embodiment of the invention, when the high performance liquid chromatography is adopted for gradient washing, the detection wavelength is preferably 230nm, the sample injection amount is preferably 50 μ L, the C18 chromatographic column is preferably YMC Triart C18 column, and the specification is 4.6mm × 250mm and 3 μm.
According to an embodiment of the invention, when the high performance liquid chromatography of the invention is used for gradient washing, the initial mobile phase a: B volume ratio is 75:25, 77:23 or 73: 27; regulating the pH of the mobile phase potassium dihydrogen phosphate solution to 2.8-3.2 by using phosphoric acid; the flow rate is 0.8-1.2 mL/min; when the column temperature is 25-35 ℃, the detection method has good durability, the detected amount of the impurity V is less than the detection limit, and the tailing factor of the V peak of the intermediate in the reference solution is 0.8-1.5.
Preferably, the mobile phase A potassium dihydrogen phosphate solution is adjusted to pH 3.0 with phosphoric acid, the flow rate is 1.0mL/min, and the column temperature is 30 ℃.
According to the embodiment of the invention, when the high performance liquid chromatography is adopted for gradient washing, in a special test of an afatinib product containing the intermediate V and known impurities, a blank solvent and the known impurities do not interfere with the detection of the intermediate V, and the separation degree between the intermediate V and an adjacent impurity peak is more than 1.5;
in a linear range test, the intermediate V has a good linear relation in a range of 8ppm to 45ppm relative to the concentration of a test 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 a repeatability test, the detected quantity RSD of 6 parts of sample intermediate V is less than or equal to 10 percent;
in the test of intermediate precision, different testers detect the same sample on different instruments according to the repetitive test operation on different dates, and the detected amount RSD of the intermediate V in 12 samples is less than or equal to 15 percent.
The invention has the beneficial effects that: compared with the prior art, the method adopts an acetonitrile-potassium dihydrogen phosphate solution gradient elution system, so that the genetic toxic impurity intermediate V, afatinib maleate and other known impurities can be effectively separated, the peak type symmetry is good, the peak emergence is fast, the genetic toxic impurity intermediate V lower than the quality control limit 1/10 can be quickly detected, meanwhile, the method has remarkable 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 the quality control of afatinib medicaments.
Drawings
FIG. 1 is a liquid chromatogram for examining a blank solvent;
FIG. 2 is a liquid chromatogram of a positioning solution for examining maleic acid;
FIG. 3 is a liquid chromatogram of a solution for examining intermediate V;
FIG. 4 is a liquid chromatogram for checking interference of impurities;
FIG. 5 is a liquid chromatogram of an examination sample solution;
FIG. 6 is a liquid chromatogram examining 8.0ppm of intermediate V;
FIG. 7 is a linear graph showing the concentration of a sample in the range of 8ppm to 45 ppm.
Detailed description of the preferred embodiments
The invention discloses a method for detecting low-limit genetic toxic impurities in an afatinib maleate product. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such substitutions and modifications which are obvious to those skilled in the art are deemed to be included herein. While the detection methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the practice of the methods described herein, as well as suitable variations and combinations thereof, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
According to the detection method, the intermediate V is mainly detected, and an external standard method is used for calculation.
In the embodiment of the detection method, the chromatograph adopted is a Saimerfin high performance liquid chromatograph, and the chromatographic column is a YMC Triart C18 column (4.6 mm multiplied by 250mm,3 mu m).
The invention is further illustrated by the following examples.
Example 1: chromatographic conditions and chromatographic system of the detection method of the invention
The instrument comprises the following steps: saimeishafing high performance liquid chromatograph
A 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 program is as follows:
according to the volume ratio, starting elution by using 75% of a mobile phase A and 25% of a mobile phase B, gradually reducing the proportion of the mobile phase A, gradually increasing the proportion of the mobile phase B until the mobile phase A reaches 35% and the mobile phase B reaches 65%, continuing elution for 10 minutes to 35 minutes, increasing the proportion of the mobile phase A, reducing the proportion of the mobile phase B, adjusting the proportion of the mobile phase A to 75% and the proportion of the mobile phase B to 25% when the time reaches 36 minutes, and continuing elution for 9 minutes.
Flow rate: 1.0mL/min
Column temperature: 30 deg.C
Detection wavelength: 230nm
Sample introduction amount: 50 μ L
Test solution: and (4) avoiding light. A proper amount of a test sample is precisely weighed, and acetonitrile-water (30: 70) is added for dissolving and diluting to prepare a solution containing about 0.5mg in each 1mL as a test sample solution.
Control solution: taking a proper amount of the intermediate V reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution containing 0.5mg of the intermediate V in every 1mL, and taking the solution as an intermediate V stock solution; an appropriate amount of intermediate V stock solution was diluted with acetonitrile-water (30: 70) to give a solution containing about 0.015. mu.g of intermediate V per 1mL, as a control solution.
The calculation method comprises the following steps:
content (%) of intermediate V of afatinib maleate =
Wherein: ai is the peak area of each impurity of the test solution;
as is the peak area of each impurity of the reference solution;
v is the dilution multiple of the test solution;
vs is the dilution factor of the control solution;
wi is the sample weighing of the sample;
ws is the weight of the reference substance.
Example 2: the detection method of the invention is a special test
Diluent agent: acetonitrile-water (30: 70)
Intermediate V solution: taking a proper amount of the intermediate V, precisely weighing, and adding a diluent to prepare a solution of about 5 mu g per 1 mL.
Maleic acid localization solution: a proper amount of maleic acid is precisely weighed and added with a diluent to prepare a solution containing about 200 mu g of maleic acid per 1mL as a maleic acid positioning solution.
Impurity interfering solution: an appropriate amount of each impurity A, B, C, D, E, F, G, H, I was added to a diluent to prepare a mixed solution containing about 0.5. mu.g of each impurity per 1mL as an impurity interfering solution.
Afatinib maleate sample solution: and taking a proper amount of the product, precisely weighing, and adding a diluent to prepare a solution containing about 0.5mg of the product in every 1mL of the product to be used as a sample solution of afatinib maleate.
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 specificity test
And (4) conclusion: 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 an adjacent impurity peak is more than 1.5.
Example 3: quantitative limit and detection limit tests of the detection method of the invention
The limit of detection (LOD) and limit of quantitation (LOQ) are determined according to a signal-to-noise ratio method. The intermediate V solution was diluted step by step, the measured signal was compared with baseline noise, and the lowest concentration that could be reliably detected was calculated, with the results 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 1/10, and as can be seen from the table 2 and the figure 6, the high performance liquid chromatography disclosed by the invention can be used for detecting the intermediate V with the concentration of 2.6-8.0 ppm, so that the high sensitivity of the invention is proved to be good.
Example 4: linear and range detection for the detection method of the invention
An appropriate amount of the intermediate V reference substance is precisely weighed, dissolved and diluted by methanol to prepare a solution containing about 1.5 mu g of the intermediate V per 1mL as a stock solution, the stock solutions are precisely weighed into measuring bottles of 0.5mL, 0.6mL, 0.8mL, 1mL, 1.5mL to 200mL, 100mL and 100mL respectively, acetonitrile-water (30: 70) is added to dilute to a scale, and the solution is shaken up to be a linear 1-linear 6 solution. The linear relationship is plotted as a function of measured peak area versus analyte concentration and a least squares linear regression is used, requiring that the value of the linear regression coefficient r should be no less than 0.990, with the results shown in table 3.
Table 3: results of linear measurement
As can be seen from Table 3 and FIG. 7, the detection method of the invention proves that the intermediate V has a good linear relation in the range of 8ppm to 45ppm relative to the concentration of the test sample, and the linear correlation coefficient r is 0.9996.
Example 5: detection of the repeatability of the detection method of the invention
The good precision of the method is verified by repeating the detection method of the embodiment 1 of the invention for 6 times by taking a sample of afatinib maleate, and the result is shown in table 4.
Table 4: repeatability test results
As can be seen from Table 4, the intermediates V were all below the detection limit after 6 times of detection, demonstrating that the method has good precision.
Example 6: detection of the accuracy of the detection method of the invention
The ratio (recovery rate) between the measured amount and the theoretical amount of the reagent for measuring the intermediate V in the standard sample is measured by a sample-adding recovery method, expressed in percentage percent, and the recovery rate is required to be 80-115 percent, so as to confirm that the method has good accuracy, and the result is shown in table 5.
Table 5: accuracy test results
As can be seen from Table 5, the recovery rate of the intermediate V is 90.57-104.08%, and the intermediate V meets the validation requirements (80-115%). The method was confirmed to have good accuracy.
Example 7: detection of the stability of the control solution in the detection method of the invention
And taking the 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 area of each intermediate V. The results are shown in Table 6.
Table 6: test results of solution stability of control
As can be seen from Table 6, the intermediate V control solution was stable well when left at room temperature for 4 days.
Example 8: the detection method of the invention detects the stability of the test solution
And taking the afatinib maleate sample solution, injecting 50 mu L of the sample solution after 0h, 5.5h, 10.5h, 13.5h, 24h and 3 days respectively, recording a chromatogram, and calculating the relative standard deviation of the peak areas of the intermediate V and the afatinib, wherein the test result is shown in Table 7.
Table 7: test result of stability of test solution
As is clear from Table 7, the test solution had good stability when left at room temperature for 3 days.
Example 9: detection of durability test by the detection method of the present invention
And evaluating the bearing degree of the measurement result which is not influenced when the measurement condition is slightly changed by changing the pH value of the mobile phase A, the proportion of the 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 values on the determination of intermediate V
Table 10: effect of different flow phase ratios on intermediate V determination
Table 11: effect of different column temperatures on intermediate V determination
Table 12: effect of different flow rates on intermediate V determination
As can be seen from tables 8-12, the pH of mobile phase A was adjusted to 2.8-3.2 with phosphoric acid, and the initial mobile phase A to B volume ratio was 75:25, 77:23 or 73: 27; the column temperature is 25-35 ℃; the flow rate is 0.8-1.2 mL/min, the detected amount of the intermediate V is less than the detection limit, and the tailing factors of the intermediate V peak in the reference solution are all 0.8-1.5. The detection method of the present application, in which the pH of the 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 only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the invention.
Claims (8)
1. The detection method of afatinib dimaleate genotoxic impurities is characterized in that high performance liquid chromatography is adopted, 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength and the sample injection amount are set, and gradient elution is carried out through a C18 chromatographic column according to the volume ratio, wherein the elution procedure is as follows: starting elution by using 75% of a mobile phase A and 25% of a mobile phase B, gradually reducing the proportion of the mobile phase A, gradually increasing the proportion of the mobile phase B until the proportion of the mobile phase A reaches 35% in 25 minutes and the proportion of the mobile phase B reaches 65%, continuing elution for 10 minutes to 35 minutes, increasing the proportion of the mobile phase A, reducing the proportion of the mobile phase B, adjusting the proportion of the mobile phase A to 75% in 36 minutes, adjusting the proportion of the mobile phase B to 25%, and continuing elution for 9 minutes; the detection method can detect 8.0ppm of genetic toxic impurities of afatinib maleate.
2. The method for detecting afatinib dimaleate genotoxic impurities as claimed in claim 1, wherein the detection wavelength in the high performance liquid chromatography is 230nm, the sample injection amount is 50 μ L, and the C18 chromatographic column is selected from YMC Triart C18 column with specification of 4.6mm x 250mm,3 μm.
3. The method for detecting genetic toxic impurities of afatinib dimaleate according to claim 1, wherein the 0.01mol/L potassium dihydrogen phosphate solution is adjusted to pH 2.8-3.2 with phosphoric acid.
4. The method for detecting genetic toxic impurities of afatinib dimaleate of claim 3, wherein the 0.01mol/L potassium dihydrogen phosphate solution is adjusted to pH 3.0 with phosphoric acid.
5. The method for detecting afatinib dimaleate genotoxic impurities according to claim 1, wherein the flow rate of the high performance liquid chromatography is 0.8-1.2 mL/min.
6. The method for detecting Afatinib dimaleate genotoxic impurities according to claim 5, wherein the flow rate of the high performance liquid chromatography is 1.0 mL/min.
7. The method for detecting afatinib dimaleate genotoxic impurities according to claim 1, wherein the column temperature of the high performance liquid chromatography is 25-35 ℃.
8. The method for detecting afatinib dimaleate genotoxic impurities according to claim 7, wherein the column temperature of the high performance liquid chromatography is 30 ℃.
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| CN113917043B (en) | 2023-10-13 |
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