A method for analyzing drug impurities

Abstract

The invention discloses an analysis method for drug impurities, comprising the following steps: S1: preparing a sample solution for standby use; S2: adopting liquid chromatography, using a known impurity external standard method and an unknown impurity main component self-comparison method to calculate the impurity content, wherein mobile phase A is a salt solution, and mobile phase B is acetonitrile; the sample solution in step S1 comprises a first impurity reference substance stock solution, a second impurity reference substance stock solution, a test solution, a reference substance solution, a sensitivity stock solution and a sensitivity solution; the chromatographic column adopted in step S2 is Phenomenex Gemini C18, the detection wavelength is 332nm (VWD), the flow rate is 1.0ml / min, the column temperature is 35°C, the injection temperature is 4°C, and the injection volume is 10μl. The invention can effectively detect the content of relevant impurities in vometinib mesylate tablets, ensure that the produced products meet the quality standards, and ensure the safety of the products.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for analyzing drug impurities. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world, of which 70-80% are non-small cell lung cancer. Due to the disease characteristics of lung cancer itself, many lung cancer patients have metastasized when the disease is diagnosed, and have lost the opportunity for surgical cure, and their survival rate is also reduced. In recent years, with the development of molecular diagnostic technology and the research and development of targeted drugs, the treatment model of lung cancer is no longer limited to traditional chemotherapy. Among them, EGFR-TKI treatment has a significant effect in lung cancer patients with EGFR gene activating mutations.

[0003] Currently, EGFR-TKIs are the first-line standard treatment for patients with lung adenocarcinoma with EGFR mutations, including first-generation EGFR-TKIs such as gefitinib, erlotinib, icotinib and second-generation EGFR-TKIs such as afatinib and dacomitinib. However, all patients still inevitably develop drug resistance.

[0004] There is an urgent clinical need for related therapeutic drugs, and vometinib mesylate, as a third-generation EGFR-TKI small molecule targeted drug for patients with non-small cell lung cancer, has the characteristics of strong brain transformation, excellent efficacy, good safety, and a wide therapeutic window.

[0005] Vometinib mesylate is abbreviated as Vometinib; trade name: Aversa. It is a national Class 1 new drug independently developed by Shanghai Elis Pharmaceutical Technology Co., Ltd. It has now become a highly selective and irreversible third-generation epidermal factor receptor tyrosine kinase inhibitor EGFR-TKI. It is used to treat disease progression during or after previous treatment with epidermal factor receptor inhibitor TKI, and has been tested to confirm the presence of EGFR T790M mutation-positive locally advanced or metastatic non-small cell lung cancer NSCLC.

[0006] During the production process of vometinib mesylate, relevant impurities will be produced. In order to ensure that the products produced meet the quality standards and ensure the safety of the products, it is necessary to effectively detect the content of relevant substances in vometinib mesylate. Summary of the invention

[0007] To solve the above problems, the present invention provides a method for analyzing drug impurities, which adopts an impurity external standard method to calculate the content of known impurities and a principal component self-comparison method to calculate the content of unknown impurities, and can effectively detect the content of relevant impurities in vometinib mesylate tablets.

[0008] A method for analyzing drug impurities comprises the following steps:

[0009] S1: prepare sample solution for use;

[0010] S2: Using liquid chromatography, the impurity content is calculated using the known impurity external standard method and the unknown impurity main component self-comparison method, wherein mobile phase A is a salt solution and mobile phase B is acetonitrile;

[0011] The sample solution in step S1 includes a first impurity reference substance stock solution, a second impurity reference substance stock solution, a test solution, a reference substance solution, a sensitivity stock solution, and a sensitivity solution;

[0012] The chromatographic column used in step S2 is Phenomenex Gemini C18, the detection wavelength is 332 nm (VWD), the flow rate is 1.0 ml / min, the column temperature is 35° C., the injection temperature is 4° C., and the injection volume is 10 μl.

[0013] Preferably, the elution procedure of mobile phase A and mobile phase B is:

[0014] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 73 27 5 73 27 25 70 30 35 70 30 45 58 42 60 58 42 68 35 65 78 35 65 80 73 27 90 73 27

[0015] Preferably, the preparation process of the salt solution is as follows: 4.949 g of potassium dihydrogen phosphate and 1.465 g of tetrabutylammonium bromide are added to 1000 ml of water to dissolve, and then the pH value is adjusted to 3.85±0.05 with a phosphoric acid solution (1→20).

[0016] Preferably, the specifications of the chromatographic column Phenomenex Gemini C18 are 250×4.6 mm, 5 μm.

[0017] Preferably, the drug is afatinib mesylate, and the impurities include impurity A, impurity B, impurity C, and impurity D, and its structural formula is:

[0018]

[0019]

[0020] Preferably, the sample solution is prepared by:

[0021] First impurity reference substance stock solution: accurately weigh 27.1 mg of impurity A reference substance, 23.5 mg of impurity B reference substance, and 30.4 mg of impurity C reference substance, respectively, place them in a 100 mL brown volumetric flask, sonicate with a diluent, dilute to the scale, and shake well;

[0022] Second impurity reference substance stock solution: accurately weigh 28.0 mg of impurity D reference substance, place in a 20 mL brown volumetric flask, sonicate with diluent, dilute to scale, and shake well;

[0023] Test solution: Take 20 tablets of afatinib mesylate, weigh accurately, grind with an agate mortar, weigh an appropriate amount, put into a 50ml brown volumetric bottle, add 40ml of diluent, sonicate for 20 minutes, shake to disperse evenly, cool, dilute to the scale with diluent, shake well, filter with a 0.45μm filter membrane, and take the filtrate as the test solution;

[0024] Reference solution: Accurately pipette 1.0 mL of the first impurity reference stock solution, the second impurity reference stock solution and the test solution into a 100 mL brown volumetric flask, dilute to the mark with diluent, and shake well;

[0025] Sensitivity stock solution: Accurately pipette 2.0 mL of the first impurity reference stock solution, 1.0 mL of the second impurity reference stock solution, and 1.0 mL of the test solution into the same 100 mL brown volumetric flask, dilute to the scale with diluent, and shake well;

[0026] Sensitivity solution: Accurately pipette 1.0 mL of the sensitivity stock solution into a 20 mL brown volumetric flask, dilute to the mark with diluent, and shake well.

[0027] Preferably, the diluent is a mixture of hydrochloric acid solution and acetonitrile in a volume ratio of 25:75.

[0028] Preferably, the liquid chromatography step is:

[0029] S201: inject one or more blank solutions;

[0030] S202: inject 1 needle of sensitivity solution and record the chromatogram;

[0031] S203: Repeat the injection of 5 reference solutions and record the spectrum;

[0032] S204: Calculate the impurity content according to the formula;

[0033] The reference substance information table of impurity A, impurity B, impurity C and impurity D is as follows:

[0034]

[0035]

[0036] The calculation formula of impurity A content is:

[0037]

[0038] The calculation formula of impurity B content is:

[0039]

[0040] The calculation formula of impurity C content is:

[0041]

[0042] The calculation formula of impurity D content is:

[0043]

[0044] Among them, A R - System suitability 5 is the average value of the impurity peak areas in the reference solution;

[0045] A S is the peak area of ​​the impurity in the test solution;

[0046] W R When preparing the stock solution, weigh the impurity reference substance, mg;

[0047] f is the purity factor of each impurity reference substance when preparing the reference substance solution;

[0048] W S When preparing the test solution, the weight of the test sample is mg;

[0049] W is the average tablet weight of 20 test tablets, mg;

[0050] 40 is the labeled amount of the test sample in terms of afatinib, mg.

[0051] 0.9212 is the conversion factor of impurity A reference substance into bases;

[0052] 0.8527 is the conversion factor of impurity B reference substance converted to base;

[0053] 0.6579 is the conversion factor of impurity C reference substance converted to base;

[0054] 0.7139 is the conversion factor of impurity D reference substance in terms of bases;

[0055] Preferably, the blank solution is the diluent.

[0056] Preferably, the content of each unknown impurity and total impurities is calculated as follows:

[0057]

[0058] Total impurities = known impurities + unknown impurities

[0059] Where: As——peak area of ​​each impurity in the test solution;

[0060] Ar——Average peak area of ​​afatinib peak in control solution.

[0061] In summary, the beneficial effects of the present invention are:

[0062] (1) The present invention uses methodological research, adopts the impurity external standard method to calculate the content of known impurities, and the main component self-comparison method to calculate the content of unknown impurities, which can effectively detect the content of relevant impurities in vometinib mesylate tablets, thereby ensuring that the produced products meet the quality standards and ensure the safety of the products.

[0063] (2) The present invention establishes a method for detecting related substances of afatinib mesylate tablets according to the characteristics of afatinib mesylate tablets. The solution has strong stability, high precision, and good durability. All peaks can be effectively separated on the chromatogram. The blank solution, excipients, and impurities have no interference with the peaks of each component. The determination results are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is a chromatogram of a stock solution of a first impurity reference substance;

[0065] Figure 2 This is the chromatogram of the stock solution of the second impurity reference substance. DETAILED DESCRIPTION

[0066] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The following examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0067] The present invention discloses a method for testing drug-related substances, which can accurately and quantitatively analyze the percentage of impurities A, B, C, and D in the drug, respectively, in the drug. The principle is to separate the main component and each impurity by reversed-phase high performance liquid chromatography, and determine the impurity content at the maximum absorption wavelength of each impurity at 332nm, using a UV detector. Through methodological research, the impurity external standard method is used to calculate the known impurity content, and the main component self-control method is used to calculate the unknown impurity content.

[0068] First, prepare the drugs and reagents: acetonitrile, potassium dihydrogen phosphate, tetrabutylammonium bromide, phosphoric acid, concentrated hydrochloric acid and afatinib mesylate.

[0069] Prepare instruments and equipment: analytical balance (one hundred thousandth), octadecylsilane bonded silica gel column (Phenomenex Gemini C18 250×4.6 mm, 5 μm), high performance liquid chromatograph, acidity meter, brown volumetric flask, pipette, ultrasonic instrument, centrifuge.

[0070] The specific method includes the following steps:

[0071] S1: prepare sample solution for use;

[0072] S2: Using liquid chromatography, the impurity content is calculated using the known impurity external standard method and the unknown impurity main component self-comparison method, wherein the mobile phase A is a salt solution and the mobile phase B is acetonitrile;

[0073] Specifically, the instrument is an Agilent 1200 high performance liquid chromatograph equipped with an ultraviolet detector.

[0074] The chromatographic conditions are:

[0075] Chromatographic column: Octadecylsilane bonded silica gel as filler (Phenomenex Gemini C18250×4.6mm, 5μm);

[0076] Detection wavelength: 332nm (VWD);

[0077] Flow rate: 1.0ml / min;

[0078] Column temperature: 35 °C;

[0079] Injection temperature: 4°C;

[0080] Injection volume: 10 μl;

[0081] Mobile phase A: saline solution (4.949 g potassium dihydrogen phosphate and 1.465 g tetrabutylammonium bromide were dissolved in 1000 ml of water, and the pH value was adjusted to 3.85 ± 0.05 with phosphoric acid solution (1 → 20));

[0082] Mobile phase B: acetonitrile. The acetonitrile in this example should be of Merck brand to avoid interference from blank solution.

[0083] Perform linear gradient elution as shown in the table below.

[0084] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 73 27 5 73 27 25 70 30 35 70 30 45 58 42 60 58 42 68 35 65 78 35 65 80 73 27 90 73 27

[0085] To prepare the sample solution, first prepare the diluent: hydrochloric acid solution-acetonitrile (V:V, 25:75). The diluent can also be used as a blank solution. The preparation process of the hydrochloric acid solution is to accurately pipette 0.7 mL of concentrated hydrochloric acid, dilute it to 1000 mL with water, and shake well.

[0086] First impurity reference substance stock solution: accurately weigh 27.1 mg of impurity A reference substance (equivalent to 25 mg of free base), 23.5 mg of impurity B reference substance (equivalent to 20 mg of free base), and 30.4 mg of impurity C reference substance (equivalent to 20 mg of free base), respectively, place them in a 100 mL brown volumetric flask, sonicate with a diluent, dilute to the scale, and shake well;

[0087] Second impurity reference substance stock solution: accurately weigh 28.0 mg of impurity D reference substance (equivalent to 20 mg of free base), place in a 20 mL brown volumetric flask, sonicate with diluent, dilute to scale, and shake well;

[0088] Test solution: Take 20 tablets of afatinib mesylate, accurately weigh, grind with an agate mortar, accurately weigh an appropriate amount (equivalent to 50 mg of afatinib), put into a 50 ml brown volumetric flask, add 40 ml of diluent, ultrasonicate for 20 minutes, shake during the period to make it evenly dispersed, cool, dilute to the scale with diluent, shake well, filter with a 0.45 μm filter membrane, and take the filtrate as the test solution;

[0089] Reference solution: Accurately pipette 1.0 mL of the first impurity reference stock solution, the second impurity reference stock solution and the test solution into a 100 mL brown volumetric flask, dilute to the mark with diluent, and shake well;

[0090] Sensitivity stock solution: Accurately pipette 2.0 mL of the first impurity reference stock solution, 1.0 mL of the second impurity reference stock solution, and 1.0 mL of the test solution into the same 100 mL brown volumetric flask, dilute to the scale with diluent, and shake well;

[0091] Sensitivity solution: Accurately pipette 1.0 mL of the sensitivity stock solution into a 20 mL brown volumetric flask, dilute to the mark with diluent, and shake well.

[0092] The steps of liquid chromatography are:

[0093] S201: Inject one or more blank solutions until the system is stable and free of interference;

[0094] S202: inject 1 needle of the sensitivity solution and record the chromatogram. The signal-to-noise ratio of impurity A, impurity B, impurity C, impurity D and afatinib should not be less than 10;

[0095] S203: Repeat the injection of 5 samples of the reference solution and record the spectrum. The separation degree of the first sample of the reference solution for impurities B and impurity C, impurity C and afatinib, afatinib and impurity A, and impurity A and impurity D is required to be ≥ 1.5, the RSD of the peak areas of impurities A, impurity B, impurity C, and impurity D shall not be greater than 5%, the RSD of the peak area of ​​afatinib shall not be greater than 5%, and the number of theoretical plates shall be not less than 10,000 based on afatinib;

[0096] S204: Calculate the impurity content according to the formula;

[0097] The impurity A content is calculated as follows:

[0098]

[0099] The impurity B content is calculated as follows:

[0100]

[0101] The impurity C content is calculated as follows:

[0102]

[0103] The impurity D content is calculated as follows:

[0104]

[0105] Where: A R - System suitability 5 is the average value of the impurity peak areas in the reference solution;

[0106] A S ——Peak area of ​​impurities in the test solution;

[0107] W R ——Weighing of impurity reference substance when preparing stock solution, mg;

[0108] f——purity factor of each impurity reference substance when preparing reference substance solution;

[0109] W S ——When preparing the test solution, the weight of the test sample, mg;

[0110] ——Average tablet weight of 20 test tablets, mg;

[0111] 40——labeled amount of the test article in terms of afatinib, mg.

[0112] 0.9212——Conversion coefficient of impurity A reference substance converted to base;

[0113] 0.8527——Conversion coefficient of impurity B reference substance converted to base;

[0114] 0.6579——Conversion coefficient of impurity C reference substance converted to base;

[0115] 0.7139——Conversion coefficient of impurity D reference substance converted to base;

[0116] The content of each unknown impurity and total impurities is calculated according to the following formula:

[0117]

[0118] Total impurities = known impurities + unknown impurities

[0119] Where: As——peak area of ​​each impurity in the test solution;

[0120] Ar——average peak area of ​​afatinib peak in control solution;

[0121] It should be noted that the total impurities are the average value of the sum of all impurities not less than 0.05% in the two test solutions.

[0122] Reporting limit (RL): 0.05%. No integration below 0.025%. Integration ≥ 0.025% to < 0.05% but not included in the calculation.

[0123] Specifically, if there are impurity peaks in the chromatogram of the test solution, impurity A shall not exceed 0.25% of the labeled amount as free base, impurity B shall not exceed 0.20% of the labeled amount as free base, impurity C shall not exceed 0.20% of the labeled amount as free base, and impurity D shall not exceed 1.0% of the labeled amount as free base. The peak area of ​​other individual impurities shall not be greater than 0.2 times (0.2%) the area of ​​the main peak of afatinib in the reference solution, and the total impurities shall not be greater than 2.0%.

[0124] The information table of impurity A, impurity B, impurity C, impurity D and reference substance of afatinib mesylate is as follows:

[0125]

[0126] On the other hand, according to the above-mentioned analysis method for impurities in afatinib mesylate tablets, the above-mentioned method was verified, and the verification contents included: specificity, linearity and range, detection limit, quantification limit, precision (injection precision, repeatability and intermediate precision), accuracy, solution stability and durability. Specifically:

[0127] 1. System adaptability

[0128] 1.1. Acceptance criteria: The blank solution has no significant interference with the test results.

[0129] Verification results: no significant interference.

[0130] 1.2. Acceptance criteria: The signal-to-noise ratio (S / N) of impurity A, impurity B, impurity C, impurity D and afatinib peaks in the sensitivity solution is ≥ 10.

[0131] Verification results:

[0132]

[0133] 1.3. Acceptance criteria: Repeat the injection of 5 samples of the reference solution, and examine the separation of impurity B and impurity C, impurity C and afatinib, afatinib and impurity A, impurity A and impurity D in the first sample solution ≥ 1.5 and the theoretical plate number of the afatinib peak ≥ 10000; and calculate the relative standard deviation (RSD) of the peak area (Area) of impurity A, impurity B, impurity C, impurity D and afatinib in the five samples of the reference solution ≤ 5%.

[0134] Verification results:

[0135] Component name Impurity B Impurity C Afatinib Impurity A Impurity D Separation / 12.0 3.7 38.1 2.6 RSD(n=5) 0.9％ 0.6％ 0.2％ 0.6％ 0.3％

[0136] 1.4. Acceptance criteria: After each validation project, inject one injection of the reference solution, and calculate the RSD values ​​of the peak areas of impurities A, B, C, D and afatinib for all injections of the reference solution and 5 injections of the system suitability reference solution. The RSD of each impurity is ≤5%, and the RSD of afatinib is ≤5%.

[0137] Verification results:

[0138] Component name Impurity B Impurity C Afatinib Impurity A Impurity D RSD (n=6) 0.8％ 0.6％ 0.2％ 0.5％ 0.5％

[0139] The test results of the above verification items show that each component can meet the set acceptance standards during the verification process of the system suitability test. The first step in all verification experiments, the system suitability test, has been passed.

[0140] 2. Exclusivity

[0141] 2.1. Acceptance criteria: All peaks can be effectively separated on the chromatogram; the peak purity factor of the DAD spectrum of each impurity stock solution and the DAD spectrum of the main peak of the test solution should be >990; the blank, excipients and impurities have no interference with the peaks of each component.

[0142] Verification results:

[0143]

[0144] 2.2 Acceptance criteria: Strong degradation: All peaks can be effectively separated on the chromatogram; the main peak purity factor should be >990; blank and impurities have no interference with the peaks of each component; the material balance of each test should be between 90% and 110%.

[0145] Verification results: Forced degradation test: After being destroyed by acid, alkali, oxidation, high temperature (solid and solution), high humidity and light (solid), the main component peak and the impurity peaks can be well separated; the peak purity of the main component peak and the impurity peaks meet the requirements, and the material is basically conserved after destruction.

[0146] 3. Solution stability

[0147] 3.1. Acceptance criteria: The impurity recovery rate of the impurity reference stock solution and the reference solution should range from 90% to 110%.

[0148] Verification results: After the impurity reference stock solution 1 was refrigerated (4°C) for 54 hours, the recoveries of impurities A, B, and C were all within the range of 90% to 110%, and the solution remained stable.

[0149] After the impurity reference substance stock solution 2 was refrigerated (4° C.) for 55.5 hours, the recovery rate of impurity D was in the range of 90% to 110%, and the solution remained stable.

[0150] 3.2. Acceptance criteria: The recovery rates of impurities A, B, C, and D in the reference solution are 90% to 110%.

[0151] Verification results: After the reference solution was refrigerated (4°C) for 58.5 hours, the recoveries of impurities A, B, C, D and the main peak of afatinib mesylate were all within the range of 90% to 110%, and the solution remained stable.

[0152] 3.3 Acceptance criteria:

[0153] Impurity levels Relative change 0.05％～0.2％ ≤20％ 0.3％～0.5％ ≤10％ 0.6％～5％ ≤7.5％

[0154] Verification results: After the test solution was left at room temperature for 60 hours, more than 0.1% of new unknown single impurity was produced, the known impurity C of the test solution was detected at 0 hour, but not detected after 60 hours at room temperature, and the relative changes in the contents of other known impurities were within the acceptable range; after the spiked test solution was left at room temperature for 61.5 hours, more than 0.1% of new unknown single impurity was produced, and the relative changes in the contents of known impurities A, B, C, and D were within the acceptable range.

[0155] After the test solution was refrigerated (4°C) for 48 hours and the spiked test solution was refrigerated (4°C) for 51 hours, no new unknown single impurity was generated, and the relative changes in the contents of known impurities A, B, C, and D were all within the acceptable range. Therefore, the test solution remained stable when placed under refrigeration (4°C) for 48 hours and the spiked test solution was placed for 51 hours.

[0156] IV. LOQ

[0157] 4.1. Acceptance criteria: RSD of peak area ≤ 10%, signal-to-noise ratio S / N ≥ 10, concentration (μg / mL) according to reporting requirements, relative concentration of the test article (%) according to reporting requirements.

[0158] Verification results:

[0159]

[0160]

[0161] 5. LOD

[0162] 5.1. Acceptance criteria: signal-to-noise ratio S / N ≥ 3

[0163] Verification results:

[0164]

[0165] 6. Linear

[0166] 6.1. Acceptance criteria: Linear range (LOQ+4 points) LOQ~200%, correlation coefficient r≥0.99, ratio of intercept b to Y at 100% X≤25%.

[0167] Verification results:

[0168]

[0169] According to the Technical Guidelines for the Study of Impurities in Chemical Drugs, under normal circumstances, when the relative response factor is within the range of 0.9 to 1.1, no correction is required and the self-control method without correction factor can be used directly for quantification; when it exceeds the range of 0.9 to 1.1, if the main component self-control method is used for quantification, it must be corrected with a correction factor, that is, the "main component self-control method with correction factor" to ensure the accuracy of impurity quantification; if the relative response factor is outside the range of 0.2 to 5.0, it is necessary to consider using appropriate impurity methods such as the impurity reference substance external standard method for quantification.

[0170] This product is a Class 1.1 innovative drug, and the related substance method is developed independently. There is no official reference for the correction factor (or response factor). The determination of the correction factor (or response factor) is easily affected by chromatographic conditions and instrument operation, and the error is large. This product has obtained an impurity reference substance and has been calibrated in a standardized manner. The external standard method is more accurate in calculating the impurity content, so the external standard method is preferred.

[0171] The test results show that the excipient solution does not interfere with the linearity test of each component. The linearity verification tests of impurities A, B, C, D and the main component afatinib all meet the set acceptance standards, and the linearity verification is passed.

[0172] 7. Precision

[0173] 7.1、Repeatability acceptance criteria:

[0174] Repeatability was verified by preparing 6 spiked sample solutions and testing them, with one injection of each solution. The RSD of each impurity peak above the reporting limit of the 6 determination results was within the required range to confirm good repeatability.

[0175] Impurity levels Relative change 0.05％～0.2％ ≤20％ 0.3％～0.5％ ≤10％ 0.6％～5％ ≤7.5％

[0176] Verification results:

[0177] Component name Impurity B Impurity C Impurity A Impurity D Unknown impurities RSD (n=6) 1.9％ 4.8％ 1.5％ 2.4％ / According to the impurity level ≤20％ ≤10％ ≤10％ ≤7.5％ /

[0178] 7.2. Intermediate precision acceptance criteria:

[0179] Intermediate precision is achieved when two analysts independently set up the system on different instruments at different times to examine the effect of random variation factors on precision. At least 6 injections and 12 injections in total are made with repeatability results. Good intermediate precision is confirmed when the RSD of each impurity peak with a content above the reporting limit is within the required range.

[0180] Impurity levels Relative change 0.05％～0.2％ ≤20％ 0.3％～0.5％ ≤10％ 0.6％～5％ ≤7.5％

[0181] 8. Accuracy / Recovery

[0182] Acceptance criteria:

[0183] Content level Recovery rate <0.05％ 60％～140％ 0.05％～0.2％ 70％～130％ 0.3％～0.5％ 80％～120％ >0.6％ 90％～110％

[0184] Content level verification results:

[0185]

[0186] Recovery verification results:

[0187]

[0188]

[0189] IX. Scope

[0190] Acceptance criteria: LOQ ~ 150%

[0191] Verification results:

[0192] Impurity A is in the concentration range of 0.25 μg / ml to 3.7 μg / ml;

[0193] Impurity B is in the concentration range of 0.20 μg / ml to 3.0 μg / ml;

[0194] Impurity C is in the concentration range of 0.20 μg / ml to 3.0 μg / ml;

[0195] The concentration of impurity D is in the range of 0.50 μg / ml to 14.8 μg / ml.

[0196] 10. Durability

[0197] 10.1. Acceptance criteria for membrane adsorption:

[0198] Adsorption to the filter membrane was evaluated using spiked test solution.

[0199] Prepare 1 portion of spiked test solution, filter it through nylon and polytetrafluoroethylene (PTFE) membrane filters (0.45 μm, discard 1 ml of the initial filtrate) and compare it with centrifugation (12,000 rpm, 5 minutes), calculate the relative change in the peak area of ​​individual impurities and afatinib, and examine the adsorption of different filter membranes on each component.

[0200]

[0201]

[0202] Verification results: The filtration method using nylon and polytetrafluoroethylene (PTFE) filter membranes had no significant adsorption on impurities and main components, and both were within the acceptable error range, indicating that the influence of the filter membrane material on the determination results of each component was negligible.

[0203] 10.2. Extraction robustness acceptance criteria:

[0204] The content of afatinib was calculated by using different ultrasonic times and the method of content determination, the extraction efficiency of the main components was investigated, and the appropriate ultrasonic time for complete extraction was selected.

[0205] Verification results: The test sample was completely extracted after 10 to 15 minutes of ultrasonic treatment. Considering the selection of a more appropriate ultrasonic time, 20 minutes was finally confirmed as the extraction operation for tablets. This method can ensure that all components are completely extracted and the measurement results are accurate and reliable.

[0206] 10.3. The influence of the change of the measurement condition parameters on the measurement results, acceptance criteria:

[0207] The pH value of the buffer solution was adjusted to 3.85±0.2; the column temperature was 35±2℃; the flow rate was 1.0±0.1mL / min; the detection wavelength was 332±2nm; different batches of chromatographic columns were replaced, and system suitability tests and test solution inspections were carried out.

[0208] Verification results: The five factors of column temperature, flow rate, detection wavelength, aqueous pH value and chromatographic column have no effect on the measurement results, and the method has good durability.

[0209] Through the verification of the above-mentioned method based on the relevant impurity inspection method of the afatinib mesylate raw material, it can be seen that the impurity analysis method provided by the present invention has strong solution stability, high precision, good durability, all peaks can be effectively separated on the chromatogram, the blank solution, excipients and impurities have no interference with the peaks of each component, the measurement results are accurate and reliable, and the content of relevant impurities in the vometinib mesylate tablets can be effectively detected, thereby ensuring that the produced products meet the quality standards and ensuring the safety of the products.

[0210] The analytical method for related substances 1 of Afatinib Mesylate Tablets of this product was verified in accordance with the relevant requirements of the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs" and the "General Rules of the Four Parts of the 2015 Edition of the Chinese Pharmacopoeia" and combined with the relevant requirements of the FDA on method validation. All items were validated and passed all the set validation requirements. The test results show that the tablet impurity (degradation product) analytical method has undergone a comprehensive validation test, proving that it is a stable, reliable, highly accurate, and repeatable impurity detection method. Since this analytical method has also undergone the test of destructive experiments (Forced Degradation), it has also been proven to be capable of detecting a series of potential degradation products of tablets (and API). Therefore, this analytical method has the characteristics of stability indication.

[0211] The above description shows and describes the preferred embodiments of the present invention. As mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for analyzing drug impurities, characterized in that: The following steps are involved: S1: prepare sample solution for use; S2: Using liquid chromatography, the impurity content is calculated using the known impurity external standard method and the unknown impurity main component self-comparison method, wherein mobile phase A is a salt solution and mobile phase B is acetonitrile; The sample solution in step S1 includes a first impurity reference substance stock solution, a second impurity reference substance stock solution, a test solution, a reference substance solution, a sensitivity stock solution, and a sensitivity solution; The chromatographic column used in step S2 is Phenomenex Gemini C18, the detection wavelength is 332 nm (VWD), the flow rate is 1.0 ml / min, the column temperature is 35° C., the injection temperature is 4° C., and the injection volume is 10 μl.

2. The method for analyzing drug impurities according to claim 1, characterized in that: The elution procedures of the mobile phase A and the mobile phase B are:

3. The method for analyzing drug impurities according to claim 1, characterized in that: The preparation process of the salt solution is as follows: 4.949 g of potassium dihydrogen phosphate and 1.465 g of tetrabutylammonium bromide are added with 1000 ml of water to dissolve them, and then the pH value is adjusted to 3.85±0.05 with a phosphoric acid solution (1→20).

4. The method for analyzing drug impurities according to claim 1, characterized in that: The specifications of the chromatographic column Phenomenex Gemini C18 are 250×4.6 mm, 5 μm.

5. The method for analyzing drug impurities according to claim 1, characterized in that: The drug is afatinib mesylate, and the impurities include impurity A, impurity B, impurity C, and impurity D, and its structural formula is:

6. The method for analyzing drug impurities according to claim 1, characterized in that: The sample solution is prepared as follows: First impurity reference substance stock solution: accurately weigh 27.1 mg of impurity A reference substance, 23.5 mg of impurity B reference substance, and 30.4 mg of impurity C reference substance, respectively, place them in a 100 mL brown volumetric flask, sonicate with a diluent, dilute to the scale, and shake well; Second impurity reference substance stock solution: accurately weigh 28.0 mg of impurity D reference substance, place in a 20 mL brown volumetric flask, sonicate with diluent, dilute to scale, and shake well; Test solution: Take 20 tablets of afatinib mesylate, weigh accurately, grind with an agate mortar, weigh an appropriate amount, put into a 50ml brown volumetric bottle, add 40ml of diluent, sonicate for 20 minutes, shake to disperse evenly, cool, dilute to the scale with diluent, shake well, filter with a 0.45μm filter membrane, and take the filtrate as the test solution; Reference solution: Accurately pipette 1.0 mL of the first impurity reference stock solution, the second impurity reference stock solution and the test solution into a 100 mL brown volumetric flask, dilute to the mark with diluent, and shake well; Sensitivity stock solution: Accurately pipette 2.0 mL of the first impurity reference stock solution, 1.0 mL of the second impurity reference stock solution, and 1.0 mL of the test solution into the same 100 mL brown volumetric flask, dilute to the scale with diluent, and shake well; Sensitivity solution: Accurately pipette 1.0 mL of the sensitivity stock solution into a 20 mL brown volumetric flask, dilute to the mark with diluent, and shake well.

7. The method for analyzing drug impurities according to claim 6, characterized in that: The diluent is a mixture of hydrochloric acid solution and acetonitrile, and the volume ratio thereof is 25:

75.

8. The method for analyzing drug impurities according to claim 6, characterized in that: The liquid chromatography steps are: S201: inject one or more blank solutions; S202: inject 1 needle of the sensitivity solution and record the chromatogram; S203: Repeat the injection of 5 reference solutions and record the spectrum; S204: Calculate the impurity content according to the formula; The reference substance information table of impurity A, impurity B, impurity C and impurity D is as follows: The calculation formula of impurity A content is: The calculation formula of impurity B content is: The calculation formula of impurity C content is: The calculation formula of impurity D content is: Among them, A R - System suitability 5 is the average value of the impurity peak areas in the reference solution; A S is the peak area of ​​the impurity in the test solution; W R When preparing the stock solution, weigh the impurity reference substance, mg; f is the purity factor of each impurity reference substance when preparing the reference substance solution; W S When preparing the test solution, the weight of the test sample is mg; W is the average tablet weight of 20 test tablets, mg; 40 is the labeled amount of the test sample in terms of afatinib, mg. 0.9212 is the conversion factor of impurity A reference substance into bases; 0.8527 is the conversion factor of impurity B reference substance converted to base; 0.6579 is the conversion factor of impurity C reference substance converted to base; 0.7139 is the conversion factor for the impurity D reference substance in terms of bases.

9. The method for analyzing drug impurities according to claim 8, characterized in that: The blank solution is a diluent.

10. The method for analyzing drug impurities according to claim 9, characterized in that: The content of each unknown impurity and total impurities is calculated according to the following formula: Total impurities = known impurities + unknown impurities Where: As——peak area of ​​each impurity in the test solution; Ar——Average peak area of ​​afatinib peak in control solution.

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