Method for detecting related impurities of imatinib mesylate intermediate

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for detecting related impurities of an imatinib mesylate intermediate. In particular to a method for detecting related substances in N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyrimidine-2-amine. The related substances in N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyrimidine-2-amine are detected by high performance liquid chromatography, and the content of each impurity in N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyrimidine-2-amine is calculated by a main component external standard method with correction factors. The methodology verification shows that the detection method provided by the invention is good in specificity and high in accuracy and sensitivity, and can be used for quality control of related substances in N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyrimidine-2-amine, so that the product quality is effectively controlled, and the medication safety of imatinib mesylate is improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, and more specifically to a method for detecting impurities in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine. Background Technology

[0002] Imatinib mesylate, chemically known as 4-[(4-methyl-1-piperazine)methyl]-N-[4-methyl-3-[[4-(3-pyridine)-2-pyrimidine]amino]phenyl]-benzamide mesylate, is a small molecule protein tyrosine kinase inhibitor used to treat Philadelphia chromosome-positive chronic myeloid leukemia (Ph+CML) in its chronic, accelerated, or blast crisis phases; to treat adult patients with unresectable and / or metastatic malignant gastrointestinal stromal tumors (GIST); and in combination with chemotherapy for the treatment of newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) in children. It is also used to treat adult patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL).

[0003] One of the synthetic routes for imatinib mesylate uses N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine as the starting material. Therefore, detecting impurities in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine is crucial for controlling the quality of imatinib mesylate and ensuring its safety. Currently, there are many studies on the determination of related substances in imatinib mesylate, but studies on the quality control of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine are limited. Analysis of the impurity structures reveals that most are isomers with similar polarities, while some impurities are highly polar, making method development challenging. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting impurities in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine. The detection method provided by this invention has high specificity, high sensitivity and high accuracy.

[0005] To achieve the above objectives, this invention employs high-performance liquid chromatography (HPLC), and the detection conditions include at least the following:

[0006] Chromatographic column: Octadecylsilane-bonded silica column;

[0007] Column temperature: 25~35℃;

[0008] Mobile phase: includes mobile phase A and mobile phase B;

[0009] The mobile phase A is a mixed solution of disodium hydrogen phosphate and acetonitrile with a volume ratio of 88-92:12-8; the concentration of the disodium hydrogen phosphate solution is 0.005-0.015 mol / L, and the pH value is 5.0-6.0; the mobile phase B is acetonitrile.

[0010] Flow rate: 0.9–1.3 mL / min;

[0011] Detection wavelength: 215–225 nm;

[0012] Elution method: gradient elution.

[0013] Furthermore, the gradient elution procedure is as follows:

[0014] 0 min, the volume fraction of the mobile phase A is maintained at 96% to 100%;

[0015] Within 0–10 min, the volume fraction of the mobile phase A decreased to 78%–82%;

[0016] The volume fraction of the mobile phase A is maintained at 78%–82% for 10–25 min.

[0017] After 25–45 minutes, the volume fraction of the mobile phase A decreased to 53%–57%.

[0018] For 45–55 minutes, the volume fraction of the mobile phase A is maintained at 53%–57%.

[0019] After 55–56 minutes, the volume of the mobile phase A increases to 96%–100%.

[0020] The volume of the mobile phase A is maintained at 96%–100% for 56–70 minutes.

[0021] Preferably, the injection volume of the method is 17–19 μL.

[0022] Furthermore, the method for detecting impurities related to imatinib mesylate intermediates includes the following steps:

[0023] I. Dissolve the imatinib mesylate intermediate sample in a methanol-acetonitrile mixed solvent to obtain the test solution;

[0024] II. Dissolve the imatinib mesylate intermediate reference standard in a methanol-acetonitrile mixed solvent to obtain the reference standard solution;

[0025] III. The test solution and the reference solution were subjected to high performance liquid chromatography under the same detection conditions to obtain the peak areas of impurities in the test sample and the peak areas of the reference solution, respectively.

[0026] IV. The mass percentage content of the impurity is calculated based on the peak area of ​​the impurity in the test sample, the peak area of ​​the reference sample, and the correction factor.

[0027] Preferably, in step I, the volume fraction of acetonitrile in the methanol-acetonitrile mixed solvent is 45-55%; the concentration of the imatinib mesylate intermediate sample in the test solution is 0.9-1.1 mg / mL; and the concentration of the imatinib mesylate intermediate reference standard in the reference solution is 3-7 μg / mL.

[0028] Furthermore, the intermediate of imatinib mesylate is N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine.

[0029] Furthermore, the impurities include 2-methyl-5-nitrophenylguanidine, 3-acetylpyridine, (E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one, 1-(2-methyl-5-nitrophenyl)-3-(4-(pyridin-3-yl)pyrimidin-2-yl)guanidine, N-(2-methyl-3-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine and N-(2-methyl-4-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine.

[0030] Furthermore, the formula for the calculation is:

[0031]

[0032] Total impurities % = % of known impurities + % of other impurities;

[0033] In the formula: W 对 W 供 : Weigh the reference standard and the test sample respectively, in grams;

[0034] P 对 %: Content of reference standard;

[0035] A 已知杂质 : Peak area of ​​each known impurity in the test solution;

[0036] A 其他最大杂质、A其他杂质总和 These are the areas of the largest single impurity peak and the sum of the areas of all other impurity peaks in the test solution, respectively.

[0037] A 对 : The area of ​​the main peak in the reference solution;

[0038] n 对 n 供 : These are the dilution factors for the reference standard and the test sample, respectively.

[0039] Furthermore, in step IV, the method for obtaining the correction factor is as follows:

[0040] (1) Establish the standard curve regression equations of concentration-peak area for N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities, respectively;

[0041] (2) Based on the regression equation of the concentration-peak area standard curve of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities, the slope of the principal component standard curve and the slope of the impurity standard curve are obtained respectively.

[0042] (3) The correction factor is obtained based on the slope of the principal component standard curve and the slope of the impurity standard curve.

[0043] Furthermore, the formula for calculating the correction factor is as follows:

[0044]

[0045] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0046] The detection method provided by this invention can achieve good separation between all impurities in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine (IM-3), with a resolution greater than 1.5. This indicates that the detection method provided by this invention has high system applicability and can effectively separate all impurities.

[0047] The detection methods provided by this invention have quantitation limits of less than 0.05% of the sample concentration and detection limits of less than 0.02% of the sample concentration, indicating that the detection methods provided by this invention have high sensitivity.

[0048] The detection method provided by this invention shows that IM-3 and various impurities exhibit a linear relationship within a certain concentration range, with a correlation coefficient R > 0.999, indicating a good linear relationship.

[0049] The detection method provided by this invention has good specificity, accuracy and sensitivity, and can be used for the quality control of related substances in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine, thereby effectively controlling product quality and improving the safety of imatinib mesylate. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0051] Figure 1 The HPLC chromatogram of the blank solvent in Example 1;

[0052] Figure 2 This is the HPLC chromatogram of the system suitability solution in Example 1;

[0053] Figure 3 The HPLC chromatogram of the test solution in Example 1;

[0054] Figure 4 The following are HPLC chromatograms of the blank solvent, system suitability solution, test solution, and various impurity positioning solutions in Example 1 (a. System suitability solution b. IM-B positioning c. IM-1 positioning d. IM-2 positioning e. IM-3-1 positioning f. IM-3-2 positioning g. IM-3-3 positioning h. Test solution i. Blank solution);

[0055] Figure 5 This is a linear relationship graph of concentration and peak area for 2-methyl-5-nitrophenylguanidine in Example 1;

[0056] Figure 6 This is a linear relationship graph of concentration-peak area for 3-acetylpyridine in Example 1;

[0057] Figure 7 The graph shows the linear relationship between concentration and peak area for (E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one in Example 1.

[0058] Figure 8 The graph shows the linear relationship between concentration and peak area for 1-(2-methyl-5-nitrophenyl)-3-(4-(pyridin-3-yl)pyrimidin-2-yl)guanidine in Example 1.

[0059] Figure 9 The graph shows the linear relationship between the concentration and peak area of ​​N-(2-methyl-3-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine in Example 1.

[0060] Figure 10 The graph shows the linear relationship between the concentration and peak area of ​​N-(2-methyl-4-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine in Example 1.

[0061] Figure 11 The graph shows the linear relationship between the concentration and peak area of ​​N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine in Example 1. Detailed Implementation

[0062] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] In this invention, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile mixed solvent is 47-53:53-47, and in a specific embodiment, it can be 50:50; the N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine sample in this invention has good solubility in the above solvent.

[0064] In this invention, the concentration of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine sample in the test solution is 0.9 to 1.1 mg / mL, and in a specific embodiment, it can be 1.0 mg / mL.

[0065] The concentration of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine reference standard in the reference solution is 3-7 μg / mL, and in specific examples, it can be 5 μg / mL.

[0066] After obtaining the test solution and the reference solution, the present invention performs high performance liquid chromatography on the test solution and the reference solution under the same detection conditions to obtain the peak area of ​​the impurity in the test solution and the peak area of ​​the reference solution, respectively.

[0067] In this invention, the injection volume of the test solution and the reference solution in the high performance liquid chromatography detection is independently 13-17 μL, and in a specific embodiment, it can be 15 μL.

[0068] In this invention, the high-performance liquid chromatograph used for detection is a Shimadzu model; the detection conditions for high-performance liquid chromatography include: the chromatographic column is an octadecylsilane-bonded silica column, and in a specific embodiment, it can be a Waters Atlantis T34.6×250mm, 5μm.

[0069] The column temperature of the chromatographic column is 25-35°C, and in specific embodiments, it can be 28°C, 30°C or 32°C.

[0070] The mobile phase system includes mobile phase A and mobile phase B.

[0071] The mobile phase A is a mixed solution of disodium hydrogen phosphate and acetonitrile, with a volume ratio of 88-92:12-8; in a specific embodiment, it can be 90:10; the concentration of the disodium hydrogen phosphate solution is 0.005-0.015 mol / L, in a specific embodiment, it can be 0.008 mol / L, 0.010 mol / L, or 0.012 mol / L; the pH value is 5.0-6.0, in a specific embodiment, it can be 5.3, 5.5, or 5.7.

[0072] The mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.9 to 1.3 mL / min, and in specific embodiments, it can be 1.0 mL / min, 1.1 mL / min or 1.2 mL / min.

[0073] The detection wavelength is 215–225 nm, and in specific embodiments, it can be 218 nm, 220 nm, or 222 nm.

[0074] In this invention, the elution method for high-performance liquid chromatography detection is gradient elution; the gradient elution procedure is as follows:

[0075] At 0 min, the volume fraction of the mobile phase A is 96% to 100%, and in a specific embodiment, it can be 98%, 99%, or 100%.

[0076] Within 0 to 10 minutes, the volume fraction of the mobile phase A decreases to 78% to 82%, and in a specific embodiment, it can be decreased to 80% at a uniform rate.

[0077] For 10 to 25 minutes, the volume fraction of the mobile phase A is maintained at 78% to 82%, and in specific embodiments, it can be 79%, 80%, or 81%.

[0078] In 25–45 minutes, the volume fraction of the mobile phase A decreases to 53%–58%, and in a specific embodiment, it can be decreased to 55% at a uniform rate.

[0079] For 45 to 55 minutes, the volume fraction of the mobile phase A is maintained at 53% to 58%, which in specific embodiments can be 54%, 55% or 56%.

[0080] After 55-56 minutes, the volume fraction of the mobile phase A increases to 96%-100%, and in a specific embodiment, it can be increased to 100%.

[0081] For 56 to 70 minutes, the volume fraction of the mobile phase A is maintained at 96% to 100%. In specific embodiments, it can be 98%, 99%, or 100%.

[0082] In this invention, the structures of the N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities are shown in Table 1.

[0083] Table 1. Structures of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine (IM-3) and its impurities

[0084]

[0085]

[0086] The N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine test solution and the N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine reference solution were detected by high performance liquid chromatography (HPLC). After obtaining the HPLC chromatograms of the reference solution and the test solution, the peak areas were calculated, and the mass percentage of the impurity was calculated using the principal component external standard method with correction factor.

[0087] In this invention, the method for obtaining the correction factor is as follows:

[0088] Regression equations for concentration-peak area standard curves of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities were established respectively.

[0089] Based on the regression equation of the concentration-peak area standard curves of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities, the slopes of the principal component standard curve and the impurity standard curves were obtained respectively.

[0090] The correction factor is obtained based on the slope of the principal component standard curve and the slope of the impurity standard curve.

[0091] In this invention, the step of establishing the standard curve regression equation for the concentration-peak area of ​​N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine includes: preparing a series of reference solutions of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine; performing high-performance liquid chromatography (HPLC) on the series of reference solutions of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine to obtain the chromatogram of the principal component; obtaining the peak area of ​​the principal component based on the chromatogram; linearly fitting the peak area of ​​the principal component with the concentration; and performing linear regression with the peak area as the ordinate and the concentration as the abscissa to obtain the standard curve regression equation of the principal component.

[0092] In this invention, the step of establishing the standard curve regression equation for the concentration-peak area of ​​impurities includes: preparing a series of impurity reference solutions; performing high-performance liquid chromatography (HPLC) on the series of impurity reference solutions to obtain impurity chromatograms; obtaining the impurity peak area based on the impurity chromatograms; linearly fitting the impurity peak area with the concentration; and performing linear regression with the peak area as the ordinate and the concentration as the abscissa to obtain the impurity standard curve regression equation.

[0093] In this invention, the formula for calculating the correction factor is:

[0094]

[0095] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a method for detecting impurities in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0096] The instruments, liquid chromatography conditions, materials, reagents, and solution preparations used in the examples are as follows:

[0097] 1. Instruments and Liquid Chromatography Conditions

[0098] Shimadzu LC-20AT high performance liquid chromatograph (LabSolutions workstation, Shimadzu Corporation); XSE105DUE analytical balance (METTLER TOLEDO, USA); AP125WD analytical balance (Shimadzu); ME104E analytical balance (METTLER TOLEDO, USA); S210 pH meter (METTLER TOLEDO, USA).

[0099] Chromatographic conditions: A Waters Atlantis T34.6 × 250 mm, 5 μm column packed with octadecylsilane-bonded silica gel; mobile phase A was 0.01 mol / L phosphate buffer (dissolved in 1000 ml of water with 1.42 g of disodium hydrogen phosphate, pH adjusted to 5.5 with phosphoric acid) - acetonitrile (90:10), and mobile phase B was acetonitrile; gradient elution was performed according to the proportions in Table 2; flow rate was 1.1 ml / min; detection wavelength was 220 nm; column temperature was 30 °C; injection volume was 15 μL.

[0100] Table 2 Elution gradient program

[0101]

[0102]

[0103] 2. Materials and Reagents

[0104] Reference substance of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine (IM-3) (Source: Beijing Biluda Medical Technology Co., Ltd., Batch number: PBS20210922-6, Content: 100.0%); Reference substance of impurity IM-3-1 (Source: Beijing Biluda Medical Technology Co., Ltd., Batch number: PBS20210922-3, Content: 100%); Reference substance of impurity IM-3-2 (Source: SINCO, Batch number: 22-04-0109, Content: 98.59%); Reference substance of impurity IM-3-3 (Source: SINCO, Batch number: 22-04-1106, Content: 98.41%); Reference substance of impurity IM-1 (Source: Beijing Biluda Medical Technology Co., Ltd., Batch number: PBS20210922-9, Content: 100%); Reference substance of impurity IM-2 (Source: Beijing Biluda Medical Technology Co., Ltd., Batch number: PBS20210922-7, Content: 99.8%); Reference substance of impurity IM-B (Source: Beijing Biluda Medical Technology Co., Ltd., Batch number: PBS20210922-8, Content: 100%); Test sample of IM-3 (3 batches of samples from Company A, Batch numbers: C374-stock 2301001, C374-stock 302001, C374-stock 2303001); Disodium hydrogen phosphate (Analytical grade, Tianjin Guangfu); Phosphoric acid (Analytical grade, Fisher); Water (Watsons ultrapure water); Acetonitrile (HPLC, FEPURE); Methanol (HPLC, FEPURE).

[0105] 3. Solution preparation

[0106] 3.1 Blank solvent: Methanol: Acetonitrile (50:50). In this embodiment, the solvents used for preparing solutions are all this blank solvent.

[0107] 3.2 Test sample solution: Precisely weigh 10 mg of IM-3 sample and place it in a 10 mL volumetric flask. Dissolve, dilute, and make up the volume with the blank solvent, shake well to obtain a test sample solution with a concentration of 1.0 mg / mL.

[0108] 3.3 Reference substance solution: Weigh 10 mg of IM-3 reference substance and place it in a 10 mL volumetric flask. Dissolve, dilute, and make up the volume with the blank solvent, shake well to obtain a stock solution (1) of IM-3 reference substance with a concentration of 1.0 mg / mL. Precisely measure 5 mL of the stock solution (1) of IM-3 reference substance and place it in a 100 mL volumetric flask. Dilute and make up the volume with the blank reagent, shake well to obtain a stock solution (2) of IM-3 reference substance with a concentration of 50 μg / mL. Precisely measure 1 mL of the stock solution (1) of IM-3 reference substance and place it in a 10 mL volumetric flask. Dilute and make up the volume with the blank reagent, shake well to obtain an IM-3 reference substance solution with a concentration of 5 μg / mL.

[0109] 3.4 System Suitability Solution: Weigh appropriate amounts of IM-3 test sample and IM-1, IM-B, IM-2, IM-3-1, IM-3-2, and IM-3-3 reference standards, dissolve, dilute, and bring to volume using the blank solvent, shake well, and obtain a system suitability solution with IM-3 concentration of 1.0 mg / mL, IM-B concentration of 0.2 μg, and other known impurities of 1 μg.

[0110] 3.5 Reference standard stock solutions and impurity localization solutions for each impurity:

[0111] Accurately weigh 10 mg of impurity IM-B reference standard and place it in a 10 mL volumetric flask. Dissolve, dilute, and bring to volume using the blank reagent, and shake well to obtain a 1 mg / mL impurity IM-B reference standard stock solution (1). Accurately measure 1 mL of impurity IM-B reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain a 100 μg / mL impurity IM-B positioning solution.

[0112] Accurately weigh 10 mg of impurity IM-1 reference standard and place it in a 10 mL volumetric flask. Dissolve, dilute and make up to volume using the blank reagent, and shake well to obtain a 1 mg / mL impurity IM-1 reference standard stock solution (1). Accurately measure 1 mL of impurity IM-1 reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and make up to volume using the blank reagent, and shake well to obtain a 100 μg / mL impurity IM-1 positioning solution.

[0113] Accurately weigh 10 mg of impurity IM-2 reference standard and place it in a 100 mL volumetric flask. Dissolve, dilute, and bring to volume using the blank reagent, and shake well to obtain a 1 mg / mL impurity IM-2 reference standard stock solution (1). Accurately measure 1 mL of impurity IM-2 reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain a 100 μg / mL impurity IM-2 positioning solution.

[0114] Accurately weigh 10 mg of impurity IM-3-1 reference standard and place it in a 100 mL volumetric flask. Dissolve, dilute, and bring to volume using the blank reagent, and shake well to obtain a 1 mg / mL impurity IM-3-1 reference standard stock solution (1). Accurately measure 1 mL of impurity IM-1 reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain a 100 μg / mL impurity IM-3-1 positioning solution.

[0115] Accurately weigh 10 mg of impurity IM-3-2 reference standard and place it in a 10 mL volumetric flask. Dissolve, dilute, and bring to volume using the blank reagent, and shake well to obtain a stock solution (1) of impurity IM-3-2 reference standard with a concentration of 1 mg / mL. Accurately measure 1 mL of impurity IM-1 reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain a positioning solution of impurity IM-3-2 with a concentration of 100 μg / mL.

[0116] Accurately weigh 10 mg of impurity IM-3-3 reference standard and place it in a 100 mL volumetric flask. Dissolve, dilute, and bring to volume using the blank reagent, and shake well to obtain a stock solution (1) of impurity IM-3-3 reference standard with a concentration of 1 mg / mL. Accurately measure 1 mL of impurity IM-1 reference standard stock solution (1) and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain a positioning solution of impurity IM-3-3 with a concentration of 100 μg / mL.

[0117] Example 1

[0118] 1-1 Specificity Verification

[0119] Accurately measure 15 μL of blank solvent, test solution, mixed control solution, system suitability solution, IM-3 positioning solution, and each impurity positioning solution, and inject them into the liquid chromatograph. Detection is performed using high-performance liquid chromatography (HPLC). The results are shown in Table 3. Figures 1-4 As shown, where, where, Figure 1 The image shows the HPLC chromatogram of the blank solvent. Figure 2 The HPLC chromatogram of the system suitability solution. Figure 3 The HPLC chromatogram of the test solution is shown below. Figure 4 The HPLC chromatograms are for the system suitability solution, the localization solutions for each impurity, the test solution, and the blank solvent.

[0120] Table 3 Results of specificity verification experiments

[0121]

[0122] From Table 3 and Figures 1-4 It can be seen that in the chromatograms of the blank solvent and the test solution, none of the unknown impurities interfere with the determination of known impurities; in the system suitability solution, IM-1, IM-B, IM-2, IM-3-1, IM-3-2, IM-3, and IM-3-3 elute sequentially, and the resolution between each chromatographic peak should meet the requirements (greater than 1.5). This indicates that the detection method provided by this invention has high system suitability and can effectively separate various impurities.

[0123] 1-2 Limit of Quantitation, Limit of Detection

[0124] The limits of detection (LOD) and quantitation (LOQ) are determined based on the signal-to-noise ratio (SNR) method. The known concentrations of the SM positioning solution and each impurity positioning solution are serially diluted. The concentration at which S / N ≈ 10 is taken as the limit of quantitation, and the concentration at which S / N ≈ 3 is taken as the limit of detection.

[0125] Accurately measure 15 μL of each limit of quantitation and limit of detection solution, inject them into the liquid chromatograph for detection, record the chromatograms, and the detection results are shown in Tables 4 and 5.

[0126] Table 4 Results of Limit of Quantitation Test

[0127]

[0128] Table 5 Results of the detection limit test

[0129]

[0130]

[0131] As shown in Tables 4 and 5, the signal-to-noise ratio (SNR) of SM and all impurities in the limit of quantitation (LOQ) solution is greater than 10, and the SNR of SM and all impurities in the limit of detection (LOD) solution is greater than 3. With six consecutive injections of the LQ solution, the RSD of all impurities, calculated by peak area, is less than 10.0%. The LQ concentrations are all less than 0.05% of the sample concentration, and the LOD concentrations are all less than 0.02% of the sample concentration, indicating that the detection method provided by this invention has high sensitivity.

[0132] 1-3 Precision

[0133] (1) Repeatability

[0134] System suitability solutions: under the same specificity category.

[0135] Preparation of repeatability solutions: Test solution: Accurately weigh 10 mg of IM-3 sample and place it in a 10 mL volumetric flask. Dissolve, dilute and make up to volume using the blank solvent, shake well to obtain a test solution with a concentration of 1.0 mg / mL; prepare 6 parallel solutions.

[0136] Reference solution: Weigh 10 mg of IM-3 reference standard into a 10 mL volumetric flask, dissolve, dilute and make up to volume with the blank solvent, shake well to obtain a 1.0 mg / mL IM-3 reference standard stock solution (1); accurately measure 5 mL of IM-3 reference standard stock solution into a 100 mL volumetric flask, dilute and make up to volume with the blank reagent, shake well to obtain a 50 μg / mL IM-3 reference standard stock solution (2); accurately measure 1 mL of IM-3 reference standard stock solution into a 10 mL volumetric flask, dilute and make up to volume with the blank reagent, shake well to obtain a 5 μg / mL IM-3 reference standard solution.

[0137] 3.4 System Suitability Solution: Weigh 15 μL each of the IM-3 test sample, the IM-precision measuring system suitability solution, the test sample solution, and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and the detection results are shown in Table 6.

[0138] Table 6 Results of Repeatability Tests

[0139]

[0140]

[0141] As shown in Table 6, the RSD of the detection amount of each impurity in the 6 samples is ≤20.0%, indicating that the detection method provided by the present invention has good repeatability.

[0142] (2) Intermediate precision

[0143] The tests were conducted by different personnel on different instruments. The personnel who performed the repeatability of step (1) are referred to as (personnel 1).

[0144] Different experimental personnel (personnel 2) prepared 15 μL of each test sample solution and their own control solution according to step (1), injected them into another liquid chromatograph, recorded the chromatograms, and the detection results are shown in Table 7.

[0145] Table 7 Results of intermediate precision test

[0146]

[0147]

[0148] As shown in Table 7, the RSD of the detection amount of each impurity in the 12 samples is ≤20.0%, indicating that the intermediate precision of the detection method provided by the present invention is good.

[0149] 1-4 accuracy

[0150] (1) Accuracy solution preparation

[0151] Mixed reference stock solution: Accurately measure 0.2 mL of the prepared impurity IM-B reference stock solution (1), 5 mL of impurity IM-3 reference stock solution (1), 1.0 mL of impurity IM-1 reference stock solution (1), IM-2 reference stock solution (1), IM-3-1 reference stock solution (1), IM-3-2 reference stock solution (1), and IM-3-3 reference stock solution (1), and place them in the same 100 mL volumetric flask. Dilute and bring to volume using the blank reagent, and shake well to obtain reference stock solutions with impurity IM-B concentration of 2 μg / mL, impurity IM-3 concentration of 50 μg / mL, impurity IM-1 concentration of 10 μg / mL, impurity IM-2 concentration of 10 μg / mL, impurity IM-3-1 concentration of 10 μg / mL, impurity IM-3-2 concentration of 10 μg / mL, and impurity IM-3-3 concentration of 10 μg / mL.

[0152] Mixed reference solution: Accurately measure 1.0 mL of the prepared mixed reference stock solution and place it in a 10 mL volumetric flask. Dilute and bring to volume using the blank reagent described above, and shake well to obtain a reference solution with impurity IM-B concentration of 0.2 μg / mL, impurity IM-3 concentration of 5 μg / mL, impurity IM-1 concentration of 1 μg / mL, impurity IM-2 concentration of 1 μg / mL, impurity IM-3-1 concentration of 1 μg / mL, impurity IM-3-2 concentration of 1 μg / mL, and impurity IM-3-3 concentration of 1 μg / mL.

[0153] Recovery solutions: Accurately weigh 10 mg of IM-3 sample (9 portions) and place them in 10 mL volumetric flasks. Accurately measure 0.5 mL, 1.0 mL, and 1.5 mL of the mixed reference stock solution and add them to the corresponding volumetric flasks (3 portions each). Dissolve, dilute, and bring to volume using the blank solvent, and shake well to obtain 50% recovery solution, 100% recovery solution, and 150% recovery solution.

[0154] Background solution: Accurately weigh 10 mg of IM-3 sample into a 10 mL volumetric flask, dissolve, dilute and make up to volume using the blank solvent, shake well to obtain a test solution with a concentration of 1.0 mg / mL.

[0155] Accurately measure 15 μL of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms and the detection results are shown in Table 8.

[0156] Table 8. Accuracy Test Results

[0157]

[0158] As shown in Table 8, the recoveries of impurities IM-B, IM-1, IM-2, IM-3-1, IM-3-2, and IM-3-3 were all in the range of 80% to 120% at low concentrations (50% recovery rate solution), medium concentrations (100% recovery rate solution), and high concentrations (150% recovery rate solution), with a recovery rate RSD ≤ 10.0%.

[0159] 1-5 Solution Stability

[0160] Accurately measure 15 μL each of the prepared test solution and mixed reference solution, and inject them into the liquid chromatograph at different time points after being placed at room temperature. Record the chromatograms. The detection results are shown in Tables 9 and 10.

[0161] Table 9 Results of the stability test of the test sample solution

[0162]

[0163] Table 10 Results of stability test of mixed reference solution

[0164]

[0165]

[0166] As shown in Tables 9 and 10, no new impurities were observed in the test solution after 48 hours of standing at room temperature, and the RSD of impurity changes was less than 20%, indicating that the sample solution had good stability within 24 hours. The reference solution, when injected within 48 hours of standing at room temperature, had a maximum peak area RSD of 1.8%, which was less than 5.0%, indicating that the reference solution had good stability after 48 hours of standing at room temperature.

[0167] 1-6 Durability

[0168] Robustness refers to the degree to which the test results remain unaffected by minor variations in test conditions. The parameters for varying chromatographic detection conditions are shown in Table 11. Accurately measure 15 μL each of the prepared system suitability solution, test solution, and reference solution, and inject them separately into the liquid chromatograph, recording the chromatograms. The test results are shown in Table 12.

[0169] Table 11 Parameters for Durability Variation of Chromatographic Conditions

[0170]

[0171] Table 12 Results of the durability test on the test specimens

[0172]

[0173]

[0174] Table 12 shows that by changing the flow rate, column temperature, pH of mobile phase A, initial flow ratio, and using different batches of the same brand of chromatographic column, the blank solvent did not interfere with the determination of known impurities. The system suitability test showed that the resolution between the main peak and adjacent impurities met the requirements. The maximum percentage of each impurity in the test sample under different chromatographic conditions was 16.6%, less than 20%, indicating good method robustness. The detection method provided by this invention has good robustness within the set range.

[0175] 1-7 Linearity and Range

[0176] (1) Six concentrations (limit of quantitation solution, 50%, 100%, 150%, 200%, and 300% linear solutions) within the range of limit of quantitation to 300% were selected as a series of linear solutions for study. These linear solutions were obtained by diluting the prepared IM-3 standard and stock solutions of each impurity with the blank reagent. 15 μL of each linear solution was accurately measured and injected into the liquid chromatograph, and the chromatograms were recorded. Linear regression analysis was performed with concentration as the abscissa (x) and peak area as the ordinate (y). The results are shown in Table 13 and... Figures 5-11 As shown, Figure 11 The graph shows the linear relationship between IM-3 and each impurity.

[0177] Table 13 Results of Linearity and Range Tests

[0178] Impurity Name Standard curve regression equation r Linear range (μg / mL) IM-1 <![CDATA[y=4.8767×10 4 x-2.7636×10 3 ]]> 0.9995 0.108～2.946 IM-B <![CDATA[y=4.3422×10 4 x-4.5832×10 2 ]]> 0.9998 0.046～0.627 IM-2 <![CDATA[y=2.6838×10 4 x-7.8281×10 2 ]]> 0.9998 0.106～2.880 IM-3-1 <![CDATA[y=5.6934×10 4 x-4.6542×10 3 ]]> 0.9999 0.112～3.057 IM-3-2 <![CDATA[y=4.8767×10 4 x-2.7636×10 3 ]]> 0.9998 0.108～2.934 IM-3-3 <![CDATA[y=3.0417×10 4 x-1.8172×10 3 ]]> 0.9998 0.105～2.855 IM-3 <![CDATA[y=4.2496×10 4 x-7.8032×10 3 ]]> 0.9997 0.117～15.930

[0179] From Table 13 and Figures 5-11 It can be seen that, within the concentration range of the limit of quantitation to 300%, the concentrations of IM-1, IM-B, IM-2, IM-3-1, IM-3-2, IM-3-3, and IM-3 show a linear relationship between concentration and peak area, with a correlation coefficient r > 0.9995, indicating a good linear relationship.

[0180] 1-8 correction factors

[0181] Using the standard curve regression equations obtained in Examples 1-7, the correction factor was calculated using the standard curve slope method. The correction factor for impurities was calculated by comparing the slope of the principal component standard curve with the slope of the impurity standard curve. That is, when the correction factor is in the range of 0.9 to 1.1, no correction was performed. When the correction factor is in the range of 0.2 to 5.0, the rounded correction factor was used for calculation. The correction factor results for each impurity are shown in Table 14.

[0182] Table 14 Results of IM-3 and Correction Factors for Each Impurity

[0183] Impurity Name slope Correction factor F After the revision of F IM-3 42495.57 -- -- IM-1 48767.05 0.87 0.9 IM-B 43421.86 0.98 1.0 IM-2 26837.79 1.58 1.6 IM-3-1 56933.70 0.75 0.8 IM-3-2 37028.29 1.15 1.2 IM-3-3 30417.34 1.40 1.4

[0184] 1-9 Sample Testing

[0185] (1) Solution preparation

[0186] Test solution: Accurately weigh 10 mg of IM-3 samples (batch numbers are C374-stock 2301001, C374-stock 2302001, C374-stock 2303001) and place them in a 10 mL volumetric flask. Dissolve, dilute, and make up the volume with the blank solvent, shake well to obtain a test solution with a concentration of 1.0 mg / mL.

[0187] Reference solution: Weigh 10 mg of IM-3 reference substance and place it in a 10 mL volumetric flask. Dissolve, dilute, and make up the volume with the blank solvent, shake well to obtain an IM-3 reference stock solution (1) with a concentration of 1.0 mg / mL. Accurately measure 5 mL of the IM-3 reference stock solution and place it in a 100 mL volumetric flask. Dilute and make up the volume with the blank reagent, shake well to obtain an IM-3 reference stock solution (2) with a concentration of 50 μg / mL; accurately measure 1 mL of the IM-3 reference stock solution and place it in a 10 mL volumetric flask. Dilute and make up the volume with the blank reagent, shake well to obtain an IM-3 reference solution with a concentration of 5 μg / mL.

[0188] System suitability solution: Weigh appropriate amounts of IM-3 test substance, and IM-1, IM-B, IM-2, IM-3-1, IM-3-2, IM-3-3 reference substances. Dissolve, dilute, and make up the volume with the blank solvent, shake well to obtain a system suitability solution with an IM-3 concentration of 1.0 mg / mL, an IM-B concentration of 0.2 μg, and 1 μg of other known impurities.

[0189] (2) Experimental method

[0190] Accurately measure 15 μL of the test solution, reference solution, and system suitability solution respectively, inject them into the liquid chromatograph, record the peak areas, and calculate the content of related substances in N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine by the external standard method of the main component with correction factor. The detection results are shown in Table 15.

[0191] Table 15 Determination results of related substances in IM-3 test substance

[0192]

[0193]

[0194] As can be seen from the above examples, the detection method provided by the present invention can effectively and accurately detect the content of related substances in the IM-3 test substance, and this detection method has good specificity, high accuracy and sensitivity.

[0195] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting impurities related to imatinib mesylate intermediates, characterized in that, When using high-performance liquid chromatography (HPLC), the detection conditions must include at least: Chromatographic column: Octadecylsilane-bonded silica column; Column temperature: 25~35℃; Mobile phase: includes mobile phase A and mobile phase B; The mobile phase A is a mixed solution of disodium hydrogen phosphate and acetonitrile with a volume ratio of 88-92:12-8; the concentration of the disodium hydrogen phosphate solution is 0.005-0.015 mol / L, and the pH value is 5.0-6.0; the mobile phase B is acetonitrile. Flow rate: 0.9–1.3 mL / min; Detection wavelength: 215–225 nm; Elution method: gradient elution.

2. The method for detecting impurities related to imatinib mesylate intermediates according to claim 1, characterized in that, The gradient elution procedure is as follows: 0 min, the volume fraction of the mobile phase A is maintained at 96% to 100%; Within 0–10 min, the volume fraction of the mobile phase A decreased to 78%–82%; The volume fraction of the mobile phase A is maintained at 78%–82% for 10–25 min. After 25–45 minutes, the volume fraction of the mobile phase A decreased to 53%–57%. For 45–55 minutes, the volume fraction of the mobile phase A is maintained at 53%–57%. After 55–56 minutes, the volume of the mobile phase A increases to 96%–100%. The volume of the mobile phase A is maintained at 96%–100% for 56–70 minutes.

3. The method for detecting impurities related to imatinib mesylate intermediates according to claim 1, characterized in that, The injection volume of the method is 17–19 μL.

4. The method for detecting impurities related to imatinib mesylate intermediates according to claim 1, characterized in that, Includes the following steps: I. Dissolve the imatinib mesylate intermediate sample in a methanol-acetonitrile mixed solvent to obtain the test solution; II. Dissolve the imatinib mesylate intermediate reference standard in a methanol-acetonitrile mixed solvent to obtain the reference standard solution; III. The test solution and the reference solution were subjected to high performance liquid chromatography under the same detection conditions to obtain the peak areas of impurities in the test sample and the peak areas of the reference solution, respectively. IV. The mass percentage content of the impurity is calculated based on the peak area of ​​the impurity in the test sample, the peak area of ​​the reference sample, and the correction factor.

5. The method for detecting impurities related to imatinib mesylate intermediates according to claim 4, characterized in that, In step I, the volume fraction of acetonitrile in the methanol-acetonitrile mixed solvent is 45-55%; the concentration of the imatinib mesylate intermediate sample in the test solution is 0.9-1.1 mg / mL; and the concentration of the imatinib mesylate intermediate reference standard in the reference solution is 3-7 μg / mL.

6. The method for detecting impurities related to imatinib mesylate intermediates according to claim 4, characterized in that, The intermediate of imatinib mesylate is N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine; The impurities include 2-methyl-5-nitrophenylguanidine, 3-acetylpyridine, (E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one, 1-(2-methyl-5-nitrophenyl)-3-(4-(pyridin-3-yl)pyrimidin-2-yl)guanidine, N-(2-methyl-3-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine and N-(2-methyl-4-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine.

7. The method for detecting impurities related to imatinib mesylate intermediates according to claim 4, characterized in that, The formula for the calculation is: Total impurities % = % of known impurities + % of other impurities; In the formula: W 对 W 供 : Weigh the reference standard and the test sample respectively, in grams; P 对 %: Content of reference standard; A 已知杂质 : Peak area of ​​each known impurity in the test solution; A 其他最大杂质、A其他杂质总和 These are the areas of the largest single impurity peak and the sum of the areas of all other impurity peaks in the test solution, respectively. A 对 : The area of ​​the main peak in the reference solution; n 对 n 供 : These are the dilution factors for the reference standard and the test sample, respectively.

8. The method for detecting impurities related to imatinib mesylate intermediates according to claim 7, characterized in that, In step IV, the method for obtaining the correction factor is as follows: (1) Establish the standard curve regression equations of concentration-peak area for N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities, respectively; (2) Based on the regression equation of the concentration-peak area standard curve of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidine-2-amine and impurities, the slope of the principal component standard curve and the slope of the impurity standard curve are obtained respectively. (3) The correction factor is obtained based on the slope of the principal component standard curve and the slope of the impurity standard curve.

9. The method for detecting impurities related to imatinib mesylate intermediates according to claim 7, characterized in that, The formula for calculating the correction factor is as follows:

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