Method for determining related substances in imatinimide by HPLC (High Performance Liquid Chromatography) method
The HPLC method for detecting impurities in imaamine solves the problem of controlling impurity components in imaamine in existing technologies, achieving efficient separation and quantification, ensuring the quality of imaamine, and thus improving the product quality of imatinib mesylate.
Patent Information
- Application Number
- CN202511488337.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2026-01-23
AI Technical Summary
The lack of effective methods in the current technology to detect related substances in imatinib mesylate makes it difficult to control its composition and affects the quality of the product.
Impurities in imamine were detected by HPLC using a Titank C18 column, with mobile phase A being 0.01 mol/L potassium dihydrogen phosphate solution (pH 3.0) and mobile phase B being acetonitrile. The detection wavelength was 200–300 nm, the flow rate was 0.5–1.5 mL/min, and the column temperature was 25–45 °C. This method can simultaneously detect impurities such as 2-methyl-5-nitroaniline, intermediate P1, and intermediate P2.
It achieves efficient separation and quantification of impurities in imaamine, with a detection limit of 0.0005%, making it suitable for rapid screening of large batches of samples, ensuring controllable imaamine quality, and improving the quality of imatinib mesylate.
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Figure CN121385136A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of chemical and pharmaceutical technology, and particularly relates to a method for detecting related substances in imatinib by HPLC. BACKGROUND
[0002] Imatinib is a key starting material for preparing imatinib mesylate. Imatinib is a protein tyrosine kinase inhibitor, which can effectively inhibit the activity of BCR-ABL tyrosine kinase. Imatinib can inhibit the proliferation of BCR-ABL positive cell lines and Philadelphia chromosome positive CML patient neoplastic cells, and induce cell apoptosis. Imatinib can also inhibit the clonal formation of peripheral blood and bone marrow samples of CML patients in vitro. Imatinib can be used for treating chronic myeloid leukemia (Ph+CML), malignant gastrointestinal stromal tumor (GIST) and acute lymphoblastic leukemia. A large number of clinical trials have shown that imatinib mesylate can significantly prolong the survival of patients, reduce the risk of tumor progression, and has relatively mild adverse reactions, simple use method, and significant clinical efficacy. Imatinib, as a key structural unit of imatinib mesylate, is an organic synthesis intermediate and a pharmaceutical intermediate, which can be used in laboratory research and development process and chemical and pharmaceutical synthesis, and has a wide application prospect in the fields of medical treatment and chemical synthesis.
[0003] The content of imatinib impurities is an important factor affecting the quality of imatinib mesylate product. At present, there is no detection method for related substances in imatinib in the prior art, so it is difficult to control the composition of impurities contained in imatinib in actual production, thereby affecting the quality of imatinib mesylate product. SUMMARY
[0004] In order to solve the technical problems in the background art, the technical scheme adopted by the present application is as follows: a method for detecting related substances in imatinib by HPLC, comprising the following detection steps:
[0005] 1) Selection of instrument: high performance liquid chromatograph; chromatographic column is Titank C18, 4.6mm*250mm, 5um or an equivalent chromatographic column; mobile phase A is 0.01mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 3.0 by phosphoric acid; mobile phase B is acetonitrile; ultraviolet detector is used, and the detection wavelength is 200-300nm; the flow rate of mobile phase is 0.5-1.5ml / min; the column temperature is 25-45℃; and the column length of chromatographic column is 100-250mm;
[0006] 2) Preparation of solution, the following solutions are prepared:
[0007] a, diluent: mobile phase A-acetonitrile (90:10);
[0008] b, blank solution: take appropriate amount of diluent;
[0009] c. System suitability solution: an appropriate amount of 2-methyl-5-nitroaniline, intermediate P1, intermediate P2 and imatinib reference substance is precisely weighed, dissolved and diluted with a diluent to prepare a mixed solution containing about 0.16 μg of 2-methyl-5-nitroaniline, 0.8 μg of intermediate P1, 0.8 μg of intermediate P2 and 0.8 mg of imatinib per 1 ml;
[0010] d. Test solution: an appropriate amount of imatinib test sample is precisely weighed, dissolved and quantitatively diluted with a diluent to prepare a solution containing about 0.8 mg per 1 ml;
[0011] e. Control solution: an appropriate amount of the test solution is precisely measured and quantitatively diluted with a diluent to prepare a solution containing 1.6 μg per 1 ml;
[0012] The samples are injected in the following order:
[0013] Name Injection needle number Blank solution ≥ 1 needle System suitability solution 1 needle Test solution 1 needle Reference solution 1 needle
[0014] The related substances in the imatinib test sample solution are calculated according to the self-control method.
[0015] ① The conditions of the high performance liquid chromatography method include: an ultraviolet detector is used, the detection wavelength is preferably 267 nm; the optimal flow rate of the mobile phase is 1.0 ml / min; the column temperature is 35°C; the chromatographic column is preferably a Titank C18, 4.6 mm x 250 mm, 5 μm; and the column length is 250 mm.
[0016] ② The impurities are 2-methyl-5-nitroaniline, intermediate P1 and intermediate P2.
[0017] The beneficial effects of the present application are as follows:
[0018] The present application can provide a method capable of simultaneously detecting the content of impurities such as 2-methyl-5-nitroaniline, intermediate P1 and intermediate P2, which is simple to operate, can separate multiple impurities, has a high degree of separation, has a detection limit of known impurities in imatinib as low as 0.0005%, can accurately quantify and monitor trace impurities that are difficult to capture in conventional analysis, has a relatively short running time, is suitable for rapid screening and release inspection of a large number of samples, effectively reduces the time and energy consumption costs, thereby ensuring the controllable quality of imatinib, and further improving the quality of imatinib mesylate prepared. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 The blank solution spectrum is shown in Figure 1;
[0020] Figure 2 The system suitability solution spectrum is shown in Figure 2;
[0021] Figure 3 The test sample solution spectrum is shown in the following table:
[0022] Figure 4 The control solution spectrum is shown in the following table. DETAILED DESCRIPTION
[0023] The present application will be further supplemented by the above-mentioned content in conjunction with the examples (drawings), but should not be understood as limiting the scope of the present application to the following examples.
[0024] Example 1
[0025] Specificity investigation, please refer to Figures 1-4 ;
[0026] A method for detecting related substances in imatinib, comprising the following steps: instrument and chromatographic conditions, instrument: high performance liquid chromatograph; chromatographic column Titank C18, 4.6mmx250mm, 5μm; mobile phase A is 0.01mol / L potassium dihydrogen phosphate solution, pH value is adjusted to 3.0 by phosphoric acid; mobile phase B is acetonitrile; wavelength: 267nm, column temperature: 35℃, flow rate: 1.0ml / min, injection volume: 20μL, running time 24min.
[0027] Gradient elution is carried out under the following table conditions:
[0028] Time (min) 0 10 15 18 19 24 B％ 10 40 70 70 10 10
[0029] Each impurity stock solution: take 20mg of impurity 2-methyl-5-nitroaniline, intermediate P1, intermediate P2, accurately weigh, respectively, in a 25ml volumetric flask, dissolve and dilute to the mark with diluent, shake well.
[0030] Each impurity positioning solution: accurately take 0.2ml, 1ml, 1ml of impurity 2-methyl-5-nitroaniline, impurity P1, impurity P2 stock solution, respectively, in different 50ml volumetric flasks, dilute to the mark with diluent, shake well; accurately take 1ml, in different 20ml volumetric flasks, dilute to the mark with diluent, shake well.
[0031] System suitability solution: take appropriate amount of 2-methyl-5-nitroaniline, intermediate P1, intermediate P2 and imatinib reference substance, accurately weigh, dissolve and dilute to prepare a mixed solution containing about 2-methyl-5-nitroaniline 0.16μg, intermediate P1 0.8μg, intermediate P2 0.8μg and imatinib 0.8mg per 1ml.
[0032] Accurately take 20μL of the blank solution, each impurity positioning solution and system suitability solution, inject into the liquid chromatograph, and record the liquid chromatogram.
[0033] The specificity test results are shown in the following table:
[0034]
[0035]
[0036] The minimum separation between each impurity and each peak of imatinib in the above table is 9.28, which is greater than 1.5, indicating that the method has good specificity.
[0037] Example 2
[0038] Limit of detection investigation, please refer to Figures 1-4 ,
[0039] Accurately pipette appropriate amount of each impurity stock solution, dilute with diluent to prepare limit of detection solution. Take 20 μL of the limit of detection solution, inject into the liquid chromatograph, and record the chromatogram.
[0040] The following table is the limit of detection test result table:
[0041] Name Concentration / pg / ml Ratio to limit Ratio to test solution concentration Signal to noise Intermediate P1 0.004 0.5％ 0.0005％ 3.6 2-methyl-5-nitroaniline 0.003 1.8％ 0.0004％ 4.3 Intermediate P2 0.001 0.2％ 0.0002％ 5.0
[0042] The limit of detection determination result shows that the detection limits of impurity 2-methyl-5-nitroaniline, intermediate P1, and intermediate P2 are all less than 0.005% of the concentration of the test sample, indicating that the method has good sensitivity.
[0043] Example 3
[0044] Accuracy investigation, please refer to Figures 1-4 ;
[0045] Accurately pipette 0.2 ml, 1 ml, and 1 ml of impurity 2-methyl-5-nitroaniline, impurity P1, and impurity P2 stock solutions, respectively, into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to serve as a mixed stock solution. Accurately weigh 16 mg of imatinib sample, a total of 9 times, and place in 20 ml volumetric flasks. Three flasks are one group, and the impurity mixed stock solution is added at low, medium, and high concentrations of 0.8 ml, 1.0 ml, and 1.2 ml, respectively. Dilute to the mark with diluent, shake well, and analyze by injection, record the chromatogram, and calculate the recovery rate.
[0046] The following table is the accuracy test result:
[0047] Limit concentration 80％ 100％ 120％ RSD Intermediate P1 99.6％ 97.3％ 96.7％ 1.6％ 2-methyl-5-nitroaniline 98.2％ 96.2％ 95.5％ 1.5％ Intermediate P2 102.1％ 100.5％ 99.8％ 1.2％
[0048] In the above table, the average recoveries of intermediate P1, 2-methyl-5-nitroaniline, and intermediate P2 are all within the range of 92% to 105%; the RSDs are all less than 5%, indicating that the method has good accuracy.
[0049] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements without departing from the principles of the present application, and these improvements should also be considered as the protection scope of the present application.
Claims
1. A method for detecting related substances in imamidine using HPLC, characterized in that: The following testing steps are included: 1) Equipment selection: High performance liquid chromatography; ultraviolet detector; chromatographic column with octadecylsilane-bonded silica gel as the packing material; 2) Preparation of diluent: Mobile phase A-acetonitrile (90:10); 3) Preparation of blank solution: Take an appropriate amount of diluent; 4) Preparation of system suitability solution: Weigh appropriate amounts of 2-methyl-5-nitroaniline, intermediate P1, intermediate P2 and imamidine reference standard, accurately weigh them, dissolve and dilute with diluent to prepare a mixed solution containing approximately 0.16 μg of 2-methyl-5-nitroaniline, 0.8 μg of intermediate P1, 0.8 μg of intermediate P2 and 0.8 mg of imamidine per 1 ml; 5) Preparation of test solution: Take an appropriate amount of imamidine test sample, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 0.8 mg per ml; 6) Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with diluent to prepare a solution containing 1.6 μg per ml; 7) Determination of substances: Accurately measure blank solution, system suitability solution, control solution and test solution and inject them into the liquid chromatograph. Record the liquid chromatogram and calculate the result by peak area according to the self-comparison method.
2. The method for HPLC detection of related substances in imamine according to claim 1, characterized in that: In step 1), the selected chromatographic column is a Titank C18 or a column with equivalent performance, and the column length is 100-250 mm.
3. The method for HPLC detection of related substances in imamine according to claim 1, characterized in that: In high-performance liquid chromatography, an ultraviolet detector is used, with a detection wavelength of 200–300 nm.
4. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: Mobile phase A is a 0.01 mol / L potassium dihydrogen phosphate solution, with the pH adjusted to 3.0 using phosphoric acid; mobile phase B is acetonitrile.
5. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: The mobile phase flow rate is 0.5–1.5 ml / min.
6. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: Gradient elution was used, and the gradient elution table is as follows:
7. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: The column temperature of the chromatographic column is 25–45℃.
8. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: The injection volume was 20 μL.
9. The method for detecting related substances in imamidine by HPLC according to claim 1, characterized in that: The substance to be determined in the substance determination step includes at least one or a combination of two or more of 2-methyl-5-nitroaniline, intermediate P1, and intermediate P2.