An analytical method for determining genotoxic impurities in nilotinib compositions

Abstract

The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a kind of analytical method for determining genetic toxic impurities in nilotinib composition.The present application has good specificity, simple operation, high sensitivity, low cost, and is suitable for frequent detection in mass production, etc., for determining impurity A in compositions such as nilotinib oral suspension, nilotinib dry suspension or nilotinib capsules.The method can effectively separate the nilotinib impurity A peak from its adjacent impurity peak, and solve the problem of easy damage of the chromatographic column during the determination of nilotinib impurity A.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and specifically relates to an analytical method for determining genotoxic impurities in nilotinib compositions. Background Technology

[0002] Nilotinib, developed by Novartis, is a BCR-ABL tyrosine kinase inhibitor approved for the treatment of adult patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic or accelerated phase who are resistant to or intolerant of prior treatments (including imatinib). It was approved in 2016 for the treatment of newly diagnosed adult patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase, and in 2019 for the treatment of chronic myeloid leukemia in children aged 2 years and older. Currently, nilotinib is marketed as a capsule formulation. The structure of impurity A in nilotinib is shown below. Impurity A has an aromatic amine warning structure and potential genotoxicity. Impurity A is both a starting material in the nilotinib synthesis process and a degradation impurity; therefore, its content must be strictly controlled to ensure patient safety.

[0003]

[0004] The existing European Pharmacopoeia discloses a method for detecting impurity A in nilotinib hydrochloride raw material. However, the inventors found that this method cannot detect the content of impurity A in nilotinib formulations. The existing technology, "Study on the Synthesis of 5-trifluoromethyl-3-(4-methyl-1H-imidazol-1-yl)-aniline," authored by Hao Yue, discloses a method for detecting 5-trifluoromethyl-3-(4-methyl-1H-imidazol-1-yl)-aniline (i.e., impurity A). However, the inventors found that the detection method in this literature has low sensitivity and is unsuitable for detecting low concentrations of impurity A. Patent CN106932522A discloses a method for determining the content of impurity compound I (i.e., impurity A) in nilotinib, which uses high-performance liquid chromatography-mass spectrometry (HPLC-MS) to determine impurity A. However, this detection method is costly and unsuitable for frequent sampling and testing in large-scale production.

[0005] There is an urgent need to establish a stable, effective, and simple method for determining impurity A in nilotinib formulations in order to achieve quality control of nilotinib combination formulations during large-scale production. Summary of the Invention

[0006] To address the problems in the detection of impurity A in nilotinib formulations in existing technologies, this solution provides a stable, effective, and simple method for detecting impurity A in nilotinib formulations. The formulations include, but are not limited to, the capsules shown in patent application number CN200780035356.7 and the suspension shown in patent application number CN202411814338.0.

[0007] Specifically, it is an analytical method for determining genotoxic impurities in a nilotinib composition, comprising at least the following steps:

[0008] (1) Preparation of the test solution:

[0009] (a) Accurately weigh the nilotinib composition and adjust the pH to 1.0 to 3.0 with organic and / or inorganic acids;

[0010] (b) Dissolve the nilotinib composition by adding a first solvent;

[0011] (c) Add an appropriate amount of the second solution to settle the suspension in the solution obtained in (b), wherein the second solution is an aqueous solution of an organic solvent, and the volume fraction of the organic solvent in the second solution is 60% to 100%.

[0012] (d) Take an appropriate amount of the supernatant of the solution obtained in (c) and add it to the first solvent again to dissolve the nilotinib composition;

[0013] (e) Dilute the solution obtained in (d) with a diluent until the volume fraction of the organic solvent in the test solution is ≤30%;

[0014] (2) The test solution was detected by liquid chromatography.

[0015] As a preferred technical solution, the present invention provides an analytical method for determining genotoxic impurities in a nilotinib composition, comprising at least the following steps:

[0016] (1) Preparation of reference solution: Accurately weigh impurity A reference standard and dissolve it in the third solution to obtain the reference solution;

[0017] (2) Preparation of the test solution:

[0018] (a) Accurately weigh the nilotinib composition and adjust the pH to 1.0 to 3.0 with organic and / or inorganic acids;

[0019] (b) Dissolve the nilotinib composition by adding the first solvent.

[0020] (c) Add an appropriate amount of the second solution to settle the suspension in the solution obtained in (b), wherein the second solution is an aqueous solution of an organic solvent, and the volume fraction of the organic solvent in the second solution is 60% to 100%.

[0021] (d) Take an appropriate amount of the supernatant of the solution obtained in (c) and add it to the first solvent again to dissolve the nilotinib composition;

[0022] (e) Dilute the solution obtained in (d) with a diluent until the volume fraction of the organic solvent in the test solution is ≤30%;

[0023] (3) The reference solution and the test solution were detected by liquid chromatography, and the content of impurity A was calculated by external standard method of reference.

[0024] As a preferred technical solution, the diluent is water or a solution using water as a solvent.

[0025] As a more preferred technical solution, the diluent is water.

[0026] As a preferred technical solution, the organic acid is selected from at least one of citric acid, trifluoroacetic acid, glacial acetic acid, and formic acid, and the inorganic acid is phosphoric acid and / or hydrochloric acid.

[0027] As a preferred technical solution, the first solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

[0028] As a preferred technical solution, the first solvent is dimethyl sulfoxide.

[0029] As a preferred technical solution, the second solution is selected from at least one of aqueous alcohol, aqueous acetone, and aqueous chloroform.

[0030] As a preferred technical solution, the second solution is an aqueous alcohol solution.

[0031] As a preferred technical solution, the third solution is selected from at least one of dimethyl sulfoxide aqueous solution, dimethyl sulfoxide salt aqueous solution, alcohol aqueous solution, alcohol salt aqueous solution, acetone aqueous solution, acetone salt aqueous solution, and chloroform aqueous solution.

[0032] As a preferred technical solution, the third solution is a dimethyl sulfoxide aqueous solution, wherein the volume ratio of water to dimethyl sulfoxide in the dimethyl sulfoxide aqueous solution is 8:2.

[0033] As a preferred technical solution, the detection conditions of the liquid chromatograph are as follows:

[0034] Detector: Ultraviolet detector;

[0035] Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0036] Column temperature: 15℃~60℃;

[0037] Mobile phase A: Phosphate buffer;

[0038] Mobile phase B: A mixture of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is (10-30):(70-90);

[0039] Gradient elution was performed using mobile phase A and mobile phase B. The gradient elution procedure is as follows:

[0040]

[0041] Flow rate: 1.0 ml / min~2.0 ml / min;

[0042] Injection volume: 100 μl;

[0043] Detection wavelength: 207nm.

[0044] As a preferred technical solution, the detection conditions of the liquid chromatograph are as follows:

[0045] Detector: Ultraviolet detector;

[0046] Chromatographic column: An Agilent Poroshell 120EC-C18 column with octadecylsilane-bonded silica gel as the packing material, 4.6 mm × 100 mm, 2.7 μm;

[0047] Column temperature: 40℃;

[0048] Mobile phase A: Phosphate buffer;

[0049] Mobile phase B: A mixture of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 20:80;

[0050] Gradient elution was performed using mobile phase A and mobile phase B. The gradient elution procedure is as follows:

[0051]

[0052]

[0053] Flow rate: 1.0 ml / min;

[0054] Injection volume: 100 μl;

[0055] Detection wavelength: 207nm.

[0056] As a preferred technical solution, the pH value of the mobile phase A phosphate buffer is between 3.5 and 3.9.

[0057] As a preferred technical solution, the phosphate buffer solution is selected from at least one of aqueous phosphate solution, potassium dihydrogen phosphate solution, and dipotassium hydrogen phosphate solution.

[0058] As a preferred technical solution, the mobile phase A phosphate buffer is a potassium dihydrogen phosphate solution with a concentration of 1.36 g / L, and the pH value of the potassium dihydrogen phosphate solution is adjusted to 3.5-3.9 by phosphoric acid.

[0059] As a preferred technical solution, the mobile phase A phosphate buffer is a potassium dihydrogen phosphate solution with a concentration of 1.36 g / L, and the pH value of the potassium dihydrogen phosphate solution is adjusted to 3.7 by phosphoric acid.

[0060] Beneficial effects

[0061] This invention provides a method for determining impurity A in nilotinib compositions, such as nilotinib oral suspension, nilotinib dry suspension, and nilotinib capsules. This method has the advantages of low detection cost, high specificity, simple operation, high sensitivity, and wide applicability. It can effectively separate the nilotinib impurity A peak from its adjacent impurity peaks and solves the problem of easy damage to the chromatographic column during the determination of impurity A in nilotinib compositions. Attached Figure Description

[0062] Figure 1 This is the liquid phase detection spectrum in Example 1.

[0063] Figure 2 This is the liquid phase detection spectrum in Example 2.

[0064] Figure 3 This is the liquid phase detection spectrum in Example 3.

[0065] Figure 4 This is the liquid phase detection spectrum in Example 4.

[0066] Figure 5 This is the liquid phase detection spectrum in Example 5.

[0067] Figure 6 This is the liquid phase detection spectrum in Example 6.

[0068] Figure 7 This is the liquid phase detection spectrum in Example 7. Detailed Implementation

[0069] To facilitate understanding of the present invention by those skilled in the art, specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0070] The liquid chromatograph used in this invention is a high-performance liquid chromatograph with an ultraviolet detector.

[0071] Example 1

[0072] Blank excipient suspension: Take one bottle of nilotinib dry suspension blank excipient (excluding nilotinib), gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0073] Preparation of blank excipient solution: Take 8g of blank excipient suspension and place it in a 20ml volumetric flask. Adjust the pH to 3.0 with citric acid solution, add 4ml of dimethyl sulfoxide, vortex to dissolve, add 6ml of anhydrous ethanol, dilute with water to the mark, shake vigorously or sonicate to mix, and centrifuge. Accurately measure 5ml of the supernatant and place it in a 10ml volumetric flask. Add 1ml of dimethyl sulfoxide, dilute with water to the mark, shake well, and filter to obtain the excipient solution.

[0074] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, dissolve it in a mixture of water and dimethyl sulfoxide, and quantitatively dilute it to prepare a solution containing 0.015 μg of impurity A per 1 ml. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0075] Chromatographic conditions: An Agilent Poroshell 120EC-C18 column, 4.6 mm × 100 mm, 2.7 μm, was used as the packing material, consisting of an octadecylsilane-bonded silica column.

[0076] Mobile phase A was a 1.36 g / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid); mobile phase B was a mixture of mobile phase A and acetonitrile, with a volume ratio of 20:80. Gradient elution was performed using mobile phases A and B as follows: flow rate 1.0 mL / min; column temperature 40 °C; detection wavelength 207 nm; injection volume 100 μL. 100 μL each of the blank excipient solution and the reference solution were accurately injected into the high-performance liquid chromatograph, and the chromatograms were recorded. The results are shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the blank excipient has an excipient peak near the peak of impurity A, interfering with the detection of impurity A. The results indicate that the blank excipient interferes with the detection of impurity A.

[0077] Linear gradient elution of mobile phase

[0078]

[0079]

[0080] Example 2

[0081] Blank excipient suspension: Take one bottle of nilotinib dry suspension blank excipient (excluding nilotinib), gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0082] Preparation of blank excipient solution: Take 8g of blank excipient suspension and place it in a 20ml volumetric flask. Adjust the pH to 3.0 with citric acid solution, add 4ml of dimethyl sulfoxide, vortex to dissolve, add 6ml of anhydrous ethanol, dilute with water to the mark, shake vigorously or sonicate to mix, and centrifuge. Accurately measure 5ml of the supernatant and place it in a 10ml volumetric flask. Add 1ml of dimethyl sulfoxide, dilute with water to the mark, shake well, and filter to obtain the excipient solution.

[0083] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, dissolve it in a mixture of water and dimethyl sulfoxide, and quantitatively dilute it to prepare a solution containing 0.015 μg of impurity A per 1 ml. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0084] Chromatographic conditions: An Agilent Poroshell 120EC-C18 column (4.6 mm × 100 mm, 2.7 μm) was used as the packing material, consisting of octadecylsilane-bonded silica gel. Mobile phase A was 1.36 g / L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid); mobile phase B was a mixture of mobile phase A and acetonitrile (volume ratio 20:80); gradient elution was performed using mobile phases A and B at a flow rate of 1.0 mL / min; column temperature was 40 °C; detection wavelength was 207 nm; injection volume was 100 μL. 100 μL each of the blank excipient solution and the reference solution were accurately injected into the HPLC system, and the chromatograms were recorded. The results are shown in the table below. Figure 2 .Depend on Figure 2 It can be seen that the peak shape in the blank excipient completely overlaps with that of impurity A, interfering with the detection of impurity A. The results indicate that the blank excipient interferes with the detection of impurity A.

[0085] Gradient elution conditions of mobile phase

[0086]

[0087] Example 3

[0088] Blank solvent: a mixed solution of water and dimethyl sulfoxide, wherein the volume ratio of water to dimethyl sulfoxide is 8:2.

[0089] Blank excipient suspension: Take one bottle of nilotinib dry suspension blank excipient (excluding nilotinib), gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0090] Preparation of blank excipient solution: Take 8g of blank excipient suspension and place it in a 20ml volumetric flask. Adjust the pH to 3.0 with citric acid solution, add 4ml of dimethyl sulfoxide, vortex to dissolve, add 6ml of anhydrous ethanol, dilute with water to the mark, shake vigorously or sonicate to mix, and centrifuge. Accurately measure 5ml of the supernatant and place it in a 10ml volumetric flask. Add 1ml of dimethyl sulfoxide, dilute with water to the mark, shake well, and filter to obtain the excipient solution.

[0091] Preparation of the test sample suspension: Take one bottle of nilotinib dry suspension, gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0092] Preparation of test solution: Take 8g of test suspension and place it in a 20ml volumetric flask. Adjust the pH to 3.0 with citric acid solution, add 4ml of dimethyl sulfoxide, vortex to dissolve, add 6ml of anhydrous ethanol, dilute with water to the mark, shake vigorously or sonicate to mix, and centrifuge. Accurately measure 5ml of the supernatant and place it in a 10ml volumetric flask. Add 1ml of dimethyl sulfoxide, dilute with water to the mark, shake well, filter, and collect the filtrate.

[0093] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, dissolve it in a mixture of water and dimethyl sulfoxide, and quantitatively dilute it to prepare a solution containing 0.015 μg of impurity A per 1 ml. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0094] Chromatographic conditions: An Agilent Poroshell 120EC-C18 column (4.6 mm × 100 mm, 2.7 μm) was used as the packing material, consisting of octadecylsilane-bonded silica gel. Mobile phase A was 1.36 g / L potassium dihydrogen phosphate solution (pH adjusted to 3.7 with phosphoric acid). Mobile phase B was a mixture of mobile phase A and acetonitrile at a volume ratio of 20:80. Linear gradient elution was performed using mobile phases A and B, with the same elution program as in Example 2, at a flow rate of 1.0 mL / min. The column temperature was 40 °C, the detection wavelength was 207 nm, and the injection volume was 100 μL.

[0095] Take 100 μl each of blank solvent, blank excipient solution, test solution, and reference solution, and inject them separately into the high-performance liquid chromatograph. Record the chromatograms, and the detection results are as follows: Figure 3 As shown in the figure. The results indicate that no peak was observed at the peak position of impurity A in either the blank solvent or the blank excipient solution, indicating that the blank solvent and blank excipient do not interfere with the detection of impurity A. Furthermore, the peak shape of impurity A in the test sample was good, demonstrating that this method is suitable for the detection of impurity A in this product.

[0096] Example 4

[0097] Prepare the test solution (prepare the solution according to the method for detecting impurity A in nilotinib hydrochloride in the European Pharmacopoeia quality standard): Take an appropriate amount of nilotinib dry suspension (equivalent to 0.3g of nilotinib hydrochloride), add 4ml of dimethyl sulfoxide to dissolve it, dilute with water to prepare a solution containing 27mg of nilotinib per ml, filter (using a 0.45μm membrane, the filtration resistance is relatively large, and filtration is difficult), and collect the filtrate.

[0098] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, add water and dimethyl sulfoxide mixed solution, dissolve and quantitatively dilute to prepare a solution containing 0.015 μg of impurity A per 1 ml, the volume ratio of water to dimethyl sulfoxide in the water and dimethyl sulfoxide mixed solution is 8:2.

[0099] The chromatographic column used was YMC-PACK ODS-AQ, 4.6 mm × 150 mm, 3 μm, and other chromatographic conditions were the same as in Example 3.

[0100] Take 100 μl each of the test solution and the reference solution in this example, inject them separately into the high-performance liquid chromatograph, record the chromatograms, and inject the reference solution once and the test solution three times. The results are shown in the figure. Figure 4 The results showed that after two injections of the test solution prepared according to the European Pharmacopoeia, there were almost no peaks of impurity A in the test solution. The content of impurity A in the raw material used in the nilotinib dry suspension in this example was detected to be 2 ppm, indicating that the detection accuracy of this method is poor. After two injections of the test solution prepared according to the European Pharmacopoeia, the chromatographic column showed abnormal peaks and was damaged.

[0101] Example 5

[0102] Blank excipient solution (prepared according to the method for detecting impurity A in nilotinib hydrochloride in the European Pharmacopoeia quality standard): Take 2.6g of blank excipient from nilotinib dry suspension, place it in a 10ml volumetric flask, add 4ml of dimethyl sulfoxide to dissolve it, dilute with water to the mark, filter (filtering with a 0.45μm membrane is difficult due to high filtration resistance), and collect the filtrate.

[0103] Column 2 was used: Agilent Poroshell 120EC-C18, 4.6 mm × 100 mm, 2.7 μm. Other chromatographic conditions were the same as in Example 3.

[0104] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, add water and dimethyl sulfoxide mixed solution, dissolve and quantitatively dilute to prepare a solution containing 0.015 μg of impurity A per 1 ml, the volume ratio of water to dimethyl sulfoxide in the water and dimethyl sulfoxide mixed solution is 8:2.

[0105] Take 100 μl each of the blank excipient solution and the reference solution, and inject them separately into the high-performance liquid chromatograph. Record the chromatograms. The injection order is: one injection of the reference solution, one injection of the blank excipient solution, and one injection of the reference solution. The detection results are shown in [the table below]. Figure 5 The results showed that after injecting one injection of a blank excipient solution prepared according to the European Pharmacopoeia, the peak of impurity A in the reference solution in the second injection showed a shoulder peak, which was different in shape and lower in height than the peak of impurity A in the first injection of the reference solution, indicating that the sensitivity was lower. This indicates that the chromatographic column was damaged after injecting one injection of a blank excipient solution prepared in a conventional manner.

[0106] Example 6

[0107] Column: An Agilent Poroshell 120EC-C18 column with octadecylsilane-bonded silica gel as the packing material, 4.6 mm × 100 mm, 2.7 μm;

[0108] Mobile phase A: Phosphate buffer (dissolve 1.36 g of potassium dihydrogen phosphate in water and dilute to 1000 ml, then adjust the pH to 3.7 with phosphate);

[0109] Mobile phase B: A mixture of mobile phase A and acetonitrile, with a volume ratio of mobile phase A to acetonitrile of 20:80;

[0110] Gradient elution conditions of mobile phase

[0111]

[0112] Flow rate: 1.0 ml / min;

[0113] Column temperature: 40℃;

[0114] Injection volume: 100 μl;

[0115] Detection wavelength: 207nm;

[0116] Blank solvent: a mixed solution of water and dimethyl sulfoxide, wherein the volume ratio of water to dimethyl sulfoxide is 8:2.

[0117] Preparation of blank excipient suspension: Take one bottle of nilotinib dry suspension blank excipient (excluding nilotinib), gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0118] Preparation of the test sample suspension: Take one bottle of nilotinib dry suspension, gently tap the sealed glass bottle to loosen the powder, add 60ml of water, and shake well before use.

[0119] Preparation of blank excipient solution: Take about 8g of blank excipient suspension and place it in a 20ml volumetric flask. Adjust the pH to 3.0 with citric acid solution, add 4ml of dimethyl sulfoxide, vortex to dissolve, add 6ml of anhydrous ethanol, dilute with water to the mark, shake vigorously or sonicate to mix, and centrifuge. Accurately measure 5ml of the supernatant and place it in a 10ml volumetric flask. Add 1ml of dimethyl sulfoxide, dilute with water to the mark, shake well, filter, and collect the filtrate.

[0120] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, dissolve it in a mixture of water and dimethyl sulfoxide, and quantitatively dilute it to prepare a solution containing 0.015 μg of impurity A per 1 ml. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0121] Preparation of the test solution: Accurately weigh an appropriate amount of the test suspension (equivalent to 200 mg of nilotinib), place it in a 20 ml volumetric flask, adjust the pH to 3.0 with phosphoric acid, add 4 ml of dimethyl sulfoxide, vortex to dissolve, add 6 ml of anhydrous ethanol, vortex to mix, dilute with water to the mark, vortex to mix, and centrifuge; accurately measure 5 ml of the supernatant, place it in a 10 ml volumetric flask, add 1 ml of dimethyl sulfoxide, dilute with water to the mark, shake well, and filter to obtain the test solution.

[0122] Accurately measure 100 μl each of blank solvent, blank excipient solution, test solution, and reference solution, and inject them separately into the high-performance liquid chromatograph. Record the chromatograms, and the detection results are shown in the figure. Figure 6 The results showed that no peak was observed at the location of impurity A in the blank excipient solution and blank solvent, indicating that the blank excipient and blank solvent did not interfere with the detection of impurity A in the test sample.

[0123] Example 7

[0124] The liquid chromatography detection conditions in this embodiment are the same as those in Example 6.

[0125] Preparation of reference solution: Take an appropriate amount of impurity A reference standard, accurately weigh it, dissolve it in a mixture of water and dimethyl sulfoxide, and quantitatively dilute it to prepare a solution containing 0.015 μg of impurity A per 1 ml. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0126] Preparation of sensitivity solution: Accurately measure 3 ml of the reference solution and place it in a 10 ml volumetric flask. Dilute to the mark with a mixture of water and dimethyl sulfoxide, and shake well. The volume ratio of water to dimethyl sulfoxide in the mixture of water and dimethyl sulfoxide is 8:2.

[0127] Accurately measure 100 μl of the sensitivity solution and inject it into the high-performance liquid chromatograph. Record the chromatogram. The detection results are shown in [the table below]. Figure 7 And Table 1. The results show that... Figure 7The peak shape of impurity A is clearly visible, and impurity A can still be detected at low concentrations, indicating good sensitivity. In Table 1, the theoretical plate number of impurity A is 21367 > 5000, and the peak height signal-to-noise ratio of impurity A is 53 > 10, which shows that the column efficiency and sensitivity of this detection method are good.

[0128] Table 1 System Applicability Test Results

[0129] name Theoretical plate number of impurity A Peak height signal-to-noise ratio of impurity A Reference solution 21367 53

[0130] Example 8

[0131] Six parallel test solutions of nilotinib dry suspension were prepared according to the method described in Example 6, and detected by high-performance liquid chromatography (HPLC) as described in Example 6. The results are shown in Table 2 below. The results indicate that the RSD of impurity A in the six parallel test solutions was 8.4% < 20%, indicating that the method has good repeatability.

[0132] Table 2 Results of Repeatability Tests

[0133]

Claims

1. An analytical method for determining genotoxic impurities in a nilotinib composition, characterized in that, At least the following steps are included: (1) Preparation of the test solution: (a) Accurately weigh the nilotinib composition and adjust the pH to 1.0 to 3.0 with organic and / or inorganic acids; (b) Dissolve the nilotinib composition by adding a first solvent; (c) Add an appropriate amount of the second solution to settle the suspension in the solution obtained in (b), wherein the second solution is an aqueous solution of an organic solvent, and the volume fraction of the organic solvent in the second solution is 60% to 100%. (d) Take an appropriate amount of the supernatant of the solution obtained in (c) and add it to the first solvent again to dissolve the nilotinib composition; (e) Dilute the solution obtained in (d) with a diluent until the volume fraction of the organic solvent in the test solution is ≤30%; (2) The test solution was detected by liquid chromatography.

2. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1, characterized in that, At least the following steps are included: (1) Preparation of reference solution: Accurately weigh impurity A reference standard and dissolve it in the third solution to obtain the reference solution; (2) Preparation of the test solution: (a) Accurately weigh the nilotinib composition and adjust the pH to 1.0 to 3.0 with organic and / or inorganic acids; (b) Dissolve the nilotinib composition by adding a first solvent; (c) Add an appropriate amount of the second solution to settle the suspension in the solution obtained in (b), wherein the second solution is an aqueous solution of an organic solvent, and the volume fraction of the organic solvent in the second solution is 60% to 100%. (d) Take an appropriate amount of the supernatant of the solution obtained in (c) and add it to the first solvent again to dissolve the nilotinib composition; (e) Dilute the solution obtained in (d) with a diluent until the volume fraction of the organic solvent in the test solution is ≤30%; (3) The reference solution and the test solution were detected by liquid chromatography, and the content of impurity A was calculated by external standard method of reference.

3. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1 or 2, characterized in that, The organic acid is selected from at least one of citric acid, trifluoroacetic acid, glacial acetic acid, and formic acid, and the inorganic acid is phosphoric acid and / or hydrochloric acid.

4. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1 or 2, characterized in that, The first solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

5. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1 or 2, characterized in that, The second solution is selected from at least one of aqueous alcohol, aqueous acetone, and aqueous chloroform.

6. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 5, characterized in that, The second solution is an aqueous solution of an alcohol.

7. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1 or 2, characterized in that, The third solution is selected from at least one of the following: dimethyl sulfoxide aqueous solution, dimethyl sulfoxide salt aqueous solution, alcohol aqueous solution, alcohol salt aqueous solution, acetone aqueous solution, acetone salt aqueous solution, and chloroform aqueous solution.

8. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 1 or 2, characterized in that, The detection conditions of the liquid chromatograph are as follows: Detector: Ultraviolet detector; Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; Column temperature: 15℃~60℃; Mobile phase A: Phosphate buffer; Mobile phase B: A mixture of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is (10-30):(70-90); Gradient elution was performed using mobile phase A and mobile phase B. The gradient elution procedure is as follows: Flow rate: 1.0 ml / min~2.0 ml / min; Injection volume: 100 μl; Detection wavelength: 207nm.

9. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 8, characterized in that, The detection conditions of the liquid chromatograph are as follows: Detector: Ultraviolet detector; Chromatographic column: An Agilent Poroshell 120EC-C18 column with octadecylsilane-bonded silica gel as the packing material, 4.6 mm × 100 mm, 2.7 μm; Column temperature: 40℃; Mobile phase A: Phosphate buffer; Mobile phase B: A mixture of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 20:80; Gradient elution was performed using mobile phase A and mobile phase B. The gradient elution procedure is as follows: Flow rate: 1.0 ml / min; Injection volume: 100 μl; Detection wavelength: 207nm.

10. The analytical method for determining genotoxic impurities in a nilotinib composition according to claim 9, characterized in that, The pH of the mobile phase A, phosphate buffer, is between 3.5 and 3.9.

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