Method for detecting genotoxic impurities in perampanel raw materials

Abstract

The present application provides a method for detecting genotoxic impurities in perampanel raw materials. The method detects five genotoxic impurities in perampanel raw materials by high performance liquid chromatography, the method comprising using a phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and using acetonitrile-mobile phase A as an impurity reference solvent, and the structures of the five genotoxic impurities are as follows: #imgabs0# The method of the present invention enables effective separation of each genotoxic impurity and between each genotoxic impurity and perampanel, and has good peak symmetry, which is conducive to the detection of each genotoxic impurity and has high specificity. The present invention shows incomparable advantages in detection limit, quantification limit, linearity and range, repeatability and durability, and has high precision.

Description

Technical Field

[0001] The present application relates to the field of chemical drug analysis and detection, and specifically to a method for detecting five genotoxic impurities in perampanel raw materials. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Perampanel (trade name Fycompa) is a new type of anti-epileptic drug developed by Eisai Pharmaceuticals (EISAI) of Japan. It was launched in the EU and the United States in 2012 and approved in China in 2019. Its mechanism of action is to inhibit the release of overexcited glutamate-mediated neurotransmitters by non-competitive antagonism of the α-amino-3-hydroxy-5-methyl-4-isoxazole receptor (AMPAR) of postsynaptic neurons, thereby reducing the occurrence of epileptic seizures. It is mainly used as an adjuvant treatment for patients with partial-onset epileptic seizures (with or without secondary generalized epileptic seizures) in children aged 12 years and above, and can also be used to treat primary generalized tonic-clonic seizures. The dosage forms currently available in clinical practice are tablets and orally disintegrating tablets.

[0004] The chemical name of perampanel is [2-(6'-oxo-1'-phenyl-1',6'-dihydro-[2,3'-bipyridyl]-5'-yl)benzonitrile] tri-tetrahydrate, and its molecular formula is C 23 H 15 N₃O·3 / 4H₂O, with a molecular weight of 362.90 g / mol and a CAS number of 380917-97-5. Its process uses o-bromophenylacetic acid and aniline as starting materials, followed by condensation, cyanation, and cyclization to produce perampanel. Genotoxic impurities primarily originate from starting materials, intermediates, reagents, and reaction byproducts during the synthesis process, and can also be produced by degradation during storage.

[0005] ICH M7(R1) is a guideline developed by the International Conference on Harmonization of Technical Requirements for Pharmaceuticals (ICH). It establishes standards and methods for the evaluation and control of DNA-reactive impurities (i.e., impurities that can react with DNA and cause mutations) that may be present in pharmaceuticals, with the goal of reducing the carcinogenic risk of these impurities to humans. The guideline provides a framework for the identification, classification, characterization, and control of mutagenic impurities, encompassing steps such as in silico prediction, laboratory testing, and risk assessment. In silico prediction uses the known relationship between chemical structure and biological activity to predict the potential presence of DNA-reactive impurities in pharmaceuticals; laboratory testing uses in vitro models such as bacteria or mammalian cells to detect known or predicted DNA-reactive impurities in pharmaceuticals; and risk assessment evaluates the carcinogenic risk of DNA-reactive impurities in pharmaceuticals based on the results of laboratory testing.

[0006] The threshold of toxicological concern (TTC) approach is typically used to control genotoxic impurities. This approach establishes a safe exposure level based on toxicological data, stipulating that the TTC value for genotoxic impurities in drug substances and drug products should not exceed 1.5 μg / day. The TTC value refers to the maximum daily dose of a compound that does not pose any appreciable risk. The maximum daily dose of perampanel is 12 mg, so the control limit for genotoxic impurities is 0.0125%.

[0007] Due to the low limits of genotoxic impurities, detection methods generally require the use of liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS). LC-MS and GC-MS combine liquid chromatography or gas chromatography with a mass spectrometer, which can simultaneously perform separation, identification, and quantitative analysis. However, these instruments are expensive and not every pharmaceutical company's QC department can use them. Conventional liquid chromatography methods use the interaction between different compounds in the mobile phase and the stationary phase to achieve separation and quantitative analysis. Compared with LC-MS or GC-MS, they have the advantages of low instrument cost, simple operation, and high sensitivity. Therefore, it is more practical to develop conventional liquid chromatography methods to detect genotoxic impurities. Summary of the Invention

[0008] The present invention provides a detection method for simultaneously detecting five genotoxic impurities in perampanel, wherein the method fully meets the standards in terms of specificity, detection limit, quantification limit, linearity and range, repeatability, accuracy, durability, etc. and has high precision, and can be used for quality control and safety assessment of perampanel.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] In a first aspect of the present invention, a method for detecting genotoxic impurities in a perampanel bulk drug substance is provided, wherein the method detects five genotoxic impurities in the perampanel bulk drug substance by high performance liquid chromatography, the method comprising using a phosphoric acid solution as mobile phase A, using acetonitrile as mobile phase B, and using acetonitrile-mobile phase A as an impurity reference solvent;

[0011] The structures and names of the five genotoxic impurities are shown in the following table:

[0012]

[0013]

[0014] In an embodiment of the present invention, the mobile phase A is a 0.08-0.12% phosphoric acid solution, preferably a 0.1% phosphoric acid solution.

[0015] In an embodiment of the present invention, the impurity reference substance solvent is a mixture of acetonitrile and mobile phase A in a volume ratio of 60:40.

[0016] In an embodiment of the present invention, the method comprises:

[0017] The test solution was prepared by dissolving the perampanel sample in acetonitrile;

[0018] Impurity L, impurity N, impurity O, impurity K, and impurity D were used as impurity reference substances. Each impurity reference substance was dissolved and diluted in a mixture of acetonitrile and mobile phase A at a volume ratio of 60:40 to prepare an impurity reference substance stock solution, which was then diluted with acetonitrile to prepare a reference substance solution.

[0019] The test solution and the reference solution were respectively injected into the liquid chromatograph for detection and the chromatograms were recorded. The contents of the five genotoxic impurities in the test solution were calculated by the peak area using the external standard method.

[0020] In an embodiment of the present invention, the detection conditions of the high performance liquid chromatograph are:

[0021] Chromatographic column: Octadecylsilane column, CAPCELL PAK ADME HR C18 (150 mm × 4.6 mm, 3 μm)

[0022] Mobile phase A: 0.08-0.12% phosphoric acid solution, preferably 0.1% phosphoric acid solution;

[0023] Mobile phase B: acetonitrile

[0024] Flow rate: 0.75-0.85 mL / min, preferably 0.8 mL / min;

[0025] Column temperature: 23-27°C, preferably 25°C;

[0026] Detection wavelength: 208-212 nm, preferably 210 nm;

[0027] Injection volume: 10 μL.

[0028] In an embodiment of the present invention, a gradient elution method is adopted, and the gradient elution program is:

[0029] The initial ratio of mobile phase A and mobile phase B was 80:20;

[0030] From 0 to 35 minutes, the proportion of mobile phase A was reduced, while the proportion of mobile phase B was increased to a ratio of 42:58;

[0031] From 35 to 45 min, the ratio of mobile phase A to mobile phase B was maintained at 42:58;

[0032] From 45 to 50 minutes, the proportion of mobile phase A was increased, while the proportion of mobile phase B was reduced until it returned to the initial ratio of 80:20;

[0033] From 50 to 60 minutes, the ratio of mobile phase A to mobile phase B was maintained at 80:20.

[0034] In an embodiment of the present invention, the linear range of impurity K is 0.2086 μg / mL to 2.0862 μg / mL, the linear range of impurity L is 0.3086 μg / mL to 2.0573 μg / mL, the linear range of impurity O is 0.2059 μg / mL to 2.0591 μg / mL, the linear range of impurity N is 0.2074 μg / mL to 2.0741 μg / mL, and the linear range of impurity D is 0.2496 μg / mL to 1.9970 μg / mL.

[0035] In an embodiment of the present invention, the detection limit of the method is as low as 0.103 μg / mL, and the quantification limit is as low as 0.2059 μg / mL.

[0036] In an embodiment of the present invention, the formula for calculating the impurity content is:

[0037] The content of each genotoxic impurity in the test solution (%) = f 平均 ×A i ×V i / m i ×100%

[0038] Where: f = m s ×c s / (V s ×A s );

[0039] Where: f is the response factor; ms is the sample amount of each genotoxic impurity in the reference solution;

[0040] c s is the content of each genotoxic impurity, %; V s A is the total dilution volume of each genotoxic impurity in the reference solution; s is the peak area of ​​each genotoxic impurity in the reference solution; f 平均 is the average value of the response factor; A i is the peak area of ​​each genotoxic impurity in the test solution; m i is the sample weight of the test sample; V i is the dilution volume of the test sample.

[0041] In some embodiments, different mobile phases A, different reference solvents, different column temperatures, different detection wavelengths, and different elution modes are explored. For example, in some embodiments, mobile phase A is replaced with 0.1% ammonium acetate solution (adjusted to pH 4.0 with glacial acetic acid) or 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid); for example, in some embodiments, the reference solvent is replaced with 50% acetonitrile; for example, in some embodiments, the test solvent is replaced with N,N-dimethylformamide; for example, in some embodiments, the detection wavelength is changed to 220 nm or above; for example, in some embodiments, the gradient elution conditions are changed or the gradient elution mode is changed to isocratic elution (for example, mobile phase A:B = 60:40); for example, in some embodiments, the column temperature is changed to 30°C or above; in these embodiments, the detection results have some unsatisfactory aspects, such as large baseline fluctuations, and / or low separation between the impurities to be detected, or even difficulty in separation, and / or difficulty in separating the impurities to be detected from unknown impurities or difficulty in dissolving the test sample, etc.

[0042] In some preferred embodiments of the present invention, the method for detecting genotoxic impurities D, K, L, N, and O in the perampanel bulk drug comprises:

[0043] 1. Chromatographic conditions:

[0044] Instrument: Agilent 1260

[0045] Chromatographic column: CAPCELL PAK ADME HR C18 (150 mm × 4.6 mm, 3 μm);

[0046] Mobile phase A: 0.1% phosphoric acid solution;

[0047] Mobile phase B: acetonitrile;

[0048] Flow rate: 0.8 mL / min;

[0049] Column temperature: 25°C;

[0050] Detection wavelength: 210nm;

[0051] Injection volume: 10 μL;

[0052] Gradient elution, the elution program is shown in Table 1:

[0053] Table 1: Gradient elution program

[0054] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 35 42 58 45 42 58 50 80 20 60 80 20

[0055] 2. Detection method

[0056] (1) Prepare the test solution: accurately weigh 80 mg of perampanel sample into a 10 mL volumetric flask, dissolve in acetonitrile and dilute to the mark, shake well, and obtain the solution.

[0057] (2) Preparation of reference solution: Take approximately 10 mg of each of impurity L, impurity N, impurity O, impurity K, and impurity D, accurately weigh them, place them in the same 10 mL volumetric flask, dissolve them in acetonitrile-mobile phase A (60:40), dilute to the mark, and shake well to prepare the reference solution stock solution; accurately measure 0.1 mL of the reference solution stock solution, place it in a 100 mL volumetric flask, dilute to the mark with acetonitrile, and shake well to prepare the reference solution stock solution;

[0058] (3) Accurately measure the test solution and reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and use the external standard method to calculate the content of impurity L, impurity N, impurity O, impurity K, and impurity D in the test solution based on the peak area. The content calculation method is the same as above.

[0059] In a second aspect of the present invention, there is provided the use of five substances selected from the group consisting of the following in the quality control of perampanel bulk drug or its preparation;

[0060]

[0061] In an embodiment of the present invention, the five substances are genotoxic impurities and are used as impurity reference substances in quality control.

[0062] Compared with the prior art, the advantages of the present invention include:

[0063] The method of the present invention enables the five genotoxic impurities of the present invention, namely, impurity D: (2-(6'-oxo-1'-phenyl-1',6'-dihydro-[2,3'-bipyridine]-5'-yl)bromobenzene, impurity K: (5'-(2-cyanophenyl)-6'-oxo-1'-phenyl-1',6'-dihydro-[2,3'-bipyridine] 1-oxide), impurity L: o-bromophenylacetic acid, impurity N: (2-(2-bromophenyl)-N-phenylacetamide) and impurity O: 2-(2-cyanophenyl)-N-phenylacetamide), to be effectively separated from each other and from perampanel, with a separation degree of more than 2.8, and good peak symmetry, which is conducive to the detection of each genotoxic impurity and has high specificity. In addition, the method described in the present invention has good linearity, a detection limit as low as 0.103 μg / mL, and a quantification limit as low as 0.2059 μg / mL. It shows incomparable advantages in detection limit, quantification limit, linearity and range, repeatability and durability, and has high precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The drawings constituting part of the present application are provided to provide a further understanding of the present application. The illustrative embodiments and their descriptions of the present application are provided to explain the present application and do not constitute an undue limitation on the present application. The following describes the implementation scheme of the present application in detail in conjunction with the drawings, wherein:

[0065] Figure 1 It is the chromatogram of the reference solution.

[0066] Figure 2 It is the chromatogram of the test solution.

[0067] Figure 3 It is the chromatogram of the test solution.

[0068] Figure 4 It is the chromatogram of the test solution.

[0069] Figure 5 is the blank solvent chromatogram.

[0070] Figure 6 It is the chromatogram of the test solution.

[0071] Figure 7 It is a chromatogram of the sample solution, where 1-impurity K; 2-impurity L; 3-impurity O; 4-impurity N; 5-impurity D.

[0072] Figure 8 This is the standard curve of impurity L.

[0073] Figure 9 This is the standard curve of impurity N.

[0074] Figure 10 This is the standard curve of impurity O.

[0075] Figure 11 This is the standard curve of impurity K.

[0076] Figure 12 This is the standard curve of impurity D. DETAILED DESCRIPTION

[0077] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0078] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in this area or in accordance with the product instructions. In addition, any methods and materials similar to or equivalent to those described herein can be applied to the present method. The preferred embodiments and materials described herein are for demonstration purposes only.

[0079] According to the detection method of the present invention, the detection of impurities L, N, O, K and D is mainly carried out using the external standard method for calculation.

[0080]

[0081] The sources of the perampanel raw materials and reference substances for impurities to be detected used in the following embodiments of the present invention are shown in the table below:

[0082]

[0083] The information of the instruments and reagents used in the detection method of the present invention is shown in the following table:

[0084]

[0085] Example 1

[0086] A method for simultaneously detecting five genotoxic impurities (impurity L, impurity N, impurity O, impurity K, and impurity D) in perampanel.

[0087] 1. Chromatographic conditions:

[0088] Instrument: Agilent 1260

[0089] Chromatographic column: CAPCELL PAK ADME HR C18 (150 mm × 4.6 mm, 3 μm);

[0090] Mobile phase A: 0.1% phosphoric acid solution;

[0091] Mobile phase B: acetonitrile;

[0092] Flow rate: 0.8 mL / min;

[0093] Column temperature: 25°C;

[0094] Detection wavelength: 210nm;

[0095] Injection volume: 10 μL.

[0096] Gradient elution, the elution program is shown in Table 1:

[0097] Table 1: Gradient elution program

[0098] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 35 42 58 45 42 58 50 80 20 60 80 20

[0099] 2. Detection method

[0100] (1) Prepare the test solution: accurately weigh 80 mg of perampanel sample into a 10 mL volumetric flask, dissolve in acetonitrile and dilute to the mark, shake well, and obtain the solution.

[0101] (2) Preparation of reference solution: Take approximately 10 mg of each of impurity L, impurity N, impurity O, impurity K, and impurity D, accurately weigh them, place them in the same 10 mL volumetric flask, dissolve them in acetonitrile-mobile phase A (60:40) and dilute to the mark, shake well, and use them as the reference solution stock solution; accurately measure 0.1 mL of the reference solution stock solution, place it in a 100 mL volumetric flask, dilute to the mark with acetonitrile, shake well, and the result is obtained.

[0102] (3) Accurately measure the test solution and reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and use the external standard method to calculate the content of impurity L, impurity N, impurity O, impurity K, and impurity D in the test solution based on the peak area.

[0103] Example 2

[0104] Compared with Example 1, except for the chromatographic conditions, the others are the same.

[0105] The chromatographic conditions are as follows:

[0106] Column: Inertisl ODS-3V, 250 mm × 4.6 mm, 5 μm; C18-145-261;

[0107] Mobile phase A: 0.1% ammonium acetate solution (adjusted to pH 4.0 with glacial acetic acid);

[0108] Mobile phase B: acetonitrile;

[0109] Detection wavelength: 210nm / 220nm / 230nm;

[0110] Flow rate: 1.0 mL / min;

[0111] Column temperature: 25°C;

[0112] Concentration: 10 μg / mL;

[0113] Injection volume: 10 μL;

[0114] Gradient elution conditions:

[0115]

[0116] Test results are shown in Figure 1 ,The test results show that the baseline fluctuation of each wavelength is ,large under the conditions of this system, which affects the ,test results.

[0117] Example 3

[0118] Compared with Example 1, the elution mode was changed to mobile phase A:B=60:40, isocratic elution, column temperature 35°C, flow rate: 1.0 mL / min, and detection wavelengths: 224 nm / 220 nm / 210 nm / 272 nm / 290 nm, respectively. Others were the same as in Example 1.

[0119] Preparation method of sample solution: Take about 80 mg of this product, place it in a 10 mL volumetric flask, accurately add 0.1 mL each of the stock solutions of impurity L, impurity N, impurity O, impurity K, and impurity D, dissolve it in acetonitrile and dilute to the scale, shake well, and obtain the solution.

[0120] Results: There was no obvious baseline fluctuation under this condition. Although the absorption of each impurity at 224nm was not as good as that at 210nm, the baseline fluctuation at 210nm was larger than that at 224nm under this condition. The impurities in the chromatogram of the mixed solution (reference solution) at 224nm wavelength did not interfere with each other, but the chromatogram of the sample solution (see Figure 2 ) has a poor peak shape of impurity O because there is an unknown impurity before impurity O that overlaps with the peak of impurity O. The unknown impurity cannot be separated from impurity O, affecting the test results.

[0121] Example 4

[0122] Compared with Example 3, the column temperature was modified to 25° C., and the rest were the same as Example 3.

[0123] Results: Under this condition, the separation degree of impurity O and adjacent unknown impurities meets the requirements. The absorption of each impurity at 210nm is higher than that at 224nm, and the baseline fluctuation difference is not large. Therefore, the detection wavelength is changed to 210nm. Figure 3 .

[0124] However, in the subsequent destruction experiment, it was found that light destruction would produce an unknown impurity, which would seriously interfere with the detection of impurity K. The flow rate was changed to 0.8 mL / min, and other detection conditions remained unchanged. There was a slight improvement, but it still seriously affected the detection of impurity K. The chromatogram of the sample solution was as follows: Figure 4 shown.

[0125] Example 5

[0126] The detection method described in Example 1 was methodologically verified.

[0127] 1. Methodological validation

[0128] 1.1 Exclusivity

[0129] Impurity location solutions: Take about 10 mg each of impurity L, impurity N, impurity O, impurity K, and impurity D reference substances, accurately weigh them, place them in different 10 mL volumetric flasks, dissolve them with acetonitrile-mobile phase A (60:40) and dilute them to the scale, shake them well, and use them as the stock solutions of each impurity; accurately measure 0.1 mL of each impurity stock solution, place them in different 100 mL volumetric flasks, dilute them to the scale with acetonitrile, shake them well, and obtain them.

[0130] Test solution: Take about 80 mg of the product, place it in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain the solution.

[0131] Add the test solution: Take about 80 mg of the product and place it in a 10 mL volumetric flask. Accurately add 0.1 mL each of the stock solutions of impurity L, impurity N, impurity O, impurity K, and impurity D. Dissolve with acetonitrile and dilute to the scale. Shake well to obtain the product.

[0132] According to the chromatographic conditions of Example 1, blank solution, i.e., acetonitrile-mobile phase A (60:40, mobile phase A is 0.1% phosphoric acid solution), each impurity location solution, test solution, and added test solution were injected respectively, and the chromatogram was recorded. Figure 5 、 Figure 6 and Figure 7 As shown in Table 2, the results show that all impurities can be well separated, and the separation degree is greater than 2.8.

[0133] Table 2: Specificity test results

[0134]

[0135]

[0136] 1.2 Limit of detection and limit of quantification

[0137] Take appropriate amounts of each reference substance for Impurity L, Impurity N, Impurity O, Impurity K, and Impurity D, gradually dilute them to low concentrations, inject them into a liquid chromatograph, and record the chromatograms. The concentration at which the signal-to-noise ratio (S / N) ≥ 3 is defined as the limit of detection, and the concentration at which the signal-to-noise ratio (S / N) ≥ 10 is defined as the limit of quantification. The results are shown in Table 3.

[0138] Table 3: Limit of detection and limit of quantification results

[0139]

[0140] The detection limit and quantification limit concentration of each impurity are both lower than the reporting limit concentration, which meet the requirements, demonstrating that the sensitivity of the present invention is good.

[0141] 1.3 Linearity and range

[0142] Linear stock solution: Accurately measure 1 mL each of impurity L, impurity N, impurity O, impurity K, and impurity D stock solutions, place them in the same 10 mL volumetric flask, dilute to the scale with acetonitrile, and shake well to obtain the product.

[0143] The limit of quantification concentration was taken as the linearity 1 solution.

[0144] Accurately measure 0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of the linear stock solution and place them in 10 mL volumetric flasks, respectively. Dilute to the scale with acetonitrile and shake well to prepare linear 2 (50%), linear 3 (100%), linear 4 (150%), and linear 5 (200%) solutions.

[0145] According to the chromatographic conditions of Example 1, with concentration (C) as the horizontal axis (X axis) and peak area (A) as the vertical axis (Y axis), linear regression analysis was performed. The linearity of each impurity was good within the concentration range of the quantitative limit to 200%, the Y-axis intercept was very small, and the systematic error was very small. The test results are shown in the instructions. Figure 9 、 Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 and Table 4.

[0146] Table 4: Linearity test results

[0147] name Quadratic equations R 25% of the 100% response value Concentration range Impurity K y=58.0677x+0.0018 0.9996 14.99 0.2086 μg / mL~2.0862 μg / mL Impurity L y=32.6806x-0.3183 1.0000 8.32 0.3086 μg / mL~2.0573 μg / mL Impurity O y=63.0058x-0.0569 0.9996 16.06 0.2059 μg / mL~2.0591 μg / mL Impurity N y=55.1904x-0.0411 0.9996 14.17 0.2074 μg / mL~2.0741 μg / mL Impurity D y=48.4201x+0.0724 0.9997 12.12 0.2496 μg / mL~1.9970 μg / mL

[0148] 1.4 Accuracy

[0149] Prepare two reference solutions in parallel according to the method described in Example 1.

[0150] 50% test solution: Take approximately 80 mg of the product and place it in a 10 mL volumetric flask. Accurately add 50 μL of each impurity stock solution. Dissolve in acetonitrile and dilute to the mark. Shake well. Prepare three replicates.

[0151] 100% test solution: Take approximately 80 mg of the product and place it in a 10 mL volumetric flask. Accurately add 100 μL of each impurity stock solution. Dissolve in acetonitrile and dilute to volume. Shake well. Prepare three replicates.

[0152] 150% test solution: Take approximately 80 mg of the product and place it in a 10 mL volumetric flask. Accurately add 150 μL of each impurity stock solution. Add acetonitrile and dilute to the mark. Shake well. Prepare three replicates.

[0153] Test solution: Take about 80 mg of this product, place it in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and obtain the product. According to the chromatographic conditions of Example 1, the recovery rate was calculated by the external standard method. The test results are shown in Table 5.

[0154] Table 5: Recovery test results

[0155]

[0156]

[0157]

[0158] As can be seen from the table above, the average sample recoveries of each impurity at the three concentration levels are all in the range of 80.0% to 120.0%, and the RSD% of the recovery results of the nine samples are all less than 10, confirming that the method has good accuracy.

[0159] 1.5 Repeatability

[0160] Prepare two reference solutions in parallel according to the method described in Example 1;

[0161] Prepare 6 test solution in parallel according to the method described in Example 1;

[0162] The detection was carried out according to the chromatographic conditions of Example 1, and the content of each impurity was calculated by the external standard method. The results are shown in Table 6.

[0163] Table 6: Repeatability test results

[0164]

[0165]

[0166] As can be seen from the above table, the RSD% of the contents of impurity L, impurity N, impurity O, impurity K, and impurity D are all less than 5.0, indicating that the method has good repeatability.

[0167] 1.6 Intermediate precision

[0168] An intermediate precision test was conducted on the same batch of samples using the repeatability test solution preparation method. Six parallel aliquots of the test solution were prepared using different instruments at different times, and the impurity content in the samples was determined. The intermediate precision of this method was evaluated based on the repeatability test results. The results are shown in Table 7.

[0169] Table 7: Intermediate precision test results

[0170]

[0171] As can be seen from the table above, the RSDs of the genotoxic impurity contents in the 12 samples were all within 8%, indicating that the intermediate precision of this method was good.

[0172] 1.7 Solution stability

[0173] The reference solution and the test solution were prepared according to the method described in Example 1, placed at room temperature, and measured at 0, 3, 6, 9, 12, and 24 hours according to the chromatographic conditions of Example 1. The results are shown in Tables 8 and 9.

[0174] Table 8: Reference solution

[0175]

[0176]

[0177] Table 9: Test solution

[0178] Time (h) Impurity K peak area Impurity L peak area Impurity O peak area Impurity N peak area Impurity D peak area 0 57.847 30.743 60.414 56.212 51.731 3 59.020 31.565 60.729 56.512 51.841 6 59.064 31.374 61.072 56.272 51.637 9 59.384 30.950 61.057 56.230 51.456 12 59.170 30.704 60.726 56.134 51.674 24 58.813 30.962 60.795 55.842 51.760 RSD (%) 1.0 1.2 0.5 0.4 0.3

[0179] The test results show that the peak area and content of impurities L, N, O, K and D in the reference solution and the sampled test solution did not change significantly within 24 hours, proving that the solution was stable within 24 hours.

[0180] 1.8 Durability

[0181] The changes in the concentrations of various genotoxic impurities in the sample solution and the resolution of the impurities were investigated under various conditions, including detection wavelength, column temperature, flow rate, phosphoric acid concentration, and the use of different chromatographic columns. Detailed results are shown in Table 10.

[0182] Table 10: Durability test results

[0183]

[0184]

[0185] The test results show that under the above detection conditions, the separation between impurities L, N, O, K and D is greater than 1.5; the RSD% of the change in the content of each impurity is within 10, indicating that the method has good durability.

[0186] The results of methodological validation show that the method for determining the contents of five genotoxic impurities in perampanel has strong specificity, high accuracy and precision, and good durability, and is suitable for accurate control of the five genotoxic impurities in perampanel.

[0187] Example 6

[0188] Sample measurement

[0189] Three batches (220916, 220919, 220920) of perampanel API were weighed respectively, and assayed according to the method described in Example 1. The chromatograms were recorded, and the contents of the five genotoxic impurities were calculated using the external standard method.

[0190] The results showed that impurity L, impurity N, impurity O, impurity K and impurity D were not detected in the three batches of perampanel raw materials.

[0191] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for detecting genotoxic impurities in perampanel raw materials, characterized in that: The method comprises detecting five genotoxic impurities in the perampanel raw material by high performance liquid chromatography, wherein the structures of the five genotoxic impurities are respectively as follows: Impurity D: ; Impurity K: ; Impurity L: ; Impurity N: ; Impurity O: ; Wherein, in the method, phosphoric acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and acetonitrile-mobile phase A is used as the solvent of the impurity reference substance; The volume ratio of acetonitrile and mobile phase A in the solvent of the impurity reference substance is 60:40; The detection conditions of the HPLC method are: Chromatographic column: C18 column Mobile phase A: 0.08-0.12% phosphoric acid solution Mobile phase B: acetonitrile Flow rate: 0.75-0.85 mL / min Column temperature: 23-27°C Detection wavelength: 208-212 nm Injection volume: 10 μL; The gradient elution method was adopted, and the gradient elution program was as follows: The initial ratio of mobile phase A and mobile phase B was 80:20; From 0 to 35 minutes, the proportion of mobile phase A was reduced, while the proportion of mobile phase B was increased to a ratio of 42:58; From min 35 to 45, the ratio of mobile phase A to mobile phase B was maintained at 42:58; From 45 to 50 minutes, the proportion of mobile phase A was increased, while the proportion of mobile phase B was decreased until it returned to the initial ratio of 80:20; From 50 to 60 minutes, the ratio of mobile phase A to mobile phase B was maintained at 80:

20.

2. The method according to claim 1, characterized in that The mobile phase A is 0.1% phosphoric acid solution.

3. The method according to any one of claims 1 to 2, characterized in that include: Take a sample of perampanel, accurately weigh it, dissolve it in acetonitrile and dilute it to prepare the test solution; Impurity L, impurity N, impurity O, impurity K, and impurity D are used as impurity reference substances, accurately weighed, placed in the same volumetric flask, dissolved in a mixture of acetonitrile and mobile phase A, and diluted to the mark to prepare a stock solution of the impurity reference substance solution; Accurately measure the reference solution stock solution into a volumetric flask and dilute to the mark with acetonitrile to prepare the reference solution; Accurately measure the test solution and reference solution, inject them into the liquid chromatograph for detection and record the chromatogram. Use the external standard method to calculate the content of the five genotoxic impurities in the test solution based on the peak area.

4. The method according to claim 3, characterized in that The linear range of impurity K was 0.2086 μg / mL~2.0862 μg / mL, the linear range of impurity L was 0.3086 μg / mL~2.0573 μg / mL, the linear range of impurity O was 0.2059 μg / mL~2.0591 μg / mL, the linear range of impurity N was 0.2074 μg / mL~2.0741 μg / mL, and the linear range of impurity D was 0.2496 μg / mL~1.9970 μg / mL.

5. Use of the method according to claim 1 in the quality control of perampanel bulk drug or its preparation.

6. The use according to claim 5, characterized in that Five substances are genotoxic impurities and are used as impurity reference substances in quality control.

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