Method for separating and determining Enzalocamide Z3 and genotoxic impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining Enzalocamide Z3 and genotoxic impurities thereof. The impurities comprise one or more of an impurity Xd, an impurity Xg and an impurity Xh. The method comprises the following steps: carrying out linear gradient elution of high performance liquid chromatography by taking octadecyl silane bonded silica gel as a chromatographic column filler, taking a trifluoroacetic acid solution as a mobile phase A and taking acetonitrile as a mobile phase B; and entering a detector for detection. And judging whether the content of each impurity in the sample is qualified or not by adopting a limit method according to the chromatogram. The method disclosed by the invention has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and determining enzalutamide Z3 and its genotoxic impurities. Background Technology

[0002] Enzalutamide, also known as enzalutamide, is an androgen receptor inhibitor jointly developed by Astellas and Medivation. Enzalutamide is primarily used to treat asymptomatic or mildly symptomatic metastatic castration-resistant prostate cancer that has failed androgen deprivation therapy and has not received chemotherapy. It inhibits the proliferation of prostate cancer cells and induces their death by competitively inhibiting the binding of androgens to androgen receptors.

[0003] Enzalutamide Z3 is a key intermediate in the synthesis of enzalutamide, and its structural formula is shown in Formula 1.

[0004]

[0005] Studies have revealed that enzalutamide Z3 may contain three genotoxic impurities with warning structures: Impurity X d Impurity X g and impurity X h To ensure the quality of active pharmaceutical ingredients and finished products, it is necessary to control these three genotoxic impurities. However, impurity X... d Impurity X g Impurity X h The analytical method is not included in any national pharmacopoeia, nor is it reported in the literature. Among existing technologies, patent CN106153772B discloses a method for detecting enzalutamide-related substances using high-performance liquid chromatography (HPLC). This patent uses pentafluorophenyl-bonded silica gel as the stationary phase and an acid-organic phase as the mobile phase with gradient elution; the mobile phase is a trifluoroacetic acid solution and acetonitrile. However, this method cannot detect impurity X in enzalutamide Z3. d Impurity X g and impurity X h Separation and detection.

[0006] Therefore, an impurity X in enzalutamide Z3 was established. d Impurity X g Impurity X h The separation and determination method is of great significance for achieving quality control of enzalutamide intermediate Z3 and enzalutamide finished product. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a method for separating enzalutamide Z3 and its genotoxic impurities based on high performance liquid chromatography, which can complete the separation of multiple substances in a short time.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A method for separating enzalutamide Z3 and its genotoxic impurities using high-performance liquid chromatography, wherein enzalutamide Z3 and the genotoxic impurities together constitute a composition, and the genotoxic impurities include impurity X. d Impurity X g Impurity X h The method comprises using octadecylsilane-bonded silica gel as the column packing material, trifluoroacetic acid solution as mobile phase A, acetonitrile as mobile phase B, and separating enzalutamide Z3 and its genotoxic impurities by linear gradient elution; the structural formula of each component in the composition is as follows:

[0010]

[0011] The separated material can be used in the next stage of production.

[0012] The aforementioned impurities can be arranged and combined in various ways.

[0013] For example, combination method 1: impurity X d and impurity X h .

[0014] For example, combination method 2: impurity X g and impurity X h .

[0015] For example, combination method 3: impurity X d Impurity X g and impurity X h .

[0016] All possible permutations and combinations will not be listed here. Theoretically, when the upper limit of the substances that this method can separate, identify and / or detect is n (where n is the number of substances), it can naturally detect 1 to n substances.

[0017] Preferably, the mobile phase A is a 0.05% trifluoroacetic acid solution.

[0018] As a preferred embodiment, an example of the preparation method for the trifluoroacetic acid solution in the mobile phase is as follows: measure 1000 ml of water, add 0.5 ml of trifluoroacetic acid, mix well, and the solution is obtained.

[0019] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0020] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 60±10 40±10 10±0.5 60±10 40±10 30±0.5 20±10 80±10 35±0.5 20±10 80±10 36±0.5 60±10 40±10 45±0.5 60±10 40±10 .

[0021] As a preferred method, the linear gradient elution procedure is as follows:

[0022] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 60±2 40±2 10 60 40±2 30 20 80 35 20 80 36 60±2 40±2 45 60±2 40±2 .

[0023] For example, at 0, 10, 36, and 45 minutes, the mobile phase A is set to 58 parts and the mobile phase B to 42 parts. Figure 6 ); or the mobile phase A accounts for 59 parts and the mobile phase B accounts for 41 parts; or the mobile phase A accounts for 60 parts and the mobile phase B accounts for 40 parts. Figure 3 ); or the mobile phase A accounts for 61 parts and the mobile phase B accounts for 39 parts; or the mobile phase A accounts for 62 parts and the mobile phase B accounts for 38 parts. Figure 7 ).

[0024] As the preferred option, the linear gradient elution procedure is as follows:

[0025] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 60 40 10 60 40 30 20 80 35 20 80 36 60 40 45 60 40 .

[0026] Furthermore, the flow rate was 0.8-1.2 mL / min; the column temperature was 20-30℃.

[0027] Preferably, the flow rate is 0.9-1.1 mL / min; the column temperature is 23-27 °C. For example, the flow rate is 0.9 mL / min ( Figure 8 ); or a flow rate of 1.0 mL / min ( Figure 3 ); or a flow rate of 1.1 mL / min ( Figure 9 For example, the column temperature is 23℃. Figure 10 ); or column temperature 24℃; or column temperature 25℃ Figure 3 ); or column temperature of 26℃; or column temperature of 27℃. Figure 11 ).

[0028] The optimal flow rate is 1.0 mL / min, and the column temperature is 25 °C.

[0029] Preferably, the chromatographic column has a size of 4.6 mm × 150 mm and a diameter of 5 μm.

[0030] As the optimal choice, the chromatographic column selected was a Shim-pack GIST C18 4.6mm×150mm, 5μm.

[0031] Preferably, the injection volume is 10 μl.

[0032] As a preferred option, the running time is 45 minutes.

[0033] Furthermore, the method is used to separate genotoxic impurities from enzalutamide Z3 and its related substances; the genotoxic impurities include impurity X. d Impurity Xg Impurity X h Any one or more of the following; the related substances include impurity X, impurity SM3, and impurity Z. 3a Impurity Z 3c Impurity Z 3g Impurity Z 3k Impurity Z 3m Impurity Z 3b Impurity Z 3l Impurity X a Impurity Z2, Impurity Z 3h Impurity Z 3q Impurity Z 3r Impurity Z 3s Impurity Z 3p Impurity Z 3j Any one or more of the following; the structural formulas of the relevant substances are as follows:

[0034]

[0035] The second objective of this invention is to provide a method for identifying enzalutamide Z3 and its genotoxic impurities, which can complete the identification of multiple substances in a short time.

[0036] To achieve the above objectives, the technical solution of the present invention is as follows:

[0037] A method for identifying enzalutamide Z3 and its genotoxic impurities involves separating the composition using the aforementioned separation method and detecting it in a detector with a detection wavelength of 275±10 nm to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains enzalutamide Z3 and its genotoxic impurities.

[0038] The ±10nm setting range for the detection wavelength is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This setting range helps ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0039] As the most preferred option, the detection wavelength of the detector is 275 nm.

[0040] Preferably, the detector is an ultraviolet detector.

[0041] As a preferred embodiment, the components in the composition can be identified according to the order of retention time, wherein the components of the composition are in ascending order as follows: impurities X g Impurity X h Impurity X d .

[0042] Retention time is used to determine the components. Retention time refers to the time required for a sample to travel from the point of entry into the chromatographic column to detection by the detector. This time is calculated based on the migration rate of the components on the column, i.e., the time interval from the start of injection to the chromatographic peak (maximum concentration) of a particular component. It is primarily used to determine the elution order and position of each component in the sample and is one of the fundamental data points in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0043] As a preferred method, a retention time of 7.9 ± 0.5 min is used to classify it as impurity X. g The retention time was 17.7 ± 0.5 min, and it was determined to be impurity X. h The retention time was 22.8 ± 0.5 min, and it was determined to be impurity X. d .

[0044] The third objective of this invention is to provide a method for detecting whether the content of genotoxic impurities in enzalutamide Z3 is up to standard. This method can determine whether the content of genotoxic impurities in enzalutamide Z3 is up to standard in a relatively short time.

[0045] To achieve the above objectives, the technical solution of the present invention is as follows:

[0046] The method for determining whether the content of genotoxic impurities in enzalutamide Z3 is within acceptable limits includes the following steps:

[0047] (1) Enzalutamide Z3 and its genotoxic impurities were separated and identified using the aforementioned identification method, and chromatograms were obtained;

[0048] (2) Based on the chromatogram obtained in step (1), determine whether the impurity content in the sample is qualified;

[0049] If impurity X in the sample is g Impurity X h and / or impurity X d If the peak area of ​​the sample is not greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is within acceptable limits; conversely, if the peak area of ​​impurity X in the sample is greater than the peak area of ​​the reference solution, it indicates that the impurity content is within acceptable limits. g Impurity X h and / or impurity X d If the peak area of ​​the impurity is larger than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is unqualified.

[0050] Furthermore, the impurity limit is 0.003%.

[0051] The aforementioned judgment method can serve as a drug quality assessment model and further as an indispensable key module in intelligent production processes. This model, through precise control of parameters such as mobile phase composition, flow rate, and column temperature, achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive drug quality assessment. In intelligent production systems, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically executing analysis tasks, and rapidly providing judgment results based on preset quality standards.

[0052] As a preferred option, the solvent for sample preparation is tetrahydrofuran.

[0053] As a preferred option, the concentration of the sample solution is 5 mg / mL.

[0054] As a preferred embodiment, the method includes the following steps:

[0055] Step 1. Take an appropriate amount of enzalutamide Z3, accurately weigh it, dissolve and dilute it with tetrahydrofuran to prepare a solution containing about 5 mg per 1 ml, as the sample solution;

[0056] Step 2. Take impurity X d Impurity X g Impurity X h Weigh an appropriate amount accurately, dissolve and dilute in tetrahydrofuran to prepare a solution containing approximately X impurities per 1 ml. d Impurity X g Impurity X h Each solution, approximately 0.15 μg / ml, was used as a reference solution;

[0057] Step 3. Set the chromatographic conditions as follows:

[0058] A chromatographic column with dimensions of 4.6 mm × 150 mm and a diameter of 5 μm, packed with octadecylsilane-bonded silica gel, was used. A linear gradient elution was performed using 0.05% trifluoroacetic acid solution as mobile phase A and acetonitrile as mobile phase B. The flow rate was 0.9–1.1 mL / min; the column temperature was 23–27 °C; the detection wavelength was 275 nm; and the injection volume was 10 μL. The linear gradient elution program was set as follows:

[0059] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 60±2 40±2 10 60 40±2 30 20 80 35 20 80 36 60±2 40±2 45 60±2 40±2 ;

[0060] Step 4. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms;

[0061] Step 5. Based on the obtained chromatogram, use the limit method to determine the impurity X in the sample. g Impurity X h and / or impurity X dWhether the content is up to standard.

[0062] The beneficial effects of this invention are as follows:

[0063] 1. Currently, there is no effective way to control impurity X. d Impurity X g Impurity X h This invention utilizes impurity X... d Impurity X g Impurity X h Using a specific UV absorption wavelength of 275 nm, selecting a reagent with ion-enhancing properties (trifluoroacetic acid), screening chromatographic columns and conditions, and establishing an HPLC method, impurity X was successfully removed. d Impurity X g Impurity X h This analytical method can effectively separate impurities such as enzalutamide Z3 and other known impurities, and can meet the requirements of impurity X. d Impurity X g Impurity X h Its detection is specific, sensitive, and durable, ensuring symmetrical peaks and no solvent effect.

[0064] 2. This invention selects tetrahydrofuran as a solvent, effectively ensuring the protection of impurity X. d Impurity X g Impurity X h This method improves solution stability while reducing solvent effects. It is highly specific, sensitive, reproducible, economical, and robust.

[0065] 3. The method of this invention has the characteristic of high sensitivity. Impurity X g The detection limit concentration is 0.0447 μg / ml; impurity X h The detection limit concentration is 0.0446 μg / ml; impurity X d The detection limit concentration is 0.0446 μg / ml. Attached Figure Description

[0066] Figure 1 The chromatogram is for a blank solution;

[0067] Figure 2 The chromatogram is of the reference solution;

[0068] Figure 3 The chromatogram is of the mixed solution;

[0069] Figure 4 The chromatogram of the test solution;

[0070] Figure 5 Chromatogram of the solution at the detection limit;

[0071] Figure 6Chromatogram of the mixed solution for the durability test - under the condition of an initial mobile phase ratio of 58:42;

[0072] Figure 7 Chromatogram of the mixed solution for a durability test with an initial mobile phase ratio of 62:38;

[0073] Figure 8 Chromatogram of the mixed solution at a flow rate of 0.9 mL / min for the durability test;

[0074] Figure 9 Chromatogram of the mixed solution at a flow rate of 1.1 mL / min for the durability test;

[0075] Figure 10 Chromatogram of the mixed solution at a column temperature of 23°C for robustness testing;

[0076] Figure 11 Chromatogram of the mixed solution at a column temperature of 27°C for robustness testing. Detailed Implementation

[0077] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0078] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figures 1-11 Visual aids are provided for understanding and explanation. If anything is unclear, the user should refer to the corresponding numbered table. Figures 1-11 The integral results correspond to Tables 1-11 respectively, for more detailed information. Conversely, if reviewing Tables 1-11... Figure 11 If any information that may cause misunderstanding or ambiguity is found during the process, the content of the corresponding numbered figure shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some text overlaps in the spectra of this application, it is still clearly visible, and the specification details the integration results of each figure. Furthermore, the numbers in the spectra do not affect the scope of protection of the claims or the full disclosure of the technical solutions in the specification.

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] Table 3

[0084]

[0085] Table 4

[0086]

[0087] Table 5

[0088]

[0089] Table 6

[0090]

[0091] Table 7

[0092]

[0093] Table 8

[0094]

[0095] Table 9

[0096]

[0097] Table 10

[0098]

[0099] Table 11

[0100]

[0101] In Tables 3 and 6 to 11 above, serial number 5 is enzalutamide Z3.

[0102] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference standards, and test items described, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to illustrate specific embodiments and not to impose a limiting interpretation.

[0103] Furthermore, all references cited in this document, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited references or similar materials and this application, particularly regarding terminology definitions, usage, or technical descriptions, the content of this application shall prevail.

[0104] the term

[0105] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement result should have a certain degree of accuracy and precision. In other words, the LQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LQ usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the analyte corresponding to a certain level of signal-to-noise ratio is taken as the LQ. Determining the LQ is crucial for ensuring the accuracy and reliability of analytical results.

[0106] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and unaffected analytical results when measurement conditions are slightly changed. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0107] The limit of detection (LOD) is the lowest concentration or amount of an analyte in a sample that can be detected. It reflects the sensitivity and noise level of the analytical method and instrument, and also indicates the level of the blank (background) value after sample processing.

[0108] A correction factor is a coefficient or parameter used to correct analytical results. It aims to improve data accuracy and reliability. In HPLC analysis, because the same detector responds differently to different substances, peak areas produced when the same mass of different substances passes through the detector may not be equal. To ensure that the peak area accurately reflects the content of the analyte, standard substances are used for correction, a correction factor is calculated, and this factor is applied to the measurement results of the sample.

[0109] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in high-performance liquid chromatography (HPLC) to evaluate the detection sensitivity and resolution of an instrument, and is an important indicator of instrument performance. Peak height refers to the signal value output by the detector when the analyte elutes from the column; noise refers to the fluctuation of the baseline signal, i.e., the signal value measured for a blank sample. The signal-to-noise ratio is the ratio of the signal measured for a sample of known concentration to the signal measured for a blank sample. A higher signal-to-noise ratio means that the instrument can more accurately separate and identify the target component when detecting samples, while also reducing interference from background noise.

[0110] In this embodiment of the invention, information on enzalutamide Z3 and its related impurities is shown in Table A.

[0111] Table A. Compound Information Table

[0112]

[0113] In this embodiment of the invention, the structural information of other impurities is shown in Table B.

[0114] Table B. Information on the structural formulas of other impurities

[0115]

[0116]

[0117] In this embodiment of the invention, the analytical method for genotoxic impurities in enzalutamide Z3 is as follows:

[0118] (1) Preparation of the test solution

[0119] Test solution: Weigh an appropriate amount of this product (enzalutamide Z3) accurately, dissolve and dilute it in tetrahydrofuran to prepare a solution containing approximately 5 mg per ml.

[0120] Reference solution: Take impurity X d Impurity X g Impurity X h Weigh an appropriate amount accurately, dissolve and dilute in tetrahydrofuran to prepare a solution containing approximately X impurities per 1 ml. d Impurity X g Impurity X h Each solution is approximately 0.15 μg / ml.

[0121] (2) Chromatographic conditions

[0122] The column was packed with octadecylsilane-bonded silica gel (Shim-pack GIST C18 4.6mm × 150mm, 5μm or equivalent performance column); mobile phase A was 0.05% trifluoroacetic acid solution (1000ml water, 0.5ml trifluoroacetic acid was added) and mobile phase B was acetonitrile, and linear gradient elution was performed according to Table C; the flow rate was 1.0ml per minute; the column temperature was 25℃; the detection wavelength was 275nm; and the injection volume was 10μl.

[0123] Table C. Gradient Elution Procedure

[0124] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 60 40 10 60 40 30 20 80 35 20 80 36 60 40 45 60 40

[0125] (3) Measurement

[0126] Accurately measure the test solution and reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0127] (4) Limits

[0128] In the chromatogram of the test solution, if there is an impurity X that is similar to that in the reference solution... d Impurity X g Impurity X h The chromatographic peaks with consistent retention times must have peak areas greater than that of impurities X in the reference solution. d Impurity X g Impurity X h The peak area is 0.003%.

[0129] In this invention, according to the FDA instructions for the related formulation, enzalutamide soft capsules, this product is a cancer treatment drug with a maximum daily dose of 160 mg / day (40 mg × 4 times) and a median survival of 18.4 months. Therefore, the TTC is calculated at 10 μg / day, and the limit is 62.5 ppm. This invention, based on ICH Q3A(R2) and M7 requirements and the EMA review report, strictly controls the content, raising the limit for warning structural impurities to 30 ppm. When impurity X in enzalutamide Z3... d Impurity X g and impurity X h When the content of [specific substance] meets the limit requirements, it indicates that the impurity X in the sample is [not specified]. d Impurity X g and impurity X h The content is within acceptable limits.

[0130] In the embodiments of this invention, if there are any chromatographic conditions not mentioned, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512).

[0131] Example 1. Specificity

[0132] (1) Preparation of the test solution

[0133] Blank solvent: Tetrahydrofuran

[0134] Impurity X g Stock solution: Accurately weigh impurity X g 15 mg, placed in a 100 ml volumetric flask, dissolved in tetrahydrofuran and diluted to the mark, then shaken well.

[0135] Impurity X h Stock solution: Accurately weigh impurity X h 15 mg, placed in a 100 ml volumetric flask, dissolved in tetrahydrofuran and diluted to the mark, then shaken well.

[0136] Impurity X d Stock solution: Accurately weigh impurity X d15 mg, placed in a 100 ml volumetric flask, dissolved in tetrahydrofuran and diluted to the mark, then shaken well.

[0137] Impurity X d Positioning solution: Accurately measure impurity X d Transfer 1.0 ml of the stock solution to a 100 ml volumetric flask, dilute to the mark with tetrahydrofuran, and mix well. (1.5 μg / ml)

[0138] Impurity X g Positioning solution: Accurately measure impurity X g Transfer 1.0 ml of the stock solution to a 100 ml volumetric flask, dilute to the mark with tetrahydrofuran, and mix well. (1.5 μg / ml)

[0139] Impurity X h Positioning solution: Accurately measure impurity X h Transfer 1.0 ml of the stock solution to a 100 ml volumetric flask, dilute to the mark with tetrahydrofuran, and mix well. (1.5 μg / ml)

[0140] Mixed impurity stock solution: Accurately measure impurity X d Stock solution, impurities X g Stock solution, impurities X h Place 1.0 ml of each stock solution into the same 100 ml volumetric flask, dilute to the mark with tetrahydrofuran, and mix well. (1.5 μg / ml)

[0141] Reference solution: Accurately measure 1.0 ml of the mixed impurities stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with tetrahydrofuran and mix well. (0.15 μg / ml)

[0142] Location of other impurities: Accurately weigh and measure impurity X, impurity SM3, and impurity Z. 3a Impurity Z 3c Impurity Z 3g Impurity Z 3k Impurity Z 3m Impurity Z 3b Impurity Z 3l Impurity X a Impurity Z2, Impurity Z 3h Impurity Z 3q Impurity Z 3r Impurity Z 3s Impurity Z 3p Impurity Z 3j Place 5 mg of each ingredient in the same 50 ml volumetric flask, dissolve and dilute with tetrahydrofuran to the mark, and shake well.

[0143] Test solution: Accurately weigh 50 mg of the test sample, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with tetrahydrofuran, and shake well. (5 mg / ml test sample)

[0144] Mixed solution: Accurately weigh 50 mg of the test sample and place it in a 10 ml volumetric flask. Accurately measure 1.0 ml of the other impurity positioning solution and 2.0 ml of the mixed impurity stock solution and place them in the same volumetric flask. Dissolve and dilute with tetrahydrofuran to the mark and shake well.

[0145] (2) Detection

[0146] Take blank solvent and impurity X respectively d Positioning solution, impurity X g Positioning solution, impurity X h Inject 10 μl each of the positioning solution, other impurity positioning solution, reference solution, test solution, and mixed solution according to the method, and record the chromatogram.

[0147] The measurement results are shown in Table D. Figures 1-4 Tables 1 to 4. The blank solvent does not interfere with the detection of the sample. The resolution between the main peak and the adjacent impurity peak, and between each known impurity and the adjacent impurity peak, is 1.72. The method specificity meets the requirements.

[0148] Table D. Results of Specificity Test

[0149]

[0150] Example 2. Detection Limit

[0151] Reference solution: Prepared according to the preparation method of the reference solution under “Example 1. Specificity”.

[0152] Detection limit solution: Accurately measure 3.0 ml of the reference solution, place it in a 10 ml volumetric flask, dilute with tetrahydrofuran to the mark, and shake well.

[0153] Inject the solution at the detection limit three times consecutively and calculate the ratio of the main peak height to the noise (signal-to-noise ratio).

[0154] The test results are shown in Table E. Figure 5 As shown in Table 5. Impurity X g The detection limit was 0.00447 μg / ml, expressed as 0.0009% in the sample, with a mean signal-to-noise ratio of 8.8; impurity X h The detection limit was 0.0446 μg / ml, which is expressed as 0.0009% in the sample, with a mean signal-to-noise ratio of 6.1; impurity X d The detection limit concentration was 0.0446 μg / ml, which is expressed as 0.0009% in the sample, and the mean signal-to-noise ratio was 6.8; meeting the requirement of S / N≥3:1 (N=3). The detection limit test met the requirements.

[0155] Table E. Results of Limit of Detection

[0156]

[0157] Example 3. Durability

[0158] Mixed solution: Prepared according to the preparation method of the mixed solution under “Example 1. Specificity”.

[0159] Take the mixed solution and test it using the normal mobile phase, different proportions of the mobile phase to be tested, column temperature, column flow rate, and different batches of the same model of chromatographic column. After the instrument system is stable, test it separately and record the resolution between each peak.

[0160] The test results are shown in Table F. Figure 3 , Figures 6 to 11 Tables 3 and 6 to 11. When there are slight fluctuations in the flow rate, column temperature, and mobile phase ratio in the chromatographic conditions, impurity X... g Impurity X h Impurity X d The separation degree between the peak and the adjacent peak is greater than 1.2, indicating that the method of the present invention has good robustness.

[0161] Table F. Results of the durability test for changes in chromatographic conditions

[0162]

Claims

1. A method for separating enzalutamide Z3 and its genotoxic impurities based on high performance liquid chromatography, characterized in that, The enzalutamide Z3 and the genotoxic impurities collectively make up a composition, the genotoxic impurities comprising one or more of impurity Xd, impurity Xg, impurity X h ; the method comprising using octadecylsilane bonded silica gel as a chromatographic column filler, trifluoroacetic acid solution as mobile phase A, acetonitrile as mobile phase B, and separating enzalutamide Z3 and its genotoxic impurities by linear gradient elution; the structural formula of each component in the composition is as follows:

2. The method of claim 1, wherein, The procedure for linear gradient elution is as follows: 。 3. The method of claim 1, wherein, The procedure for linear gradient elution is as follows: 。 4. The method of claim 1, wherein, The flow rate is 0.8-1.2 mL / min; the column temperature is 20-30℃.

5. The method of claim 1, wherein, Genotoxic impurities in enzalutamide Z3 and related substances thereof isolated using the method; the genotoxic impurities include any one or more of impurity X d , impurity X g , impurity X h ; the related substances include any one or more of impurity X, impurity SM3, impurity Z 3a , impurity Z 3c , impurity Z 3g , impurity Z 3k , impurity Z 3m , impurity Z 3b , impurity Z 3l , impurity X a , impurity Z2, impurity Z 3h , impurity Z 3q , impurity Z 3r , impurity Z 3s , impurity Z 3p , impurity Z 3j ; the structures of the related substances are as follows:

6. A method of discriminating between amisulpride Z3 and its genotoxic impurities, characterized in that, The composition is separated using the method described in any one of claims 1-4 and detected by a detector with a detection wavelength of 275±10nm to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains enzalutamide Z3 and its genotoxic impurities.

7. The method of claim 56, wherein, In order of retention time, the components of the composition can be identified as follows: impurity X g , impurity X h , impurity X d .

8. The method of claim 6, wherein, Retention time 7.9 ± 0.5 min, identified as impurity X g Retention time 17.7 ± 0.5 min, identified as impurity X h Retention time 22.8 ± 0.5 min, identified as impurity X d .

9. A method for determining whether the content of genotoxic impurities in enzalutamide Z3 is up to standard, characterized in that, Includes the following steps: (1) Enzalutamide Z3 and its genotoxic impurities are separated and identified by the method described in any one of claims 6-8, and a chromatogram is obtained; (2) Based on the chromatogram obtained in step (1), determine whether the impurity content in the sample is qualified; If the peak area of impurity X g , impurity X h and / or impurity X d in the test sample is not greater than the peak area of the corresponding impurity in the reference solution, it indicates that the impurity content is qualified; otherwise, if the peak area of impurity X g , impurity X h and / or impurity X d in the test sample is greater than the peak area of the corresponding impurity in the reference solution, it indicates that the impurity content is unqualified.

10. The method according to claim 9, characterized in that, The solvent used to prepare the sample was tetrahydrofuran.

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