HPLC (High Performance Liquid Chromatography) method for separating and determining Enzalocamide SM3 and related impurities thereof

Abstract

The invention belongs to the technical field of chemical analysis, and particularly relates to an HPLC (High Performance Liquid Chromatography) method for separating and determining Enzalocamide SM3 and related impurities thereof. According to the self-built high performance liquid chromatography method, octadecylsilane chemically bonded silica is adopted as a chromatographic column filler, a mobile phase A and a mobile phase B are adopted as mobile phases, and an impurity SM3c, an impurity SM3d, an impurity SM3b, an enzalocamide SM3, an impurity SM3j, an impurity SM3h, an impurity SM3f, an impurity SM3a, an impurity SM3i, an impurity SM3g and an impurity SM3e are sequentially separated through gradient elution; then entering a detector for detection; wherein the mobile phase A is a trifluoroacetic acid aqueous solution with the volume fraction of 0.05%, and the mobile phase B is acetonitrile; the detection wavelength is 254 nm. The analysis method provided by the invention has the characteristics of strong specificity, high sensitivity and good durability.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and specifically relates to an HPLC method for separating and determining enzalutamide SM3 and related impurities thereof. Background Art

[0002] Enzalutamide is an androgen receptor inhibitor, mainly used to treat advanced castration-resistant prostate cancer, and can effectively prolong the survival of patients. Enzalutamide can competitively inhibit the binding of androgens to receptors, and can inhibit the nuclear transport of androgen receptors and the interaction between the receptor and DNA.

[0003] SM3 is the starting material for the synthesis of enzalutamide. Its chemical name is 4-amino-2-(trifluoromethyl)benzonitrile, its molecular formula is C8H5F3N2, and its structural formula is shown in Formula I. Studies have found that the impurity SM3 may exist in the starting material of enzalutamide. 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i 、Impurity SM 3j 、Impurity SM 3k and impurity SM 2a There are 12 impurities in total, and the presence of these impurities directly affects the yield and purity of the final product enzalutamide.

[0004]

[0005] In the previous study, we explored the analytical methods of other impurities in enzalutamide and enzalutamide raw material SM3 and applied for patents, including invention patents with application numbers 202211214015.9 and 202211214058.7. Among them, patent 202211214015.9 proposed to use water as mobile phase A and methanol as mobile phase B to separate and determine enzalutamide raw material SM3 and impurity SM 3k and 10 other known impurities; Patent 202211214058.7 uses trifluoroacetic acid aqueous solution as mobile phase A and a mixed solution of acetonitrile / methanol / tetrahydrofuran as mobile phase B to separate and determine 26 impurities in enzalutamide and / or enzalutamide Z3. Although the above patents disclose enzalutamide raw material SM3 and its analysis method, it is impossible to achieve the impurity SM3 in enzalutamide raw material SM3. 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and impurity SM 3j Separation determination.

[0006] Currently, there is no method to convert the enzalutamide starting material SM3 into SM 3a , SM 3b , SM 3c There is no statutory analytical method for separating impurities such as SM3 and other impurities, and no relevant literature analysis data has been retrieved. Therefore, it is necessary to further explore a simple and effective method to achieve the separation of SM3 in the starting material of enzalutamide. 3a , SM 3b , SM 3c Separation and determination of impurities. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating enzalutamide SM3 and its related impurities using an HPLC method, which can simultaneously separate the enzalutamide starting material SM3 and the impurity SM 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and impurity SM 3j The method has high sensitivity and good durability, providing technical support for the subsequent qualitative and quantitative analysis of related impurities.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Method for separating enzalutamide SM3 and its related impurities using HPLC method, wherein the related impurities are impurities SM 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and impurity SM 3j Any one or more of; the structural formula of the enzalutamide SM3 is shown in Formula I; the impurity SM 3aThe structural formula is shown in Formula II, and the impurity SM 3b The structural formula is shown in Formula III, and the impurity SM 3c The structural formula is shown in Formula IV, and the impurity SM 3d The structural formula is shown in Formula V, and the impurity SM 3e The structural formula of is shown in Formula VI, the impurity SM 3f The structural formula is shown in Formula VII, and the impurity SM 3g The structural formula is shown in Formula VIII, the impurity SM 3h The structural formula is shown in Formula IX, and the impurity SM 3i The structural formula is shown in Formula X, and the impurity SM 3j The structural formula is shown in Formula Ⅺ; the HPLC method uses octadecylsilane bonded silica gel as the stationary phase, mobile phase A and mobile phase B as the mobile phase, and the impurity SM is sequentially removed by the gradient elution. 3c 、Impurity SM 3d 、Impurity SM 3b , Enzalutamide SM3, Impurity SM 3j 、Impurity SM 3h 、Impurity SM 3f 、Impurity SM 3a 、Impurity SM 3i 、Impurity SM 3g and impurity SM 3e For separation; the mobile phase A is trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile;

[0010]

[0011]

[0012] Further, the gradient elution program is:

[0013] 0min, the volume ratio of the mobile phase A to the mobile phase B is 79-81:21-19;

[0014] 3min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0015] 15min, the volume ratio of the mobile phase A to the mobile phase B is 60:40;

[0016] 40min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0017] 45min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0018] 46min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0019] 55min, the volume ratio of the mobile phase A to the mobile phase B is 80:20.

[0020] Preferably, the gradient elution procedure is:

[0021] 0min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0022] 3min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0023] 15min, the volume ratio of the mobile phase A to the mobile phase B is 60:40;

[0024] 40min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0025] 45min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0026] 46min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0027] 55min, the volume ratio of the mobile phase A to the mobile phase B is 80:20.

[0028] Furthermore, the volume fraction of the trifluoroacetic acid aqueous solution is 0.05%.

[0029] Furthermore, the flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min, preferably 1.0 ml / min; the column temperature of the chromatographic column is 28°C-32°C, preferably 30°C.

[0030] Furthermore, the injection volume was 20 μl.

[0031] The second object of the present invention is to provide a method for identifying enzalutamide SM3 and its related impurities based on the method described in the first object, which realizes the separation of enzalutamide starting material SM3 and impurity SM 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and / or impurities SM 3j Qualitative identification of.

[0032] To achieve the above object, the present invention adopts the following technical solutions:

[0033] A method for identifying enzalutamide SM3 and its related impurities, comprising separating the enzalutamide SM3 and its related impurities using the method described in the first objective, and detecting them using a detector; the detection wavelength of the detector is 254 nm.

[0034] The retention times of the 11 components from short to long are as follows: Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3b , Enzalutamide SM3, Impurity SM 3j 、Impurity SM 3h 、Impurity SM 3f 、Impurity SM 3a 、Impurity SM 3i 、Impurity SM 3g and impurity SM 3e ; The qualitative identification of enzalutamide SM3 and its related impurities can be achieved based on the length of retention time.

[0035] As a preferred technical solution, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume fraction of 0.05%, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 1.0 ml / min, the column temperature of the chromatographic column is 30° C.; the detection wavelength is 254 nm; the specifications of the chromatographic column are 4.6 mm×150 mm×3 μm; elution is performed according to a gradient elution program and a chromatogram is obtained, and the gradient elution program is set as follows:

[0036] 0min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0037] 3min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0038] 15min, the volume ratio of the mobile phase A to the mobile phase B is 60:40;

[0039] 40min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0040] 45min, the volume ratio of the mobile phase A to the mobile phase B is 30:70;

[0041] 46min, the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0042] 55min, the volume ratio of the mobile phase A to the mobile phase B is 80:20.

[0043] Furthermore, when the retention time was 2.7±0.5min, it was judged to be impurity SM 3c; When the retention time is 9.0±0.5min, it is judged to be impurity SM 3d ; When the retention time is 15.0±0.5min, it is judged to be impurity SM 3b ; When the retention time is 18.6±0.5min, it is judged to be enzalutamide SM3; when the retention time is 21.4±0.5min, it is judged to be impurity SM 3j ; When the retention time is 21.9±0.5min, it is judged to be impurity SM 3h ; When the retention time is 23.0±0.5min, it is judged to be impurity SM 3f ; When the retention time is 26.2±0.5min, it is judged to be impurity SM 3a ; When the retention time is 28.4±0.5min, it is judged to be impurity SM 3i ; When the retention time is 30.3±0.5min, it is judged to be impurity SM 3g ; When the retention time is 30.9±0.5min, it is judged to be impurity SM 3e .

[0044] The third object of the present invention is to provide a method for detecting the content of enzalutamide SM3 and its related impurities. The method of the present invention can detect the content of enzalutamide starting material SM3 and impurity SM3 within 55 minutes or more. 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and / or impurities SM 3j The method is simple, rapid, specific and sensitive.

[0045] To achieve the above object, the present invention adopts the following technical solutions:

[0046] The method for detecting the content of enzalutamide SM3 and its related impurities comprises the following steps:

[0047] (1) Separation: Separating enzalutamide SM3 and its related impurities using the method described in objective 1;

[0048] (2) Identification: Detect enzalutamide SM3 and its related impurities using the method described in Objective 2 and obtain a chromatogram;

[0049] (3) Content calculation: Based on the chromatogram obtained in step (2), the content of each impurity is calculated using the peak area using the principal component self-comparison method with the addition of a correction factor.

[0050] Furthermore, before separation, a solvent is used to prepare a test solution, a control solution and a system suitability solution; the solvent is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water in the solvent is 50:50; the specific method is as follows:

[0051] 1. Prepare sample solution: Take an appropriate amount of this product, weigh it accurately, dissolve it in solvent and dilute it to make a solution with a concentration of about 1 mg / ml as the sample solution.

[0052] 2. Prepare control solution: accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, shake well, as the control solution.

[0053] 3. Detection: Accurately measure the test solution and control solution, such as 20 μl, and inject them into the liquid chromatograph respectively, and record the chromatogram; then use the principal component self-reference method with correction factor to calculate the content of each impurity using the peak area.

[0054] Further, the impurity content calculation formula is as follows:

[0055] 1.

[0056] 2.

[0057] In the formula, A 杂质 is the impurity peak area;

[0058] A 对照 is the peak area of ​​the self-control solution;

[0059] f is the correction factor for known impurities.

[0060] Furthermore, the impurity SM 3c The correction factor is 0.57, and the impurity SM 3d The correction factor is 0.80, the impurity SM 3b The correction factor is 2.4, the impurity SM 3j The correction factor is 0.65, and the impurity SM 3h The correction factor is 0.48, and the impurity SM 3a The correction factor is 0.40, the impurity SM 3g The correction factor is 0.71, and the impurity SM 3e The correction factor is 1.2, the impurity SM 3f The correction factor is 0.64, and the impurity SM 3i The correction factor is 1.9.

[0061] The beneficial effects of the present invention are:

[0062] The method of the present invention adopts a high-sensitivity and high-durability chromatographic method, which can well separate the main peak of the test sample and 10 known impurities that may exist in the test sample within 55 minutes, and provides the problem of separation and determination of the enzalutamide starting material SM3 and possible impurities that cannot be solved in the prior art; the analytical method proposed by the present invention is short in time, high in sensitivity, strong in specificity, good in reproducibility, simple and feasible in operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is the chromatogram of the blank solution;

[0064] Figure 2 is the chromatogram of the test solution;

[0065] Figure 3 is the chromatogram of the mixed solution;

[0066] Figure 4 is the chromatogram of the quantitation limit solution;

[0067] Figure 5 is the chromatogram of the detection limit solution;

[0068] Figure 6 For durability - mixed solution - chromatogram under normal conditions;

[0069] Figure 7 This is the chromatogram under the conditions of durability-mixed solution-flow rate of 0.9 ml / min;

[0070] Figure 8 This is the chromatogram under the conditions of durability-mixed solution-flow rate of 1.1 ml / min;

[0071] Fig. 9 This is the chromatogram under the conditions of durability-mixed solution-column temperature of 28°C;

[0072] Fig.10 This is the chromatogram under the conditions of durability-mixed solution-column temperature of 32°C;

[0073] Fig.11 This is the chromatogram under the condition of durability-mixed solution-mobile phase initial ratio of 81:19;

[0074] Fig.12 This is the chromatogram under the condition of the initial ratio of the robustness-mixed solvent-mobile phase being 79:21. DETAILED DESCRIPTION

[0075] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0076] In the embodiment of the present invention, the solvent is a mixed solution of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 50:50.

[0077] In the embodiment of the present invention, the preparation method of 0.05% trifluoroacetic acid aqueous solution is as follows: 1000 ml of water is measured, and 0.5 ml of trifluoroacetic acid is added to obtain the solution.

[0078] Example 1

[0079] 1. Configure the sample to be tested

[0080] Test solution: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and dilute to make a solution with a concentration of about 1 mg / ml.

[0081] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, shake well.

[0082] System suitability solution: Weigh the enzalutamide SM3 system suitability reference (containing enzalutamide SM3, impurity SM 3c 、Impurity SM 3d 、Impurity SM 3b 、Impurity SM 3j 、Impurity SM 3h 、Impurity SM 3a 、Impurity SM 3g ) is added with a solvent to dissolve and dilute to prepare a solution with a concentration of about 1 mg / ml.

[0083] 2. Chromatographic conditions

[0084] Use octadecylsilane bonded silica gel as filler (YMC-Triart C18 ExRS 4.6mm×150mm, 3μm, or chromatographic column with equivalent performance), 0.05% trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and perform linear gradient elution according to Table 1; the flow rate is 1.0ml per minute; the column temperature is 30℃; the detection wavelength is 254nm; the injection volume is 20μl.

[0085] Table 1. Gradient elution program

[0086] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 3 80 20 15 60 40 40 30 70 45 30 70 46 80 20 55 80 20

[0087] 3. Detection

[0088] Determination method: Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0089] It should be noted that in the system suitability solution chromatogram, impurity SM 3j With impurity SM 3h The peak separation should not be less than 1.0. In the chromatogram of the test solution, except for the solvent peak and the gradient elution peak, the impurity content should comply with the provisions of Table 2 according to the main component self-reference method with the correction factor, and the total amount of impurities should not exceed 1.0%. The chromatographic peaks in the chromatogram of the test solution that are less than 0.25 times the main peak area of ​​the reference solution are ignored (0.05%).

[0090] Table 2. Impurity content requirements

[0091] name Relative retention time Correction Factor limit <![CDATA[Impurity SM 3c > 0.15 0.57 0.15％ <![CDATA[Impurity SM 3d > 0.49 0.80 0.15％ <![CDATA[Impurity SM 3b > 0.81 2.4 0.15％ <![CDATA[Impurity SM 3j > 1.16 0.65 0.15％ <![CDATA[Impurity SM 3h > 1.18 0.48 0.15％ <![CDATA[Impurity SM 3a > 1.42 0.40 0.15％ <![CDATA[Impurity SM 3g > 1.64 0.71 0.15％ Other single impurities - 1.0 0.2％

[0092] Note: The relative retention times in the above table are for reference only. The peaks should be located using the chromatogram of the system suitability solution.

[0093] Impurity SM 3f 、Impurity SM 3i 、Impurity SM 3e As a potential impurity, it is only studied in this analytical method and not included in the standard. 3f The relative retention time is 1.24, the correction factor is 0.64, and the limit is 0.15%; the impurity SM 3i The relative retention time is 1.53, the correction factor is 1.9, and the limit is 0.15%; the impurity SM 3e The relative retention time is 1.67, the correction factor is 1.2, and the limit is 0.15%.

[0094] Example 2. Specificity

[0095] Impurity SM 3a Stock solution: Accurately weigh impurity SM 3a 25.09 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0096] Impurity SM 3b Stock solution: Accurately weigh the impurity SM 3b 25.52 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0097] Impurity SM 3c Stock solution: Accurately weigh the impurity SM 3c10.37 mg, put in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0098] Impurity SM 3d Stock solution: Accurately weigh the impurity SM 3d 25.15 mg, put into a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0099] Impurity SM 3e Stock solution: Accurately weigh the impurity SM 3e 25.44 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0100] Impurity SM 3f Stock solution: Accurately weigh the impurity SM 3f 26.36 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0101] Impurity SM 3g Stock solution: Accurately weigh impurity SM 3g 25.27 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0102] Impurity SM 3h Stock solution: Accurately weigh the impurity SM 3h 25.04 mg, put into a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0103] Impurity SM 3i Stock solution: Accurately weigh the impurity SM 3i 25.34 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0104] Impurity SM 3j Stock solution: Accurately weigh the impurity SM 3j 25.12 mg, put in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0105] Each impurity location solution: accurately measure the impurity SM 3j Take 1 ml of the stock solution and 0.3 ml of each of the remaining impurities, place them in different 100 ml volumetric flasks, dilute to the scale with solvent, shake well, and you have it.

[0106] Mixed impurity stock solution (concentration 50μg / ml): Accurately measure the impurity SM 3a Stock solution, impurities SM 3b Stock solution, impurities SM 3cStock solution, impurities SM 3d Stock solution, impurities SM 3e Stock solution, impurities SM 3f Stock solution, impurities SM 3g Stock solution, impurities SM 3h Stock solution, impurities SM 3i Place 1.0 ml of each stock solution in a 20 ml volumetric flask, add solvent to dilute to the mark, shake well, and you have it.

[0107] Test sample solution: Accurately weigh 10.03 mg of the test sample (batch number: BK100K210605), place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0108] Mixed solution: Accurately weigh 10.27 mg of the test sample (batch number: BK100K210605) and place it in a 10 ml volumetric flask. Accurately measure 0.6 ml of the mixed impurity stock solution and the impurity SM 3j Prepare 0.1 ml of the stock solution by placing it in a volumetric flask, adding solvent to dissolve and dilute to the mark, shaking well, and the solution is ready.

[0109] Take 20 μl of each blank solution (solvent), each impurity location solution, test solution, and mixed solution, inject them into the liquid chromatograph, and record the chromatogram. The determination results are shown in Table 3 and Figure 1-Figure 3 As shown, Figure 2 and Figure 3 The integration results are shown in Table 4 and Table 5. The results show that the blank solution does not interfere with the detection of the known impurities and main components, the separation between the main peak and the adjacent impurity peaks is greater than 1.5, and the separation between the known impurities and the adjacent impurity peaks is greater than 1.2, and the method specificity meets the requirements.

[0110] Table 3. Specificity test results

[0111]

[0112] Table 4. Figure 2 The integral result of

[0113]

[0114] Table 5. Figure 3 The integral result of

[0115]

[0116]

[0117] Example 3. Limit of quantitation and limit of detection

[0118] Linear stock solution 1: Accurately measure the impurity SM under the "Specificity" item of Example 23a Stock solution, impurities SM 3b Stock solution, impurities SM 3c Stock solution, impurities SM 3d Stock solution, impurities SM 3e Stock solution, impurities SM 3f Stock solution, impurities SM 3g Stock solution, impurities SM 3h Stock solution, impurities SM 3i Place 5 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0119] Linear stock solution 2: Accurately measure the impurity SM under the "Specificity" item of Example 2 3j Place 5 ml of the stock solution and the test solution in the same 100 ml volumetric flask, dilute to the mark with solvent, shake well, and the solution is ready.

[0120] Quantitation limit solution: Accurately measure 0.6 ml of linear stock solution 1 and linear stock solution 2, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0121] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0122] The quantitative limit solution was sampled 6 times continuously, and the detection limit solution was sampled 3 times continuously. The ratio of the main peak height to the noise (signal-to-noise ratio) was calculated and the chromatogram was recorded. The test results are shown in Tables 6 and 7. Some chromatograms are shown in Figure 4 and Figure 5 shown.

[0123] Table 6. Quantitation limit determination results

[0124]

[0125]

[0126]

[0127] Table 7. Detection limit determination results

[0128]

[0129]

[0130] Results: As shown in Table 6, impurity SM 3c The quantitative limit concentration is 0.302μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 1.5%, and the average signal-to-noise ratio is 79.6; impurity SM 3dThe quantitative limit concentration is 0.299μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 2.1% and the average signal-to-noise ratio is 51.8; impurity SM 3b The quantitative limit concentration is 0.305μg / ml, which is expressed as 0.031% in the sample. After 6 consecutive injections, the RSD value of the peak area is 3.6%, and the mean signal-to-noise ratio is 18.9; impurity SM 3j The quantitative limit concentration is 0.301μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 0.7% and the mean signal-to-noise ratio is 68.6; impurity SM 3h The quantitative limit concentration is 0.300 μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 0.7% and the mean signal-to-noise ratio is 94.7. Impurity SM 3a The quantitative limit concentration is 0.300 μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 0.8% and the average signal-to-noise ratio is 99.8; impurity SM 3g The quantitative limit concentration is 0.297μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 0.7% and the average signal-to-noise ratio is 54.4; impurity SM 3f The quantitative limit concentration is 0.301μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 0.8% and the average signal-to-noise ratio is 68.8; impurity SM 3i The quantitative limit concentration is 0.299μg / ml, which is expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area is 3.9% and the mean signal-to-noise ratio is 21.9. Impurity SM 3e The quantitative limit concentration was 0.299 μg / ml, expressed as 0.030% in the sample. After 6 consecutive injections, the RSD value of the peak area was 1.2%, and the average signal-to-noise ratio was 31.1. The quantitative limit concentration of enzalutamide SM3 was 0.308 μg / ml. After 6 consecutive injections, the RSD value of the peak area was 1.1%, and the average signal-to-noise ratio was 76.6. The signal-to-noise ratios met the S / N ≥ 10:1 requirement.

[0131] As shown in Table 7, impurity SM 3c The detection limit concentration is 0.091μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 26.3; impurity SM 3d The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 16.8; impurity SM3b The detection limit concentration is 0.092μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 6.2; impurity SM 3j The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 22.3; impurity SM 3h The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 30.2. Impurity SM 3a The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 32.2; impurity SM 3g The detection limit concentration is 0.089 μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 17.5. Impurity SM 3f The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 22.6; impurity SM 3i The detection limit concentration is 0.090μg / ml, which is expressed as 0.009% in the sample. The average signal-to-noise ratio of three consecutive injections is 7.4. Impurity SM 3e The detection limit concentration is 0.090μg / ml, expressed as 0.009% in the sample, and the average signal-to-noise ratio of three consecutive injections is 10.5; the detection limit concentration of enzalutamide SM3 is 0.092μg / ml, and the average signal-to-noise ratio of three consecutive injections is 19.9; the signal-to-noise ratios meet the S / N≥3:1 requirement.

[0132] Example 4. Chromatographic Conditions Robustness

[0133] Take the mixed solution in Example 2, use normal chromatographic conditions, different column temperatures, column flow rates, and mobile phase starting ratios for the predetermined tests, test them separately after the instrument system is stable, record the separation between the peaks, and investigate the changes in the relative retention time and normalized content of each known impurity. The test results are shown in Tables 8 to 10, which show that when the chromatographic conditions have slight fluctuations, the separation between the main peak and the adjacent impurity peaks is greater than 1.5, the separation between each impurity is greater than 1.2, the relative retention time and normalized content of each component are basically the same, and the method durability meets the requirements.

[0134] Table 8. Results of relative retention time determination of impurities in the experiment of changing chromatographic conditions

[0135]

[0136] Table 9. Results of normalized content determination of each component in the chromatographic condition variation test

[0137]

[0138] Table 10. Results of separation test for chromatographic condition variation

[0139]

Claims

1. A method for separating enzalutamide SM3 and its related impurities using HPLC, characterized in that: The relevant impurity is impurity SM 3a 、Impurity SM 3b 、Impurity SM 3c 、Impurity SM 3d 、Impurity SM 3e 、Impurity SM 3f 、Impurity SM 3g 、Impurity SM 3h 、Impurity SM 3i and impurity SM 3j Any one or more of; the structural formula of the enzalutamide SM3 is shown in Formula I; the impurity SM 3a The structural formula is shown in Formula II, and the impurity SM 3b The structural formula is shown in Formula III, and the impurity SM 3c The structural formula is shown in Formula IV, and the impurity SM 3d The structural formula is shown in Formula V, and the impurity SM 3e The structural formula of is shown in Formula VI, the impurity SM 3f The structural formula is shown in Formula VII, and the impurity SM 3g The structural formula is shown in Formula VIII, the impurity SM 3h The structural formula is shown in Formula IX, and the impurity SM 3i The structural formula is shown in Formula X, and the impurity SM 3j The structural formula is shown in Formula Ⅺ; the HPLC method uses octadecylsilane bonded silica gel as the stationary phase, mobile phase A and mobile phase B as the mobile phase, and the impurity SM is sequentially removed by gradient elution. 3c 、Impurity SM 3d 、Impurity SM 3b , Enzalutamide SM3, Impurity SM 3j 、Impurity SM 3h 、Impurity SM 3f 、Impurity SM 3a 、Impurity SM 3i 、Impurity SM 3g and impurity SM 3e For separation; the mobile phase A is trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile; 2. The method according to claim 1, characterized in that The gradient elution program was: 0min, the volume ratio of the mobile phase A to the mobile phase B is 79-81:21-19; 3min, the volume ratio of the mobile phase A to the mobile phase B is 80:20; 15min, the volume ratio of the mobile phase A to the mobile phase B is 60:40; 40min, the volume ratio of the mobile phase A to the mobile phase B is 30:70; 45min, the volume ratio of the mobile phase A to the mobile phase B is 30:70; 46min, the volume ratio of the mobile phase A to the mobile phase B is 80:20; 55min, the volume ratio of the mobile phase A to the mobile phase B is 80:

20.

3. The method according to claim 1, characterized in that The volume fraction of the trifluoroacetic acid aqueous solution is 0.05%.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min, and the column temperature of the chromatographic column is 28° C.-32° C.

5. A method for identifying enzalutamide SM3 and its related impurities based on the method described in any one of claims 1 to 4, characterized in that: The enzalutamide SM3 and its related impurities are separated by the method described in any one of claims 1 to 4, and detected by a detector; the detection wavelength of the detector is 254 nm.

6. The method according to claim 1, characterized in that The mobile phase A is a trifluoroacetic acid aqueous solution with a volume fraction of 0.05%, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 1.0 ml / min, the column temperature of the chromatographic column is 30° C.; the detection wavelength is 254 nm; the specification of the chromatographic column is 4.6 mm×150 mm×3 μm; elution is performed according to the gradient elution program and a chromatogram is obtained, and the gradient elution program is set as follows: 0min, the volume ratio of the mobile phase A to the mobile phase B is 80:20; 3min, the volume ratio of the mobile phase A to the mobile phase B is 80:20; 15min, the volume ratio of the mobile phase A to the mobile phase B is 60:40; 40min, the volume ratio of the mobile phase A to the mobile phase B is 30:70; 45min, the volume ratio of the mobile phase A to the mobile phase B is 30:70; 46min, the volume ratio of the mobile phase A to the mobile phase B is 80:20; 55min, the volume ratio of the mobile phase A to the mobile phase B is 80:

20.

7. The method according to claim 1, characterized in that When the retention time is 2.7±0.5min, it is judged to be impurity SM 3c ; When the retention time is 9.0±0.5min, it is judged to be impurity SM 3d ; When the retention time is 15.0±0.5min, it is judged to be impurity SM 3b ; When the retention time is 18.6±0.5min, it is judged to be enzalutamide SM3; when the retention time is 21.4±0.5min, it is judged to be impurity SM 3j ; When the retention time is 21.9±0.5min, it is judged to be impurity SM 3h ; When the retention time is 23.0±0.5min, it is judged to be impurity SM 3f ; When the retention time is 26.2±0.5min, it is judged to be impurity SM 3a ; When the retention time is 28.4±0.5min, it is judged to be impurity SM 3i ; When the retention time is 30.3±0.5min, it is judged to be impurity SM 3g ; When the retention time is 30.9±0.5min, it is judged to be impurity SM 3e .

8. A method for detecting the content of enzalutamide SM3 and its related impurities, characterized in that: The following steps are involved: (1) separation: separating enzalutamide SM3 and its related impurities by the method described in any one of claims 1 to 4; (2) Identification: Detecting enzalutamide SM3 and its related impurities using the method described in any one of claims 5 to 7, and obtaining a chromatogram; (3) Content calculation: Based on the chromatogram obtained in step (2), the content of each impurity is calculated using the peak area using the principal component self-comparison method with the addition of a correction factor.

9. The method according to claim 8, characterized in that Before separation, a test solution, a control solution and a system suitability solution were prepared with a solvent; the solvent was a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water in the solvent was 50:

50.

10. The method according to claim 8, characterized in that The impurity SM 3c The correction factor is 0.57, and the impurity SM 3d The correction factor is 0.80, the impurity SM 3b The correction factor is 2.4, the impurity SM 3j The correction factor is 0.65, and the impurity SM 3h The correction factor is 0.48, and the impurity SM 3a The correction factor is 0.40, the impurity SM 3g The correction factor is 0.71, and the impurity SM 3e The correction factor is 1.2, the impurity SM 3f The correction factor is 0.64, and the impurity SM 3i The correction factor is 1.9.

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