Method for separating and detecting impurities of apattamide

Abstract

The invention provides a separation and detection method of an apattamide impurity, and belongs to the field of pharmaceutical analysis. The separation and detection method adopts high performance liquid chromatography, takes a C18 column as a chromatographic column, and takes acetonitrile and a phosphate aqueous solution as a mobile phase. The separation and detection method is good in specificity, good in durability, simple to operate and capable of rapidly and effectively separating five impurities of apattamide.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and particularly to a method for separating and detecting impurities of apalutamide. Background Art

[0002] Apalutamide, with the CAS number 956104-40-8, has a chemical structure as shown in Formula I below:

[0003]

[0004] During the production of apalutamide, quality control is required. However, currently, apalutamide is not included in the United States Pharmacopeia (USP), the European Pharmacopeia (EP), or the Chinese Pharmacopeia (Ch.P.). In order to better and more accurately control the related substances in the product and ensure the quality of the active pharmaceutical ingredient, a method for detecting apalutamide impurities with good specificity, good durability, and simple operation needs to be developed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for separating and detecting apalutamide impurities. The method of the present invention can quickly and effectively separate 5 impurities of apalutamide. The method is simple, fast, highly sensitive, and the results are accurate and reliable, and can be used for the quality control of apalutamide active pharmaceutical ingredient and preparations.

[0006] A method for separating and detecting apalutamide impurities, characterized by comprising the following steps:

[0007] (1) Prepare a test solution;

[0008] (2) Set instrument parameters: chromatographic column, flow rate of the mobile phase, column temperature;

[0009] (3) Take a certain amount of the test solution prepared in step (1) and inject it into a high-performance liquid chromatograph to complete the separation and detection of apalutamide impurities.

[0010] In some embodiments, the mobile phase of the high-performance liquid chromatography is divided into mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of ammonium acetate, and mobile phase B is acetonitrile.

[0011] In some embodiments, the pH of mobile phase A is 2-7. In some embodiments, the pH of mobile phase A is 3-6. In some examples, the pH of mobile phase A is approximately 2, 3, 4, 5, 6, or 7.

[0012] In some embodiments, the concentration of the phosphoric acid aqueous solution is 0.1% (V / V), and the concentration of the ammonium acetate aqueous solution is 10 mmol / L. In some embodiments, the initial volume ratio of mobile phase A and mobile phase B is 95:5 to 60:40. In some embodiments, the elution mode of high performance liquid chromatography is gradient elution. In some embodiments, the initial volume ratio of mobile phase A and mobile phase B is 85:15 to 70:30. In some embodiments, the initial volume ratio of mobile phase A and mobile phase B is 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, or 60:40. The initial volume ratio refers to the volume ratio of mobile phase A and mobile phase B at 0 min of gradient elution.

[0013] In some embodiments, the flow rate of the mobile phase is 0.5 ml / min to 2.0 ml / min. In some embodiments, the flow rate of the mobile phase is 0.9 ml / min to 1.1 ml / min. In some embodiments, the flow rate of the mobile phase is 0.9 ml / min. In some embodiments, the flow rate of the mobile phase is 1.0 ml / min. In some embodiments, the flow rate of the mobile phase is 1.1 ml / min.

[0014] In some embodiments, the column temperature is 15°C to 40°C. In some embodiments, the column temperature is 20°C to 35°C. In some embodiments, the column temperature is 20°C. In some embodiments, the column temperature is 25°C. In some embodiments, the column temperature is 30°C. In some embodiments, the column temperature is 35°C.

[0015] In some embodiments, the injection volume of the test solution is 2 μl to 20 μl. In some embodiments, the injection volume of the test solution is 5 μl to 10 μl. In some embodiments, the injection volume of the test solution is 5 μl.

[0016] In some embodiments, the preparation of the test solution comprises the following steps: dissolving apalutamide with a diluent. In some embodiments, the preparation of the test solution comprises the following steps: dissolving apalutamide with a diluent and then diluting it to an apalutamide solution of 0.6 mg / ml.

[0017] In some embodiments, the diluent / blank solution is a mixed solvent of acetonitrile and an aqueous solution of EDTA-2Na, and the volume ratio of acetonitrile to the aqueous solution of EDTA-2Na is 30:70 to 70:30. In some examples, the volume ratio of acetonitrile to the aqueous solution of EDTA-2Na is 50:50, 40:60 or 60:40. In some examples, the concentration of the aqueous solution of EDTA-2Na is 5 mmol / L.

[0018] In some embodiments, the chromatographic column is selected from Waters XBridge BEH Phenyl, or YMC MeteoricCore C18, or ACE Excel 3C18-PFP.

[0019] The chemical structural formulas of 5 impurities are shown as follows:

[0020]

[0021]

[0022] The high performance liquid chromatograph can be an Agilent 1260 high performance liquid chromatography system and workstation in the United States or other suitable and feasible systems.

[0023] In the content above or below, whether the words "about" or "approximately" are used or not, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the numerical value of each number may have differences of ±1%, ±2%, ±5%, ±7%, ±8% or ±10%, etc. Description of the Drawings

[0024] Figure 1 , Figure 2 is the high performance liquid chromatogram of Example 1;

[0025] Figure 3 is the high performance liquid chromatogram of Example 2;

[0026] Figure 4 is the high performance liquid chromatogram of Example 3;

[0027] Figure 5 is the high performance liquid chromatogram of Example 4;

[0028] Figure 6 is the high performance liquid chromatogram of Example 5;

[0029] Figure 7 is the high performance liquid chromatogram of Example 6;

[0030] Figure 8 is the high performance liquid chromatogram of Example 7;

[0031] Figure 9 It is the high performance liquid chromatography (HPLC) chromatogram of Example 8;

[0032] Figure 10 It is the high performance liquid chromatography (HPLC) chromatogram of the test solution in Example 9 using diluent 1 (acetonitrile: water = 50:50 (V:V));

[0033] Figure 11 It is the high performance liquid chromatography (HPLC) chromatogram of the test solution in Example 9 using diluent (acetonitrile: EDTA-2Na aqueous solution = 50:50 (V:V)).

[0034] Figure 12 It is the high performance liquid chromatography (HPLC) chromatogram of Comparative Example 2(1);

[0035] Figure 13 It is the high performance liquid chromatography (HPLC) chromatogram of Comparative Example 2(2);

[0036] Figure 14 It is the high performance liquid chromatography (HPLC) chromatogram of Comparative Example 2(3);

[0037] Figure 15 It is the high performance liquid chromatography (HPLC) chromatogram of the blank solution in Example 10. Detailed implementation manners

[0038] In the present invention, APT is the English abbreviation of apalutamide, and EDTA-2Na refers to disodium ethylenediaminetetraacetate;

[0039] V refers to volume, mmol / L refers to millimole per liter, μm refers to micrometer, nm refers to nanometer, mm refers to millimeter, μl refers to microliter, mL refers to milliliter, min refers to minute, °C refers to degree Celsius, mg refers to milligram, and ml / min refers to milliliter per minute.

[0040] The general method of the examples of the present invention is as follows:

[0041] 1. Instruments and conditions

[0042] Instruments: Agilent 1260 type high performance liquid chromatography system and workstation from the United States; automatic injection.

[0043] Chromatographic column 1: Waters XBridge BEH Phenyl, 4.6×100 mm, 2.5 μm; Chromatographic column 2: YMC Meteoric Core C18, 4.6×100 mm, 2.7 μm; Chromatographic column 3: ACE Excel 3, C18-PFP, 4.6×100 mm, 3 μm as the separation column;

[0044] Detector: ultraviolet;

[0045] Detection wavelength: 268 nm;

[0046] Flow rate: 0.9 ml / min, or 1.0 ml / min, or 1.1 ml / min;

[0047] Column temperature: 20 °C, or 25 °C, or 30 °C, or 35 °C;

[0048] Sample injection volume: 5 μl;

[0049] Running time: 32 min;

[0050] Mobile phase A1: Aqueous phosphoric acid solution with a concentration of 0.1% (V / V), Mobile phase A2: Aqueous ammonium acetate solution with a concentration of 10 mmol / L;

[0051] Mobile phase B: Acetonitrile;

[0052] Gradient elution 1:

[0053] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 80 20 18 35 65 22 5 95 27 5 95 27.1 80 20 32 80 20

[0054] Gradient elution 2:

[0055] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 75 25 20 20 80 25 5 95 30 5 95 30.1 75 25 35 75 75

[0056] Gradient elution 3:

[0057]

[0058]

[0059] 2. Preparation of mobile phase

[0060] Aqueous phosphoric acid solution with a concentration of 0.1% (Mobile phase A1): Take 1 ml of phosphoric acid and add it to 1000 ml of ultrapure water, mix well to obtain;

[0061] Aqueous ammonium acetate solution with a concentration of 10 mmol / L (Mobile phase A2): Weigh 0.77 g of ammonium acetate into 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix well to obtain.

[0062] 3. Preparation of solutions

[0063] (1) Test solution: Take about 30 mg of apalutamide test substance, accurately weigh it into a 50 ml brown volumetric flask, dissolve it ultrasonically with the diluent, dilute it to the scale with the diluent, and shake well to obtain.

[0064] (2) Spiked test solution 1: Take about 30 mg of apalutamide test substance, accurately weigh it, and transfer it to a 50 ml volumetric flask; Pipette 2 ml of the linear stock solution into this volumetric flask, dilute it to the scale with the diluent, and shake well to obtain a 0.6 mg / ml apalutamide solution and a 0.6 μg / ml impurity solution.

[0065] Linear stock solution: Accurately pipette 5 ml of the linear stock solution into a 100-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain it.

[0066] Linear stock solution: Weigh accurately about 15 mg of reference substances A, B, C, D, and F respectively, transfer them to a 50-ml volumetric flask, dissolve with acetonitrile, sonicate for about 1 min, dilute to the mark with acetonitrile, and shake well.

[0067] (3) Spiked test solution 2: Weigh accurately about 50 mg of apalutamide test sample (which already contains impurity C) and transfer it to a 100-ml brown volumetric flask. Dissolve it by sonication with the diluent. Add about 4 mg of impurity A, 3 mg of impurity B, 5 mg of impurity D, and 2 mg of impurity F to the volumetric flask, dilute to the mark with the diluent, and shake well to obtain it.

[0068] (4) Diluent / blank solution: Acetonitrile: EDTA-2Na aqueous solution = 50:50 (V:V). Measure acetonitrile and EDTA-2Na aqueous solution according to the volume ratio of 50:50, mix well to obtain it.

[0069] Preparation of EDTA-2Na aqueous solution: Weigh about 1.86 g of EDTA-2Na dihydrate and transfer it to 1000 ml of ultrapure water, dissolve and shake well to obtain a 5 mmol / L EDTA-2Na aqueous solution.

[0070] (5) Control solution: Accurately pipette 5 ml of the control stock solution into a 25-ml volumetric flask, dilute and make up the volume with the diluent, and shake well to obtain it.

[0071] Control stock solution: Accurately pipette 1 ml of test solution 1 into a 100-ml volumetric flask, dilute and make up the volume with the diluent, and shake well to obtain it.

[0072] (6) System suitability solution: Use spiked test solution 1 as the system suitability solution.

[0073] (7) Sensitivity solution: Accurately pipette 5 ml of the control stock solution into a 100-ml volumetric flask, make up the volume with the diluent to obtain a sensitivity solution with a concentration level of 0.05%.

[0074] Example 1

[0075] Prepare spiked test solution 1 according to the general method 3 of the example, and perform high-performance liquid chromatography analysis according to the instrument and conditions of the general method 1 of the example (wherein, use chromatographic column 1, the flow rate is 1.0 ml / min, the column temperature is 30 °C, the mobile phase is A1 + B, and gradient elution 1 is adopted). Inject samples separately using 2 chromatographic columns of the same model, record the chromatograms, and the results are shown in Figure 1 and Figure 2 .

[0076] The results showed that the resolution of the 5 impurity peaks was greater than or equal to 4.8, indicating good resolution.

[0077] Example 2

[0078] Prepare the test sample spiked solution 1 according to the general method 3 of the examples, and perform high performance liquid chromatography analysis according to the instruments and conditions of the general method 1 of the examples (wherein, chromatographic column 1 is used, the flow rate is 1.0 ml / min, the column temperature is 25 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the results are shown in Figure 3 .

[0079] The results showed that the resolution of the 5 impurity peaks was greater than or equal to 5.0, indicating good resolution.

[0080] Example 3

[0081] Prepare the test sample spiked solution 1 according to the general method 3 of the examples, and perform high performance liquid chromatography analysis according to the instruments and conditions of the general method 1 of the examples (wherein, chromatographic column 1 is used, the flow rate is 1.0 ml / min, the column temperature is 35 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the results are shown in Figure 4 .

[0082] The results showed that the resolution of the 5 impurity peaks was greater than or equal to 4.5, indicating good resolution.

[0083] Example 4

[0084] Prepare the test sample spiked solution 1 according to the general method 3 of the examples, and perform high performance liquid chromatography analysis according to the instruments and conditions of the general method 1 of the examples (wherein, chromatographic column 1 is used, the flow rate is 0.9 ml / min, the column temperature is 30 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the results are shown in Figure 5 .

[0085] The results showed that the resolution of the 5 impurity peaks was greater than or equal to 4.8, indicating good resolution.

[0086] Example 5

[0087] Prepare the test sample spiked solution 1 according to the general method 3 of the examples, and perform high performance liquid chromatography analysis according to the instruments and conditions of the general method 1 of the examples (wherein, chromatographic column 1 is used, the flow rate is 1.1 ml / min, the column temperature is 30 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the results are shown in Figure 6 .

[0088] The results showed that the resolution of the 5 impurity peaks was greater than or equal to 4.7, indicating good resolution.

[0089] Example 6

[0090] Prepare the spiked test solution 1 according to the general method 3 of the examples. Perform high-performance liquid chromatography analysis according to the instrument and conditions of the general method 1 of the examples (wherein, chromatographic column 2 is used, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A1 + B, and gradient elution 3 is adopted), record the chromatogram, and the results are shown in Figure 7 .

[0091] The results show that the resolution of the 5 impurity peaks is greater than or equal to 2.8, and the resolution is good.

[0092] Example 7

[0093] Prepare the test solution according to the general method 3 of the examples. Perform high-performance liquid chromatography analysis according to the instrument and conditions of the general method 1 of the examples (wherein, chromatographic column 2 is used, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A2 + B, and gradient elution 3 is adopted), record the chromatogram, and the results are shown in Figure 8 .

[0094] The results show that when using the mobile phase A2 + B (gradient elution 3) and the mobile phase A1 + B (gradient elution 3), the retention times of the main peak and impurities in the chromatogram are exactly the same.

[0095] Example 8

[0096] Prepare the spiked test solution 2 according to the general method 3 of the examples. Perform high-performance liquid chromatography analysis according to the instrument and conditions of the general method 1 of the examples (wherein, chromatographic column 3 is used, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A1 + B, and gradient elution 2 is adopted), record the chromatogram, and the results are shown in Figure 9 .

[0097] The results show that the resolution between impurity B and impurity C is 3.2, and the resolution is good.

[0098] Example 9 (Comparative Example 1)

[0099] Diluent 1: Acetonitrile: Water = 50:50 (V:V). Measure acetonitrile and water in a volume ratio of 50:50 and mix well to obtain it.

[0100] Test solution 1: Take about 30 mg of apalutamide test sample, accurately weigh it into a 50 ml brown volumetric flask, dissolve it ultrasonically with diluent 1, and then dilute it to the scale with diluent 1 and shake well to obtain test solution 1.

[0101] Test solution 2: Take about 30 mg of apalutamide test sample, accurately weigh it into a 50 ml brown volumetric flask, dissolve it ultrasonically with the diluent prepared according to the general method 3 of the examples, and then dilute it to the scale with the diluent and shake well to obtain test solution 2.

[0102] Perform high performance liquid chromatography analysis according to the instrument and conditions of General Method 1 of the examples (wherein, Chromatographic Column 1 is used, the flow rate is 1.0 / min, the column temperature is 30 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the results of Test Solution 1 are shown in Figure 10 , and the results of Test Solution 2 are shown in Figure 11 .

[0103] The results show that Figure 10 compared with Figure 11 , there are significantly two more abnormal peaks at RT 4.326 and RT 12.771, which proves that using acetonitrile and EDTA-2Na aqueous solution as diluents can effectively avoid the appearance of abnormal peaks.

[0104] Comparative Example 2

[0105] (1) Prepare Test Solution Spiked Solution 2 according to General Method 3 of the examples. Except for the different chromatographic columns (Waters XBridgeShield RP18, 4.6 * 100 mm, 3.5 μm), other conditions are the same as General Method 1 of the examples (wherein, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A1 + B, and gradient elution 2 is used) for high performance liquid chromatography analysis, record the chromatogram, and the results are shown in Figure 12 .

[0106] The results show that the resolution between Impurity B and Impurity C is 1.6, and the resolution is poor.

[0107] (2) Prepare Test Solution Spiked Solution 2 according to General Method 3 of the examples. Except for the different chromatographic columns (Waters Xselect CSH Phenyl Hexyl, 4.6 * 100 mm, 2.5 μm), other conditions are the same as General Method 1 of the examples (wherein, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A1 + B, and gradient elution 2 is used) for high performance liquid chromatography analysis, record the chromatogram, and the results are shown in Figure 13 .

[0108] The results show that the resolution between Impurity B and Impurity C is 1.6, and the resolution is poor.

[0109] (3) Prepare Test Solution according to General Method 3 of the examples. Except for the different chromatographic columns (YMC-Triart C18, 4.6 * 100 mm, 3 μm), other conditions are the same as General Method 1 of the examples (wherein, the flow rate is 1.0 ml / min, the column temperature is 20 °C, the mobile phase is A1 + B, and gradient elution 2 is used) for high performance liquid chromatography analysis, record the chromatogram, and the results are shown in Figure 14 .

[0110] The results show that the resolution between Impurity B and Impurity C is 1.2, and the resolution is poor.

[0111] Method Verification of Example 10

[0112] Perform high performance liquid chromatography analysis according to the general method 1-3 of the examples (wherein, chromatographic column 1 is used, the flow rate is 1.0 ml / min, the column temperature is 30 °C, the mobile phase is A1 + B, and gradient elution 1 is used), record the chromatogram, and the chromatogram of the blank solution is shown in Figure 15 , and other results are shown in Table 1.

[0113] Table 1

[0114]

[0115]

[0116]

[0117]

[0118] Note: “—” means that there is no chromatographic peak at this position, so there is no corresponding relevant data.

[0119] The results of the examples show that the method of the present invention can quickly and effectively separate 5 impurities of apalutamide, the resolution is greater than 1.5 and baseline separation can be achieved. The method has high accuracy and good specificity. Under the conditions of changing the chromatographic column or mobile phase (and the gradient elution method of the mobile phase), changing the flow rate or column temperature, the resolution of the 5 impurities is still very good, which proves that the method has good durability and is suitable for the quality control of apalutamide raw materials.

[0120] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

[0121] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "some implementation manners", "some implementation schemes", "examples", "specific examples", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0122] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for separating and detecting apalutamide impurities, characterized in that, It includes the following steps: (1) Prepare the test solution; (2) Set the instrument parameters: chromatographic column, flow rate of the mobile phase, column temperature; (3) Take a certain amount of the test solution in step (1), inject it into the high performance liquid chromatograph, and complete the separation and detection of apalutamide impurities.

2. The method according to claim 1, wherein The mobile phase is divided into mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of ammonium acetate, and mobile phase B is acetonitrile.

3. The method according to claim 2, wherein The pH of the mobile phase A is 2 to 7; the concentration of the aqueous phosphoric acid solution is 0.1%, and the concentration of the aqueous ammonium acetate solution is 10 mmol / L.

4. The method according to claim 2 or 3, characterized in that, The initial volume ratio of the mobile phase A to the mobile phase B is 95:5 to 60:

40.

5. The method according to any one of claims 1 to 4, characterized in that The flow rate of the mobile phase is 0.5 ml / min to 2.0 ml / min.

6. The method according to any one of claims 1-5, characterized in that The column temperature is 15°C to 40°C.

7. According to the method described in any one of claims 1-6, characterized in that, The injection volume of the test solution is 2 μl to 20 μl.

8. The method according to any one of claims 1 to 7, characterized in that The preparation of the test solution includes the following steps: dissolve apalutamide with a diluent.

9. The method according to any one of claims 1-8, characterized in that, The diluent is a mixed solvent of acetonitrile and an aqueous solution of EDTA-2Na, and the volume ratio of acetonitrile to the aqueous solution of EDTA-2Na is 30:70 to 70:

30.

10. The method according to any one of claims 1-9, characterized in that, The chromatographic column is selected from Waters XBridge BEHPhenyl, or YMC Meteoric Core C18, or ACE Excel 3C18-PFP.

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