Liquid chromatography for content determination or impurity determination of apalutamide, salt thereof or preparation thereof

Abstract

The invention provides a liquid chromatography for content determination or impurity determination of apalutamide, a salt thereof or a preparation thereof, and belongs to the field of analytical chemistry. According to the liquid chromatography, octadecylsilane chemically bonded silica is adopted as a chromatographic column of a filler, an acid-containing aqueous solution and acetonitrile are adopted as mobile phases, and gradient elution is carried out. The liquid chromatography has the advantages of high detection efficiency, high accuracy, good linearity, high sensitivity, good durability, good repeatability and reproducibility, high precision and the like, and compared with other chromatographic conditions, the liquid chromatography also has the advantages of large quantity of separated impurities, good separation effect and the like.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and particularly to a liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations. Background Art

[0002] Apalutamide, chemically named 4-[7-(6-cyano-5-trifluoromethylpyridin-3-yl)-8-oxo-6-thia-5,7-diazaspiro[3.4]octan-5-yl]-2-fluoro-N-methylbenzamide, is an androgen receptor (AR) inhibitor developed by the University of California, USA.

[0003] During the production process of apalutamide and its preparations, quality control is required. However, currently, apalutamide tablets are not included in the United States Pharmacopeia (USP), the European Pharmacopeia (EP), or the Chinese Pharmacopeia (Ch.P.), and there are very few literature reports on the detection methods for related substances of apalutamide.

[0004] Therefore, there is an urgent need to develop a method for the determination of the content or impurities of apalutamide, its salts or its preparations. Summary of the Invention

[0005] To solve the above problems, the present invention provides a liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations.

[0006] A liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations, comprising: a chromatographic column filled with octadecylsilane-bonded silica gel, and using an aqueous acid solution and acetonitrile as the mobile phase for gradient elution.

[0007] In some embodiments, the elution program of the gradient elution includes: gradient elution method a:

[0008] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 33 45 55 33.1 80 20 40 80 20 。

[0009] In some embodiments, the elution program of the gradient elution includes: gradient elution method b:

[0010] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60 50 20 80 。

[0011] In some embodiments, the elution program of the gradient elution includes: gradient elution method c:

[0012] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60 。

[0013] In some embodiments, the packing material of the chromatographic column is Chromcore Polar C18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18.

[0014] In some embodiments, the length of the chromatographic column is 100 mm - 250 mm. In some embodiments, the length of the chromatographic column is 100 mm.

[0015] In some embodiments, the inner diameter of the chromatographic column is 2 mm - 5 mm. In some embodiments, the inner diameter of the chromatographic column is 4 mm - 5 mm. In some embodiments, the inner diameter of the chromatographic column is 4.6 mm.

[0016] In some embodiments, the particle size of the packing material of the chromatographic column is 2 μm - 5 μm. In some embodiments, the particle size of the packing material of the chromatographic column is 2.5 μm - 5 μm. In some embodiments, the particle size of the packing material of the chromatographic column is 3 μm. In some embodiments, the particle size of the packing material of the chromatographic column is 2.7 μm.

[0017] In some embodiments, the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 100 mm - 250 mm, and the particle size of the packing material of the chromatographic column is 2.5 μm - 3 μm.

[0018] In some embodiments, the aqueous acid solution is an aqueous solution containing trifluoroacetic acid. In some embodiments, the aqueous acid solution is an aqueous solution containing 0.025% vol - 0.1% vol trifluoroacetic acid. In some embodiments, the aqueous acid solution is an aqueous solution containing 0.05% vol - 0.1% vol trifluoroacetic acid. In some embodiments, the aqueous acid solution is an aqueous solution containing 0.025% vol - 0.05% vol trifluoroacetic acid.

[0019] In some embodiments, the detection wavelength of the liquid chromatography method is 241 nm - 245 nm. In some embodiments, the detection wavelength of the liquid chromatography method is 241 nm, 242 nm, 243 nm, 244 nm or 245 nm.

[0020] In some embodiments, the flow rate of the liquid chromatography method is 0.8 ml / min - 1.2 ml / min. In some embodiments, the flow rate of the liquid chromatography method is 0.9 ml / min - 1.1 ml / min. In some embodiments, the flow rate of the liquid chromatography method is 0.95 ml / min - 1.05 ml / min. In some embodiments, the flow rate of the liquid chromatography method is 1.0 ml / min.

[0021] In some embodiments, the column temperature of the liquid chromatography is 28°C - 35°C. In some embodiments, the column temperature of the liquid chromatography is 29°C - 32°C. In some embodiments, the column temperature of the liquid chromatography is 30°C - 31°C. In some embodiments, the column temperature of the liquid chromatography is 29°C, 30°C, 31°C or 32°C.

[0022] In some embodiments, the injection volume of the liquid chromatography is 2 μL - 100 μL. In some embodiments, the injection volume of the liquid chromatography is 5 μL - 30 μL. In some embodiments, the injection volume of the liquid chromatography is 10 μL - 25 μL. In some embodiments, the injection volume of the liquid chromatography is 10 μL - 20 μL.

[0023] In some embodiments, the liquid chromatography further includes preparing apalutamide, its salt or its preparation into a test solution, and the diluent of the test solution is acetonitrile or a mixture of acetonitrile and solution A, where solution A is an aqueous solution containing citric acid and / or phosphate.

[0024] In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:9 - 10:0. In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:5 - 5:1. In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:3 - 3:1. In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:2 - 2:1. In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:1.5 - 1.5:1. In some embodiments, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:1.

[0025] In some embodiments, the pH of solution A is 6 - 8. In some embodiments, the pH of solution A is 7. In some embodiments, the pH of solution A is 7.4.

[0026] In some embodiments, the pH of solution A is adjusted with ammonia water or sodium hydroxide.

[0027] In some embodiments, for apalutamide, its salt or its preparation, calculated as apalutamide, the content of apalutamide in the test solution is 0.10 mg / ml - 1.00 mg / ml. In some embodiments, for apalutamide, its salt or its preparation, calculated as apalutamide, the content of apalutamide in the test solution is 0.10 mg / ml - 0.50 mg / ml. In some embodiments, for apalutamide, its salt or its preparation, calculated as apalutamide, the content of apalutamide in the test solution is 0.15 mg / ml - 0.40 mg / ml. In some embodiments, for apalutamide, its salt or its preparation, calculated as apalutamide, the content of apalutamide in the test solution is 0.20 mg / ml - 0.30 mg / ml. In some embodiments, for apalutamide, its salt or its preparation, calculated as apalutamide, the content of apalutamide in the test solution is 0.25 mg / ml.

[0028] In some embodiments, a liquid chromatography method for the determination of the content or impurities of apalutamide, its salt or its preparation, includes: preparing apalutamide, its salt or its preparation into a test solution, using a chromatographic column filled with octadecylsilane-bonded silica gel, the packing material of the chromatographic column being Chromcore Polar C18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18, the length of the chromatographic column being 100 mm, the inner diameter of the chromatographic column being 4.6 mm, the particle size of the packing material of the chromatographic column being 2.7 μm - 3 μm, using an aqueous acid solution and acetonitrile as the mobile phase, the aqueous acid solution being an aqueous solution containing 0.025% vol - 0.1% vol trifluoroacetic acid, the detection wavelength of the liquid chromatography method being 241 nm - 245 nm, the flow rate of the liquid chromatography method being 0.8 ml / min - 1.2 ml / min; the column temperature of the liquid chromatography method being 28°C - 35°C; the injection volume of the liquid chromatography method being 5 μL - 30 μL, and subjecting the test solution to gradient elution, the gradient elution program including: Gradient elution method a:

[0029] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 33 45 55 33.1 80 20 40 80 20

[0030] Or gradient elution method b:

[0031] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60 50 20 80

[0032] Or gradient elution method c:

[0033] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60

[0034] In some embodiments, a liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations includes: preparing apalutamide, its salts or its preparations into a test solution, using a chromatographic column filled with octadecylsilane-bonded silica gel, and the packing material of the chromatographic column is Chromcore Polar C18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18. The length of the chromatographic column is 100 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the packing material of the chromatographic column is 2.7 μm - 3 μm. An aqueous acid solution and acetonitrile are used as the mobile phase. The aqueous acid solution is an aqueous solution containing 0.025% vol - 0.1% vol trifluoroacetic acid. The detection wavelength of the liquid chromatography method is 241 nm - 245 nm or 243 nm, and the flow rate of the liquid chromatography method is 1 ml / min; the column temperature of the liquid chromatography method is 30 °C; the injection volume of the liquid chromatography method is 10 μL - 20 μL, and the test solution is subjected to gradient elution. The gradient elution program includes: Gradient elution method a:

[0035] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 33 45 55 33.1 80 20 40 80 20

[0036] Or gradient elution method b:

[0037] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60 50 20 80

[0038] Or gradient elution method c:

[0039] Time (minutes) Aqueous acid solution (%) Acetonitrile (%) 0 80 20 10 60 40 40 40 60 。

[0040] In some embodiments, the liquid chromatography method may further include a post-run after the gradient elution of gradient elution method b or gradient elution method c. The purpose of the post-run is to change the ratio of the aqueous acid solution and acetonitrile in the chromatographic column to the initial ratio of the gradient elution, which is beneficial to improving the reproducibility of the elution time and resolution of the impurity peaks and main peaks in each chromatogram.

[0041] In some embodiments, the post-run time can be 5 minutes or more. In some embodiments, the post-run time can be 7 minutes or more. In some embodiments, the post-run time can be 8 minutes or more. In some embodiments, the post-run time can be 9 minutes or more. In some embodiments, the post-run time can be 10 minutes or more. In some embodiments, the post-run time can be 5 minutes - 30 minutes. In some embodiments, the post-run time can be 5 minutes - 20 minutes. In some embodiments, the post-run time can be 5 minutes - 15 minutes. In some embodiments, the post-run time can be 7 minutes - 15 minutes. In some embodiments, the post-run time can be 8 minutes - 15 minutes. In some embodiments, the post-run time can be 8 minutes - 15 minutes. In some embodiments, the post-run time can be 5 minutes - 10 minutes. In some embodiments, the post-run time can be 7 minutes - 15 minutes. In some embodiments, the post-run time can be 8 minutes - 10 minutes. In some embodiments, the post-run time can be 10 minutes - 30 minutes. In some embodiments, the post-run time can be 10 minutes - 20 minutes. In some embodiments, the post-run time can be 10 minutes - 15 minutes. After the post-run of about 5 minutes for a chromatographic column with a specification of 100 mm × 4.6 mm, the ratio of the aqueous acid solution and acetonitrile in the chromatographic column can be changed to the initial ratio of gradient elution; after the post-run of about 7 minutes for a chromatographic column with a specification of 250 mm × 4.6 mm, the ratio of the aqueous acid solution and acetonitrile in the chromatographic column can be changed to the initial ratio of gradient elution.

[0042] Beneficial effects

[0043] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0044] (1) This method has been comprehensively verified, with high detection efficiency, high accuracy, good linearity, high sensitivity, good durability, good repeatability and reproducibility, and high precision, and can be used for the related substance detection of apalutamide or its salt or its preparation.

[0045] (2) Compared with other types of chromatographic columns and gradient elution conditions, when using the chromatographic column and gradient elution conditions provided by the present invention to detect the related substances of apalutamide or its salt or its preparation, the tailing factor of the main peak in the obtained chromatogram is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak of the sensitivity solution is high, and the resolution between each impurity peak and the adjacent peak of the test solution is good.

[0046] (3) When detecting the related substances of apalutamide or its salts or its preparations using the chromatographic column and gradient elution conditions provided by the present invention, compared with other types of chromatographic columns, the separation between impurity peaks and between impurity peaks and the main peak in the obtained chromatogram is better; while for other types of chromatographic columns, there will be problems such as the main peak embedding the subsequent impurity peaks, fewer impurity peaks, and poor separation effect.

[0047] (4) When detecting the related substances of apalutamide or its salts or its preparations using the gradient elution conditions provided by the present invention, compared with other gradient elution conditions, the separation between impurity peaks and between impurity peaks and the main peak in the obtained chromatogram is better; while when using other types of gradient elution conditions, there will be problems such as fewer impurity peaks, inability to separate impurity peaks, and poor resolution. Description of the Drawings

[0048] Figure 1 It is the high performance liquid chromatogram of each solution in Example 1.

[0049] Figure 2 It is the high performance liquid chromatogram of the injection of the blank solution, blank excipient solution, reference solution STD1, and test solution in Example 10.

[0050] Figure 3 It is the linear relationship diagram of the apalutamide concentration and peak area in Example 11.

[0051] Figure 4 It is the chromatogram of the EB-A-3513 chromatographic column in the chromatographic column investigation of Example 15.

[0052] Figure 5 It is the chromatogram of the EB-A-5067 chromatographic column in the chromatographic column investigation of Example 15.

[0053] Figure 6 It is the chromatogram of the EB-A-4934 chromatographic column in the chromatographic column investigation of Example 15.

[0054] Figure 7 It is the chromatogram of the EB-A-6115 chromatographic column in the chromatographic column investigation of Example 15.

[0055] Figure 8 It is the chromatogram of the EB-A-5061 chromatographic column in the chromatographic column investigation of Example 15.

[0056] Figure 9 It is the chromatogram of the EB-A-4733 chromatographic column in the chromatographic column investigation of Example 15.

[0057] Figure 10 It is the high performance liquid chromatogram of gradient 2 and gradient 3 in the gradient investigation of Example 16.

[0058] Figure 11 It is the high performance liquid chromatography (HPLC) chromatogram of gradient 4 and gradient 3 in the gradient investigation of Example 16.

[0059] Figure 12 It is the high performance liquid chromatography (HPLC) chromatogram of gradient 5 and gradient 3 in the gradient investigation of Example 16.

[0060] Figure 13 It is the high performance liquid chromatography (HPLC) chromatogram using chromatographic column EB-A-4762 and chromatographic column EB-A-4505 in Comparative Example 1.

[0061] Figure 14 It is the high performance liquid chromatography (HPLC) chromatogram using chromatographic condition I in Comparative Example 2.

[0062] Figure 15 It is the high performance liquid chromatography (HPLC) chromatogram using chromatographic condition II in Comparative Example 2. Detailed implementation mode

[0063] 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 regarded as included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0064] To further understand the present invention, the present invention will be described in detail below in conjunction with embodiments.

[0065] Specifications of the instrument and chromatographic column:

[0066] Agilent 1260 type high performance liquid chromatography system and workstation in the United States; automatic injection.

[0067] Unless otherwise specified, the apalutamide raw material drug described in the present invention is: manufacturer: Dr.Reddy’s; batch number: AONH000539; grade: pharmaceutical grade.

[0068] Example 1: Sample detection

[0069] 1. Instrument and chromatographic conditions

[0070] Instrument: Agilent 1260 type high performance liquid chromatography system and workstation in the United States; equipped with an automatic sampler;

[0071] Chromatographic column: Chromcore Polar C18, 4.6mm x 100mm, 3μm;

[0072] Detection wavelength: 243nm;

[0073] Mobile phase: 0.05% trifluoroacetic acid was used as mobile phase A, and acetonitrile was used as mobile phase B.

[0074] Gradient elution:

[0075]

[0076]

[0077] Flow rate: 1.0 ml / min;

[0078] Column temperature: 30 °C;

[0079] Injection volume: 10 μL.

[0080] 2. Experimental procedures

[0081] 2.1 Solution preparation:

[0082] Solution A: Weigh 9.62 g of anhydrous citric acid, dissolve it in 900 mL of ultrapure water, adjust the pH value to 7.4 with ammonia water, and add water to make the total volume 1000 mL.

[0083] Diluent / blank solvent: Acetonitrile - Solution A = 1:1 ratio (V / V);

[0084] Related substances reference stock solution I: Take 25 mg of apalutamide raw material medicine, accurately weigh it, place it in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, and dilute to the mark with diluent, then shake well. The concentration is about 0.25 mg / mL. Prepare two portions in parallel.

[0085] Related substances reference stock solution II: Accurately pipette 2 ml of related substances reference stock solution I into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration is about 0.01 mg / ml. Prepare two portions in parallel.

[0086] Related substances reference solution: Accurately pipette 2.5 ml of related substances reference stock solution II into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration is about 0.0005 mg / ml. Prepare two portions in parallel, namely STD1 and STD2.

[0087] Related substances sensitivity solution: Accurately pipette 2.5 ml of related substances reference stock solution II into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration is about 0.00025 mg / ml.

[0088] Test solution: Take 10 tablets of apalutamide tablets, weigh the total weight and grind them. Take the tablet powder equivalent to 25 mg of apalutamide, weigh accurately, place it in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, dilute to the mark with diluent, and mix well. The concentration is about 0.25 mg / mL.

[0089] 2.2 Injection operation and result calculation

[0090] Inject 1 injection of the sensitivity solution SST and record its signal-to-noise ratio S / N; inject 3 injections of the reference solution STD1 and record the RSD of its peak area; inject 1 injection of the reference solution STD2 and calculate the system recovery rate; inject 1 injection of the test solution and record the chromatogram. Calculate the impurity content in the sample according to the following formula. The results are shown in Table 1 and Figure 1 .

[0091]

[0092] Where

[0093] A i : Peak area of each impurity peak in the test solution;

[0094] M STD : Sampling amount of the reference substance, mg;

[0095] P STD : Purity of the reference substance;

[0096] M: Total tablet weight, mg;

[0097] D i : Dilution factor of the test solution;

[0098] D STD : Dilution factor of the reference solution;

[0099] M i : Sampling amount of the test substance;

[0100] n: Number of ground tablets, tablets;

[0101] L: Labeled amount, mg;

[0102] A STD : Average value of the peak areas of the reference solution for the front and back needles of the test solution.

[0103] Table 1: Test results of the sample

[0104]

[0105] Conclusion: The tailing factor of the main peak in the chromatogram obtained by the method provided by the present invention is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak of the sensitivity solution is high, and the resolution between each impurity peak of the test solution and the adjacent peak is good.

[0106] Example 2: Investigation of Detection Wavelength (241 nm)

[0107] 1. Instruments and Chromatographic Conditions

[0108] Except that the detection wavelength is 241 nm, other chromatographic conditions and instruments are the same as those in Example 1.

[0109] 2. Experimental Procedures

[0110] 2.1 Solution Preparation

[0111] The same as in Example 1.

[0112] 2.2 Injection Operation and Result Calculation

[0113] The same as in Example 1.

[0114] Refer to the calculation formula in Example 1, and the results are shown in Table 2.

[0115] Table 2: Detection Results at a Detection Wavelength of 241 nm

[0116]

[0117]

[0118] Conclusion: In the chromatogram obtained by the detection method with a detection wavelength of 241 nm, the tailing factor of the main peak is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak of the sensitivity solution is high, and the resolution between the impurity peaks of the test solution and the adjacent peaks is good.

[0119] Example 3: Investigation of Detection Wavelength (245 nm)

[0120] 1. Instrument Chromatographic Conditions

[0121] Except that the detection wavelength is 245 nm, other chromatographic conditions and instruments are the same as those in Example 1.

[0122] 2. Experimental Procedures

[0123] 2.1 Solution Preparation

[0124] The same as in Example 1.

[0125] 2.2 Injection Operation and Result Calculation

[0126] The same as in Example 1.

[0127] Refer to the calculation formula in Example 1, and the results are shown in Table 3.

[0128] Table 3: Results at a Wavelength of 245 nm

[0129]

[0130]

[0131] Conclusion: In the chromatogram obtained by the detection method with a detection wavelength of 245 nm, the tailing factor of the main peak is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak in the sensitivity solution is high, and the resolution between each impurity peak and the adjacent peak in the test solution is good.

[0132] Example 4: Investigation of column temperature (28 °C)

[0133] 1. Instruments and chromatographic conditions

[0134] Except that the column temperature is 28 °C, other chromatographic conditions and instruments are the same as those in Example 1.

[0135] 2. Experimental procedures

[0136] 2.1 Solution preparation

[0137] The same as in Example 1.

[0138] 2.2 Injection operation and result calculation

[0139] The same as in Example 1.

[0140] Refer to the calculation formula in Example 1, and the results are shown in Table 4.

[0141] Table 4: Results at column temperature of 28 °C

[0142]

[0143] Conclusion: In the chromatogram obtained by the detection method at 28 °C, the tailing factor of the main peak is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak in the sensitivity solution is high, and the resolution between each impurity peak and the adjacent peak in the test solution is good.

[0144] Example 5: Investigation of column temperature (35 °C)

[0145] 1. Instruments and chromatographic conditions

[0146] Except that the column temperature is 35 °C, other chromatographic conditions and instruments are the same as those in Example 1.

[0147] 2. Experimental procedures

[0148] 2.1 Solution preparation

[0149] The same as in Example 1.

[0150] 2.2 Injection operation and result calculation

[0151] The same as in Example 1.

[0152] Refer to the calculation formula in Example 1, and the results are shown in Table 5.

[0153] Table 5: Results at Column Temperature of 35°C

[0154]

[0155] Conclusion: In the chromatogram obtained by the detection method at 35°C, the tailing factor of the main peak is good, the RSD of the peak area of the reference solution is low, the signal-to-noise ratio of the main peak of the sensitivity solution is high, and the resolution between each impurity peak of the test solution and the adjacent peak is good.

[0156] Example 6: Investigation of Flow Rate (0.8 ml / min)

[0157] 1. Instruments and Chromatographic Conditions

[0158] Except that the flow rate is 0.8 ml / min, other chromatographic conditions and instruments are the same as those in Example 1.

[0159] 2. Experimental Procedures

[0160] 2.1 Solution Preparation

[0161] Same as Example 1.

[0162] 2.2 Injection Operation and Result Calculation

[0163] Same as Example 1.

[0164] Refer to the calculation formula in Example 1, and the results are shown in Table 6.

[0165] Table 6: Results at Flow Rate of 0.8 mL / min

[0166]

[0167]

[0168] Example 7: Investigation of Flow Rate (1.2 ml / min)

[0169] 1. Instruments and Chromatographic Conditions

[0170] Except that the flow rate is 1.2 ml / min, other chromatographic conditions and instruments are the same as those in Example 1.

[0171] 2. Experimental Procedures

[0172] 2.1 Solution Preparation

[0173] Same as Example 1.

[0174] 2.2 Injection Operation and Result Calculation

[0175] Same as Example 1.

[0176] Refer to the calculation formula in Example 1, and the results are shown in Table 7.

[0177] Table 7: Results at a flow rate of 1.2 mL / min

[0178]

[0179] Example 8: Investigation of mobile phase A (using 0.025% trifluoroacetic acid as mobile phase A)

[0180] 1. Instruments and chromatographic conditions

[0181] Mobile phase: Use 0.025% trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B.

[0182] Other chromatographic conditions and instruments are the same as in Example 1.

[0183] 2. Experimental procedures

[0184] 2.1 Solution preparation

[0185] Same as in Example 1.

[0186] 2.2 Injection operation and result calculation

[0187] Same as in Example 1.

[0188] Refer to the calculation formula in Example 1, and the results are shown in Table 8.

[0189] Table 8: Results with mobile phase A = 0.025% trifluoroacetic acid

[0190]

[0191] Example 9: Mobile phase investigation (using 0.1% trifluoroacetic acid as mobile phase A)

[0192] 1. Instruments and chromatographic conditions

[0193] Mobile phase: Use 0.1% trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B.

[0194] Other chromatographic conditions and instruments are the same as in Example 1.

[0195] 2. Experimental procedures

[0196] 2.1 Solution preparation

[0197] Same as in Example 1.

[0198] 2.2 Injection operation and result calculation

[0199] Same as in Example 1.

[0200] Refer to the calculation formula in Example 1, and the results are shown in Table 9.

[0201] Table 9: Results with mobile phase A = 0.1% trifluoroacetic acid

[0202]

[0203]

[0204] Conclusion: From the results of Examples 1 - 9, it can be seen that the detection method using the chromatographic condition range provided by the present invention (aqueous solution of 0.025% vol - 0.1% vol trifluoroacetic acid as mobile phase A, detection wavelength of the liquid chromatography method is 241 nm - 245 nm, flow rate of the liquid chromatography method is 0.8 ml / min - 1.2 ml / min; column temperature of the liquid chromatography method is 28 °C - 35 °C) has the advantages of high sensitivity, good durability, good resolution between each impurity peak and adjacent peaks, and comprehensive impurity detection results.

[0205] Example 10: Specificity investigation

[0206] 1. Instruments and chromatographic conditions

[0207] Same as Example 1.

[0208] 2. Experimental procedures

[0209] 2.1 Solution preparation

[0210] The preparation of Solution A, diluent / blank solvent, related substance reference stock solution I, related substance reference stock solution II, related substance reference solution, related substance sensitivity solution, and test solution is the same as in Example 1.

[0211] Blank excipient solution: Take 100 mg of blank excipients of apalutamide (equivalent to the excipients of tablet powder containing 25 mg of apalutamide), accurately weigh, place in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, dilute to the mark with diluent, and mix well. Take an appropriate amount, centrifuge (10000 rpm, 10 min), and use the supernatant as the blank excipient solution.

[0212] 2.2 Injection operation and result calculation

[0213] After the system suitability is qualified, inject the blank solution, blank excipient solution, reference solution STD1, and test solution for high - performance liquid chromatography analysis under the above conditions, and record the chromatogram and peak purity factor. The results are shown in Table 10 and Figure 2 :

[0214] Table 10: Results of specificity (main component) investigation

[0215]

[0216]

[0217] Conclusion: The detection method of the present invention has good specificity.

[0218] Example 11: Linearity Investigation

[0219] 1. Instruments and Chromatographic Conditions

[0220] Same as Example 1.

[0221] 2. Experimental Procedures

[0222] 2.1 Solution Preparation

[0223] The preparation of Solution A, diluent / blank solvent, stock solution of related substance reference standard I, stock solution of related substance reference standard II, related substance reference solution, and related substance sensitivity solution is the same as that in Example 1.

[0224] Linear stock solution of the main component: Weigh accurately an amount equivalent to 25 mg of apalutamide raw material drug, place it in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, dilute to the mark with diluent, and mix well. The concentration is about 0.25 mg / mL. Pipette 5 mL of the above solution accurately into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well to obtain. The concentration is about 0.0125 mg / mL.

[0225] Linear solution: Take the linear stock solution of the main component according to the following table, pipette the corresponding volume of the stock solution accurately into the corresponding volumetric flask, dilute to the mark with diluent, and mix well to obtain the linear solutions at each concentration level.

[0226]

[0227] 2.2 Injection Operation and Result Calculation

[0228] After the system is balanced, inject the above linear solutions respectively under the above chromatographic conditions, record the corresponding chromatograms and peak areas. Take the peak areas against the concentrations at each of the above concentration points for linear regression, calculate and report the linear correlation coefficient r, the Y-axis intercept, and the sum of squared residuals, the residual plot, and the absolute value of the Y-axis intercept / peak area corresponding to the limit concentration level. The results are shown in Table 11 and Figure 3 :[[]]

[0229] Table 11: Results of Linearity Investigation

[0230]

[0231]

[0232] Conclusion: The detection method provided by the present invention has good linearity.

[0233] Example 12: Accuracy Investigation

[0234] 1. Instruments and Chromatographic Conditions

[0235] Same as Example 1.

[0236] 2. Experimental procedures

[0237] 2.1 Solution preparation

[0238] The preparation of Solution A, diluent / blank solvent, Stock solution of related substance reference standard I, Stock solution of related substance reference standard II, Related substance reference solution, and Related substance sensitivity solution is the same as that in Example 1.

[0239] Accuracy stock solution: Weigh accurately an amount equivalent to 25 mg of apalutamide raw material drug, place it in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, dilute to the mark with diluent, and mix well. The concentration is about 0.25 mg / mL. Pipette 5 mL of the above solution accurately into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the solution. The concentration is about 0.0125 mg / mL.

[0240] 0.05%, 0.2%, 0.5% Accuracy test sample solutions: Weigh the corresponding blank excipients (excipients in apalutamide tablets) as shown in the following table into the corresponding volumetric flasks, moisten with diluent, then pipette the corresponding volume of stock solution accurately into the volumetric flasks, shake mechanically for 30 min, dilute to the mark with diluent, and mix well. Take an appropriate amount of the solution, centrifuge (10000 rpm, 10 min), and take the supernatant to obtain 0.05%, 0.2%, 0.5% Accuracy test sample solutions.

[0241] Prepare three parallel portions of each concentration solution.

[0242]

[0243] 2.2 Injection operation and result calculation

[0244] After the system is balanced, take each concentration of accuracy test sample solution respectively under the above chromatographic conditions, record the chromatogram, and calculate the actually measured value, theoretical value, single recovery rate, and 95% confidence interval of the average recovery rate (n = 9) of the component to be measured. The results are shown in Table 12.

[0245] Table 12: Results of accuracy investigation

[0246]

[0247]

[0248] Conclusion: The detection method provided by the present invention has high accuracy.

[0249] Example 13: Precision investigation

[0250] 1. Instruments and chromatographic conditions

[0251] Same as Example 1.

[0252] 2. Experimental Procedures

[0253] 2.1 Solution Preparation

[0254] The preparation of Solution A, diluent / blank solvent, Stock Solution I of related substance reference standard, Stock Solution II of related substance reference standard, Related Substance Reference Solution, Related Substance Sensitivity Solution, and Test Solution was the same as that in Example 1.

[0255] Precision Stock Solution: Weigh accurately an amount equivalent to 25 mg of apalutamide raw material drug, place it in a 100 mL volumetric flask, add about 60 mL of diluent, shake for 30 min, dilute to the mark with diluent, and mix well. The concentration is about 0.25 mg / mL. Pipette 5 mL of the above solution accurately into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the solution. The concentration is about 0.0125 mg / mL.

[0256] 0.2% Precision Test Solution: Weigh 50 mg of blank excipient into a 50 mL volumetric flask, moisten it with diluent, then pipette 2 mL of the stock solution accurately into the volumetric flask, shake mechanically for 30 min, dilute to the mark with diluent, and mix well. Take an appropriate amount of the solution, centrifuge (10000 rpm, 10 min), and take the supernatant as the precision test solution. Prepare 6 parallel samples.

[0257] 2.2 Injection Operation and Result Calculation

[0258] After the system is balanced, take the 0.2% precision test solution respectively, inject samples according to the above chromatographic conditions, record the chromatogram, and calculate the recovery rate of the component to be measured and the 95% confidence interval of the recovery rate (n = 6). The results are shown in Table 13.

[0259] Table 13: Results of Precision (Analytical Repeatability) Investigation

[0260]

[0261] Conclusion: The detection method provided by the present invention has good precision.

[0262] Example 14: Sensitivity Investigation

[0263] 1. Instruments and Chromatographic Conditions

[0264] The same as in Example 1.

[0265] 2. Experimental Procedures

[0266] 2.1 Solution Preparation

[0267] The preparation of Solution A, diluent / blank solvent, Stock Solution I of related substance reference standard, Stock Solution II of related substance reference standard, Related Substance Reference Solution, and Related Substance Sensitivity Solution was the same as that in Example 1.

[0268] Sensitivity solution: Accurately pipette 3 ml of the related substances sensitivity solution into a 50-ml volumetric flask, dilute to the mark with the diluent, and mix well. The concentration is approximately 0.000075 mg / ml (0.03% of the 0.25 mg / mL test solution).

[0269] 2.2 Injection operation and result calculation

[0270] Inject the sensitivity solution (quantitation limit solution) 3 times, record the signal-to-noise ratio, peak area, and RSD of the peak area. The results are shown in Table 14.

[0271] Table 14: Results of sensitivity investigation

[0272]

[0273] Example 15: Chromatographic column screening

[0274] 1. Instruments and chromatographic conditions:

[0275] Except for the chromatographic column, the others are the same as in Example 1;

[0276] Chromatographic column 1 (internal number: EB-A-3513): Kromasil 100-5-C18, 100×4.6 mm, 5 μm.

[0277] Chromatographic column 2 (internal number: EB-A-5067): ACE Excel 3C18-Amide, 100×4.6 mm, 3 μm.

[0278] Chromatographic column 3 (internal number: EB-A-4934): ACE Excel 3C18-PFP, 100×4.6 mm, 3 μm.

[0279] Chromatographic column 4 (internal number: EB-A-6115): Poroshell 120EC-C18, 100×4.6 mm, 2.7 μm.

[0280] Chromatographic column 5 (internal number: EB-A-5061): YMC-Triart C18, 100×4.6 mm, 3 μm.

[0281] Chromatographic column 6 (internal number: EB-A-4733): Chromcore 120C18, 100×4.6 mm, 3 μm.

[0282] 2. Experimental procedure

[0283] Test solution: The same as in Example 1.

[0284] Inject the test solution once under the conditions of different chromatographic columns, and record the chromatogram. The results are shown in Table 15 and Figures 4 - 9 as follows.

[0285] Results:

[0286] Table 15: Results of Chromatographic Column Investigation

[0287]

[0288] Example 16: Investigation of Gradient Elution Program

[0289] Apparatus and chromatographic conditions: Except for the elution program, others are the same as in Example 1. Gradient elution program 1 (abbreviated as Gradient 1):

[0290]

[0291] Gradient elution program 2 (abbreviated as Gradient 2):

[0292]

[0293] Gradient elution program 3 (abbreviated as Gradient 3):

[0294]

[0295] Elution program 4 (abbreviated as Gradient 4):

[0296]

[0297] Gradient elution program 5 (abbreviated as Gradient 5):

[0298]

[0299] Experimental procedure

[0300] Test solution: The same as in Example 1.

[0301] Inject the test solution once under the conditions of different chromatographic columns, and record the chromatogram. The results are as Figures 10 - 12 , shown in Table 16 and Table 17.

[0302] Results:

[0303] Table 16: Detection Results of Elution Programs Using Gradients 1, 2, and 3

[0304]

[0305] Table 17: Detection Results of Elution Program Using Gradient 5

[0306]

[0307] Result analysis:

[0308] (1) In the chromatograms obtained by using the elution programs of gradient 1 and gradient 2, the resolution between the main peak and unknown impurity 4 is small, and the separation effect is poor.

[0309] (2) In the chromatogram obtained by using the elution program of gradient 3, the two impurities (i.e., unknown impurity 7 and unknown impurity 8) with retention times after 35 min cannot be separated.

[0310] (3) Compared with the elution program of gradient 3, in the chromatogram obtained by using the elution program of gradient 4 ( Figure 11 ), all impurity peaks elute relatively late, and unknown impurity 7 and unknown impurity 8 elute completely together and cannot be separated.

[0311] (4) In the chromatogram obtained by using the elution program of gradient 5, unknown impurity 7 and unknown impurity 8 (the two peaks with retention times of about 35.731 and 36.265 in the chromatogram obtained by using the elution program of gradient 5) can be separated, and the resolution between other impurity peaks is greater than 1.5, and the separation effect is good.

[0312] (5) Since there are no impurity peaks exceeding the detection limit between 40 min and 50 min in the chromatogram obtained by using the elution program of gradient 5, therefore, the elution program between 40 min and 50 min in the elution program of gradient 5 can be deleted.

[0313] Comparative Example 1: Investigation of chromatographic column

[0314] 1. Instruments and chromatographic conditions:

[0315] Except for the chromatographic column, others are the same as in Example 1.

[0316] Chromatographic column 7 (internal number: EB-A-4762): Waters Symmetry C18, 4.6 mm x 150 mm, 3.5 μm.

[0317] Chromatographic column 8 (the type of chromatographic column used in the present invention, internal number: EB-A-4505): chromcore polarC18, 4.6 mm x 100 mm, 3 μm.

[0318] 2. Experimental procedure

[0319] Test solution: The same as in Example 1.

[0320] Take the test solution and inject it once under the conditions of different chromatographic columns, and record the chromatograms. The results of chromatographic column 7 and chromatographic column 8 are as Figure 13 shown.

[0321] Results:

[0322] Result analysis: As can be seen from Figure 13 , under the condition that the chromatographic columns are different and other chromatographic conditions are the same, compared with the chromatographic column type provided by the present invention (for example, chromcore polar C18, 4.6 mm x 100 mm, 3 μm), only three unknown impurities can be separated and detected by using chromatographic column 7, and fewer impurities can be detected; while using the chromatographic column type provided by the present invention (for example, chromcore polar C18, 4.6 mm x 100 mm, 3 μm) can detect more impurities, and the resolution between peaks is better.

[0323] Comparative example 2: Investigation of different gradient elutions

[0324] 1. Instruments and chromatographic conditions

[0325] The instruments are the same as those in Example 1, and the following chromatographic conditions are respectively used for investigation:

[0326] Chromatographic condition I: Except for the chromatographic column, other conditions are the same as those in Example 1; Chromatographic column: Agilent Zorbax SB-CN, 4.6 mm x 250 mm, 5 μm (chromatographic column 9).

[0327] Chromatographic condition II: As shown in the following table:

[0328]

[0329]

[0330] Experimental procedure

[0331] Test solution: The same as that in Example 1.

[0332] Take the test solution and inject it once under the conditions of different chromatographic columns, and record the chromatogram. The results are shown in Table 18, Table 19 and Figures 14 - 15 as shown.

[0333] Results:

[0334] Table 18: Detection results using chromatographic condition I

[0335]

[0336] Table 19: Detection results using chromatographic condition II

[0337]

[0338]

[0339] Result analysis: The results show that under the condition of using the same chromatographic column, compared with other chromatographic conditions, the chromatographic conditions provided by the present invention have better separation effects and can detect more impurities.

[0340] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations 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 parameters for implementation. It should be particularly pointed out that all similar substitutions and alterations are obvious to those skilled in the art, and they are all regarded as included in the present invention.

Claims

1. A liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations, comprising: A chromatographic column filled with octadecylsilyl-bonded silica gel, using an aqueous acid solution and acetonitrile as the mobile phase, for gradient elution.

2. The liquid chromatography method according to claim 1, wherein the elution program of the gradient elution comprises: Gradient elution method a: Or gradient elution method b: Or gradient elution method c: 。 3. The liquid chromatography method according to any one of claims 1-2, wherein the packing material of the chromatographic column is Chromcore PolarC18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18.

4. The liquid chromatography method according to any one of claims 1-3, wherein the length of the chromatographic column is 100 mm - 250 mm, preferably 100 mm; optionally, the inner diameter of the chromatographic column is 2 mm - 5 mm, or 4 mm - 5 mm, or 4.6 mm; Optionally, the particle size of the packing material of the chromatographic column is 2 μm - 5 μm, or 2.5 μm - 5 μm, or 2.7 μm or 3 μm; Optionally, the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 100 mm - 250 mm, and the particle size of the packing material of the chromatographic column is 2.5 μm - 3 μm.

5. The liquid chromatography method according to any one of claims 1-4, wherein the aqueous acid solution is an aqueous solution containing trifluoroacetic acid; Optionally, the aqueous acid solution is an aqueous solution containing 0.025% vol - 0.1% vol trifluoroacetic acid.

6. The liquid chromatography method according to any one of claims 1-5, wherein the detection wavelength of the liquid chromatography method is 241 nm - 245 nm; Optionally, the flow rate of the liquid chromatography method is 0.8 ml / min - 1.2 ml / min; Optionally, the column temperature of the liquid chromatography method is 28 °C - 35 °C.

7. The liquid chromatography method according to any one of claims 1-6, wherein the liquid chromatography method further comprises preparing apalutamide, its salt or its preparation into a test solution, and the diluent of the test solution is acetonitrile or a mixture of acetonitrile and solution A, and solution A is an aqueous solution containing citric acid and / or phosphate; Optionally, the volume ratio of acetonitrile to solution A in the mixture of acetonitrile and solution A is 1:9 - 10:0 or 1:1; Optionally, the pH of solution A is 6 - 8, 7 or 7.4; Optionally, the pH of solution A is adjusted with ammonia water or sodium hydroxide.

8. The liquid chromatography method according to claim 7, wherein apalutamide, its salt or its preparation is calculated as apalutamide, and the content of apalutamide in the test solution is 0.10 mg / ml - 1.00 mg / ml.

9. A liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations, which comprises: Prepare apalutamide, its salt or its preparation into a test solution. Use a chromatographic column filled with octadecylsilyl silica gel. The packing material of the chromatographic column is Chromcore Polar C18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18. The length of the chromatographic column is 100 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the packing material of the chromatographic column is 3 μm. Use an aqueous acid solution and acetonitrile as the mobile phase. The aqueous acid solution is an aqueous solution containing 0.025% vol to 0.1% vol trifluoroacetic acid. The detection wavelength of the liquid chromatography method is 241 - 245 nm, and the flow rate of the liquid chromatography method is 0.8 ml / min - 1.2 ml / min; the column temperature of the liquid chromatography method is 28°C - 35°C; the injection volume of the liquid chromatography method is 5 μL - 30 μL. Perform gradient elution on the test solution. The gradient elution program includes: Gradient elution method a: Or gradient elution method b: Or gradient elution method c: 。 10. A liquid chromatography method for the determination of the content or impurities of apalutamide, its salts or its preparations, comprising: Prepare apalutamide, its salt or its preparation into a test solution. Use a chromatographic column filled with octadecylsilyl silica gel. The packing material of the chromatographic column is Chromcore Polar C18, Poroshell 120EC-C18, YMC-Triart C18 or Chromcore 120C18. The length of the chromatographic column is 100 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the packing material of the chromatographic column is 3 μm. Use an aqueous acid solution and acetonitrile as the mobile phase. The aqueous acid solution is an aqueous solution containing 0.025% vol to 0.1% vol trifluoroacetic acid. The detection wavelength of the liquid chromatography method is 243 nm, and the flow rate of the liquid chromatography method is 1 ml / min; the column temperature of the liquid chromatography method is 30°C; the injection volume of the liquid chromatography method is 10 μL - 20 μL. Perform gradient elution on the test solution. The gradient elution program includes: Gradient elution method a: Or gradient elution method b: Or gradient elution method c: 。

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