Method for detecting impurity content in abiraterone acetate tablet by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for detecting impurity content in abiraterone acetate tablets by utilizing HPLC (High Performance Liquid Chromatography), and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: 1) preparing a test solution and a reference solution; 2) setting high performance liquid detection conditions; a chromatographic column taking octadecyl bonded silica gel as a filling agent is adopted; the mobile phase is a mixed solution of a mobile phase A and a mobile phase B, the mobile phase A is a methanol-ammonium acetate solution, and the mobile phase B is methanol-acetonitrile; carrying out gradient elution by adopting a mobile phase; and 3) respectively taking the test solution and the reference solution, injecting into a liquid chromatograph, recording and analyzing a chromatogram, drawing a standard curve, and calculating the impurity content. The impurities in the abiraterone acetate tablet can be accurately and efficiently detected by utilizing the high performance liquid chromatography, the product quality of the abiraterone acetate tablet is improved, and the medication safety of a patient is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting the impurity content in abiraterone acetate tablets by utilizing HPLC. Background Art

[0002] Prostate cancer is the most common malignant tumor of the male reproductive system. It is reported to be the second leading cause of cancer death in men, second only to lung cancer. Abiraterone acetate is used in combination with prednisone to treat metastatic castration-resistant prostate cancer. Currently, the indications of abiraterone acetate have been expanded from prostate cancer patients who have failed chemotherapy to the treatment of prostate cancer patients before chemotherapy, and have been widely used in clinical practice.

[0003] Abiraterone acetate, chemically known as (3β)-17-(3-pyridyl)-androsta-5,16-diene-3-acetate, is a highly selective, orally effective, irreversible inhibitor of 17α-hydroxylase / C17,20-lyase (CYP17). It is metabolized into abiraterone in the body and exerts its effect. Its inhibitory activity on CYP17 is 10 to 30 times that of ketoconazole. It is used in combination with prednisone in clinical treatment of castration-resistant prostate cancer. Abiraterone is a potent, irreversible, and selective 17α-hydroxylase / C17,20-lyase (CYP17) inhibitor. CYP17 is an enzyme expressed in the testis, adrenal gland, and prostate tumor tissues that regulates the biosynthesis of androgens. It is used to treat metastatic castration-resistant prostate cancer and hormone-sensitive high-risk metastatic prostate cancer. However, due to the poor oral bioavailability of abiraterone and its susceptibility to hydrolysis by esterases, abiraterone acetate has been developed as an orally bioavailable prodrug with greater stability and absorption.

[0004] Abiraterone acetate belongs to the class of organic compounds such as androgens and their derivatives, and is a 3-hydroxylated C19 steroid hormone. In the process of preparing intermediates, the process route of abiraterone acetate will produce process by-product impurities 7-Ketoabiraterone Acetate (7-ketoabiraterone acetate), 3-Deoxy-3-Acetyl Abiraterone-3-ene (abiraterone dehydrated 3-acetyl impurity), α-Enantiomer (α enantiomer), Abiraterone Ethyl Ether (abiraterone ethyl ether), 3β-Propionyloxy (abiraterone propionate), Abiraterone Isopropyl Ether (abiraterone isopropyl ether), AnhydroAbiraterone (abiraterone dehydrated impurity), 3-Deoxy-3-Chloroabiraterone (3-deoxy-3-chloroabiraterone), O-Chlorobutylabiraterone (o-chlorobutylabiraterone); the final product abiraterone acetate tablets will also produce the impurity α-Epoxyabiraterone during production and storage. Acetate (α-Epoxyabiraterone acetate), β-EpoxyabirateroneAcetate (β-Epoxyabiraterone acetate), Abiraterone (Abiraterone).

[0005] Therefore, it is necessary to provide a method for detecting the impurity content in abiraterone acetate tablets using HPLC. Summary of the invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for detecting the impurity content in abiraterone acetate tablets using HPLC, which is used to accurately and efficiently detect the impurities in abiraterone acetate tablets, improve the product quality of abiraterone acetate tablets, and improve the safety of medication.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] A method for detecting the impurity content in abiraterone acetate tablets using HPLC, the specific steps are as follows:

[0009] 1) Prepare test solution and reference solution;

[0010] 2) Setting high performance liquid phase detection conditions; using a chromatographic column with octadecyl bonded silica gel as a filler; the mobile phase is a mixed solution of mobile phase A and mobile phase B, wherein mobile phase A is a methanol-ammonium acetate solution and mobile phase B is methanol-acetonitrile; gradient elution is performed using the mobile phase;

[0011] 3) Take the test solution and the reference solution respectively, inject them into the liquid chromatograph, record and analyze the chromatogram, draw the standard curve, and calculate the impurity content.

[0012] Furthermore, in step 1), the method for preparing the test solution is: accurately weighing the abiraterone acetate tablet sample and diluting it with acetonitrile to form a solution containing 0.6 mg of abiraterone acetate per 1 mL; the method for preparing the reference solution is: accurately measuring the test solution and diluting it with acetonitrile to form a solution containing 0.6 μg of abiraterone acetate per 1 mL.

[0013] Furthermore, in step 2), the volume ratio of methanol to ammonium acetate solution in mobile phase A is 2:3.

[0014] Furthermore, the concentration of the ammonium acetate is 0.02 mol / L.

[0015] Furthermore, in step 2), the volume ratio of methanol to acetonitrile in mobile phase B is 9:1.

[0016] Furthermore, in step 2), the gradient elution method is: 0 to 15 min, eluting with mobile phase A and mobile phase B in a volume ratio of 50:50; 55 to 70 min, eluting with mobile phase B; 75 to 90 min, eluting with mobile phase A and mobile phase B in a volume ratio of 50:50, and the elution is completed, wherein the elution flow rate is 1.0 mL / min.

[0017] Furthermore, in step 2), the specification of the chromatographic column is 4.6 mm×250 mm, and the filler particle size is 2.7 μm.

[0018] Furthermore, in step 2), the column temperature of the chromatographic column is 30°C.

[0019] Furthermore, in step 3), the injection volume of the test solution and the reference solution is 10 μL.

[0020] Furthermore, in step 3), the detection wavelength of the liquid chromatograph is 254 nm.

[0021] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0022] (1) In the present invention, at a detection wavelength of 254 nm, abiraterone acetate and various impurities can be well separated, the auxiliary material peak and the solvent peak do not interfere with the detection of the main component and various known impurities, and the peak purity test of the samples destroyed under various conditions shows that the spectral purity is greater than 990, and the impurities generated by the destruction under various conditions do not interfere with the detection of various known impurities and the main component, and the detection specificity of related substances is good.

[0023] (2) Compared with the prior art, the present invention has no influence on the separation and inspection results of impurities related to abiraterone acetate tablets when slight changes occur in chromatographic condition parameters, and has good durability.

[0024] (3) The high performance liquid chromatography method used in the present invention can accurately and efficiently detect impurities in abiraterone acetate tablets, which is beneficial to improving the product quality of abiraterone acetate tablets and improving the drug safety of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a chromatogram of the system applicability of the method for analyzing related substances of Abiraterone Acetate Tablets of the present invention;

[0026] Figure 2 It is a chromatogram of each destruction condition test in the destructive test of abiraterone acetate tablets of the present invention;

[0027] Figure 3 This is the standard curve of impurity 7-KetoabirateroneAcetate;

[0028] Figure 4 This is the standard curve of impurity α-Epoxyabiraterone Acetate;

[0029] Figure 5 This is the standard curve of impurity β-EpoxyabirateroneAcetate;

[0030] Figure 6 This is the standard curve of impurity Abiraterone;

[0031] Figure 7 It is the standard curve diagram of impurity 3-Deoxy-3-AcetylAbiraterone-3-ene;

[0032] Figure 8 This is the standard curve of impurity α-Enantiomer;

[0033] Fig. 9 This is the standard curve of impurity Abiraterone Ethyl Ether;

[0034] Fig.10 This is the standard curve of AbirateroneAcetate;

[0035] Fig.11 This is the standard curve of impurity 3β-Propionyloxy;

[0036] Fig.12 This is the standard curve of the impurity Abiraterone Isopropyl Ether;

[0037] Fig.13 This is the standard curve of impurity AnhydroAbiraterone;

[0038] Fig.14 This is the standard curve of impurity 3-Deoxy-3-Chloroabiraterone;

[0039] Fig.15 This is the standard curve of impurity O-Chlorobutylabiraterone;

[0040] Fig.16 This is the system suitability chromatogram for detecting related substances of Abiraterone Acetate Tablets using the raw material method;

[0041] Fig.17 This is the spectrum of abiraterone acetate tablets and raw materials, blank excipients, and blank solvent. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments.

[0043] The HPLC detection conditions used in the following examples are as follows:

[0044] Chromatographic column: Agilent ZORBAX Bonus-RP C18 column 4.6mm×250mm, particle size 2.7μm;

[0045] Flow rate: 1.0 mL / min;

[0046] Column temperature: 30°C;

[0047] Mobile phase: Mobile phase A is a mixed solution of methanol and 0.02 mol / L ammonium acetate solution in a volume ratio of 2:3; Mobile phase B is a mixed solution of methanol and acetonitrile in a volume ratio of 9:1;

[0048] Detection wavelength: 254nm;

[0049] Injection volume: The injection volume of the test solution and the reference solution is 10 μL.

[0050] The method of gradient elution using mobile phase is: the method of gradient elution is: 0-15min, mobile phase A and mobile phase B are eluted in a volume ratio of 50:50; 55-70min, mobile phase B is used for elution; 75-90min, mobile phase A and mobile phase B are eluted in a volume ratio of 50:50, and elution is completed, wherein the elution flow rate is 1.0mL / min.

[0051] The structures of the impurities described in the following examples are as follows:

[0052]

[0053]

[0054]

[0055] Example 1

[0056] A method for detecting the impurity content in abiraterone acetate tablets using HPLC is as follows:

[0057] 1) Prepare the test solution and the reference solution; accurately weigh the abiraterone acetate tablet sample and dilute it with acetonitrile to a solution containing 0.6 mg of abiraterone acetate per 1 mL; the reference solution is prepared by accurately measuring the test solution and diluting it with acetonitrile to a solution containing 0.6 μg of abiraterone acetate per 1 mL.

[0058] 2) Setting the HPLC detection conditions; using a chromatographic column with octadecyl bonded silica gel as a filler; the column temperature of the chromatographic column is 30°C; the mobile phase is a mixed solution of mobile phase A and mobile phase B, wherein mobile phase A is a mixed solution of methanol and 0.02 mol / L ammonium acetate solution in a volume ratio of 2:3; mobile phase B is a mixed solution of methanol and acetonitrile in a volume ratio of 9:1; the method of gradient elution using the mobile phase is: first, mobile phase A and mobile phase B are eluted at a volume ratio of 50:50 for 15 min, then mobile phase B is eluted for 70 min, and finally mobile phase A and mobile phase B are eluted at a volume ratio of 50:50 for 90 min, and the elution is completed, wherein the elution flow rate is 1.0 mL / min.

[0059] 3) Take 10 μL of the test solution and the reference solution respectively, inject them into the liquid chromatograph with the detection wavelength of 254 nm, record and analyze the chromatogram, draw the standard curve, and calculate the impurity content.

[0060] Example 2

[0061] 1. System applicability

[0062] Accurately weigh appropriate amounts of abiraterone acetate reference substance and impurity 7-Ketoabiraterone Acetate, α-Epoxyabiraterone Acetate, β-Epoxyabiraterone Acetate, Abiraterone, 3-Deoxy-3-Acetyl Abiraterone-3-ene, α-Enantiomer, Abiraterone Ethyl Ether, 3β-Propionyloxy, Abiraterone Isopropyl Ether, Anhydro Abiraterone, 3-Deoxy-3-Chloroabiraterone, and O-Chlorobutylabiraterone reference substances, dissolve them in acetonitrile and dilute them into a mixed solution containing 0.6 mg of abiraterone acetate, approximately 1.8 μg each of α-Epoxyabiraterone Acetate and β-Epoxyabiraterone Acetate, and 0.6 μg of each other known impurities per 1 mL as the system suitability solution.

[0063] 10 μL of each was injected into a high performance liquid chromatograph, and the chromatogram was recorded at a detection wavelength of 254 nm to test and examine the separation of each component. The results are shown in Table 1 and Figure 1 shown.

[0064] Table 1 Specificity results

[0065] Product Name System Suitability Solutions Separation Blank solvent —— —— Blank Accessories —— —— 7-KetoabirateroneAcetate 9.259 6.414 α-EpoxyabirateroneAcetate 12.284 10.054 β-EpoxyabirateroneAcetate 17.996 4.005 Abiraterone 20.568 2.154 3-Deoxy-3-AcetylAbiraterone-3-ene 22.036 13.748 α-Enantiomer 31.123 3.709 AbirateroneAcetate 33.366 7.399 AbirateroneEthylEther 37.431 8.321 3β-Propionyloxy 41.578 3.077 AbirateroneIsopropylEther 43.007 4.267 AnhydroAbiraterone 44.933 3.054 3-Deoxy-3-Chloroabiraterone 46.376 1.720 O-Chlorobutylabiraterone 48.826 1.859

[0066] Table 1 shows the specificity results. Figure 1 The chromatogram of the system applicability of the method for analyzing the related substances of Abiraterone Acetate Tablets of the present invention is shown in Table 1 and Figure 1It can be seen that at the detection wavelength of 254nm, the peak order of the mixed solution is 7-KetoabirateroneAcetate, α-EpoxyabirateroneAcetate, β-EpoxyabirateroneAcetate, Abiraterone, 3-Deoxy-3-AcetylAbiraterone-3-ene, α-Enantiomer, Abiraterone EthylEther, 3β-Propionyloxy, Abiraterone Isopropyl Ether, Anhydro Abiraterone, 3-Deoxy-3-Chloroabiraterone, O-Chlorobutylabiraterone, and there is sufficient separation between AbirateroneAcetate and each known impurity, and the solvent peak, excipient peak and other impurities do not interfere with the determination of AbirateroneAcetate and each known impurity.

[0067] 2. Destructive testing

[0068] Undamaged: Take an appropriate amount of abiraterone acetate tablets, add an appropriate amount of acetonitrile, dissolve by ultrasonication, and then dilute to a fixed volume to make a solution containing 0.6 mg of abiraterone acetate per 1 mL.

[0069] Acid destruction: Weigh 30 mg of abiraterone acetate, place in a 50 mL volumetric flask, add 5 mL of 0.1 mol / L hydrochloric acid solution, place in a 60°C water bath for 16 hours, take out, cool to room temperature, add 5 mL of 0.1 mol / L sodium hydroxide solution to neutralize, add appropriate amount of solvent to dissolve by ultrasonication, dilute to the scale, shake well, and use as the acid destruction solution.

[0070] Alkali destruction: Weigh 30 mg of abiraterone acetate, place in a 50 mL volumetric flask, add 5 mL of 0.1 mol / L sodium hydroxide solution, place in a 60°C water bath for 16 hours, take out, cool to room temperature, add 5 mL of 0.1 mol / L hydrochloric acid solution to neutralize, add appropriate amount of solvent to dissolve by ultrasonication, dilute to the scale, shake well, and use as the alkali destruction solution.

[0071] Oxidative destruction: Weigh 30 mg of abiraterone acetate, place in a 50 mL volumetric flask, add 5 mL of 3% hydrogen peroxide solution, place in a 60°C water bath for 16 hours, take out, cool to room temperature, add appropriate amount of solvent to dissolve by ultrasonication, dilute to the mark, shake well, and use as the oxidative destruction solution.

[0072] Light damage: Weigh 30 mg of abiraterone acetate into a 50 mL volumetric flask, add a small amount of solvent to disperse, and place at 4000 lx, UV-100 μw / cm 2After being placed under light for 24 hours, take it out, add appropriate amount of solvent to dissolve it by ultrasonication, make up to the scale, and shake well to use as the light destruction solution.

[0073] Pyrolytic destruction: Weigh 30 mg of abiraterone acetate, place in a 50 mL volumetric flask, add a small amount of solvent to disperse, place in a 90 ° C oven for 16 hours, take out, cool to room temperature, add appropriate amount of solvent to dissolve by ultrasonication, dilute to the scale, shake well, and use as the pyrolytic destruction solution.

[0074] Take 10 μL of the above test solution and inject it into the chromatograph to record the chromatogram. The results are shown in Table 2, Table 3 and Figure 2 shown.

[0075] (1) Peak purity

[0076] The peak purity of the test solution under various destruction conditions was determined by diode array measurement. The results are shown in Table 2 below.

[0077] Table 2 Peak purity results

[0078] Destruction conditions Unbroken Acid damage Alkali damage Oxidative damage Light damage Pyrolysis Spectral purity 0.99991 0.999990 0.999990 0.99992 0.999990 0.999991

[0079] (2) Material balance

[0080] Table 3 Peak purity results

[0081]

[0082] Tables 2 and 3 are the peak purity results. It can be seen from the table that the peak purity of the samples destroyed under various conditions is greater than 0.990 after peak purity detection, the impurities are well separated from the main peak, and there is no impurity in the main peak, indicating that the method has good specificity.

[0083] Figure 2 The chromatograms of the various destruction conditions in the destructive test of the Abiraterone acetate tablets of the present invention are shown in the figure. It can be seen from the figure that in the degradation test, under the conditions of acid destruction, light destruction, oxidation destruction, alkali destruction, and thermal destruction, degradation impurities are generated, and the degradation products can be well separated from the solvent peak and the main peak; the peak purity of the destroyed samples under each condition is greater than 990 after peak purity detection, and the materials are basically balanced before and after destruction. This analytical method can effectively detect the impurity content in the Abiraterone acetate tablets.

[0084] 3. Limit of quantification and limit of detection

[0085] Take appropriate amounts of 7-KetoabirateroneAcetate, α-EpoxyabirateroneAcetate, β-Epoxyabiraterone Acetate, Abiraterone, 3-Deoxy-3-Acetyl Abiraterone-3-ene, 3α-acetoxy-17-(3-pyrridyl)androsta-5,16-diene, Abiraterone Acetate, AbirateroneEthyl Ether, Abiraterone propionate, Abiraterone Isopropyl Ether, AnhydroAbiraterone, 3-Deoxy-3-Chloroabiraterone and O-Chlorobutylabiraterone, weigh them accurately, add solvents to dissolve them respectively and dilute them to prepare stock solutions of each impurity containing about 1 mg of AbirateroneAcetate and each known impurity in each mL. The limit of quantification (S / N≥10) and detection limit (S / N≥3) were measured by the dilution method. The preparation method of the sensitivity solution was as follows: 1 mL of the control solution was accurately measured and placed in a 10 mL volumetric flask, and acetonitrile was added to dilute to the scale and shaken to prepare the sensitivity solution. The results are shown in Tables 4 and 5 below.

[0086] Table 4 Quantitation limit results

[0087] Sample name Limit of quantification (ng) Ratio of concentration to test product S / N 7-KetoabirateroneAcetate 0.28 0.004％ 13.4 α-EpoxyabirateroneAcetate 0.29 0.005％ 12.4 β-EpoxyabirateroneAcetate 0.22 0.003％ 14.8 Abiraterone 0.38 0.006％ 10.4 3-Deoxy-3-AcetylAbiraterone-3-ene 0.29 0.005％ 13.3 α-Enantiomer 0.30 0.005％ 12.9 AbirateroneAcetate 0.48 0.008％ 12.4 AbirateroneEthylEther 0.32 0.005％ 12.4 3β-Propionyloxy 0.29 0.005％ 15.3 AbirateroneIsopropylEther 0.31 0.005％ 12.9 AnhydroAbiraterone 0.29 0.005％ 15.3 3-Deoxy-3-Chloroabiraterone 0.30 0.005％ 13.5 O-Chlorobutylabiraterone 0.30 0.005％ 11.1

[0088] Table 5 Detection limit results

[0089]

[0090]

[0091] Table 4 is the results of the limit of quantification, and Table 5 is the results of the limit of detection. It can be seen from the table that under the chromatographic conditions of the present invention, the limits of quantification and the limits of detection of 7-Ketoabiraterone Acetate, α-Epoxyabiraterone Acetate, β-EpoxyabirateroneAcetate, Abiraterone, 3-Deoxy-3-Acetyl Abiraterone-3-ene, α-Enantiomer, Abiraterone Acetate, Abiraterone Ethyl Ether, 3β-Propionyloxy, AbirateroneIsopropyl Ether, Anhydro Abiraterone, 3-Deoxy-3-Chloroabiraterone and O-Chlorobutyl abiraterone meet the inspection requirements. The results show that the sensitivity of the method meets the requirements.

[0092] 4. Linearity and range

[0093] Take appropriate amount of 7-KetoabirateroneAcetate, α-EpoxyabirateroneAcetate, β-Epoxyabiraterone Acetate, Abiraterone, 3-Deoxy-3-Acetyl Abiraterone-3-ene, α-Enantiomer,16-diene, Abiraterone Acetate, Abiraterone Ethyl Ether, 3β-Propionyloxy, Abiraterone Isopropyl Ether, Anhydro Abiraterone, 3-Deoxy-3-Chloroabiraterone and O-Chlorobutyl abiraterone, weigh accurately, add solvent to dissolve and dilute to prepare stock solutions of various impurities. Take appropriate amount of each impurity stock solution and prepare 1 mL of mixed stock solution containing 60 μg of 7-Ketoabiraterone Acetate, α-Epoxyabiraterone Acetate, β-Epoxyabiraterone Acetate, Abiraterone, 3-Deoxy-3-Acetyl Abiraterone-3-ene, α-Enantiomer, Abiraterone Acetate, Abiraterone EthylEther, 3β-Propionyloxy, Abiraterone Isopropyl Ether, Anhydro Abiraterone, 3-Deoxy-3-Chloroabiraterone and O-Chlorobutylabiraterone respectively as linear stock solution. Take the above solutions and dilute them with solvents according to Table 6 to prepare linear solutions of various concentrations.

[0094] Table 6 Linear solution preparation

[0095]

[0096] Accurately measure 10 μL of each of the above solutions and inject them into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Table 7 and Figures 3 to 15 shown.

[0097] Table 7 Linearity test results

[0098]

[0099]

[0100] Figure 3 This is the standard curve of impurity 7-Ketoabiraterone Acetate; Figure 4 This is the standard curve of impurity α-Epoxyabiraterone Acetate; Figure 5 This is the standard curve of impurity β-EpoxyabirateroneAcetate; Figure 6 This is the standard curve of impurity Abiraterone; Figure 7 It is the standard curve of impurity 3-Deoxy-3-Acetyl Abiraterone-3-ene; Figure 8 This is the standard curve of impurity α-Enantiomer; Fig. 9 This is the standard curve of impurity Abiraterone Ethyl Ether; Fig.10 This is the standard curve of AbirateroneAcetate;

[0101] Fig.11 This is the standard curve of impurity 3β-Propionyloxy; Fig.12 This is the standard curve of impurity Abiraterone IsopropylEther; Fig.13 This is the standard curve of impurity Anhydro Abiraterone; Fig.14 This is the standard curve of impurity 3-Deoxy-3-Chloroabiraterone; Fig.15The standard curve of impurity O-Chlorobutylabiraterone is shown in the figure. According to the chromatogram, the linear regression equation is made with the peak area A as the ordinate and the concentration C as the abscissa. The linear equation of AbirateroneAcetate is A=29625C-34.89, the linear equation of impurity 7-KetoabirateroneAcetate is A=34976C-118.83, the linear equation of impurity α-EpoxyabirateroneAcetate is A=8256.1C+498.9, the linear equation of impurity β-EpoxyabirateroneAcetate is A=8044.9C-60.99, the linear equation of impurity Abiraterone is A=32341C-78.776, the linear equation of impurity 3-Deoxy-3-Acetyl The linear equation of Abiraterone-3-ene is A=49276C-4.6569, the linear equation of the impurity α-Enantiomer is A=31700C-5.5755, the linear equation of the impurity Abiraterone Ethyl Ether is A=31875C-43.138, the linear equation of the impurity 3β-Propionyloxy is A=30209C-50.303, the linear equation of the impurity Abiraterone Isopropyl Ether is A=32563C-103.98, the linear equation of the impurity Anhydro Abiraterone is A=43652C+6.735, the linear equation of the impurity 3-Deoxy-3-Chloroabiraterone is A=30518C-2.0925, and the linear equation of the impurity O-Chlorobutylabiraterone is A=27248C-49.92.

[0102] The calculation results of the known impurity correction factors are shown in Table 8 below, and the calculation formula is as follows:

[0103] (f)

[0104] k AnhydroAbiraterone : Slope of the standard curve of abiraterone acetate;

[0105] k 杂质 : Slope of impurity standard curve.

[0106] Table 8 Correction factors for known impurities

[0107] name Relative correction factor 7-KetoabirateroneAcetate 0.85 α-EpoxyabirateroneAcetate 3.59 β-EpoxyabirateroneAcetate 3.68 Abiraterone 0.92 3-Deoxy-3-AcetylAbiraterone-3-ene 0.60 α-Enantiomer 0.93 AbirateroneEthylEther 0.93 3β-Propionyloxy 0.98 AbirateroneIsopropylEther 0.91 AnhydroAbiraterone 0.68 3-Deoxy-3-Chloroabiraterone 0.97 O-Chlorobutylabiraterone 1.09

[0108] The relative correction factors of all known impurities are in the range of 0.2 to 5, and the impurity content can be calculated by the self-control method with the correction factor added.

[0109] Table 7 is the linear test results, Table 8 is the known impurity correction factors, as shown in the table, Abiraterone Acetate is in the range of 0.068~3.396μg / mL, impurity 7-Ketoabiraterone Acetate is in the range of 0.056~2.804μg / mL, impurity α-Epoxyabiraterone Acetate is in the range of 0.059~2.949μg / mL, impurity β-Epoxyabiraterone Acetate is in the range of 0.073~3.632μg / mL, impurity Abiraterone is in the range of 0.075~3.757μg / mL, impurity 3-Deoxy-3-Acetyl Abiraterone-3-ene is in the range of 0.058~2.885μg / mL, impurity α-Enantiomer is in the range of 0.060~3.006μg / mL, impurity Abiraterone Ethyl The impurity ether was in the range of 0.064~3.189μg / mL, the impurity 3β-Propionyloxy was in the range of 0.061~3.065μg / mL, the impurity Abiraterone Isopropyl Ether was in the range of 0.061~3.057μg / mL, the impurity Anhydro Abiraterone was in the range of 0.062~3.120μg / mL, the impurity 3-Deoxy-3-Chloroabiraterone was in the range of 0.060~3.024μg / mL, the impurity O-Chlorobutylabiraterone was in the range of 0.061~3.031μg / mL, and the peak area showed a good linear relationship with the measured concentration.

[0110] 5. Injection precision

[0111] Take an appropriate amount of abiraterone acetate tablets and grind them into fine powder, add acetonitrile to dissolve, sonicate, and then dilute to a constant volume to make a solution containing about 0.6 mg of abiraterone acetate per 1 mL, take 10 μL and inject it into the liquid chromatograph, inject 6 times continuously, and record the peak area. The results are shown in Table 9 below.

[0112] Table 9 Injection precision test results

[0113]

[0114]

[0115] Table 9 shows the results of the injection precision test. It can be seen from the table that the injection precision of each impurity in the test solution is good after 6 consecutive injections.

[0116] 6. Solution stability

[0117] 6.1 Stability of reference solution

[0118] A solution containing approximately 0.9375 μg of abiraterone acetate per 1 ml was prepared and injected at 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, and 8 hours to investigate its intraday stability.

[0119] Table 10 Results of stability test of reference solution

[0120]

[0121]

[0122] Conclusion: The reference solution has good stability within 8 hours.

[0123] 6.2 Stability of impurity reference solution

[0124] The system suitability solutions containing various impurities were sampled and measured at room temperature and away from light for 0 h, 4 h, 8 h, 12 h and 24 h, at room temperature and normal light for 24 h, and refrigerated and away from light for 24 h to investigate the stability of the impurity reference solution.

[0125] Table 11 Results of stability test of impurity reference solution

[0126]

[0127]

[0128] The results showed that after the impurity reference solution was placed at room temperature and light, room temperature and away from light, and refrigerated and away from light for 24 hours, except for the decrease in the peak area of ​​α-Enantiomer impurity, the other components did not undergo significant changes, indicating that the impurities were not sensitive to light and had good stability at room temperature.

[0129] 6.3 Stability of test solution

[0130] The preparation methods of blank solvent, system suitability solution, sensitivity solution, test solution and control solution are the same as those under system suitability study.

[0131] The test solution was stored at room temperature away from light for 8h, 16h, 24h, 48h, and 72h, at room temperature and normal light for 72h, and refrigerated away from light for 72h.

[0132] Table 12 Test solution stability test results

[0133]

[0134]

[0135] The results showed that the total impurities of abiraterone acetate related substances changed by 0.06% within 72 hours of room temperature away from light, room temperature and normal light, and refrigerated away from light; the changes of the two specific impurities were both 0.02%, indicating that the abiraterone acetate test solution had good stability at room temperature away from light, room temperature and normal light, and refrigerated away from light for 72 hours.

[0136] 7. Repeatability

[0137] Grind the abiraterone acetate tablets into fine powder, accurately weigh an appropriate amount, add an appropriate amount of acetonitrile, ultrasonically dissolve, and dilute to the scale to make a solution containing about 0.6 mg of abiraterone acetate per 1 mL. Repeat the determination 6 times according to the relevant substance inspection method. The results are shown in Table 13 below.

[0138] Table 13 Repeatability test results

[0139]

[0140]

[0141] Table 13 shows the results of the repeatability test. It can be seen from the table that, calculated by the peak area normalization method, there is no significant difference in the results of 6 tests of known impurities, the difference between the maximum and minimum values ​​is less than 0.01%, and the difference between other single impurities and total impurities does not exceed 0.2%, indicating that the method has good repeatability.

[0142] 8. Sample recovery rate

[0143] Impurity mixed solution: Accurately pipette the mixed impurity stock solution under the linear item and dilute it with acetonitrile to prepare a solution containing about 6 μg of each impurity as the impurity mixed solution.

[0144] Test solution for sample addition: Take 10 tablets of the homemade preparation, weigh the total weight, grind it into powder, weigh an appropriate amount of fine powder (equivalent to about 30 mg of abiraterone acetate), place it in a 10 mL volumetric flask, add acetonitrile to dissolve it ultrasonically and dilute it to the scale, shake it well, centrifuge it, take the supernatant, accurately measure 2 mL, place it in a 10 mL volumetric flask, add 1 mL of the impurity mixed solution, dilute it to the scale with acetonitrile, shake it well, and prepare 6 portions in the same way. (Contains about 0.6 mg / mL of abiraterone acetate and about 0.6 μg / mL of each impurity)

[0145] 10 μL of each of the above samples was injected, the peak area of ​​each known impurity was recorded, and the recovery rate and RSD of each known impurity were calculated. The results are shown in Table 14 below.

[0146] Table 14 Sample recovery test results

[0147]

[0148]

[0149]

[0150] Table 14 shows the experimental results of sample recovery rate. It can be seen from the table that except for the low recovery rates of β-Epoxyabiraterone Acetate and Abiraterone impurities, the average recovery rates of the other 10 impurities are between 90% and 110%, indicating that the accuracy of the method is good.

[0151] 9. Durability

[0152] The durability of the method for related substances of Abiraterone Acetate Tablets was verified mainly from four aspects: different proportions of mobile phase, different flow rates, different column temperatures and different wavelengths. The durability of the method was evaluated based on the separation of each impurity and main component in the system applicability solution, the theoretical plate number of the main peak, the tailing factor, etc. The results are shown in Tables 15 to 17 below.

[0153] Table 15 Durability test results (changes in main peak parameters under different conditions)

[0154]

[0155] Table 16 Durability test results (retention time changes of each component under different conditions)

[0156]

[0157]

[0158]

[0159] Table 17 Durability test results (separation results under different conditions)

[0160]

[0161]

[0162] Table 15 shows the results of the durability test (changes in the main peak parameters under different conditions), Table 16 shows the results of the durability test (changes in the retention time of each component under different conditions), and Table 17 shows the results of the durability test (separation results under different conditions). It can be seen from the table that after the flow rate changes by about ±10%, the column temperature changes by ±2°C, the detection wavelength changes by ±2nm, and the mobile phase ratio is adjusted by ±5%, the separation of each impurity and API in the system suitability solution, the theoretical plate number and tailing factor of the API all meet the expected requirements, indicating that the method has good durability. However, after the flow rate and mobile phase ratio change, the retention time and relative retention time of each component change greatly. In the later stage of sample testing, the stability of the flow rate of the liquid chromatograph should be focused on to ensure that the test is carried out using instruments that meet the requirements, and careful operation should be performed when preparing the mobile phase. In addition, when the chromatographic system changes, it is necessary to re-position the two key impurities α-Epoxyabiraterone Acetate and β-Epoxyabiraterone Acetate.

[0163] 10. Intermediate precision

[0164] The same batch of samples were checked for related substances by the same operator at different times using the repeatability test method; the same batch of samples were checked for related substances by different operators at different times using the repeatability test method; the same batch of samples were checked for related substances by the same operator using different instruments at different times. The results are shown in Table 18 below.

[0165] Table 18 Intermediate precision test results

[0166]

[0167]

[0168] Table 18 shows the results of the intermediate precision test. It can be seen from the table that according to the peak area normalization method, there is no significant difference in the 6 test results of known impurities and other single impurities in the second test, and the difference between the maximum and minimum values ​​is less than 0.01%, and the difference between total impurities does not exceed 0.2%, indicating that the method has good repeatability. After summarizing the 12 results of the two tests, it shows that there is no significant difference in the test results of known impurities, the change is more than 0.01%, and the change of single known impurities and total impurities does not exceed 0.2%, indicating that the intermediate precision of the method is good.

[0169] Comparative Example 1

[0170] According to the ICH guidelines and from the perspective of drug safety, the limits are initially set as follows: the impurities α-Epoxyabiraterone Acetate and β-Epoxyabiraterone Acetat should not exceed 0.3%, and the impurities 7-Ketoabiraterone Acetate, 3-Deoxy-3-Acetyl Abiraterone-3-ene, α-Enantiomer, Abiraterone Ethyl Ether, 3β-Propionyloxy, Abiraterone Isopropyl Ether, AnhydroAbiraterone, 3-Deoxy-3-Chloroabiraterone, O-Chlorobutylabiraterone, and Abiraterone should not exceed 0.1%.

[0171] The present invention was compared with the raw material related substances, and the detection was carried out according to the method in Table 19 below.

[0172] Table 19 Comparison of the methods of the present invention and raw material related substances

[0173]

[0174] The method of raw material gradient elution is: from 0 to 15 minutes, mobile phase A, mobile phase B and mobile phase C are eluted at a volume ratio of 25:25:50; from 37 to 65 minutes, mobile phase B and mobile phase C are eluted at a volume ratio of 50:50, and elution is completed.

[0175] The system suitability of related substances of Abiraterone Acetate Tablets was tested using the raw material method. The results are as follows Fig.16 As shown, the chromatograms of Abiraterone Acetate Tablets and the raw materials are as follows Fig.17 shown.

[0176] Fig.16 This is the system suitability chromatogram for detecting related substances of Abiraterone Acetate Tablets using the raw material method. As can be seen from the figure, the raw material analysis method cannot effectively separate the impurities, and the peaks of the impurity Anhydro Abiraterone and the impurity 3-Deoxy-3-Chloroabiraterone interfere with each other. Therefore, the raw material analysis method cannot accurately quantify the content of each impurity.

[0177] Fig.17 The above is the spectrum of abiraterone acetate tablets and raw materials, blank excipients, and blank solvent. It can be seen from the figure that in the developed method, the solvent peak, excipient peak and other impurities do not interfere with the determination of abiraterone acetate and various known impurities. Therefore, this analytical method can effectively detect the impurity content in abiraterone acetate and tablets.

Claims

1. A method for detecting the impurity content in abiraterone acetate tablets using HPLC, characterized in that: The specific steps are as follows: 1) Prepare test solution and reference solution; 2) Setting high performance liquid phase detection conditions; using a chromatographic column with octadecyl bonded silica gel as a filler; the mobile phase is a mixed solution of mobile phase A and mobile phase B, wherein mobile phase A is a methanol-ammonium acetate solution and mobile phase B is methanol-acetonitrile; gradient elution is performed using the mobile phase; 3) Take the test solution and the reference solution respectively, inject them into the liquid chromatograph, record and analyze the chromatogram, draw the standard curve, and calculate the impurity content.

2. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 1), the test solution is prepared by accurately weighing the abiraterone acetate tablet sample and diluting it with acetonitrile to form a solution containing 0.6 mg of abiraterone acetate per 1 mL; the reference solution is prepared by accurately measuring the test solution and diluting it with acetonitrile to form a solution containing 0.6 μg of abiraterone acetate per 1 mL.

3. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 2), the volume ratio of methanol to ammonium acetate solution in mobile phase A is 2:

3.

4. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 3, characterized in that: The concentration of the ammonium acetate is 0.02 mol / L.

5. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 2), the volume ratio of methanol to acetonitrile in mobile phase B is 9:

1.

6. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 2), the gradient elution method is: 0-15 min, eluting with mobile phase A and mobile phase B in a volume ratio of 50:50; 55-70 min, eluting with mobile phase B; 75-90 min, eluting with mobile phase A and mobile phase B in a volume ratio of 50:50, and the elution is completed, wherein the elution flow rate is 1.0 mL / min.

7. The method for detecting the impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 2), the specification of the chromatographic column is 4.6 mm×250 mm, and the filler particle size is 2.7 μm.

8. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 2), the column temperature of the chromatographic column is 30°C.

9. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 3), the injection volume of the test solution and the reference solution was 10 μL.

10. The method for detecting impurity content in abiraterone acetate tablets by HPLC according to claim 1, characterized in that: In step 3), the detection wavelength of the liquid chromatograph is 254 nm.

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