An analytical method for determining abiraterone-related impurities in abiraterone oral emulsion

Abstract

The invention discloses a high performance liquid chromatography analysis method for determining abiraterone-related impurities in abiraterone oral emulsion. The chromatographic conditions are as follows: the chromatographic column is an octadecylsilane bonded silica gel column, the acetate buffer-mobile phase B volume ratio is (45:55) to (55:45) as mobile phase A, the methanol-acetonitrile volume ratio is (1.5:8.5) to (2.5:7.5) as mobile phase B, gradient elution, the concentration of acetate buffer is 0.005 to 0.015 mol / L, the column temperature is 35 to 45 ° C, the flow rate is 1.1 to 1.3 ml / min, the detection wavelength is 254 nm, and the injection volume is 10 μl. The assay method of the present invention can quickly and accurately detect the content of abiraterone-related impurities in abiraterone oral emulsion, is simple to operate, has good reproducibility, is highly sensitive, can better control abiraterone oral emulsion intermediates and formulation impurities, and provides a guarantee for formulation process optimization.

Description

Technical Field

[0001] The present invention relates to an analysis method for abiraterone oral emulsion, in particular to a method for determination using a high performance liquid chromatography gradient method, and belongs to the field of drug analysis and detection. Background Art

[0002] The abiraterone acetate of the present invention is (3β)-17-(3-pyridyl)-androsta-5,16-diene-3-ol acetate, with a molecular formula of C 26 H 33 NO2, whose chemical structural formula is shown in (I).

[0003]

[0004] Abiraterone oral emulsion is a new and improved dosage form. Currently, no standards for related preparations are included in pharmacopoeias or patent literature worldwide.

[0005] This article achieved the purpose of determining the related substances in abiraterone oral emulsion for the first time by examining different mobile phase ratios, different chromatographic columns, and different buffer salts, which well ensured the quality control of abiraterone oral emulsion. Summary of the Invention

[0006] The present invention aims to provide a method for determining abiraterone-related impurities in abiraterone oral emulsion, which can be used to control related substances in the preparation process and final product of abiraterone oral emulsion. The abiraterone-related impurities mentioned in the present invention are impurities 1 to 10, as shown in the following table.

[0007] The technical solution of the present invention is:

[0008] A method for determining abiraterone and other related substances in abiraterone oral emulsion using high performance liquid chromatography. The names, molecular formulas, and structural formulas of abiraterone acetate and its related substances are shown in the following table.

[0009]

[0010]

[0011] Preferably, the detection method is used to determine the content of abiraterone and other impurities in abiraterone oral emulsion, comprising the following steps:

[0012] (1) System suitability test and sensitivity test

[0013] Prepare system suitability solution: Mix the Abiraterone system suitability reference mixture with a solvent to make a system suitability solution with a concentration of 0.625 mg / ml; wherein the solvent is a mixture of methanol and acetonitrile in a volume ratio of 50:50.

[0014] Prepare a sensitivity test solution: take abiraterone acetate reference substance and quantitatively dilute it with the solvent to prepare a sensitivity test solution with a concentration of 0.4 μg / ml;

[0015] Performing a system suitability test and a sensitivity test using the system suitability solution and the sensitivity test solution under the following chromatographic conditions, respectively, to calibrate the detection system;

[0016] The chromatographic conditions are as follows: the chromatographic column is an octadecylsilane bonded silica gel column, the mobile phase A is an acetate buffer-mobile phase B volume ratio of 50:50, the mobile phase B is a mixed solution of methanol and acetonitrile with a volume ratio of 20:80, gradient elution, the concentration of the acetate buffer is 0.005 mol / L to 0.015 mol / L, the column temperature of the chromatographic column is 35°C to 45°C, the flow rate is 1.1 ml / min to 1.3 ml / min, and the detection wavelength is 254 nm;

[0017] Accurately measure 10 μl of the system suitability solution and 10 μl of the sensitivity test solution, inject them into the liquid chromatograph, record the chromatogram, and calibrate the detection system until the separation between the abiraterone isopropyl ether peak and the dehydrated abiraterone peak is greater than 1.0, and the tailing factor of the abiraterone acetate peak is less than 1.5; the signal-to-noise ratio of the abiraterone acetate peak in the chromatogram of the sensitivity test solution is not less than 10.

[0018] (2) Preparation of test solution and reference solution

[0019] Take about 1250 mg of the abiraterone oral emulsion sample to be tested (equivalent to about 100 mg of abiraterone acetate) and add the solvent to prepare a test solution with a concentration of 2.0 mg / ml (based on abiraterone acetate);

[0020] Take the abiraterone acetate reference substance and add the solvent to prepare a reference substance solution with a concentration of 4 μg / ml;

[0021] (3) Determine the content of abiraterone and other impurities in the test solution by high performance liquid chromatography;

[0022] The reference solution is injected into the liquid chromatograph calibrated in step (1), the detection sensitivity is adjusted, and then the reference solution and the test solution are injected into the liquid chromatograph, and high performance liquid chromatography is performed under the chromatographic conditions described in step (1), and the chromatogram is recorded.

[0023] The chromatographic conditions are as follows: the chromatographic column is an octadecylsilane bonded silica gel column, the mobile phase A is acetate buffer-mobile phase B (45:55) to (55:45), the mobile phase B is methanol-acetonitrile (1.5:8.5) to (2.5:7.5), gradient elution, the concentration of acetate buffer is 0.005 to 0.015 mol / L, the column temperature is 35 to 45°C, the flow rate is 1.1 to 1.3 ml / min, the detection wavelength is 254 nm, and the injection volume is 10 μl;

[0024] (4) Calculate the content of abiraterone-related impurities 1 to 10 in the abiraterone oral emulsion sample to be tested; calculate the content of abiraterone and other impurities by peak area using the external standard method. If impurity peaks are shown in the chromatogram of the test solution (except for the butylated hydroxytoluene excipient peak with a relative retention time of approximately 0.81 to the abiraterone acetate peak), calculate using the main component external standard method with a correction factor:

[0025] The abiraterone content of qualified abiraterone emulsion shall not exceed 0.40% (W / W), the content of other individual impurities shall not exceed 0.20% (W / W), and the total amount of all impurities shall not exceed 2.0% (W / W).

[0026] Preferably, the gradient elution is as follows: from 0 minutes to 13 minutes, linear gradient elution, the proportion of mobile phase A changes from 87% to 74%, and the proportion of mobile phase B changes from 13% to 26%; from 13 minutes to 31 minutes, linear gradient elution, the proportion of mobile phase A changes from 74% to 30%, and the proportion of mobile phase B changes from 26% to 70%; from 31 minutes to 36 minutes, isocratic elution, the proportion of mobile phase A remains unchanged at 30%, and the proportion of mobile phase B remains unchanged at 70%; from 36 minutes to 37 minutes, mobile phases A and B return to their initial proportions; from 37 minutes to 45 minutes, the chromatographic column is re-equilibrated.

[0027] Preferably, the above-mentioned acetate is selected from ammonium acetate.

[0028] Preferably, the acetate buffer solution is an ammonium acetate solution with a concentration of 0.01 mol / L.

[0029] Preferably, the chromatographic column is an octadecylsilane bonded silica gel column with a size of 4.6 mm×150 mm and a filler particle size of 3.5 μm.

[0030] More preferably, the chromatographic column is a Waters X-Bridge Shield RP18 chromatographic column.

[0031] Preferably, the abiraterone oral emulsion sample to be tested is abiraterone oral emulsion.

[0032] Preferably, the abiraterone oral emulsion sample to be tested comprises the active ingredient abiraterone acetate, a solubilizer, an emulsifier, and an antioxidant.

[0033] Preferably, the solubilizer is selected from any one of caprylic capric mono- and diglycerides, propylene glycol monocaprylate, or a mixture of caprylic capric mono- and diglycerides and propylene glycol monocaprylate, the emulsifier is selected from polyoxyethylene 40 hydrogenated sesame oil, and the antioxidant is selected from butylated hydroxytoluene.

[0034] The present invention uses a high-performance liquid chromatography gradient method to determine the content of abiraterone-related impurities 1 to 10 in abiraterone oral emulsion. By exploring chromatographic conditions, the present invention determines chromatographic conditions that are effective for the determination of abiraterone and other impurities. This method, for the first time, achieves the determination of related substances in abiraterone oral emulsion, with excellent specificity, resolution, sensitivity, and reproducibility. It is of great significance for evaluating the quality of formulation processes, controlling final product quality, and testing stability samples.

[0035] The method of the present invention is suitable for detecting abiraterone oral emulsion. The method of the present invention is accurate, rapid, and can fully meet the requirements for the determination of related substances. It effectively controls specific and non-specific impurities in samples, ensuring product quality and having strong practicality in actual quality control work. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the system suitability solution chromatogram of ammonium acetate buffer in Example 2.1;

[0037] Figure 2 This is the system suitability solution chromatogram of the ammonium bicarbonate buffer in Example 2.1;

[0038] Figure 3 This is the system suitability solution chromatogram of the potassium dihydrogen phosphate buffer in Example 2.1;

[0039] Figure 4 This is the system suitability solution chromatogram of 40 mmol / L ammonium acetate buffer in Example 2.2;

[0040] Figure 5 This is the chromatogram of the system suitability solution for the Waters XBridge Phenyl, 4.6×150 mm, 3.5 μm column in Example 2.3;

[0041] Figure 6 This is the chromatogram of the system suitability solution for the Waters XBridge shield RP18, 4.6×150 mm, 3.5 μm column in Example 2.3;

[0042] Figure 7Overlay chromatograms of the system suitability solution of methanol-acetonitrile (3:7) and the spiked test solution in Example 2.4;

[0043] Figure 8 Overlay chromatograms of the methanol-acetonitrile (2:8) system suitability solution and the spiked test solution in Example 2.4;

[0044] Figure 9 This is the system suitability solution chromatogram for gradient ① in Example 2.5;

[0045] Figure 10 This is the system suitability solution chromatogram for gradient ② in Example 2.5;

[0046] Figure 11 This is the system suitability solution chromatogram for gradient ③ in Example 2.5;

[0047] Figure 12 This is the system suitability solution chromatogram at a flow rate of 1.0 ml / min in Example 2.6;

[0048] Figure 13 This is the chromatogram of the undamaged test solution in Example 3;

[0049] Figure 14 This is the chromatogram of the undamaged blank excipient solution in Example 3;

[0050] Figure 15 The chromatogram of the alkali-destroyed test solution in Example 3 is shown;

[0051] Figure 16 The chromatogram of the acid-destroyed test solution in Example 3 is shown;

[0052] Figure 17 The chromatogram of the test solution destroyed by high temperature in Example 3;

[0053] Figure 18 This is a chromatogram of the light-destroyed test solution in Example 3;

[0054] Figure 19 The chromatogram of the oxidative destruction test solution in Example 3;

[0055] Figure 20 The chromatogram of the blank auxiliary material solution oxidized and destroyed in Example 3;

[0056] Figure 21 is the chromatogram of the system suitability solution in Example 4;

[0057] Figure 22 It is the chromatogram of the sensitivity solution in Example 4. DETAILED DESCRIPTION

[0058] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0059] In the following specific embodiments, the reference substances, samples, reagents and instruments used are:

[0060] Instrument: Agilent 1260 series high performance liquid chromatograph;

[0061] Reagents: ammonium acetate (mass spectrometry grade), hydrochloric acid (analytical grade), sodium hydroxide (analytical grade), acetonitrile (chromatographic grade, Merck), methanol (chromatographic grade, Merck); ammonium bicarbonate (analytical grade); potassium dihydrogen phosphate (analytical grade).

[0062] Chromatographic column:

[0063] Waters X-Bridge Shield RP18, 4.6mm×150mm, 3.5μm;

[0064] Waters X-Bridge Shield RP18, 4.6mm×100mm, 3.5μm;

[0065] Waters X-Bridge Phenyl, 4.6mm×150mm, 3.5μm;

[0066] The solvent mentioned in the present invention, unless otherwise specified, is ultrapure water.

[0067] Abiraterone acetate API, source: Industrias Quimicas Falcon De Mexico, SA de C.V. Batch number: ANMA000296.

[0068] Abiraterone oral emulsion, source: Shenzhen Neptunus Pharmaceutical Technology Research Institute Co., Ltd., main ingredient: abiraterone acetate, specification: 80mg / ml. Batch number: 20200601, its preparation method is:

[0069] (1) After heating the solubilizer and emulsifier to 60° C., stir them evenly to obtain solution 1;

[0070] (2) adding an antioxidant to solution 1 and stirring to dissolve to obtain solution 2;

[0071] (3) Add the active ingredient to solution 2 and stir to dissolve, thereby obtaining the abiraterone oral emulsion.

[0072] For specific preparation methods, please refer to Chinese patent application CN111012745A.

[0073] Chemical name, structural formula and source of the standard product:

[0074] Abiraterone acetate reference substance:

[0075] Molecular formula: C 26 H 33 NO2

[0076] Molecular weight: 391.55

[0077] Batch number: R078C0

[0078] Content: 99.9%

[0079] Source: USP

[0080] Packaging: Brown penicillin bottle Storage: Light-proof, sealed Chemical name: Abiraterone acetate

[0081] Structural formula:

[0082]

[0083] Abiraterone reference substance:

[0084] Molecular formula: C 24 H 31 NO

[0085] Molecular weight: 349.52

[0086] Batch number: HW09G2201-3

[0087] Content: 96.7%

[0088] Source: Beijing Huawei Ruike Chemical Co., Ltd. Packaging: Brown vial Storage: Light-proof, sealed Chemical name: Abiraterone

[0089] Structural formula:

[0090]

[0091] Abiraterone system suitability mixed reference substance: Batch number: R103R0

[0092] Source: USP

[0093] Packaging: brown glass bottle

[0094] Storage: Keep away from light and sealed

[0095] Solution composition:

[0096] Table 1 Information of compounds in Abiraterone system suitability solution

[0097]

[0098] [Example 1] Preparation of detection solution

[0099] 1.1 System Suitability Solution

[0100] Take about 3.125 mg of the Abiraterone system mixed reference substance, accurately weigh it, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, centrifuge and transfer to a liquid phase injection vial.

[0101] 1.2 Preparation of reference solution

[0102] Abiraterone reference substance stock solution (0.625 mg / ml): Take about 12.5 mg of abiraterone reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, shake it well, and filter it.

[0103] Abiraterone reference solution (0.006 mg / ml): Accurately measure 1 ml of the Abiraterone reference stock solution and place it in a 100 ml volumetric flask. Add solvent to dilute to the mark, shake well, and filter to obtain the solution.

[0104] BHT reference substance stock solution (0.625 mg / ml): Take about 6.25 mg of BHT reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, shake it well, filter it, and you are done.

[0105] Butylated hydroxytoluene reference solution (0.4 mg / ml): Take about 4 mg of butylated hydroxytoluene reference, accurately weigh it, place it in a 10 ml volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, shake it well, filter it, and you are done.

[0106] Reference substance solution (4 μg / ml): Take approximately 4 mg of abiraterone acetate reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, shake it well, filter it, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake it well, and make a reference substance solution containing approximately 4 μg of abiraterone acetate per 1 ml.

[0107] Sensitivity solution (0.4 μg / ml): Accurately measure 2 ml of the reference solution and place it in a 20 ml volumetric flask. Add diluent to dilute to the mark and shake well to make a solution containing approximately 0.4 μg of abiraterone acetate per 1 ml.

[0108] 1.3 Excipient test solution

[0109] PG8 localization solution (5mg / ml): Take about 250mg of the auxiliary material PG-8, accurately weigh it, and place it in a 50ml volumetric flask. Add solvent to dissolve and dilute to the scale, shake well, and filter to obtain the solution.

[0110] MCM localization solution (5 mg / ml): Take about 250 mg of the auxiliary material MCM, accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, filter, and obtain.

[0111] RH40 positioning solution (5mg / ml): Take about 250mg of auxiliary material RH40, accurately weigh it, place it in a 50ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, filter, and obtain.

[0112] 1.4 Preparation of test solution

[0113] Take about 1250 mg of abiraterone acetate oral microemulsion solution (containing about 100 mg of abiraterone acetate), accurately weigh it, place it in a 50 ml volumetric flask, add appropriate amount of solvent to dilute to the scale, shake well, filter, and obtain the product.

[0114] 1.5 Preparation of spiked test solution

[0115] Take about 62.5 mg of abiraterone acetate raw material, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately measure 1 ml of butylated hydroxytoluene reference stock solution (0.625 mg / ml) and abiraterone reference stock solution (0.625 mg / ml) and place them in a volumetric flask. Add appropriate amount of solvent to dilute to the scale, shake well, filter, and obtain.

[0116] 1.6 Preparation of blank excipient solution

[0117] Take about 1150 mg of blank excipient of abiraterone acetate oral microemulsion solution, accurately weigh it, place it in a 50 ml volumetric flask, add appropriate amount of solvent to dilute to the scale, shake well, filter, and obtain.

[0118] [Example 2] Chromatographic Condition Screening

[0119] 2.1 Selection of buffer salt

[0120] Buffer (0.04 mol / L ammonium acetate): Dissolve approximately 3.08 g of ammonium acetate in 1000 ml of water to prepare a 0.04 mol / L ammonium acetate buffer solution.

[0121] Buffer (0.04 mol / L ammonium bicarbonate): Dissolve approximately 3.16 g of ammonium acetate in 1000 ml of water to prepare a 0.04 mol / L ammonium bicarbonate buffer solution.

[0122] Buffer solution (0.04 mol / L potassium dihydrogen phosphate): Dissolve 5.44 g of potassium dihydrogen phosphate in 1000 ml of water to prepare 0.04 mol / L potassium dihydrogen phosphate buffer solution.

[0123] The system suitability solution prepared in Example 1 was divided into three groups, and the three buffer salts were used to detect the elution gradient in Table 2 and the following chromatographic conditions. The chromatograms were obtained as shown in the attached figure. Figures 1 to 3 shown.

[0124] Table 2 Elution gradient

[0125]

[0126] Column: Waters XBridge shield RP18, 4.6 × 100 mm, 3.5 μm

[0127] Mobile phase A: 0.04 mol / L buffer-mobile phase B (50:50);

[0128] Mobile phase B: methanol-acetonitrile (30:70);

[0129] Flow rate: 1.2 ml / min, column temperature: 40 °C, detection wavelength: 254 nm, injection volume: 10 μl

[0130] The above chromatographic columns were compared using the retention time of the main peak and each impurity peak, the number of theoretical plates, the tailing factor, and the separation degree from adjacent peaks as evaluation indicators. The results are shown in Table 3 and the attached table. Figure 1 ~Attachment Figure 3 .

[0131] Table 3 Detection results of different mobile phase buffer salts

[0132]

[0133] The results showed that there was no significant difference in the separation degree, tailing factor, theoretical plate number and impurity detection ability among the three salts of ammonium acetate, ammonium bicarbonate and potassium dihydrogen phosphate. Since ammonium acetate is more soluble and has the lowest risk of salting out in the organic phase, ammonium acetate was finally determined to be the buffer salt type.

[0134] 2.2 Selection of buffer salt concentration

[0135] Take the system suitability solution of Example 1, use the following ammonium acetate buffer mobile phase of different concentrations, and detect according to the chromatographic conditions of Example 2.1, wherein ammonium acetate is selected as the buffer salt, and obtain the chromatogram as shown below Figure 1 Figure 4 Table 4 shows the detection results using different concentrations of buffer salt mobile phase.

[0136] Table 4 System applicability of buffer solution with different concentrations and separation of impurities layer by layer

[0137]

[0138] The results showed that there was no significant difference in the separation degree, tailing factor, theoretical plate number and impurity detection ability between the two salt concentrations of 10mM ammonium acetate and 40mM ammonium acetate. Considering the risk of salting out in the high proportion of organic phase, the buffer salt concentration was determined to be 10mmol / L.

[0139] 2.3 Column selection

[0140] The system suitability solution prepared in Example 1 was divided into three groups and tested using the chromatographic columns shown in Table 5 according to the chromatographic conditions of Example 2.2, wherein 0.01 mol / L ammonium acetate was selected as the buffer salt. The chromatograms obtained were as shown in the attached figure. Figure 1 、 Figure 5 and Figure 6 Table 6 shows the test results using different chromatographic columns.

[0141] Table 5 Chromatographic column information

[0142]

[0143] Table 6 System suitability for separation of main components and impurities in solution under different chromatographic columns

[0144]

[0145] The results showed that under the conditions of chromatographic column 2, the main peak had severe tailing, with a tailing factor of 1.24, and the separation between impurity 4 and impurity 1 became smaller. When replaced with chromatographic column 3, the separation between impurity 8 and impurity 7 increased, and the separation of each impurity was optimal. Therefore, the chromatographic column was determined to be XBridge shield RP18 (4.6×150mm, 3.5μm).

[0146] 2.4 Selection of mobile phase B composition

[0147] The system suitability solution and spiked test solution prepared in Example 1 were divided into two groups, and different ratios of methanol and acetonitrile were selected for detection according to the chromatographic conditions of Example 2.3. The chromatographic column was XBridge shield RP18, and the chromatograms were obtained as shown in the attached figure. Figures 7 and 8 shown.

[0148] Table 7 shows the test results using different mobile phase B compositions.

[0149] Table 7 Retention time and peak width of impurity 5 in system suitability solution and BHT in spiked test solution

[0150]

[0151] The results showed that when the composition of mobile phase B was adjusted to methanol:acetonitrile = 3:7, impurity 5 and BHT could not be completely separated, so the composition of mobile phase B was determined to be methanol-acetonitrile (20:80).

[0152] 2.5 Selection of elution gradient

[0153] The system suitability solution of Example 1 was divided into three groups and tested using the gradient elution program in Table 8 according to the chromatographic conditions of Example 2.4, wherein the mobile phase B was methanol-acetonitrile (20:80) and the injection volume was 10 μl.

[0154] The system suitability solution prepared in Example 1 was divided into three groups and tested using the elution gradient shown in Table 8 according to the following chromatographic conditions. The chromatograms obtained were as shown in the attached figure. Figures 9-11 shown.

[0155] Table 8 Elution gradient information

[0156]

[0157] Column: Waters XBridge shield RP18, 4.6 × 150 mm, 3.5 μm

[0158] Mobile phase A: 0.01 mol / L buffer-mobile phase B (50:50);

[0159] Mobile phase B: methanol-acetonitrile (20:80);

[0160] Flow rate: 1.2 ml / min, column temperature 40°C, detection wavelength: 254 nm

[0161] The above elution gradients were compared using the retention time of the main peak and each impurity peak, the number of theoretical plates, the tailing factor, and the separation degree from adjacent peaks as evaluation indicators. The results are shown in Table 9.

[0162] Table 9 System suitability under different gradients Separation of main components and impurities in solution

[0163]

[0164] Under the conditions of elution gradient ③, the separation between impurities 7, 8 and 9 increased, and the elution gradient was finally determined to be elution gradient ③.

[0165] 2.6 Flow rate selection

[0166] The system suitability solution prepared in Example 1 was divided into two groups and tested according to the chromatographic conditions of Example 2.5 at mobile phase flow rates of 1.0 ml / min and 1.2 ml / min, respectively. Gradient elution was performed using gradient ③. The chromatograms obtained were as shown in the attached figure. Figures 11-12 Table 10 shows the test results using different mobile phase flow rates.

[0167] Table 10 Separation of main components and impurities in solutions at different flow rates

[0168]

[0169] When the flow rate is 1.0 ml / min, the retention time of the main peak is delayed to 23.588 min, and the separation between impurities 7, 8 and 9 is even smaller, which are 8.90, 1.60 and 2.35 respectively, so the flow rate is still set at 1.2 ml / min.

[0170] 2.7 Summary

[0171] In summary, the preferred chromatographic conditions for detecting related substances in abiraterone oral emulsion of the present invention are: chromatographic column Waters XBridge shield RP18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: acetate buffer (about 0.77 g of ammonium acetate is dissolved in 1000 ml of water) - mobile phase B (50:50), mobile phase B: methanol-acetonitrile (20:80); flow rate 1.2 ml / min, column temperature 40°C, detection wavelength 254 nm, gradient elution: 0 min to 13 min, linear gradient elution, the proportion of mobile phase A changes from 87% to 74% %, the proportion of mobile phase B changed from 13% to 26%; from 13 minutes to 31 minutes, linear gradient elution, the proportion of mobile phase A changed from 74% to 30%, and the proportion of mobile phase B changed from 26% to 70%; from 31 minutes to 36 minutes, isocratic elution, the proportion of mobile phase A remained unchanged at 30%, and the proportion of mobile phase B remained unchanged at 70%; from 36 minutes to 37 minutes, mobile phases A and B returned to their initial proportions; from 37 minutes to 45 minutes, the chromatographic column was re-equilibrated.

[0172] The chromatographic method of the present invention can effectively determine the content of related substances in abiraterone oral emulsion, can fully meet the requirements of related substance inspection and decomposition product determination, better control specific impurities and non-specific impurities in the sample, ensure product quality, and has strong practicality in actual quality control work.

[0173] [Example 3] Forced degradation test

[0174] After the product is destroyed by high temperature, acid, alkali, oxidation, light and other severe conditions, the relevant substances are determined to examine whether the selected chromatographic conditions can detect the possible degradation products of the product, as follows:

[0175] (1) Unspoiled test solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, and place it in a 50 ml volumetric flask. Dissolve it in solvent and dilute it to the mark. Shake well, filter, and use the filtrate as the test solution. Simultaneously, prepare a blank excipient and perform the same operation in parallel.

[0176] (2) Preparation of high temperature destruction test solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve, shake well, place it in a 90 ° C water bath for 24 hours, dilute it to the scale with solvent, shake well, filter, and use the filtrate as the test solution. Simultaneously, prepare a blank excipient and perform the same operation.

[0177] (3) Light-induced destruction of the test solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, and place it in a 50 ml volumetric flask. Dissolve it in solvent and dilute it to the mark. Shake it well and place it in a light box (5000 lux, UV 85 μw / cm2) for 16 days. Remove it, shake it well, filter it, and use the filtrate as the test solution. Simultaneously, take a blank excipient and perform the same operation.

[0178] (4) Acid-destroyed test solution: Take approximately 1250 mg of the product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve it, add 1 ml of 1 mol / L HCl solution, let it stand at room temperature for 6 h, add 1 ml of 1 mol / L NaOH to neutralize it, then add solvent to the mark, shake well, and filter to obtain the product. Simultaneously, prepare a blank excipient and perform the same operation.

[0179] (5) Alkali-destroyed test solution: Take approximately 1250 mg of the product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve it, add 1 ml of 0.1 mol / L NaOH solution to dissolve it, let it stand at room temperature for 30 min, add 1 ml of 0.1 mol / L HCl to neutralize it, add solvent to the scale, shake well, and filter to obtain the product. Simultaneously, take a blank excipient and perform the same operation.

[0180] (6) Oxidative Destruction Test Solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh it, place it in a 50 ml volumetric flask, dissolve it in solvent, add 1 ml of 30% H2O2 solution, dilute to the mark with solvent, let it stand at room temperature for 12 hours, shake it well, and filter it. Simultaneously, prepare blank solvent and blank excipients and perform the same operation.

[0181] The solutions of (1) to (6) were tested using the chromatographic conditions summarized in Example 2.7, and the chromatograms were recorded, which were Figures 13 to 20 , the test results are shown in Table 11.

[0182] Table 11 Forced degradation test results

[0183]

[0184]

[0185] Results: Under acid, alkaline, and high temperature conditions, the main degradation product was the abiraterone peak, which was well resolved from the abiraterone acetate peak. Under light and oxidation conditions, the abiraterone acetate peak was significantly separated from adjacent chromatographic peaks, with tailing factors of the abiraterone acetate peak less than 1.5, and the resolution between impurities was no less than 1.5, indicating good separation. The purity of the abiraterone acetate peak under all decomposition conditions was greater than 990, and no interfering peaks were detected at the corresponding position of the abiraterone acetate peak in the blank excipient solutions, indicating that this method has good specificity for the determination of related substances in this product.

[0186] [Example 4] System suitability test

[0187] Take about 3 mg of Abiraterone system suitability mixed reference substance, accurately weigh it, place it in a 5 ml volumetric flask, add diluent to dissolve it and dilute it to the scale, shake it well, and add solvent to dilute it to the scale as the system suitability solution, shake it well, and the system suitability solution is obtained.

[0188] Take 10 μl of the system suitability solution and inject it into the high performance liquid chromatograph. Detect according to the chromatographic conditions summarized in Example 2.7 and record the chromatogram. Figures 21-22 and Table 12.

[0189] Table 12 System suitability test results

[0190]

[0191]

[0192] In the system suitability solution chromatogram, the separation between the abiraterone isopropyl ether peak and the dehydrated abiraterone peak was 1.83, the tailing factor of the main peak was 0.97, and the sensitivity solution signal-to-noise ratio was 20.36, all of which met the system suitability requirements.

[0193] [Example 5] Blank interference test

[0194] Take 10 μl of blank solvent, system suitability solution, blank excipient solution and test solution, inject them into liquid chromatograph, and detect according to the chromatographic conditions summarized in Example 2.7. Record the chromatogram. The chromatographic detection results are shown in Figure 22 .

[0195] The results showed that no chromatographic peaks were detected in the blank solvent and blank excipients at the main peak, abiraterone and other impurity peaks, and they did not interfere with the determination of related substances of this product.

[0196] [Example 6] Determination of Quantitation Limit and Detection Limit

[0197] Accurately weigh approximately 5 mg of abiraterone acetate reference substance into a 100 ml volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the mark. Shake well. Continue diluting the solution and accurately measure 10 μl of the solution before injecting it into a liquid chromatograph. Record the chromatogram and calculate the signal-to-noise ratio. The limit of detection was determined at the concentration corresponding to a signal-to-noise ratio of approximately 3:1; the limit of quantification was determined at the concentration corresponding to a signal-to-noise ratio of approximately 10:1. Five consecutive injections were made, and the RSD of the peak area was ≤10%. The results are shown in Tables 13 and 14.

[0198] Table 13 Abiraterone acetate quantification limit

[0199]

[0200] Table 14 Limits of quantification and detection of abiraterone acetate

[0201]

[0202] Results: After testing and calculation, the minimum detection limit of abiraterone acetate was 0.04994 μg / ml, equivalent to 0.0025% of the test sample concentration; the minimum quantification limit was 0.14982 μg / ml, equivalent to 0.0075% of the test sample concentration. The peak area RSD of repeated injections was 9% after 5 times of injection, which met the requirements of quantitative detection.

[0203] [Example 7] Linearity and range

[0204] (1) Preparation of linear solution

[0205] Abiraterone acetate reference substance stock solution: Take 25 mg of abiraterone acetate reference substance, place it in a 25 ml volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain;

[0206] Linear series solutions: Accurately measure an appropriate amount of the stock solution of the abiraterone acetate reference substance to prepare solutions containing approximately 1, 2, 4, 10, 20, and 100 μg of abiraterone acetate per ml (equivalent to 0.05%, 0.1%, 0.2%, 0.5%, 1%, and 5% of the test sample concentration, respectively). The quantification limit solution and the above 6 solutions are regarded as the linear series solutions.

[0207] Table 15 Linear solution preparation list

[0208]

[0209] (2) Determination

[0210] Accurately measure 10 μl of each of the above linear series solutions and inject them into a liquid chromatograph. Record the chromatogram and plot the peak area (A) on the ordinate against the abiraterone acetate concentration (C) on the abscissa. Calculate the regression equation and correlation coefficient. The results are shown in Table 16.

[0211] Table 16 Linearity test results

[0212]

[0213]

[0214] Results: The linear equation correlation coefficient R of abiraterone acetate was greater than 0.990, the intercept was less than 25%, and the residual standard deviation was less than 10%, indicating that abiraterone acetate had a good linear relationship in the concentration range of 0.1508 μg / ml to 100.5314 μg / ml (equivalent to 0.0075% to 5% of the related substance test sample).

[0215] [Example 8] Precision test

[0216] A reference solution and spiked test solution were prepared according to the method in Example 1. Accurately measure 10 μl of the system suitability solution and inject it into the liquid chromatograph. Testing was performed according to the chromatographic conditions summarized in Example 2.7. The test results are shown in Table 20. The contents of abiraterone, the largest single impurity, and total impurities were calculated using the principal component external standard method and used as the reproducibility test results. The results are shown in Table 17.

[0217] Table 17 Repeatability test results

[0218] Related substances 1 2 3 4 5 6 RSD% Abiraterone (%) 0.44 0.44 0.43 0.43 0.44 0.44 1.0 Total impurities (%) 0.44 0.44 0.43 0.43 0.44 0.44 1.0

[0219] Results: In the 6 test solutions, the RSD of abiraterone content was less than 3%, and the RSD of total impurity content was less than 2%, indicating that the method had good repeatability.

[0220] This product was tested using the repeatability method by different individuals, using different equipment, and on different dates. The concentrations of abiraterone, the largest single impurity, and total impurities were calculated as the intermediate precision test results. The results of the repeatability and intermediate precision tests were combined to calculate the RSDs for the twelve samples. The results are shown in Table 18.

[0221] Table 18 Intermediate precision results

[0222]

[0223] Results: A and B were the operators. In 12 test solutions, the RSD of abiraterone content and total impurity content were less than 6% and 4%, respectively, indicating that the intermediate precision of this method was good.

[0224] [Example 9] Accuracy test

[0225] (1) Preparation of solution

[0226] System suitability solution: Take approximately 3 mg of Abiraterone system suitability mixed reference substance, accurately weigh it, place it in a 5 ml volumetric flask, add appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain the solution;

[0227] Reference solution: Take about 4 mg of abiraterone acetate reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0228] Abiraterone reference substance stock solution: Take about 20 mg of Abiraterone reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the scale. Shake well to obtain the solution.

[0229] 50% Test Solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh, and place in a 50 ml volumetric flask. Dissolve with solvent. Accurately add 1 ml of the abiraterone reference stock solution, dilute to the mark with solvent, shake well, filter, and use the filtrate as the test solution. Prepare three replicates.

[0230] 100% test solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh, and place in a 50 ml volumetric flask. Dissolve in solvent. Accurately add 2 ml of the abiraterone reference stock solution, then dilute to the mark with solvent. Shake well, filter, and use the filtrate as the test solution. Prepare three replicates.

[0231] 150% test solution: Take approximately 1250 mg of this product (equivalent to 100 mg of abiraterone acetate), accurately weigh, and place in a 50 ml volumetric flask. Dissolve in solvent. Accurately add 3 ml of the abiraterone reference stock solution, dilute to the mark with solvent, shake well, filter, and use the filtrate as the test solution. Prepare three replicates.

[0232] Measure 10 μl of each reference solution and test solution, inject them into a liquid chromatograph, and record the chromatogram. Calculate the abiraterone recovery using the principal component external standard method. The results are shown in Table 19.

[0233] Calculation formula:

[0234] Where A 供 is the peak area of each impurity in the chromatogram of the test solution;

[0235] A 对 is the main peak area in the chromatogram of the reference solution;

[0236] m 对is the sample weight of the reference substance, mg;

[0237] w is the content of the reference substance, %;

[0238] V 供 is the dilution multiple of the test sample;

[0239] V 对 is the dilution multiple of the reference substance;

[0240]

[0241] Table 19 Accuracy results

[0242]

[0243] Results: The recovery rate of abiraterone was 102%, within the range of 90% to 108%. The RSD value of the recovery rate of abiraterone in each concentration of the test sample was 2.6%, which was less than 10%, indicating that the method had good accuracy.

[0244] [Example 10] Durability test

[0245] The durability test was conducted on different batches of chromatographic columns with column temperature ±2°C, flow rate ±0.1ml / min, ammonium acetate buffer salt concentration in the mobile phase ±0.005mol / L.

[0246] The system suitability solution and sensitivity solution were prepared according to the preparation method of Example 1. 10 μl was accurately measured and injected into the liquid chromatograph. The test was carried out according to the chromatographic conditions summarized in Example 2.7. The test results are shown in Table 20.

[0247] Table 20 Durability test results

[0248]

[0249]

[0250] The results showed that when all parameters changed slightly, the theoretical plate number of the abiraterone acetate peak, the resolution between the abiraterone isopropyl ether peak and the dehydrated abiraterone peak, the main peak tailing factor, and the signal-to-noise ratio of the main peak of the sensitivity solution all met the system suitability requirements.

Claims

1. A method for simultaneously determining abiraterone-related impurities 1 to 10 in an abiraterone oral emulsion using high performance liquid chromatography, wherein the active ingredient of the abiraterone oral emulsion is abiraterone acetate, having the structural formula shown in formula (I). The chemical names and structural formulas of the abiraterone-related impurities 1 to 10 are shown in the following table: It is characterized in that The method comprises the following steps: (1) System suitability test and sensitivity test Prepare system suitability solution: Mix the Abiraterone system suitability reference mixture with a solvent to make a system suitability solution with a concentration of 0.625 mg / ml; wherein the solvent is a mixture of methanol and acetonitrile in a volume ratio of 50:

50. Prepare a sensitivity test solution: take abiraterone acetate reference substance and quantitatively dilute it with the solvent to prepare a sensitivity test solution with a concentration of 0.4 μg / ml; Performing a system suitability test and a sensitivity test using the system suitability solution and the sensitivity test solution under the following chromatographic conditions, respectively, to calibrate the detection system; The chromatographic conditions are as follows: mobile phase A with a volume ratio of acetate buffer to mobile phase B of (45:55) to (55:45), mobile phase B with a volume ratio of methanol to acetonitrile of (15:85) to (25:75), gradient elution, a concentration of acetate buffer of 0.005 mol / L to 0.015 mol / L, a column temperature of 35° C. to 45° C., a flow rate of 1.1 ml / min to 1.3 ml / min, and a detection wavelength of 254 nm; the chromatographic column is a Waters X-Bridge Shield RP18 column; Accurately measure 10 μl of the system suitability solution and 10 μl of the sensitivity test solution, inject them into the liquid chromatograph, record the chromatogram, and calibrate the detection system until the resolution between the abiraterone isopropyl ether peak and the dehydrated abiraterone peak is greater than 1.0, and the tailing factor of the abiraterone acetate peak is less than 1.5; the signal-to-noise ratio of the abiraterone acetate peak in the chromatogram of the sensitivity test solution is not less than 10; (2) Preparation of test solution and reference solution Take about 1250 mg of the abiraterone oral emulsion sample to be tested, equivalent to about 100 mg of abiraterone acetate, and add the solvent to prepare a test solution with a concentration of 2.0 mg / ml based on abiraterone acetate; Take the abiraterone acetate reference substance and add the solvent to prepare a reference substance solution with a concentration of 4 μg / ml; (3) determining the content of abiraterone-related impurities 1-10 in the test solution by high performance liquid chromatography; The reference solution is injected into the liquid chromatograph calibrated in step (1), the detection sensitivity is adjusted, and then the reference solution and the test solution are injected into the liquid chromatograph, and high performance liquid chromatography is performed under the chromatographic conditions described in step (1) with an injection volume of 10 μl, and the chromatogram is recorded; (4) Calculating the content of abiraterone and other impurities in the abiraterone oral emulsion sample to be tested; calculating the content of abiraterone and other impurities by peak area according to the external standard method; The abiraterone oral emulsion sample to be tested contains the active ingredient abiraterone acetate, a solubilizer, an emulsifier, and an antioxidant; the gradient elution is: From minute 0 to minute 13, linear gradient elution was used, with the proportion of mobile phase A changing from 87% to 74% and the proportion of mobile phase B changing from 13% to 26%; From minute 13 to minute 31, linear gradient elution was performed, with the proportion of mobile phase A changing from 74% to 30% and the proportion of mobile phase B changing from 26% to 70%; From minute 31 to minute 36, isocratic elution was performed, with the proportion of mobile phase A remaining constant at 30% and the proportion of mobile phase B remaining constant at 70%; From minute 36 to minute 37, the ratio of mobile phases A and B returned to the initial ratio; From minute 37 to minute 45, the column was re-equilibrated.

2. The method according to claim 1, wherein The acetate is selected from ammonium acetate.

3. The method according to claim 1, wherein The acetate buffer has a concentration of 0.01 mol / L.

4. The method according to claim 1, wherein The mobile phase B is methanol-acetonitrile in a volume ratio of 20:80, and the mobile phase A is 0.01 mol / L ammonium acetate buffer-mobile phase B in a volume ratio of 50:

50.

5. The method according to claim 1, wherein The chromatographic conditions are as follows: flow rate of 1.2 ml / min, column temperature of 40° C., and injector temperature of room temperature.

6. The method according to claim 1, wherein The solubilizer is selected from any one of caprylic capric mono- and diglycerides, propylene glycol monocaprylate, or a mixture of caprylic capric mono- and diglycerides and propylene glycol monocaprylate; the emulsifier is selected from polyoxyethylene 40 hydrogenated castor oil; and the antioxidant is selected from butylated hydroxytoluene.

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