Method for separating, identifying and quantifying fenerenone Z3 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating, identifying and quantifying fenerenone Z3 and impurities thereof. The impurities comprise any one or more of an impurity SM3, an impurity Z3f, an impurity Z2, an impurity Z3e, an impurity Z3b and an impurity Z3a. According to the method, a phosphoric acid solution is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, octadecylsilane chemically bonded silica is taken as a chromatographic column filler, linear gradient elution of high performance liquid chromatography is carried out, and a product enters a detector for detection; and then calculating the content of each impurity by adopting a main component self-contrast method multiplied by a correction factor according to the chromatogram. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating, identifying and quantifying finerenone Z3 and its impurities. Background Art

[0002] Finerenone is a nonsteroidal selective mineralocorticoid receptor antagonist, mainly used to treat chronic kidney disease (DKD) associated with type 2 diabetes. Finerenone exerts anti-inflammatory and anti-fibrotic effects by highly selectively blocking excessive activation of mineralocorticoid receptors (MR), thereby providing dual protection of the kidneys and cardiovascular system. Finerenone has a unique molecular structure. Compared with traditional steroidal MRAs, it has higher MR selectivity and stronger affinity, and can accurately inhibit MR overactivation, reduce inflammation and fibrosis. Existing studies have shown that finerenone can effectively delay the decline of renal function and reduce cardiovascular events in patients with DKD, especially in reducing proteinuria and oxidative stress-related tubular damage. In addition, finerenone has a low incidence of adverse drug reactions. Therefore, finerenone provides a new treatment option for patients with chronic kidney disease associated with type 2 diabetes, which has important clinical value.

[0003] Finerenone Z3 is a key intermediate in the synthesis of finerenone, and its structural formula is shown in Formula I. Studies have found that the following six impurities are easily introduced during the synthesis of finerenone Z3: impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a Among them, impurity Z2 is the raw material for synthesizing finerenone Z3, and it is easy to have residues; and impurity Z2 will be introduced into its raw material SM3, and impurity SM3 will continue to participate in the reaction, thereby generating impurity Z 3f At the same time, during the reaction, finerenone Z3 is easily aromatized to form impurity Z 3a , or transesterify with trace amounts of ethanol in the reaction system to produce impurity Z 3b , or react with trace amounts of methanol to produce Z 3e Therefore, in order to ensure the quality controllability of intermediate Z3 and to better prepare subsequent intermediates and finished products, it is necessary to control the six related impurities in finerenone Z3.

[0004]

[0005] At present, there is no literature or patent information reporting on the separation and determination methods of the six impurities in finerenone Z3. In addition, there are many difficulties in separating finerenone Z3 and its six impurities in a set of HPLC methods: 1) Due to the presence of impurities SM3 and Z 3fThe polarity is relatively large and the structure is similar, so it is difficult to retain and separate in reverse phase; 2) Impurity Z 3a The polarity is small and it is difficult to elute on the reverse phase; and impurity Z 3f 、Impurity Z 3a The peak shape is poor on other chromatographic columns; 3) Finerenone Z3 and impurity Z 3e The structures are also extremely similar, making them difficult to separate and method development difficult.

[0006] In the prior art, patent CN117630224A discloses a method for separating and determining enantiomers of finerenone by HPLC, which uses silica gel coated with amylose-tris (3,5-dimethylphenylcarbamate) as a filler, uses lower alkanes as mobile phase A and lower alcohols as mobile phase B, performs isocratic elution, the flow rate is 0.6-0.8 ml / min, and the column temperature is 30-40°C; an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 245-255 nm. However, this method cannot achieve the separation of impurities SM3 and impurities Z3 in finerenone Z3. 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a effective determination. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating finerenone Z3 and its impurities based on high performance liquid chromatography, which method can complete the separation of multiple substances in a relatively short time.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] A method for separating finerenone Z3 and its impurities based on high performance liquid chromatography, wherein the finerenone Z3 and the impurities together form a composition, and the impurities include impurities SM3, impurities Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a Any one or more of; the structural formula of each component in the composition is as follows:

[0010]

[0011] In the high performance liquid chromatography, the mobile phase: mobile phase A is phosphoric acid solution, mobile phase B is acetonitrile; the stationary phase:

[0012] The chromatographic column used octadecylsilane bonded silica as filler and performed linear gradient elution.

[0013] After separation and testing, it is used for the next production step.

[0014] The above impurities can be arranged and combined in various ways. For example, combination 1: impurity SM3, impurity Z 3f For example, combination 2: impurity SM3, impurity Z 3f , impurity Z2. For another example, combination 3: impurity Z 3f , impurity Z2, impurity Z 3e .

[0015] Theoretically, when the upper limit of the substances to be separated, identified and / or detected by the method is n (number), 1-n substances can be detected naturally.

[0016] Further, the procedure of the linear gradient elution is as follows:

[0017] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 75±10 25±10 3±0.5 75±10 25±10 25±0.5 60±10 40±10 30±0.5 25±10 75±10 40±0.5 25±10 75±10 41±0.5 75±10 25±10 50±0.5 75±10 25±10 .

[0018] As a preferred solution, the procedure of the linear gradient elution is as follows:

[0019] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 74～76 24～26 3 74～76 24～26 25 60 40 30 25 75 40 25 75 41 74～76 24～26 50 74～76 24～26 .

[0020] For example, at 0, 3, 41, and 50 minutes, the mobile phase A accounts for 74 parts and the mobile phase B accounts for 26 parts ( Fig.15 ); or the mobile phase A accounts for 75 parts and the mobile phase B accounts for 25 parts ( Figure 2 ); or the mobile phase A accounts for 76 parts and the mobile phase B accounts for 24 parts ( Fig.14 ).

[0021] Most preferably, the procedure of the linear gradient elution is as follows:

[0022] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 75 25 3 75 25 25 60 40 30 25 75 40 25 75 41 75 25 50 75 25 .

[0023] Furthermore, the concentration of the phosphoric acid solution is 0.2% to 0.3%, preferably 0.23% to 0.27%, and most preferably 0.25%.

[0024] Furthermore, the flow rate is 0.5-1.5 mL / min; the column temperature is 20-40°C, and the injector temperature is 4-6°C.

[0025] Preferably, the flow rate is 0.9-1.1 mL / min; the column temperature is 28-32°C. For example, the flow rate is 0.9 mL / min ( Fig.10 ); or flow rate is 1.0 mL / min ( Figure 2 ); or flow rate of 1.1 mL / min ( Fig.11 ). For example, the column temperature is 28°C ( Fig.12); or column temperature is 29°C; or column temperature is 30°C ( Figure 2 ); or column temperature is 31°C; or column temperature is 32°C ( Fig.13 ).

[0026] Preferably, the running time is 50 minutes.

[0027] Preferably, the injector temperature is 5°C.

[0028] Preferably, the injection volume is 10 μl.

[0029] Preferably, the chromatographic column has a specification of 4.6 mm×250 mm and 5 μm.

[0030] Most preferably, the chromatographic column is SHIMADZU Shim-pack Scepter C18-120, 4.6 mm×250 mm, 5 μm.

[0031] Furthermore, the sample preparation solvent was 50% acetonitrile.

[0032] As a preferred solution, before separation, a sample solution, a control solution and a system suitability solution are prepared using a solvent, as follows:

[0033] 1) Take an appropriate amount of the finerenone Z3 sample to be tested, weigh it accurately, dissolve it in a solvent and dilute it to prepare a solution with a concentration of 1 mg / ml as the sample solution.

[0034] 2) Accurately measure an appropriate amount of the sample solution and add solvent to quantitatively dilute it into a solution with a concentration of 5 μg / ml as the control solution.

[0035] 3) Take Z3 system suitability reference (containing finerenone Z3, impurity SM3, impurity Z 3f , impurity Z2, impurity Z3e, impurity Z 3b 、Impurity Z 3a ) is accurately weighed, dissolved in solvent and diluted to make a solution containing about 1 mg per 1 ml as the system suitability solution.

[0036] The second object of the present invention is to provide a method for identifying finerenone Z3 and its impurities, which method can complete the identification of multiple substances in a relatively short time.

[0037] To achieve the above object, the technical solution of the present invention is:

[0038] The method for identifying finerenone Z3 and its impurities comprises separating the composition by the aforementioned separation method, and entering a detector for detection to obtain a chromatogram; and determining whether the test sample contains finerenone Z3 and its impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0039] As a preferred solution, the detection wavelength of the detector is 250±10nm. The setting range of ±10nm is a comprehensive consideration based on multiple factors such as error considerations, method superiority, and practical application requirements. This setting range helps to ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0040] Most preferably, the detection wavelength of the detector is 250 nm.

[0041] As a preferred solution, the components in the composition can be identified according to the order of relative retention time. The components of the composition are in ascending order: impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e , Finerenone Z3, Impurity Z 3b 、Impurity Z 3a .

[0042] Generally, relative retention time is used to describe the relative retention of different components in a mixture on a chromatographic column. It refers to the ratio of the retention time of a component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on a chromatographic column and is an important parameter for positioning, qualitative and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0043] As a preferred solution, finerenone Z3 is used as the reference peak, and the relative retention time is 0.11, which is determined to be impurity SM3; the relative retention time is 0.13, which is determined to be impurity Z 3f ; The relative retention time is 0.78, which is determined to be impurity Z2; the relative retention time is 0.96, which is determined to be impurity Z 3e ; The relative retention time is 1.32, which is determined to be impurity Z 3b ; The relative retention time is 1.70, which is determined to be impurity Z 3a ; The relative retention time of each component fluctuates within the range of ±5%.

[0044] In addition to relative retention time, retention time can also be used to determine components. Retention time refers to the time required for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the movement speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic apex (maximum concentration) of a certain component. It is mainly used to determine the peak order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0045] The third object of the present invention is to provide a method for determining the content of finerenone Z3 and its impurities, which method can complete the identification and content determination of multiple substances in a relatively short time.

[0046] To achieve the above object, the technical solution of the present invention is:

[0047] The method for determining the content of finerenone Z3 and its impurities comprises the following steps: using the aforementioned identification method to separate and identify finerenone Z3 and its impurities to obtain a chromatogram; and according to the obtained chromatogram, using the principal component self-comparison method with a correction factor to calculate the content of each impurity.

[0048] Through content determination, we can further determine whether finerenone Z3 and its impurity content are qualified.

[0049] If impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a If the corrected peak area of ​​any one or more impurities in the reference solution is greater than 0.3 times the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is unqualified; on the contrary, if impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a The corrected peak area of ​​any one or more impurities in the solution is not greater than 0.3 times the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is qualified.

[0050] Furthermore, the correction factor of the impurity SM3 is 0.63; the correction factor of the impurity Z2 is 1.2; 3a The correction factor is 0.74; the impurity Z 3f The correction factor is 0.70; the impurity Z 3b and impurity Z 3e The correction factor is 1.

[0051] The above determination method can be used as a drug quality determination model, and further as an indispensable key module in the intelligent production process. This model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as mobile phase composition, flow rate, column temperature, etc., providing a scientific basis for the comprehensive evaluation of drug quality. In the intelligent production system, this model is seamlessly integrated, able to receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feedback the determination results based on preset quality standards.

[0052] The beneficial effects of the present invention are:

[0053] 1. The present invention provides a method for separating and determining finerenone Z3 and its impurities based on high performance liquid chromatography, wherein the impurities include impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a The method of the present invention can effectively separate finerenone Z3 and its six impurities in a set of HPLC methods without being interfered by blank and other unknown impurities, and the separation degree between known impurities is greater than 1.5, which has good specificity.

[0054] 2. The method of the present invention can achieve effective separation of finerenone Z3 and its six impurities within 50 minutes, and has the characteristic of short separation time.

[0055] 3. The method of the present invention has the advantages of being simple, rapid, highly sensitive, durable, highly accurate, highly practical, and having accurate and reliable detection results, and is of great significance for achieving quality control of finerenone intermediates and finerenone finished products. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is the chromatogram of the blank solution;

[0057] Figure 2 is the chromatogram of the mixed solution;

[0058] Figure 3 is the chromatogram of the test solution;

[0059] Figure 4 is the chromatogram of the control solution;

[0060] Figure 5 The chromatograms of the quantitative limit solutions of the remaining impurities are as follows;

[0061] Figure 6 is the chromatogram of the quantification limit solution of Z3;

[0062] Figure 7 is the chromatogram of the detection limit solution of the remaining impurities;

[0063] Figure 8 is the chromatogram of the Z3 detection limit solution;

[0064] Fig. 9 This is the chromatogram of the 100% spiked test solution in the accuracy experiment;

[0065] Fig.10 This is the chromatogram of the mixed solution at a flow rate of 0.9 ml / min in the durability experiment;

[0066] Fig.11This is the chromatogram of the mixed solution at a flow rate of 1.1 ml / min in the durability experiment;

[0067] Fig.12 This is the chromatogram of the mixed solution in the durability experiment at a column temperature of 28°C;

[0068] Fig.13 This is the chromatogram of the mixed solution in the durability experiment at a column temperature of 32°C;

[0069] Fig.14 This is the chromatogram of the mixed solution in the durability experiment under the condition of the initial ratio of the mobile phase A:B = 76:24;

[0070] Fig.15 This is the chromatogram of the mixed solution in the durability experiment under the condition of the initial ratio of the mobile phase A:B = 74:26;

[0071] Fig.16 This is the chromatogram of the mixed solution in the durability experiment under the condition of mobile phase A with a phosphoric acid solution concentration of 0.23%;

[0072] Fig.17 This is the chromatogram of the mixed solution in the durability experiment under the condition of mobile phase A with a phosphoric acid solution concentration of 0.27%. DETAILED DESCRIPTION

[0073] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0074] Supplementary table to the drawings in the specification. Figure 1-Figure 17 Visual aids are provided for the purpose of understanding and interpretation. In the event of any ambiguity, the user should refer to the corresponding numbered tables (Tables 1-17) and data for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found during the review of Tables 1-17, the content in the corresponding numbered figures shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some of the characters in the spectrum of this application document overlap, they can still be viewed clearly, and the integration results of each figure are recorded in detail in the specification. In addition, the numbers in the spectrum have no effect on the scope of protection of the claims and the full disclosure of the technical solutions in the specification.

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079] Table 3

[0080]

[0081] Table 4

[0082]

[0083] Table 5

[0084]

[0085] Table 6

[0086]

[0087] Table 7

[0088]

[0089] Table 8

[0090]

[0091] Table 9

[0092]

[0093] Table 10

[0094]

[0095] Table 11

[0096]

[0097] Table 12

[0098]

[0099] Table 13

[0100]

[0101] Table 14

[0102]

[0103] Table 15

[0104]

[0105] Table 16

[0106]

[0107] Table 17

[0108]

[0109] In Tables 10 to 17, No. 1 is impurity SM3, No. 2 is impurity Z 3f , No. 4 is impurity Z2, No. 5 is impurity Z 3e , No. 6 is phenaretone Z3, No. 8 is impurity Z 3b , No. 12 is impurity Z 3a .

[0110] In Tables 1 to 17, other impurities not marked or described are unknown impurities.

[0111] In order to enhance the understanding of the present invention, some key technologies and scientific terms are clearly defined below. Unless specifically defined herein, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference substances, and test substances described, but reasonable flexibility and adjustment in these aspects are allowed. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments rather than to be interpreted restrictively.

[0112] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0113] If there are any unmentioned conditions in the chromatographic conditions, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part 4 0512).

[0114] the term

[0115] The limit of quantitation refers to the lowest amount of the substance to be measured in the sample, and the measurement result should have a certain degree of accuracy and precision. In other words, the limit of quantitation is the lowest level at which the analytical method can accurately and reliably measure the concentration of the substance to be measured in the sample. In HPLC, the determination of the limit of quantitation usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the substance to be measured corresponding to a certain level of signal-to-noise ratio is used as the limit of quantitation. The determination of the limit of quantitation is crucial to ensure the accuracy and reliability of the analysis results.

[0116] The durability of chromatographic conditions refers to the ability of the chromatographic analysis system to maintain stable analytical performance and not significantly affect the measurement results when there is a slight change in the measurement conditions. This durability is crucial to ensure the reliability, repeatability and stability of the analysis results.

[0117] The limit of detection (LOD) refers to the lowest concentration or amount of the substance in the sample that can be detected. It reflects the sensitivity and noise of the analytical method and instrument, and also indicates the level of the blank (background) value after the sample is processed.

[0118] The correction factor is a coefficient or parameter used to correct the results during the analysis process. It is intended to improve data accuracy and reliability. In HPLC analysis, since the same detector has different response values ​​for different substances, when different substances of the same mass pass through the detector, the peak areas generated are not necessarily equal. In order to make the peak area accurately reflect the content of the component to be measured, standard substances are used for correction, the correction factor is calculated, and it is applied to the measurement results of the sample to be measured.

[0119] The ratio of peak height to noise (S / N, i.e. signal-to-noise ratio) is used in HPLC to evaluate the detection sensitivity and resolution of the instrument and is an important indicator for measuring instrument performance. Among them: peak height refers to the signal value output by the detector when the component to be tested is eluted from the column; and noise refers to the fluctuation of the baseline signal, that is, the signal value measured by the blank sample. The signal-to-noise ratio is the ratio of the signal measured by a sample of known concentration to the signal measured by a blank sample. The larger the signal-to-noise ratio, the more accurately the instrument can separate and identify the target component when detecting the sample, while also reducing the interference of background noise.

[0120] In the examples of the present invention, information on finerenone Z3 and its related impurities is shown in Table A.

[0121] Table A. Compound information table

[0122]

[0123]

[0124] Example 1. Method for separating and determining finerenone Z3 and its related substances

[0125] (1) Prepare the test solution

[0126] Solvent: 50% acetonitrile.

[0127] Test solution: Take an appropriate amount of the product (Fenerenone Z3), weigh accurately, dissolve in solvent and dilute to make a solution containing about 1 mg per 1 ml.

[0128] Control solution: Accurately measure an appropriate amount of the test sample solution and add solvent to quantitatively dilute it into a solution containing approximately 5 μg per 1 ml.

[0129] System suitability solution: Take Z3 system suitability reference substance (containing finerenone Z3, impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a ) is accurately weighed, dissolved in solvent and diluted to make a solution containing about 1 mg per 1 ml.

[0130] (2) Chromatographic conditions

[0131] The analysis was performed using a Thermo Fisher / Shimadzu high performance liquid chromatograph. Octadecylsilane bonded silica gel was used as a filler (SHIMADZU Shim-pack Scepter C18-120, 4.6 mm × 250 mm, 5 μm or a chromatographic column of equivalent performance); 0.25% phosphoric acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to Table B; the detection wavelength was 250 nm; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the injector temperature was 5°C; and the injection volume was 10 μl.

[0132] Table B. Gradient elution schedule

[0133] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 75 25 3 75 25 25 60 40 30 25 75 40 25 75 41 75 25 50 75 25

[0134] (3) Determination

[0135] Accurately measure the system suitability solution, test solution and reference solution, inject them into the liquid chromatograph respectively, record the chromatogram; and calculate the content of each impurity using the principal component self-reference method with correction factor.

[0136] System suitability requirements: In the system suitability solution chromatogram, according to impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e , Z3, impurity Z 3b 、Impurity Z 3a The peaks are extracted sequentially, and the separation between the peaks of each component should meet the requirements.

[0137] Limit: If there are impurity peaks in the test solution, impurity SM3, impurity Z2, impurity Z 3a 、Impurity Z 3f The corrected peak area (multiplied by the correction factors 0.63, 1.2, 0.73, and 0.70, respectively) should not be greater than 0.3 times (0.15%) the main peak area of ​​the control solution. 3b 、Impurity Z 3e (Impurity Z 3b 、Impurity Z3e The correction factor is 1.0) The peak area shall not be greater than 0.3 times the main peak area of ​​the control solution (0.15%), the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the control solution (0.5%), the sum of the impurity peak areas after correction shall not be greater than 4 times the main peak area of ​​the control solution (2.0%), and the peaks less than 0.1 times the main peak area of ​​the control solution shall be ignored (0.05%). The spectrum of the control solution is as follows Figure 4 The integration results are shown in Table 4.

[0138] Example 2. Specificity

[0139] (1) Prepare the test solution

[0140] Solvent: 50% acetonitrile.

[0141] Impurity SM3 stock solution: Accurately weigh 24.87 mg of impurity SM3, place it in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0142] Impurity Z2 stock solution: Accurately weigh 25.14 mg of Z2, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0143] Impurity Z 3a Stock solution: Accurately weigh impurity Z 3a 25.05 mg, put in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0144] Impurity Z 3b Stock solution: Accurately weigh impurity Z 3b 24.88 mg, put in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0145] Impurity Z 3e Stock solution: Accurately weigh impurity Z 3e 24.90 mg, put into a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0146] Impurity Z 3f Stock solution: Accurately weigh impurity Z 3f 24.95 mg, put in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0147] Z3 reference substance stock solution: Accurately weigh 24.89 mg of Z3 reference substance, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0148] Impurity localization solution: Accurately measure 1.5 ml of the above impurity stock solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0149] Test solution: Accurately weigh 24.93 mg of intermediate Z3, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0150] Self-control solution: Accurately pipette 0.5 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0151] Mixed solution: Accurately weigh 24.96 mg of intermediate Z3 and place it in a 25 ml volumetric flask. Then accurately weigh impurities SM3, Z2, and Z 3a , Z 3b , Z 3e , Z 3f Place 2.5 ml of each positioning solution in the bottle, add solvent to dissolve and dilute to the scale, shake well, and you are done.

[0152] (2) Detection

[0153] Accurately measure 10 μl of each of the blank solvent, positioning solution, test solution and mixed solution, inject them separately, and record the chromatogram.

[0154] The test results are shown in Table C. Figure 1 to Figure 3 , Table 1 to Table 3. The blank solution and the test solution did not interfere with the detection of related substances; the separation between the impurity peaks and between the impurity peaks and the main peak was greater than 1.5, and the specificity of this method was good.

[0155] Table C. Specificity test results

[0156]

[0157] Example 3. System adaptability

[0158] System suitability solution: that is, the self-control solution under the "specificity" item.

[0159] Accurately measure 10 μl of the system suitability solution, inject it into the liquid chromatograph, test it 6 times continuously, and record the chromatogram.

[0160] The test results are shown in Table D. In the system suitability solution, the RSD of the main peak area was 0.40%, less than 5.0%, the tailing factor was less than 2.0, and the number of theoretical plates was greater than 5000, all meeting the requirements.

[0161] Table D. System suitability test results

[0162]

[0163]

[0164] Example 4. Limit of Quantitation

[0165] Z3 reference substance stock solution: Accurately weigh 25.16 mg of Z3 reference substance, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0166] Z3 linear stock solution: Accurately pipette 5.0 ml of Z3 reference stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, shake well, and the solution is ready.

[0167] Z3 quantitative limit solution: Accurately measure 0.3 ml of Z3 linear stock solution, place it in a 50 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0168] The remaining impurities quantitative limit stock solution: take impurities SM3, impurity Z2, impurity Z 3a 、Impurity Z 3b 、Impurity Z 3e and impurity Z 3f Place 1.5 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0169] Quantitative limit solution of other impurities: Accurately pipette 1.0 ml of the quantitative limit stock solution of other impurities into a 50 ml volumetric flask, dilute to the scale with solvent, shake well, and the solution is ready.

[0170] The above-mentioned Z3 quantitative limit solution and the quantitative limit solutions of other impurities were sampled continuously for 6 times, the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded.

[0171] The formula for calculating the limit of quantitation is as follows:

[0172]

[0173] The test results are shown in Table E. Figure 5-6 , Table 5-Table 6. The data show that the quantitative limit of impurity SM3 is 0.297 μg / ml, the concentration in the sample is 0.030%, the mean signal-to-noise ratio is 327.7, and the peak area RSD is 0.4%; impurity Z 3f The quantitative limit concentration of impurity Z2 is 0.298 μg / ml, the concentration in the sample is 0.030%, the mean signal-to-noise ratio is 52.0, and the peak area RSD is 2.1%. 3e The quantitative limit concentration of Z3 was 0.993 μg / ml, the mean signal-to-noise ratio was 190.6, and the peak area RSD was 1.9%. The impurity Z 3bThe quantitative limit of impurity Z was 0.289 μg / ml, the concentration in the sample was 0.029%, the mean signal-to-noise ratio was 52.1, and the peak area RSD was 3.6%. 3a The quantitative limit concentration is 0.295μg / ml, the concentration in the sample is 0.029%, the signal-to-noise ratio mean is 116.0, and the peak area RSD is 0.7%; the signal-to-noise ratio of the chromatographic peak of the quantitative limit concentration of each component is greater than 10, and the peak area RSD is less than 10%, which meets the requirements for the determination of related substances, indicating that the impurities can be accurately quantified at this level.

[0174] Table E. Quantitation limit test results

[0175]

[0176]

[0177] Example 5. Detection Limit

[0178] Z3 detection limit solution: Accurately measure 3.3 ml of Z3 quantification limit solution under the "quantification limit" item, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0179] Detection limit solution of other impurities: Accurately measure 5.0 ml of the quantification limit solution of other impurities under the "quantification limit" item, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0180] The above-mentioned Z3 detection limit solution and other impurity detection limit solutions were sampled continuously for 3 times, the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded.

[0181] The detection limit is calculated as follows:

[0182]

[0183] The test results are shown in Table F. Figure 7-8 , Table 7 to Table 8. The data show that the detection limit concentration of impurity SM3 is 0.149 μg / ml, the concentration in the sample is 0.015%, and the average signal-to-noise ratio is 185.9; impurity Z 3f The detection limit concentration of impurity Z2 is 0.149 μg / ml, the concentration in the sample is 0.015%, and the mean signal-to-noise ratio is 136.6; the detection limit concentration of impurity Z2 is 0.149 μg / ml, the concentration in the sample is 0.015%, and the mean signal-to-noise ratio is 27.4; impurity Z 3e The detection limit concentration of Z3 is 0.496 μg / ml, the concentration in the sample is 0.050%, and the signal-to-noise ratio is 99.6.3b The detection limit concentration is 0.144μg / ml, the concentration in the sample is 0.014%, and the mean signal-to-noise ratio is 27.4; impurity Z 3a The detection limit concentration is 0.147μg / ml, the concentration in the sample is 0.015%, and the average signal-to-noise ratio is 60.2; the signal-to-noise ratio of the chromatographic peak of the detection limit concentration of each component is greater than 3, which meets the requirements for the determination of related substances, indicating that impurities can be effectively detected at this level.

[0184] Table F. Detection limit determination results

[0185]

[0186]

[0187] Example 6. Accuracy

[0188] (1) Prepare the test solution

[0189] Solvent (blank solution): 50% acetonitrile.

[0190] Reference stock solution: Take impurities SM3, Z2, Z 3a , Z 3b , Z 3e , Z 3f 1.5 ml of each stock solution and 5.0 ml of Z3 reference substance stock solution are placed in the same 100 ml volumetric flask, diluted to the mark with solvent, shake well, and the result is obtained. Reference substance solution: accurately measure 2.5 ml of reference substance stock solution, place in a 25 ml volumetric flask, dilute to the mark with solvent, shake well, and the result is obtained.

[0191] Unspiked test sample solution: Use the test sample solution under “Specificity”.

[0192] Spiked test solution 1#: Accurately weigh 25 mg of the test sample, place it in a 25 ml volumetric bottle, add 2.0 ml of the reference stock solution, dilute to the mark with solvent, shake well, and you have it. (Prepare three times in parallel) (weigh 24.87 mg, 24.83 mg, 24.85 mg)

[0193] Spiked test solution 2#: Accurately weigh 25 mg of the test sample, place it in a 25 ml volumetric flask, add 2.5 ml of the reference stock solution, dilute to the mark with solvent, shake well, and you have it. (Prepare three times in parallel) (weigh 25.10 mg, 25.11 mg, 25.20 mg)

[0194] Spiked test solution 3#: Accurately weigh 25 mg of the test sample, place it in a 25 ml volumetric flask, add 3.0 ml of the reference stock solution, dilute to the mark with solvent, shake well, and you have it. (Prepare three times in parallel) (weigh 25.00 mg, 24.94 mg, 25.00 mg)

[0195] Self-control solution: Accurately measure 0.5 ml of each spiked test solution and unspecified test solution, place them in 100 ml volumetric flasks respectively, dilute to the scale with solvent, shake well, and obtain the corresponding self-control solution.

[0196] (2) Detection

[0197] Accurately measure 10 μl of each of the blank solution, reference solution, unspiked test solution, self-control solution, and spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the impurity content and recovery rate according to the external standard method and the self-control method with correction factor.

[0198] The test results are shown in Table G. Fig. 9 , Table 9. The recovery rates of 9 spiked solutions (3 concentrations) were determined using the external standard method and the self-control method with correction factors, and the impurities were all between 90% and 108%, meeting the requirements.

[0199] Table G. Accuracy test results

[0200]

[0201]

[0202]

[0203] Example 7. Chromatographic Conditions Robustness

[0204] Take the mixed solution under the "Specificity" item, use normal chromatographic conditions and the following changed chromatographic conditions (initial ratio of mobile phase ±1, flow rate ±0.1ml / min, column temperature ±2℃, phosphoric acid concentration of mobile phase A ±0.02%), test them respectively after the instrument system is stable, and record the separation between each peak.

[0205] The results are shown in Table H. Figure 10 to Figure 17 As shown in Tables 10 to 17, the chromatographic conditions were fine-tuned, and the separation between the impurities and between the main peak and the impurities was greater than 1.5, indicating that this method had good specificity.

[0206] Table H. Chromatographic Conditions Durability Test Results (Separation)

[0207]

Claims

1. A method for separating finerenone Z3 and its impurities based on high performance liquid chromatography, characterized in that: The finerenone Z3 and the impurities together form a composition, and the impurities include impurities SM3, impurities Z 3f , impurity Z2, impurity Z 3e 、Impurity Z 3b 、Impurity Z 3a Any one or more of; the structural formula of each component in the composition is as follows: In the high performance liquid chromatography method, the mobile phases: mobile phase A is a phosphoric acid solution, and mobile phase B is acetonitrile; Stationary phase: The chromatographic column uses octadecylsilane bonded silica gel as a filler and performs linear gradient elution.

2. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: 。 3. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: 。 4. The method according to claim 1, characterized in that: The concentration of the phosphoric acid solution is 0.2% to 0.3%.

5. The method according to claim 1, characterized in that The flow rate is 0.5-1.5 mL / min; the column temperature is 20-40°C, and the injector temperature is 4-6°C.

6. A method for identifying finerenone Z3 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 5, and is detected in a detector to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample with those of the reference sample, it is determined whether the test sample contains finerenone Z3 and its impurities.

7. The method according to claim 6, characterized in that The detection wavelength of the detector is 250±10 nm.

8. The method according to claim 6, characterized in that According to the order of relative retention time, the components in the composition can be identified. The components of the composition are in ascending order: impurity SM3, impurity Z 3f , impurity Z2, impurity Z 3e , Finerenone Z3, Impurity Z 3b 、Impurity Z 3a .

9. The method according to claim 8, characterized in that Using finerenone Z3 as the reference peak, the relative retention time was 0.11, and it was determined to be impurity SM3; The relative retention time is 0.13, which is determined to be impurity Z 3f ; The relative retention time is 0.78, which is determined to be impurity Z2; The relative retention time is 0.96, which is determined to be impurity Z 3e ; The relative retention time is 1.32, which is determined to be impurity Z 3b ; The relative retention time is 1.70, which is determined to be impurity Z 3a ; The relative retention time of each component fluctuates within the range of ±5%.

10. A method for determining finerenone Z3 and its impurity content, characterized in that: The method according to any one of claims 6 to 9 is used to separate and identify finerenone Z3 and its impurities to obtain a chromatogram; and the content of each impurity is calculated based on the obtained chromatogram using the principal component self-reference method with a correction factor.

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