Analysis and detection method of fenerenone enantiomer

Abstract

The invention relates to an analysis and detection method of a fenerenone enantiomer. According to the method, a normal-phase high-performance liquid chromatography is adopted, methyl tertiary butyl ether-acetonitrile-diethylamine is used as a mobile phase to detect the fenerenone enantiomer, and the specific chromatographic conditions are as follows: a chromatographic column adopts silica gel surface covalent bonding cellulose-tri (3, 5-dichlorophenyl carbamate) as a filling agent (# imgabs0 # IC, 4.6 * 250mm, 5.0 mu m); mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V); the flow rate is 0.8 ml / min; the column temperature is 30 DEG C; the sample size is 20 [mu] L; the detection wavelength is 254 nm; the elution gradient is isocratic elution. By adopting the method disclosed by the invention, complete separation of the fenerenone and the enantiomer thereof can be realized, and the separation degree of the main component and the enantiomer is greater than 3.0.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a high performance liquid chromatography method for analyzing and separating finerenone enantiomers. Background Art

[0002] Finerenone is a non-steroidal selective mineralocorticoid receptor (MR) antagonist developed by Bayer. MR is expressed in the kidneys, heart, and blood vessels. Finerenone can reduce inflammation and fibrosis mediated by excessive MR activation. Finerenone has been approved for marketing in the United States (July 2021), the European Union (February 2022), Japan (February 2022), and China (June 2022). The drug is suitable for adult patients with chronic kidney disease associated with type 2 diabetes.

[0003] Finerenone has a chiral center. In chemical synthesis, it is obtained by splitting the finerenone racemate. Finerenone enantiomers are commonly present in samples and are listed as process impurities of finerenone, affecting the quality of finerenone raw materials and their preparations; therefore, the analysis and detection of this impurity plays an important role in the quality control of the final product finerenone. In order to better control the quality of finerenone products, it is necessary to establish a simple, reliable, stable and effective method to detect the enantiomers of this product. Among them, the chemical structural formulas of finerenone and finerenone enantiomers are as follows:

[0004] Summary of the invention

[0005] The object of the present invention is to provide a method for analyzing and detecting enantiomers of finerenone, specifically a high performance liquid chromatography method for analyzing enantiomers of finerenone, thereby realizing the determination of finerenone and its enantiomers.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention adopts normal phase high performance liquid chromatography, using methyl tert-butyl ether-acetonitrile-diethylamine as the mobile phase to detect the enantiomers of finerenone, specifically:

[0008] A method for analyzing and detecting finerenone enantiomers comprises the following steps:

[0009] (1) Preparation of system suitability solution: Weigh appropriate amounts of finerenone and its enantiomer, respectively, and add a diluent to prepare a mixed solution containing finerenone and its enantiomer as the system suitability solution.

[0010] (2) Preparation of test solution: Weigh an appropriate amount of finerenone and add diluent to prepare a solution as the test solution.

[0011] (3) Determination: Take an appropriate amount of blank diluent, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of test solution, inject it into the liquid chromatograph, and record the chromatogram;

[0012] (4) Content calculation: Based on the obtained chromatogram, the enantiomer content of finerenone in the test sample was calculated by the area normalization method;

[0013] Wherein, the structural formula of the finerenone and the finerenone enantiomers is as follows:

[0014]

[0015] In step (3), the determination is performed by normal phase high performance liquid chromatography; the chromatographic conditions are as follows: chromatographic column: a chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel as a filler; mobile phase: methyl tert-butyl ether-acetonitrile-diethylamine as the mobile phase for elution.

[0016] The chromatographic conditions are specifically:

[0017] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0018] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0019] Flow rate: 0.8ml / min;

[0020] Column temperature: 30°C;

[0021] Injection volume: 20 μL;

[0022] Detection wavelength: 254nm;

[0023] Elution gradient: isocratic.

[0024] The diluent is a mobile phase. Preferably, the diluent is a mixed solution of methyl tert-butyl ether, acetonitrile and diethylamine, and the volume ratio of methyl tert-butyl ether, acetonitrile and diethylamine is 100:900:0.5.

[0025] The specific method of step (1) is: weigh appropriate amounts of finerenone and its enantiomer, respectively, add a diluent to prepare a mixed solution containing 250 μg / ml of finerenone and 2.5 μg / ml of the enantiomer of finerenone, as a system suitability solution.

[0026] The specific method of step (2) is: weigh an appropriate amount of finerenone, add a diluent to prepare a 250 μg / ml solution as the test solution.

[0027] In step (3), the amount of blank diluent, system suitability solution, and test solution is 20 μl. That is, 20 μl of blank diluent is injected into the liquid chromatograph to record the chromatogram; 20 μl of system suitability solution is injected into the liquid chromatograph to record the chromatogram; 20 μl of test solution is injected into the liquid chromatograph to record the chromatogram.

[0028] Unless otherwise specified, the abbreviations in this invention have the following meanings:

[0029] min: minutes

[0030] ml: milliliters

[0031] μl: microliter

[0032] mg: milligram

[0033] LOQ: Limit of quantitation refers to the lowest amount of the analyte in the sample that can be quantitatively determined. The measurement result should have a certain degree of accuracy. The limit of quantitation reflects whether the analytical method has sensitive quantitative detection capabilities.

[0034] LOD: Limit of detection, also known as detection limit, refers to the corresponding amount of 3 times the value of the instrument background signal generated by the matrix blank, or the average value of the background signal generated by the matrix blank plus 3 times the mean standard deviation.

[0035] RSD: Relative standard deviation, also known as standard deviation coefficient, coefficient of variation, coefficient of change, etc., is the value obtained by dividing the standard deviation by the corresponding mean value multiplied by 100%, which can be used to analyze the precision of the results in inspection and testing work.

[0036] S / N: Signal-to-noise ratio refers to the ratio of signal to noise in an electronic device or electronic system. The signal here refers to the electronic signal from outside the device that needs to be processed by this device, and the noise refers to the irregular additional signal (or information) that does not exist in the original signal after passing through the device, and this signal does not change with the change of the original signal.

[0037] Compared with the prior art, the advantages of the present invention are:

[0038] The invention provides a high performance liquid chromatography method for analyzing finerenone enantiomers. The method of the invention can achieve complete separation of finerenone and its enantiomers, and the separation degree between the main component and the isomer is greater than 3.0.

[0039] The present invention effectively ensures that the isomer quality of the reactants in the subsequent pharmaceutical process is controllable by detecting and analyzing the isomers of the finerenone compound, and provides data support and reference for the formulation of the finerenone quality standard and the screening of the preparation process, which has important practical significance in production and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : The chromatogram of the finerenone test solution measured in Example 2;

[0041] Figure 2 : Chromatogram of enantiomer-specific solution determination in Example 3;

[0042] Figure 3 : Chromatogram of the system suitability solution determination of Example 3;

[0043] Figure 4 : The standard curve of finerenone in Example 5.

[0044] Figure 5 : Standard curve of finerenone enantiomers in Example 5. DETAILED DESCRIPTION

[0045] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0046] Example 1: Optimization of chromatographic conditions

[0047] Finerenone and finerenone enantiomer samples were used in the development of the finerenone analysis method, and the mobile phase and chromatographic column were preliminarily optimized to obtain the following detection conditions. Under this method, finerenone and its finerenone enantiomer peaks showed chromatographic peak tailing, which affected its sensitivity and daily detection needs. In order to effectively ensure that the quality of the finerenone enantiomer of the reactant in the subsequent pharmaceutical process is controllable, the method was optimized and subsequently tested for durability to meet the accurate detection of finerenone enantiomers.

[0048] The detection conditions are:

[0049] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0050] Mobile phase: methyl tert-butyl ether: acetonitrile = 100:900 (V:V);

[0051] Flow rate: 0.8ml / min;

[0052] Column temperature: 30°C;

[0053] Injection volume: 20 μL;

[0054] Detection wavelength: 254nm;

[0055] Diluent: Acetonitrile

[0056] Elution gradient: isocratic elution;

[0057] By adjusting the mobile phase and adding an appropriate amount of diethylamine, the tailing phenomenon of the chromatographic peak was significantly optimized. The specific investigation is as follows:

[0058] The system suitability solution was tested without adding diethylamine. The system suitability results are shown in the table below.

[0059]

[0060] 0.05% diethylamine was added to measure the system suitability solution. The system suitability results are shown in the table below.

[0061]

[0062] 0.1% diethylamine was added to measure the system suitability solution. The system suitability results are shown in the table below.

[0063]

[0064] From the above investigation, it can be seen that by adding an appropriate amount of diethylamine, the chromatographic peak tailing phenomenon is significantly optimized. Preferably, the detection conditions are:

[0065] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0066] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0067] Flow rate: 0.8ml / min;

[0068] Column temperature: 30°C;

[0069] Injection volume: 20 μL;

[0070] Detection wavelength: 254nm;

[0071] Diluent: acetonitrile;

[0072] Elution gradient: isocratic.

[0073] The diluent was optimized, and the diluent was changed from acetonitrile to mobile phase, and the system interference peaks were significantly reduced; preferably, the detection conditions are:

[0074] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0075] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0076] Flow rate: 0.8ml / min;

[0077] Column temperature: 30°C;

[0078] Injection volume: 20 μL;

[0079] Detection wavelength: 254nm;

[0080] Diluent: mobile phase;

[0081] Elution gradient: isocratic.

[0082] The robustness of this method was investigated to ensure accurate detection of finerenone enantiomers.

[0083] The specific operation method of the durability investigation experiment is as follows:

[0084] (1) Solution preparation

[0085] System suitability solution: Precisely measure finerenone and finerenone enantiomers respectively. Dilute with diluent to prepare a mixed solution containing about 250 μg of finerenone and 2.5 μg of finerenone enantiomers per 1 ml. (2) Determination: Take 20 μl of the prepared system suitability solution respectively and perform determination based on the above chromatographic conditions. During the determination, adjust the flow rate range.

[0086] The results of the durability test are as follows:

[0087] The flow rate was 0.6 ml / min, the column temperature was 30°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0088]

[0089]

[0090] The flow rate was 0.8 ml / min, the column temperature was 30°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0091]

[0092] The flow rate was 1.0 ml / min, the column temperature was 30°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0093]

[0094] The flow rate was 0.8 ml / min, the column temperature was 25°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0095]

[0096] The flow rate was 0.8 ml / min, the column temperature was 35°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0097]

[0098] Based on the above system suitability solution results, it can be seen that this method has good durability and the following method can meet daily testing requirements.

[0099] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0100] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0101] Flow rate: 0.6~1.0ml / min;

[0102] Column temperature: 25-35°C;

[0103] Injection volume: 20 μL;

[0104] Detection wavelength: 254nm;

[0105] Diluent: mobile phase;

[0106] Elution gradient: isocratic.

[0107] In particular, the following conditions are the best chromatographic conditions:

[0108] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0109] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0110] Flow rate: 0.8ml / min;

[0111] Column temperature: 30°C;

[0112] Injection volume: 20 μL;

[0113] Detection wavelength: 254nm;

[0114] Diluent: mobile phase;

[0115] Elution gradient: isocratic.

[0116] The optimal chromatographic conditions were used in subsequent experiments.

[0117] Example 2: Determination of test solution:

[0118] The test solution was measured under the optimized best chromatographic conditions.

[0119] (1) Preparation of the test solution: Weigh an appropriate amount of finerenone and add a diluent to prepare a 250 μg / ml solution as the test solution, wherein the diluent is the mobile phase.

[0120] (2) Determination: First, inject 20 μl of the blank diluent into the liquid chromatograph, record the chromatogram, balance the instrument, and confirm the position of the solvent peak in the diluent. Next, inject 20 μl of the test solution into the liquid chromatograph and record the chromatogram; wherein the chromatographic conditions used in step (2) are as follows:

[0121] Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm);

[0122] Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V);

[0123] Flow rate: 0.8ml / min;

[0124] Column temperature: 30°C;

[0125] Injection volume: 20 μL;

[0126] Detection wavelength: 254nm;

[0127] Diluent: mobile phase;

[0128] Elution gradient: isocratic.

[0129] Sample results see Figure 1 , showing that the blank has no interference, the separation degree between finerenone and finerenone enantiomers meets the requirements, and meets the impurity control requirements.

[0130] Experimental Example 3: Specificity and system suitability experiment:

[0131] The blank solution, finerenone enantiomer-specific solution, test solution and system suitability solution were measured under the optimized best chromatographic conditions.

[0132] (1) Preparation of solution:

[0133] The diluent described below is: mobile phase.

[0134] Preparation of test solution: Take 12.5 mg of the test sample (phenarezone), accurately weigh it, put it in a 50 ml volumetric flask, dilute it to 50 ml with diluent, shake well, and obtain.

[0135] Enantiomer-specific solution: Accurately measure the enantiomer of finerenone and dilute it with a diluent to prepare a solution containing 2.5 μg of the enantiomer of finerenone per 1 ml.

[0136] System suitability solution: Accurately measure finerenone and finerenone enantiomers respectively. Dilute with diluent to make a mixed solution containing about 250 μg of finerenone and 2.5 μg of finerenone enantiomers per 1 ml. (2) Determination: Take 20 μl of blank solvent, enantiomer-specific solution, test solution and system suitability solution respectively, and inject into the HPLC;

[0137] The experimental results show that the diluent does not interfere with the detection of each finerenone enantiomer, the separation between the main component and the isomer is greater than 3.0, and the analytical method has good specificity. Figure 2 , system suitability solution map see Figure 3 The system suitability index results are shown in Table 1.

[0138] Table 1. System suitability results

[0139]

[0140] Example 4: Limit of detection and limit of quantitation:

[0141] Solution preparation: Take an appropriate amount of finerenone and finerenone enantiomers, accurately weigh them, dilute them step by step with a diluent to form a series of solutions, dilute them step by step with a diluent to form a series of solutions and measure them, measure them according to the chromatographic conditions described in Example 2, and determine the quantification limit (LOQ) and detection limit (LOD) of this product by the signal-to-noise ratio (S / N) method based on the measurement results, and prepare 6 parallel portions of the quantification limit solution for testing respectively, inject and analyze them under the chromatographic conditions described in Example 2, examine the RSD of the peak area, and confirm the precision of the method at the quantification limit concentration.

[0142] The signal-to-noise ratio S / N ≥ 10:1 was used to calculate the corresponding test sample concentration as the quantitative limit; the signal-to-noise ratio S / N ≥ 3:1 was used to calculate the corresponding test sample concentration as the detection limit. The results are shown in Tables 2 and 3.

[0143] Table 2. Quantitation limit results

[0144]

[0145] Table 3. Detection limit results

[0146]

[0147] The results showed that the RSD of the peak area of ​​the quantification limit of finerenone enantiomers was no more than 10.0%; calculated at a test sample concentration of 250 μg / ml, the detection limits of finerenone and finerenone enantiomers were 0.05% and 0.05%, respectively, and the detection limits of finerenone and finerenone enantiomers were 0.02% and 0.01%, respectively. The results showed that the method had high sensitivity and could fully meet the requirements for the detection of finerenone enantiomers.

[0148] Experimental Example 5: Linearity and Range Experiment

[0149] Diluent: Mobile phase.

[0150] Solution preparation: Take appropriate amount of finerenone and finerenone enantiomers, weigh accurately, add diluent to dissolve and dilute to prepare a series of linear solutions containing each component at a concentration ranging from the limit concentration to 200% of the limit concentration. Accurately measure 20 μl of each of the above linear solutions, inject into the liquid chromatograph, inject and analyze under the chromatographic conditions described in Example 1, and record the chromatogram as shown in Figure 4 , 5 As shown in Table 4, a standard curve was drawn with concentration (μg / ml) as the horizontal axis and peak area as the vertical axis, and the regression equation was calculated.

[0151] Table 4. Linearity test results

[0152] name Range (μg / ml) Equivalent to the percentage of limit concentration Linear equations Correlation coefficient Finerenone 0.112～5.492 5～200％ y=72816.0193x+7174.6566 0.9993 Finerenone enantiomers 0.124～5.380 5～200％ y=74300.4816x+7619.2465 0.9995

[0153] The results showed that finerenone and finerenone enantiomers had a good linear relationship with the peak area in the concentration range of 0.112-5.492 μg / ml, and the correlation coefficient r was not less than 0.995. The relative response factor of finerenone enantiomers and finerenone was 0.98, which can be considered that their responses are equivalent.

[0154] Example 6: Accuracy

[0155] Diluent: Mobile phase.

[0156] Test solution: Take about 12.5 mg of phenerenone compound, accurately weigh it, place it in a 50 ml volumetric flask, dilute it to 50 ml with solvent, and shake well.

[0157] Enantiomer stock solution: Take an appropriate amount of finerenone enantiomer, accurately weigh it, place it in a volumetric flask, add a diluent to dissolve and dilute it to make a solution containing about 250 μg of the enantiomer per 1 ml, as the isomer stock solution.

[0158] Isomer quantification limit stock solution: Take an appropriate amount of finerenone isomer, accurately weigh it, put it in a volumetric flask, add diluent to dissolve and dilute it to make a solution containing about 1.1 μg / ml of isomer per 1 ml, which is used as the isomer quantification limit stock solution.

[0159] LOQ% accuracy solution: Take about 12.5 mg of phenerenone compound, weigh accurately, place in a 50 ml volumetric flask, accurately add 0.5 ml of isomer stock solution, dilute to 50 ml with diluent, shake well. Prepare three copies in the same way.

[0160] 100% accuracy solution: Take about 12.5 mg of phenerenone compound, weigh accurately, place in a 50 ml volumetric flask, accurately add 0.5 ml of isomer stock solution, dilute to 50 ml with diluent, shake well. Prepare three copies in the same way.

[0161] 200% accuracy solution: Take about 12.5 mg of phenerenone compound, weigh accurately, place in a 50 ml volumetric flask, accurately add 1.0 ml of isomer stock solution, dilute to 50 ml with diluent, shake well. Prepare three copies in the same way.

[0162] Accurately measure 20 μl of each of the above solutions, inject into a liquid chromatograph, measure according to the chromatographic conditions of Example 2, and record the chromatogram. The test results are shown in Table 5.

[0163] Table 5. Recovery test results

[0164]

[0165] The results showed that within the range of the quantitative limit concentration of finerenone enantiomers to the limit concentration of 200%, the isomer recoveries were between 80% and 120%, and the RSD of the recoveries was less than 10.0%, indicating that the method had good accuracy.

[0166] The above-described embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments. The method of the present invention is also not limited to the analysis and separation of finerenone and finerenone enantiomers, and any separation and determination of finerenone and its impurities using the method of the present invention falls within the scope of the present invention.

Claims

1. A method for analyzing and detecting enantiomers of finerenone, characterized in that: The following steps are involved: (1) Preparation of system suitability solution: Weigh appropriate amounts of finerenone and its enantiomer, respectively, and add a diluent to prepare a mixed solution containing finerenone and its enantiomer as the system suitability solution. (2) Preparation of test solution: Weigh an appropriate amount of finerenone and add diluent to prepare a solution as the test solution. (3) Determination: Take an appropriate amount of blank diluent, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of test solution, inject it into the liquid chromatograph, and record the chromatogram; (4) Content calculation: Based on the obtained chromatogram, the enantiomer content of finerenone in the test sample was calculated by the area normalization method; Wherein, the structural formula of the finerenone and the finerenone enantiomers is as follows: In step (3), the determination is performed by normal phase high performance liquid chromatography; the chromatographic conditions are as follows: chromatographic column: a chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel as a filler; mobile phase: methyl tert-butyl ether-acetonitrile-diethylamine as the mobile phase for elution.

2. The analysis and detection method according to claim 1, characterized in that: The chromatographic conditions are specifically: Chromatographic column: Silica gel surface covalently bonded cellulose-tris (3,5-dichlorophenyl carbamate) as filler ( IC, 4.6*250mm, 5.0μm); Mobile phase: methyl tert-butyl ether: acetonitrile: diethylamine = 100: 900: 0.5 (V: V: V); Flow rate: 0.8ml / min; Column temperature: 30°C; Injection volume: 20 μL; Detection wavelength: 254nm; Elution gradient: isocratic.

3. The analysis and detection method according to claim 1 or 2, characterized in that: The diluent is the mobile phase.

4. The analysis and detection method according to claim 3, characterized in that: The diluent is a mixed solution of methyl tert-butyl ether, acetonitrile and diethylamine, and the volume ratio of methyl tert-butyl ether, acetonitrile and diethylamine is 100:900:0.

5.

5. The analysis and detection method according to claim 1, characterized in that: The specific method of step (1) is: weigh appropriate amounts of finerenone and its enantiomer, respectively, add a diluent to prepare a mixed solution containing 250 μg / ml of finerenone and 2.5 μg / ml of the enantiomer of finerenone, as a system suitability solution.

6. The analysis and detection method according to claim 1, characterized in that: The specific method of step (2) is: weigh an appropriate amount of finerenone, add a diluent to prepare a 250 μg / ml solution as the test solution.

7. The analysis and detection method according to claim 1, characterized in that: In step (3), the amount of blank diluent, system suitability solution, and test solution taken is 20 μl.

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