Method for analyzing and detecting related substances of rosmeltirol

Abstract

The invention relates to an analysis and detection method of a rosmeltirol related substance. According to the method, a reversed-phase high-performance liquid chromatography method is adopted for determination; chromatographic conditions are as follows: octadecyl silane bonded silica gel is taken as a filling agent (Agilent Eclipse XDB-C18, 4.6 * 150 mm, 5 [mu] m) of a chromatographic column; an ultraviolet detector is adopted as a detector; the column temperature is 35 DEG C; the flow velocity is 1.0 ml / min; the detection wavelength is 302nm; the sample size is 5 microliters; a mobile phase A is a 0.05% phosphoric acid aqueous solution, a mobile phase B is acetonitrile, and gradient elution is carried out. According to the analysis and detection method for the related substances of the rosmeltirol, the complete separation of the rosmeltirol from the impurity A and the impurity B can be realized, and the separation degree of the main component and each impurity is greater than 3.0. According to the present invention, the compound rosmeltirol is detected and analyzed, such that the controllable quality of the related substance of the reactant during the subsequent pharmaceutical process is effectively ensured;

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and specifically relates to an analysis and detection method for resmetiro-related substances, which is used for analyzing and separating resmetiro-related substances. Background Art

[0002] Resmetirom (MGL 3196) is a thyroid hormone receptor β (THRβ) agonist under development by the American biotechnology company Madrigal. As the first successful Phase III clinical drug in the NASH field, it is used to treat patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) and achieved the primary endpoint and key secondary endpoints.

[0003] In the process of synthesizing Resmetiro, two process impurities, impurity A and impurity B, will be formed. According to the forced destruction test, impurity B is also a degradation impurity, and both impurities will exist in Resmetiro. In order to ensure the quality of Resmetiro, it is necessary to purify the synthesized Resmetiro and determine the content of impurity A and impurity B in the obtained product. However, there is no method for analyzing and detecting impurities A and impurity B in Resmetiro in the art. This leads to the inability to accurately assess the purity and quality of Resmetiro, which may affect the safety and effectiveness of the drug. In addition, the lack of an analytical method for the related substances of Resmetiro will also bring difficulties to drug production and quality control, increasing production costs and time. In the prior art, although there are some general drug impurity analysis methods, these methods may not be suitable for the detection of Resmetiro and its specific impurities. For example, some methods may not be able to effectively separate Resmetiro from impurities A and impurity B, resulting in inaccurate test results. In addition, some existing methods may not be sensitive enough to detect trace impurities, thereby affecting the evaluation of drug quality. In view of the above problems, it is urgent to develop a detection method for analyzing and separating resmetirol-related substances. The chemical structural formulas of resmetirol, impurity A and impurity B are as follows:

[0004] Summary of the invention

[0005] The purpose of the present invention is to provide a method for analyzing and detecting related substances of resmetiro, so as to achieve the determination of related substances and purity of resmetiro. The method has the advantages of being able to accurately evaluate the purity and quality of resmetiro, improve the safety and effectiveness of the drug, provide a standardized analytical method for drug production and quality control, and reduce production costs and time.

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

[0007] A method for analyzing and detecting resmetiro-related substances comprises the following steps:

[0008] (1) Preparation of system suitability solution: Weigh appropriate amounts of resmetiro and its impurity A and impurity B respectively, add diluent to prepare a mixed solution containing resmetiro, impurity A and impurity B as the system suitability solution;

[0009] (2) Preparation of test solution: Weigh an appropriate amount of resmetirol and add a diluent to prepare a solution as the test solution;

[0010] (3) Determination: Take an appropriate amount of blank diluent, inject it into the liquid chromatograph, record the chromatogram, balance the instrument, and confirm the position of the solvent peak in the diluent; 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;

[0011] (4) Content calculation: Based on the obtained chromatogram, the content of impurity A and impurity B in the test sample was calculated using the area normalization method;

[0012] Wherein, the structural formulas of the resmetirol are as follows:

[0013]

[0014] The structural formulas of impurities A and B are shown in the following table:

[0015]

[0016]

[0017] In step (3), the determination is performed by reverse phase high performance liquid chromatography; the chromatographic conditions are: chromatographic column: a chromatographic column with C18 bonded silica gel as the chromatographic column filler; mobile phase: 0.05% phosphoric acid aqueous solution and acetonitrile as the mobile phase for gradient elution.

[0018] Preferably, the chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm); the detector is an ultraviolet detector; the column temperature is 35°C; the flow rate is 1.0ml / min; the detection wavelength is 302nm; the injection volume is 5μl; the mobile phase A is 0.05% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution is performed in the following manner:

[0019]

[0020] Preferably, the diluent is ethanol.

[0021] The specific method for preparing the system suitability solution in step (1) is as follows: weigh appropriate amounts of resmetiro and its impurity A and impurity B, respectively, and add a diluent to prepare a mixed solution containing 1 mg / ml of resmetiro, 2 μg / ml of impurity A, and 2 μg / ml of impurity B as the system suitability solution;

[0022] The specific method of preparing the test solution in step (2) is as follows: weigh an appropriate amount of resmetirol, add a diluent to prepare a 1 mg / ml solution as the test solution;

[0023] In step (3), the amount of blank diluent, system suitability solution, and test solution taken was 5 μl.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] 1. The present application provides a method for analyzing and detecting related substances of Resmetiro, including the configuration of system suitability solution, the configuration of test solution, determination and content calculation, and the determination is carried out by reversed-phase high performance liquid chromatography. When determining, 0.05% phosphoric acid aqueous solution and acetonitrile are used as mobile phases for gradient elution and combined with other detection parameters (such as column temperature, flow rate and injection volume, etc.) and the impurity content is calculated using the area normalization method. This method has good separation ability, can effectively separate Resmetiro from impurities A and B, improve the accuracy and sensitivity of detection, so as to accurately evaluate the purity and quality of Resmetiro, improve the safety and effectiveness of drugs, and provide standardized analytical methods for drug production and quality control, reducing production costs and time.

[0026] 2. The analytical detection method for resmetiro-related substances established in the present invention can achieve complete separation of resmetiro from impurities A and impurity B, and the degree of separation between the main component and each impurity is greater than 3.0.

[0027] 3. The present invention effectively ensures that the quality of related substances of reactants in the subsequent pharmaceutical process is controllable by detecting and analyzing the compound resmetiro, and provides data support and reference for the formulation of resmetiro quality standards and the screening of preparation processes, which has important practical significance in production and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a chromatogram of the determination of the resmetiro test solution in Example 2 of the present invention.

[0029] Figure 2 It is a chromatogram of the impurity A-specific solution of Example 3 of the present invention.

[0030] Figure 3 It is a chromatogram of the impurity B-specific solution of Example 3 of the present invention.

[0031] Figure 4 This is a typical chromatogram of the impurity mixed solution of resmetiro in Example 3 of the present invention.

[0032] Figure 5 This is a typical chromatogram of the resmetiro system suitability solution of Example 3 of the present invention.

[0033] Figure 6 This is the standard curve of resmetirol in Example 5 of the present invention.

[0034] Figure 7 It is the standard curve of impurity A in Example 5 of the present invention.

[0035] Figure 8 It is the standard curve of impurity B in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0037] Example 1: Optimization of chromatographic conditions

[0038] The forced degradation test samples were used for the development of the analytical method for Resmetiro, while taking into account the detection of related substance impurities A and B, and the chromatographic conditions were preliminarily optimized to obtain the following detection conditions. Under this method, the forced degradation impurities, impurities A, impurities B and the main peak have good separation, which meets the daily detection needs and effectively ensures that the quality of related substances of the reactants in the subsequent pharmaceutical process is controllable. Therefore, the method was subsequently subjected to a durability investigation to meet the accurate detection of related substance impurities A and B.

[0039] The detection conditions are:

[0040] Chromatographic column: Octadecylsilane bonded silica gel as filler (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm);

[0041] Mobile phase A: 0.05% phosphoric acid water. Preparation method: accurately pipette 0.5 ml of phosphoric acid into 1000 ml of water, and ultrasonicate;

[0042] Mobile phase B: acetonitrile;

[0043] Flow rate: 1.0ml / min;

[0044] Column temperature: 35°C;

[0045] Injection volume: 5 μL;

[0046] Detection wavelength: 302nm;

[0047] Perform gradient elution according to the following table:

[0048]

[0049] The specific operation method of the durability investigation experiment is as follows:

[0050] (1) Solution preparation

[0051] Impurity A reference substance stock solution: Accurately weigh 2.62 mg of impurity A reference substance, place it in a 100 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0052] Impurity B reference substance stock solution: Accurately weigh 2.86 mg of impurity B reference substance, place in a 100 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0053] System suitability solution: Accurately weigh 25.17 mg of resmetiro, accurately pipette 2 ml of impurity B reference stock solution, accurately pipette 2 ml of impurity A reference stock solution, place in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0054] (2) Determination: Take 5 μl of the prepared system suitability solution and perform the determination based on the above chromatographic conditions. During the determination, adjust the flow rate and column temperature.

[0055] The results of the durability test are as follows:

[0056] 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.

[0057]

[0058] The flow rate was 1.2 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.

[0059]

[0060] 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.

[0061]

[0062] The flow rate was 1.0 ml / min, the column temperature was 40°C, and the system suitability solution was measured. The system suitability results are shown in the table below.

[0063]

[0064] The flow rate was 1.0 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.

[0065]

[0066]

[0067] Based on the above system suitability solution results and the impurity separation of the test solution under various conditions, it can be seen that this method has good durability and the following method can meet the needs of daily testing.

[0068] Chromatographic column: Octadecylsilane bonded silica gel as filler (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm);

[0069] Mobile phase A: 0.05% phosphoric acid aqueous solution, which is prepared by accurately pipetting 0.5 ml of phosphoric acid into 1000 ml of water and sonicating;

[0070] Mobile phase B: acetonitrile;

[0071] Flow rate: 0.8~1.2ml / min;

[0072] Column temperature: 30-40°C;

[0073] Injection volume: 5 μL;

[0074] Detection wavelength: 302nm;

[0075] Perform gradient elution according to the following table:

[0076]

[0077] In particular, the following conditions are the best chromatographic conditions:

[0078] Chromatographic column: Octadecylsilane bonded silica gel as filler (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm);

[0079] Mobile phase A: 0.05% phosphoric acid aqueous solution, which is prepared by accurately pipetting 0.5 ml of phosphoric acid into 1000 ml of water and sonicating;

[0080] Mobile phase B: acetonitrile;

[0081] Flow rate: 1.0ml / min;

[0082] Column temperature: 35°C;

[0083] Injection volume: 5 μL;

[0084] Detection wavelength: 302nm;

[0085] Perform gradient elution according to the following table:

[0086]

[0087] The optimal chromatographic conditions were used in subsequent experiments.

[0088] Example 2: Determination of test solution:

[0089] The test solution was measured under the optimized best chromatographic conditions.

[0090] (1) Preparation of the test solution: Weigh an appropriate amount of resmetirol and add a diluent to prepare a 1 mg / ml solution as the test solution. The diluent is ethanol.

[0091] (2) Determination: First, inject 5 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 5 μl of the test solution into the liquid chromatograph and record the chromatogram; wherein the chromatographic conditions used in step (2) are as follows:

[0092] Chromatographic column: Octadecylsilane bonded silica gel as filler (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm);

[0093] Mobile phase A: 0.05% phosphoric acid aqueous solution, which is prepared by accurately pipetting 0.5 ml of phosphoric acid into 1000 ml of water and sonicating;

[0094] Mobile phase B: acetonitrile;

[0095] Flow rate: 1.0ml / min;

[0096] Column temperature: 35°C;

[0097] Injection volume: 5 μL;

[0098] Detection wavelength: 302nm;

[0099] Perform gradient elution according to the following table:

[0100]

[0101] Sample results see Figure 1 , showing that the blank has no interference, the separation degree of each impurity meets the requirements, and meets the impurity control requirements.

[0102] Experimental Example 3: Specificity Experiment:

[0103] The impurity A specific solution, impurity B specific solution, impurity mixed solution and system suitability solution were tested under the optimized best chromatographic conditions.

[0104] (1) Preparation of solution:

[0105] The diluent described below is: ethanol

[0106] Impurity A reference substance stock solution: Accurately weigh 2.62 mg of impurity A reference substance, place it in a 100 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0107] Impurity B reference substance stock solution: Accurately weigh 2.86 mg of impurity B reference substance, place in a 100 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0108] Impurity A specific solution: Accurately pipette 2 ml of the impurity A reference stock solution into a 25 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0109] Impurity B specific solution: Accurately pipette 2 ml of the impurity B reference stock solution into a 25 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0110] Impurity mixed solution: Accurately pipette 2 ml of impurity B reference stock solution and 2 ml of impurity A reference stock solution, place in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0111] System suitability solution: Accurately weigh 25.17 mg of resmetiro, accurately pipette 2 ml of impurity B reference stock solution, accurately pipette 2 ml of impurity A reference stock solution, place in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0112] (2) Determination: Take 5 μl of the prepared impurity A specific solution, impurity B specific solution, impurity mixed solution and system suitability solution respectively, and perform determinations respectively using the chromatographic conditions described in Example 2.

[0113] The experimental results obtained are as follows Figure 2-5 The experimental results show that the diluent does not interfere with the detection of known impurities, the separation of each impurity is good, and the analytical method has good specificity. The specific solution spectrum of impurity A is shown in Figure 2 , impurity B specific solution spectrum see Figure 3 , impurity mixed solution spectrum see Figure 4 , system suitability solution spectrum see Figure 5 .

[0114] Example 4: Limit of detection and limit of quantitation:

[0115] Diluent: ethanol.

[0116] Solution preparation: Take appropriate amounts of resmetirol, impurity B, and impurity A, accurately weigh them, 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 quantitation limit (LOQ) and detection limit (LOD) of this product by the signal-to-noise ratio (S / N) method according to the measurement results, and prepare 6 parallel portions of the quantitation limit solution for separate testing, sample analysis under the chromatographic conditions described in Example 2, examine the RSD of the peak area, and confirm the precision of the method at the quantitation limit concentration.

[0117] The signal-to-noise ratio S / N ≥ 10:1 was used to calculate the corresponding test sample concentration as the quantification 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 1 and 2.

[0118] Table 1. Quantitation limit results

[0119]

[0120]

[0121] Table 2. Detection limit results

[0122]

[0123] The results showed that the RSD of the peak area of ​​the quantitative limit of resmetiro and its impurity A and impurity B was no more than 10.0%; calculated at a test sample concentration of 1 mg / ml, the quantitative limit detection limits of resmetiro and its impurity A and impurity B were 0.04%, 0.02%, and 0.02%, respectively, and the detection limits of resmetiro and its impurity A and impurity B were 0.012%, 0.006%, and 0.006%, respectively. The results showed that the method had high sensitivity and could fully meet the requirements for the detection of related substances of resmetiro.

[0124] Experimental Example 5: Linearity and Range Experiment

[0125] Diluent: ethanol.

[0126] Solution preparation: Take appropriate amounts of resmetirol and impurity A and impurity B, weigh them 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 250% of the limit concentration. Accurately measure 5 μl of each of the above linear solutions, inject into a liquid chromatograph, perform sample analysis under the chromatographic conditions described in Example 2, record the chromatogram, and draw a standard curve with concentration (μg / ml) as the horizontal axis and peak area as the vertical axis. Figure 6-8 The results are shown in Table 3.

[0127] Table 3. Linearity test results

[0128] name Range (μg / ml) Equivalent to the percentage of limit concentration Linear equations Correlation coefficient Resmetirol 0.407～2.589 20～250％ y=7745.3191x-1066.1001 0.9995 Impurity A 0.202～5.038 10～250％ y=7,813.2233x+559.5246 0.9996 Impurity B 0.207～5.171 10～250％ y=7,148.1009x-69.1067 0.9996

[0129] The results showed that resmetiro and each impurity had a good linear relationship with the peak area within the above range, and the correlation coefficient r was not less than 0.998. The relative response factors of impurity A and impurity B to resmetiro were 0.99 and 1.08, respectively, and their responses could be considered equivalent.

[0130] Example 6: Accuracy

[0131] Diluent: ethanol.

[0132] Test solution: Take about 25 mg of resmetiro, accurately weigh it, place it in a 25 ml volumetric flask, dilute it to 25 ml with diluent, and shake well.

[0133] Impurity quantitative limit stock solution: Take appropriate amount of impurity A and impurity B, weigh accurately, place in a volumetric flask, add diluent to dissolve and dilute to make a solution containing approximately 2.0 μg / ml of impurity A and 2.0 μg / ml of impurity B per 1 ml, as the impurity quantitative limit stock solution.

[0134] Impurity stock solution: Take appropriate amount of impurity A and impurity B, weigh accurately, place in a volumetric flask, add diluent to dissolve and dilute to make a solution containing approximately 10μg / ml of impurity A and 10μg / ml of impurity B per 1ml, as the impurity stock solution.

[0135] LOQ% accuracy solution: Take about 25 mg of resmetiro, weigh accurately, place in a 25 ml volumetric bottle, accurately add 2.5 ml of impurity quantitation limit stock solution, dilute to 25 ml with diluent, shake well. Prepare three copies in the same way.

[0136] 100% accuracy solution: Take about 25 mg of resmetiro, weigh accurately, place in a 25 ml volumetric bottle, accurately add 5 ml of impurity stock solution, dilute to 25 ml with diluent, shake well. Prepare three portions in the same way.

[0137] 200% accuracy solution: Take about 25 mg of resmetiro, weigh accurately, place in a 25 ml volumetric bottle, accurately add 10 ml of impurity stock solution, dilute to 25 ml with diluent, shake well. Prepare three portions in the same way.

[0138] Accurately measure 5 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 Tables 4 and 5.

[0139] Table 4. Impurity A recovery test results

[0140]

[0141]

[0142] Table 5. Impurity B recovery test results

[0143]

[0144] The results showed that within the range of the quantitative limit concentration to 200% of the limit concentration of impurity A and impurity B of resmetiro, the recoveries were between 80% and 120%, and the RSD of the recoveries was less than 10.0%, indicating that the method had good accuracy.

[0145] Example 7: Repeatability

[0146] Diluent: ethanol

[0147] Test solution: Prepare 6 portions in parallel with the 100% accuracy solution in Example 6.

[0148] Accurately measure 5 μl of each of the above, inject into the liquid chromatograph, test according to the chromatographic conditions described in Example 2, record the chromatogram, and calculate the content of each impurity. The results are shown in Table 6.

[0149] Table 6 Repeatability test results

[0150]

[0151] The results showed that the RSD of each impurity content in the 6 sample solutions was less than 10.0%, and the method had good repeatability.

[0152] The above-described embodiments can enable technicians in this field to understand the present invention more comprehensively, 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 resmetirol and impurity A and impurity B. Any separation and determination of resmetirol 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 related substances of resmetiro, characterized in that: The following steps are involved: (1) Preparation of system suitability solution: Weigh appropriate amounts of resmetiro and its impurity A and impurity B respectively, add diluent to prepare a mixed solution containing resmetiro, impurity A and impurity B as the system suitability solution; (2) Preparation of test solution: Weigh an appropriate amount of resmetirol and add a 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 content of impurity A and impurity B in the test sample was calculated using the area normalization method; Among them, the structural formulas of resmetiro, impurity A and impurity B are as follows: In step (3), the determination is performed by reverse phase high performance liquid chromatography; the chromatographic conditions are: chromatographic column: a chromatographic column with C18 bonded silica gel as the chromatographic column filler; mobile phase: 0.05% phosphoric acid aqueous solution and acetonitrile as the mobile phase for gradient elution.

2. The analysis and detection method according to claim 1, characterized in that: The chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel (Agilent Eclipse XDB-C18, 4.6×150mm, 5μm); the detector is an ultraviolet detector; the column temperature is 35°C; the flow rate is 1.0ml / min; the detection wavelength is 302nm; the injection volume is 5μl; the mobile phase A is 0.05% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution is performed in the following manner:

3. The analysis and detection method according to claim 1, characterized in that: The diluent is ethanol.

4. The analysis and detection method according to claim 1, characterized in that: The specific method of step (1) is: weigh appropriate amounts of resmetiro and its impurity A and impurity B respectively, add a diluent to prepare a mixed solution containing 1 mg / ml of resmetiro, 2 μg / ml of impurity A, and 2 μg / ml of impurity B as a system suitability solution.

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

6. 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 was 5 μl.

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