Detection method of related substances of NASH therapeutic drug resmetarom
Gradient elution and detection of NASH therapeutic drug resmetirom through high performance liquid chromatography solves the problem of lack of effective detection methods in the prior art, and efficient separation and detection of resmetirom and its related substances is achieved, ensuring the quality and safety of the drug.
Patent Information
- Application Number
- CN202411733120.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-28
AI Technical Summary
There is a lack of effective detection methods in the prior art to analyze relevant substances in the NASH therapeutic drug resmetirom, which makes it difficult to guarantee the stability and efficacy of the drug.
High performance liquid chromatography is used to perform gradient elution through C18 reverse phase chromatography column and use an ultraviolet detector to detect the eluent. The specific steps include selecting the appropriate solvent, detection wavelength, column and gradient elution program to achieve efficient separation and detection of resmetirom and its related substances.
This method can effectively detect resmetirom and its related substances, showing high specificity, sensitivity, good linear relationship and repetition, ensuring the quality control and safety of the drug.
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Figure CN119534699A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for detecting substances related to resmetirom, a NASH therapeutic drug. Background Art
[0002] Nonalcoholic steatohepatitis (NASH) is a progressive liver disease characterized by hepatic steatosis, inflammation, hepatocellular damage, and fibrosis progression. When NASH progresses to advanced fibrosis, the risk of adverse clinical outcomes increases significantly, especially in patients with type 2 diabetes. The prevalence of NASH is closely related to the growing obesity epidemic, and although lifestyle changes can help manage associated metabolic disorders, they are often difficult to implement and maintain.
[0003] On March 14, 2024, local time, the U.S. Food and Drug Administration approved resmetirom as a treatment for adults with non-cirrhotic NASH with advanced liver fibrosis. The drug resmetirom selectively activates THR-β, and its selectivity for THR-β is about 28 times that of THR-α. Clinical studies have shown that the improvement in fibrosis in patients treated with resmetirom may be due to a reduction in metabolic damage rather than a direct anti-fibrotic effect. In addition, resmetirom has good safety and low toxicity in preclinical studies, and generally does not affect thyroid function, so it is well tolerated by patients.
[0004] Related substances refer to impurities in drugs other than the main ingredients. Most of the impurities in drugs have potential biological activity and will have adverse effects on the stability and efficacy of the drugs. Some impurities are not only harmful to the human body, but also produce new side effects. Therefore, the research and control of related substances is an important factor in ensuring the safety and effectiveness of drugs.
[0005] At present, there are few literatures on the quality research of the drug resmetirom, especially the detection method of the drug resmetirom-related substances has not been reported. Summary of the invention
[0006] The purpose of the present invention is to provide a method for detecting related substances of the drug resmetirom, using high performance liquid chromatography to detect the substance to be detected in C 18 Gradient elution was performed in a reverse phase chromatographic column, and the eluent was detected by a UV detector;
[0007] The analyte contains resmetirom and / or resmetirom-related substances;
[0008] The mobile phases used in the gradient elution are mobile phase A and mobile phase B, mobile phase A is a mixed solution of trifluoroacetic acid aqueous solution and acetonitrile, and mobile phase B is acetonitrile;
[0009] The procedure of the gradient elution is: 0-2 min, 10% B→10% B; 2-20 min, 10% B→50% B; 20-30 min, 50% B→54% B; 30-35 min, 54% B→54% B; 35-36 min, 54% B→10% B; 36-45 min, 10% B→10% B.
[0010] Furthermore, the C 18 The chromatographic column was Agilent 5HC C 18 (2) (250 mm × 4.6 mm, 5 μm) column.
[0011] Furthermore, the flow rate of the gradient elution was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was 35° C.
[0012] Furthermore, the detection wavelength of the ultraviolet detector is 220 nm.
[0013] Furthermore, the volume ratio of trifluoroacetic acid aqueous solution to acetonitrile in the mobile phase A is 90:10, and the trifluoroacetic acid aqueous solution is an aqueous solution containing 0.045% trifluoroacetic acid.
[0014] Furthermore, the solvent of the analyte is 75% methanol aqueous solution.
[0015] The present invention establishes a high performance liquid chromatography method for effectively detecting related substances of the NASH therapeutic drug resmetirom, and verifies the detection method. The results show that the method has good specificity, high sensitivity, good linear relationship and good repeatability. The present invention can provide a valuable reference for the subsequent research of the NASH therapeutic drug resmetirom. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 A chemical synthesis route for resmetirom.
[0017] Figure 2 The chemical structural formula of resmetirom and its related substances.
[0018] Figure 3 UV-Vis spectra of resmetirom and its related substances.
[0019] Figure 4 To optimize the chromatogram under liquid chromatography conditions and the chromatogram of the mixed impurity solution, wherein: A is isocratic elution condition 1, B is isocratic elution condition 2, C is isocratic elution condition 3, D is gradient elution condition 1, E is the final gradient elution condition, and F is the chromatogram of the mixed impurity solution.
[0020] Figure 5 HPLC chromatograms of undegraded (A), acid-degraded (B), alkaline-degraded (C), oxidatively-degraded (D), thermally-degraded (E), and photo-degraded (F) samples. DETAILED DESCRIPTION
[0021] The chemical synthesis route of the drug resmetirom is as follows Figure 1 The inventors have previously determined four main related substances of resmetirom through forced degradation studies: imp-A, imp-B, imp-C and imp-D. The chemical structures of resmetirom and imp-A, imp-B, imp-C and imp-D are as follows: Figure 2 As shown. Imp-A, imp-B, imp-C and imp-D are connected with Figure 1 The synthetic route was compared with Figure 1 Intermediates 6, 7, and 9 (identified as imp-C, imp-D, and imp-B, respectively) were classified as process-related impurities and degradation products, while imp-A was an oxidative degradation product of resmetirom.
[0022] Resmetirom, imp-B, imp-C and imp-D used in the present invention were purchased from Shanghai Kalulan Technology Co., Ltd.; imp-A was prepared by the inventors themselves.
[0023] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0026] Example 1
[0027] Establishment of detection method
[0028] 1. Selection of solvent
[0029] Dissolution tests showed that resmetirom was insoluble in water, slightly soluble in acetonitrile and methanol; the solubility of resmetirom in methanol was higher than that in acetonitrile. In addition, resmetirom (1.0 mg / mL) remained stable in methanol, and no significant changes were observed at 12, 24, 48, and 72 h. In view of the potential impact of methanol on the environment, methanol aqueous solutions of different concentrations were investigated as solvents. The results showed that 50% methanol aqueous solution could induce the precipitation of the active ingredient resmetirom, while 75% methanol aqueous solution could increase the solubility of resmetirom. Therefore, considering the solubility, stability, and cost-effectiveness of resmetirom in different solvents, water-methanol (25 / 75, v / v) solution was selected as the best solvent.
[0030] 2. Selection of detection wavelength
[0031] In order to determine the optimal detection wavelength, UV-visible spectra of resmetirom and its related substances imp-A, imp-B, imp-C, and imp-D (at a concentration of 10 μg / mL) were obtained in the wavelength range of 200 to 500 nm. The UV-visible spectra were recorded as follows: Figure 3 As shown in the figure, these five substances have high absorption near 220nm. Therefore, 220nm is selected as the detection wavelength of this method.
[0032] 3. Selection of chromatographic column
[0033] The mobile phase system of water and acetonitrile was used for the C 18 The key information related to the columns used in the screening studies is summarized in Table 1.
[0034] Table 1 HPLC columns used for screening
[0035]
[0036] The separation of the main peak and impurity peaks was improved by using a 250 mm column compared to a 150 mm column. 18 The comparison of the chromatograms of the Agilent 5HC C 18 (2) The column (250 mm × 4.6 mm, 5 μm) produced more peaks in the water-acetonitrile mobile phase setting, with improved resolution and reduced back pressure.
[0037] 4. Optimization of liquid chromatography conditions
[0038] Preliminary structural analysis showed that adding a trace amount of trifluoroacetic acid to the mobile phase can improve the chromatographic efficiency. Therefore, the mobile phase A was finally selected as a water / acetonitrile (90 / 10, v / v) solution with 0.045% trifluoroacetic acid added, and the mobile phase B was acetonitrile.
[0039] Since the impurity content in the system suitability solution is high, the number of impurities is large, and there are key peak pairs that are difficult to separate, the system suitability solution is used here to optimize the elution method. First, three isocratic elution methods were investigated, gradually increasing the initial proportion of mobile phase A from 10% to 50%. Figure 4 The results shown in Figures AC indicate that the separation efficiency of resmetirom and its related substances is improved, and the number of impurity peaks increases. Despite the improvement, it is still difficult to achieve effective separation of all components. Subsequently, a variety of gradient elution methods were investigated. Initially, gradient elution condition 1 (0-2 min, 10% B→10% B; 2-20 min, 10% B→90% B; 20-30 min, 90% B→90% B; 30-31 min, 90% B→10% B; 31-40 min, 10% B→10% B) was used, and the minimum separation between adjacent impurity peaks was 0.97, which was lower than the specified value of 1.2 ( Figure 4 D). After that, the elution gradient was continuously optimized to improve the quality of experimental data. After many experiments, the best liquid chromatography conditions were finally obtained.
[0040] Example 2
[0041] The chromatographic conditions used in this implementation are as follows:
[0042] Column: Agilent 5HC C 18 (2) Column (250 mm × 4.6 mm, 5 μm); mobile phase A: water-acetonitrile (90 / 10, v / v) solution containing 0.045% trifluoroacetic acid; mobile phase B: acetonitrile; gradient elution (0-2 min, 10% B→10% B; 2-20 min, 10% B→50% B; 20-30 min, 50% B→54% B; 30-35 min, 54% B→54% B; 35-36 min, 54% B→10% B; 36-45 min, 10% B→10% B); detection wavelength: 220 nm; flow rate: 1.0 mL / min; injection volume: 10 μL, column temperature: 35°C.
[0043] The preparation methods of various solutions are as follows:
[0044] Dissolve 25 mg of resmetirom in water-methanol (25 / 75, v / v) solution, transfer the mixed liquid to a 100 mL volumetric flask and dilute to the mark to obtain a resmetirom standard stock solution with a concentration of 0.25 mg / mL.
[0045] Accurately weigh 25 mg of imp-A, imp-B, imp-C or imp-D respectively, and dilute it to a concentration of 0.25 mg / mL with water-methanol (25 / 75, v / v) solution to obtain the standard stock solutions of the respective impurities.
[0046] Accurately weigh 25 mg of resmetirom and place it in a 100 mL volumetric flask. Then add 1 mL of imp-A, imp-B, imp-C and imp-D impurity standard stock solutions respectively. Add water-methanol (25 / 75, v / v) solution to dilute to 100 mL mark to obtain a mixed solution of resmetirom and its related substances.
[0047] The system suitability solution was prepared with the resmetirom oxidative degradation product and imp-B standard stock solution, and 15% hydrogen peroxide solution was added to the resmetirom solution at 55°C. After reacting for 5 hours, the oxidative degradation product was obtained, and then an appropriate amount of imp-B was added to obtain the system suitability solution.
[0048] Accurately weigh 25 mg of resmetirom or imp-A, imp-B, imp-C, imp-D and mix with water-methanol (25 / 75, v / v) solution to obtain test solution with a concentration of 0.25 mg / mL.
[0049] 1. System suitability test
[0050] The specificity of the method is crucial to the successful development of HPLC technology. Therefore, according to the above HPLC conditions, the mixed solution and the system suitability solution were analyzed to evaluate the system suitability of the method.
[0051] The chromatogram of the system suitability solution is as follows: Figure 4 As shown in E. The resolution between the main peak and the impurity peak is 3.75 and 4.88, both exceeding 1.5. The minimum resolution between the impurity peaks is 1.84, exceeding 1.2. Therefore, the resolution meets the requirements.
[0052] The chromatogram of a mixed solution containing four impurities is as follows Figure 4As shown in Figure F, the marked peaks correspond to imp-A, resmetirom, imp-B, imp-C and imp-D from left to right. The resolution of the main peak of resmetirom and its nearest impurity peak are 14.69 and 4.88, respectively, both exceeding 1.5; among all the impurity peaks, the minimum resolution is 5.81, exceeding 1.2. Therefore, the specificity of this method is good.
[0053] 2. Forced degradation test
[0054] Accurately weigh 25 mg of resmetirom and place it in a 100 mL volumetric flask, then dissolve it in a water-methanol (25 / 75, v / v) solution. The above samples were subjected to acid degradation, alkaline degradation, oxidative degradation, thermal degradation and photodegradation respectively. The specific conditions include acid degradation with 2 mol / L HCl at room temperature for 7 days; alkaline degradation with 0.1 mol / L NaOH at room temperature for 0.3 h; oxidative degradation with 15% hydrogen peroxide at 55°C for 4.3 h; thermal degradation at 100°C for 7 days; and photodegradation with LED lamps at a light intensity of 4500 lux for 30 days. After the respective treatments, the sample was diluted to 100 mL with a water-methanol (25 / 75, v / v) solution, and analyzed according to the above liquid chromatography conditions. The results are shown in Table 2. Figure 5 .
[0055] The stability of resmetirom under various forced degradation conditions was investigated based on the amount of impurities, the main peak content, the minimum separation between the main peak and the impurity peaks, and the minimum separation between the impurity peaks. The results are shown in Table 2.
[0056] Table 2 Results of forced degradation test
[0057]
[0058] Resmetirom is relatively stable under acid, heat and light conditions, but is easily degraded under alkaline and oxidative conditions. The minimum separation between the main peak and the impurity peak is greater than 1.5, and the separation between the impurity peaks is greater than 1.2, which meets the standard. The material balance range is 95.8% to 103.6%. Usually the material balance rate should be within the range of 95% to 105%, so the material of this method is basically balanced.
[0059] 3. Limit of detection and limit of quantification
[0060] The limit of detection (LOD) represents the minimum analytical level that can be qualitatively detected by the method, while the limit of quantification (LOQ) is the minimum concentration that can be quantified by the specified recovery rate and precision level. The standard stock solution and the reference standard of the relevant substance were gradually diluted, 10 μL of the sample was injected, and the ratio of the main peak height to the baseline noise (S / N) was calculated. After 6 parallel tests, the concentrations with S / N ratios of 3:1 and 10:1 were determined as the LOD and LOQ of this method, respectively. The results are shown in Table 3.
[0061] Table 3 Linearity, LOD and LOQ of resmetirom and its related substances
[0062]
[0063] 4. Linearity and range
[0064] This example investigates the linear relationship of resmetirom and its related substances in the concentration range from the quantitative limit concentration to the expected analytical level of 2.0% (about 0.25 mg / mL). The imp-A, imp-B, imp-C, and imp-D samples were dissolved in a water-methanol (25 / 75, v / v) solution in the concentration range of 0.025 to 5 μg / mL, and 10 μL of each of the above solutions was measured for injection analysis and the chromatogram was recorded. The linearity of the method was then evaluated based on the chromatogram.
[0065] The linear regression equations of resmetirom and related substances imp-A, imp-B, imp-C, and imp-D are given in Table 3, where x represents the mass concentration (μg / mL) of resmetirom and its impurities, and y represents the peak area. The results show that the correlation coefficients of the linear regression equations of resmetirom and the four related substances are all higher than 0.99, showing good linearity and meeting the test requirements.
[0066] 5. System precision test
[0067] In order to evaluate the precision, the mixed solution used for stability study was measured 6 times under the above-mentioned liquid chromatography conditions, and the retention time and peak area of resmetirom and imp-A, imp-B, imp-C, and imp-D were recorded. As shown in the results, the relative standard deviations (RSDs) of the retention time of resmetirom and its related substances imp-A, imp-B, imp-C, and imp-D were 0.07%, 0.08%, 0.08%, 0.10%, and 0.11%, respectively. The RSDs of the peak areas were 0.66%, 1.23%, 1.27%, 1.94%, and 1.71%, respectively. The RSDs were all below 2%, indicating that the precision of this method was good.
[0068] 6. Repeatability test
[0069] To evaluate repeatability, six replicate sample solutions were created and analyzed. 10 μL of the test solution was accurately measured and injected, and the chromatogram was recorded. The RSDs of resmetirom, imp-A, imp-B, imp-C, and imp-D were 1.05%, 1.33%, 1.41%, 1.30%, and 1.47%, all below 2%, meeting the test requirements, indicating that this method has good repeatability.
[0070] 7. Recovery rate
[0071] The recoveries of the four related substances were evaluated at three concentration levels: 50%, 100%, and 150%. Related substance solutions were added to the sample solutions at different levels, and 3 replicates were prepared for each concentration. The samples were then injected and the recoveries were determined.
[0072] Table 4 shows the recovery results of the four related substances. The recovery results are between 95% and 110%, which meets the requirements. The results show that this method has good accuracy.
[0073] Table 4 Recovery test results
[0074]
[0075]
[0076] 8. Durability test
[0077] The effects of the initial organic phase ratio of the mobile phase, detection wavelength, column temperature, flow rate, and column type on the chromatographic method were investigated. The durability test conditions are shown in Table 5. The system suitability sample solutions were taken and analyzed using the conditions in Table 5.
[0078] Table 5 Durability test conditions
[0079] Liquid chromatography conditions Parameter variation range Initial ratio of AB mobile phase (%) 92:8；90:10；88:12 Detection wavelength (nm) 215；220；225 Column temperature (℃) 30；35；40 Flow rate (mL / min) 0.9；1.0；1.1 Chromatographic column model <![CDATA[Gemini NX C 18 ;Agilent 5HC C 18 (2);XBridge C 18 ]]>
[0080] The results showed that under acceptable change conditions, the minimum separation between resmetirom and its adjacent impurity peaks was 2.44, greater than 1.5, confirming adequate separation. For other impurity peaks, except for the significant effect of changing the chromatographic column, the minimum separation between impurity peaks under other conditions was 1.26, greater than 1.2, and there was no significant change in the number and content of impurities, indicating that the separation effect was good.
[0081] Therefore, changes in chromatographic parameters such as the initial ratio of the organic phase in the mobile phase, detection wavelength, column temperature, flow rate, column type, etc. have little effect on the detection of related substances. Slight adjustments to the above detection parameters will basically not have a significant effect on the determination results, indicating that the method has good durability.
Claims
1. A method for detecting related substances of the drug resmetirom, characterized in that: Using high performance liquid chromatography, the analyte was 18 Gradient elution was performed in a reverse phase chromatographic column, and the eluent was detected by a UV detector; The analyte contains resmetirom and / or resmetirom-related substances; The mobile phases used in the gradient elution are mobile phase A and mobile phase B, mobile phase A is a mixed solution of trifluoroacetic acid aqueous solution and acetonitrile, and mobile phase B is acetonitrile; The gradient elution procedure is: 0-2 min, 10% B→10% B; 2-20 min, 10% B→50% B; 20-30 min, 50% B→54% B; 30-35 min, 54% B→54% B; 35-36 min, 54% B→10% B; 36-45 min, 10% B→10% B.
2. The detection method according to claim 1, characterized in that The C 18 The chromatographic column was Agilent 5 HC C 18 (2) Column.
3. The detection method according to claim 2, characterized in that The flow rate of the gradient elution was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was 35 °C.
4. The detection method according to claim 1, characterized in that The detection wavelength of the ultraviolet detector is 220 nm.
5. The detection method according to claim 1, characterized in that: The volume ratio of trifluoroacetic acid aqueous solution to acetonitrile in the mobile phase A is 90:10, and the trifluoroacetic acid aqueous solution is an aqueous solution containing 0.045% trifluoroacetic acid.
6. The detection method according to claim 1, characterized in that: The solvent of the analyte is 75% methanol aqueous solution.