A method for determining the content of ambrisentan by volumetric method

Abstract

The invention relates to a method for determining the content of ambrisentan by volumetric method. 0.2 g of ambrisentan is accurately weighed, 50 mL of a mixed solvent is added to dissolve the ambrisentan product, and the solution is used as a test solution. The solution is titrated to a second-order differential endpoint with a 0.1 mol / L tetrabutylammonium hydroxide titration solution. In view of the poor solubility and weak acidity of ambrisentan, a non-aqueous titration solution, tetrabutylammonium hydroxide titration solution, is used to facilitate the dissociation of acid radicals, and the content of ambrisentan can be quantitatively detected, so that the content of the raw material can be effectively controlled by the invention, which is of indispensable significance for the quality control of ambrisentan.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for determining the content of ambrisentan by volumetric method. Background Art

[0002] Ambrisentan is a receptor antagonist that is highly bound to ETA (Ki=0.011nM) and has high selectivity for ETA compared to ETB (>4000 times). The clinical impact of its high selectivity for ETA is still unknown.

[0003] Endothelin-1 (ET-1) is a potent autocrine and paracrine peptide. Two receptor subtypes (ETA and ETB) co-regulate the effects of ET-1 in vascular smooth muscle and endothelial cells. The main effects of ETA are vasoconstriction and cell proliferation, while the main effects of ETB are vasodilation, inhibition of proliferation, and scavenging of ET-1.

[0004] In patients with pulmonary hypertension, plasma ET-1 concentrations increased 10-fold and correlated with increases in mean right atrial pressure and disease severity. ET-1 and ET-1 mRNA concentrations increased 9-fold in lung tissue of patients with pulmonary hypertension, mainly concentrated in pulmonary artery endothelial cells. These findings suggest that ET-1 may play an important role in the onset and progression of pulmonary hypertension.

[0005] Ambrisentan was developed by Myogen and later by Gilead. GlaxoSmithKline obtained the market license for this product outside the United States under the trade name "LETAIRIS®". It was approved by the FDA on June 15, 2007. The approved dosage form is oral tablets with 5mg and 10mg tablets. It is clinically used to treat PAH of WHO cardiac function grade I-III to improve activity and delay clinical deterioration.

[0006] According to literature search, the content of ambrisentan raw materials is usually determined by high performance liquid chromatography. Although this method has high specificity, it requires professional equipment and standardized reference substances, which is costly and the work of standardizing reference substances for generic companies is complicated and expensive.

[0007] Therefore, the present invention proposes a method for determining the content of ambrisentan by volumetric method to solve the above problems. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for determining the content of ambrisentan by volumetric method, which can quantitatively detect the content of ambrisentan, thereby effectively controlling the content of ambrisentan raw materials.

[0009] In order to solve the above technical problems, the technical solution of the present invention is: a method for determining the content of ambrisentan by volumetric method, the innovation of which is: the method comprises: taking 0.2 g of ambrisentan, accurately weighing it, adding 50 mL of a mixed solvent to dissolve the ambrisentan product, using the mixed solvent as the test solution, and titrating it with 0.1 mol / L tetrabutylammonium hydroxide titration solution to the second-order differential endpoint.

[0010] Furthermore, the mixed solvent comprises a mixed solution of N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide, and the purity of N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide are all analytically pure.

[0011] Furthermore, the volume ratio of N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide is 50:20:30.

[0012] Furthermore, the specific operating steps of the volumetric method for determining the content of ambrisentan are: S1. Take 0.2 g of ambrisentan, weigh accurately, add 50 mL of mixed solvent to dissolve the ambrisentan product, and use the solution as the test solution. Place the test solution on a potentiometric titrator; S2. Titrate with 0.1 mol / L tetrabutylammonium hydroxide titrant, and record the volume V consumed by the titrant and the potential value E; S3. According to the potential value E obtained from the titration experiment and the corresponding consumption volume V, the first-order differential ΔE / ΔV and the second-order differential Δ 2 E / ΔV 2 Values, list the measured values ​​(E, V) and calculated values, list the calculated values ​​ΔE / ΔV or Δ 2 E / ΔV 2 As the ordinate, the corresponding titration liquid consumption volume V is plotted as the abscissa, and the second-order derivative Δ 2 E / ΔV 2 The volume of titrant consumed when it equals zero is the titration endpoint; S4. Calculate the ambrisentan content according to the volume of titrant consumed at the titration endpoint and the titer according to Formula 1: Formula 1: Among them, V 0 is the volume of titrant consumed at the titration endpoint, 37.84 is the titration degree, W 供试 is the sample weight of the test product.

[0013] Furthermore, the volume of the titrant consumed at the titration endpoint is calculated by linear interpolation of the coordinates of the two points before and after the curve crosses zero in the figure, and the volume of the titrant consumed at the titration endpoint is calculated according to Formula 2: Formula 2: Wherein, a is the absolute value of the second derivative before the curve passes through zero, b is the absolute value of the second derivative after the curve passes through zero, V is the volume of the titrant consumed corresponding to point a, and ΔV is the volume of the titrant added from point a to point b.

[0014] The advantages of the present invention are: (1) In view of the poor solubility and weak acidity of ambrisentan, the present invention adopts a non-aqueous titrant, tetrabutylammonium hydroxide titrant, to facilitate the dissociation of the acid radical, so as to quantitatively detect the content of ambrisentan. Thus, the present invention can effectively control the content of the raw materials, which is of indispensable significance for the quality control of ambrisentan.

[0015] (2) The present invention starts from the compound structure and, according to the generalized proton acid theory, the acidity of organic weak acids will be enhanced under alkaline solvent conditions, so that groups that were originally unable to be titrated can be titrated with alkaline titrants. Alkaline non-aqueous solvents N,N-dimethylformamide, N,N-dimethylacetamide, and N,N-diethylformamide were selected as solvents. Through a large number of experiments, the mixing ratio of each solvent was optimized to form a volumetric method system, and the optimized method was methodologically verified. The verification results showed that all indicators of this method met the requirements of the current "Chinese Pharmacopoeia". BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 This is the titration curve of Experiment 4 of the present invention.

[0018] Figure 2 This is the titration curve of Experiment 6 of the present invention. DETAILED DESCRIPTION

[0019] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0020] The structure of the compound ambrisentan is as follows: Ambrisentan has a molecular weight of 378.4 and contains a phenylpropionic acid group in its structure. According to the generalized proton acid theory, it is possible to store it in acid-base titration. However, due to the poor solubility and weak acidity of ambrisentan, in order to facilitate the dissociation of the acid radical, the present invention selects a non-aqueous titration system and uses a non-aqueous titrant—tetrabutylammonium hydroxide titrant.

[0021] The import standard uses high performance liquid chromatography to determine the content of ambrisentan. Although this method has high specificity, it requires professional equipment and standardized reference substances, which is costly and the work of standardizing reference substances for counterfeit enterprises is relatively complicated and expensive. Therefore, the present invention explores the method of determining the content of ambrisentan by volumetric method based on the compound structure through a large number of experiments, especially screening the solvent and the solvent mixing ratio, optimizing the volumetric method system, and conducting methodological verification on the optimized method.

[0022] (1)Solvent selection According to the generalized proton acid theory, the acidity of organic weak acids will be enhanced under alkaline solvent conditions, so that groups that were originally unable to be titrated can be titrated by alkaline titrants. Based on this theory, the present invention screens alkaline non-aqueous solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, and N,N-diethylformamide; The experimental steps are as follows: S1. Take 0.2 g of ambrisentan, weigh accurately, add 50 mL of solvent to dissolve the ambrisentan product, and use it as the test solution. Place the test solution on a potentiometric titrator; S2. Titrate with 0.1 mol / L tetrabutylammonium hydroxide titrant, and record the volume V consumed by the titrant and the potential value E; S3. According to the potential value E obtained from the titration experiment and the corresponding consumption volume V, the first-order differential ΔE / ΔV and the second-order differential Δ 2 E / ΔV 2 Values, list the measured values ​​(E, V) and calculated values, list the calculated values ​​ΔE / ΔV or Δ 2 E / ΔV 2 As the ordinate, the corresponding titration liquid consumption volume V is plotted as the abscissa, and the second-order derivative Δ 2 E / ΔV 2 The volume of titrant consumed when the value is equal to zero is the titration endpoint. The volume of titrant consumed at the titration endpoint can be calculated by linear interpolation of the coordinates of the two points before and after the curve passes through zero in the figure. The volume of titrant consumed at the titration endpoint is calculated according to formula 2: Formula 2: Wherein, a is the absolute value of the second derivative before the curve passes through zero, b is the absolute value of the second derivative after the curve passes through zero, V is the volume of the titrant consumed corresponding to point a, and ΔV is the volume of the titrant added from point a to point b; S4. Calculate the ambrisentan content according to the volume of titrant consumed at the titration endpoint and the titer according to Formula 1: Formula 1: Among them, V 0 is the volume of titrant consumed at the titration endpoint, 37.84 is the titration degree, W 供试is the sample weight of the test product.

[0023] ① Experiment 1 In this experiment, the solvent used was N,N-dimethylformamide, which was analytically pure. The titration results were as follows: no clear titration endpoint.

[0024] ② Experiment 2 In this experiment, the solvent used was N,N-dimethylacetamide, which was analytically pure. The above steps were followed and the titration results were as follows: after adding the titrant, the solution became turbid, the sample precipitated, and there was a potential jump, but according to calculations, the titration endpoint had not been reached.

[0025] ③ Experiment 3 In this experiment, the solvent used was N,N-diethylformamide, which was analytically pure. The above steps were followed and the titration results were as follows: after adding the titrant, the solution became turbid, the sample precipitated, and there was a potential jump, but according to calculations, the titration endpoint had not been reached.

[0026] According to the titration results, in Experiment 1, the sample could not reach the titration endpoint in N,N-dimethylformamide, and the analysis was related to the dissociation strength; in Experiments 2 and 3, although there were potential jumps in N,N-dimethylacetamide and N,N-diethylformamide, the subsequent titration reaction was affected due to the change in sample solubility.

[0027] Therefore, it is considered to use a mixed solvent, adding N,N-dimethylacetamide and N,N-diethylformamide to N,N-dimethylformamide to enhance the dissociation ability of the solvent.

[0028] ④ Experiment 4 In this experiment, the solvent used was a binary mixture of N,N-dimethylformamide and N,N-dimethylacetamide, where N,N-dimethylformamide: N,N-dimethylacetamide = 50:50, and the purity of both was analytically pure. According to the above steps, the titration results were: the titration curve was improved, but the titration endpoint was not obvious. The titration curve was as follows Figure 1 shown.

[0029] ⑤ Experiment 5 In this experiment, the solvent used was a binary mixture of N,N-dimethylformamide and N,N-diethylformamide, wherein N,N-dimethylformamide: N,N-diethylformamide = 50:50, and the purity of both was analytical grade. The above steps were followed and the titration results were as follows: after adding the titrant, the solution became turbid, the sample precipitated, and there was a potential jump, but according to calculation, the titration endpoint had not been reached.

[0030] ⑥ Experiment 6 In this experiment, the solvent used was a ternary mixture of N,N-diethylformamide, N,N-dimethylacetamide and N,N-diethylformamide, wherein N,N-dimethylformamide: N,N-dimethylacetamide: N,N-diethylformamide = 50:20:30, and the purity of each was analytical grade. According to the above steps, the titration results were: there was an obvious potential jump, no precipitation, and the titration curve was as follows Figure 2 shown.

[0031] According to the titration results, in the binary mixed liquid system, in Experiment 4, the mixture of N,N-dimethylformamide and N,N-dimethylacetamide can improve the titration curve, but the titration endpoint is not obvious and is not suitable for titration; in Experiment 5, the mixture of N,N-dimethylformamide and N,N-diethylformamide is similar to the case of using N,N-diethylformamide alone in Experiment 3, which is not suitable for titration; in the ternary mixed liquid system, in Experiment 6, the combined action of N,N-dimethylacetamide and N,N-diethylformamide improves the dissociation degree of the sample, but because the proportion of N,N-diethylformamide decreases, no sample precipitation occurs, the titration can be completed, and the titration endpoint is consistent with the theoretical calculated value.

[0032] (2) Methodological validation According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, the method proposed in this invention was methodologically validated. The results of the methodological validation are as follows: ① Titration curve Ambrisentan was titrated with 0.1 mol / L tetrabutylammonium hydroxide titrant. There was an obvious potential jump at the titration end point, and the selected indicator had an obvious color change. The content measured by titration was consistent with that by HPLC (high performance liquid chromatography), indicating that the selected titration method was suitable for the determination of ambrisentan.

[0033] ② Solution stability The test solution was placed at room temperature for 2 hours, and there was no significant difference in the test results, indicating that the test solution was stable at room temperature for 2 hours and met the test requirements.

[0034] ③Linear The sampling amount of ambrisentan was in the range of 0.16~0.22g, and there was a good linear relationship between the sampling amount and the titration volume, the linear equation was y=26.2298x-0.0169, and the correlation coefficient was r=1.0000; The slope of the linear equation obtained experimentally is 26.2298, and the relative deviation from the theoretical value of the titration constant (26.2956) is 0.25%, which is less than 0.3%; The intercept of the linear equation obtained experimentally was 0.0169, which was 0.32% of the target titration volume (5.26 ml) and less than 0.4%; The slope and intercept evaluation results show that the linear experimental results meet the requirements and the method has good linearity.

[0035] ④Repeatability The same batch of ambrisentan samples were measured in parallel 6 times, and the RSD of the 6 results was 0.06%, which was less than 1.0%, which was in line with the regulations. This method has good repeatability.

[0036] ⑤Intermediate precision The same batch of ambrisentan samples were tested by different personnel at different times. The average content of the 12 test results was 99.27%, and the RSD was 0.07%, which was less than 1.5%, meeting the requirements. The intermediate precision of this method was good.

[0037] The above verification results show that all indicators of this method meet the requirements of the 2020 edition of the "Chinese Pharmacopoeia" and are suitable for testing.

[0038] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for determining the content of ambrisentan by volumetric method, characterized in that: The method is as follows: 0.2 g of ambrisentan is accurately weighed, 50 mL of a mixed solvent is added to dissolve the ambrisentan product, the solution is used as a test solution, and 0.1 mol / L tetrabutylammonium hydroxide titration solution is used to titrate to a second-order differential endpoint.

2. The method for determining the content of ambrisentan by volumetric method according to claim 1, characterized in that: The mixed solvent comprises a mixed solution of N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide, and the purity of N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide are all analytically pure.

3. The method for determining the content of ambrisentan by volumetric method according to claim 2, characterized in that: The volume ratio of the N,N-dimethylformamide, N,N-dimethylacetamide and N,N-diethylformamide is 50:20:

30.

4. The method for determining the content of ambrisentan by volumetric method according to claim 1, characterized in that: The specific operation steps of the volumetric method for determining the content of ambrisentan are as follows: S1. Take 0.2 g of ambrisentan, weigh accurately, add 50 mL of mixed solvent to dissolve the ambrisentan product, and use the solution as the test solution. Place the test solution on a potentiometric titrator; S2. Titrate with 0.1 mol / L tetrabutylammonium hydroxide titrant, and record the volume V consumed by the titrant and the potential value E; S3. According to the potential value E obtained from the titration experiment and the corresponding consumption volume V, the first-order differential ΔE / ΔV and the second-order differential Δ 2 E / ΔV 2 Values, list the measured values ​​(E, V) and calculated values, list the calculated values ​​ΔE / ΔV or Δ 2 E / ΔV 2 As the ordinate, the corresponding titration liquid consumption volume V is plotted as the abscissa, and the second-order derivative Δ 2 E / ΔV 2 The volume of titrant consumed when it equals zero is the titration endpoint; S4. Calculate the ambrisentan content according to the volume of titrant consumed at the titration endpoint and the titer according to Formula 1: Formula 1: Where V0 is the volume of titrant consumed at the titration endpoint, 37.84 is the titration degree, and W 供试 is the sample weight of the test product.

5. The method for determining the content of ambrisentan by volumetric method according to claim 4, characterized in that: The volume of the titrant consumed at the titration endpoint is calculated by linear interpolation of the coordinates of the two points before and after the curve crosses zero in the figure, and the volume of the titrant consumed at the titration endpoint is calculated according to Formula 2: Formula 2: Wherein, a is the absolute value of the second derivative before the curve passes through zero, b is the absolute value of the second derivative after the curve passes through zero, V is the volume of the titrant consumed corresponding to point a, and ΔV is the volume of the titrant added from point a to point b.

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