Method for simultaneously determining aniline and phenylazo malononitrile impurities in riociguat

Abstract

The invention relates to the technical field of drug detection, in particular to a method for simultaneously determining aniline and phenylazo malononitrile impurities in riociguat. The impurities are aniline and phenylazo malononitrile; the detection method is carried out by using a high performance liquid chromatograph, a mobile phase is composed of a mobile phase A and a mobile phase B, and gradient elution is adopted. According to the structural characteristics of products and the properties of compounds, the invention provides a detection method for simultaneously determining aniline and phenylazo malononitrile impurities in riociguat by adopting a high performance liquid chromatograph. Gradient elution chromatography conditions are adopted, aniline and phenylazo malononitrile impurities can be further measured and separated at the same time, aniline and phenylazo malononitrile can be effectively separated from riociguat, the detection sensitivity and accuracy of all components are further improved by optimizing the conditions, the limit requirements of all the impurities are met, and the method is suitable for industrial production. And the detection cost and the operation difficulty are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and more specifically, to a method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat. Background Art

[0002] Riociguat, the first new class of soluble guanylate cyclase agonists developed and marketed by Bayer, is the world's first drug approved for the treatment of chronic thromboembolic pulmonary hypertension (CTEPH) and the only drug currently with dual indications for pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH).

[0003] Aniline and phenylazomalononitrile are materials used in the synthesis process of Riociguat. Aniline is a genotoxic carcinogen, and some animal in vitro and in vivo genotoxicity tests have positive results. It has been listed in the appendix of ICH M7. Therefore, this impurity needs to be strictly controlled, and its limit should not exceed 0.1%. Phenylazomalononitrile has a warning structure of an azo group. According to the requirements of ICHM7, this impurity needs to be strictly controlled. According to the TTC method, the limit of this impurity is 0.02%.

[0004] Therefore, it is necessary to develop a fast and efficient liquid chromatography method to improve detection sensitivity, shorten detection time and reduce costs, thereby facilitating the popularization and use of the method and drugs. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of one aspect of the present invention is to provide a method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat.

[0006] The mutagenic impurities of the present invention are aniline and phenylazomalononitrile; the detection method is carried out using a high performance liquid chromatograph, the mobile phase consists of mobile phase A and mobile phase B, and gradient elution is used. The chemical structural formula of the impurity is as follows:

[0007]

[0008] The present invention finds that when high performance liquid chromatography is used to detect aniline and phenylazomalononitrile, the selection of mobile phase has a great influence on the detection of impurities. After a large number of attempts, it is found that when the above elution method is used, the detection sensitivity of aniline and phenylazomalononitrile can be significantly improved.

[0009] The gradient elution is adopted, and the total volume of the mobile phase is 100%.

[0010] In the 0th to 20th minute, the volume of the mobile phase A decreases from 64% to 66% to 49% to 51%, and the volume of the mobile phase B increases from 36% to 34% to 51% to 49%;

[0011] At 20-30 minutes, the volume of the mobile phase A decreases from 49%-51% to 29%-31%, and the volume of the mobile phase B increases from 51%-49% to 71%-69%;

[0012] At 30-40 minutes, the volume of the mobile phase A is 29%-31%, and the volume of the mobile phase B is 71%-69%;

[0013] At 40.1 to 50 minutes, the volume of the mobile phase A is 64% to 66%, and the volume of the mobile phase B is 36% to 34%.

[0014] Preferably, the gradient elution is adopted, and the total volume of the mobile phase is 100%,

[0015] From 0 to 20 minutes, the volume of the mobile phase A decreases from 65% to 50%, and the volume of the mobile phase B increases from 35% to 50%;

[0016] At 20-30 minutes, the volume of the mobile phase A is gradually reduced from 50% to 30%, and the volume of the mobile phase B is gradually increased from 50% to 70%;

[0017] At 30-40 minutes, the volume of the mobile phase A is 30%, and the volume of the mobile phase B is 70%;

[0018] At 40.1-50 min, the volume of the mobile phase A was 65%, and the volume of the mobile phase B was 35%.

[0019] Preferably, the specific operation steps of the detection method are:

[0020] S1. Test solution: Take Riociguat, weigh accurately, dissolve in solvent and quantitatively dilute to make 1 mg / ml test solution;

[0021] S2. Reference solution: accurately weigh the reference substances of aniline and phenylazomalononitrile, and quantitatively dilute them with a solvent to prepare a reference solution of 1 μg / ml aniline and 0.2 μg / ml phenylazomalononitrile;

[0022] S3 impurity localization solution: take aniline reference substance, accurately weigh it, add solvent to quantitatively dilute it to make 10μg / ml aniline localization solution; take phenylazomalononitrile reference substance, accurately weigh it, add solvent to quantitatively dilute it to make 2μg / ml phenylazomalononitrile localization solution;

[0023] S4. System suitability solution: Take riociguat, aniline and phenylazomalononitrile reference substances, accurately weigh them, and quantitatively dilute them with a solvent to prepare a mixed solution of 1 mg / ml riociguat, 1 μg / ml aniline and 0.2 μg / ml phenylazomalononitrile reference substance as the system suitability solution;

[0024] S5. Adjust the instrument: the chromatographic column uses octadecylsilane bonded silica gel as filler, mobile phase A, mobile phase B, adjust the column temperature, adjust the mobile phase flow rate, adjust the detection wavelength, and use gradient elution;

[0025] S6. Determination: Accurately measure the solutions prepared in S1 to S4, inject them into liquid chromatograph respectively, and record the chromatograms.

[0026] Preferably, the concentration of the test solution in S1 is 0.9-1.1 mg / ml.

[0027] Preferably, in the reference substance solution of S2: the concentration of the aniline reference substance is 0.9-1.1 μg / ml, and the concentration of the phenylazomalononitrile reference substance is 0.18-0.22 μg / ml.

[0028] Preferably, in the impurity localization solution of S3: the concentration of the aniline localization solution is 9.0-11.0 μg / ml; the concentration of the phenylazomalononitrile localization solution is 1.8-2.2 μg / ml.

[0029] Preferably, in the system suitability solution of S4: the concentration of the riociguat reference solution is 0.9 to 1.1 mg / ml; the concentration of the aniline reference solution is 0.9 to 1.1 μg / ml; and the concentration of the phenylazomalononitrile reference solution is 0.18 to 0.22 μg / ml.

[0030] Preferably, the solvent is mobile phase A-mobile phase B, and the mass ratio of mobile phase A-mobile phase B is (64% to 66%): (36% to 34%).

[0031] Preferably, the chromatographic column in S5 uses octadecylsilane bonded silica gel as a filler, and the specifications are Shim-packGIST C18, 4.6mm×250mm, 5μm.

[0032] Preferably, the mobile phase A is 0.01 mol / L potassium dihydrogen phosphate solution, the pH value of which is adjusted to 2.5 with phosphoric acid, and the mobile phase B is methanol-acetonitrile, and the mass ratio of methanol-acetonitrile is (0.99-1.01):(9.01-8.99).

[0033] Preferably, the column temperature is adjusted to 28-32° C., the flow rate of the mobile phase is adjusted to 0.9-1.1 ml / min, and the detection wavelength is adjusted to 235 nm.

[0034] The beneficial effects of the present invention are as follows:

[0035] According to the product structure characteristics and compound properties, the present invention provides a detection method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat using a high performance liquid chromatograph. By using gradient elution chromatography conditions, aniline and phenylazomalononitrile impurities can be further simultaneously determined and separated, and each impurity can be effectively separated from riociguat. By optimizing the conditions, the sensitivity and accuracy of the detection of each component are further improved, and the limit requirements of each impurity are met, and the detection cost and operation difficulty are greatly reduced.

[0036] The method of the present invention uses simple equipment, fast analysis speed, good specificity, low detection cost, high accuracy, and can quickly, simply and accurately control the amount of impurities and reduce drug safety risks. The method of the present invention has strong specificity and high reproducibility, is convenient for quality detection and impurity monitoring of riociguat, is conducive to the popularization of riociguat impurity detection methods, and is conducive to the safe promotion and application of riociguat.

[0037] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0039] Figure 1 This is the blank solution chromatogram in Example 1 of the present invention.

[0040] Figure 2 This is a solution chromatogram of the system suitability in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0043] This embodiment provides a method for detecting aniline and phenylazomalononitrile impurities in riociguat using a high performance liquid chromatograph, and the determination is performed using the following conditions:

[0044] Chromatographic column: octadecylsilane bonded silica gel is used as filler, and the specifications of the chromatographic column are Shim-pack GIST C18, 4.6mm×250mm, 5μm; mobile phase A: 0.01mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.5 with phosphoric acid; mobile phase B: methanol-acetonitrile (0.99~1.01): (9.01~8.99); flow rate: 0.9~1.1ml / min; column temperature: 28~32℃; detection wavelength 235nm; gradient elution is adopted, and the total volume of the mobile phase is 100%.

[0045] In the 0th to 20th minute, the volume of the mobile phase A decreases from 64% to 66% to 49% to 51%, and the volume of the mobile phase B increases from 36% to 34% to 51% to 49%;

[0046] At 20-30 minutes, the volume of the mobile phase A decreases from 49%-51% to 29%-31%, and the volume of the mobile phase B increases from 51%-49% to 71%-69%;

[0047] At 30-40 minutes, the volume of the mobile phase A is 29%-31%, and the volume of the mobile phase B is 71%-69%;

[0048] At 40.1 to 50 minutes, the volume of the mobile phase A is 64% to 66%, and the volume of the mobile phase B is 36% to 34%.

[0049] Preferably, from 0 to 20 minutes, the volume of the mobile phase A decreases from 65% to 50%, and the volume of the mobile phase B increases from 35% to 50%;

[0050] At 20-30 minutes, the volume of the mobile phase A is gradually reduced from 50% to 30%, and the volume of the mobile phase B is gradually increased from 50% to 70%;

[0051] At 30-40 minutes, the volume of the mobile phase A is 30%, and the volume of the mobile phase B is 70%;

[0052] At 40.1-50 min, the volume of the mobile phase A was 65%, and the volume of the mobile phase B was 35%.

[0053] Test solution: Take Riociguat, weigh accurately, dissolve in solvent and quantitatively dilute to make 1 mg / ml test solution.

[0054] Reference solution: Take appropriate amount of aniline and phenylazomalononitrile reference substances, weigh accurately, and quantitatively dilute with solvent to make a mixed solution containing about 1 μg of aniline and about 0.2 μg of phenylazomalononitrile per 1 ml.

[0055] Impurity localization solution: take aniline reference substance, accurately weigh it, add solvent to quantitatively dilute it to make aniline localization solution with a concentration of 10 μg / ml; take phenylazomalononitrile reference substance, accurately weigh it, add solvent to quantitatively dilute it to make aniline localization solution with a concentration of 2 μg / ml.

[0056] System suitability solution: Take riociguat, aniline and phenylazomalononitrile reference substances, accurately weigh them, and quantitatively dilute them with solvent to make a mixed solution containing approximately 1 mg riociguat, 1 μg aniline, and 0.2 μg phenylazomalononitrile per 1 ml.

[0057] The specific preparation process of each solution and the experimental verification process and results of the method of the present invention are shown in Experimental Examples 1 to 5.

[0058] Embodiment 1

[0059] Specificity test

[0060] Solvent: Mobile phase A-Mobile phase B (65:35)

[0061] Impurity localization solution: take aniline reference substance, accurately weigh it, add solvent to quantitatively dilute it to make a 10μg / ml solution, as aniline localization solution; take phenylazomalononitrile reference substance, accurately weigh it, add solvent to quantitatively dilute it to make a 2μg / ml solution, as phenylazomalononitrile localization solution.

[0062] System suitability solution: Take riociguat, aniline and phenylazomalononitrile reference substances, accurately weigh them, and quantitatively dilute them with a solvent to prepare a mixed solution containing riociguat 1 mg / ml, aniline 2 μg / ml and phenylazomalononitrile 0.2 μg / ml as the system suitability solution;

[0063] Determination: The chromatographic column uses octadecylsilane bonded silica gel as filler, the mobile phase A is 0.01 mol / L potassium dihydrogen phosphate solution (take 1.361 g of potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH value to 2.5 with 1 phosphoric acid), the mobile phase B is methanol-acetonitrile (1:9), the column temperature is 30°C, the flow rate is 1 ml per minute, the detection wavelength is 235 nm, and gradient elution is used.

[0064] Accurately measure 50 μl of each of the above solutions, inject into the HPLC, and record the chromatogram. The results are shown in Table 1. The chromatograms of the blank solvent and the system suitability solution are shown in Figure 1 and Figure 2 shown.

[0065] Table 1. Specificity test results

[0066]

[0067]

[0068] Conclusion: The solvent does not interfere with the determination of known impurities in the test solution. The impurities and the impurities and the main peak are well separated, meeting the requirements for the determination of related substances.

[0069] Embodiment 2

[0070] Linearity and range tests

[0071] Preparation of linear solutions: Take an appropriate amount of aniline reference substance, accurately weigh it, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 1 mg per 1 ml as aniline stock solution; take an appropriate amount of phenylazomalononitrile reference substance, accurately weigh it, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 0.2 mg per 1 ml as phenylazomalononitrile stock solution. Accurately measure an appropriate amount of the above stock solutions and dilute them with solvent (mobile phase A-mobile phase B (65:35)) to make a series of linear solutions.

[0072] Accurately measure 50 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram; the results are shown in Table 2.

[0073] Table 2. Linearity and range test results

[0074]

[0075] Conclusion: (1) Aniline has a good linear relationship in the concentration range of 0.100-2.002 μg / ml (equivalent to 0.010%-0.2% of the detection concentration). The linear regression equation is y=41326.4626x-24.6530, r=0.9999, and the y-axis intercept is equivalent to 0.06% of the 100% concentration response value.

[0076] (2) Phenyl azomalononitrile has a good linear relationship in the concentration range of 0.048 to 0.386 μg / ml (equivalent to 0.005% to 0.04% of the detection concentration), and the linear regression equation is y=106685.0068x+32.4806, r=1.0000, and the percentage of the y-axis intercept equivalent to 100% concentration response value is 0.16%.

[0077] Embodiment 3

[0078] Limit of Quantitation and Limit of Detection Tests

[0079] Quantitation limit solution: Take appropriate amount of aniline and phenylazomalononitrile reference substances, dissolve them in acetonitrile and dilute with solvent to make a solution containing approximately 0.1 μg of aniline and 0.048 μg of phenylazomalononitrile per 1 ml. Shake well and use this as the quantitation limit solution.

[0080] Detection limit solution: Take appropriate amount of aniline and phenylazomalononitrile reference substances, dissolve them in acetonitrile and dilute with solvent to make a solution containing about 0.03 μg of aniline and 0.014 μg of phenylazomalononitrile per 1 ml, shake well and use this as the detection limit solution.

[0081] Accurately measure 50 μl of each of the above solutions and inject them into the liquid chromatograph. The results are shown in Tables 3-4.

[0082] Table 3. Limit of quantitation and limit of detection results

[0083] sample Limit of quantitation (μg / ml) Equivalent to detection concentration% Signal-to-Noise Ratio aniline 0.100 0.010 25.7 Phenyl azomalononitrile 0.048 0.005 12.5 sample Detection limit (μg / ml) Equivalent to detection concentration% Signal-to-Noise Ratio aniline 0.030 0.003 8.0 Phenyl azomalononitrile 0.014 0.001 3.6

[0084] Table 4. Quantitation limit repeatability results

[0085]

[0086]

[0087] Conclusion: The signal-to-noise ratio of the quantitative limit solution was greater than 10. After 6 repeated injections, the retention time RSD was less than 1.0%, and the peak area RSD was less than 10%. The signal-to-noise ratio of the detection limit solution was greater than 3, and the method had good sensitivity.

[0088] Embodiment 4

[0089] Recovery test

[0090] Reference solution: Take appropriate amount of aniline and phenylazomalononitrile reference substances, dissolve in acetonitrile and dilute with solvent to make a solution containing about 10μg aniline and 2μg phenylazomalononitrile per 1ml, shake well, and use it as reference stock solution. Accurately measure 2ml of reference stock solution, place in a 20ml volumetric flask, dilute to scale with solvent, and shake well. Prepare 2 copies in parallel.

[0091] Preparation of accuracy solution:

[0092] 50% accuracy solution: Take about 20 mg of this product, weigh it accurately, put it in a 20 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately measure 1 ml of the reference stock solution, put it in the same volumetric flask, add solvent to dilute to the scale, shake well, and obtain; prepare 3 parallel portions.

[0093] 100% accuracy solution: Take about 20 mg of this product, weigh it accurately, put it in a 20 ml volumetric bottle, add an appropriate amount of solvent to dissolve it, accurately measure 2 ml of the reference stock solution, put it in the same volumetric bottle, add solvent to dilute to the scale, shake well, and obtain; prepare 3 parallel portions.

[0094] 150% accuracy solution: Take about 20 mg of this product, weigh it accurately, put it in a 20 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately measure 3 ml of the reference stock solution, put it in the same volumetric flask, add solvent to dilute to the scale, shake well, and obtain; prepare 3 parallel portions.

[0095] Preparation of background solution:

[0096] Take about 20 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0097] Accurately measure 50 μl of each of the above solutions and inject them into the liquid chromatograph. The results are shown in Tables 3-4.

[0098] Table 5. Related substances method validation - aniline recovery results

[0099]

[0100] Table 6. Related substances method validation - phenylazomalononitrile recovery results

[0101]

[0102] Conclusion: The recovery test results show that the recoveries of 9 accuracy samples of aniline and phenylazomalononitrile are between 80.0% and 120.0%, and the average recoveries are 98.5% and 102.8%, respectively. It can be seen from Tables 5 and 6 that the recovery rates of various impurities meet the determination requirements of this product for each known impurity, indicating that this method has good accuracy.

[0103] Embodiment 5

[0104] Durability test

[0105] The system suitability solution in Experimental Example 1 was taken and tested at column temperatures of 28°C and 32°C, flow rates of 0.9 ml / min and 1.1 ml / min, respectively, with the chromatographic column replaced. The results are shown in Table 7.

[0106] Table 7. Durability test results (separation)

[0107]

[0108] Conclusion: Under the fine-tuning chromatographic conditions, the separation between impurities and between impurities and main peaks in the system suitability solution meets the requirements, indicating that this method has good durability.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat, characterized in that: The impurities are aniline and phenylazomalononitrile; the detection method is carried out using a high performance liquid chromatograph, the mobile phase consists of mobile phase A and mobile phase B, and gradient elution is used; The gradient elution is adopted, and the total volume of the mobile phase is 100%. In the 0th to 20th minute, the volume of the mobile phase A decreases from 64% to 66% to 49% to 51%, and the volume of the mobile phase B increases from 36% to 34% to 51% to 49%; At 20-30 minutes, the volume of the mobile phase A decreases from 49%-51% to 29%-31%, and the volume of the mobile phase B increases from 51%-49% to 71%-69%; At 30-40 minutes, the volume of the mobile phase A is 29%-31%, and the volume of the mobile phase B is 71%-69%; At 40.1 to 50 minutes, the volume of the mobile phase A is 64% to 66%, and the volume of the mobile phase B is 36% to 34%.

2. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 1, characterized in that: The specific operation steps of the detection method are: S1. Test solution: Take Riociguat, weigh accurately, dissolve in solvent and quantitatively dilute to prepare test solution; S2. Reference solution: Take aniline and phenylazomalononitrile reference substances, accurately weigh them, and quantitatively dilute them with a solvent to prepare aniline and phenylazomalononitrile reference solutions; S3. Impurity localization solution: Take aniline reference substance, accurately weigh, and dilute with solvent to prepare aniline localization solution; take phenylazomalononitrile reference substance, accurately weigh, and dilute with solvent to prepare phenylazomalononitrile localization solution; S4. System suitability solution: Take riociguat, aniline and phenylazomalononitrile reference substances, accurately weigh them, and quantitatively dilute them with a solvent to prepare a mixed solution of riociguat, aniline and phenylazomalononitrile reference substances as the system suitability solution; S5. Adjust the instrument: the chromatographic column uses octadecylsilane bonded silica gel as the filler, mobile phase A, mobile phase B, adjust the column temperature, adjust the mobile phase flow rate, adjust the detection wavelength, and use gradient elution; S6. Determination: Accurately measure the solutions prepared in S1 to S4, inject them into liquid chromatograph respectively, and record the chromatograms.

3. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: The concentration of the test solution in S1 is 0.9-1.1 mg / ml.

4. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: In the reference substance solution of S2, the concentration of the aniline reference substance is 0.9-1.1 μg / ml, and the concentration of the phenylazomalononitrile reference substance is 0.18-0.22 μg / ml.

5. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: In the impurity localization solution of S3, the concentration of the aniline localization solution is 9.0-11.0 μg / ml; the concentration of the phenylazomalononitrile localization solution is 1.8-2.2 μg / ml.

6. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: In the system suitability solution of S4: the concentration of the riociguat reference solution is 0.9-1.1 mg / ml; the concentration of the aniline reference solution is 0.9-1.1 μg / ml; and the concentration of the phenylazomalononitrile reference solution is 0.18-0.22 μg / ml.

7. The method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat according to any one of claims 2 to 6, characterized in that: The solvent is mobile phase A-mobile phase B, and the mass ratio of mobile phase A-mobile phase B is (64% to 66%): (36% to 34%).

8. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: The chromatographic column in S5 uses octadecylsilane bonded silica gel as a filler, and the specifications are Shim-pack GISTC18, 4.6 mm×250 mm, 5 μm.

9. The method for simultaneously determining aniline and phenylazomalononitrile impurities in riociguat according to claim 2 or 8, characterized in that: The mobile phase A is 0.01 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.5 with phosphoric acid. The mobile phase B is methanol-acetonitrile, and the mass ratio of methanol-acetonitrile is (0.99-1.01): (9.01-8.99).

10. The method for simultaneous determination of aniline and phenylazomalononitrile impurities in riociguat according to claim 2, characterized in that: The column temperature was adjusted to 28-32°C, the flow rate of the mobile phase was adjusted to 0.9-1.1 ml / min, and the detection wavelength was adjusted to 235 nm.

Images