Analysis method of related substances in macitentan bulk drug
The gradient elution conditions were optimized by high-performance liquid chromatography, and the problem of impurities separation in mascetitan raw materials was solved, and the effective separation and detection of impurities was achieved.
Patent Information
- Application Number
- CN202510578034.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art has not yet provided an effective method to analyze impurities in mascetitan raw materials, which affects its widespread application.
High performance liquid chromatography was used, and a silane-bonded silica gel chromatography column was used, and gradient elution was performed. The detection wavelength was 200-250 nm. Mobile phase A was a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 90:10, and mobile phase B was a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 10:90. Gradient conditions were optimized to separate impurities.
Effective separation of multiple impurities is achieved, the retention time of each impurity peak is moderate, and the separation between the impurity peak and the massititan peak meets the requirements and meets the detection needs.
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Figure CN120446365A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of chemical drug analysis methods, in particular to an analysis method for related substances in a macitentan raw material drug. Background Art
[0002] Endothelin (ET)-1 and its receptors (ETA and ETB) mediate various deleterious effects such as vasoconstriction, fibrosis, hyperplasia, hypertrophy and inflammation. In disease states such as PAH, the local ET system is upregulated and is involved in vascular hypertrophy and organ damage. Macitentan is an endothelin receptor antagonist that prevents the binding of ET-1 to two receptors, ETA and ETB. Macitentan shows high affinity and sustained occupancy for the ET receptors of human pulmonary artery smooth muscle cells. A metabolite of macitentan also has pharmacological activity at ET receptors, with an estimated potency of approximately 20% of the parent drug in vitro. Macitentan tablets were first developed by Actelion Pharmaceuticals and are an endothelin receptor antagonist. It received marketing approval from the US FDA on October 18, 2013, and was approved for marketing by the European Union on December 20, 2013, with a strength of 10 mg and a trade name. It is used once daily for the long-term treatment of pulmonary arterial hypertension (PAH) in adults. It is not currently available for sale in China. PAH is a relatively rare disease, with an incidence of approximately 15 to 25 people per million. Clinical manifestations are characterized by elevated pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR). If not treated promptly, it can easily lead to right heart function impairment or even death. However, the macitentan API contains certain impurities. Developing an analytical method for the relevant substances in the macitentan API is of great significance for the widespread application of the macitentan API.
[0003] Summary of the Invention
[0004] Based on the technical problems existing in the background technology, the present invention proposes a method for analyzing related substances in macitentan raw materials.
[0005] The present invention provides a method for analyzing related substances in a macitentan raw material drug, which adopts a high performance liquid chromatography method. The chromatographic conditions include: a silane bonded silica gel chromatographic column; gradient elution using an eluent, and a detection wavelength of 200 to 250 nm;
[0006] The eluent includes mobile phase A and mobile phase B; a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 90:10 is used as mobile phase A; a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 10:90 is used as mobile phase B.
[0007] Preferably, the gradient elution includes: within 0 to 2 minutes, the volume ratio of mobile phase A and mobile phase B is 96:4; within 2 to 40 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes from 96:4 to 22:78 at a uniform rate; within 40 to 50 minutes, the volume ratio of mobile phase A and mobile phase B is 22:78; within 50 to 50.01 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes from 22:78 to 96:4 at a uniform rate; within 50.01 to 60 minutes, the volume ratio of mobile phase A and mobile phase B is 96:4.
[0008] Preferably, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column.
[0009] More preferably, the chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.
[0010] Preferably, the column temperature of the chromatographic column is 25-35°C.
[0011] Preferably, the volume concentration of the trifluoroacetic acid solution is 0.05-0.1%.
[0012] Preferably, the flow rate of the chromatographic column is 0.5-2.0 ml / min.
[0013] Preferably, the related substances include at least one of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6. The structural formulas of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6 are as follows: impurity 1: Impurity 2: Impurity 3: Impurity 4: Impurity 5: Impurity 6:
[0014] The analysis method of related substances in the macitentan raw material drug of the present invention specifically comprises the steps of preparing a blank solution, a system solution and an impurity reference substance stock solution and performing sampling and detection.
[0015] Preferably, the system solution comprises macitentan solution, impurity 1 reference stock solution, impurity 2 reference stock solution, impurity 3 reference stock solution, impurity 4 reference stock solution, impurity 5 reference stock solution, and impurity 6 reference stock solution.
[0016] More preferably, the macitentan solution comprises macitentan and methanol.
[0017] More preferably, the preparation of the reference stock solution of impurity 3 comprises first dissolving impurity 3 in acetonitrile and then adding methanol to make up the volume.
[0018] Preferably, the injection volume is 10 to 20 μl.
[0019] The beneficial effects of the present invention are:
[0020] The analytical method for related substances in the macitentan raw material drug proposed by the present invention can detect multiple impurities, the retention time of each impurity peak is moderate, and the separation between each impurity peak and between a macitentan peak and an adjacent impurity peak meets the requirements. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The chromatogram of the crude macitentan in the present invention's exploratory experiment 1 is shown.
[0022] Figure 2 This is the chromatogram of the blank solvent in Experiment 2 of the present invention.
[0023] Figure 3 The chromatogram of the crude macitentan product in the present invention's exploration experiment 2 is shown.
[0024] Figure 4 This is the chromatogram of starting material 3 in Experiment 2 of the present invention.
[0025] Figure 5 This is the chromatogram of impurity A in starting material 3 in Experiment 2 of the present invention.
[0026] Figure 6 This is the chromatogram of the blank solvent in Experiment 3 of the present invention.
[0027] Figure 7 The chromatogram of the crude macitentan in the present invention's exploration experiment 3 is shown.
[0028] Figure 8 This is the chromatogram of impurity A in starting material 3 in experiment 3 of the present invention.
[0029] Figure 9 This is the chromatogram of the blank solvent in Experiment 4 of the present invention.
[0030] Figure 10 This is the chromatogram of the system solution in Experiment 4 of the present invention.
[0031] Figure 11 This is the chromatogram of the blank solvent in Experiment 5 of the present invention.
[0032] Figure 12 This is the chromatogram of the system solution in Experiment 5 of the present invention.
[0033] Figure 13 4 is a chromatogram of a blank solvent in an embodiment of the present invention.
[0034] Figure 14 4 is a chromatogram of the system solution in the embodiment of the present invention. DETAILED DESCRIPTION
[0035] The technical solution of the present invention is described in detail through specific embodiments.
[0036] Unless otherwise specified, the materials, reagents, etc. used in the following examples and comparative examples can be obtained from commercial sources.
[0037] The inventors conducted chromatographic analysis of the macitentan API and impurities by screening a suitable mobile phase and optimizing the ratio of each component, as well as screening other suitable chromatographic conditions, and determined the analytical method of the present invention. The specific process is as follows:
[0038] Exploratory Experiment 1: Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler (250 × 4.6 mm, 5 μm); mobile phase A was 0.03 M ammonium dihydrogen phosphate solution-acetonitrile (90:10), and mobile phase B was 0.03 M ammonium dihydrogen phosphate solution-acetonitrile (30:70); column temperature was 30°C; flow rate was 1.0 ml / min; detection wavelength was 220 nm; elution was performed according to the gradient conditions in Table 1:
[0039] Table 1
[0040] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 70 30 10 70 30 35 10 90 50 10 90 50.01 70 30 60 70 30
[0041] Place approximately 12.5 mg of crude macitentan in a 25 ml volumetric flask, add an appropriate amount of methanol to dissolve and dilute to the mark, shake well, and set aside. Separately, place approximately 12.5 mg each of starting materials 1 and 3 and intermediates I and II in separate 25 ml volumetric flasks, add an appropriate amount of methanol to dissolve and dilute to the mark, shake well, and prepare the stock solutions. Accurately measure 0.25 ml of the stock solution in the same 25 ml volumetric flask, dilute to the mark with methanol, and shake well to prepare the locating solutions for each sample.
[0042] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph to record the chromatogram. The test results are shown in Table 2 and Figure 1 shown.
[0043] Table 2
[0044] sample Retention time (min) Starting material 1 39.451 Starting material 3 15.215 Intermediate I 34.712 Intermediate II 27.892 Macitentan 40.316
[0045] Under these chromatographic conditions, the separation between each starting material and intermediate, as well as between the macitentan peak and the adjacent impurity peak, all met the requirements. However, considering that impurity A in starting material 3 may be produced during the strong degradation process of the final product, impurity A in starting material 3 was added to the system, and the gradient conditions were optimized based on the above chromatographic conditions.
[0046] Exploratory experiment 2: Combined with the detection method of starting material 3, based on Exploratory experiment 1, the retention of the initial organic phase under the gradient conditions was reduced. The specific chromatographic conditions are as follows:
[0047] Mobile phase A was 0.03 M ammonium dihydrogen phosphate solution-acetonitrile (90:10), and mobile phase B was 0.03 M ammonium dihydrogen phosphate solution-acetonitrile (30:70); column temperature was 30°C; flow rate was 1.0 ml / min; detection wavelength was 220 nm; elution was performed according to the gradient conditions in Table 3:
[0048] Table 3
[0049]
[0050]
[0051] Take approximately 12.5 mg of crude macitentan 2 and place it in a 25 ml volumetric flask. Add an appropriate amount of methanol to dissolve and dilute to the mark. Shake well to prepare the test solution. Separately, take approximately 12.5 mg of starting material 3, its impurity A, and intermediate II and place them in 25 ml volumetric flasks. Add an appropriate amount of methanol to dissolve and dilute to the mark. Shake well to prepare the stock solution. Accurately measure 0.25 ml of the stock solution and place it in a 25 ml volumetric flask. Add methanol to dilute to the mark. Shake well to prepare the locating solution for each sample.
[0052] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph to record the chromatogram. The test results are shown in Table 4 and Figures 2 to 5 shown.
[0053] Table 4
[0054] sample Retention time (min) Starting material 3 impurity A 4.622 Starting material 3 22.693 Intermediate II 38.263 Macitentan 53.401
[0055] Figure 2 This is the chromatogram of the blank solvent in Experiment 2 of the present invention. Figure 3 The chromatogram of the crude product of macitentan in Experiment 2 of the present invention is as follows: retention time is 53.401 min, area is 11232841, height is 903953, area is 89.7%, USP peak width is 0.333, resolution is 8.969, theoretical plates are 411087.991, and tailing factor is 0.989. Figure 4 This is the chromatogram of the starting material 3 in Experiment 2 of the present invention; the retention time is 22.693 min, the area is 175165, the height is 12388, the area is 100%, the USP peak width is 0.384, the theoretical plates are 55793.239, and the tailing factor is 1.180. Figure 5This is the chromatogram of impurity A in the starting material 3 in Experiment 2 of the present invention; retention time is 4.622 min, area is 198443, height is 31586, area is 100%, USP peak width is 0.169, theoretical plates are 11966.461, and tailing factor is 1.385.
[0056] Under these chromatographic conditions, the resolution between each starting material and intermediate, as well as between the macitentan peak and adjacent impurity peaks, met the requirements. However, the blank solvent peak at position 3 of the starting material interfered and was relatively complex. Based on these chromatographic conditions, the phosphate system was replaced with a trifluoroacetic acid system to optimize the chromatographic conditions.
[0057] Exploratory Experiment 3: Based on Exploratory Experiment 2, the mobile phase system was changed from phosphate system to trifluoroacetic acid system. The specific chromatographic conditions are as follows:
[0058] 0.1% trifluoroacetic acid solution was used as mobile phase A, and 0.1% trifluoroacetic acid solution-acetonitrile (20:80) was used as mobile phase B; the column temperature was 30°C; the flow rate was 1.0 ml / min; the detection wavelength was 220 nm; and the elution was performed according to the gradient conditions in Table 5:
[0059] Table 5
[0060] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 80 20 5 80 20 40 10 90 50 10 90 50.01 80 20 60 80 20
[0061] Approximately 12.5 mg of crude macitentan 3 was placed in a 25 ml volumetric flask. An appropriate amount of methanol was added to dissolve the mixture and dilute to the mark. The mixture was shaken to prepare the test solution. Approximately 12.5 mg of starting material 3, impurity A, and intermediate II were placed in separate 25 ml volumetric flasks. An appropriate amount of methanol was added to dissolve the mixture and dilute to the mark. The mixture was shaken to prepare stock solution 1. Approximately 12.5 mg of a process impurity was placed in a 25 ml volumetric flask. An appropriate amount of acetonitrile was added to dissolve the mixture and the mixture was diluted to the mark with methanol. The mixture was shaken to prepare stock solution 2. Accurately measure 0.25 ml of the above stock solutions and place them in separate 25 ml volumetric flasks. The mixture was diluted to the mark with methanol and shaken to prepare the impurity location solutions.
[0062] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph to record the chromatogram. The test results are shown in Table 6 and Figures 6-8 shown.
[0063] Table 6
[0064]
[0065]
[0066] Figure 6 This is the chromatogram of the blank solvent in Experiment 3 of the present invention. Figure 7The chromatogram of the crude product of macitentan in Experiment 3 of the present invention is as follows: retention time 40.189 min, area 13098671, height 1270164, area 97.7%, USP peak width 0.275, resolution 5.944, theoretical plates 341888.608, and tailing factor 1.024. Figure 8 This is the chromatogram of impurity A in starting material 3 in experiment 3 of the present invention; retention time is 4.130 min, area is 199315, height is 46634, area is 100%, USP peak width is 0.133, theoretical plates are 15414.297, and tailing factor is 1.253.
[0067] Exploratory Experiment 4: Based on Exploratory Experiment 3, the initial organic phase ratio of the gradient conditions was reduced. The specific chromatographic conditions were as follows:
[0068] 0.1% trifluoroacetic acid solution was used as mobile phase A, and 0.1% trifluoroacetic acid solution-acetonitrile (20:80) was used as mobile phase B; the column temperature was 30°C; the flow rate was 1.0 ml / min; the detection wavelength was 220 nm; and the elution was performed according to the gradient conditions in Table 7:
[0069] Table 7
[0070]
[0071] About 12.5 mg of starting material 1, starting material 3 and its impurity A, intermediate I, intermediate II and strong acid destruction product were taken respectively, placed in 25 ml volumetric flasks, added an appropriate amount of methanol to dissolve and dilute to the scale, and shaken to prepare stock solution 1; about 12.5 mg of process impurity was taken, placed in a 25 ml volumetric flask, added an appropriate amount of acetonitrile to dissolve, and then added methanol to dilute to the scale, and shaken to prepare stock solution 2; about 12.5 mg of macitentan secondary refined product was taken separately, placed in a 25 ml volumetric flask, added an appropriate amount of methanol to dissolve, accurately measured 0.25 ml of each of the above stock solutions and placed in the same 25 ml volumetric flask, added methanol to dilute to the scale, and shaken to prepare the system solution.
[0072] Accurately measure 10μl of the system solution, inject it into the liquid chromatograph, and record the chromatogram. The test results are shown in Table 8 and Figures 9-10 shown.
[0073] Table 8
[0074]
[0075] Figure 9 This is the chromatogram of the blank solvent in Experiment 4 of the present invention. Figure 10 This is the chromatogram of the system solution in Experiment 4 of the present invention.
[0076] Exploratory Experiment 5: Based on Exploratory Experiment 4, the concentration of trifluoroacetic acid was changed to investigate the effect of trifluoroacetic acid concentration on baseline and impurity separation. The specific chromatographic conditions are as follows:
[0077] 0.05% trifluoroacetic acid solution was used as mobile phase A, and 0.05% trifluoroacetic acid solution-acetonitrile (20:80) was used as mobile phase B; the column temperature was 30°C; the flow rate was 1.0 ml / min; the detection wavelength was 220 nm; and the elution was performed according to the gradient conditions in Table 9:
[0078] Table 9
[0079] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 83 17 2 83 17 40 10 90 50 10 90 50.01 83 17 60 83 17
[0080] Prepare the system solution in the same way as in Experiment 4. Accurately measure 10 μl of the system solution, inject it into the liquid chromatograph, and record the chromatogram. The test results are shown in Table 10 and Figures 11-12 shown.
[0081] Table 10
[0082]
[0083] Figure 11 This is the chromatogram of the blank solvent in Experiment 5 of the present invention. Figure 12 This is the chromatogram of the system solution in Experiment 5 of the present invention.
[0084] The above results indicate that decreasing the trifluoroacetic acid concentration stabilizes the baseline at higher concentrations, but the presence of impurity peaks increases significantly. Under these chromatographic conditions, the retention times of the impurity peaks are moderate, and the resolution between each impurity peak and between the macitentan peak and its adjacent impurity peaks meets the requirements. However, there is interference from the blank solvent at the position of the process impurities. Therefore, based on a 0.1% trifluoroacetic acid solution mobile phase system, the gradient conditions were fine-tuned to ensure that the analytical method met the detection requirements.
[0085] Example
[0086] Based on exploratory experiment 5, the mobile phase system was adjusted from 0.05% trifluoroacetic acid solution to 0.1% trifluoroacetic acid solution. The specific chromatographic conditions are as follows:
[0087] Octadecylsilane bonded silica gel was used as the filler (250×4.6 mm, 5 μm); 0.1% trifluoroacetic acid solution-acetonitrile (90:10) was used as mobile phase A, and 0.1% trifluoroacetic acid solution-acetonitrile (10:90) was used as mobile phase B; the detection wavelength was 220 nm; the column temperature was 30°C; the flow rate was 1.0 ml / min; and the gradient conditions in Table 11 were used for elution:
[0088] Table 11
[0089]
[0090] Take about 12.5 mg of the reference substance of starting material 3 impurity A (impurity 1), starting material 3 (impurity 2), intermediate II (impurity 4), intermediate I (impurity 5), and starting material 1 (impurity 6), accurately weigh them, place them in a 25 ml volumetric flask, add an appropriate amount of methanol to dissolve them and dilute them to the scale, shake them well, and use them as impurity reference substance stock solution one; take about 12.5 mg of the process impurity (impurity 3) reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add an appropriate amount of acetonitrile to dissolve them, and then add methanol to dilute them to the scale, shake them well, and use them as impurity reference substance stock solution two; take about 12.5 mg of macitentan, accurately weigh it, place it in a 25 ml volumetric flask, add an appropriate amount of methanol to dissolve them, then accurately measure 0.25 ml of the above-mentioned impurity reference substance stock solutions and place them in the same 25 ml volumetric flask, add methanol to dilute them to the scale, shake them well, and use them as system solution. Accurately measure 10 μl of the system solution, inject it into the liquid chromatograph, and record the chromatogram. The test results are shown in Table 12 and Figures 13-14 shown.
[0091] The chemical structures of Macitentan and impurities 1 to 6 are shown below. Macitentan: Impurity 1: Impurity 2: Impurity 3: Impurity 4: Impurity 5: Impurity 6:
[0092] Table 12
[0093]
[0094] Figure 13 4 is a chromatogram of a blank solvent in an embodiment of the present invention. Figure 14 4 is a chromatogram of the system solution in the embodiment of the present invention.
[0095] The above results demonstrate that under these chromatographic conditions, the retention times of the impurity peaks are moderate, and the separation between each impurity peak and between the macitentan peak and adjacent impurity peaks meets the requirements. Impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, and macitentan peaks elute in sequence, with separation meeting the requirements. The number of theoretical plates calculated based on the macitentan peak must be no less than 3000.
[0096] In summary, the analysis method of related substances in the macitentan bulk drug provided by the present invention has the separation degree of each impurity meeting the requirements.
[0097] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A method for analyzing related substances in a macitentan bulk drug, characterized in that: High performance liquid chromatography (HPLC) was used, and the chromatographic conditions included: a silane bonded silica gel chromatographic column; gradient elution was performed using an eluent, and a detection wavelength was 200 to 250 nm; The eluent includes mobile phase A and mobile phase B; a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 90:10 is used as mobile phase A; a trifluoroacetic acid solution-acetonitrile mixed solution with a volume ratio of 10:90 is used as mobile phase B.
2. The analysis method according to claim 1, characterized in that The gradient elution includes: within 0 to 2 minutes, the volume ratio of mobile phase A and mobile phase B is 96:4; within 2 to 40 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes from 96:4 to 22:78 at a uniform speed; within 40 to 50 minutes, the volume ratio of mobile phase A and mobile phase B is 22:78; within 50 to 50.01 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes from 22:78 to 96:4 at a uniform speed; within 50.01 to 60 minutes, the volume ratio of mobile phase A and mobile phase B is 96:
4.
3. The analysis method according to claim 1, characterized in that The chromatographic column is an octadecylsilane bonded silica gel chromatographic column.
4. The analysis method according to any one of claims 1 or 3, characterized in that The chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.
5. The analysis method according to claim 1, characterized in that The column temperature of the chromatographic column is 25-35°C.
6. The analysis method according to claim 1, characterized in that The volume concentration of the trifluoroacetic acid solution is 0.05-0.1%.
7. The analysis method according to claim 1, characterized in that The flow rate of the chromatographic column is 0.5-2.0 ml / min.
8. The analysis method according to claim 1, characterized in that The related substances include at least one of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6. The structural formulas of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6 are as follows: impurity 1: Impurity 2: Impurity 3: Impurity 4: Impurity 5: Impurity 6: