Method for determination of related substances in selexipag raw materials by high performance liquid chromatography

Abstract

The present invention discloses a method for determining related substances in selexipag raw materials by high performance liquid chromatography, wherein the analysis conditions of the method are as follows: a phenylsilane bonded silica gel column is used as a chromatographic column; a mixed solvent of trifluoroacetic acid solution and acetonitrile is used as a mobile phase for gradient elution. The method can be used to quickly and efficiently separate related substances in selexipag raw materials, and effectively control the quality of the raw materials. The detection method has strong specificity, high precision, high accuracy, and convenient operation, and can effectively control product quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular, to a method for determining related substances in selexipag raw materials by high performance liquid chromatography. Background Art

[0002] The chemical name of Selexipag is 2-[4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butoxy]-N-(methylsulfonyl)acetamide, and its structural formula is as follows. It is an orally effective, highly selective and long-acting non-prostaglandin prostacyclin receptor agonist that can relax vascular wall smooth muscle, dilate blood vessels, and reduce pulmonary artery pressure. It is used to treat pulmonary arterial hypertension in adults and has the advantages of high selectivity, long duration of action, safety and tolerability.

[0003]

[0004] Selexipag API may contain synthetic raw materials, by-products and degradation impurities. The known impurities are:

[0005] Impurity 1 (raw material impurity): 5-chloro-2,3-diphenylpyrazine;

[0006] Impurity 2 (intermediate): 4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)-1-butanol;

[0007] Impurity 3 (intermediate): tert-butyl 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetate;

[0008] Impurity 4 (intermediates and degradation products): 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetic acid;

[0009] Impurity 5 (byproduct): 5-(isopropyl(4-(2-(methylsulfonamido)-2-oxoethoxy)butyl)amino)-2,3-diphenylpyrazine 1-oxide;

[0010] Impurity 6 (by-product): 5-((4-(carboxymethoxy)butyl)(isopropyl)amino)-2,3-diphenylpyrazine 1-oxide.

[0011] At present, there is no publicly available method for analyzing these process impurities and degradation impurities in China. For example, the method disclosed in the literature Determination of the content and related substances of the pulmonary arterial hypertension drug Selexipag [J], Ma Huili, Pharmaceutical Science, 2016, (30): 69, does not locate each impurity, cannot effectively separate all impurities, and cannot be used in actual drug production. Summary of the invention

[0012] One object of the present invention is to provide a method for rapidly analyzing selexipag-related substances so as to fully separate and display the target product from process impurities and degradation impurities.

[0013] In one aspect of the present invention, the present invention provides a method for determining related substances in selexipag raw materials by high performance liquid chromatography. The chromatographic conditions of the method are: the chromatographic column is a phenylsilane bonded silica gel column; the mobile phase is a mixed solvent of trifluoroacetic acid solution and acetonitrile.

[0014] Thus, the related substances in the Selexipag API can be separated quickly and efficiently, and the quality of the API can be effectively controlled. The detection method has high sensitivity, strong specificity, high precision, high accuracy, and convenient operation, and can effectively control the quality of the product.

[0015] The method for determining related substances in selexipag raw materials by high performance liquid chromatography according to some embodiments of the present invention may also have the following additional technical features:

[0016] According to a specific embodiment of the present invention, the conditions for gradient elution are:

[0017] Time / min Mobile phase A / % Mobile phase B / % 0-5 75～55 25-45 10-30 55～52 45-48 41-45 40～25 60-75 46-60 15 85 61-78 55～75 45-25

[0018] Using the gradient program under the chromatographic column conditions selected in the present invention, various substances can be well separated.

[0019] According to a preferred embodiment of the present invention, the conditions of the gradient elution are:

[0020] Time / min Trifluoroacetic acid solution / % Acetonitrile / % 0 60 40 5 60 40 10 55 45 31 52 48 42 40 60 45 25 75 47 15 85 61 15 85 63 60 40 78 60 40

[0021] According to some embodiments of the present invention, the length of the chromatographic column is 15 cm to 25 cm, preferably 25 cm, thereby further improving the separation degree.

[0022] According to some embodiments of the present invention, the concentration of the trifluoroacetic acid solution is 0.01% to 0.5%, and most preferably 0.05%. Thus, the peak shape and blank baseline interference can be further improved.

[0023] According to an embodiment of the present invention, the analysis method of the present invention further comprises:

[0024] The sample to be tested was prepared into a sample solution using a mixture of methanol / acetonitrile and water, and the sample solution was injected into a high performance liquid chromatograph to complete the determination of related substances in the selexipag raw material.

[0025] According to an embodiment of the present invention, the volume ratio of methanol / acetonitrile and water in the mixture of methanol / acetonitrile and water is (50%:50%) to (90%:10%), preferably acetonitrile:water=80%:20%.

[0026] According to some embodiments of the present invention, the volume of the sample solution injected into the high performance liquid chromatograph is 5 μL to 50 μL, preferably 20 μL.

[0027] According to some embodiments of the present invention, the detection wavelength of the high performance liquid chromatograph is 210 nm to 365 nm, preferably 300 nm, thereby significantly improving the detection sensitivity.

[0028] According to an embodiment of the present invention, the flow rate of the mobile phase is 0.8 mL / min to 1.2 mL / min, preferably 1.0 mL / min. Thus, the separation degree can be further improved.

[0029] According to some embodiments of the present invention, the column temperature of the chromatographic column is 25° C. to 50° C., preferably 45° C. Thus, the separation between adjacent chromatographic peaks can be significantly improved.

[0030] According to a specific embodiment of the present invention, the method for determining related substances in selexipag API can be implemented according to the following steps:

[0031] (1) Take an appropriate amount of the sample to be tested and dissolve it in a mixture of acetonitrile and water (volume ratio is 80:20) to prepare a sample solution containing about 0.6 mg / ml per 1 ml.

[0032] (2) The column temperature of the chromatographic column is set at 25° C. to 50° C., preferably 45° C. The flow rate of the mobile phase is 0.8 to 1.2 mL / min, preferably 1.0 ml / min, and the detection wavelength is 210 nm to 365 nm, preferably 300 nm.

[0033] (3) Take 5 μL to 50 μL of the sample solution of (1), preferably 20 μL, and inject it into a high performance liquid chromatograph to complete the determination of related substances in the selexipag raw material.

[0034] The present invention can completely separate the components of related substances in the selexipag raw material drug, the impurity peaks before and after the main peak are well separated from the main peak, the operability is strong, the analysis method has strong specificity, high precision, high accuracy, and convenient operation, and the quality of the selexipag raw material drug can be effectively controlled.

[0035] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : HPLC chromatogram of blank solution obtained under the conditions of Example 1;

[0037] Figure 2 : HPLC chromatogram of the system suitability solution obtained under the conditions of Example 1;

[0038] Figure 3 : HPLC chromatogram of the impurity mixed solution obtained under the conditions of Example 1;

[0039] Figure 4 : HPLC chromatogram of the spiked test solution obtained under the conditions of Example 1;

[0040] Figure 5 : HPLC chromatogram of the test solution obtained under the conditions of Example 2;

[0041] Figure 6 : HPLC chromatogram of the control solution obtained under the conditions of Example 2;

[0042] Figure 7 : HPLC chromatogram of mixed solution 1 obtained according to the conditions of Example 3;

[0043] Figure 8 : HPLC chromatogram of mixed solution 1 obtained according to the conditions of Example 4;

[0044] Fig. 9 : HPLC chromatogram of mixed solution 2 obtained according to the conditions of Example 5;

[0045] Fig.10 : HPLC chromatogram of mixed solution 2 obtained according to the conditions of Comparative Example 1;

[0046] Fig.11 : HPLC chromatogram of mixed solution 2 obtained according to the conditions of Comparative Example 2. DETAILED DESCRIPTION

[0047] The following examples are provided to further illustrate the present invention and its embodiments. The specific details given in the examples are for illustrative purposes only and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0048] The selexipag API used in the embodiments of the present invention is prepared by the applicant according to the above-mentioned synthesis route.

[0049] Example 1

[0050] Instrument: High performance liquid chromatography with diode array detector

[0051] Chromatographic column: Supelcosil LC-DP (4.6 mm × 250 mm, 5 μm)

[0052] Mobile phase A: 0.05% trifluoroacetic acid solution

[0053] Mobile phase B: acetonitrile

[0054] Gradient elution is shown in the following table:

[0055] Time / min Mobile phase A / % Mobile phase B / % 0 60 40 5 60 40 10 55 45 31 52 48 42 40 60 45 25 75 47 15 85 61 15 85 63 60 40 78 60 40

[0056] Flow rate: 1.0mL / min

[0057] Detection wavelength: 300nm

[0058] Column temperature: 45°C

[0059] Injection volume: 20 μL

[0060] Solvent: acetonitrile: water = 80:20 (v / v)

[0061] Running time: 78 minutes

[0062] 1. Determination of detection wavelength

[0063] Selexipag and its impurities 1 to 6 were scanned by ultraviolet spectrum between 200 and 400 nm using an ultraviolet-visible spectrophotometer. The results showed that Selexipag and its impurities had strong absorption at a wavelength of 300 nm, so 300 nm was selected as the detection wavelength.

[0064] 2. Methodological specificity

[0065] Blank solution: acetonitrile-water (80:20)

[0066] Take appropriate amount of each reference substance of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5 and impurity 6, dissolve them in solvent and dilute them to make impurity reference substance stock solutions with concentrations of approximately 15μg / ml, 15μg / ml, 15μg / ml, 75μg / ml, 150μg / ml and 150μg / ml, respectively.

[0067] Take an appropriate amount of Selexipag reference substance, dissolve it in solvent and dilute it to make a stock solution with a concentration of about 75 μg / ml.

[0068] System suitability solution: Take about 15 mg of this product, weigh it accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well; accurately measure 1 ml, place it in a 50 ml volumetric flask, accurately add 2 ml each of impurity 4 stock solution, impurity 5 stock solution, and impurity 6 stock solution, add solvent to dilute to the scale, and shake well.

[0069] Impurity mixed solution: Accurately measure 2 ml of impurity 1 stock solution, 2 ml of impurity 2 stock solution, 2 ml of impurity 3 stock solution, 0.6 ml of impurity 4 stock solution, 0.1 ml of impurity 5 stock solution, and 0.1 ml of impurity 6 stock solution, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0070] Spiked test solution: Take about 30 mg of the product, accurately weigh it, put it in a 50 ml volumetric flask, accurately add 0.6 ml of impurity 4 stock solution, 0.1 ml of impurity 5 stock solution, and 0.1 ml of impurity 6 stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0071] Accurately measure 20 μL each of the blank solution, system suitability solution, impurity mixed solution, and spiked test solution, inject them into the HPLC, and record the chromatogram.

[0072] The results show:

[0073] (1) Blank solution (see Figure 1 ) has no interference with the detection of each peak.

[0074] (2) System suitability solution (see Figure 2 ) in the chromatography peaks with retention times of 13.520min, 15.456min, 23.679min and 26.977min are the chromatographic peaks of impurity 6, impurity 5, impurity 4 and selexipag, respectively, wherein the separation degree of selexipag and impurity 4 is 5.906, the separation degree of impurity 5 and impurity 6 is 5.370, the tailing factor of the selexipag peak is 1.039, and the theoretical plate number is 37268.

[0075] (3) Impurity mixed solution (see Figure 3 ) in the chromatographic peaks with retention times of 13.588min, 15.495min, 17.634min, 21.597min, 23.799min and 47.338min are the chromatographic peaks of impurity 6, impurity 5, impurity 1, impurity 2, impurity 4 and impurity 3, respectively, wherein the minimum resolution between the peaks is 4.367 (between impurity 2 and impurity 4), and the resolution between the peaks is greater than 1.5.

[0076] (4) Spiked test solution (see Figure 4 ) in the chromatography peaks with retention times of 13.532min, 15.460min, 23.665min and 26.469min are the chromatographic peaks of impurity 6, impurity 5, impurity 4 and selexipag, respectively. Among them, the separation between the unknown impurity peak at 22.577min and impurity 4 is the smallest (separation is 2.023), and the separation between each peak is greater than 1.5.

[0077] The above results show that the analytical method has strong specificity.

[0078] 3. Precision test (repeatability test)

[0079] Accurately weigh about 15 mg of selexipag raw material, place it in a 25 ml volumetric flask, add acetonitrile-water (80:20), ultrasonically dissolve it and dilute it to the scale, shake well, and use it as the test solution (prepare 6 copies in parallel).

[0080] Accurately measure 20 μL of each of the 6 test solution, inject into the high performance liquid chromatograph, determine the area normalized content of the known impurities, the largest unknown single impurity and the main peak, record the chromatogram, and the results are shown in Table 1. As can be seen from Table 1, the RSD of the main peak content results in the 6 test solution is 0.001%, and the RSD of the content of impurity 4 and the largest unknown single impurity are 2.4% and 1.1%, respectively, indicating that the detection method of the related substances has good repeatability.

[0081] Table 1: Repeatability test results

[0082] Serial number 1 2 3 4 5 6 average value RSD(%) Impurity 4(%) 0.016 0.017 0.017 0.017 0.017 0.017 0.017 2.4 Maximum unknown single impurity (%) 0.038 0.039 0.038 0.038 0.038 0.038 0.038 1.1 Main peak (%) 99.904 99.904 99.904 99.904 99.903 99.903 99.90 0.001

[0083] Example 2

[0084] Instrument: High performance liquid chromatograph with UV detector

[0085] Chromatographic column: Supelcosil LC-DP (4.6 mm × 250 mm, 5 μm)

[0086] Mobile phase A: 0.05% trifluoroacetic acid solution

[0087] Mobile phase B: acetonitrile

[0088] Gradient elution is shown in the following table:

[0089]

[0090]

[0091] Flow rate: 1.0mL / min

[0092] Detection wavelength: 300nm

[0093] Column temperature: 45°C

[0094] Injection volume: 20 μL

[0095] Solvent: acetonitrile: water = 80:20 (v / v)

[0096] Running time: 78 minutes

[0097] Experimental steps:

[0098] Test solution: Take about 15 mg of the product, weigh accurately, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0099] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0100] Accurately measure 20 μL of the test solution and the control solution, inject them into the high performance liquid chromatograph, and record the chromatogram. Figure 5 and Figure 6 .

[0101] The principal component self-comparison method was used to calculate the content of each impurity in the selexipag raw material. The results showed that each impurity was ≤0.1%, the total impurity was ≤0.5%, and the test results of related substances of the raw material met the quality standards.

[0102] Example 3

[0103] Instrument: High performance liquid chromatograph with UV detector

[0104] Chromatographic column: Supelcosil LC-DP (4.6 mm × 250 mm, 5 μm)

[0105] Mobile phase A: 0.05% trifluoroacetic acid solution

[0106] Mobile phase B: acetonitrile

[0107] The elution conditions are:

[0108]

[0109]

[0110] Flow rate: 1.0mL / min

[0111] Detection wavelength: 300nm

[0112] Column temperature: 35°C

[0113] Injection volume: 20 μL

[0114] Running time: 70 minutes

[0115] Experimental steps:

[0116] Preparation of mixed solution 1: Take appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, and selexipag reference substance, add methanol-water (80:20) to dissolve and dilute to prepare mixed solutions with a concentration of approximately 2.5 μg / ml.

[0117] Take mixed solution 1, perform HPLC analysis under the above chromatographic conditions, record the chromatogram, and the results are shown in the attached Figure 7 .

[0118] Figure 7 The chromatographic peaks with retention times of 11.234min, 13.759min, 17.831min, 22.837min, 26.589min, 32.044min and 45.714min are the chromatographic peaks of impurity 6, impurity 5, impurity 1, impurity 2, impurity 4, selexipag and impurity 3, respectively. The separation degree between selexipag and impurity 4 is 6.176, and the separation degree between impurity 6 and impurity 5 is 5.605. The separation degrees between the other peaks are all greater than 4.0.

[0119] The above chromatograms show that the related substances in the selexipag raw material can be well separated.

[0120] Example 4

[0121] Instrument: High performance liquid chromatograph with UV detector

[0122] Chromatographic column: Supelcosil LC-DP (4.6 mm × 250 mm, 5 μm)

[0123] Mobile phase A: 0.1% trifluoroacetic acid solution

[0124] Mobile phase B: acetonitrile

[0125] Gradient elution is shown in the following table:

[0126]

[0127]

[0128] Flow rate: 1.0mL / min

[0129] Detection wavelength: 300nm

[0130] Column temperature: 30°C

[0131] Injection volume: 20 μL

[0132] Running time: 66 minutes

[0133] Experimental steps:

[0134] Preparation of mixed solution 1: Take appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, and selexipag reference substance, add methanol-water (80:20) to dissolve and dilute to prepare mixed solutions with a concentration of approximately 2.5 μg / ml.

[0135] Take mixed solution 1, perform HPLC analysis under the above chromatographic conditions, record the chromatogram, and the results are shown in the attached Figure 8 .

[0136] Figure 8 The chromatographic peaks with retention times of 22.248min, 23.620min, 25.218min, 28.124min, 28.994min, 30.191min and 37.829min are the chromatographic peaks of impurity 6, impurity 5, impurity 1, impurity 2, impurity 4, selexipag and impurity 3, respectively. The separation degree between selexipag and impurity 4 is 3.954, and the separation degree between impurity 2 and impurity 4 is 2.865. The separation degrees between the other peaks are all greater than 2.0.

[0137] The above chromatograms show that the related substances in the selexipag raw material can be well separated.

[0138] Example 5

[0139] Instrument: High performance liquid chromatograph with UV detector

[0140] Chromatographic column: Supelcosil LC-DP (4.6 mm × 250 mm, 5 μm)

[0141] Mobile phase A: 0.1% trifluoroacetic acid solution

[0142] Mobile phase B: acetonitrile

[0143] Gradient elution is shown in the following table:

[0144]

[0145]

[0146] Flow rate: 1.0mL / min

[0147] Detection wavelength: 300nm

[0148] Column temperature: 30°C

[0149] Injection volume: 20 μL

[0150] Running time: 56 minutes

[0151] Experimental steps:

[0152] Preparation of mixed solution 2: Take appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, and selexipag reference substance, add methanol-water (80:20) to dissolve and dilute to prepare mixed solutions with a concentration of approximately 2.5 μg / ml.

[0153] Take mixed solution 2 and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Fig. 9 .

[0154] Fig. 9The chromatographic peaks with retention times of 21.990min, 23.385min, 23.842min, 24.638min and 29.553min are the chromatographic peaks of impurity 1, impurity 2, impurity 4, selexipag and impurity 3, respectively. Among them, the separation between impurity 2 and impurity 4 is the smallest (the separation is 1.953), and the separation between each peak is greater than 1.5.

[0155] The above chromatograms show that the related substances in the selexipag raw material can be well separated.

[0156] Comparative Example 1

[0157] Instrument: High performance liquid chromatograph with detector

[0158] Chromatographic column: Wondasil C18 (250mm×4.6mm, 5μm)

[0159] Mobile phase A: 0.1% trifluoroacetic acid solution

[0160] Mobile phase B: acetonitrile

[0161] Gradient elution is shown in the following table:

[0162] Time / min Mobile phase A / % Mobile phase B / % 0 75 25 5 75 25 25 20 80 45 20 80 46 75 25 56 75 25

[0163] Flow rate: 1.0mL / min

[0164] Detection wavelength: 300nm

[0165] Column temperature: 30°C

[0166] Injection volume: 20 μL

[0167] Running time: 56 minutes

[0168] Experimental steps:

[0169] Preparation of mixed solution 2: Take appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, and selexipag reference substance, add methanol-water (80:20) to dissolve and dilute to prepare mixed solutions with a concentration of approximately 2.5 μg / ml.

[0170] Take mixed solution 2 and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Fig.10 .

[0171] Fig.10 The chromatographic peaks with retention times of 25.161min, 25.347min, 25.919min, 27.963min and 34.614min are the chromatographic peaks of impurity 4, impurity 2, selexipag, impurity 1 and impurity 3, respectively, among which impurity 4 and impurity 2 are not separated.

[0172] The above chromatograms show that by changing the chromatographic column type on the basis of Example 5, the related substances in the selexipag raw material cannot be well separated.

[0173] Comparative Example 2

[0174] Instrument: High performance liquid chromatograph with UV detector

[0175] Chromatographic column: Wondasil C18 (250mm×4.6mm, 5μm)

[0176] Mobile phase A: 0.1% trifluoroacetic acid solution

[0177] Mobile phase B: acetonitrile

[0178] Gradient elution is shown in the following table:

[0179] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 5 70 30 15 50 50 25 50 50 35 30 70 48 30 70 52 70 30 65 70 30

[0180] Flow rate: 1.0mL / min

[0181] Detection wavelength: 300nm

[0182] Column temperature: 30°C

[0183] Injection volume: 20 μL

[0184] Running time: 65 minutes

[0185] Experimental steps:

[0186] Preparation of mixed solution 2: Take appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, and selexipag reference substance, add methanol-water (80:20) to dissolve and dilute to prepare mixed solutions with a concentration of approximately 2.5 μg / ml.

[0187] Take mixed solution 2 and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Fig.11 .

[0188] Fig.11 The chromatographic peaks with retention times of 33.060min, 33.448min, 35.409min and 38.244min are the chromatographic peaks of impurity 2, impurity 4, selexipag and impurity 1, respectively. Impurity 2 and impurity 4 were not separated, and impurity 3 was not eluted.

[0189] The above chromatograms show that by changing the gradient elution procedure on the basis of Comparative Example 1, the related substances in the selexipag raw material cannot be well separated, nor can the related substances be accurately determined.

[0190] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for determining related substances in selexipag raw materials by high performance liquid chromatography, characterized in that: The chromatographic conditions are: The chromatographic column was Supelcosil LC-DP, with specifications of 4.6 mm × 250 mm, 5 μm; 0.01% to 0.5% trifluoroacetic acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, gradient elution was adopted, the detection wavelength was 300nm, the mobile phase flow rate was 0.8mL / min to 1.2mL / min, and the column temperature was 25℃ to 50℃; The conditions of the gradient elution are: or or Wherein, the related substances are impurity 1, impurity 2, impurity 3, impurity 4, impurity 5 and impurity 6, and their names are: Impurity 1: 5-chloro-2,3-diphenylpyrazine; Impurity 2: 4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)-1-butanol; Impurity 3: tert-butyl 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetate; Impurity 4: 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetic acid; Impurity 5: 5-(isopropyl(4-(2-(methylsulfonamido)-2-oxoethoxy)butyl)amino)-2,3-diphenylpyrazine 1-oxide; Impurity 6: 5-((4-(carboxymethoxy)butyl)(isopropyl)amino)-2,3-diphenylpyrazine 1-oxide.

2. The method according to claim 1, characterized in that The concentration of the trifluoroacetic acid solution is 0.05%.

3. The method according to claim 1, characterized in that Further including: The sample to be tested is prepared into a sample solution by using a mixed solution of methanol and water or a mixed solution of acetonitrile and water; The sample solution was injected into the high performance liquid chromatograph to complete the determination of related substances in the selexipag raw material.

4. The method according to claim 3, characterized in that The volume of the sample solution injected into the high performance liquid chromatograph is 5 μL to 50 μL.

5. The method according to claim 4, characterized in that The volume of the sample solution injected into the HPLC was 20 μL.

6. The method according to claim 1, characterized in that The flow rate of the mobile phase was 1.0 mL / min.

7. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 45°C.

Images