A method for analyzing the related substances of a benzamide compound starting material

Abstract

The application belongs to the technical field of pharmaceutical analysis, and discloses a method for analyzing related substances of a benzamide compound starting material. The method can quickly and accurately perform qualitative and quantitative analysis on related substances of a test sample, and ensures controllability of quality.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for related substance analysis of benzamide starting materials. Background Technology

[0002] A benzamide compound, compound I, has the following structure:

[0003]

[0004] This drug has been approved for marketing in Japan and China. The preparation route of compound I disclosed in the literature (Chinese Journal of New Drugs, 2004, Vol. 6, pp. 536-538) and Examples 1 and 2 of patent CN1284772C is as follows:

[0005]

[0006] The route involves two starting materials:

[0007]

[0008] Currently, there is a lack of methods to accurately separate and detect related substances from the starting material of Compound I. Summary of the Invention

[0009] On one hand, the present invention provides an analytical method for related substances of compound S1, characterized in that the method is high-performance liquid chromatography (HPLC) using a reversed-phase column, with mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is an aqueous solution of dipotassium hydrogen phosphate and mobile phase B is acetonitrile, eluting isocratic or gradient, and the structural formula of compound S1 is...

[0010]

[0011] In some embodiments, the concentration of the dipotassium hydrogen phosphate aqueous solution is selected from 0.0213 to 0.0288 mol / L; preferably, the concentration of the dipotassium hydrogen phosphate aqueous solution is selected from 0.0225 to 0.0275 mol / L; more preferably, the concentration of the phosphoric acid aqueous solution is 0.025 mol / L.

[0012] In some embodiments, mobile phase A and mobile phase B are eluted in a gradient manner according to the following procedure:

[0013]

[0014] Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

[0015] In some embodiments, the flow rate of the eluent is 0.9 to 1.1 mL / min; preferably, the flow rate of the eluent is 1 mL / min.

[0016] In some embodiments, the reversed-phase column uses a nonpolar stationary phase as packing material; preferably, the reversed-phase column uses octadecylsilane-bonded silica gel as packing material; more preferably, the reversed-phase column is selected from Waters Xterra MS C18, Agilent Extend C18, Waters Xbridge C18 or YMC Triart C18; even more preferably, the reversed-phase column is YMC Triart C18 with dimensions of 4.6 mm × 250 mm and 5 μm.

[0017] In some embodiments, the detection method is performed on a high-performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high-performance liquid chromatograph using an ultraviolet detector with a detection wavelength of 258 nm to 262 nm; more preferably 260 nm.

[0018] In some embodiments, the column temperature of the reversed-phase chromatographic column is 20–40°C; preferably, the column temperature of the reversed-phase chromatographic column is 25–35°C; more preferably, the column temperature of the reversed-phase chromatographic column is 30°C.

[0019] On the other hand, the present invention provides an analytical method for related substances of compound S1, characterized in that the method is high-performance liquid chromatography (HPLC), employing a reversed-phase column, a UV-Vis detector with a detection wavelength of 260 nm, and a column temperature of 30 °C; the analytical method uses mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is a 0.025 mol / L dipotassium hydrogen phosphate aqueous solution, and mobile phase B is acetonitrile; gradient elution is performed according to the following program:

[0020]

[0021] Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

[0022] The eluent flow rate was 1.0 mL / min; the test solution, reference solution, and system suitability solution of compound S1 were injected separately; the reference solution was a solution containing compound S1; the system suitability solution contained compound S1 and one or more compounds selected from S1-1, S1-2, S1-3, S1-4, and / or compound S1-5; the contents of compounds S1-1, S1-2, S1-3, S1-4, and / or compound S1-5 in the test sample were calculated by the external standard method of principal component; wherein the structures of compounds S1-1, S1-2, S1-3, S1-4, and compound S1-5 are as follows:

[0023]

[0024] In some typical embodiments, the present invention provides an analytical method for related substances of compound S1, characterized by comprising the following steps:

[0025] Optionally, the preparation of the positioning solution of compound S1-1 includes step (1): accurately weigh the reference standard of compound S1-1, dissolve and dilute it in a mixed solvent of methanol and water, make up to volume, and shake well to obtain the solution.

[0026] The compound S1-1 has the following structural formula:

[0027] Optionally, step (2) involves the preparation of a localization solution for compound S1-2: accurately weigh compound S1-2 reference standard, dissolve and dilute it in a mixed solvent of methanol and water, make up to volume, and shake well to obtain the solution.

[0028] The compound S1-2 has the following structural formula:

[0029] Optionally, step (3) involves the preparation of the target solution for compound S1-3: accurately weigh compound S1-3 reference standard, dissolve and dilute it in a mixed solvent of methanol and water, make up to volume, and shake well to obtain the solution.

[0030] The compound S1-3 has the following structural formula:

[0031] Optionally, step (4) involves the preparation of the target solution for compound S1-4: accurately weigh compound S1-4 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0032] The compound S1-4 has the following structural formula:

[0033] Optionally, step (5) involves the preparation of the target solution for compound S1-5: accurately weigh compound S1-5 reference standard, dissolve and dilute it in a mixed solvent of methanol and water, make up to volume, and shake well to obtain the solution.

[0034] The compound S1-5 has the following structural formula:

[0035] (6) Preparation of system suitability solution: Take an appropriate amount of compound S1 reference standard, dissolve it in a mixed solvent of methanol and water, and mix it with one or more of the reference standards of compounds S1-1, S1-2, S1-3, S1-4 and S1-5 that have been dissolved and diluted in a mixed solvent of methanol and water to prepare a system suitability solution.

[0036] (7) Preparation of test solution: Take an appropriate amount of test sample, weigh it accurately, add solvent to dissolve and dilute it, make up to volume, shake well, and the solution is ready;

[0037] (8) Preparation of reference solution: Take an appropriate amount of compound S1 reference standard, accurately weigh it, dissolve and dilute it with solvent, make up to volume, and shake well to obtain the solution;

[0038] (9) Determination of test sample: Accurately measure the test sample solution, reference solution and system suitability solution, and inject them into the liquid chromatograph respectively. Record the chromatograms. The chromatographic conditions are as follows:

[0039] The chromatographic column was a YMC Triart C18, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The column temperature was 30 °C. Mobile phase A was 0.025 mol / L dipotassium hydrogen phosphate aqueous solution, and mobile phase B was acetonitrile. The flow rate was 1.0 mL / min, the detection wavelength was 260 nm, and the injection volume was 5 μl. Gradient elution was performed according to the following program:

[0040]

[0041] (10) Content calculation:

[0042]

[0043] In the formula: C R Indicates the concentration of the reference solution; A R Indicates the peak area of ​​the reference solution; A t Indicates the peak area of ​​a known impurity; F represents the correction factor for the impurity and the reference standard; D represents the dilution factor; m represents the sample weight; A max ∑A represents the area of ​​the largest impurity peak other than the known impurities; ∑A represents the sum of the areas of all other impurity peaks other than the known impurities.

[0044] The method includes at least any one of steps (1) to (5).

[0045] In some implementations, the analysis method includes steps (1) to (5) simultaneously.

[0046] Those skilled in the art will readily understand that, in implementing this invention, the order of the above steps can be appropriately adjusted without affecting the implementation of the assay method, such as the order of preparation of the positioning solution, reference solution, system suitability solution, and test solution.

[0047] In some embodiments, the content of compound S1-1 in the test solution is not higher than 1.5%, preferably not higher than 1.0%; the content of compound S1-2 is not higher than 0.5%, preferably not higher than 0.3%; the content of compound S1-3 is not higher than 5.0%, preferably not higher than 3.0%; the content of compound S1-4 is not higher than 0.5%, preferably not higher than 0.3%; and the content of compound S1-5 is not higher than 5.0%, preferably not higher than 4.0%.

[0048] In some embodiments, the concentration of compounds S1-1, S1-2, S1-3, S1-4 or S1-5 in the system suitability solution is selected from 1.0 μg / mL to 2.0 μg / mL; preferably 1.25 μg / mL to 1.75 μg / mL; more preferably 1.5 μg / mL.

[0049] In some embodiments, the concentration of compound S1 in the system suitability solution is selected from 0.25 mg / mL to 0.75 mg / mL; preferably 0.375 mg / mL to 0.625 mg / mL; more preferably 0.5 mg / mL.

[0050] In some embodiments, the concentration of the reference solution is selected from 0.13 μg / mL to 6.68 μg / mL; preferably 1.0 μg / mL to 2.0 μg / mL; more preferably 1.5 μg / mL.

[0051] On the other hand, the present invention provides the use of compounds S1-1, S1-2, S1-3, S1-4 or S1-5 for preparing impurity reference standards for related substance analysis methods of compound S1.

[0052] Furthermore, this invention provides an analytical method for related substances of compound S2, characterized in that the method is high-performance liquid chromatography (HPLC), employing a reversed-phase column and using mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is a mixed solution of acetonitrile and methanol, eluting isocratic or gradient; wherein the structural formula of compound S2 is...

[0053]

[0054] In some embodiments, the concentration of the ammonium acetate aqueous solution is selected from 9 to 11 mmol / L; preferably, the concentration of the ammonium acetate aqueous solution is 10 mmol / L.

[0055] In some embodiments, the volume ratio of acetonitrile to methanol in the mobile phase B is selected from 48:52 to 52:48; preferably, the volume ratio of acetonitrile to methanol is 50:50.

[0056] In some embodiments, mobile phase A and mobile phase B are eluted in a gradient manner according to the following procedure:

[0057]

[0058] Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

[0059] In some embodiments, the flow rate of the eluent is 0.9 to 1.1 mL / min; in some typical embodiments, the flow rate of the eluent is 1 mL / min.

[0060] In some embodiments, the reversed-phase column uses a nonpolar stationary phase as packing material; in some typical embodiments, the reversed-phase column uses octadecylsilane-bonded silica gel as packing material; in some more typical embodiments, the reversed-phase column is YMC Triart C18 with dimensions of 4.6 mm × 250 mm and 5 μm.

[0061] In some embodiments, the detection method is performed on a high-performance liquid chromatograph using a diode array detector or an ultraviolet detector; in some typical embodiments, the analysis method is performed on a high-performance liquid chromatograph using an ultraviolet detector with a detection wavelength of 230 nm to 234 nm; preferably 232 nm.

[0062] In some embodiments, the column temperature of the reversed-phase chromatographic column is 20–40°C; in some typical embodiments, the column temperature of the reversed-phase chromatographic column is 25–35°C; and in some more typical embodiments, the column temperature of the reversed-phase chromatographic column is 30°C.

[0063] On the other hand, the present invention provides an analytical method for related substances of compound S2, characterized in that the method is high-performance liquid chromatography (HPLC), employing a reversed-phase column, using an ultraviolet absorption detector with a detection wavelength of 232 nm, and a column temperature of 30 °C; the analytical method uses mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is a 10 mmol / L ammonium acetate aqueous solution, and mobile phase B is a mixed solution of acetonitrile and methanol, eluted according to the following gradient program:

[0064]

[0065] Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

[0066] The eluent flow rate is 1.0 mL / min; the reference solution is a solution containing compound S2; the system suitability solution contains compound S2 and a mixture of one or more compounds selected from S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and / or compound S2-9; wherein the structures of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and compound S2-9 are as follows:

[0067]

[0068] In some specific embodiments, the present invention provides an analytical method for related substances of compound S2, characterized by comprising the following steps:

[0069] Optionally, the preparation of the positioning solution of compound S2-1 includes step (1): accurately weigh the reference standard of compound S2-1, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0070] The compound S2-1 has the following structural formula:

[0071] Optionally, step (2) involves the preparation of a localization solution for compound S2-2: accurately weigh compound S2-2 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0072] The compound S2-2 has the following structural formula:

[0073]

[0074] Optionally, step (3) involves the preparation of the target solution for compound S2-4: accurately weigh compound S2-4 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0075] The compound S2-4 has the following structural formula:

[0076] Optionally, step (4) involves the preparation of the target solution for compound S2-5: accurately weigh compound S2-5 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0077] The compound S2-5 has the following structural formula:

[0078] Optionally, step (5) involves the preparation of the target solution for compound S2-6: accurately weigh compound S2-6 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0079] The compound S2-6 has the following structural formula:

[0080] Optionally, step (6) involves the preparation of the target solution for compound S2-7: accurately weigh compound S2-7 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0081] The compound S2-7 has the following structural formula:

[0082] Optionally, step (7) involves the preparation of the target solution for compound S2-8: accurately weigh compound S2-8 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0083] The compound S2-8 has the following structural formula:

[0084] Optionally, step (8) involves the preparation of the positioning solution for compound S2-9: accurately weigh compound S2-9 reference standard, dissolve and dilute it in methanol, make up to volume, and shake well to obtain the solution.

[0085] The compound S2-9 has the following structural formula:

[0086] (9) Preparation of system suitability solution: Weigh appropriate amounts of one or more reference standards selected from compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and S2-9 into a volumetric flask, dilute to the mark with solvent, and shake well to prepare impurity stock solution I; transfer impurity reference standard stock solution I and dilute to volume with solvent to prepare impurity stock solution II; take an appropriate amount of S2 reference standard, dissolve it in solvent, add impurity stock solution II, and dilute quantitatively with solvent to prepare system suitability solution;

[0087] (10) Preparation of test solution: Take an appropriate amount of test sample, weigh it accurately, add solvent to dissolve and dilute it, make up to volume, shake well, and the solution is ready;

[0088] (11) Preparation of reference solution: Take an appropriate amount of compound S2 reference standard, accurately weigh it, and dilute it with solvent to prepare a solution containing about 22.5 μg per 1 mL, which is used as the reference solution;

[0089] (12) Determination of test sample: Accurately measure the test sample solution, reference solution and system suitability solution, and inject them into the liquid chromatograph respectively. Record the chromatograms. The chromatographic conditions are as follows:

[0090] The chromatographic column was packed with octadecylsilane-bonded silica gel, model YMC Triart C18, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. Mobile phase A was 10 mmol / L ammonium acetate solution, and mobile phase B was a mixture of acetonitrile and methanol at a volume ratio of 50:50. Linear gradient elution was performed according to the table below. The flow rate was 1 mL / min; the column temperature was 30 °C; the detection wavelength was 232 nm; the injection volume was 10 μL; and the gradient elution was performed according to the following program:

[0091]

[0092]

[0093] (13) Content calculation:

[0094]

[0095] The method includes at least any one of steps (1) to (8).

[0096] In some implementations, the analysis method includes steps (1) to (8) simultaneously.

[0097] Those skilled in the art will readily understand that, in implementing this invention, the order of the above steps can be appropriately adjusted without affecting the implementation of the assay method, such as the order of preparation of the positioning solution, reference solution, system suitability solution, and test solution.

[0098] In some embodiments, the content of compound S2-1, S2-2, S2-6 or compound S2-8 in the test solution is not higher than 0.3%, preferably not higher than 0.15%; and the content of compound S2-4, S2-5, S2-7 or compound S-9 is not higher than 0.45%, preferably not higher than 0.3%.

[0099] In some embodiments, the concentrations of compounds S2-1, S2-4, S2-6, S2-9 and S2-5 in the system suitability solution are selected from 4.05 μg / mL to 4.95 μg / mL; preferably 4.275 μg / mL to 4.725 μg / mL; more preferably 4.5 μg / mL.

[0100] In some embodiments, the concentrations of compounds S2-2, S2-7, and S2-8 in the system suitability solution are selected from 2.03 μg / mL to 2.46 μg / mL; preferably 2.14 μg / mL to 2.36 μg / mL; more preferably 2.25 μg / mL.

[0101] In some embodiments, the concentration of the reference solution is selected from 0.091 μg / mL to 15.05 μg / mL; preferably 6.75 μg / mL to 8.25 μg / mL; more preferably 7.5 μg / mL.

[0102] On the other hand, the present invention provides the use of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and S2-9 for preparing impurity reference standards for related substance analysis methods of compound S2.

[0103] In this invention, unless otherwise stated, the compound S1 used for “test preparation” includes, but is not limited to, newly prepared or stored compound S1 or pharmaceutical compositions containing compound S1, preferably newly prepared or stored compound S1.

[0104] In this invention, unless otherwise stated, the compound S2 used for “test preparation” includes, but is not limited to, newly prepared or stored compound S2 or pharmaceutical compositions containing compound S2, preferably newly prepared or stored compound S2.

[0105] In this invention, related substances are also referred to as impurities.

[0106] In this invention, "appropriate amount" means that, according to the experimental purpose, the amount of each compound is within the detection limit or quantitation limit of its high-performance liquid chromatograph.

[0107] In this invention, "ppm" is a concentration expressed as parts per million (ppm) of a certain compound component relative to the total mass of the test sample; it is also called parts per million concentration.

[0108] In this invention, "mL" refers to milliliters; "mg" refers to milligrams; "μg" refers to micrograms; and "min" refers to minutes.

[0109] In this invention, "V:V" or "V / V" refers to a volume ratio.

[0110] The method of the present invention is not limited to the aforementioned substances. Any separation and determination of compound S1 and related substances using the method of the present invention falls within the protection scope of the present invention, particularly the separation and determination of compound S1 and any one of compounds S1-1, S1-2, S1-3, S1-4, S1-5, S1-6 and S1-7 described in the present invention; any separation and determination of compound S2 and related substances using the method of the present invention falls within the protection scope of the present invention, particularly the separation and determination of compound S2 and any one of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and S2-9 described in the present invention.

[0111] The beneficial effects of this invention are:

[0112] The method provided by this invention can accurately determine the identified impurities (compounds S1-1, S1-2, S1-3, S1-4, S1-5, S1-6, and S1-7) in the sample of compound S1, with the resolution between the main peak and adjacent impurity peaks, as well as the resolution between each impurity, being greater than 1.5, and the chromatographic baseline remaining stable. This method can rapidly and accurately perform qualitative and quantitative analysis of related substances in the sample, ensuring quality control.

[0113] The method provided by this invention can accurately determine the identified impurities (compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and S2-9) in the sample of compound S2. The resolution between the main peak and adjacent impurity peaks, as well as the resolution between each impurity, are all greater than 1.5, and the chromatographic baseline is stable. This method can rapidly and accurately perform qualitative and quantitative analysis of related substances in the sample, ensuring quality control. Attached Figure Description

[0114] Figure 1 This is the chromatogram of the blank solution from Example 1.

[0115] Figure 2 This is a chromatogram of the system suitability solution from Example 1.

[0116] Figure 3 This is the chromatogram of the blank solution from Example 9.

[0117] Figure 4 This is a chromatogram of the system suitability solution from Example 9. Detailed Implementation

[0118] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0119] Unless otherwise specified, all reagents and compounds used in the following examples of the present invention are commercially available.

[0120] In the following examples, the structures of compounds S1-1, S1-2, S1-3, S1-4 and S1-5 are as follows:

[0121]

[0122] In the following examples, the structures of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and compound S2-9 are as follows:

[0123]

[0124] Example 1: Analytical method for determination of related substances of compound S1

[0125] 1. Solution preparation:

[0126] The following solution is prepared using a mixed solvent of methanol and water (volume ratio 10:90).

[0127] 1) Test solution: Take an appropriate amount of compound S1, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 0.5 mg per 1 mL.

[0128] 2) Reference solution: Take an appropriate amount of compound S1 reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 1.5 μg per 1 mL.

[0129] 3) System suitability solution: Take an appropriate amount of compound S1 reference standard, add an appropriate amount of solvent to dissolve it, accurately measure an appropriate amount of the above impurity reference standard stock solution, and quantitatively dilute it with solvent to prepare a mixed solution containing approximately 0.5 mg of compound S1 and 1.5 μg of each impurity per 1 mL.

[0130] 4) Blank solvent: A mixture of methanol and water (volume ratio 10:90)

[0131] 2. Chromatographic conditions:

[0132] Chromatographic column: YMC Triart C18 (250mm × 4.6mm, 5μm);

[0133] Mobile phase A: 0.025 mol / L dipotassium hydrogen phosphate aqueous solution;

[0134] Mobile phase B: Acetonitrile;

[0135] Column temperature: 30℃;

[0136] Gradient elution procedure:

[0137]

[0138] Flow rate: 1.0 mL / min

[0139] Detection wavelength: 260nm

[0140] Injection volume: 5 μL

[0141] 3. Determination method:

[0142] Calculation process:

[0143]

[0144] In the formula: C R Indicates the concentration of the reference solution; A R Indicates the peak area of ​​the reference solution; A t Indicates the peak area of ​​a known impurity; F represents the correction factor for the impurity and the reference standard; D represents the dilution factor; m represents the sample weight; A max ∑A represents the area of ​​the largest impurity peak other than the known impurities; ∑A represents the sum of the areas of all other impurity peaks other than the known impurities.

[0145] Take one batch of samples and test them according to the above method. The results are shown in the figure. Figures 1-2 The results of the determination of related substances of compound S1 are shown in Table 1.

[0146] Table 1. Results of determination of related substances of compound S1

[0147] Impurity Name content(%) Compound S1-1 0.00 Compound S1-2 0.00 Compound S1-4 0.00 Compound S1-3 0.47 Compound S1-5 0.34 Other largest single hybrids 0.02 Total impurities 0.84

[0148] Example 2: Specificity test of the analytical method for determining related substances of compound S1

[0149] Solution preparation:

[0150] (1) Solvent: A mixture of methanol and water (volume ratio 10:90)

[0151] (2) Stock solutions of each impurity reference standard: Take appropriate amounts of each of the reference standards of compounds S1-1, S1-2, S1-3, S1-4 and S1-5, dissolve them in solvent and dilute quantitatively to prepare a solution containing about 0.5 mg per 1 mL.

[0152] (3) Impurity positioning solution: Take an appropriate amount of each impurity reference standard stock solution and dilute it with water to prepare a solution containing about 1.5 μg per 1 mL, which shall be used as the impurity positioning solution.

[0153] (4) System suitability: Solution preparation is the same as in Example 1.

[0154] Accurately measure 5 μL each of blank solvent, each impurity localization solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatograms. The specificity test results are shown in Table 2.

[0155] Table 2. Results of specificity test

[0156]

[0157] Note: " / " indicates that it is not applicable.

[0158] Example 3: Linearity and Range Tests of the Analytical Method for the Determination of Related Substances

[0159] Sample preparation:

[0160] (1) Solvent: A mixture of methanol and water (volume ratio 10:90)

[0161] (2) Impurity reference stock solution: Same as in Example 2 (concentration of each impurity: 0.5 mg / mL).

[0162] (3) Linear stock solution: Accurately measure 1 mL of each impurity reference stock solution, place it in the same 10 mL volumetric flask, dilute to the mark with solvent, and shake well (concentration of each impurity: 50 μg / mL).

[0163] (4) Take an appropriate amount of the above linear stock solution and prepare a series of linear solutions with concentrations of 0.10 to 5.08 μg / mL for impurity compound S1-1, 0.11 to 5.42 μg / mL for compound S1-2, 0.10 to 40.99 μg / mL for compound S1-3, 0.11 to 5.33 μg / mL for compound S1-4, 0.12 to 55.40 μg / mL for compound S1, and 0.13 to 6.68 μg / mL for compound S1.

[0164] (5) Accurately measure 5 μL of the above linear solution and inject it into the liquid chromatograph, recording the chromatogram. Plot a standard curve with concentration (μg / mL) on the x-axis and peak area on the y-axis, and calculate the regression equation. The results are shown in Table 3.

[0165] Table 3. Results of Linearity and Range Tests

[0166]

[0167]

[0168] Example 4: Limit of Quantitation and Limit of Detection Tests for Related Substances Determination of Compound S1

[0169] (1) Solvent: A mixture of methanol and water (volume ratio 10:90)

[0170] (2) Reference stock solution: Same as in Example 2.

[0171] (3) Based on the response values ​​of each impurity reference solution, take an appropriate amount of each impurity reference standard, accurately weigh it, place it in the same volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and make the peak height response values ​​of each component basically consistent, and use it as the stock solution. Take an appropriate amount of the stock solution, dilute it stepwise, and use the solution with a signal-to-noise ratio ≥10:1 as the limit of quantitation solution; use the solution with a signal-to-noise ratio of approximately ≥3:1 as the limit of detection solution. The results are shown in Tables 4 and 5.

[0172] Table 4. Limit of Quantification Results

[0173]

[0174] Table 5. Detection Limit Results

[0175]

[0176]

[0177] Example 5: Accuracy test of the analytical method for determining related substances of compound S1

[0178] The solution is prepared as follows:

[0179] (1) Solvent: A mixture of methanol and water (volume ratio 10:90)

[0180] (2) Reference stock solution: Weigh 15 mg of each of impurity S-1, impurity S1-2 and impurity S1-4, place them in a 100 mL volumetric flask, and dilute to the mark with solvent. Weigh 25 mg of each of impurity S1-3 and impurity S1-5, place them in a 50 mL volumetric flask, and dilute to the mark with solvent.

[0181] (3) Mixed impurity stock solution: Accurately measure 5 mL of each of the impurity compound S-1, S1-2 and S1-4 control stock solutions, place them in the same 50 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0182] (4) Reference solution: Prepared in the same way as in Example 2.

[0183] (5) Test solution: Prepared in the same way as in Example 2.

[0184] (6) Recovery solution: Weigh approximately 10 mg of the test sample accurately, place it in a 20 mL volumetric flask, add an appropriate amount of impurity stock solution, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0185] Accurately measure 5 μL of each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. Calculate the recovery rate by peak area using the principal component external standard method with correction factors. The recovery rates of each impurity at concentrations equivalent to 50%, 100%, and 150% of the limit concentration are shown in Table 6 below.

[0186] Table 6. Recovery Rate Results

[0187]

[0188] Example 6: Precision test of the analytical method for determining related substances of compound S1

[0189] 1. Sample injection precision

[0190] The reference solution from Example 2 was injected six times consecutively, and the chromatograms were recorded. The peak areas and retention times RSDs were calculated. The results are shown in Table 7.

[0191] Table 7. Results of the injection precision test

[0192]

[0193] 2. Repeatability

[0194] Reference solution: Take the reference solution from Example 5.

[0195] 100% spiked test solution: Take the 100% concentration level recovery solution under the "Accuracy" section.

[0196] Accurately measure 5 μL each of the reference solution and the 100% spiked test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity using the principal component external standard method with correction factors. The results are shown in Table 8.

[0197] Table 8. Repeatability test results of the test sample (%)

[0198] name content(%) RSD (%) Compound S1-1 0.23 2.9 Compound S1-2 0.21 2.9 Compound S1-4 0.23 3.0 Compound S1-3 2.85 6.1 Compound S1-5 3.31 2.5

[0199] 3. Intermediate precision

[0200] To investigate the effect of random variations on precision, another analyst independently established a system, prepared six new spiked test solutions, and conducted tests on different dates using different instruments.

[0201] Accurately measure 5 μL each of the reference solution and the 100% spiked test solution under the "Repeatability" section, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity using the principal component external standard method with correction factors. Statistical analysis of the intermediate precision and repeatability results for 12 samples is shown in Table 9.

[0202] Table 9. Results of intermediate precision test of the test sample

[0203] name content(%) RSD (%) Compound S1-1 0.22 5.4 Compound S1-2 0.21 3.8 Compound S1-4 0.23 4.0 Compound S1-3 2.87 4.5 Compound S1-5 3.27 3.8

[0204] Example 7 Solution stability test of related substances determination method for compound S1

[0205] Reference solution: Same as the reference solution in Example 2.

[0206] Test solution: Same as the test solution in Example 2.

[0207] Accurately measure 5 μL of the reference solution and the test solution, and inject them into the liquid chromatograph at the corresponding time points under ambient light conditions at room temperature. Record the chromatograms. The results are shown in Tables 10 and 11.

[0208] Table 10. Results of stability test of reference solution

[0209]

[0210] Note: " / " indicates that it is not applicable.

[0211] Table 11. Results of the stability test of the test sample solution

[0212]

[0213] Note: " / " indicates that it is not applicable.

[0214] Example 8. Robustness test of the analytical method for determining related substances of compound S1

[0215] Under different experimental conditions, 5 μL of the system suitability solution was injected into the liquid chromatograph, and the chromatograms were recorded. The resolution between the components in the system suitability solution was examined. The results are shown in Tables 12 and 13.

[0216] Table 12. Robust Chromatographic Conditions

[0217]

[0218] Note: Column 1 and Column 2 are the same model but different batches of chromatographic columns, model YMC Triart C18 (4.6mm×250mm, 5μm).

[0219] Table 13. Durability Results - System Suitability

[0220]

[0221] Note: " / " indicates that it is not applicable.

[0222] Comparative Example 1

[0223] 1. Investigation of mobile phase pH

[0224] The chromatographic column was a Waters Xterra MS C18 (250 × 4.6 mm, 5 μm); the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; the wavelength was 260 nm; mobile phase A was ①: 0.01 mol / L potassium dihydrogen phosphate aqueous solution (pH 3.0) or ②: 0.02 mol / L ammonium acetate aqueous solution (pH 6.0) or ③: 0.01 mol / L dipotassium hydrogen phosphate aqueous solution (pH 9.0); mobile phase B was acetonitrile; gradient elution was performed according to the following program:

[0225]

[0226] The solution was prepared in the same manner as in Example 1. Under alkaline conditions (dipotassium hydrogen phosphate solution, pH = 9), the peak shapes of compound S1 and impurities were better, while the peak shapes were worse under conditions ① and ②.

[0227] 2. Column Observation

[0228] To improve the separation between impurities, different types of chromatographic columns were screened. The column types are as follows:

[0229] ①Waters Xterra MS C18 (250×4.6mm, 5μm)

[0230] ②YMC Triart C18(250×4.6mm,5μm)

[0231] ③Agilent Extend C18 (250×4.6mm, 5μm)

[0232] ④Waters XBridge C18 (250×4.6mm, 5μm)

[0233] ⑤Agilent XDB Phenyl(250×4.6mm,5μm)

[0234] ⑥YMC Triart PFP(250×4.6mm,5μm)

[0235] Other conditions are the same as in "1. Investigation of mobile phase pH", and the results are shown in Table 14.

[0236] Table 14. Separation effect of different chromatographic columns

[0237]

[0238] Example 9. Analytical method for determining related substances of compound S2

[0239] 1. Solution preparation:

[0240] The following solution is prepared using a mixed solvent of methanol and water (volume ratio 10:90).

[0241] 1) Test solution: Take about 15 mg of compound S2 and place it in a 10 mL volumetric flask. Add solvent to dissolve it and then dilute it quantitatively with water to prepare a solution containing about 1.5 mg per mL.

[0242] 2) Reference solution: Weigh approximately 9 mg of compound S2 reference standard accurately, place it in a 100 mL volumetric flask, dissolve it in solvent, and dilute quantitatively with water to prepare a solution containing approximately 90 μg per mL; transfer 5 mL to a 20 mL volumetric flask and dilute with solvent to prepare a solution containing approximately 22.5 μg per mL.

[0243] 3) System Suitability Solution: Take appropriate amounts of impurity reference standards S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and S2-9, dissolve them in solvent, and quantitatively dilute to prepare a mixed solution containing approximately 450 μg each of impurity S2-1, S2-4, S2-6, S2-9, and S2-5 per mL, and 225 μg each of impurity S2-2, S2-7, and S2-8 per mL, as impurity stock solution I. Transfer 1 mL of impurity reference standard stock solution I to a 10 mL volumetric flask and dilute to volume with solvent to prepare impurity stock solution II. Take 15 mg of compound S2 reference standard and place it in a 10 mL volumetric flask. Add 1 mL of solvent to dissolve it, then add 1 mL of impurity stock solution II and quantitatively dilute with water to prepare a solution containing approximately S2 per mL. A mixed solution containing 1.5 mg of impurities S2-1, S2-4, S2-6, S2-9 and S2-5, 4.5 μg each of impurities S2-2, S2-7 and S2-8, and 2.25 μg each of impurities S2-2, S2-7 and S2-8.

[0244] 2. Chromatographic conditions:

[0245] The chromatographic column was a YMC Triart C18, 4.6 mm × 250 mm, 5 μm; the mobile phase A was 10 mmol / L ammonium acetate aqueous solution, and the mobile phase B was a mixed solution of acetonitrile and methanol at a volume ratio of 50:50; the flow rate was 1 mL / min; the column temperature was 30 ℃; the detection wavelength was 232 nm; the injection volume was 10 μL; and gradient elution was performed according to the following program:

[0246]

[0247]

[0248] 3. Determination method:

[0249] Calculation process: Principal component external standard method with correction factor

[0250]

[0251] In the formula: C R Indicates the concentration of the reference solution; A R Indicates the peak area of ​​the reference solution; A t Indicates the peak area of ​​a known impurity; F represents the correction factor for the impurity and the reference standard; D represents the dilution factor; m represents the sample weight; A max ∑A represents the area of ​​the largest impurity peak other than the known impurities; ∑A represents the sum of the areas of all other impurity peaks other than the known impurities.

[0252] Take one batch of samples and determine them according to the above method. The chromatogram is shown in the figure. Figures 3-4 The results of the determination of related substances of compound S2 are shown in Table 15.

[0253] Table 15. Results of determination of related substances of compound S2

[0254] Impurity Name content(%) S2-1 Not detected S2-2 Not detected S2-4 0.04 S2-5 Not detected S2-6 Not detected S2-7 Not detected S2-8 Not detected S2-9 0.13 Other single-mixed 0.04 Total impurities 0.32

[0255] Example 10 Specificity test of the analytical method for determining related substances of compound S2

[0256] Solution preparation:

[0257] (1) Solvent: A mixture of methanol and water (volume ratio 10:90)

[0258] (2) Stock solution of impurity reference standards: Accurately weigh appropriate amounts of impurity S2-1, impurity S2-4, impurity S2-6, impurity S2-9 and impurity S2-5 reference standards, add solvent, sonicate to dissolve, and quantitatively dilute to prepare a solution containing approximately 225 μg per mL. Accurately weigh appropriate amounts of impurity S2-2, impurity S2-7 and impurity S2-8 reference standards, add solvent, sonicate to dissolve, and quantitatively dilute to prepare a solution containing approximately 112.5 μg per mL.

[0259] (3) Mixed stock solutions: Accurately transfer 5 mL of each stock solution into a 50 mL volumetric flask, dilute the solvent to the mark, and shake well.

[0260] (4) System suitability solution: Take an appropriate amount of compound S2 reference standard, add an appropriate amount of solvent and sonicate to dissolve it. Then add an appropriate amount of solution (3) and solution (4) respectively, and dilute quantitatively with solvent to prepare a mixed solution containing approximately 1.5 mg of compound S2, 4.5 μg each of impurities S2-1, S2-4, S2-6, S2-9 and S2-5, and 2.25 μg each of impurities S2-2, S2-7 and S2-8 per 1 mL. This solution is used as the system suitability solution.

[0261] (5) Specific solutions of impurity reference standards: Accurately pipette appropriate amounts of the stock solutions of impurity reference standards S2-1, S2-4, S2-6, S2-9 and S2-5, respectively, and quantitatively dilute them with solvent to prepare solutions containing approximately 9.0 μg per mL. Accurately pipette appropriate amounts of the stock solutions of impurity reference standards S2-2, S2-7 and S2-8, respectively, and quantitatively dilute them with solvent to prepare solutions containing approximately 4.5 μg per mL.

[0262] (6) The preparation of the test solution is the same as in Example 9.

[0263] Accurately measure 10 μL each of blank solvent, specific solutions for each impurity, system suitability solution, and test solution, and inject them into the liquid chromatograph. Record the chromatograms. The results are shown in Table 16.

[0264] Table 16. Specificity test results of compound S2

[0265]

[0266] Note: "N / A" indicates that it is not applicable;

[0267] Example 11 Solution stability test of related substance determination method for compound S2

[0268] Solvent: A mixture of methanol and water (volume ratio 10:90)

[0269] Reference solution: Accurately pipette an appropriate amount of the stock solution of compound S2 reference standard, dilute it with a solvent to prepare a solution containing approximately 7.5 μg per 1 mL, which is used as the reference solution.

[0270] 100% limit spiked test sample: Accurately weigh an appropriate amount of compound S2 test sample, dissolve it in an appropriate amount of solvent, and then accurately add an appropriate amount of the mixed stock solution under Example 10. Dilute quantitatively with solvent to prepare a mixed solution containing approximately 1.5 mg of compound S2, 4.5 μg each of impurities S2-1, S2-4, S2-6, S2-9 and S2-5, and 2.25 μg each of impurities S2-2, S2-7 and S2-8 per 1 mL.

[0271] The solution was placed at 4℃. At the corresponding time points, 10 μL of the reference solution and the 100% limit spiked test solution were accurately measured and injected into the liquid chromatograph, and the chromatograms were recorded. The results are shown in Tables 17 to 19.

[0272] Table 17. Results of stability test of reference solution

[0273]

[0274] Table 18. Results of Stability Tests on Spiked Test Sample Solutions

[0275]

[0276] Note: " / " indicates that it is not applicable.

[0277] Example 12: Detection limit and quantitation limit experiments of the analytical method for determining related substances of compound S2.

[0278] Take appropriate amounts of stock solutions of compound S2 reference standard and each impurity reference standard, and dilute them stepwise. Prepare six parallel solutions with a signal-to-noise ratio (S / N) ≥ 10 as the limit of quantitation, and calculate the RSD of the peak area. Use a signal-to-noise ratio (S / N) ≥ 3 as the limit of detection. Accurately inject 10 μL of the diluted solution into the liquid chromatograph and record the chromatogram. The results are shown in Tables 19 and 20.

[0279] Table 19. Detection limits of related substances in compound S2 as determined by analytical methods

[0280]

[0281]

[0282] Table 20. Limits of Quantitation (LOQ) Results of Analytical Methods for the Determination of Related Substances in Compound S2

[0283]

[0284] Example 13: Linearity and range experiments of the analytical method for determining related substances of compound S2.

[0285] Solvent: A mixture of methanol and water (volume ratio 10:90)

[0286] Take appropriate amounts of stock solutions of compound S2, impurities S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and compound S2-9, dissolve and dilute them with solvent to prepare a series of linear solutions containing compounds S2, impurities S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and impurity S2-9 at concentrations ranging from the limit of quantitation to 200% of the limit concentration. Accurately measure 10 μL of each of the above linear solutions and inject them into the liquid chromatograph. Record the chromatograms, plot a standard curve with peak area as the ordinate and concentration (μg / mL) as the abscissa, and calculate the regression equation. The results are shown in Table 21.

[0287] Table 21. Results of Linearity and Range Tests

[0288]

[0289]

[0290] Example 14 Precision Experiment of Related Substance Determination Method for Compound S2

[0291] 1. Repetitiveness

[0292] Solvent: A mixture of methanol and water (volume ratio 10:90)

[0293] Reference solution: Weigh approximately 9 mg of compound S2 reference standard accurately and place it in a 100 mL volumetric flask. Add solvent to dissolve the compound and dilute quantitatively with water to prepare a solution containing approximately 90 μg per mL. Transfer 5 mL of the solution to a 20 mL volumetric flask and dilute with solvent to prepare a solution containing approximately 22.5 μg per mL.

[0294] 100% limit spiked test solution: Prepare 100% limit spiked test solution according to Example 11, and prepare 6 parallel solutions.

[0295] Accurately measure 10 μL each of the reference solution and the 100% limit spiked test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity using the principal component external standard method with correction factor. The results are shown in Table 22.

[0296] Table 22. Results of Repeatability Experiments

[0297] name mean RSD Impurity S2-8 0.14％ 5.3％ Impurity S2-2 0.12％ 4.9％ Impurity S2-5 0.52％ 2.6％ Compound S2 / / Impurity S2-7 0.19％ 5.4％ Impurity S2-6 0.32％ 5.2％ Impurity S2-4 0.36％ 3.9％ Impurity S2-9 0.30％ 4.6％ Impurity S2-1 0.27％ 5.2％ Other single-mixed 0.88％ 3.2％ General Miscellaneous 3.71％ 3.3％

[0298] Note: " / " indicates that it is not applicable.

[0299] 2. Intermediate precision

[0300] Prepare the reference solution according to Example 11 and prepare 6 parallel 100% limit spiked test solutions.

[0301] Accurately measure 10 μL each of the reference solution and the 100% limit spiked test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity using the principal component external standard method with correction factor. The results of 12 100% limit spiked test solutions are shown in Table 23.

[0302] Table 23. Intermediate Precision Results

[0303] name mean RSD Range Impurity S2-8 0.16％ 4.4％ / Impurity S2-2 0.14％ 4.3％ / Impurity S2-5 0.32％ 5.9％ / Compound S2 / / / Impurity S2-7 0.18％ 5.5％ / Impurity S2-6 0.41％ 3.6％ / Impurity S2-4 0.37％ 6.3％ / Impurity S2-9 0.35％ 3.5％ / Impurity S2-1 0.31％ 2.9％ / Other single-mixed 0.23％ / 0.13％ General Miscellaneous 2.88％ 9.5％ /

[0304] Note: " / " indicates that it is not applicable.

[0305] 3. Sample injection precision

[0306] The reference solution was prepared according to Example 11. 10 μL of the reference solution was accurately measured and injected into the liquid chromatograph. Six consecutive injections were performed, and the chromatograms were recorded. The retention times and peak areas are shown in Table 24.

[0307] Table 24. Results of Injection Precision Test

[0308] Mean of retention time RSD mean peak area RSD 12.114min 0.1％ 165780 1.5％

[0309] Example 15: Accuracy Experiment of Related Substance Determination Method for Compound S2

[0310] Solvent: A mixture of methanol and water (volume ratio 10:90)

[0311] Test solution: Take about 15 mg of compound S2 and place it in a 10 mL volumetric flask. Add 1 mL of solvent and sonicate to dissolve. Then dilute quantitatively with water to prepare a solution containing about 1.5 mg per mL.

[0312] The reference solution and the 100% limit spiked test solution were prepared according to the "Reproducibility" section of Example 14. 10 μL each of the reference solution, test solution, and 100% limit spiked test solution were accurately measured and injected into the liquid chromatograph. The chromatograms were recorded, and the content of each impurity was calculated using the external standard method. The results are shown in Table 25.

[0313] Table 25. Accuracy Results

[0314]

[0315]

[0316] Example 16 Robustness test of the analytical method for determining related substances of compound S2

[0317] The system suitability solution was prepared according to Example 10. Under different robustness test conditions (Table 26), 10 μL each of blank solvent and system suitability solution were injected into the liquid chromatograph, and the chromatograms were recorded. The flow rate, column temperature, and pH of mobile phase A were adjusted appropriately in the chromatographic conditions to examine the separation of the main peak and impurity peaks in the system suitability solution after the chromatographic conditions were changed. The results are shown in Table 27.

[0318] Table 26. Robust Chromatographic Conditions

[0319]

[0320] Table 27. Durability Results - System Suitability

[0321]

[0322] Note: "-" indicates that it is not applicable.

[0323] Comparative Example 2

[0324] In the following comparative examples, the column temperature was 30℃, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The separation of related substances of compound S2 was tested under different mobile phases, chromatographic columns, and elution gradient conditions (gradient elution conditions 1-3 are shown in Tables 28-30, respectively), and the results are shown in Table 31.

[0325] Table 28. Gradient elution conditions 1

[0326] T / min 0 20 25 25.1 35 A％ 95 20 20 95 95 B％ 5 80 80 5 5

[0327] Table 29. Gradient elution conditions 2

[0328] T / min 0 20 25 25.1 35 A％ 100 100 20 100 100 B％ 0 0 80 0 0

[0329] Table 30. Gradient elution conditions 3

[0330] T / min 0 25 26 40 A％ 95 20 95 95 B％ 5 80 5 5

[0331] Table 31. Analytical results under different chromatographic conditions

[0332]

Claims

1. A method for analyzing related substances of a compound S1, characterized in that, The method described is high-performance liquid chromatography (HPLC), employing a reversed-phase column and using mobile phase A and / or mobile phase B as eluents. Mobile phase A is an aqueous solution of dipotassium hydrogen phosphate, and mobile phase B is acetonitrile. Elution is performed isocratic or gradient elution. The structural formula of compound S1 is [insert structural formula here].

2. The method of analysis of claim 1, wherein, The concentration of the dipotassium hydrogen phosphate aqueous solution is selected from 0.0213 to 0.0288 mol / L; preferably, the concentration of the dipotassium hydrogen phosphate aqueous solution is selected from 0.0225 to 0.0275 mol / L; more preferably, the concentration of the phosphoric acid aqueous solution is 0.025 mol / L.

3. The method of analysis of claim 1, wherein, The mobile phase A and mobile phase B are eluted according to the following gradient procedure: Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

4. A method for analyzing related substances of a compound S1, characterized in that, The method is high performance liquid chromatography (HPLC), which uses a reversed-phase column and an ultraviolet absorption detector with a detection wavelength of 260 nm; the column temperature is 30 °C; the analytical method uses mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is a 0.025 mol / L dipotassium hydrogen phosphate aqueous solution and mobile phase B is acetonitrile; Elute using the following gradient procedure: Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent. The eluent flow rate was 1.0 mL / min; the test solution of compound S1, the reference solution, and the system suitability solution were injected separately; the reference solution was a solution containing compound S1. The system suitability solution comprises compound S1 and a mixture of one or more compounds selected from S1-1, S1-2, S1-3, S1-4 and / or S1-5; the content of compounds S1-1, S1-2, S1-3, S1-4 and / or S1-5 in the test sample is calculated by the principal component external standard method; wherein, the structures of compounds S1-1, S1-2, S1-3, S1-4 and S1-5 are as follows:

5. Use of compound S1-1, S1-2, S1-3, S1-4 or compound S1-5 for the preparation of an impurity reference standard for the related substance analytical method of compound S1; wherein, The structures of compounds S1, S1-1, S1-2, S1-3, S1-4 and S1-5 are as follows:

6. A method for analyzing related substances of a compound S2, characterized in that, The method described is high-performance liquid chromatography (HPLC). This method employs a reversed-phase column and uses mobile phase A and / or mobile phase B as eluents. Mobile phase A is an aqueous solution of ammonium acetate, and mobile phase B is a mixed solution of acetonitrile and methanol. Elution is performed isocratic or gradient elution. The structural formula of compound S2 is...

7. The method of analysis of claim 6, wherein, The concentration of the ammonium acetate aqueous solution is selected from 9 to 11 mmol / L; preferably, the concentration of the ammonium acetate aqueous solution is 10 mmol / L.

8. The method of analysis of claim 6, wherein, The mobile phase A and mobile phase B are eluted according to the following gradient procedure: Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

9. A method for analyzing related substances of a compound S2, characterized in that, The method described is high-performance liquid chromatography (HPLC), employing a reversed-phase column and a UV-Vis detector with a detection wavelength of 232 nm; the column temperature is 30 °C; the analytical method uses mobile phase A and / or mobile phase B as eluents, wherein mobile phase A is a 10 mmol / L ammonium acetate aqueous solution, and mobile phase B is a mixed solution of acetonitrile and methanol, eluted according to the following gradient program: Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent. The eluent flow rate is 1.0 mL / min; the reference solution is a solution containing compound S2; the system suitability solution contains compound S2 and a mixture of one or more compounds selected from S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and / or compound S2-9; wherein the structures of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8 and compound S2-9 are as follows:

10. The uses of compounds S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and S2-9 for preparing impurity reference standards for the analytical method related to compound S2. The structures of compounds S2, S2-1, S2-2, S2-4, S2-5, S2-6, S2-7, S2-8, and S2-9 are as follows:

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