Analysis method for related substances of atavalopag intermediate

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to an analysis method for related substances of an atavalopag intermediate. The method can accurately determine the content of related substances in a test sample, the solvent does not interfere with impurity detection, the method is good in specificity, and the detection method is simple, convenient, rapid, accurate, high in sensitivity, good in repeatability and good in accuracy.

Description

Field of the Invention

[0001] The present invention belongs to the technical field of drug analysis, and particularly relates to an analysis method for related substances of an avatrombopag intermediate. Background of the Invention

[0002] Avatrombopag is an orally bioavailable, small-molecule thrombopoietin receptor agonist that stimulates the proliferation and differentiation of megakaryocytes in the bone marrow, thereby increasing platelet production. The product was approved for marketing by the FDA in May 2018.

[0003] The synthetic route of Avatrombopag reported in patent application CN107383000A is as follows:

[0004]

[0005] In this route, the preparation of the intermediate compound I involves the following related substances: compound I-1, compound I-2, compound I-3, and compound I-4, which may affect product quality. Currently, there are no relevant literature or patents reporting analytical methods for compound I and its related substances. Therefore, in order to control the quality of the intermediate compound I, an analytical method for the related substances in compound I, namely compound I-1, compound I-2, compound I-3, and compound I-4, is urgently needed.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides a method for analyzing related substances of Compound I, characterized in that: the method is a high performance liquid chromatography method using a reverse phase chromatography column, an eluent comprising a mixed solution of aqueous phosphoric acid and methanol and acetonitrile, and eluting in an isocratic or gradient manner, wherein the structural formula of Compound I is

[0008] In some embodiments, the concentration of the phosphoric acid aqueous solution is selected from 0.09 to 0.11%; in some typical embodiments, the concentration of the phosphoric acid aqueous solution is 0.1%.

[0009] In some embodiments, mobile phase A and / or mobile phase B are used as eluent, wherein mobile phase A is a mixed solution of 0.1% aqueous phosphoric acid and methanol, and mobile phase B is acetonitrile.

[0010] In some embodiments, the volume ratio of the 0.1% phosphoric acid aqueous solution to methanol in the mobile phase A is 88-92:8-12; in some typical embodiments, the volume ratio of the 0.1% phosphoric acid aqueous solution to methanol in the mobile phase A is 90:10.

[0011] In some embodiments, the mobile phase A and mobile phase B are gradient eluted according to the following program:

[0012]

[0013] ; and during the gradient elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein 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 mobile phase B to the total volume of the eluent.

[0014] 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.

[0015] In some embodiments, the reverse phase chromatographic column uses a non-polar stationary phase as a filler; in some typical embodiments, the reverse phase chromatographic column uses octadecylsilane bonded silica gel as a filler; in some more typical embodiments, the reverse phase chromatographic column is Waters T3, its specifications are 4.6mm×250mm, 5μm.

[0016] 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, and its detection wavelength is 200nm to 220nm; preferably 205nm.

[0017] In some embodiments, the column temperature of the reverse phase chromatography column is 30-40°C; in some typical embodiments, the column temperature of the reverse phase chromatography column is 35°C.

[0018] In another aspect, the present invention provides a method for analyzing related substances of Compound I, characterized in that: the method is a high performance liquid chromatography method, the method uses a reverse phase chromatography column, the detector is an ultraviolet absorption detector, the detection wavelength is 205 nm; the column temperature is 35°C;

[0019] The analytical method uses mobile phase A and / or mobile phase B as eluent, wherein mobile phase A is a mixed solution of 0.1% phosphoric acid aqueous solution and methanol, wherein the volume ratio of 0.1% phosphoric acid aqueous solution to methanol is 90:10; mobile phase B is acetonitrile; and gradient elution is performed according to the following program:

[0020]

[0021] During gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of the eluent;

[0022] The eluent flow rate was 1.0 ml / min;

[0023] Inject the Compound I test solution, reference solution, and system suitability solution respectively;

[0024] The reference solution is a solution containing Compound I;

[0025] The system suitability solution comprises compound I and a mixture of one or more selected from compound I-1, compound I-2, compound I-3 and / or compound I-4;

[0026] The content of compound I-1, compound I-2, compound I-3 and / or compound I-4 in the test sample was calculated by the principal component external standard method.

[0027] Among them, the structures of compound I-1, compound I-2, compound I-3 and compound I-4 are as follows:

[0028]

[0029] In some specific embodiments, the present invention provides a method for analyzing related substances of Compound I, characterized in that it comprises the following steps:

[0030] Optionally, the method comprises the steps of (1) preparing a solution for localizing compound I-1: taking a reference compound I-1, accurately weighing it, dissolving it in methanol and diluting it, fixing the volume, and shaking it well to obtain:

[0031] Wherein, the compound I-1 has the structural formula:

[0032] Optionally, the method comprises the following steps: preparing a solution for localizing compound I-2: taking a reference compound I-2, accurately weighing it, dissolving it in methanol, diluting it, fixing the volume, and shaking it well to obtain:

[0033] Wherein, the compound I-2 has the structural formula:

[0034] Optionally, the method comprises the following steps: preparing a solution for positioning the compound I-3: taking a reference compound of formula III, accurately weighing it, dissolving it in water and diluting it, fixing the volume, and shaking it well to obtain:

[0035] Among them, the compound I-3 has the structural formula:

[0036] Optionally, the method comprises the following steps: preparing a solution for the localization of compound I-4: taking a reference compound I-4, accurately weighing it, dissolving it in methanol, diluting it, fixing the volume, and shaking it well to obtain:

[0037] Among them, the compound I-4 has the structural formula:

[0038] (5) Preparation of Compound I localization solution: Take Compound I reference substance, accurately weigh it, dissolve and dilute it with methanol, make up to volume, and shake well to obtain Compound I, wherein the structural formula of Compound I is:

[0039] (6) Preparation of system suitability solution: Take appropriate amounts of the reference substance of Compound I-1, Compound I-2, and Compound I-4, accurately weigh them, dissolve them in methanol, and dilute them to prepare a solution as standard stock solution A; take appropriate amounts of the reference substance of Compound I-3, accurately weigh them, dissolve them in water, and dilute them to prepare a solution as standard stock solution B; take appropriate amounts of the reference substance of Compound I, dissolve them in methanol, then add appropriate amounts of the above-mentioned standard stock solution A and standard stock solution B, and dilute them with water to prepare a solution as system suitability solution;

[0040] (7) Preparation of test solution: Take an appropriate amount of test sample, weigh accurately, dissolve and dilute with methanol as solvent, make up to volume, shake well, and obtain the solution.

[0041] (8) Preparation of reference solution: Take an appropriate amount of Compound I reference substance, accurately weigh it, add approximately 70% of the volume of the volumetric flask to dissolve it, and dilute it with water to make a solution containing approximately 5 μg per 1 ml. This is the reference solution;

[0042] (9) Test sample determination: Accurately measure 10 μl of each of the test sample solution, reference solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows:

[0043] Chromatographic column: Waters T3 (4.6mm×250mm, 5μm)

[0044] Mobile phase A: a mixture of 0.1% aqueous phosphoric acid and methanol in a volume ratio of 90:10;

[0045] Mobile phase B: acetonitrile

[0046] Column temperature: 35°C;

[0047] Gradient elution program:

[0048]

[0049] Flow rate: 1.0ml / min

[0050] Detection wavelength: 205nm

[0051] Injection volume: 10 μl;

[0052] (10) Calculation of content:

[0053]

[0054]

[0055]

[0056]

[0057] Where: f represents the correction factor of the reference solution of compound I;

[0058] C R Indicates the concentration of Compound I reference solution, mg / ml;

[0059] A R represents the peak area of ​​Compound I reference solution;

[0060] A t Indicates the peak area of ​​each impurity in the test solution;

[0061] A max Indicates the area of ​​the other largest single impurity peak in the chromatogram of the test sample;

[0062] ∑A t It represents the sum of the peak areas of unknown impurities in the chromatogram of the test sample;

[0063] W represents the sample weight, mg; D represents the dilution factor of the sample; F represents the correction factor of each impurity, among which the correction factors of compound I-1, compound I-2, compound I-3, and compound I-4 are 0.67, 1.20, 2.13, and 0.79, respectively.

[0064] The method comprises at least any one of steps (1) to (5).

[0065] In some embodiments, the analysis method comprises steps (1) to (5) simultaneously.

[0066] Those skilled in the art will readily appreciate that, in the implementation of the present invention, the order of the above steps may be appropriately adjusted without affecting the implementation of the assay method, such as the order of preparing the positioning solution, reference solution, system suitability solution, and test solution.

[0067] In some embodiments, in the test solution, the content of compound I-1 is not higher than 1.5%, preferably not higher than 1.0%; the content of compound I-2 is not higher than 0.45%, preferably not higher than 0.3%; the content of compound I-3 is not higher than 0.75%, preferably not higher than 0.5%; and the content of compound I-4 is not higher than 0.75%, preferably not higher than 0.5%.

[0068] In some embodiments, the concentration of Compound 1-1 in the system suitability solution is selected from 2 μg / mL to 20 μg / mL; preferably 5 μg / mL to 15 μg / mL; more preferably 10 μg / mL.

[0069] In some embodiments, the concentration of Compound 1-2 in the system suitability solution is selected from 0.6 μg / mL to 6 μg / mL; preferably 1.5 μg / mL to 4.5 μg / mL; more preferably 3 μg / mL.

[0070] In some embodiments, the concentration of Compound I-3 or Compound I-4 in the system compatibility solution is independently 1 μg / mL to 10 μg / mL; preferably 2.5 μg / mL to 7.5 μg / mL; more preferably 5 μg / mL.

[0071] In some embodiments, the concentration of the reference solution is selected from 1 μg / mL to 10 μg / mL; preferably 2.5 μg / mL to 7.5 μg / mL; more preferably 5 μg / mL.

[0072] In another aspect, the present invention provides compound I-4,

[0073] , its structural formula is:

[0074] In another aspect, the present invention provides the use of compound I-4 for preparing an impurity reference substance for the analysis method of related substances of compound I.

[0075] In another aspect, the present invention provides use of Compound I-1 for preparing an impurity reference substance for an analysis method of related substances of Compound I.

[0076] On the other hand, the present invention provides the use of compound I2 for preparing an impurity reference substance for the analysis method of related substances of compound I.

[0077] In another aspect, the present invention provides the use of compound I-3 for preparing an impurity reference substance for the analysis method of related substances of compound I.

[0078] In the present invention, unless otherwise specified, Compound I used for "test sample preparation" includes but is not limited to newly prepared or stored Compound I or a pharmaceutical composition comprising Compound I, preferably newly prepared or stored Compound I.

[0079] In the present invention, related substances are also referred to as impurities.

[0080] In the present invention, an appropriate amount means that the amount of each compound is within the detection limit or quantification limit of its high performance liquid chromatography according to the experimental purpose.

[0081] In the present invention, the 0.1% phosphoric acid aqueous solution refers to a solution in which the volume ratio of phosphoric acid to water is 0.1%.

[0082] In the present invention, "ppm" is the concentration expressed as the mass of a certain compound component relative to the mass of the total test mass, also known as parts per million concentration;

[0083] In the present invention, "mL" means milliliter; "mg" means milligram; "μg" means microgram; "min" means minute;

[0084] In the present invention, "V:V" means a volume ratio.

[0085] The method of the present invention is not limited to the above-mentioned four related substances. Any separation and determination of compound I and its related substances using the method of the present invention falls within the scope of protection of the present invention, especially the separation and determination of compound I and any of compound I-1, compound I-2, compound I-3 and compound I-4 described in the present invention.

[0086] The technical problem to be solved by the present invention is to provide a high performance liquid chromatography analysis method that can accurately determine the content of related substances in a test sample of Compound I. In this detection method, the solvent does not interfere with the detection of impurities, the method has good specificity, and the detection method is simple, rapid, accurate, highly sensitive, reproducible and accurate.

[0087] The method provided by the present invention can accurately determine the above-identified impurities (Compound I-1, Compound I-2, Compound I-3, and Compound I-4) in the Compound I test sample, with the separation between the main peak and adjacent impurity peaks and the separation between each impurity greater than 1.0, and the chromatogram baseline is stable. This method can quickly and accurately perform qualitative and quantitative analysis of related substances in the test sample, ensuring the controllability of the sample quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Chromatogram of blank solution of Example 2

[0089] Figure 2 Chromatogram of the system suitability solution of Example 2

[0090] Figure 3 Chromatogram of Example 2 test solution

[0091] Figure 4Example 2: Localized solution chromatogram of impurity compound I-1

[0092] Figure 5 Example 2: Localized solution chromatogram of impurity compound I-2

[0093] Figure 6 Example 2: Localized solution chromatogram of impurity compound I-3

[0094] Figure 7 Example 2: Localized solution chromatogram of impurity compound I-4

[0095] Figure 8 Chromatogram of the system suitability solution of the comparative example DETAILED DESCRIPTION

[0096] The following examples may enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the examples.

[0097] Unless otherwise specified, the reagents and compounds used in the following examples of the present invention are commercially available.

[0098] Compound I-4 was isolated by column chromatography:

[0099] DMF (26.44 kg) was added to a 100L enamel reactor, and compound I-1 (3.50 kg) was added under stirring. NBS (2.87 kg) was added in batches at a temperature of -5 to 5°C. After the addition, the temperature was controlled at -5 to 5°C for reaction, and HPLC was monitored until the reaction endpoint. The temperature was controlled at -5 to 10°C, and a 5% aqueous solution of sodium thiosulfate (4.62 kg of sodium thiosulfate was dissolved in 54.43 kg of purified water) was added dropwise to the reaction system. After the addition was completed, the temperature was controlled at -5 to 10°C, and the mixture was stirred for crystallization for 1 hour. Centrifugation was performed, and the filter cake was rinsed with water and centrifuged. After the obtained filter cake was dried, a sample was taken for column chromatography. The silica gel column had a diameter of 6 cm and a column height of 12 cm. Compound I was first eluted with EA:PE=1:2, and then compound I-4 was eluted with DCM:MeOH=15:1. Characterization data of compound I-4: 1 HNMR (500MHz, DMSO-d6): δ (ppm): 7.515 (s, 4H), 7.584 ~ 7.587 (d, J = 1.5Hz, 2H), 7.634 ~ 7.637 (d, J = 1.5Hz, 2H), 13 CNMR (500 MHz, DMSO-d6): δ (ppm): 107.948, 121.727, 123.721, 125.686, 137.855, 145.391, 168.593. [M+H]+ m / z: 462.9.

[0100] In the following examples, the structures of compound I-1, compound I-2, compound I-3 and compound I-4 are as follows:

[0101]

[0102] Example 1 Related Substance Determination Method

[0103] 1. Solution configuration:

[0104] 1) Stock solutions of various impurities: Take an appropriate amount of the reference substance of compound I-1, the reference substance of compound I-2, and the reference substance of compound I-4, accurately weigh them, dissolve them in methanol, and dilute them to prepare solutions containing approximately 100 μg of compound I-1, 30 μg of compound I-2, and 50 μg of compound I-4 per 1 ml, as the stock solutions of the reference substances of compound I-1, compound I-2, and compound I-4; take an appropriate amount of the reference substance of compound I-3, accurately weigh them, dissolve them in water, and dilute them to prepare solutions containing approximately 50 μg per 1 ml, as the stock solution of the reference substance of compound I-3.

[0105] 2) Impurity localization solution: Take 1 ml of each reference substance stock solution and place it in a 10 ml volumetric flask. Add solvent to dilute to the scale and shake well to use as the localization solution for each impurity.

[0106] 3) Test solution: Take an appropriate amount of the product, accurately weigh it, add methanol about 70% of the volume of the volumetric flask to dissolve it, and dilute it with water to make a solution containing about 1.0 mg per 1 ml, which is used as the test solution.

[0107] 4) Reference solution: Take an appropriate amount of this product, weigh accurately, add methanol approximately 70% of the volume of the flask to dissolve it, and dilute with water to make a solution containing approximately 5 μg per 1 ml, which is used as the reference solution.

[0108] 5) System suitability solution: Take appropriate amounts of the reference substance of Compound I-1, Compound I-2, and Compound I-4, accurately weigh them, dissolve them in methanol, and dilute them to prepare a solution containing approximately 100 μg of Compound I-1, 30 μg of Compound I-2, and 50 μg of Compound I-4 per 1 ml as standard stock solution A; take appropriate amounts of the reference substance of Compound I-3, accurately weigh them, dissolve them in water, and dilute them to prepare a solution containing approximately 50 μg per 1 ml as standard stock solution B; take appropriate amounts of the reference substance of Compound I, dissolve it in methanol at approximately 60% of the volume of the volumetric flask, then add appropriate amounts of the above-mentioned standard stock solution A and standard stock solution B, and dilute them with water to prepare a solution containing approximately 1.0 mg of Compound I, 10 μg of Compound I-1, 3 μg of Compound I-2, 5 μg of Compound I-3, and 5 μg of Compound I-4 per 1 ml as the system suitability solution.

[0109] 6) Blank solvent: methanol-water (volume ratio 70:30)

[0110] 2. Chromatographic conditions:

[0111] Chromatographic column: Waters T3 (4.6mm×250mm, 5μm)

[0112] Mobile phase A: 0.1% phosphoric acid aqueous solution-methanol (volume ratio 90:10);

[0113] Mobile phase B: acetonitrile

[0114] Column temperature: 35°C;

[0115] Gradient elution program:

[0116]

[0117] Flow rate: 1.0ml / min

[0118] Detection wavelength: 205nm

[0119] Injection volume: 10 μl

[0120] 3. Determination method: Accurately measure 10 μl of test solution, reference solution and system suitability solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0121] Calculation process:

[0122]

[0123]

[0124]

[0125]

[0126] Where: f represents the correction factor of the reference solution of compound I;

[0127] C R Indicates the concentration of Compound I reference solution, mg / ml;

[0128] A R represents the peak area of ​​Compound I reference solution;

[0129] A t Indicates the peak area of ​​each impurity in the test solution;

[0130] A max Indicates the area of ​​the other largest single impurity peak in the chromatogram of the test sample;

[0131] ∑A tIt represents the sum of the peak areas of unknown impurities in the chromatogram of the test sample;

[0132] W represents the sample weight, mg; D represents the dilution factor of the sample; F represents the correction factor of each impurity, among which the correction factors of compound I-1, compound I-2, compound I-3, and compound I-4 are 0.67, 1.20, 2.13, and 0.79, respectively.

[0133] Three batches of samples were taken and assayed according to the above method. The assay results of related substances of Compound I are shown in Table 1.

[0134] Table 1 Determination results of related substances of compound I

[0135]

[0136] Note: ND stands for Not Detected

[0137] Example 2 Specificity Experiment

[0138] Solvent: methanol-water (volume ratio 70:30)

[0139] Stock solutions of various impurities: Take an appropriate amount of the reference substance of compound I-1, the reference substance of compound I-2 and the reference substance of compound I-4, accurately weigh them, dissolve them in methanol and dilute them to make solutions containing approximately 100 μg of compound I-1, 30 μg of compound I-2 and 50 μg of compound I-4 per 1 ml, as the stock solutions of the reference substances of compound I-1, compound I-2 and compound I-4; take an appropriate amount of the reference substance of compound I-3, accurately weigh it, dissolve it in water and dilute it to make solutions containing approximately 50 μg per 1 ml, as the stock solution of the reference substance of compound I-3.

[0140] Impurity localization solution: Take 1 ml of each reference substance stock solution and place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and use it as the localization solution for each impurity.

[0141] System suitability solution: Accurately weigh approximately 10 mg of the Compound I reference substance in a 10 ml volumetric flask. Dissolve the solution in methanol (approximately 60% of the flask volume). Add an appropriate amount of each impurity stock solution and dilute with water to prepare a solution containing approximately 1.0 mg of Compound I, 10 μg of Compound I-1, 3 μg of Compound I-2, 5 μg of Compound I-3, and 5 μg of Compound I-4 per 1 ml. This solution is used as the system suitability solution.

[0142] Reference solution: Take an appropriate amount of Compound I reference substance, accurately weigh it, add methanol (about 70% of the volume of the flask) to dissolve it, and dilute it with water to make a solution containing about 5 μg per 1 ml, which is used as the reference solution.

[0143] Test solution: Take an appropriate amount of Compound I sample, accurately weigh it, add methanol approximately 70% of the volume of the volumetric flask to dissolve it, and dilute it with water to make a solution containing approximately 1.0 mg per 1 ml, which is used as the test solution.

[0144] Accurately measure 10 μl of blank solvent, each impurity location solution and system suitability solution, inject them into the liquid chromatograph, record the chromatogram, and the results are shown in Figures 1 to 7 The specificity test results are shown in Table 2.

[0145] Table 2 Specificity test results

[0146]

[0147] Note: N / A means no resolution.

[0148] The results showed that there was no interference in the blank. In the system suitability solution chromatogram, the minimum separation between the main component and adjacent impurity peaks was 8.0, and the minimum separation between adjacent impurity peaks was 1.1, both of which were greater than 1.0. The method had good specificity.

[0149] Example 3 Linearity and Range

[0150] Reference substance stock solution: Take an appropriate amount of the compound I-1 reference substance, the compound I-2 reference substance and the compound I-4 reference substance, accurately weigh them, dissolve them in methanol and dilute them to make a solution containing approximately 1 mg of compound I-1, 300 μg of compound I-2 and 500 μg of compound I-4 per 1 ml, as the reference substance stock solutions of compounds I-1, I-2 and I-4; take an appropriate amount of the compound I-3 reference substance, accurately weigh it, dissolve it in water and dilute it to make a solution containing approximately 500 μg per 1 ml, as the reference substance stock solution of compound I-3; take an appropriate amount of the compound I reference substance, add methanol approximately 70% of the volume of the volumetric flask to dissolve it, dilute it with water to make a solution containing approximately 500 μg of compound I per 1 ml, as the reference substance stock solution of compound I.

[0151] Linear stock solution: Accurately measure an appropriate amount of each reference substance stock solution and quantitatively dilute with solvent to prepare a solution containing approximately 50 μg of compound I, 100 μg of compound I-1, 30 μg of compound I-2, 50 μg of compound I-3, and 50 μg of compound I-4 per 1 ml, as the linear stock solution.

[0152] Take appropriate amounts of the above linear stock solution to prepare a series of linear solutions with a quantitation limit up to 200% of the limiting concentration. Accurately measure 10 μl of each linear solution and inject it into a liquid chromatograph. Record the chromatogram. Plot a standard curve with concentration (μg / ml) as the abscissa and peak area as the ordinate, and calculate the regression equation. The results are shown in Table 3.

[0153] Table 3 Linearity and range test results

[0154]

[0155] The results showed that the correlation coefficient r was ≥ 0.998, the response factor RSD was ≤ 10.0%, the y-intercept deviation was ≤ 25%, and there was a good linear relationship between the peak area (y) and the concentration (x).

[0156] Example 4 Quantitation Limit and Detection Limit

[0157] An appropriate amount of Compound I reference substance and each impurity reference substance was accurately weighed and dissolved in solvent to prepare a solution of a desired concentration. The solution was then diluted in series. Accurately aliquot 10 μl of the diluted solution was injected into a liquid chromatograph and the chromatogram recorded. The limit of quantification (LOQ) was determined based on a signal-to-noise ratio (S / N) ≥ 10, and the limit of detection (LOD) was determined based on a signal-to-noise ratio (S / N) ≥ 3. Six consecutive injections of the LOQ solution were performed, and the RSD of the peak area was calculated. The results are shown in Tables 4 and 5.

[0158] Table 4 Quantitation limit results

[0159]

[0160] Note: “ / ” means no calculation is required here

[0161] Table 5 Detection limit results

[0162]

[0163] Note: “ / ” means no calculation is required here

[0164] The results in Tables 3 and 4 indicate that the signal-to-noise ratio (S / N) of the limit of quantification (LOQ) is ≥10, the RSD of the peak retention time of each component in the LOQ solution is no greater than 1.0%, the RSD of the peak area is no greater than 10.0%, and the signal-to-noise ratio (S / N) of the detection limit (LOD) is ≥3. The LOQ and detection limits meet the validation requirements, indicating that the method has good sensitivity.

[0165] Example 5 Accuracy

[0166] Solvent: methanol-water (70:30)

[0167] Reference substance stock solution: Prepare reference substance stock solution as described under "Linearity and Range".

[0168] Mixed impurity stock solution: Accurately measure an appropriate amount of each reference substance stock solution (except compound I) and quantitatively dilute with solvent to prepare a solution containing approximately 100 μg of compound I-1, 30 μg of compound I-2, 50 μg of compound I-3 and 50 μg of compound I-4 per 1 ml, as the mixed impurity stock solution.

[0169] System suitability solution: Accurately weigh approximately 10 mg of the reference compound I into a 10 ml volumetric flask. Dissolve the mixture in methanol (approximately 60% of the flask volume). Add an appropriate amount of the mixed impurity stock solution and dilute with water to prepare a solution containing approximately 1.0 mg of compound I, 10 μg of compound I-1, 3 μg of compound I-2, 5 μg of compound I-3, and 5 μg of compound I-4 per 1 ml. This is the system suitability solution.

[0170] Reference solution: Take an appropriate amount of Compound I reference substance, accurately weigh it, add methanol (about 70% of the volume of the flask) to dissolve it, and dilute it with water to make a solution containing about 5 μg per 1 ml, which is used as the reference solution.

[0171] Test solution: Take an appropriate amount of Compound I sample, accurately weigh it, add methanol approximately 70% of the volume of the volumetric flask to dissolve it, and dilute it with water to make a solution containing approximately 1.0 mg per 1 ml, which is used as the test solution.

[0172] Recovery solution: Accurately weigh approximately 10 mg of Compound I sample and place in a 10 ml volumetric flask. Dissolve in methanol (approximately 70% of the flask volume). Accurately add 0.5 ml, 1.0 ml, and 1.5 ml of the mixed impurity stock solution. Dilute to volume with water and shake well. These solutions are prepared as 50%, 100%, and 150% recovery solutions, respectively. (Prepare three replicates for each concentration level.)

[0173] Accurately measure 10 μl of each of the above solutions, inject them into a liquid chromatograph, record the chromatogram, and calculate the recovery rate based on the peak area of ​​Compound I using the main component external standard method with the addition of a correction factor.

[0174] The recovery results of each impurity at concentration levels equivalent to 50%, 100%, and 150% of the limit concentration are shown in Table 6:

[0175] Table 6 Recovery results

[0176] name Recovery rate (%) Average recovery rate (%) RSD (%) Compound I-1 99.6～102.5 100.7 1.0 Compound I-2 98.3～102.5 100.4 1.3 Compound I-3 100.4～104.0 102.4 1.2 Compound I-4 95.0～101.6 99.2 1.9

[0177] The results showed that within the limit concentration range of 50% to 150%, the recoveries of all impurities were between 80% and 120%, and the RSDs of the recoveries were all less than 10.0%, indicating that the method had good accuracy.

[0178] Example 6 Precision

[0179] 1. Injection precision

[0180] A solution at the 100% limit concentration under "Linearity and Range of Example 3" was injected six times in succession, the chromatogram was recorded, and the RSDs of the peak areas and retention times were calculated. The results are shown in Table 7.

[0181] Table 7 Injection precision test results

[0182]

[0183] The results showed that the peak area RSD was less than 2.0%, the retention time RSD was less than 1.0%, and the instrument injection precision was good.

[0184] 2. Repeatability

[0185] System suitability solution: Same as the system suitability solution under “Accuracy in Example 5”.

[0186] Reference solution: Same as the reference solution under “Accuracy in Example 5”.

[0187] Test solution: Prepare 6 portions in parallel according to the test solution under "Accuracy of Example 5".

[0188] Accurately measure 10 μl each of the reference solution, test solution, and system suitability solution and inject them into the liquid chromatograph. Record the chromatogram and calculate the content of each impurity using the principal component external standard method with the addition of a correction factor. The results are shown in Table 8.

[0189] Table 8 Repeatability test results of test samples

[0190]

[0191] Note: “ / ” means it cannot be calculated

[0192] The results showed that the RSD of each known impurity detected was less than 10%, the range of other single impurities detected was less than 0.05%, and the RSD of the total impurities was less than 10.0%. The method had good repeatability.

[0193] 3. Intermediate precision

[0194] Different operators, at different times, on different instruments, prepared various solutions for testing under the repeatability criteria. The results are shown in Table 9.

[0195] Table 9 Intermediate precision test results of test samples

[0196]

[0197]

[0198] Note: “ / ” means it cannot be calculated

[0199] The results showed that different experimenters, using different types of instruments at different times, had a range of less than 0.05% for each known impurity detected, a range of less than 0.05% for other individual impurities detected, and a RSD of less than 10.0% for the total amount of impurities. This method had good intermediate precision.

[0200] Example 7 Solution Stability

[0201] Reference solution: same as the reference solution under “Specificity of Example 2”.

[0202] Test solution: Same as the test solution under “Specificity of Example 2”.

[0203] Accurately measure 10 μl of each reference solution and test solution and inject them into a liquid chromatograph at the corresponding time points under room temperature. Record the chromatogram. The results are shown in Tables 10 and 11.

[0204] Table 10 Reference solution stability test results

[0205]

[0206] Note: “ / ” means it cannot be calculated

[0207] Table 11 Test solution stability test results

[0208]

[0209]

[0210] Note: “ / ” means cannot be calculated; “ND” means not detected

[0211] The results show that: the reference solution is placed at room temperature for 32 hours, and at different time points, the percentage of the peak area of ​​each known impurity chromatographic peak compared with that at 0h meets the verification requirements of 90.0% to 110.0%, that is, the reference solution is placed at room temperature for at least 32h and the solution stability is good; the test solution is placed at room temperature for 32 hours, and at different time points, compared with the detection amount at 0h, the total impurity range value is <0.05%, and no new impurities of not less than 0.05% are detected, that is, the test solution is placed at room temperature for at least 32h and the solution stability is good.

[0212] Example 8 Durability

[0213] System suitability solution: the same as the system suitability solution under “Example 6 2. Repeatability”.

[0214] Under different durability test conditions (Table 12), 10 μl of each system suitability solution was injected into a liquid chromatograph, and the chromatograms were recorded. The separation between the components in the system suitability solution was examined. The test results are shown in Table 13.

[0215] Table 12 Durability Chromatographic Conditions

[0216]

[0217] Note: Column 1 and Column 2 are the same model but different batches, both of which are Waters T3 (4.6 mm × 250 mm, 5 μm).

[0218] Table 13 Durability results - system suitability

[0219]

[0220] Note: “-” means no separation;

[0221] The results showed that under the condition of slight changes in system parameters, the separation between each known impurity and its adjacent impurities was greater than 1.0, and the determination method had good durability.

[0222] Comparative Example

[0223] Chromatographic column: Waters T3 (4.6mm×250mm, 5μm)

[0224] Mobile phase A: 0.1% phosphoric acid aqueous solution

[0225] Mobile phase B: methanol

[0226] Flow rate: 1.0 ml / min;

[0227] Detection wavelength: 205nm,

[0228] Column temperature: 40℃

[0229] Injection volume: 10 μL

[0230] Gradient elution program:

[0231]

[0232] Sample preparation:

[0233] Solvent: methanol-water (60:40)

[0234] Test solution: Take an appropriate amount of compound I, dissolve it in methanol-water (60:40) and dilute it to make a solution containing approximately 1 mg per 1 ml.

[0235] Compound I-1 localization solution: Take an appropriate amount of compound I-1, dissolve it in methanol and dilute it to make a solution containing about 500 μg per 1 ml.

[0236] Compound I-2 localization solution: Take an appropriate amount of compound I-2, dissolve it in methanol and dilute it to make a solution containing about 100 μg per 1 ml.

[0237] Compound I-3 localization solution: Take an appropriate amount of compound I-3, dissolve it in water and dilute it to make a solution containing about 100 μg per 1 ml.

[0238] System suitability solution: Take appropriate amounts of Compound I and the above impurity localization solutions, dissolve in solvent and dilute to prepare a solution containing 1 mg of Compound I, 10 μg of Compound I-1, 3 μg of Compound I-2, and 5 μg of Compound I-3 per 1 ml.

[0239] Take appropriate amounts of the blank solvent, system suitability solution, each component positioning solution and the test solution, inject them into the liquid chromatograph and record the chromatogram. The chromatogram of the system suitability solution is as follows: Figure 8 As shown: The gradient changes significantly in the figure, and the baseline drifts upward.

Claims

1. A method for analyzing related substances of Compound I, characterized in that: The method is a high performance liquid chromatography method, which uses a reverse phase chromatography column, an eluent comprising a mixed solution of phosphoric acid aqueous solution and methanol and acetonitrile, and isocratic or gradient elution, wherein the structural formula of compound I is 2. The analysis method according to claim 1, wherein: The concentration of the phosphoric acid aqueous solution is selected from 0.09 to 0.11%; preferably, the concentration of the phosphoric acid aqueous solution is 0.1%.

3. The analysis method according to claim 1, wherein: Mobile phase A and / or mobile phase B are used as eluents, wherein mobile phase A is a mixed solution of 0.1% phosphoric acid aqueous solution and methanol, and mobile phase B is acetonitrile.

4. The analysis method according to claim 3, wherein: The volume ratio of the 0.1% phosphoric acid aqueous solution and methanol in the mobile phase A is 88-92:8-12; preferably, the volume ratio of the 0.1% phosphoric acid aqueous solution and methanol in the mobile phase A is 90:

10.

5. The analysis method according to claim 3, wherein: The mobile phase A and mobile phase B were eluted by the following gradient procedure: ； During gradient elution, the sum of the mobile phase A ratio and the mobile phase B ratio is 100%; the mobile phase A ratio refers to the percentage of the mobile phase A volume in the total eluent volume, and the mobile phase B ratio refers to the percentage of the mobile phase B volume in the total eluent volume.

6. The analysis method according to claim 1, wherein: The flow rate of the eluent is 0.9-1.1 ml / min; preferably, the flow rate of the eluent is 1 ml / min.

7. The analysis method according to claim 1, wherein: The reverse phase chromatographic column uses a non-polar stationary phase as a filler; preferably, the reverse phase chromatographic column uses octadecylsilane bonded silica gel as a filler; more preferably, the reverse phase chromatographic column is Waters T3, its specifications are 4.6mm×250mm, 5μm.

8. A method for analyzing related substances of Compound I, characterized by: The method is a high performance liquid chromatography method, which uses a reverse phase chromatography column, a detector is an ultraviolet absorption detector, a detection wavelength is 205nm, and a column temperature is 35°C. The analytical method uses mobile phase A and / or mobile phase B as eluent, wherein mobile phase A is a mixed solution of 0.1% phosphoric acid aqueous solution and methanol, wherein the volume ratio of 0.1% phosphoric acid aqueous solution to methanol is 90:10, and mobile phase B is acetonitrile, and the gradient elution is performed according to the following program: During gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B in the total volume of the eluent; The eluent flow rate was 1.0 ml / min; Inject the Compound I test solution, reference solution, and system suitability solution respectively; The reference solution is a solution containing Compound I; The system suitability solution comprises compound I and a mixture of one or more selected from compound I-1, compound I-2, compound I-3 and / or compound I-4; The content of compound I-1, compound I-2, compound I-3 and / or compound I-4 in the test sample was calculated by the principal component external standard method. Among them, the structures of compound I-1, compound I-2, compound I-3 and compound I-4 are as follows:

9. A method for analyzing related substances of compound I, characterized in that: The following steps are involved: Optionally, the method comprises the steps of (1) preparing a solution for localizing compound I-1: taking a reference compound I-1, accurately weighing it, dissolving it in methanol and diluting it, fixing the volume, and shaking it well to obtain: Wherein, the compound I-1 has the structural formula: Optionally, the method comprises the following steps: preparing a solution for localizing compound I-2: taking a reference compound I-2, accurately weighing it, dissolving it in methanol, diluting it, fixing the volume, and shaking it well to obtain: Wherein, the compound I-2 has the structural formula: Optionally, the method comprises the following steps: preparing a solution for positioning the compound I-3: taking a reference compound of formula III, accurately weighing it, dissolving it in water and diluting it, fixing the volume, and shaking it well to obtain: Among them, the compound I-3 has the structural formula: Optionally, the method comprises the following steps: preparing a solution for localizing compound I-4: taking a reference compound I-4, accurately weighing it, dissolving it in methanol, diluting it, fixing the volume, and shaking it well to obtain: Among them, the compound I-4 has the structural formula: (5) Preparation of Compound I localization solution: Take Compound I reference substance, weigh accurately, dissolve and dilute with methanol, make up to volume, and shake well to obtain: Wherein, the compound I has the structural formula: (6) Preparation of system suitability solution: Take appropriate amounts of the reference substance of Compound I-1, Compound I-2, and Compound I-4, accurately weigh them, dissolve them in methanol, and dilute them to prepare a solution as standard stock solution A; take appropriate amounts of the reference substance of Compound I-3, accurately weigh them, dissolve them in water, and dilute them to prepare a solution as standard stock solution B; take appropriate amounts of the reference substance of Compound I, dissolve them in methanol, then add appropriate amounts of the above-mentioned standard stock solution A and standard stock solution B, and dilute them with water to prepare a solution as system suitability solution; (7) Preparation of test solution: Take an appropriate amount of test sample, weigh accurately, dissolve and dilute with methanol as solvent, make up to volume, shake well, and obtain the solution. (8) Preparation of reference solution: Take an appropriate amount of Compound I reference substance, accurately weigh it, add approximately 70% of the volume of the volumetric flask to dissolve it, and dilute it with water to make a solution containing approximately 5 μg per 1 ml. This is the reference solution; (9) Test sample determination: Accurately measure 10 μl of each of the test sample solution, reference solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: Waters T3 (4.6mm×250mm, 5μm) Mobile phase A: a mixture of 0.1% aqueous phosphoric acid and methanol in a volume ratio of 90:10; Mobile phase B: acetonitrile Column temperature: 35°C; Gradient elution program: Flow rate: 1.0ml / min Detection wavelength: 205nm Injection volume: 10 μl; (10) Calculation of content: Where: f represents the correction factor of the reference solution of compound I; C R Indicates the concentration of Compound I reference solution, mg / ml; A R represents the peak area of ​​Compound I reference solution; A t Indicates the peak area of ​​each impurity in the test solution; A max Indicates the area of ​​the other largest single impurity peak in the chromatogram of the test sample; ∑A t It represents the sum of the peak areas of unknown impurities in the chromatogram of the test sample; W represents the sample weight, mg; D represents the dilution factor of the sample; F represents the correction factor of each impurity, among which the correction factors of compound I-1, compound I-2, compound I-3, and compound I-4 are 0.67, 1.20, 2.13, and 0.79, respectively.

10. Compound I-4, , its structural formula is:

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