HPLC detection method for LXH-1211 and impurities thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an HPLC (High Performance Liquid Chromatography) detection method for LXH-1211 and impurities thereof. Respectively injecting the control solution, the impurity control solution, the blank solution and the test solution into a liquid chromatograph, recording chromatograms, calculating the content of LXH-1211 in the test solution according to a peak area normalization method, and calculating the content of impurities in the test solution according to a peak area according to an external standard method; wherein the impurities are one or more of ZZ1, ZZ2, ZZ3 or ZZ4. The method is good in reproducibility and stability and excellent in precision.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an HPLC detection method for LXH-1211 and its impurities. Background Art

[0002] LXH-1211, chemical name is 2-(3-methyl-6-((2-methylbenzyl)oxy)-1-phenyl-1 H -pyrazolo[3,4-b]pyridin-5-yl)pyrimidine-4,6-diamine, molecular formula: C 25 H 23 N7O, molecular weight: 437.51, its structural formula is: LXH-1211 is an off-white solid used to treat pulmonary arterial hypertension.

[0003] Pulmonary arterial hypertension (PAH) is a chronic, progressive cardiopulmonary disease. The pathogenesis of PAH is accompanied by cellular proliferation and fibrosis of the pulmonary arterioles caused by various mechanisms and etiologies, leading to persistently elevated pulmonary vascular resistance, pulmonary vascular remodeling, increased cardiac workload, and right ventricular hypertrophy, ultimately causing right heart failure and even death. Although the pathogenesis of PAH begins with abnormal pulmonary circulation, right heart failure is the main cause of mortality from PAH. The clinical manifestation of PAH is a resting mean pulmonary artery pressure (mPAP) ≥25 mmHg as measured by right cardiac catheterization.

[0004] According to statistics, approximately 1% of the global population suffers from PAH, with the incidence rate reaching 5-10% in people over 65. PAH poses a serious threat to human life and health, being referred to as "cancer of the cardiovascular system." Without prompt treatment, the five-year survival rate is only 34%, making the treatment of PAH a persistent medical challenge.

[0005] The advantage of LXH-1211 is that a single drug can simultaneously activate sGC and inhibit AMPK, thereby achieving the dual function of "treating both the symptoms and the root cause."

[0006] A variety of impurities are produced during the synthesis of LXH-1211, so the quality of the product needs to be controlled. Summary of the Invention

[0007] The purpose of the present invention is to provide an HPLC detection method for LXH-1211 and its impurities with good reproducibility and stability and excellent precision.

[0008] The HPLC detection method for LXH-1211 and its impurities described in the present invention is to inject a control solution, an impurity reference solution, a blank solution and a test solution into a liquid chromatograph respectively, record the chromatogram, calculate the content of LXH-1211 in the test solution by the peak area normalization method, and calculate the content of impurities in the test solution by the peak area according to the external standard method; wherein the impurity is one or more of ZZ1, ZZ2, ZZ3 or ZZ4; The HPLC chromatographic conditions include the following: Chromatographic column: Pentafluorophenyl bonded silica gel as filler; Detector: PDA or UV detector; Detection wavelength: 261-265nm; Mobile phase: A mixture of acetate buffer and methanol was used as mobile phase A, and methanol was used as mobile phase B.

[0009] The structural formula of LXH-1211 is as follows: ; The structural formula of ZZ1 is as follows: ; The structural formula of ZZ2 is as follows: ; The structural formula of ZZ3 is as follows: ; The structural formula of ZZ4 is as follows: .

[0010] The column temperature is 28-32°C; the chromatographic column is a PFP column with a column length of 150-250 mm, a column inner diameter of 4.0-4.6 mm, and a filler particle size of 3.5-5 μm; the total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min; the chromatographic conditions of HPLC also include a gradient elution program, which is as follows: 0-3 minutes, mobile phase A accounts for 65-75% of the total volume of the mobile phase, and mobile phase B accounts for 25-35% of the total volume of the mobile phase; 3-25 minutes, mobile phase A accounts for 10% to 15% of the total volume of the mobile phase. Mobile phase A accounts for 45-55% of the total volume of mobile phase, and mobile phase B accounts for 45-55% of the total volume of mobile phase; 25-45 minutes, mobile phase A accounts for 45-55% of the total volume of mobile phase, and mobile phase B accounts for 45-55% of the total volume of mobile phase; 45-45.1 minutes, mobile phase A accounts for 65-75% of the total volume of mobile phase, and mobile phase B accounts for 25-35% of the total volume of mobile phase; 45.1-55 minutes, mobile phase A accounts for 65-75% of the total volume of mobile phase, and mobile phase B accounts for 25-35% of the total volume of mobile phase.

[0011] The volume ratio of acetate buffer to methanol in the mixed solution of acetate buffer and methanol is 47.5-52.5:47.5-52.5.

[0012] The preparation method of acetate buffer is to dissolve ammonium acetate in water, then add glacial acetic acid and triethylamine, and adjust the pH value with phosphoric acid to obtain the acetate buffer; the concentration of the acetate buffer is 0.02-0.05 mol / L.

[0013] The control solution is prepared by precisely measuring the test solution and diluting it with methanol; the concentration of LXH-1211 in the control solution is 2 to 3 μg / mL.

[0014] The impurity reference solution is prepared by precisely measuring the impurity stock solution and diluting it with methanol; the concentrations of ZZ1, ZZ2, ZZ3 and ZZ4 in the impurity reference solution are all 2 to 3 μg / mL.

[0015] The impurity stock solution is prepared by mixing ZZ1 reference substance, ZZ2 reference substance, ZZ3 reference substance and ZZ4 reference substance, dissolving and diluting the mixture with methanol; the concentrations of ZZ1, ZZ2, ZZ3 and ZZ4 in the impurity stock solution are all 40-60 μg / mL.

[0016] The blank solution was methanol.

[0017] The test solution is prepared by weighing the LXH-1211 sample, dissolving and diluting it with methanol; the concentration of LXH-1211 in the test solution is 0.4 to 0.6 mg / mL.

[0018] The beneficial effects of the present invention are as follows: The present invention can simultaneously detect the content of the main component of LXH-1211 and its impurities, thereby facilitating quality control of LXH-1211. The present invention has good reproducibility and stability, excellent precision, and high separation degree of LXH-1211 and its impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the HPLC chromatogram of the test sample spiked solution in Example 1.

[0020] Figure 2 This is a comparison chart of the HPLC chromatograms of the impurity localization solutions in Example 1. From bottom to top, the chart includes the HPLC chromatogram of the impurity localization solution ZZ1, the HPLC chromatogram of the impurity localization solution ZZ2, the HPLC chromatogram of the impurity localization solution ZZ3, and the HPLC chromatogram of the impurity localization solution ZZ4.

[0021] Figure 3 It is the HPLC chromatogram of the test solution in Example 1.

[0022] Figure 4 is the HPLC chromatogram of the control solution in Example 1.

[0023] Figure 5 It is the HPLC chromatogram of the impurity reference substance solution in Example 1.

[0024] Figure 6 It is the HPLC chromatogram of the blank solution in Example 1.

[0025] Figure 7 This is the HPLC chromatogram of the test sample spiked solution in Example 2.

[0026] Figure 8 This is the HPLC chromatogram of the test sample spiked solution in Example 3.

[0027] Figure 9 This is the HPLC chromatogram of the test sample spiked solution in Example 4.

[0028] Figure 10 This is the HPLC chromatogram of the test sample spiked solution in Example 5.

[0029] Figure 11 This is the HPLC chromatogram of the test sample spiked solution in Example 6.

[0030] Figure 12 This is the HPLC chromatogram of the test sample spiked solution in Example 7.

[0031] Figure 13 This is the HPLC chromatogram of the test sample spiked solution in Example 8.

[0032] Figure 14 This is the HPLC chromatogram of the test sample spiked solution in Example 9.

[0033] Figure 15 This is the HPLC chromatogram of the test sample spiked solution in Example 10.

[0034] Figure 16 This is the HPLC chromatogram of the test sample spiked solution in Example 11.

[0035] Figure 17 This is the H NMR spectrum of LXH-1211.

[0036] Figure 18 This is the NMR carbon spectrum of LXH-1211.

[0037] Figure 19 This is the H NMR spectrum of ZZ1.

[0038] Figure 20 This is the NMR carbon spectrum of ZZ1.

[0039] Figure 21 This is the H NMR spectrum of ZZ2.

[0040] Figure 22 This is the NMR carbon spectrum of ZZ2.

[0041] Figure 23 This is the H NMR spectrum of ZZ3.

[0042] Figure 24 This is the C-NMR spectrum of ZZ3.

[0043] Figure 25 This is the H NMR spectrum of ZZ4.

[0044] Figure 26 This is the NMR carbon spectrum of ZZ4.

[0045] Figure 27 It is the HPLC chromatogram of the test sample spiked solution in Comparative Example 1.

[0046] Figure 28 It is the HPLC chromatogram of the test sample spiked solution in Comparative Example 2.

[0047] Figure 29 It is the HPLC chromatogram of the test sample spiked solution in Comparative Example 3.

[0048] Figure 30 It is the HPLC chromatogram of the test sample spiked solution in Comparative Example 4. DETAILED DESCRIPTION

[0049] The present invention is further described below with reference to the following examples.

[0050] Example 1 (1) Solution preparation: Blank solution: methanol; Test solution: Weigh the LXH-1211 sample, dissolve it in methanol and dilute it to obtain the sample solution; the concentration of LXH-1211 in the test solution is 0.5 mg / mL; Control solution: Accurately measure the test solution and dilute it with methanol to obtain the control solution; the concentration of LXH-1211 in the control solution is 2.5 μg / mL; Impurity stock solution: ZZ1 reference substance, ZZ2 reference substance, ZZ3 reference substance and ZZ4 reference substance are mixed, dissolved and diluted with methanol to obtain the impurity stock solution; the concentration of ZZ1, ZZ2, ZZ3 and ZZ4 in the impurity stock solution is 50μg / mL; Impurity reference solution: Accurately measure the impurity stock solution and dilute it with methanol to obtain the impurity reference solution; the concentrations of ZZ1, ZZ2, ZZ3, and ZZ4 in the impurity reference solution are all 2.5 μg / mL; Test sample spike solution: Weigh LXH-1211 sample and place it in a volumetric flask. Accurately measure the impurity stock solution and add it to the volumetric flask. Dissolve and dilute with methanol to obtain the solution. The concentration of LXH-1211 in the test sample spike solution is 0.5 mg / mL, and the concentrations of ZZ1, ZZ2, ZZ3, and ZZ4 are all 2.5 μg / mL. Each impurity localization solution: ZZ1 reference substance, ZZ2 reference substance, ZZ3 reference substance and ZZ4 reference substance were diluted with methanol to obtain ZZ1 impurity localization solution, ZZ2 impurity localization solution, ZZ3 impurity localization solution and ZZ4 impurity localization solution, the concentration of ZZ1 in ZZ1 impurity localization solution was 2.5 μg / mL, the concentration of ZZ1 in ZZ2 impurity localization solution was 2.5 μg / mL, the concentration of ZZ1 in ZZ3 impurity localization solution was 2.5 μg / mL, and the concentration of ZZ1 in ZZ4 impurity localization solution was 2.5 μg / mL; The structural formulas of impurities ZZ1, ZZ2, ZZ3, and ZZ4 are shown in Table 1;

[0051] (2) Chromatographic conditions: Instrument: Shimadzu liquid chromatograph LC-20AT with UV detector; Chromatographic column: Welch Ultimate ® PFP 4.6×250mm 5μm; Detection wavelength: 263nm; Mobile phase A: A mixture of acetate buffer and methanol, wherein the volume ratio of acetate buffer to methanol is 50:50. Acetate buffer is prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water, adding 25 ml of glacial acetic acid and 5 ml of triethylamine, and adjusting the pH to 3.0 with phosphoric acid. The concentration of acetate buffer is 0.05 mol / L. Mobile phase B: methanol; Flow rate: 1.0 mL / min; Column temperature: 30°C; Injection volume: 10 μL; The gradient elution program is shown in Table 2;

[0052] (3) Determination method: Inject 10 μL of each of the control solution, each impurity location solution, impurity reference solution, blank solution, test solution and test solution spiked solution into the liquid chromatograph and record the chromatogram. The results are shown in Figure 1-6 , Table 3 and Table 4; the separation between LXH-1211 and adjacent impurities and between each impurity is greater than 1.5, indicating that this method has good specificity.

[0053] Depend on Figure 1It can be seen that ZZ4, ZZ1, LXH-1211, ZZ3, and ZZ2 appear in order, and the peak area data are shown in Table 3. The resolution between each chromatographic peak is greater than 1.5, indicating good specificity.

[0054]

[0055]

[0056] (4) Limit of quantification and limit of detection: Weigh 5 mg each of ZZ1, ZZ2, ZZ3, and ZZ4, place them in a 100 ml volumetric flask, dissolve them in methanol, dilute to the mark, and shake well to prepare the impurity stock solution; weigh 5 mg of the LXH-1211 reference substance, dissolve them in methanol, dilute to 100 ml, and shake well to prepare the LXH-1211 stock solution. Accurately measure 1.0 ml each of the impurity stock solution and the LXH-1211 stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with methanol, and shake well to prepare the reference solution. Take the reference solution, dilute it stepwise to different concentrations, accurately measure 10 μL, and inject it into the liquid chromatograph until the measured chromatographic peak response value is approximately 10 times and 3 times higher than the noise signal. This is the limit of quantification and limit of detection of each impurity. The test results are shown in Table 5.

[0057]

[0058] (5) Linearity: Take the impurity stock solution and LXH-1211 stock solution and place them in the same volumetric flask, dilute them gradually with methanol to the quantitative limit concentration, and use them as linearity test solution ①; take 0.5 ml of the impurity stock solution and LXH-1211 stock solution, place them in a 20 ml volumetric flask, add methanol to dilute to the scale, shake well, and use them as linearity test solution ②; take 0.8 ml of the impurity stock solution and LXH-1211 stock solution, place them in a 20 ml volumetric flask, add methanol to dilute to the scale, shake well, and use them as linearity test solution ③; take 1.0 ml of the impurity stock solution and LXH-1211 stock solution, place them in a 20 ml volumetric flask, add methanol to dilute to the scale, shake well, and use them as linearity test solution ④; take 1.2 ml of the impurity stock solution and LXH-1211 stock solution, place them in a 20 ml volumetric flask, add methanol to dilute to the scale, shake well, and use them as linearity test solution ⑤. Accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph respectively. Plot a curve of peak area versus concentration, and calculate the regression equation and correlation coefficient. The results are shown in Table 6.

[0059]

[0060] (6) Repeatability: Prepare an impurity reference solution, a control solution, and six parallel sample spiked solutions for determination. In the control solution, the RSD of the LXH-1211 chromatographic peak area should not exceed 5.0%. In the sample spiked solution, the range of each impurity result should not exceed 0.1%, and the range of the total impurity result should not exceed 0.2%. The repeatability test results are shown in Table 7.

[0061]

[0062] (7) Intermediate precision: The impurity reference solution, control solution, and 6 test sample spiked solutions were re-prepared for testing. In the control solution, the RSD of the LXH-1211 chromatographic peak area was no more than 5.0%. In the test sample spiked solution, the range of each impurity result was no more than 0.05%, and the range of the results of the 12 repeatability results was no more than 0.05%. The range of the results of the total impurity content and LXH-1211 content was no more than 0.1%, and the range of the results of the 12 repeatability results was no more than 0.1%. The experimental results are shown in Table 8.

[0063]

[0064] (8) Accuracy: Prepare impurity stock solution, impurity reference solution and test solution. Accurately weigh about 10 mg of LXH-1211, add 1.0 ml of intermediate stock solution (take 1.0 ml of impurity stock solution and place it in a 20 ml volumetric flask, add methanol to dilute to the mark, shake well), place it in a 20 ml volumetric flask, dissolve it with methanol and dilute to the mark, shake well, prepare 3 copies in parallel, as the accuracy test solution 1 for related substances; accurately weigh about 10 mg of LXH-1211, add 0.5 ml of impurity stock solution and place it in a 20 ml volumetric flask, dissolve it with methanol and dilute to the mark, shake well, prepare it in parallel 3 portions were prepared as related substance accuracy test solution 2; approximately 10 mg of LXH-1211 was accurately weighed, added to 1.0 ml of the impurity stock solution, and placed in a 20 ml volumetric flask. The solution was dissolved with methanol and diluted to the mark. The solution was shaken and prepared in parallel for 3 portions as related substance accuracy test solution 3; approximately 10 mg of LXH-1211 was accurately weighed, added to 1.2 ml of the impurity stock solution, and placed in a 20 ml volumetric flask. The solution was dissolved with methanol and diluted to the mark. The solution was shaken and prepared in parallel for 3 portions as related substance accuracy test solution 4. The above impurity reference solution, test solution, and related substance accuracy test solution were taken separately, injected according to the chromatographic conditions, and the chromatograms were recorded. The peak area RSD of the impurity reference solution injected 6 times was ≤5.0%. The recovery rates of the related substance accuracy test solutions were all between 80% and 120%. The experimental results are shown in Tables 9-12.

[0065]

[0066]

[0067]

[0068]

[0069] (9) Solution stability: The control solution, impurity reference solution, and test sample spiked solution were measured at 0, 9, 28, 46, 70, 95, and 120 hours, respectively. If the RSD of the average peak area of ​​the LXH-1211 chromatographic peak in the control solution and the impurity reference solution is no more than 5.0%; the range of the known impurity content in the test sample spiked solution is less than 0.05%, and the range of the total impurity content and LXH-1211 content is less than 0.1%, then the control solution, impurity reference solution, and test sample spiked solution are considered stable within 120 hours. The results of the solution stability test are shown in Tables 13-14. It can be seen from Tables 13-14 that the control solution, impurity reference solution, and test sample spiked solution are stable for at least 120 hours.

[0070]

[0071]

[0072] Example 2 (1) Solution preparation: The preparation methods of the control solution, impurity reference solution, blank solution and test sample spiked solution were the same as those in Example 1; (2) Chromatographic conditions: Detection wavelength: 261 nm; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μL of each of the reference solution, impurity reference solution, blank solution and test sample spiked solution and inject them into the liquid chromatograph. The impurity reference solution is injected twice and the chromatogram is recorded. The results are shown in Figure 7 As shown in Tables 15-16, the separation between LXH-1211 and adjacent impurities and between each impurity in the chromatogram of the spiked solution of the test sample was greater than 1.5, indicating good durability.

[0073]

[0074]

[0075] Example 3 Detection wavelength: 265nm; other steps are the same as in Example 2, and the results are shown in Figure 8 And Tables 17-18.

[0076]

[0077]

[0078] Example 4 Column temperature: 28°C; other steps are the same as in Example 2, and the results are shown in Figure 9 And Tables 19-20.

[0079]

[0080]

[0081] Example 5 Column temperature: 32°C; other steps are the same as in Example 2, and the results are shown in Figure 10 And Tables 21-22.

[0082]

[0083]

[0084] Example 6 Flow rate: 0.95 mL / min; other steps are the same as in Example 2, and the results are shown in Figure 11 And Tables 23-24.

[0085]

[0086]

[0087] Example 7 Flow rate: 1.05 mL / min; other steps are the same as in Example 3, and the results are shown in Figure 12 And Tables 25-26.

[0088]

[0089]

[0090] Example 8 The volume ratio of acetate buffer to methanol was 52.5:47.5; the other steps were the same as in Example 2, and the results were shown in FIG. Figure 13 And Tables 27-28.

[0091]

[0092]

[0093] Example 9 The volume ratio of acetate buffer to methanol was 47.5:52.5; the other steps were the same as in Example 2, and the results were shown in FIG. Figure 14 And Tables 29-30.

[0094]

[0095]

[0096] Example 10 Acetate buffer solution was prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water, adding 25 ml of glacial acetic acid and 5 ml of triethylamine, and adjusting the pH to 2.8 with phosphoric acid. The other steps were the same as in Example 2. The results are shown in FIG. Figure 15 And Tables 31-32.

[0097]

[0098]

[0099] Example 11 Acetate buffer solution was prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water, adding 25 ml of glacial acetic acid and 5 ml of triethylamine, and adjusting the pH to 3.2 with phosphoric acid. The other steps were the same as those in Example 2. The results are shown in FIG. Figure 16 And Tables 33-34.

[0100]

[0101]

[0102] The NMR spectra of LXH-1211, ZZ1, ZZ2, ZZ3 and ZZ4 are shown in Figure 17-26 .

[0103] Comparative Example 1 (1) Solution preparation: Test sample spiked solution: Weigh LXH-1211 sample and place it in a volumetric flask. Accurately measure the impurity stock solution and add it to the volumetric flask. Dissolve and dilute with methanol to obtain the solution. The concentration of LXH-1211 in the test sample spiked solution is 0.5 mg / mL, the concentrations of ZZ1 and ZZ2 are both 2.5 μg / mL, and the concentrations of ZZ3 and ZZ4 are both 5 μg / mL. The preparation method of other solutions is the same as that of Example 1. (2) Chromatographic conditions: Mobile phase: a mixed solution of acetate buffer and methanol, wherein the volume ratio of acetate buffer to methanol is 30:70; the acetate buffer is prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water, adding 25 ml of glacial acetic acid and 5 ml of triethylamine, and adjusting the pH to 3.0 with phosphoric acid; the concentration of the acetate buffer is 0.05 mol / L; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μL of the test sample spiked solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Figure 27 , Figure 27It is shown in the figure that under these experimental conditions, the retention time of the ZZ4 chromatographic peak is too short, the retention time of the ZZ2 chromatographic peak is too long, the chromatographic peak of the main component of LXH-1211 has a tailing phenomenon, the peak shape is poor, and the quantification is inaccurate, indicating that the method of comparative example 1 is not applicable.

[0104] Comparative Example 2 (1) Solution preparation: Test sample spiked solution: Weigh LXH-1211 sample and place it in a volumetric flask. Accurately measure the impurity stock solution and add it to the volumetric flask. Dissolve and dilute with methanol to obtain the solution. The concentration of LXH-1211 in the test sample spiked solution is 0.5 mg / mL, the concentrations of ZZ1 and ZZ2 are both 2.5 μg / mL, and the concentrations of ZZ3 and ZZ4 are both 5 μg / mL. The preparation method of other solutions is the same as that of Example 1. (2) Chromatographic conditions: The gradient elution program is shown in Table 35;

[0105] Other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μL of the test sample spiked solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Figure 28 , Figure 28 It is shown in the figure that under these experimental conditions, the retention time of the ZZ4 chromatographic peak is too short, the separation degree between the LXH-1211 main component chromatographic peak and its adjacent impurity peak is less than 1.5, the baseline separation is not achieved, and the quantification is inaccurate, indicating that the method of comparative example 2 is not applicable.

[0106] Comparative Example 3 (1) The solution was prepared in the same manner as in Example 1; (2) Chromatographic conditions: Acetate buffer was prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water, adding 25 ml of glacial acetic acid and 5 ml of triethylamine, and adjusting the pH to 2.6 with phosphoric acid. The gradient elution procedure was shown in Table 35. Other chromatographic conditions were the same as in Example 1. (3) Determination method: Accurately measure 10 μL of the test sample spiked solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Figure 29 , Figure 29 It is shown in the figure that under these experimental conditions, the chromatographic peak of the main component of LXH-1211 has a tailing phenomenon and a poor peak shape. The separation degree between the chromatographic peak of the main component of LXH-1211 and the subsequent adjacent impurity peak is less than 1.5, and baseline separation is not achieved. The quantification is inaccurate, indicating that the method of comparative example 3 is not applicable.

[0107] Comparative Example 4 (1) The solution was prepared in the same manner as in Example 1; (2) Chromatographic conditions: Acetate buffer was prepared by adding 100 ml of glacial acetic acid and 20 ml of triethylamine to 1000 ml of water, and adjusting the pH to 2.8 with phosphoric acid. The gradient elution procedure is shown in Table 35. Other chromatographic conditions were the same as in Example 1. (3) Determination method: Accurately measure 10 μL of the test sample spiked solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Figure 30 , Figure 30 It is shown in the figure that under these experimental conditions, the chromatographic peak of the main component of LXH-1211 has a tailing phenomenon and a poor peak shape. The detection sensitivity of the chromatographic peak of the main component of LXH-1211 and the impurity peaks is low, indicating that the method of Comparative Example 4 is not applicable.

Claims

1. A HPLC detection method for LXH-1211 and its impurities, characterized in that The control solution, impurity reference solution, blank solution and test solution were respectively injected into the liquid chromatograph, and the chromatogram was recorded. The content of LXH-1211 in the test solution was calculated by the peak area normalization method, and the content of impurities in the test solution was calculated by the peak area according to the external standard method; wherein the impurity is one or more of ZZ1, ZZ2, ZZ3 or ZZ4; The HPLC chromatographic conditions include the following: Chromatographic column: Pentafluorophenyl bonded silica gel as filler; Detector: PDA or UV detector; Detection wavelength: 261-265nm; Mobile phase: A mixture of acetate buffer and methanol was used as mobile phase A, and methanol was used as mobile phase B.

2. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The structural formula of LXH-1211 is as follows: ； The structural formula of ZZ1 is as follows: ； The structural formula of ZZ2 is as follows: ； The structural formula of ZZ3 is as follows: ； The structural formula of ZZ4 is as follows: 。 3. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The column temperature is 28-32°C; the chromatographic column is a PFP column with a column length of 150-250 mm, a column inner diameter of 4.0-4.6 mm, and a filler particle size of 3.5-5 μm; the total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min; the chromatographic conditions of HPLC also include a gradient elution program, which is as follows: 0-3 minutes, mobile phase A accounts for 65-75% of the total volume of the mobile phase, and mobile phase B accounts for 25-35% of the total volume of the mobile phase; 3-25 minutes, mobile phase A accounts for 10% to 15% of the total volume of the mobile phase. Mobile phase A accounts for 45-55% of the total volume of mobile phase, and mobile phase B accounts for 45-55% of the total volume of mobile phase; 25-45 minutes, mobile phase A accounts for 45-55% of the total volume of mobile phase, and mobile phase B accounts for 45-55% of the total volume of mobile phase; 45-45.1 minutes, mobile phase A accounts for 65-75% of the total volume of mobile phase, and mobile phase B accounts for 25-35% of the total volume of mobile phase; 45.1-55 minutes, mobile phase A accounts for 65-75% of the total volume of mobile phase, and mobile phase B accounts for 25-35% of the total volume of mobile phase.

4. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The volume ratio of acetate buffer to methanol in the mixed solution of acetate buffer and methanol is 47.5-52.5: 47.5-52.

5.

5. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The preparation method of acetate buffer is to dissolve ammonium acetate in water, then add glacial acetic acid and triethylamine, and adjust the pH value with phosphoric acid to obtain the acetate buffer; the concentration of the acetate buffer is 0.02-0.05 mol / L.

6. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The control solution is prepared by precisely measuring the test solution and diluting it with methanol; the concentration of LXH-1211 in the control solution is 2 to 3 μg / mL.

7. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The impurity reference solution is prepared by precisely measuring the impurity stock solution and diluting it with methanol; the concentrations of ZZ1, ZZ2, ZZ3 and ZZ4 in the impurity reference solution are all 2 to 3 μg / mL.

8. The HPLC detection method of LXH-1211 and its impurities according to claim 7, characterized in that The impurity stock solution is prepared by mixing ZZ1 reference substance, ZZ2 reference substance, ZZ3 reference substance and ZZ4 reference substance, dissolving and diluting the mixture with methanol; the concentrations of ZZ1, ZZ2, ZZ3 and ZZ4 in the impurity stock solution are all 40-60 μg / mL.

9. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The blank solution was methanol.

10. The HPLC detection method of LXH-1211 and its impurities according to claim 1, characterized in that The test solution is prepared by weighing the LXH-1211 sample, dissolving and diluting it with methanol; the concentration of LXH-1211 in the test solution is 0.4 to 0.6 mg / mL.

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