HPLC method for simultaneous qualitative and quantitative determination of LXH-2103 and its impurities

Abstract

The present invention belongs to the technical field of drug detection, and in particular to a HPLC method for simultaneously qualitatively and quantitatively detecting LXH‑2103 and its impurities. Prepare a test solution, an impurity reference solution and a control solution, inject a blank solution, a test solution, an impurity reference solution and a control solution into a liquid chromatograph respectively, record a chromatogram, calculate the content of LXH‑2103 in the test solution by peak area normalization method, and calculate the content of impurities in the test solution by peak area according to an external standard method; wherein, the impurity is one or more of LXH‑ZZ1, LXH‑ZZ2, LXH‑ZZ3, LXH‑ZZ4 or LXH‑ZZ5. The present invention has good separation between the main peak and each impurity peak, high precision, good stability and good reproducibility, and provides analytical means for LXH‑2103 quality control.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to an HPLC method for simultaneously qualitatively and quantitatively detecting LXH-2103 and its impurities. Background Art

[0002] LXH-2103, chemical name is (±)-3-[(1 RS ,2 RS )-2-[( N , N -dimethylamino)methylene]-1-hydroxycyclohexyl]phenol hydrochloride, molecular formula: C 15 H 23 NO2•HCl, molecular weight: 285.81, its structural formula is: .

[0003] LXH-2103 is a white solid, a weak opioid, used to treat various pains.

[0004] At present, the most commonly used analgesics in clinical practice are non-steroidal anti-inflammatory analgesics and strong opioid analgesics. Although they have achieved good clinical therapeutic effects, due to the limitations of the mechanism of action of non-steroidal anti-inflammatory analgesics, their analgesic effect is weak, and they all have adverse reactions to varying degrees, such as gastrointestinal reactions, gastric ulcers, gastric bleeding and allergic reactions; strong opioids are greatly limited in their clinical application due to adverse reactions such as addiction, abuse, tolerance and withdrawal reactions.

[0005] LXH-2103 can produce analgesic effects without undergoing biological metabolism, which can overcome the problems of weak analgesic effects of non-steroidal anti-inflammatory analgesics and large adverse reactions of strong opioid analgesics, and has extremely high clinical application value.

[0006] There are a variety of process impurities in the synthesis and preparation process of LXH-2103, which play a key role in the quality of the product, so quality control is particularly important. Summary of the invention

[0007] The purpose of the present invention is to provide an HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities, with good separation between the main peak and each impurity peak, high precision, good stability and good reproducibility, so as to provide an analytical means for quality control of LXH-2103.

[0008] The HPLC method for simultaneously qualitatively and quantitatively detecting LXH-2103 and its impurities of the present invention comprises preparing a test solution, an impurity reference solution and a control solution, respectively injecting a blank solution, a test solution, an impurity reference solution and a control solution into a liquid chromatograph, recording a chromatogram, calculating the content of LXH-2103 in the test solution by a peak area normalization method, and calculating the content of impurities in the test solution by peak area by an external standard method; wherein the impurities are one or more of LXH-ZZ1, LXH-ZZ2, LXH-ZZ3, LXH-ZZ4 or LXH-ZZ5;

[0009] The structural formula of LXH-2103 is as follows:

[0010]

[0011] The structural formula of LXH-ZZ1 is as follows:

[0012]

[0013] The structural formula of LXH-ZZ2 is as follows:

[0014]

[0015] The structural formula of LXH-ZZ3 is as follows:

[0016]

[0017] The structural formula of LXH-ZZ4 is as follows:

[0018]

[0019] The structural formula of LXH-ZZ5 is as follows:

[0020] .

[0021] The test solution is prepared by weighing a LXH-2103 sample, dissolving and diluting it with a solvent to prepare a test solution; the concentration of LXH-2103 in the test solution is 1.6-2.4 mg / mL.

[0022] The preparation method of the impurity reference solution is to mix LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution, and then dilute them with a solvent to prepare an impurity reference solution; the concentration of LXH-ZZ1 in the impurity reference solution is 3.2-4.8 μg / mL, and the concentrations of LXH-ZZ2, LXH-ZZ3, LXH-ZZ4 and LXH-ZZ5 are all 1.6-2.4 μg / mL.

[0023] The preparation method of the LXH-ZZ1 impurity stock solution is to weigh the LXH-ZZ1 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ1 impurity stock solution; the concentration of LXH-ZZ1 in the LXH-ZZ1 impurity stock solution is 80-120 μg / mL;

[0024] The preparation method of the LXH-ZZ2 impurity stock solution is to weigh the LXH-ZZ2 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ2 impurity stock solution; the concentration of LXH-ZZ2 in the LXH-ZZ2 impurity stock solution is 40-60 μg / mL;

[0025] The preparation method of the LXH-ZZ3 impurity stock solution is to weigh the LXH-ZZ3 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ3 impurity stock solution; the concentration of LXH-ZZ3 in the LXH-ZZ3 impurity stock solution is 40-60 μg / mL;

[0026] The preparation method of the LXH-ZZ4 impurity stock solution is to weigh the LXH-ZZ4 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ4 impurity stock solution; the concentration of LXH-ZZ4 in the LXH-ZZ4 impurity stock solution is 40-60 μg / mL;

[0027] The preparation method of the LXH-ZZ5 impurity stock solution is to weigh the LXH-ZZ5 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ5 impurity stock solution; the concentration of LXH-ZZ5 in the LXH-ZZ5 impurity stock solution is 40-60 μg / mL.

[0028] The control solution is prepared by precisely measuring the test solution, diluting it with a solvent, and preparing the control solution; the concentration of LXH-2103 in the control solution is 1.6-2.4 μg / mL.

[0029] The solvent is a mixed solution of acetate buffer and methanol, the volume ratio of acetate buffer to methanol is 65-75:25-35, and the acetate buffer is prepared from sodium acetate, glacial acetic acid and water.

[0030] The blank solution is a mixed solution of acetate buffer and methanol, the volume ratio of acetate buffer to methanol is 65-75:25-35, and the acetate buffer is prepared from sodium acetate, glacial acetic acid and water.

[0031] The chromatographic conditions in the HPLC method include a chromatographic column, a detector, a detection wavelength and a mobile phase; the chromatographic column is a C18 column, and the specifications of the chromatographic column are: a column length of 150-250 mm, a column inner diameter of 4.0-4.6 mm, a filler particle size of 3.5-5 μm, octadecylsilane bonded silica gel as a filler, and a column temperature of 28-32°C; the detector is a PDA detector or an ultraviolet detector, and the detection wavelength is 269-273 nm; the mobile phase is acetate buffer as mobile phase A, and methanol as mobile phase B; the total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min.

[0032] The chromatographic conditions also include a gradient elution program, which is as follows: 0-11 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; 11-16 minutes, mobile phase A accounts for 45-55% of the total volume of the mobile phase, and mobile phase B accounts for 45-55% of the total volume of the mobile phase; 16-36 minutes, mobile phase A accounts for 45-55% of the total volume of the mobile phase, and mobile phase B accounts for 45-55% of the total volume of the mobile phase; 36-36.1 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; 36.1-45 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.

[0033] The five process impurities of LXH-2103 are shown in Table 1.

[0034]

[0035] The beneficial effects of the present invention are as follows:

[0036] (1) The stationary phase used in the present invention is octadecylsilane bonded silica gel, which can simultaneously detect the content of LXH-2103 and its impurities, achieving the purpose of dual use in one method.

[0037] (2) The present invention has high separation degree, high accuracy and precision, and good stability and reproducibility for LXH-2103 and its impurities.

[0038] (3) The present invention is simple and easy to implement, and is convenient for effectively controlling the quality of the product LXH-2103 during the process and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The HPLC chromatogram of the test sample spiked solution in Example 1.

[0040] Figure 2This is a comparison chart of the HPLC chromatograms of the impurity localization solutions in Example 1, in which, from bottom to top, they are the HPLC chromatogram of the impurity localization solution of LXH-ZZ1, the HPLC chromatogram of the impurity localization solution of LXH-ZZ2, the HPLC chromatogram of the impurity localization solution of LXH-ZZ3, the HPLC chromatogram of the impurity localization solution of LXH-ZZ4, and the HPLC chromatogram of the impurity localization solution of LXH-ZZ5.

[0041] Figure 3 It is the HPLC chromatogram of the test solution in Example 1.

[0042] Figure 4 It is a chromatogram comparison diagram of the blank solution, the control solution and the impurity reference solution in Example 1. From top to bottom in the figure, they are the chromatogram of the blank solution, the chromatogram of the control solution, and the chromatogram of the impurity reference solution.

[0043] Figure 5 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ①.

[0044] Figure 6 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ②.

[0045] Figure 7 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ③.

[0046] Figure 8 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ④.

[0047] Fig. 9 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ⑤.

[0048] Fig.10 This is the HPLC chromatogram of the test sample spiked solution in method robustness experiment ⑥.

[0049] Fig.11 This is the HPLC chromatogram of the spiked solution of the test sample in method robustness experiment ⑦.

[0050] Fig.12 This is the HPLC chromatogram of the test sample spiked solution in the method robustness experiment ⑧.

[0051] Fig.13 This is the HPLC chromatogram of the test sample spiked solution in the method robustness comparison experiment.

[0052] Fig.14 This is the H NMR spectrum of LXH-2103.

[0053] Fig.15This is the NMR carbon spectrum of LXH-2103.

[0054] Fig.16 This is the H NMR spectrum of LXH-ZZ1.

[0055] Fig.17 This is the NMR carbon spectrum of LXH-ZZ1.

[0056] Fig.18 This is the H NMR spectrum of LXH-ZZ2.

[0057] Fig.19 This is the NMR carbon spectrum of LXH-ZZ2.

[0058] Fig. 20 This is the H NMR spectrum of LXH-ZZ3.

[0059] Fig.21 This is the NMR carbon spectrum of LXH-ZZ3.

[0060] Fig. 22 This is the H NMR spectrum of LXH-ZZ4.

[0061] Fig.23 This is the NMR carbon spectrum of LXH-ZZ4.

[0062] Fig.24 This is the H NMR spectrum of LXH-ZZ5.

[0063] Fig.25 This is the NMR carbon spectrum of LXH-ZZ5. DETAILED DESCRIPTION

[0064] The present invention is further described below with reference to the embodiments.

[0065] The instruments and reagents used in the following examples are all commercially available products: methanol: Beijing Bailingwei Technology Co., Ltd., HPLC grade; sodium acetate: Sinopharm Chemical Reagent Co., Ltd., analytical grade; glacial acetic acid: Sinopharm Chemical Reagent Co., Ltd., analytical grade; sodium hydroxide: Sinopharm Chemical Reagent Co., Ltd., analytical grade.

[0066] Example 1

[0067] (1) Solution preparation:

[0068] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 70:30 as a blank solution;

[0069] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 70:30 as a solvent;

[0070] Weigh the LXH-2103 sample, dissolve it in a solvent and dilute it to make a solution containing 2.0 mg LXH-2103 per 1 mL as the test solution;

[0071] Accurately measure 1.0 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, shake well, then accurately measure 1.0 ml from the 50 ml solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, shake well, as the control solution;

[0072] Weigh the LXH-ZZ1 reference substance, dissolve it in a solvent and dilute it to make a solution containing 100 μg LXH-ZZ1 per 1 mL as the LXH-ZZ1 impurity stock solution;

[0073] Weigh the LXH-ZZ2 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 50 μg of LXH-ZZ2 per 1 mL, which is used as the LXH-ZZ2 impurity stock solution;

[0074] Weigh the LXH-ZZ3 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 50 μg of LXH-ZZ3 per 1 mL, which is used as the LXH-ZZ3 impurity stock solution;

[0075] Weigh the LXH-ZZ4 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 50 μg of LXH-ZZ4 per 1 mL, which is used as the LXH-ZZ4 impurity stock solution;

[0076] Weigh the LXH-ZZ5 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 50 μg of LXH-ZZ5 per 1 mL, which is used as the LXH-ZZ5 impurity stock solution;

[0077] Accurately measure 1 mL of LXH-ZZ1 impurity stock solution, place it in a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to use as the LXH-ZZ1 impurity location solution;

[0078] Accurately measure 1 mL of LXH-ZZ2 impurity stock solution, place it in a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to use as the LXH-ZZ2 impurity location solution;

[0079] Accurately measure 1 mL of LXH-ZZ3 impurity stock solution, place it in a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to use as the LXH-ZZ3 impurity location solution;

[0080] Accurately measure 1 mL of LXH-ZZ4 impurity stock solution, place it in a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to use as the LXH-ZZ4 impurity location solution;

[0081] Accurately measure 1 mL of LXH-ZZ5 impurity stock solution, place it in a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to use as the LXH-ZZ5 impurity location solution;

[0082] Accurately measure 1 mL of LXH-ZZ1 impurity stock solution, 1 mL of LXH-ZZ2 impurity stock solution, 1 mL of LXH-ZZ3 impurity stock solution, 1 mL of LXH-ZZ4 impurity stock solution, and 1 mL of LXH-ZZ5 impurity stock solution, place them in the same 25 mL volumetric flask, dilute to the scale with solvent, shake well, and use as impurity reference solution;

[0083] Weigh 50 mg of LXH-2103 sample and place it in a 25 mL volumetric flask. Add 1 mL of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution, dissolve with solvent and dilute to the scale. Shake well to prepare the sample spike solution.

[0084] (2) Chromatographic conditions:

[0085] Instrument: Shimadzu liquid chromatograph LC-20AT with PDA detector;

[0086] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0087] Detection wavelength: 271nm;

[0088] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0089] Mobile phase B: methanol;

[0090] Flow rate: 1.0 mL / min;

[0091] Column temperature: 30°C;

[0092] Injection volume: 20 μL;

[0093] The gradient elution program is shown in Table 2;

[0094]

[0095] (3) Determination method: Accurately measure 20 μL of blank solution, test solution, each impurity location solution, impurity reference solution, control solution and test solution spiked solution and inject them into the liquid chromatograph respectively. Record the chromatogram. The results are shown in Figure 1-4 , Table 3 and Table 4. The separation between each impurity and between LXH-2103 and adjacent impurities was greater than 1.5, indicating that the method had good specificity.

[0096] Figure 1 is the HPLC chromatogram of the test sample spiked solution, Figure 1 It can be seen that LXH-ZZ3, LXH-2103, LXH-ZZ1, LXH-ZZ4, LXH-ZZ2 and LXH-ZZ5 are chromatographic peaks in sequence, and the peak area data are shown in Table 3. The separation between each chromatographic peak is greater than 1.5, indicating that the method of the present invention has good specificity.

[0097] Figure 2 This is a comparison chart of the HPLC chromatograms of each impurity localization solution. From bottom to top in the chart, they are the HPLC chromatogram of the impurity localization solution of LXH-ZZ1, the HPLC chromatogram of the impurity localization solution of LXH-ZZ2, the HPLC chromatogram of the impurity localization solution of LXH-ZZ3, the HPLC chromatogram of the impurity localization solution of LXH-ZZ4, and the HPLC chromatogram of the impurity localization solution of LXH-ZZ5.

[0098] Figure 3 is the HPLC chromatogram of the test solution, consisting of Figure 3 It can be seen that the test solution contains LXH-ZZ1 and LXH-ZZ2, and the peak area data are shown in Table 4.

[0099]

[0100]

[0101] The methodology validation process is as follows:

[0102] 1. Limit of quantification and limit of detection: Accurately weigh 2.5 mg of LXH-2103 reference substance, dissolve it in solvent and dilute it to 50 ml, shake well, and use it as LXH-2103 stock solution. Accurately measure 1.0 ml of each impurity stock solution and LXH-2103 stock solution and place them in the same 25 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as reference solution. Take the reference solution, dilute it step by step to different concentrations, accurately measure 20 μL, and inject it into the liquid chromatograph until the measured chromatographic peak response value is about 10 times and 3 times higher than the noise signal, which is the limit of quantification and detection limit of each impurity. The test results are shown in Table 5.

[0103]

[0104] 2. Linearity: Take LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution, LXH-ZZ5 impurity stock solution and LXH-2103 stock solution, put them in the same volumetric bottle and dilute them with solvent to the quantitative limit concentration as linear test solution ①; take LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ 3 impurity stock solution, LXH-ZZ4 impurity stock solution, LXH-ZZ5 impurity stock solution and LXH-2103 stock solution 0.5ml each, put them in a 25ml volumetric bottle, add solvent to dilute to the scale, shake well, as the linear test solution ②; take LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution, LXH-ZZ5 0.8 ml of impurity stock solution and LXH-2103 stock solution are placed in a 25 ml volumetric flask, diluted to the mark with solvent, shaken, and linear test solution ③; 1.0 ml of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution, LXH-ZZ5 impurity stock solution and LXH-2103 stock solution are placed in a 25 ml volumetric flask, diluted to the mark with solvent, shaken, and linear test solution ③; 1 ml volumetric bottle, add solvent to dilute to scale, shake well, as linear test solution ④; take 1.2 ml of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution, LXH-ZZ5 impurity stock solution and LXH-2103 stock solution, put them in a 25 ml volumetric bottle, add solvent to dilute to scale, shake well, as linear test solution ⑤. Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph respectively, draw a curve of peak area versus concentration, calculate the regression equation and correlation coefficient, and the results are shown in Table 6.

[0105]

[0106] Accurately weigh 40.25 mg, 45.10 mg, 50.25 mg, 55.23 mg, and 60.19 mg of LXH-2103 reference substance, respectively, and place them in 25 ml volumetric bottles, add solvent to dissolve and dilute to the scale, shake well, and use them as the linear test solution for LXH-2103 content determination. Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, draw a curve based on peak area versus concentration, calculate the regression equation and correlation coefficient, and the results are shown in Table 7.

[0107]

[0108] 3. Repeatability: Prepare impurity reference solution, control solution and 6 test sample spiked solutions for determination. In the control solution, the RSD of the peak area of ​​LXH-2103 chromatographic peak is not greater than 5.0%; in the test sample spiked solution, the range of each impurity result is not greater than 0.05%, and the range of the total impurity content and LXH-2103 content results is not greater than 0.1%. The repeatability test results are shown in Table 8.

[0109]

[0110] 4. Intermediate precision: On different dates from repeatability, 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-2103 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 intermediate precision and repeatability results for 12 times was no more than 0.05%; the range of the total impurity content and LXH-2103 content results was no more than 0.1%, and the range of the intermediate precision and repeatability results for 12 times was no more than 0.1%. The experimental results are shown in Table 9.

[0111]

[0112] 5. Accuracy: Take 1.0 ml of each of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution and place them in a 25 ml volumetric bottle, dilute to the mark with solvent, shake well, and use them as impurity reference solution. Weigh about 50 mg of LXH-2103 sample, place it in a 25 ml volumetric bottle, dissolve it with solvent and dilute to the mark, shake well, and use it as the test solution. Weigh about 50 mg of LXH-2103 sample, place it in a 25 ml volumetric flask, add 1.0 ml of the intermediate stock solution (take 1.0 ml each of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution and place them in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well), dissolve it with solvent and dilute to the scale, shake well, and prepare 3 copies in parallel as accuracy test solution 1. Weigh about 50 mg of LXH-2103 sample, add 0.5 ml of each of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution, place them in a 25 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, prepare 3 portions in parallel, and use them as accuracy test solution 2. Weigh about 50 mg of LXH-2103 sample, add 1.0 ml of each of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution, place them in a 25 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, prepare 3 portions in parallel, and use them as accuracy test solution 3. Weigh about 50 mg of LXH-2103 sample, add 1.2 ml of LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution, place in a 25 ml volumetric bottle, dissolve with solvent and dilute to scale, shake well, prepare 3 portions in parallel, as accuracy test solution 4. Take the above impurity reference solution, test solution and each accuracy test solution, inject according to the chromatographic conditions, record the chromatogram, the impurity reference solution is injected 6 times, the peak area RSD is ≤5.0%, and the recovery rate of each accuracy test solution is between 80-120%. The results are shown in Tables 10-14.

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] 6. Solution stability: Take the control solution, impurity reference solution, and test sample spiked solution for determination at 0, 8, 24, 48, 72, 96, and 120 hours respectively. If the RSD of the average peak area of ​​the LXH-2103 chromatographic peak in the control solution and the impurity reference solution is not greater 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-2103 content is less than 0.1%, then it can be considered that the control solution, impurity reference solution, and test sample spiked solution are stable within 120 hours. The results of the solution stability experiment are shown in Tables 15-16, and the results show that the test sample spiked solution is stable for at least 120 hours.

[0119]

[0120]

[0121] 7. Method durability:

[0122] Method durability test①

[0123] (1) Solution preparation:

[0124] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0125] (2) Chromatographic conditions:

[0126] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0127] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0128] Detection wavelength: 269nm;

[0129] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0130] Mobile phase B: methanol;

[0131] Flow rate: 1.0 mL / min;

[0132] Column temperature: 30°C;

[0133] Injection volume: 20 μL;

[0134] The gradient elution procedure was the same as in Example 1;

[0135] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Figure 5 As shown in Tables 17-18, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0136]

[0137]

[0138] Method durability test②

[0139] (1) Solution preparation:

[0140] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0141] (2) Chromatographic conditions:

[0142] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0143] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0144] Detection wavelength: 273nm;

[0145] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0146] Mobile phase B: methanol;

[0147] Flow rate: 1.0 mL / min;

[0148] Column temperature: 30°C;

[0149] Injection volume: 20 μL;

[0150] The gradient elution procedure was the same as in Example 1;

[0151] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Figure 6As shown in Tables 19-20, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0152]

[0153]

[0154] Method durability test ③

[0155] (1) Solution preparation:

[0156] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0157] (2) Chromatographic conditions:

[0158] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0159] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0160] Detection wavelength: 271nm;

[0161] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0162] Mobile phase B: methanol;

[0163] Flow rate: 1.0 mL / min;

[0164] Column temperature: 28°C;

[0165] Injection volume: 20 μL;

[0166] The gradient elution procedure was the same as in Example 1;

[0167] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Figure 7 As shown in Tables 21-22, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0168]

[0169]

[0170] Method durability test ④

[0171] (1) Solution preparation:

[0172] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1.

[0173] (2) Chromatographic conditions:

[0174] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0175] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0176] Detection wavelength: 271nm;

[0177] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0178] Mobile phase B: methanol;

[0179] Flow rate: 1.0 mL / min;

[0180] Column temperature: 32°C;

[0181] Injection volume: 20 μL;

[0182] The gradient elution procedure was the same as in Example 1;

[0183] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Figure 8 As shown in Tables 23-24, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0184]

[0185]

[0186] Method durability test⑤

[0187] (1) Solution preparation:

[0188] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0189] (2) Chromatographic conditions:

[0190] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0191] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0192] Detection wavelength: 271nm;

[0193] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0194] Mobile phase B: methanol;

[0195] Flow rate: 0.95 mL / min;

[0196] Column temperature: 30°C;

[0197] Injection volume: 20 μL;

[0198] The gradient elution procedure was the same as in Example 1;

[0199] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Fig. 9 As shown in Tables 25-26, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0200]

[0201]

[0202] Method durability test ⑥

[0203] (1) Solution preparation:

[0204] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1.

[0205] (2) Chromatographic conditions:

[0206] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0207] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0208] Detection wavelength: 271nm;

[0209] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0210] Mobile phase B: methanol;

[0211] Flow rate: 1.05 mL / min;

[0212] Column temperature: 30°C;

[0213] Injection volume: 20 μL;

[0214] The gradient elution procedure was the same as in Example 1;

[0215] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Fig.10 As shown in Tables 27-28, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0216]

[0217]

[0218] Method durability test ⑦

[0219] (1) Solution preparation:

[0220] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0221] (2) Chromatographic conditions:

[0222] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0223] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0224] Detection wavelength: 271nm;

[0225] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0226] Mobile phase B: methanol;

[0227] Flow rate: 1.0 mL / min;

[0228] Column temperature: 30°C;

[0229] Injection volume: 20 μL;

[0230] The gradient elution program is shown in Table 29;

[0231]

[0232] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Fig.11 As shown in Tables 30-31, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0233]

[0234]

[0235] Method durability test⑧

[0236] (1) Solution preparation:

[0237] The preparation methods of blank solution, control solution, impurity reference solution and test sample spiked solution are the same as those in Example 1;

[0238] (2) Chromatographic conditions:

[0239] Chromatograph: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0240] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0241] Detection wavelength: 271nm;

[0242] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0243] Mobile phase B: methanol;

[0244] Flow rate: 1.0 mL / min;

[0245] Column temperature: 30°C;

[0246] Injection volume: 20 μL;

[0247] The gradient elution program is shown in 32;

[0248]

[0249] (3) Determination method: Accurately measure 20 μL of blank solution, control solution, impurity reference solution and test sample spiked solution and inject them into the liquid chromatograph respectively. The impurity reference solution is injected twice and the chromatogram is recorded. The results of the method robustness test are shown in Fig.12 As shown in Tables 33-34, the results show that the separation between the impurities in the chromatogram of the spiked solution of the test sample and between LXH-2103 and the adjacent impurities are all greater than 1.5. The experimental results show that this method has good durability.

[0250]

[0251]

[0252] Method durability comparison experiment

[0253] (1) Solution preparation:

[0254] The preparation method of the test sample spiked solution is the same as that of Example 1;

[0255] (2) Chromatographic conditions:

[0256] Chromatograph: Shimadzu liquid chromatograph LC-20AT with PDA detector;

[0257] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0258] Detection wavelength: 271nm;

[0259] Mobile phase: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution)-methanol (65:35);

[0260] Flow rate: 1.0 mL / min;

[0261] Column temperature: 30°C;

[0262] Injection volume: 20 μL;

[0263] The elution procedure used isocratic elution with mobile phase;

[0264] Determination method: Accurately measure 20 μL of the test sample spiked solution and inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Fig.13 As shown in Table 35, under this experimental condition, the separation degree of the chromatographic peaks of LXH-ZZ3 and LXH-2103 in the chromatogram of the test sample spiked solution can only reach 1.41, and the complete separation of the chromatographic peak baselines is not achieved. In addition, the retention times of the chromatographic peaks of LXH-ZZ4 and LXH-ZZ5 are approximately 35 minutes and 50 minutes, respectively. The peak elution time is too late and is not suitable for quantitative analysis, indicating that the isocratic elution method using the mobile phase is not applicable.

[0265]

[0266] The NMR spectra of LXH-2103, LXH-ZZ1, LXH-ZZ2, LXH-ZZ3, LXH-ZZ4 and LXH-ZZ5 are shown in Figure 14-25 , among which, the H NMR spectrum of LXH-2103 is shown in Fig.14 , the NMR carbon spectrum of LXH-2103 is shown in Fig.15 ; The H NMR spectrum of LXH-ZZ1 is shown in Fig.16 , the NMR carbon spectrum of LXH-ZZ1 is shown in Fig.17 ; The H NMR spectrum of LXH-ZZ2 is shown in Fig.18 , the NMR carbon spectrum of LXH-ZZ2 is shown in Fig.19 ; The H NMR spectrum of LXH-ZZ3 is shown in Fig. 20 , the NMR carbon spectrum of LXH-ZZ3 is shown in Fig.21 ; The H NMR spectrum of LXH-ZZ4 is shown in Fig. 22 , the NMR carbon spectrum of LXH-ZZ4 is shown in Fig.23 ; The H NMR spectrum of LXH-ZZ5 is shown in Fig.24 , the NMR carbon spectrum of LXH-ZZ5 is shown in Fig.25 .

[0267] Example 2

[0268] (1) Solution preparation:

[0269] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 65:35 as a blank solution;

[0270] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 65:35 as a solvent;

[0271] Weigh the LXH-2103 sample, dissolve it in a solvent and dilute it to make a solution containing 1.6 mg of LXH-2103 per 1 mL as the test solution;

[0272] Accurately measure 1.0 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, shake well, then accurately measure 1.0 ml from the 50 ml solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, shake well, as the control solution;

[0273] Weigh the LXH-ZZ1 reference substance, dissolve it in a solvent and dilute it to make a solution containing 80 μg of LXH-ZZ1 per 1 mL, which is used as the LXH-ZZ1 impurity stock solution;

[0274] Weigh the LXH-ZZ2 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 40 μg of LXH-ZZ2 per 1 mL, which is used as the LXH-ZZ2 impurity stock solution;

[0275] Weigh the LXH-ZZ3 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 40 μg of LXH-ZZ3 per 1 mL, which is used as the LXH-ZZ3 impurity stock solution;

[0276] Weigh the LXH-ZZ4 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 40 μg of LXH-ZZ4 per 1 mL, which is used as the LXH-ZZ4 impurity stock solution;

[0277] Weigh the LXH-ZZ5 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 40 μg of LXH-ZZ5 per 1 mL, which is used as the LXH-ZZ5 impurity stock solution;

[0278] Accurately measure 1 mL of LXH-ZZ1 impurity stock solution, 1 mL of LXH-ZZ2 impurity stock solution, 1 mL of LXH-ZZ3 impurity stock solution, 1 mL of LXH-ZZ4 impurity stock solution, and 1 mL of LXH-ZZ5 impurity stock solution, place them in the same 25 mL volumetric flask, dilute to the scale with solvent, shake well, and use as impurity reference solution;

[0279] (2) Chromatographic conditions:

[0280] Instrument: Shimadzu liquid chromatograph LC-20AT with UV detector;

[0281] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0282] Detection wavelength: 269nm;

[0283] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0284] Mobile phase B: methanol;

[0285] Flow rate: 0.95 mL / min;

[0286] Column temperature: 28°C;

[0287] Injection volume: 20 μL;

[0288] The gradient elution program is shown in Table 36;

[0289]

[0290] (3) Determination method: Accurately measure blank solution, test solution, impurity reference solution and control solution and inject them into liquid chromatograph respectively. Record the chromatogram. Calculate the content of LXH-2103 in the test solution by peak area normalization method. Calculate the content of impurities in the test solution by peak area using external standard method. The results are shown in Table 37.

[0291]

[0292] Example 3

[0293] (1) Solution preparation:

[0294] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 75:25 as a blank solution;

[0295] Take a solution of acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution) and methanol in a volume ratio of 75:25 as a solvent;

[0296] Weigh the LXH-2103 sample, dissolve it in a solvent and dilute it to make a solution containing 2.4 mg of LXH-2103 per 1 mL as the test solution;

[0297] Accurately measure 1.0 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, shake well, then accurately measure 1.0 ml from the 50 ml solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, shake well, as the control solution;

[0298] Weigh the LXH-ZZ1 reference substance, dissolve it in a solvent and dilute it to make a solution containing 120 μg of LXH-ZZ1 per 1 mL, which is used as the LXH-ZZ1 impurity stock solution;

[0299] Weigh the LXH-ZZ2 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 60 μg of LXH-ZZ2 per 1 mL, which is used as the LXH-ZZ2 impurity stock solution;

[0300] Weigh the LXH-ZZ3 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 60 μg of LXH-ZZ3 per 1 mL, which is used as the LXH-ZZ3 impurity stock solution;

[0301] Weigh the LXH-ZZ4 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 60 μg of LXH-ZZ4 per 1 mL, which is used as the LXH-ZZ4 impurity stock solution;

[0302] Weigh the LXH-ZZ5 reference substance, dissolve and dilute it with a solvent to prepare a solution containing 60 μg of LXH-ZZ5 per 1 mL, which is used as the LXH-ZZ5 impurity stock solution;

[0303] Accurately measure 1 mL of LXH-ZZ1 impurity stock solution, 1 mL of LXH-ZZ2 impurity stock solution, 1 mL of LXH-ZZ3 impurity stock solution, 1 mL of LXH-ZZ4 impurity stock solution, and 1 mL of LXH-ZZ5 impurity stock solution, place them in the same 25 mL volumetric flask, dilute to the scale with solvent, shake well, and use as impurity reference solution;

[0304] (2) Chromatographic conditions:

[0305] Instrument: Shimadzu liquid chromatograph LC-20AT with PDA detector;

[0306] Chromatographic column: Agilent SB-C18 4.6×150mm 5μm;

[0307] Detection wavelength: 273nm;

[0308] Mobile phase A: acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, dilute to 1000ml with water, and then adjust the pH value to 4.5 with glacial acetic acid or 0.1-1mol / L sodium hydroxide solution);

[0309] Mobile phase B: methanol;

[0310] Flow rate: 1.05 mL / min;

[0311] Column temperature: 32°C;

[0312] Injection volume: 20 μL;

[0313] The gradient elution program is shown in Table 38;

[0314]

[0315] (3) Determination method: Accurately measure blank solution, test solution, impurity reference solution and control solution and inject them into liquid chromatograph respectively. Record the chromatogram. Calculate the content of LXH-2103 in the test solution by peak area normalization method. Calculate the content of impurities in the test solution by peak area using external standard method. The results are shown in Table 39.

[0316]

Claims

1. A HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities, characterized in that Prepare a test solution, an impurity reference solution and a control solution, inject the blank solution, the test solution, the impurity reference solution and the control solution into a liquid chromatograph respectively, record the chromatogram, calculate the content of LXH-2103 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 LXH-ZZ1, LXH-ZZ2, LXH-ZZ3, LXH-ZZ4 or LXH-ZZ5; The structural formula of LXH-2103 is as follows: The structural formula of LXH-ZZ1 is as follows: The structural formula of LXH-ZZ2 is as follows: The structural formula of LXH-ZZ3 is as follows: The structural formula of LXH-ZZ4 is as follows: The structural formula of LXH-ZZ5 is as follows: ； The chromatographic conditions in the HPLC method include mobile phases; the mobile phases are acetate buffer as mobile phase A and methanol as mobile phase B; The chromatographic conditions also include a gradient elution program, which is as follows: 0-11 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; 11-16 minutes, mobile phase A accounts for 45-55% of the total volume of the mobile phase, and mobile phase B accounts for 45-55% of the total volume of the mobile phase; 16-36 minutes, mobile phase A accounts for 45-55% of the total volume of the mobile phase, and mobile phase B accounts for 45-55% of the total volume of the mobile phase; 36-36.1 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; 36.1-45 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.

2. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 1, characterized in that The test solution is prepared by weighing a LXH-2103 sample, dissolving and diluting it with a solvent to prepare a test solution; the concentration of LXH-2103 in the test solution is 1.6-2.4 mg / mL.

3. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 1, characterized in that The method for preparing the impurity reference solution is to mix LXH-ZZ1 impurity stock solution, LXH-ZZ2 impurity stock solution, LXH-ZZ3 impurity stock solution, LXH-ZZ4 impurity stock solution and LXH-ZZ5 impurity stock solution and then dilute them with a solvent to prepare an impurity reference solution; The concentration of LXH-ZZ1 in the impurity reference solution was 3.2-4.8 μg / mL, and the concentrations of LXH-ZZ2, LXH-ZZ3, LXH-ZZ4 and LXH-ZZ5 were all 1.6-2.4 μg / mL.

4. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 3, characterized in that The preparation method of the LXH-ZZ1 impurity stock solution is to weigh the LXH-ZZ1 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ1 impurity stock solution; the concentration of LXH-ZZ1 in the LXH-ZZ1 impurity stock solution is 80-120 μg / mL; The preparation method of the LXH-ZZ2 impurity stock solution is to weigh the LXH-ZZ2 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ2 impurity stock solution; the concentration of LXH-ZZ2 in the LXH-ZZ2 impurity stock solution is 40-60 μg / mL; The preparation method of the LXH-ZZ3 impurity stock solution is to weigh the LXH-ZZ3 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ3 impurity stock solution; the concentration of LXH-ZZ3 in the LXH-ZZ3 impurity stock solution is 40-60 μg / mL; The preparation method of the LXH-ZZ4 impurity stock solution is to weigh the LXH-ZZ4 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ4 impurity stock solution; the concentration of LXH-ZZ4 in the LXH-ZZ4 impurity stock solution is 40-60 μg / mL; The preparation method of the LXH-ZZ5 impurity stock solution is to weigh the LXH-ZZ5 reference substance, dissolve and dilute it with a solvent to prepare the LXH-ZZ5 impurity stock solution; the concentration of LXH-ZZ5 in the LXH-ZZ5 impurity stock solution is 40-60 μg / mL.

5. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 1, characterized in that The control solution is prepared by precisely measuring the test solution, diluting it with a solvent, and preparing the control solution; the concentration of LXH-2103 in the control solution is 1.6-2.4 μg / mL.

6. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to any one of claims 2 to 5, characterized in that The solvent is a mixed solution of acetate buffer and methanol, and the volume ratio of acetate buffer to methanol is 65-75:25-35.

7. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 6, characterized in that Acetate buffer is prepared from sodium acetate, glacial acetic acid and water.

8. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 1, characterized in that The blank solution is a mixed solution of acetate buffer and methanol, the volume ratio of acetate buffer to methanol is 65-75:25-35, and the acetate buffer is prepared from sodium acetate, glacial acetic acid and water.

9. The HPLC method for simultaneous qualitative and quantitative detection of LXH-2103 and its impurities according to claim 1, characterized in that The chromatographic conditions in the HPLC method include a chromatographic column, a detector and a detection wavelength; the chromatographic column is a C18 column with octadecylsilane bonded silica gel as a filler, and the column temperature of the chromatographic column is 28-32°C; the detector is a PDA detector or an ultraviolet detector, and the detection wavelength is 269-273nm; the total flow rate of mobile phase A and mobile phase B is 0.95-1.05mL / min.

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