Dual-wavelength-HPLC (High Performance Liquid Chromatography) method for simultaneously determining pyridine, N, N-dimethylformamide and N-methylpyrrolidone in mesylate lenvatinib

Abstract

The invention discloses a dual-wavelength-HPLC (High Performance Liquid Chromatography) method for simultaneously determining pyridine, DMF (Dimethyl Formamide) and NMP (N-Methyl Pyrrolidone) in lenvatinib mesylate, which comprises the following steps: dissolving pyridine, DMF and NMP in a solvent to obtain a reference solution; dissolving a sample in a solvent, standing, and filtering to obtain a test solution; and injecting the reference substance solution and the test solution into a liquid chromatograph, detecting pyridine at the wavelength of 254 nm, detecting DMF and NMP at the wavelength of 210 nm, and calculating the content of each solvent according to the peak area by an external standard method. The invention belongs to the technical field of analysis and detection, and particularly relates to a dual-wavelength-HPLC (High Performance Liquid Chromatography) method for pyridine, DMF (Dimethyl Formamide) and NMP (N-Methyl Pyrrolidone) in lenvatinib mesylate, which is accurate in detection method, simple and convenient to operate, good in specificity and reproducibility and high in detection sensitivity, can fully meet the requirement of simultaneously measuring the residual quantity of pyridine, DMF and NMP in lenvatinib mesylate, ensures the product quality, and is suitable for industrial production. And the practicability in quality control work is high.

Description

Technical Field

[0001] The invention belongs to the technical field of determination and analysis, and in particular relates to a dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide and N-methylpyrrolidone in lenvatinib mesylate. Background Art

[0002] Lenvatinib mesylate is a new type of molecular targeted anti-tumor drug and a first-line treatment for hepatocellular carcinoma. This product is suitable for: 1. Patients with unresectable hepatocellular carcinoma who have not received systemic treatment in the past. 2. Patients with progressive, locally advanced or metastatic differentiated thyroid cancer that is refractory to radioactive iodine.

[0003] Lenvatinib mesylate is a multi-target drug. It is an oral multi-RTK inhibitor developed by Eisai. In addition to inhibiting other RTKs related to pro-angiogenic and oncogenic signaling pathways involved in tumor proliferation (including platelet-derived growth factor [PDGF] receptors PDGFR, KIT and RET), it can also selectively inhibit the kinase activity of vascular endothelial growth factor (VEGF) receptors (VEGFR1, VEGFR2, VEGFR3) and fibroblast growth factor (FGF) receptors (FGFR1, FGFR2, FGFR3, FGFR3).

[0004] The solvents used in the synthesis process include ethanol, acetone, acetonitrile, n-propanol, isopropyl acetate, pyridine, dimethyl sulfoxide, N-methylpyrrolidone, etc. Among them, ethanol, acetone, acetonitrile, n-propanol, isopropyl acetate and other solvents can be detected by gas chromatography.

[0005] Pyridine, N,N-dimethylformamide, and N-methylpyrrolidone have high boiling points and require higher injection port and column temperatures. Due to the poor solubility of lenvatinib mesylate and the poor thermal stability of the solution, the use of gas chromatography will result in additional degradation that interferes with the determination of the target compound. In addition, the limit of pyridine is low, and gas chromatography (FID detector) does not meet the sensitivity requirements.

[0006] Therefore, it is necessary to invent an HPLC determination method for pyridine, N,N-dimethylformamide and N-methylpyrrolidone in lenvatinib mesylate to solve the above problems.

[0007] Pyridine, N, N-dimethylformamide, and N-methylpyrrolidone can be separated on a common reverse phase column. Therefore, we selected octadecyl bonded silica gel as the column for separation. The maximum absorption wavelengths of the three compounds are different, so we selected the detection wavelength with a stronger response for each compound. Pyridine was detected at a wavelength of 254nm, and N, N-dimethylformamide and N-methylpyrrolidone were detected at a wavelength of 210nm. The methanesulfonic acid in the test sample affects the detection of pyridine, making the pyridine peak shape poor, and the theoretical plate number is only about 500. Therefore, when preparing the test sample, sodium hydroxide solution was added to the diluent to neutralize the methanesulfonic acid and eliminate the effect of methanesulfonic acid on pyridine.

[0008] In order to more objectively reflect the pyridine, N,N-dimethylformamide and N-methylpyrrolidone residues in lenvatinib mesylate, the present invention attempts to establish a new HPLC method. Summary of the invention

[0009] In view of the above problems, the present invention provides a dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide and N-methylpyrrolidone in lenvatinib mesylate. The present invention adopts a reversed-phase high performance liquid chromatography dual wavelength method to simultaneously detect the contents of pyridine, N,N-dimethylformamide and N-methylpyrrolidone in lenvatinib mesylate.

[0010] The technical solution adopted by the present invention is as follows: a dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate, comprising the following steps:

[0011] Step 1: Take lenvatinib mesylate, add methanol, add sodium hydroxide solution, sonicate to dissolve, then dilute to scale with sodium hydroxide solution, shake well, let stand for 1 hour and then filter, and take the filtrate as the test solution;

[0012] Step 2: Take about 50 mg of pyridine, about 220 mg of DMF, and about 130 mg of NMP, accurately weigh them, place them in the same 100 ml volumetric flask, dissolve them with purified water and dilute them to the mark, shake them well; accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the mark with a diluent, and shake them well as a reference stock solution;

[0013] Step 3: Accurately measure 5 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well as the reference solution;

[0014] Step 4: Accurately measure 10 μl of blank solvent, reference solution and test solution, inject into liquid chromatograph, perform linear gradient elution, and record chromatogram. Detect pyridine at 254 nm, and detect DMF and NMP at 210 nm. In the chromatogram of reference solution, the theoretical plate number of each chromatographic peak should be no less than 2000, S / N should be no less than 30, and the separation between adjacent chromatographic peaks at 210 nm should be no less than 1.5. Calculate the content of each solvent in the test sample by peak area according to the external standard method.

[0015] Furthermore, the mass concentration of the sodium hydroxide solution in step one is 0.5-2.0 g / L.

[0016] Furthermore, the diluent in step 2 and step 3 is 1.0 g / L methanol sodium hydroxide solution.

[0017] Furthermore, the blank solution in step 4 is a diluent.

[0018] Furthermore, in step 4, the liquid chromatograph uses an octadecylsilane bonded silica gel chromatographic column as a filler, and the chromatographic column model and specification are Inertsil ODS-3, 4.0 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0019] Furthermore, the mobile phase used in the liquid chromatograph in step 4 is a mixture of mobile phase A and mobile phase B, mobile phase A is potassium dihydrogen phosphate buffer, and mobile phase B is acetonitrile.

[0020] Furthermore, in step 4, the wavelength of the liquid chromatograph is 200-280 nm; the flow rate is 0.5-1.5 mL / min; the column temperature is 25-35° C.; and the ultrasonic dissolution time is 5-10 min.

[0021] Furthermore, the concentration of the potassium dihydrogen phosphate buffer is 0.01-0.05 mol / L, and the pH value is 3.0-6.0.

[0022] Furthermore, the concentration of the mobile phase A potassium dihydrogen phosphate buffer is 0.02 mol / L; the pH value is 4.0, the wavelengths in the liquid chromatograph are 210 nm and 254 nm; the flow rate is 0.8 mL / min; and the column temperature is 30°C.

[0023] After adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0024] 1. The present invention uses an Inertsil ODS-3, 4.0 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance for separation, which can well separate pyridine, DMF, NMP and other impurities from lenvatinib mesylate, thereby ensuring the specificity of the method;

[0025] 2. The present invention adopts 210nm where DMF and NMP have strong absorption and 254nm where pyridine has strong absorption as detection wavelengths, which can improve sensitivity and detect simultaneously, quickly and efficiently;

[0026] 3. The present invention adopts 0.02 mol / L potassium dihydrogen phosphate buffer (pH 4.0)-acetonitrile system to make pyridine, DMF, NMP chromatographic peak shape good, separation degree good, and detect by gradient elution program;

[0027] 4. The present invention innovatively adopts the method of first adding a large amount of methanol and a small amount of sodium hydroxide solution as solvents to dissolve the sample, so that the insoluble lenvatinib mesylate can be completely dissolved, thereby avoiding incomplete extraction during the detection process due to the poor solubility of the sample. After the sample is completely dissolved, a sufficient amount of sodium hydroxide solution is added to neutralize the methanesulfonic acid in the solution, eliminating the interference of methanesulfonic acid on pyridine;

[0028] 5. The detection method of the present invention is accurate, easy to operate, has good specificity and reproducibility, and has high detection sensitivity. It can fully meet the requirements of simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate, better control special impurities in samples, ensure product quality, and has strong practicality in quality control work;

[0029] 6. The present invention innovatively adopts dual-wavelength-HPLC method to detect multiple compounds. The detection method of the present invention has simple and convenient sample processing, accurate and reliable test results, high sensitivity, conventional reagents, and simple preparation of diluents and mobile phases, which reduces the detection cost and saves time, and has good economic value for routine detection of pharmaceutical manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 HPLC charts of blank solvent at 254 nm and 210 nm wavelengths of the embodiments of the present invention;

[0031] Figure 2 HPLC charts of the reference substance at 254 nm and 210 nm wavelengths according to the embodiment of the present invention;

[0032] Figure 3 HPLC graphs of the samples of the present invention at wavelengths of 254 nm and 210 nm;

[0033] Figure 4 It is a linear standard curve diagram of pyridine in an embodiment of the present invention;

[0034] Figure 5 This is a linear standard curve diagram of N,N-dimethylformamide according to an embodiment of the present invention;

[0035] Figure 6 This is a linear standard curve of N-methylpyrrolidone according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] The overall technical solution of the present invention is first described below.

[0038] The present invention provides the following technical solution: a dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate, wherein the method uses a reversed-phase high performance liquid chromatography dual wavelength method to simultaneously detect the contents of pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate, and the chromatographic conditions are as follows:

[0039] The chromatographic column uses octadecylsilane bonded silica gel as the filler.

[0040] A potassium dihydrogen phosphate buffer-acetonitrile mixed solution system was used as the mobile phase for linear gradient elution.

[0041] The mobile phase is a mixture of mobile phase A and mobile phase B, wherein mobile phase A is potassium dihydrogen phosphate buffer, mobile phase B is acetonitrile, the total volume percentage of mobile phase A and mobile phase B is 100%, the mobile phase A is 100% to 10%, and the mobile phase B is 0% to 90%.

[0042] The concentration of the mobile phase A potassium dihydrogen phosphate buffer is 0.01-0.05 mol / L, preferably 0.02 mol / L, and the pH value is 3.0-6.0, preferably 4.0.

[0043] The volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 90:10 to 98:2, and the preferred volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 95:5.

[0044] The mobile phase elution mode is gradient elution, and the values ​​of linear gradient elution are shown in the following table:

[0045] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 20 95 5 21 20 80 40 20 80 41 95 5 55 95 5

[0046] The chromatographic column with octadecylsilane bonded silica gel as filler has a model specification of Inertsil ODS-3, 4.0 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0047] The wavelength selected for detection in the reverse liquid chromatography method is 200-280 nm, preferably 210 nm and 254 nm, the flow rate of the mobile phase is 0.5-1.5 ml / min, preferably 0.8 ml / min, and the temperature of the chromatographic column is 25-35° C., preferably 30° C.

[0048] A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate is characterized in that it specifically comprises the following steps:

[0049] Step 1: Test solution: Take an appropriate amount of the product, weigh it accurately, put it in a volumetric flask, add an appropriate amount of methanol, add an appropriate amount of sodium hydroxide solution, dissolve it by ultrasonication, then dilute it to the scale with sodium hydroxide solution, shake well, let it stand and then filter, and take the filtrate as the test solution.

[0050] Step 2: Reference substance stock solution: Take appropriate amount of pyridine, DMF, and NMP, weigh them accurately, place them in the same volumetric flask, dissolve and dilute to the scale with purified water, and shake well; accurately measure an appropriate amount and place it in a volumetric flask, dilute to the scale with diluent, and shake well.

[0051] Step 3: Reference solution: Accurately measure an appropriate amount of reference stock solution and place it in a volumetric bottle, dilute to the scale with diluent, and shake well.

[0052] Step 4: Determination: Accurately measure the blank solvent, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram. Detect pyridine at a wavelength of 254nm, and detect DMF and NMP at a wavelength of 210nm. In the chromatogram of the reference solution, the theoretical plate number of each chromatographic peak should be no less than 2000, the S / N should be no less than 30, and the separation between adjacent chromatographic peaks at a wavelength of 210nm should be no less than 1.5. Calculate the content of each solvent in the test sample by peak area according to the external standard method.

[0053] Chromatographic conditions: select dual-wavelength UV detector and octadecylsilane bonded silica gel chromatographic column with hydrophobic effect as the main separation principle, use potassium dihydrogen phosphate buffer-acetonitrile system as mobile phase, and perform linear gradient elution; flow rate is 0.8 ml per minute; detection wavelength is 210nm and 254nm; column temperature is 30℃;

[0054] The method is applicable to lenvatinib mesylate day 0 samples, influencing factor samples, and stability samples that need to be tested by pyridine, DMF, and NMP.

[0055] Next, the present invention is described in detail through specific embodiments.

[0056] Example 1

[0057] A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate comprises the following steps:

[0058] Diluent: methanol-1.0g / L sodium hydroxide solution (50:50)

[0059] Test solution: Take about 125 mg of this product, weigh accurately, place in a 25 ml volumetric flask, add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution, dissolve by ultrasonic, then dilute to the scale with 1.0 g / L sodium hydroxide solution, shake well, let stand for 1 hour and then filter, and take the filtrate as the test solution.

[0060] Reference substance stock solution: take about 50 mg of pyridine, about 220 mg of DMF, and about 130 mg of NMP, accurately weigh them, place them in a 100 ml volumetric flask, dissolve and dilute to the scale with purified water, and shake well; accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0061] Reference substance solution: Accurately measure 5 ml of reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0062] Chromatographic conditions: select dual-wavelength UV detector and octadecylsilane bonded silica gel chromatographic column with hydrophobic effect as the main separation principle, use potassium dihydrogen phosphate buffer-acetonitrile system as mobile phase, and perform linear gradient elution; flow rate is 0.8 ml per minute; detection wavelength is 210nm and 254nm; column temperature is 30℃;

[0063] Wherein, the gradient elution process is as follows:

[0064] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 20 95 5 21 20 80 40 20 80 41 95 5 55 95 5

[0065] Determination: accurately measure blank solvent, reference solution, and test solution, inject into liquid chromatograph, and record chromatogram. Detect pyridine at 254nm wavelength, and detect DMF and NMP at 210nm wavelength. In the reference solution chromatogram, the theoretical plate number of each chromatographic peak should be no less than 2000, S / N should be no less than 30, and the separation between adjacent chromatographic peaks at 210nm wavelength should be no less than 1.5. Calculate the content of each solvent in the test sample by peak area according to the external standard method.

[0066] Example 2

[0067] This example verifies the specificity of this method:

[0068] Blank solvent: diluent.

[0069] Test solution: Accurately weigh 0.12491 g of the product, place in a 25 ml volumetric flask, add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution, dissolve by ultrasonication, then dilute to the scale with 1.0 g / L sodium hydroxide solution, shake well, let stand for 1 hour and then filter, and take the filtrate as the test solution.

[0070] Stock solutions of each solvent: accurately weigh 0.05026 g of pyridine, 0.22050 g of DMF, and 0.12898 g of NMP, place them in 100 ml volumetric flasks, dissolve and dilute to the mark with purified water, and shake well.

[0071] Reference substance stock solution: Accurately measure 2 ml of each solvent stock solution, place in the same 100 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0072] Reference substance solution: Accurately measure 5 ml of reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0073] Positioning solution: Accurately measure 100 μl of each solvent stock solution, place in 50 ml volumetric flasks, dilute to the scale with diluent, and shake well.

[0074] Mixed solutions of other impurities: Take 0.2 ml each of impurity 1 stock solution, impurity 2 stock solution, impurity 3 stock solution, impurity 4 stock solution, impurity 5 stock solution, and 2 ml of impurity 6 / impurity 7 / impurity 8 / impurity 9 mixed solution, place them in the same 10 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0075] Mixed solution of other residual solvents: Accurately measure 10 μl of DMSO and glacial acetic acid, place them in the same 100 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0076] Accurately measure 10 μl of blank solvent, test solution, reference solution, each solvent positioning solution, other impurity mixed solution and other residual solvent mixed solution, inject them into liquid chromatograph and record the chromatogram.

[0077] Specificity test results

[0078]

[0079] Example 3

[0080] This example verifies the system applicability of this method:

[0081] Take 10 μl of the reference solution under the item “Specificity” of Example 2, inject it 5 times continuously, and record the chromatogram.

[0082] System suitability test results

[0083]

[0084] Example 4

[0085] This example verifies the quantitative limits of pyridine, DMF, and NMP.

[0086] Quantitation limit solution: Accurately measure 10 ml of pyridine positioning solution, 1 ml of DMF positioning solution, and 5 ml of NMP positioning solution under the "Specificity" item of Example 2, place them in the same 100 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0087] Accurately measure 10 μl of the quantitative limit solution, inject it 6 times continuously, record the chromatogram, and calculate the relative standard deviation of each peak area.

[0088] Quantitation limit test results

[0089]

[0090] Example 5

[0091] This example verifies the detection limit of "pyridine, DMF, NMP".

[0092] Accurately measure 5 ml of the quantitation limit solution under the "quantitation limit" item, place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and use this as the detection limit solution. Take the detection limit solution and inject it continuously for 3 times.

[0093] Detection limit test results

[0094]

[0095] Example 6

[0096] This example verifies the detection limit addition of pyridine and DMF that were not detected in the test sample.

[0097] Weigh 0.12501g of this product accurately, place it in a 25ml volumetric flask, add 6ml of methanol, then accurately add 12.5ml of the quantitative limit solution under the "quantitative limit" item, dissolve it by ultrasonication, dilute it to the scale with 1.0g / L sodium hydroxide solution, shake it well, let it stand for 1h, then filter it, and take the filtrate as the detection limit addition solution. Take the detection limit addition solution for sampling and detection, record the chromatogram, repeat the injection for 2 injections, and calculate the signal-to-noise ratio.

[0098] Detection limit addition test results

[0099]

[0100] Example 7

[0101] This example verifies the linear range of "pyridine, DMF, NMP".

[0102] The quantitative limit solution under the item “Quantitative limit” in Example 4 was taken as linear solution 1.

[0103] Take the reference substance stock solution under the "Specificity" item of Example 2 as the linear stock solution.

[0104] Linear solution: Accurately measure 1 ml, 2 ml, 5 ml, 7 ml, and 10 ml of the linear stock solution respectively, place them in 50 ml volumetric flasks, add diluent to dilute to the scale, shake well, and use them as linear solutions 2, 3, 4, 5, and 6 respectively.

[0105] Accurately measure 10 μl of each concentration linear solution, inject it into the liquid chromatograph, record the chromatogram, use the peak area as the ordinate and the concentration (C) as the abscissa, perform linear regression, and calculate the correlation coefficient.

[0106] Pyridine linear relationship test results

[0107]

[0108] DMF linear relationship test results

[0109]

[0110] NMP linear relationship test results

[0111]

[0112] Example 8

[0113] In this example, six samples were measured in parallel according to the description of the above experimental method to verify the precision.

[0114] Blank solvent: diluent.

[0115] Test solution: Accurately weigh 0.12517g, 0.12473g, 0.12587g, 0.12499g, 0.12544g, and 0.12510g of the product, place them in 25ml volumetric flasks, add 12.5ml of methanol and 2ml of 1.0g / L sodium hydroxide solution, dissolve them by ultrasonication, then dilute to the scale with 1.0g / L sodium hydroxide solution, shake well, let it stand for 1h, then filter and take the filtrate as the test solution.

[0116] Stock solutions of each solvent: Accurately weigh 0.0505 g of pyridine, 0.2210 g of DMF, and 0.1331 g of NMP, place them in 100 ml volumetric flasks, dissolve and dilute to the mark with purified water, and shake well.

[0117] Reference substance stock solution: Accurately measure 1 ml of each solvent stock solution and place it in the same 50 ml volumetric flask. Dilute to the scale with diluent and shake well.

[0118] Reference substance solution: Accurately measure 5 ml of reference substance stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0119] Determination steps: accurately measure blank solvent, reference solution, and test solution, inject into liquid chromatograph, and record chromatogram. Detect pyridine at 254nm wavelength, and detect DMF and NMP at 210nm wavelength. In the chromatogram of reference solution, the theoretical plate number of each chromatographic peak should be no less than 2000, S / N should be no less than 30, and the separation between adjacent chromatographic peaks at 210nm wavelength should be no less than 1.5. Calculate the content of each solvent in the test sample by peak area according to the external standard method, and calculate the range of the determination results.

[0120] Repeatability test results

[0121] serial number 1 2 3 45 6 average value Very bad Pyridine(%) Not detected Not detected Not detected Not Detected Not Detected Not detected Not detected —— DMF(%) Not detected Not detected Not detected Not Detected Not Detected Not detected Not detected —— NMP(%) 0.003 0.003 0.003 0.0030.003 0.003 0.003 0

[0122] Example 9

[0123] This example demonstrates solution stability.

[0124] Blank solvent: diluent.

[0125] Reference substance stock solution: Accurately weigh 0.05027 g of pyridine, 0.21999 g of DMF, and 0.13009 g of NMP, place them in the same 100 ml volumetric flask, dissolve and dilute to the scale with purified water, and shake well; accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0126] Reference substance solution: Accurately measure 5 ml of reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0127] Mixed solution: Accurately weigh 0.12499 g of this product, place it in a 25 ml volumetric flask, add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution, dissolve it by ultrasonication, then accurately add 2.5 ml of the reference stock solution, and then dilute to the scale with 1.0 g / L sodium hydroxide solution, shake well, let it stand for 1 hour, then filter and take the filtrate as the mixed solution.

[0128] Test solution: Accurately weigh 0.12530 g of the product, place in a 25 ml volumetric flask, add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution, dissolve by ultrasonication, then dilute to the scale with 1.0 g / L sodium hydroxide solution, shake well, let stand for 1 hour and then filter, and take the filtrate as the test solution.

[0129] Accurately measure 10 μl each of the blank solvent, the reference solution placed at room temperature without protection from light for 0 h, 3.5 h, and 6 h, the mixed solution placed at room temperature without protection from light for 0 h, 3.5 h, 6 h, and 24 h, the test solution, and the reference solution refrigerated for 24 h, inject and detect, and record the chromatogram.

[0130] Solution Stability Results - Reference Solution

[0131]

[0132] Solution Stability Results - Mixed Solutions

[0133]

[0134] Solution Stability Results - Test Solution

[0135]

[0136] Example 10

[0137] This example verifies the accuracy of the HPLC method of the present invention through a recovery experiment. Three solutions of each component at 80%, 100%, and 120% and a blank control are selected, and the recovery rate of each component is determined according to the description of the above experimental method.

[0138] Recovery stock solution: Take the reference substance stock solution under the "Specificity" item of Example 2 as the recovery stock solution.

[0139] Preparation of test solution: Accurately weigh 0.12526 g, 0.12496 g, 0.12497 g, 0.12480 g, 0.12496 g, 0.12528 g, 0.12498 g, 0.12496 g, 0.12503 g, and 0.12490 g of the product and place them in 25 ml volumetric flasks. Add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution respectively, dissolve by ultrasound, add 2 ml of recovery stock solution to 3 parts, add 2.5 ml of recovery stock solution to 3 parts, add 3 ml of recovery stock solution to 3 parts, then add 1.0 g / L sodium hydroxide solution to dilute to the scale, directly add 1.0 g / L sodium hydroxide solution to another part and dilute to the scale, shake well, let stand for 1 hour and then filter, take the filtrate as the limit concentration of 80%, 100%, 120% and background test solution respectively.

[0140] Reference solution: Accurately measure 5 ml of the recovery stock solution, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0141] Accurately measure 10 μl of blank solvent, reference solution, and test solution, inject into liquid chromatograph, record chromatogram, and calculate by peak area according to external standard method.

[0142] Pyridine recovery test results

[0143]

[0144] DMF recovery test results

[0145]

[0146] NMP recovery test results

[0147]

[0148] Embodiment 11

[0149] This example verifies the robustness of the HPLC method of the present invention. Multiple parameter levels were selected for verification.

[0150] The flow rate (0.8±0.1ml / min), column temperature (30±5℃), mobile phase initial ratio (96:4 / 94:6), mobile phase A salt concentration (0.02±0.01mol / L), mobile phase A buffer salt pH (4.0±0.2) and different chromatographic columns were adjusted respectively.

[0151] Blank solvent: diluent.

[0152] Reference stock solution: accurately weigh 0.05014g of pyridine, 0.21749g of DMF, and 0.12944g of NMP, place them in a 100ml volumetric flask, dissolve and dilute to the mark with purified water, and shake well; accurately measure 1ml, place it in a 50ml volumetric flask, dilute to the mark with diluent, and shake well. (For normal conditions, column temperature, flow rate, and salt concentration)

[0153] Reference stock solution: accurately weigh 0.04995g of pyridine, 0.21771g of DMF, and 0.13093g of NMP, place them in a 100ml volumetric flask, dissolve and dilute to the mark with purified water, and shake well; accurately measure 1ml, place it in a 50ml volumetric flask, dilute to the mark with diluent, and shake well. (For pH and mobile phase starting ratio (96:4))

[0154] Reference stock solution: accurately weigh 0.04995g of pyridine, 0.21771g of DMF, and 0.13093g of NMP, place them in the same 100ml volumetric flask, dissolve and dilute to the mark with purified water, and shake well; accurately measure 1ml, place it in a 50ml volumetric flask, dilute to the mark with diluent, and shake well. (For the starting ratio of mobile phase (94:6), different chromatographic columns)

[0155] Reference substance solution: Accurately measure 5 ml of reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0156] Test solution: Accurately weigh 0.12529 g of this product (for normal conditions, column temperature, flow rate, and salt concentration), 0.12523 g (for pH and mobile phase starting ratio (96:4)), and 0.12431 g (for mobile phase starting ratio (94:6) and different chromatographic columns), place in a 25 ml volumetric flask, add 12.5 ml of methanol and 2 ml of 1.0 g / L sodium hydroxide solution, dissolve by ultrasound, then accurately add 2.5 ml of reference substance stock solution, and dilute to the scale with 1.0 g / L sodium hydroxide solution, shake well, let stand for 1 hour, then filter, and take the filtrate as the test solution.

[0157] Precisely measure 10 μl of blank solvent, reference solution, and test solution, respectively, and inject and test under the above conditions, and record the chromatogram. Detect pyridine at a wavelength of 254 nm, and detect DMF and NMP at a wavelength of 210 nm. Calculate the solvent content in the test sample by peak area using the external standard method.

[0158] Durability Test Results – Chromatographic System Durability

[0159]

[0160] Durability test results - impurity content

[0161]

[0162] The present invention and its embodiments are described above, but such description is not restrictive. What is shown in the specification is only part of the embodiments of the present invention, and the actual method is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate, characterized in that: The following steps are involved: Step 1: Take lenvatinib mesylate, add methanol, add sodium hydroxide solution, sonicate to dissolve, then dilute to scale with sodium hydroxide solution, shake well, let stand for 1 hour and then filter, and take the filtrate as the test solution; Step 2: Take 50 mg of pyridine, 220 mg of DMF, and 130 mg of NMP, accurately weigh them, place them in the same 100 ml volumetric flask, dissolve them with purified water and dilute them to the mark, shake them well; accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the mark with a diluent, and shake them well as a reference stock solution; Step 3: Accurately measure 5 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well as the reference solution; Step 4: Accurately measure 10 μl of blank solvent, reference solution and test solution, inject into liquid chromatograph, perform linear gradient elution, record chromatogram, detect pyridine at 254 nm, detect DMF and NMP at 210 nm, in the chromatogram of reference solution, the theoretical plate number of each chromatographic peak should be not less than 2000, S / N should be not less than 30, the separation between adjacent chromatographic peaks at 210 nm should be not less than 1.5, calculate the content of each solvent in the test sample by peak area according to the external standard method.

2. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: The mass concentration of the sodium hydroxide solution in step 1 is 0.5-2.0 g / L.

3. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: The diluent in step 2 and step 3 is 1.0 g / L methanol sodium hydroxide solution.

4. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: The blank solution in step 4 is the diluent.

5. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: In step 4, the liquid chromatograph uses an octadecylsilane bonded silica gel chromatographic column as a filler, and the chromatographic column model and specification are Inertsil ODS-3, 4.0 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

6. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: The mobile phase used in the liquid chromatograph in step 4 is a mixture of mobile phase A and mobile phase B, wherein mobile phase A is potassium dihydrogen phosphate buffer and mobile phase B is acetonitrile.

7. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 1, characterized in that: In step 4, the wavelength of the liquid chromatograph is 200-280 nm; the flow rate is 0.5-1.5 mL / min; the column temperature is 25-35° C.; and the ultrasonic dissolution time is 5-10 min.

8. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 6, characterized in that: The concentration of the potassium dihydrogen phosphate buffer is 0.01-0.05 mol / L, and the pH value is 3.0-6.

0.

9. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 7, characterized in that: The concentration of the mobile phase A potassium dihydrogen phosphate buffer was 0.02 mol / L; the pH value was 4.0; the wavelengths in the liquid chromatograph were 210 nm and 254 nm; the flow rate was 0.8 mL / min; and the column temperature was 30° C.

10. A dual-wavelength-HPLC method for simultaneously determining pyridine, N,N-dimethylformamide, and N-methylpyrrolidone in lenvatinib mesylate according to claim 6, characterized in that: The volume ratio of the potassium dihydrogen phosphate buffer solution to acetonitrile is 90:10 to 98:2, and the volume ratio of the potassium dihydrogen phosphate buffer solution to acetonitrile is 95:5.

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