Content determination and analysis method for macloxvir tablets

Abstract

The invention discloses a method for determining and analyzing the content of macloxvir tablets, and relates to the field of medicine detection. The method for determining the content of the macarovir tablet specifically provides a method for detecting the content of macarovir by using a high performance liquid detection method, and adopts a chromatographic column which takes octadecylsilane chemically bonded silica as a filling agent (a chromatographic column with 4.6 * 100 mm, 3 microns or equivalent efficiency); a solution B (a 0.1% trifluoroacetic acid solution containing 0.18-0.22 mmol / L of ethylene diamine tetraacetic acid disodium salt)-acetonitrile (45: 55-55: 45) is used as a mobile phase; the column temperature is 30-40 DEG C; the flow velocity is 0.8 ml / min to 1.2 ml / min; the sample size is 5 microliters; the concentration of the sample is 0.28 mg / ml to 0.52 mg / ml. The method has the advantages of being high in general applicability, high in efficiency, good in stability and the like, and is suitable for quality control of macloxvir tablet content determination.

Description

Technical Field

[0001] The present invention relates to the field of medical testing, and in particular to a method for determining the content of mabaloxavir tablets. Background Art

[0002] Mabaloxavir is the first small molecule anti-influenza virus drug developed by Japan that targets Cap-dependent endonucleases. It inhibits the cap-snatching mechanism of influenza viruses, resulting in the obstruction of viral replication, thereby specifically blocking the proliferation of influenza A and B viruses. Mabaloxavir has the advantages of a single-dose administration regimen and no cross-resistance. Clinical studies have found that the effect of mabaloxavir in treating influenza is better than that of traditional anti-influenza virus drugs such as oseltamivir. Influenza viruses resistant to oseltamivir are still sensitive to mabaloxavir. Mabaloxavir, as an anti-influenza virus drug with a new mechanism of action, is also quite different from hemagglutinin inhibitors, M2 ion channel inhibitors and NAIs. Mabaloxavir has an inhibitory effect on various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2), and also exhibits anti-SARS-CoV-2 virus activity in vitro. The new mechanism of action and wide range of action make it an alternative treatment for drug-resistant influenza viruses.

[0003] Content determination testing is an important test item for quality control in the process of drug development, and it plays a decisive role in quality control. Currently, no relevant reports have been found on the content determination test method of Mabaloxavir Tablets. Therefore, developing a method for content determination of Mabaloxavir Tablets is of great significance in the process of drug quality control. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the content of mabaloxavir tablets with high general applicability, high efficiency, high precision and good stability.

[0005] The present invention discloses a method for determining the content of mabaloxavir tablets. The method for determining the content of mabaloxavir tablets is to analyze the main component mabaloxavir by HPLC. The specific operation is as follows:

[0006] Step 1: Preparation of test solution

[0007] Accurately weigh mabaloxavir and add it to a volumetric flask, add 10-20% of the volume of solution B to mix, then add 45-50% of the volume of acetonitrile to mix, ultrasonically mix, dilute to scale with a diluent, shake well, centrifuge, take the supernatant, and prepare a solution containing about 0.28-0.52 mg of mabaloxavir per 1 mL; wherein solution B is composed of 0.18-0.22 mmol / L disodium ethylenediaminetetraacetic acid and 0.1-0.2% trifluoroacetic acid solution by volume;

[0008] Step 2: Preparation of reference solution

[0009] Take the mabaloxavir reference substance, weigh it accurately, add the diluent to dissolve it and quantitatively dilute it to make a solution containing about 0.28-0.52 mg of mabaloxavir per 1 mL;

[0010] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler; solution B-acetonitrile as mobile phase, isocratic elution; detection wavelength is 259 nm, flow rate is 0.8 mL to 1.2 mL per minute; column temperature is 30°C to 40°C; injection volume is 5 μL; sample concentration is 0.28 mg / mL to 0.52 mg / mL;

[0011] The diluents described in step 1 and step 2 are both prepared by mixing acetonitrile and solution B in a volume ratio of 2 to 4:1.

[0012] Furthermore, the chromatographic column is 4.6×100 mm, 3 μm or a chromatographic column with equivalent performance.

[0013] Furthermore, the 4.6×100 mm, 3 μm octadecylsilane bonded silica gel chromatographic column as filler is a CAPCELL PAK MGⅡ series chromatographic column or a BDS Hypersil TM Series of chromatographic columns.

[0014] Furthermore, the volume ratio of solution B to acetonitrile is 45:55-55:45.

[0015] Furthermore, the reference solution is accurately measured and injected into a high performance liquid chromatograph, and the chromatogram is recorded. The number of theoretical plates is not less than 4000 based on the mabaloxavir peak: the symmetry factor of the mabaloxavir peak is not greater than 1.5: the injection is repeated 5 times, and the relative standard deviation of the mabaloxavir peak area is not greater than 1.0%.

[0016] The present invention has the following beneficial effects:

[0017] The present invention detects the content of mabaloxavir, a main component in mabaloxavir tablets, and establishes for the first time an analytical method for content determination of mabaloxavir tablets using high performance liquid chromatography. The present invention has the advantages of high detection efficiency, high detection sensitivity, good reproducibility, high stability, etc., and is suitable for mabaloxavir content determination inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Full wavelength scanning spectrum of the reference solution between 200nm and 400nm UV wavelength;

[0019] Figure 2 Full wavelength scanning spectrum of the test solution between 200nm and 400nm UV wavelength;

[0020] Figure 3 is the reference solution spectrum;

[0021] Figure 4 This is the detection spectrum using CAPCELL PAK MGⅡ series chromatographic columns;

[0022] Figure 5 To adopt BDS Hypersil TM Detection profiles of series chromatographic columns;

[0023] Figure 6 This is the detection spectrum at a column temperature of 30°C;

[0024] Figure 7 This is the detection spectrum at a column temperature of 40°C;

[0025] Figure 8 The detection spectrum is the mobile phase ratio: solution B-acetonitrile 45:55;

[0026] Fig. 9 The detection spectrum is the mobile phase ratio: solution B-acetonitrile 55:45;

[0027] Fig.10 Detection profile of washing at a flow rate of 0.8 ml per minute;

[0028] Fig.11 The detection profile is eluted at a flow rate of 1.2 ml per minute. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0030] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0031] Example 1

[0032] 1) Preparation of test solution

[0033] Take 20 tablets of this product, weigh them accurately, calculate the average tablet weight, take the test sample containing 80 mg of mabaloxavir, put it in a 200 ml volumetric bottle, add 32 ml of solution B, shake vigorously to disintegrate the tablets, add 96 ml of acetonitrile, ultrasonically treat, dilute to the scale with diluent, and shake well. Take an appropriate amount of the above solution, centrifuge, and take the supernatant.

[0034] 2) Preparation of reference solution

[0035] Take about 10 mg of mabaloxavir reference substance, weigh accurately, place in a 25 ml volumetric flask, add appropriate amount of diluent, dissolve by ultrasonication, dilute to the scale with diluent, and shake well.

[0036] Example 2

[0037] This example is the optimization and determination of the chromatographic conditions used in the analytical method for determining the content of Mabaloxavir Tablets.

[0038] 1) Determination of detection wavelength

[0039] The test solution and the reference solution (both diluted to 0.016 mg) were scanned at full wavelength between 200 nm and 400 nm under ultraviolet light. The maximum ultraviolet absorption of mabaloxavir is 259-260 nm, so the detection wavelength was set at 259 nm.

[0040] 2) Optimal chromatographic conditions

[0041] Chromatographic column: Use octadecylsilane bonded silica as filler (4.6×100mm, 3μm or equivalent chromatographic column), use OSAKA SODA CAPCELL PAK MGⅡC18 (250mm×4.6mm, 5μm) or Thermo BDS Hypersil TM C18 (250mm×4.6mm, 5μm); solution B (0.1% trifluoroacetic acid solution containing 0.2mmo / L disodium ethylenediaminetetraacetate)-acetonitrile (1:1) as the mobile phase; detection wavelength is 259nm, flow rate is 1.0ml per minute; column temperature is 35℃; injection volume is 5μl.

[0042] The test solution and the reference solution of Example 1 were measured using the above-mentioned optimal chromatographic conditions.

[0043] Accurately measure 5 μL of blank solution, test solution and reference solution and inject them into the HPLC. Record the chromatogram and calculate its retention time, peak height, theoretical plate number and separation degree. The test results are shown in Table 1.

[0044] According to Table 1, it can be seen that after 5 consecutive injections of the reference solution, the average retention time of the main peak is 4.012min, the relative standard deviation is 0.027%, the average number of theoretical plates is 12647, the peak shape is good, and it meets the standard; the main peak retention time of the test solution is 4.010min, the theoretical plate number is 12607, the peak shape is good, and it meets the standard.

[0045] pass Figures 1 to 11 The above-mentioned optimal detection wavelength and optimal chromatographic conditions can be determined.

[0046] 3) Methodological validation

[0047] The above-mentioned optimal chromatographic conditions were used to verify the test sample preparation method of Example 1 and the optimal chromatographic conditions of Example 2.

[0048] ① Specificity: Weigh 103 mg of blank auxiliary material into a 50 ml volumetric flask, prepare it according to the preparation method of the test solution under the content determination method, and take the diluent at the same time. Use the chromatographic conditions described in the present invention to perform the determination. The blank solvent peak, auxiliary material peak and main peak are completely separated, and there is no interfering peak at the main component peak position.

[0049] ② Linearity: 6 mg, 5 mg, 4 mg, 3 mg and 2 mg of mabaloxavir reference substance were respectively placed in 5 10 mL volumetric flasks, diluted to the scale with diluent, and used as linear solutions with concentration levels of 150%, 125%, 100%, 75% and 50%. The chromatographic conditions of the present invention were used for determination, and linear regression was performed with concentration C as the abscissa and peak area A as the ordinate. The regression equation was A=133.9422C+0.4775, and the correlation coefficient R=1.000.

[0050] ③ Precision The precision was measured six times on different dates, by different analysts, using different equipment according to the above test conditions. The relative standard deviation of the test solution results for 12 times was calculated. The results are shown in Table 2. The relative standard deviation was less than 2.0%.

[0051] ④ Accuracy: Add a known amount of reference substance to the blank excipient in the prescription amount, prepare samples at 70%, 100%, and 130% concentration levels, prepare 3 test solutions for each concentration, and evaluate with the test results of 9 samples. The results are shown in Table 3. The average recovery rate is between 98% and 101%, and the relative standard deviation of the 9 recovery rate data is no more than 2.0%.

[0052] ⑤ Room temperature solution stability Under the chromatographic conditions described in the present invention, the test solution with a concentration level of 100% prepared under the accuracy item was placed at room temperature, and samples were injected and checked at 0, 16.2, 21.5, and 26.0 hours. The results are shown in Table 4. It can be seen from Table 4 that when the Mabaloxavir Tablets content test solution was placed at room temperature for 26.0 hours, the content result did not change significantly, indicating that the content test solution can be stable for 26.0 hours; the reference solution was taken and placed at room temperature, and samples were injected and checked at 0, 20.2, 25.5, and 30.1 hours. The results are shown in Table 5. It can be seen from Table 5 that when the Mabaloxavir Tablets reference solution was placed at room temperature for 30.1 hours, there was no significant change, indicating that the reference solution can be stable for 30.1 hours.

[0053] ⑥ Stability of reference solution at 2-8°C The reference solution under item 4) was placed in a refrigerator at 2-8°C, and sampled and checked at 98.3 hours under the chromatographic conditions of the present invention. The results are shown in Table 6. It can be seen from Table 6 that there was no obvious change in the reference solution of Mabaloxavir Tablets when it was placed at 2-8°C for 98.3 hours, indicating that the reference solution can be stable for 30.1 hours.

[0054] ⑦ Durability The content reference solution of Mabaloxavir Tablets and the test solution were taken, and the durability of the chromatographic conditions described in the present invention was investigated by making small changes in the column temperature, flow rate, mobile phase ratio, chromatographic column brand and liquid phase brand. The results showed that slight changes in the above chromatographic conditions did not affect the content determination of Mabaloxavir Tablets.

[0055] Table 1 Results of suitability of the assay system

[0056]

[0057]

[0058] Table 2 Precision results

[0059]

[0060] Table 3 Accuracy results

[0061]

[0062]

[0063] Table 4 Room temperature reference solution stability results

[0064] Time (h) Peak area (%) Recovery rate (%) - 54.9875 - 16.2 55.0620 100.1 21.5 55.0763 100.2 26.0 55.2104 100.4

[0065] Table 5 Room temperature reference solution stability results

[0066] Time (h) Peak area (%) Recovery rate (%) 0 58.4577 - 20.2 58.4267 99.9 25.5 58.5098 100.1 30.1 58.4677 100.0

[0067] Table 6 2-8℃ reference solution stability results

[0068] Time (h) Recovery rate (%) 98.3 99.8 .

Claims

1. A method for determining the content of mabaloxavir tablets, characterized in that The method for determining the content of mabaloxavir tablets is to analyze mabaloxavir using HPLC, and the specific operation is as follows: Step 1: Preparation of test solution Accurately weigh mabaloxavir and add it to a volumetric flask, add 10-20% of the volume of solution B to mix, then add 45-50% of the volume of acetonitrile to mix, ultrasonically mix, dilute to scale with a diluent, shake well, centrifuge, take the supernatant, and prepare a solution containing about 0.28-0.52 mg of mabaloxavir per 1 mL; wherein solution B is composed of 0.18-0.22 mmol / L disodium ethylenediaminetetraacetic acid and 0.1-0.2% trifluoroacetic acid solution by volume; Step 2: Preparation of reference solution Take the mabaloxavir reference substance, weigh it accurately, add the diluent to dissolve it and quantitatively dilute it to make a solution containing about 0.28-0.52 mg of mabaloxavir per 1 mL; The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler; solution B-acetonitrile as mobile phase, isocratic elution; detection wavelength is 259 nm, flow rate is 0.8 mL to 1.2 mL per minute; column temperature is 30°C to 40°C; injection volume is 5 μL; sample concentration is 0.28 mg / mL to 0.52 mg / mL; The diluents described in step 1 and step 2 are both prepared by mixing acetonitrile and solution B in a volume ratio of 2 to 4:

1.

2. The method for determining the content of mabaloxavir tablets according to claim 1, characterized in that The chromatographic column is 4.6×100 mm, 3 μm or a chromatographic column of equivalent performance.

3. The method for determining the content of mabaloxavir tablets according to claim 2, characterized in that The 4.6×100 mm, 3 μm octadecylsilane bonded silica gel column is a CAPCELL PAK MGⅡ series column or a BDS Hypersil TM Series of chromatographic columns.

4. The method for determining the content of mabaloxavir tablets according to claim 1, characterized in that The volume ratio of solution B to acetonitrile is 45:55-55:

45.

5. The method for determining the content of mabaloxavir tablets according to claim 1, characterized in that Accurately measure the reference solution, inject it into the high performance liquid chromatograph, record the chromatogram, the theoretical plate number is not less than 4000 based on the mabaloxavir peak: the symmetry factor of the mabaloxavir peak is not greater than 1.5: repeat the injection 5 times, and the relative standard deviation of the mabaloxavir peak area is not greater than 1.0%.

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