Method for determining three methanesulfonate genotoxic impurities in prasefovir mesylate tablets by gas chromatography-mass spectrometry

Abstract

The invention relates to a method for determining three methanesulfonate genotoxic impurities in a prasefovir mesylate tablet by gas chromatography-mass spectrometry, and belongs to the technical field of pharmaceutical analysis. The invention relates to a method for determining three methanesulfonate genotoxic impurities in prasefovir mesylate tablets by gas chromatography-mass spectrometry. The method comprises the following steps: (1) preparing three single-standard stock solutions, a mixed standard solution and a mixed standard series solution; (2) preparing a test solution; (3) rapid qualitative screening; and (4) quantitative determination. The gas chromatography-mass spectrometry method (GC-MS method) has remarkable advantages in the aspects of operation simplicity, specificity, sensitivity, accuracy and the like, the method is simple in operation process, sample pretreatment is easy to implement, the detection time is greatly shortened, and the detection efficiency is improved.

Description

Technical Field

[0001] This application relates to the technical field of pharmaceutical analysis, and particularly to a method for determining three kinds of methylsulfonate genotoxic impurities in prefurcavir mesylate tablets by gas chromatography-mass spectrometry. Background Art

[0002] Prefurcavir mesylate tablets are a class 1 innovative drug independently developed by Xi'an Glaxo New Tong Pharmaceutical Co., Ltd., which was just approved for marketing on October 28, 2024, and can be regarded as a new force in the field of anti-hepatitis B virus. The antiviral efficacy of this drug in phase I to III clinical trials is comparable to that of the first-line drug tenofovir disoproxil fumarate tablets, and it avoids the common renal toxicity risks of traditional nucleoside drugs. It provides a new treatment option with both efficacy and safety for patients with chronic hepatitis B.

[0003] This drug is a newly launched drug. After a comprehensive search of a large number of literatures and patents, at present, the research on prefurcavir mesylate tablets mainly focuses on the synthesis, purification process and pharmacodynamics fields, and there are obvious deficiencies in the research on impurities, especially genotoxic impurities. According to the prefurcavir mesylate drug synthesis and purification processes disclosed in Chinese patents with publication numbers CN102827206A and CN112010905A, it is found that low-grade alcohols such as methanol and ethanol used as nucleophiles are prone to nucleophilic substitution side reactions with methanesulfonic acid or its derivatives, generating methyl, ethyl, and isopropyl methanesulfonate impurities. In addition, the methanesulfonic acid salt formation reaction is at the end of the drug synthesis process. If the starting material of methanesulfonic acid itself carries methylsulfonate impurities, during the salt formation process, these impurities will directly enter the drug end product along with the reaction process, ultimately resulting in the risk of residual methylsulfonate impurities in the drug. Methylsulfonate impurities are DNA reactive substances and still have the risk of mutagenesis and carcinogenesis at extremely low levels. According to the guiding principles of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the current 2020 edition of the Chinese Pharmacopoeia, strict control of impurities that may cause genotoxicity during the drug synthesis and purification processes is required. However, there is currently a lack of effective detection methods for such genotoxic impurities in prefurcavir mesylate tablets. Therefore, it is urgent to establish a qualitative and quantitative method for methylsulfonate genotoxic impurities in prefurcavir mesylate tablets at the trace level for daily inspection or drug quality control. Summary of the Invention

[0004] In view of this, this application provides a method for determining three kinds of methylsulfonate genotoxic impurities in prefurcavir mesylate tablets by gas chromatography-mass spectrometry. This detection method can efficiently and accurately determine the methylsulfonate genotoxic impurities in prefurcavir mesylate tablets, and can effectively overcome the defects existing in the above-mentioned prior art.

[0005] The present application provides a method for determining three kinds of methylsulfonate genotoxic impurities in prefurcavir mesylate tablets by gas chromatography-mass spectrometry, which comprises the following steps:

[0006] (1) Weigh precisely three kinds of methylsulfonate genotoxic impurities and place them separately in volumetric flasks. Dissolve them with ethyl acetate and dilute to the scale, then shake well to obtain three single-standard stock solutions. Precisely measure the three single-standard stock solutions and place them in the same volumetric flask, dilute to the scale with ethyl acetate, and shake well to obtain a mixed standard solution. Pipette an appropriate amount of the mixed standard solution and dilute it with ethyl acetate to form a series of mixed standard series solutions with different mass concentrations.

[0007] (2) Preparation of test solution: Weigh precisely an appropriate amount of the fine powder of prefurcavir mesylate tablets, place it in a centrifuge tube, add ethyl acetate, shake well, extract by ultrasonic treatment, and filter through an organic filter membrane to obtain the test solution.

[0008] (3) Rapid qualitative screening: Take the test solution and the mixed standard series solutions, and determine them under the same test conditions. If chromatographic peaks of quantitative ions and qualitative ions appear in the test solution, the retention time of the characteristic ion peak of the analyte is consistent with the retention time of the corresponding standard solution at the same concentration, and the maximum deviation of the relative abundance ratio of the selected qualitative ions from the relative abundance ratio of the qualitative ions of the corresponding standard solution at the same concentration should not exceed the corresponding standard, then it can be determined that the corresponding detection component exists in the test solution.

[0009] (4) Quantitative determination: Take the mixed standard series solutions and determine them in turn. Use the series concentrations of the analytes as the abscissa and the peak areas of the analytes as the ordinate to perform linear regression and establish a standard curve. Take the test solution and determine it. Substitute the peak area of the corresponding quantitative ion chromatographic peak into the linear regression equation to calculate the content of each component in the test solution.

[0010] Preferably, the gas chromatography conditions are as follows:

[0011] Chromatographic column: DB-35ms; injection port temperature: 260 °C; temperature programming: maintain at 50 °C for 1 min, increase to 260 °C at a rate of 15 °C / min and maintain for 5 min; injection mode: direct injection, splitless; injection volume: 1 μL; carrier gas: high-purity helium gas.

[0012] Preferably, the size of the DB-35ms chromatographic column is 30 m × 0.32 μm × 0.25 mm, and the DB-35ms chromatographic column is a capillary column filled with (35%-phenyl)-methyl polysiloxane.

[0013] Preferably, the mass spectrometry conditions are as follows:

[0014] Ion source type: EI source, 70 eV; Interface temperature: 280 °C; Ion source temperature: 230 °C; Quadrupole temperature: 150 °C; Acquisition mode: Selected ion monitoring mode SIM, monitoring fragment ions.

[0015] Preferably, the three kinds of methyl sulfonate genotoxic impurities are methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate.

[0016] Preferably, the detected concentrations of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are 0.00529 μg / mL, 0.00535 μg / mL and 0.00509 μg / mL respectively.

[0017] Preferably, the quantitative concentrations of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are 0.0159 μg / mL, 0.0161 μg / mL and 0.0153 μg / mL respectively.

[0018] Preferably, in step (1), the series of mass concentrations of the mixed standard series solution are 0.010, 0.020, 0.050, 0.12, 0.20 μg / mL respectively.

[0019] Preferably, in step (2), the ultrasonic extraction time is 10 min, and the pore size of the organic filter membrane is 0.22 μm.

[0020] Preferably, in step (3), the corresponding criteria are:

[0021] When the relative ion abundance ratio k > 50%, the maximum allowable deviation is ±20%; when 20% < relative ion abundance ratio k ≤ 50%, the maximum allowable deviation is ±25%; when 10% < relative ion abundance ratio k ≤ 20%, the maximum allowable deviation is ±30%; when the relative ion abundance ratio k ≤ 10%, the maximum allowable deviation is ±50%.

[0022] This application constructs a specific detection method for the genotoxic impurities - methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate that may be generated during the synthesis and purification of the new drug prefuroviral mesylate tablets. This method has many advantages: First, it fills the blank in the field of genotoxic impurity detection of this new drug, providing key support for the safety evaluation and quality control of this drug; second, it can accurately monitor the alkyl methanesulfonate impurities in the drug, helping pharmaceutical companies effectively prevent potential medication risks during the production process and significantly improving the drug quality; third, the operation process of this method is simple, the sample pretreatment is easy to carry out, greatly shortening the detection time and improving the detection efficiency, and it can be stably and reliably applied to actual detection.

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] 1. This study focuses on the detection of genotoxic impurities, namely alkyl sulfonates, during the synthesis of the newly launched anti-hepatitis B virus drug (Prevefovir Mesylate Tablets), filling the research gap in this field and improving the comprehensive understanding of this drug. Through analysis, it can be seen that the alkyl sulfonate impurities in Prevefovir Mesylate Tablets mainly come from two aspects: one is side reactions, and the other is the starting material, methanesulfonic acid. These alkyl sulfonate impurities are genotoxic warning substances. By deeply analyzing the drug synthesis process and establishing a detection method for genotoxic impurities, from the perspective of medication safety, this is of great significance for strictly controlling drug quality. It can effectively reduce the possibility of potential health risks caused by patients taking drugs containing such impurities, providing a strong guarantee for the medication safety of patients. From the perspective of the conditions for impurity generation, studying the genotoxic impurities that are likely to be generated in the main drug synthesis route is also of positive significance for the analysis of main component impurities.

[0025] 2. According to the ICH guidelines and the regulations of the European Medicines Agency on genotoxic impurities, combined with the maximum daily dosage of this drug, the limit standards for alkyl sulfonates in Prevefovir Mesylate Tablets were clarified (both shall not exceed 0.003%). This application established a GC-MS method for the detection of sulfonate impurities to ensure that the drug R & D and production processes meet the strict requirements of global drug regulatory authorities for the control of genotoxic impurities.

[0026] 3. The gas chromatography-mass spectrometry (GC-MS) method established in this experiment has significant advantages in terms of simplicity of operation, specificity, sensitivity, and accuracy. The operation process of this method is simple, and the sample pretreatment is easy to implement, greatly shortening the detection time and improving the detection efficiency. By using the selected ion monitoring mode (SIM) for qualitative and quantitative analysis of target compounds, it effectively avoids the interference of the matrix and co-eluting substances, ensuring the reliability of the detection results. In terms of sensitivity, this method has low detection limits and quantification limits for methyl sulfonate, ethyl sulfonate, and isopropyl sulfonate, and can accurately detect extremely low levels of alkyl sulfonate impurities, ensuring the rigor of drug quality control. In addition, the relative standard deviation (RSD) of the repeatability test is less than 5%, and the average recoveries at different concentration levels are between 92.47% and 110.31%, with RSD of 3.66% - 4.11%, indicating that this method has good repeatability and high accuracy, and can be stably and reliably used for actual detection.

[0027] 4. In summary, the GC-MS method established in this application for the determination of three genotoxic sulfonate impurities in Prevefovir Mesylate Tablets is simple to operate, has good specificity, high sensitivity, and good accuracy. It is applicable to the simultaneous detection of 3 sulfonate genotoxic impurities in Prevefovir Mesylate Tablets, providing strong technical support for the quality control and standard establishment of this new drug, and helping to ensure the medication safety of patients. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the present application or the prior art, the drawings required for use in the description of the present application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 TIC diagram for blank solvent determination;

[0030] Figure 2 Determine the TIC diagram for the test solution;

[0031] Figure 3 Determine the TIC diagram for the mixed standard solution. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in this application will be clearly and completely described below in conjunction with the drawings in this application. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0033] The detection method of this application is the standard curve method specified in the Pharmacopoeia. If the following experimental processes and test methods are not described in detail, standard operating procedures shall be adopted, such as the operation methods of solution preparation and system suitability test.

[0034] Instruments: Agilent 7890B-5977B gas chromatograph-mass spectrometer (origin: the United States); AE-240 electronic balance (Mettler, Switzerland, accuracy 0.01 mg); KM-1030C ultrasonic cleaner (Guangzhou Kejiemeng Instrument Co., Ltd.);

[0035] Reagents and drugs: ethyl acetate (TEDIA; batch number: 411086, chromatographic grade); methyl methanesulfonate (Yuanye Biotechnology; batch number: M09S8K43406; purity 98%); ethyl methanesulfonate (B&K; batch number: LE40M36; purity 98%); isopropyl methanesulfonate (Shanghai Haohong Biotechnology Co., Ltd.; batch number Lb0321016; purity 98%); Prefovir mesylate tablets (batch numbers are 180303; 180304; 180305; 180306; specifications are 30 mg; 45 mg; 60 mg; 75 mg respectively); ultrapure water.

[0036] Chromatographic conditions: Chromatographic column: DB-35ms (30m × 0.32μm × 0.25mm), using (35%-phenyl)-methyl polysiloxane as the packing material; Injection port temperature: 260°C; Interface temperature: 280°C; Temperature programming: Hold at 50°C for 1 min; Increase to 260°C at a rate of 15°C / min and hold for 5 min; Carrier gas: High-purity helium; Injection method: Direct injection, splitless; Injection volume: 1 μL; Ion source type: EI source, 70 eV; Ion source temperature: 230°C; Quadrupole temperature: 150°C; Acquisition mode: Selected ion monitoring mode (SIM); The reference monitoring ions of each compound are shown in Table 1 below:

[0037] Table 1 Characteristic ion table of components to be measured

[0038] Number Component CAS No. Quantitative ion Qualitative ion Qualitative ion 1 Methyl methanesulfonate 66-27-3 80 79 65 2 Ethyl methanesulfonate 62-50-0 79 109 97 3 Isopropyl methanesulfonate 926-06-7 123 97 79

[0039] Example 1:

[0040] Preparation of test solution: Take an appropriate amount of the fine powder of this product (equivalent to about 20 mg of paritaprevir), weigh accurately, place it in a 10 mL centrifuge tube, add 5 mL of ethyl acetate, shake well, extract ultrasonically for 10 min, and filter through a 0.22 μm organic filter membrane to obtain the test solution.

[0041] Preparation of standard solution:

[0042] Preparation of single standard stock solution: Take about 50 mg each of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate, weigh accurately, place them in 50 mL volumetric flasks respectively, dissolve with ethyl acetate and dilute to the mark, shake well, and obtain;

[0043] Preparation of mixed standard solution: Accurately measure 0.5 mL each of the above three single standard stock solutions, place them in the same 100 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well, and obtain;

[0044] Preparation of mixed standard series solution: Absorb an appropriate amount of the mixed standard solution, and dilute it with ethyl acetate to prepare mixed standard series solutions with mass concentrations of about 0.010, 0.020, 0.050, 0.12, and 0.20 μg / mL.

[0045] Rapid qualitative screening:

[0046] Take the test solution and the mixed standard series solution, and measure them under the same test conditions. If chromatographic peaks of quantitative ions and qualitative ions appear in the test solution, the retention time of the characteristic ion peak of the tested component is consistent with the retention time of the corresponding concentration standard solution, and the maximum deviation of the relative abundance ratio of the selected qualitative ions from the relative abundance ratio of the qualitative ions of the corresponding concentration standard solution should not exceed the provisions of Table 2, then it can be determined that the corresponding test component exists in the test solution;

[0047] Table 2 Maximum allowable deviation of relative abundance ratio of ions during qualitative determination

[0048] Ion relative abundance (k) k＞50％ 50％≥k＞20％ 20％≥k＞10％ k≤10％ Maximum allowable deviation ±20％ ±25％ ±30％ ±50％

[0049] Quantitative determination: Sequentially measure the above-mentioned mixed standard series solutions. Using the series concentrations of the components to be measured as the abscissa and the peak areas of the components to be measured as the ordinate, perform linear regression to establish a standard curve; measure the test solution, substitute the corresponding quantitative ion chromatographic peak area into the linear regression equation, and calculate the contents of each component in the test solution.

[0050] Results: Take 2 portions each of samples of various specifications (30 mg; 45 mg; 60 mg; 75 mg), prepare the test solutions according to the above method, inject samples for determination under the above chromatographic conditions, record the peak areas, and calculate the contents. In the 4 batches of samples of different specifications, methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were not detected, and all 4 batches of samples met the requirements.

[0051] Example 2: Methodology investigation

[0052] Conduct methodology investigations on 3 detection methods for methanesulfonate impurities, including specificity, repeatability, accuracy, detection limit, and quantification limit. The specific investigation methods are as follows:

[0053] Preparation of mixed standard solution: The blank solvent is ethyl acetate; Standard solution: Take about 50 mg each of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate, accurately weigh them, place them in a 50 mL volumetric flask, dissolve with ethyl acetate and dilute to the scale for volume fixation, shake well, and obtain single standard stock solutions with mass concentrations of about 1.0 mg / mL respectively, and store at 4°C. Accurately measure 0.5 mL of each single standard stock solution respectively, place them in the same 100 mL volumetric flask, dilute to the scale with ethyl acetate to prepare a mixed standard solution with a mass concentration of about 5 μg / mL, and store at 4°C, as shown in Table 3.

[0054] Table 3 Preparation and concentration of mixed standard stock solution:

[0055]

[0056] Specificity test: Inject one portion each of the blank solvent, the test solution, and the mixed standard solution (with a mass concentration of 0.12 μg / mL) respectively, and perform injection and determination according to the above chromatographic conditions method, and record the chromatograms. The results show that other impurity peaks in the blank solvent and the test solution do not interfere with the detection of the target peaks of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate, the target peaks are completely separated and the resolution is greater than 1.5, indicating good specificity, as shown in Figures 1-3 .

[0057] Calibration curve: Appropriate amount of the mixed standard solution was taken and diluted with ethyl acetate to prepare a series of mixed standard solutions with mass concentrations of approximately 0.010, 0.020, 0.050, 0.12, and 0.20 μg / mL. The determination was carried out under the above chromatographic conditions, and the corresponding peak areas were measured. The concentration of the standard solution was used as the abscissa, and the peak area was used as the ordinate to plot the calibration curve, and the mathematical equation and linear range of the calibration curve were listed. The results showed that methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate had good linear relationships with the peak areas in the ranges of 0.01059 - 0.2118 μg / mL, 0.01070 - 0.2139 μg / mL, and 0.01019 - 0.2037 μg / mL, respectively, as shown in Table 4.

[0058] Table 4 Linear relationship, correlation coefficient, and linear range

[0059] Substance name Linear equation Correlation coefficient r Linear range (μg / mL) Methyl methanesulfonate Y = 254530X - 823.50 0.9984 0.01059～0.2118 Ethyl methanesulfonate Y = 373267X - 765.55 0.9989 0.01070～0.2139 Isopropyl methanesulfonate Y = 382109X - 883.69 0.9991 0.01019～0.2037

[0060] Detection limit and quantification limit: The samples were injected and determined under the above chromatographic conditions. The response values at a signal-to-noise ratio (S / N) of 3:1 and 10:1 were used as the detection limit and quantification limit, respectively. The measured detection limits of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were 0.00529 μg / mL, 0.00535 μg / mL, and 0.00509 μg / mL, respectively. The quantification limits of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were 0.0159 μg / mL, 0.0161 μg / mL, and 0.0153 μg / mL, respectively.

[0061] Instrument precision: The mixed standard solution with a mass concentration of 0.12 μg / mL under the linear relationship investigation item was taken and continuously injected and determined 6 times under the above chromatographic conditions, and the peak areas were recorded. The RSDs of the peak areas of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were 1.73%, 2.69%, and 2.55% (n = 6), respectively, indicating good instrument precision.

[0062] Table 5 Results of precision test

[0063] Component 1 2 3 4 5 6 RSD% Methyl methanesulfonate 30119 30535 31272 31639 30921 30910 1.73 Ethyl methanesulfonate 45056 46039 48677 46920 47650 47190 2.69 Isopropyl methanesulfonate 43818 44875 46434 45452 46959 46308 2.55

[0064] Stability test: Appropriate amounts of the mixed standard solution with a mass concentration of 0.12 μg / mL and the spiked solution (mass concentration: 0.12 μg / mL) under the linear relationship investigation item were taken. They were respectively placed at room temperature for 0, 2, 4, 8, 12, and 24 h, and then injected for determination according to the above chromatographic conditions. The chromatogram peak areas were recorded, and the contents were calculated. As a result, the RSDs of the areas of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate in the mixed standard solution were 1.58%, 2.39%, and 2.08% (n = 6) respectively. The RSDs of the areas of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate in the spiked solution were 3.03%, 2.09%, and 2.39% (n = 6) respectively, indicating that the stability of the mixed standard solution and the spiked solution was good within 12 h at room temperature.

[0065] Repeatability test: Six portions of the test sample were accurately weighed, and 5 mL of the 0.12 μg / mL standard solution was accurately added to each. They were prepared and determined according to the sample method, and the average content was calculated. As a result, the RSDs of the areas of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were 4.44%, 4.02%, and 3.22% (n = 6) respectively, indicating that the repeatability of the method was good.

[0066] Table 6 Results of repeatability test

[0067] Component 1 2 3 4 5 6 RSD% Methyl methanesulfonate 30843 30330 33547 30747 33299 31069 4.44 Ethyl methanesulfonate 49543 45881 49050 45221 49063 49331 4.02 Isopropyl methanesulfonate 48625 45734 48694 46358 45842 48878 3.22

[0068] Recovery test: Appropriate amount (equivalent to about 20 mg of pradefovir) of the sample with batch number 180304 was accurately weighed and placed in a 10 mL volumetric flask. 5 mL of each of the spiked solutions (low concentration 0.084 μg / mL, medium concentration 0.12 μg / mL, high concentration 0.156 μg / mL) was added, shaken well, ultrasonically extracted for 10 min, and filtered through a 0.22 μm organic filter membrane to obtain the spiked test sample solution; it was injected for determination according to the above chromatographic conditions, the peak areas were recorded, and the recoveries were calculated. As a result, the average recoveries of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate were 97.02%, 100.38%, and 103.63% respectively, and the RSDs were 4.11%, 4.04%, and 3.66% (n = 9) respectively, indicating that the accuracy of the method was good. The results are shown in Table 7.

[0069] Table 7 Results of recovery test

[0070]

[0071] The method established in the solution of this application is simple to operate, highly sensitive, specific, efficient and fast. It can be used for the determination of the contents of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in prefuroviral mesylate tablets, providing a reference for the risk assessment of this preparation and an effective quality control method for the production of qualified drugs. The research of this application helps pharmaceutical manufacturers to control genotoxic impurities in the production process and provides technical support for drug supervision.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for determining three methylsulfonate genotoxic impurities in pradefovir mesylate tablets by gas chromatography-mass spectrometry, characterized in that, It includes the following steps: (1) Weigh accurately three kinds of genotoxic impurities of mesylate esters respectively and place them in volumetric flasks. Dissolve them with ethyl acetate and dilute to the scale. After shaking well, three single-standard stock solutions are obtained. Accurately measure the three single-standard stock solutions respectively and place them in the same volumetric flask. Dilute with ethyl acetate to the scale. After shaking well, a mixed standard solution is obtained. Pipette an appropriate amount of the mixed standard solution and dilute it with ethyl acetate to form a series of mixed standard series solutions with different mass concentrations; (2) Preparation of test solution: Weigh accurately an appropriate amount of the fine powder of Prevefovir Mesylate Tablets and place it in a centrifuge tube. Add ethyl acetate, shake well, extract by ultrasonic wave, and filter through an organic filter membrane to obtain the test solution; (3) Rapid qualitative screening: Take the test solution and the mixed standard series solutions and measure them under the same test conditions. If chromatographic peaks of quantitative ions and qualitative ions appear in the test solution, the retention time of the characteristic ion peak of the analyte is consistent with the retention time corresponding to the standard solution of the equivalent concentration, and the maximum deviation of the relative abundance ratio of the selected qualitative ions from the relative abundance ratio of the qualitative ions of the standard solution of the equivalent concentration should not exceed the corresponding standard, then it can be determined that the corresponding detected component exists in the test solution; (4) Quantitative determination: Take the mixed standard series solutions and measure them in turn. Using the series concentrations of the analyte as the abscissa and the peak areas of the analyte as the ordinate, perform linear regression to establish a standard curve; Take the test solution for measurement, substitute the peak area of the corresponding quantitative ion chromatogram into the linear regression equation, and calculate the content of each component in the test solution.

2. The method for determining three kinds of methylsulfonate genotoxic impurities in preforvir mesylate tablets by gas chromatography-mass spectrometry according to claim 1, characterized in that The gas chromatography conditions are as follows: Chromatographic column: DB-35ms; Injection port temperature: 260 °C; Temperature programming: Hold at 50 °C for 1 min, raise to 260 °C at a rate of 15 °C / min and hold for 5 min; Injection mode: Direct injection, splitless; Injection volume: 1 μL; Carrier gas: High-purity helium gas.

3. The method for determining three methylsulfonate genotoxic impurities in prefurcatinib mesylate tablets by gas chromatography-mass spectrometry according to claim 2, characterized in that, The size of the DB-35ms chromatographic column is 30 m × 0.32 μm × 0.25 mm, and the DB-35ms chromatographic column is a capillary column filled with (35%-phenyl)-methyl polysiloxane.

4. The method for determining three methylsulfonate genotoxic impurities in prefurcatinib mesylate tablets by gas chromatography-mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions are as follows: Ion source type: EI source, 70 eV; Interface temperature: 280 °C; Ion source temperature: 230 °C; Quadrupole temperature: 150 °C; Acquisition mode: Selected ion monitoring mode SIM, monitoring fragment ions.

5. The method for determining three kinds of methylsulfonate genotoxic impurities in previfovir mesylate tablets by gas chromatography-mass spectrometry according to claim 1, characterized in that, The three kinds of genotoxic impurities of mesylate esters are methyl mesylate, ethyl mesylate and isopropyl mesylate.

6. The method for determining three kinds of methylsulfonate genotoxic impurities in prevofovir mesylate tablets by gas chromatography-mass spectrometry according to claim 5, characterized in that, The detected concentrations of methyl mesylate, ethyl mesylate and isopropyl mesylate are 0.00529 μg / mL, 0.00535 μg / mL and 0.00509 μg / mL respectively.

7. The method for determining three methylsulfonate genotoxic impurities in prefurcavir mesylate tablets by gas chromatography-mass spectrometry according to claim 5, characterized in that, The quantitative concentrations of methyl mesylate, ethyl mesylate and isopropyl mesylate are 0.0159 μg / mL, 0.0161 μg / mL and 0.0153 μg / mL respectively.

8. The method for determining three methylsulfonate genotoxic impurities in preforwett tablet by gas chromatography-mass spectrometry according to claim 1, characterized in that, In step (1), the series of mass concentrations of the mixed standard series solutions are 0.010, 0.020, 0.050, 0.12, 0.20 μg / mL respectively.

9. The method for determining three methylsulfonate genotoxic impurities in pradefovir mesylate tablets by gas chromatography-mass spectrometry according to claim 1, wherein In step (2), the ultrasonic extraction time is 10 min, and the pore size of the organic filter membrane is 0.22 μm.

10. The method for determining three methylsulfonate genotoxic impurities in prefurcavir mesylate tablets by gas chromatography-mass spectrometry according to claim 1, characterized in that, In step (3), the corresponding criteria are as follows: When the relative ion abundance ratio k > 50%, the maximum allowable deviation is ±20%; when 20% < relative ion abundance ratio k ≤ 50%, the maximum allowable deviation is ±25%. When 10% < relative ion abundance ratio k ≤ 20%, the maximum allowable deviation is ±30%; when the relative ion abundance ratio k ≤ 10%, the maximum allowable deviation is ±50%.

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