Morinonidazole raw material, potential genotoxic impurity in preparation of morphonidazole raw material, and preparation method and application of potential genotoxic impurity

Abstract

The invention relates to the technical field of medicines, in particular to a morphonidazole raw material, a potential genotoxic impurity in a preparation of the morphonidazole raw material and a preparation method and application of the morphonidazole raw material. The potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole, and the potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole. According to the invention, a novel diazole compound 2-methyl-5-nitro-1-nitrosoimidazole is found in a synthesis process of a morphonidazole raw material for the first time, and the compound contains a nitrosamine warning structure, so that the compound is identified as a potential genotoxic impurity. According to the invention, the ultra-high performance liquid chromatography-mass spectrometry analysis method of the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole is established for the first time, and the method has important significance on quality control of the morphonidazole raw material and the preparation thereof. The detection method provided by the invention provides reference for quality evaluation of other starting materials, intermediates and bulk drugs capable of producing 2-methyl-5-nitro-1-nitrosoimidazole.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to a potential genotoxic impurity in a morinidazole raw material and its preparation, a preparation method thereof, and an application thereof. Background Art

[0002] Morinidazole, with the chemical name of 1-[3-(4-morpholinyl)-2-hydroxypropyl]-2-methyl-5-nitro-1H-imidazole, has the following structural formula: , is the first third-generation nitroimidazole antibacterial drug in China with independent intellectual property rights. Its anti-anaerobic activity is stronger than that of metronidazole, tinidazole, and ornidazole, and it has better safety. After entering susceptible microbial cells, the nitro group in the molecular structure of morinidazole is reduced to a cytotoxic amino group by an electron transfer protein under anaerobic or hypoxic conditions at a relatively low redox potential, destroying the double helix structure of DNA, degrading the synthesized DNA, inhibiting the DNA synthesis and transcription of cells, and causing the death of pathogen cells. At present, the marketed drugs with morinidazole as the active ingredient mainly include morinidazole sodium chloride injection, which is clinically used for the treatment of gastroenteritis, pelvic inflammatory disease, cholecystitis, pneumonia, appendicitis, etc.

[0003] The synthetic process route of morinidazole is as follows: .

[0004] The starting material molecule of morinidazole contains a secondary amine structure. Therefore, in the synthesis process of morinidazole, there is a possibility of generating impurities containing a nitrosamine genotoxic warning structure. Potential genotoxic impurities can cause gene mutations in cells or in vivo mutagenesis at trace levels, causing direct or indirect damage to the human genetic material, and then leading to genetic toxicity such as carcinogenesis and teratogenesis. Nitrosamine potential genotoxic impurities are one of the recognized strong carcinogens. Among the carcinogen lists published by the International Agency for Research on Cancer (IARC), 18 nitrosamine compounds are included, including 2 category 1 substances, 5 category 2A substances, and 11 category 2B substances.

[0005] At present, the European EMA, the US FDA, and regulatory agencies in various countries around the world have more explicit requirements for potential genotoxic impurities. More and more pharmaceutical companies have focused on the control and detection of potential genotoxic impurities in the new drug R & D process. Therefore, the discovery of new potential genotoxic impurities and how to achieve high-sensitivity and high-selectivity analysis of such impurities have become challenges in the field of drug R & D. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies existing in the prior art and provide a newly discovered potential genotoxic impurity in the raw material of morinidazole and its preparations. This impurity is a new diazole compound, 2-methyl-5-nitro-1-nitrosoimidazole.

[0007] The present invention further provides a preparation method for the above-mentioned potential genotoxic impurity.

[0008] Another object of the present invention is to provide the application of the above-mentioned potential genotoxic impurity in the quality control of the raw material of morinidazole and its preparations; an ultra-high performance liquid chromatography-mass spectrometry combined analysis method is adopted. This method is simple, fast, highly specific, sensitive, and has good reproducibility. It can be applied to the quality control of the raw material of morinidazole and its preparations, and can also be used for the quality evaluation of other starting materials or intermediates that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a potential genotoxic impurity in the raw material of morinidazole and its preparations. The potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole, and its structural formula is as follows: 。

[0010] The present invention also provides a method for separating and purifying the potential genotoxic impurity in the raw material of morinidazole and its preparations, including the following steps: (1) Take the raw material of morinidazole, add hydrogen peroxide solution, heat it, then add methanol-water to dissolve and dilute, shake well, and filter; (2) Use semi-preparative liquid chromatography for chromatographic separation, collect the eluate of the 2-methyl-5-nitro-1-nitrosoimidazole chromatographic peak, concentrate it by nitrogen blowing, and obtain the concentrated solution of 2-methyl-5-nitro-1-nitrosoimidazole; (3) Subject the collected concentrated solution of 2-methyl-5-nitro-1-nitrosoimidazole to isocratic elution with acetonitrile-water on a high-performance preparative liquid chromatography reverse-phase C8 column and isocratic elution with methanol-water on a reverse-phase C18 column respectively. Collect and combine the eluate of the chromatographic peak, and freeze-dry it to obtain purified 2-methyl-5-nitro-1-nitrosoimidazole.

[0011] Further, in step (1), the ratio of the morinidazole to the hydrogen peroxide solution is 10 mg:1 mL; the volume concentration of the hydrogen peroxide solution is 1% - 3%; the heating treatment is carried out in a 60 °C water bath for 24 h; the volume ratio of methanol to water is 1:1.

[0012] Further, in step (2), the chromatographic conditions of the semi-preparative liquid chromatography are as follows: an Agilent 1200 semi-preparative liquid chromatograph, an Agilent Eclipse XDB C18 (9.4×250 mm, 5 µm) preparative column, with methanol - 0.1% phosphoric acid solution as the mobile phase, gradient elution, the volume ratio of methanol to 0.1% phosphoric acid solution is 20:80 to 100:0, the column temperature is 40°C, the detection wavelength is 254 nm, the flow rate is 2 mL / min, and the injection volume is 400 µL.

[0013] Further, in step (3), the volume ratio of acetonitrile to water is 50:50; the volume ratio of methanol to water is 60:40.

[0014] The present invention also provides the application of the above-mentioned potential genotoxic impurities in the quality control of morinidazole raw materials and their preparations. An ultra-high performance liquid chromatography - mass spectrometry combined analysis method is adopted, which specifically includes the following steps: (1) Preparation of the standard curve solution Precisely measure the reference substance of the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole, dissolve it with a solvent and quantitatively dilute it to prepare solutions with different concentrations to obtain the standard curve solution; (2) Preparation of the test solution Take the morinidazole raw material, first heat and extract it with solvent 1, then add solvent 2 and mix and dissolve it, shake well, filter to obtain the test solution; (3) Determination Precisely measure the solvent, the standard curve solution and the test solution respectively, use an ultra-high performance liquid chromatography - mass spectrometry combined instrument, under gradient elution conditions, perform chromatographic separation on morinidazole and potential genotoxic impurities, and use a mass spectrometry detector for analysis and detection; calculate the content of potential genotoxic impurities in the test solution according to the standard curve method with the peak area.

[0015] Further, in step (1), the concentration of the standard curve solution is 0.45 - 2.25 ng / mL; the solvent is methanol - water, and the volume ratio of methanol to water is 40:60 to 60:40.

[0016] Further, in step (2), the solvent 1 is methanol; the solvent 2 is water; the volume ratio of methanol to water is 40:60 to 60:40; the heating treatment is carried out under a water bath condition of 40 - 60°C; the concentration of the test solution is 50 mg / mL.

[0017] Further, the liquid chromatography conditions described in step (3) are as follows: chromatographic column: using pentafluorophenylsilane-bonded silica gel as the stationary phase, with the specification model of Agilent Poroshell 120 PFP, 3.0×100 mm, 1.9 μm; mobile phase: A: 0.1% - 0.5% formic acid aqueous solution; B: methanol; gradient elution; flow rate: 0.3 - 0.5 mL / min; column temperature: 35 - 45 °C; injection volume: 1 μL; The gradient elution program is as follows: 。

[0018] The conditions of the mass spectrometry are as follows: Ion source: Agilent Jet Stream Electrospray Ionization Source (AJS ESI); Scanning mode: positive ion scanning mode; Monitoring mode: Multiple Reaction Monitoring (MRM); The ion source parameters are as follows: 。

[0019] The monitored ion pairs of the compound are as follows: 。

[0020] Further, if the 2-methyl-5-nitro-1-nitrosoimidazole peak is detected in the chromatogram of the test solution, calculated by the standard curve method, the content of 2-methyl-5-nitro-1-nitrosoimidazole shall not exceed 18 ppb.

[0021] The present invention adopts ultra-high performance liquid chromatography-mass spectrometry coupling technology, which can realize the trace analysis of the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole in the raw material of morinidazole. Through the pretreatment method provided by the present invention, a high extraction recovery rate of 2-methyl-5-nitro-1-nitrosoimidazole and the stability of the test solution are achieved; secondly, by selecting a pentafluorophenylsilane-bonded silica gel chromatographic column and using methanol as the organic phase, sufficient chromatographic separation of the 2-methyl-5-nitro-1-nitrosoimidazole chromatographic peak and the morinidazole chromatographic peak is realized, and formic acid is added to the mobile phase to enhance the mass spectrometry signal of 2-methyl-5-nitro-1-nitrosoimidazole; through full scan analysis of the first-level mass spectrometry, screening of quantitative and qualitative ion pairs by scanning of the second-level fragment ions, and optimizing the collision energy of each ion pair by MassHunter software; further, optimizing the mass spectrometry parameters such as the drying gas temperature, sheath gas temperature, and capillary voltage, finally realizing the trace detection and accurate quantification of 2-methyl-5-nitro-1-nitrosoimidazole.

[0022] The present invention provides an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) technique for the separation and quantitative determination of the diazole new compound 2-methyl-5-nitro-1-nitrosoimidazole in the raw material of morinidazole, effectively realizing the quality control of potential genotoxic impurities in the raw material of morinidazole, reducing the occurrence of adverse drug reactions in patients, and ensuring the medication safety of patients to a certain extent. At the same time, it provides a reference for the quality evaluation of other starting materials or intermediates that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

[0023] The beneficial effects of the present invention are as follows: 1. The present invention first discovers the diazole new structural compound 2-methyl-5-nitro-1-nitrosoimidazole in the synthesis process of the raw material of morinidazole. This compound is identified as a potential genotoxic impurity due to the presence of the nitrosamine warning structure.

[0024] 2. The present invention first establishes an ultra-high performance liquid chromatography-mass spectrometry analysis method for the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole, which is of great significance for the quality control of the raw material of morinidazole and its preparations.

[0025] 3. The detection method of the present invention provides a reference for the quality evaluation of other starting materials, intermediates and bulk drugs that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

[0026] 4. The detection method of the present invention has high sensitivity and can effectively detect the impurity 2-methyl-5-nitro-1-nitrosoimidazole. The detection limit concentration is 2.7 ppb, and the quantitation limit concentration is 9.1 ppb.

[0027] 5. The detection method of the present invention has strong specificity, is accurate and reliable. The recovery rate of the impurity 2-methyl-5-nitro-1-nitrosoimidazole is 96.3%; the repeatability RSD is 4.7%; the precision RSD is 2.9%. Description of the Drawings

[0028] Figure 1 is the 1 H-NMR spectrum of 2-methyl-5-nitro-1-nitrosoimidazole; Figure 2 is the 1 C-NMR spectrum of 2-methyl-5-nitro-1-nitrosoimidazole; Figure 3 is the high-resolution mass spectrum of 2-methyl-5-nitro-1-nitrosoimidazole; Figure 4 is the ultraviolet spectrum of 2-methyl-5-nitro-1-nitrosoimidazole; Figure 5 is the blank solvent chromatogram of the specificity verification experiment; a: quantitative ion pair m / z128.0→82.0 Extracted chromatogram, b: Qualitative ion pair m / z 128.0→42.0 Extracted chromatogram; Figure 6 Chromatogram of the mixed solution for the specificity verification experiment; a: Extracted ion chromatogram of morinidazole; b: Quantitative ion pair of MNIMZ m / z 128.0→82.0 Extracted chromatogram; c: Qualitative ion of MNIMZ m / z 128.0→42.0 Extracted chromatogram; Figure 7 Chromatogram of the test solution for the method accuracy verification experiment; a: Quantitative ion pair m / z 128.0→82.0 Extracted chromatogram, b: Qualitative ion pair m / z 128.0→42.0 Extracted chromatogram; Figure 8 Standard curve of impurity 2-methyl-5-nitro-1-nitrosoimidazole Detailed implementation mode

[0029] The present invention will be further described below in conjunction with specific embodiments. The specific examples are used to better illustrate the content of the present invention rather than limit the present invention. Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels. Therefore, those skilled in the art who make non-essential improvements and adjustments based on the above-mentioned invention content still fall within the protection scope of the present invention.

[0030] Example 1 Separation and purification of impurities (1) Degradation test Take about 50 mg of morinidazole raw material, accurately weigh it, place it in a 50 mL volumetric flask, accurately add 5 mL of 3% hydrogen peroxide solution (take 1 mL of 30% hydrogen peroxide solution and dilute it to 10 mL with purified water), heat it in a water bath at 60 °C for 24 hours, then add methanol-water (50:50) to dissolve and dilute to the mark, shake well, and filter; (2) Separation and preparation of impurities by semi-preparative liquid chromatography: Use an Agilent 1200 semi-preparative liquid chromatograph, an Agilent Eclipse XDB C18 (9.4×250 mm, 5 µm) preparation column, use methanol-0.1% phosphoric acid solution as the mobile phase, gradient elution, the volume ratio of methanol to 0.1% phosphoric acid solution is 20:80 to 100:0, the column temperature is 40 °C, the detection wavelength is 254 nm, the flow rate is 2 mL / min, the injection volume is 400 μL, collect the eluate of the chromatographic peak of impurity 2-methyl-5-nitro-1-nitrosoimidazole, and concentrate it by nitrogen blowing; (3) Purification: The concentrated solution of the impurity 2-methyl-5-nitro-1-nitrosoimidazole collected in step (2) is subjected to isocratic elution with high-performance preparative liquid chromatography using a reverse-phase C8 column with acetonitrile-water (50:50) and a reverse-phase C18 column with methanol-water (60:40). The eluates of the chromatographic peaks are collected and combined, and then freeze-dried to obtain purified 2-methyl-5-nitro-1-nitrosoimidazole.

[0031] For the separated 2-methyl-5-nitro-1-nitrosoimidazole 1 The 1H-NMR spectrum is as Figure 1 shown 1 The 13C-NMR spectrum is as Figure 2 shown; the mass spectrum of 2-methyl-5-nitro-1-nitrosoimidazole is as Figure 3 shown; the ultraviolet spectrum of 2-methyl-5-nitro-1-nitrosoimidazole is as Figure 4 shown.

[0032] Example 2 Quality Control Method (1) Preparation of standard curve solution Accurately measure an appropriate amount of the reference substance of 2-methyl-5-nitro-1-nitrosoimidazole, dissolve it with the solvent [methanol-water (50:50)], and quantitatively dilute it to prepare a solution containing about 0.45, 0.6, 0.9, 1.5, and 2.25 ng per 1 mL.

[0033] (2) Preparation of test solution Take about 500 mg of the raw material of morinidazole, accurately weigh it, place it in a 10 mL volumetric flask, first add 6 mL of methanol and heat for extraction at 40 °C, then add 4 mL of water to dissolve and dilute to the mark, shake well, and filter.

[0034] (3) Determination Accurately measure 1 μL of the solvent, standard curve solution, and test solution respectively, inject them into an ultra-high performance liquid chromatography-mass spectrometry instrument, and record the chromatogram. Calculate the content of 2-methyl-5-nitro-1-nitrosoimidazole in the test solution according to the standard curve method based on the peak area.

[0035] The chromatographic conditions are as follows: Use a chromatographic column with pentafluorophenyl silane-bonded silica gel as the stationary phase, Agilent Poroshell 120 PFP (3.0×100 mm, 1.9 μm); use 0.1% - 0.5% formic acid aqueous solution as mobile phase A and methanol as mobile phase B, and perform linear gradient elution according to the program in Table 1 below; the flow rate is 0.3 - 0.5 mL / min, the column temperature is 35 - 45 °C, and the injection volume is 1 μL.

[0036] Table 1

[0037] The mass spectrometry conditions were as follows: electrospray ionization source (AJS ESI) was used, in positive ion scan mode, with multiple reaction monitoring (MRM). The quantitative ion pair of 2-methyl-5-nitro-1-nitrosoimidazole was 128→82, the collision energy was 20 V, the qualitative ion pair was 128→42, and the collision energy was 42 V. The ion source parameters are shown in Table 2.

[0038] Table 2

[0039] Example 3 The method provided in Example 2 was subjected to specificity verification, detection limit verification, repeatability verification, accuracy verification, and durability verification.

[0040] (1) Specificity verification Blank solvent: methanol-water (50:50) Impurity 2-methyl-5-nitro-1-nitrosoimidazole positioning solution: 90 ng / mL reference substance solution.

[0041] Blank test solution: Take 500 mg of the test substance, accurately weigh it, place it in a 10 mL volumetric flask, first add 6 mL of methanol and heat for extraction at 40 °C, then add 4 mL of water to dissolve and dilute to the scale, and shake well.

[0042] Mixed solution: Take 500 mg of the test substance, accurately weigh it, place it in a 10 mL volumetric flask, accurately add 1 mL of 90 ng / mL impurity 2-methyl-5-nitro-1-nitrosoimidazole reference substance solution and 6 mL of methanol, heat for extraction at 40 °C, then add 4 mL of water to dissolve and dilute to the scale, and shake well to obtain a mixed sample solution.

[0043] Precisely measure 1 μL of each of the above solutions and inject them into an ultra-high performance liquid chromatography-mass spectrometry instrument, and record the chromatogram. The results are shown in Table 3, Figure 5 、 Figure 6 as shown.

[0044] Table 3 Specificity test results

[0045] Conclusion: The blank solvent and morinidazole do not interfere with the detection of the impurity 2-methyl-5-nitro-1-nitrosoimidazole, and the specificity verification meets the requirements.

[0046] (2) Detection limit / quantitation limit verification Quantitation limit solution: Take 0.45 ng / mL reference substance solution as the quantitation limit solution.

[0047] Detection limit solution: Precisely measure 3 mL of the quantitation limit solution, place it in a 10 mL volumetric flask, dilute it to the scale with water, and shake well.

[0048] Precisely measure 1 μL of each of the above solutions, inject it into an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) instrument, and record the chromatogram. The results are shown in Tables 4 and 5, Figure 7 as follows.

[0049] Table 4 Results of the detection limit test

[0050] Table 5 Results of the quantification limit test

[0051] Conclusion: The detection limit of impurity 2-methyl-5-nitro-1-nitrosoimidazole is 2.7 ppb, and the quantification limit is 9.1 ppb.

[0052] (3) Verification of linearity and range Precisely measure 1 μL of the standard curve solution, inject it into an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) instrument, record the chromatogram, and establish a linear regression equation with concentration versus peak area. The correlation coefficient r ≥ 0.995. The specific results are shown in Table 6, Figure 8 .

[0053] Table 6 Results of the linearity test

[0054] Conclusion: In the concentration range of 0.45 ng / mL to 2.27 ng / mL, impurity 2-methyl-5-nitro-1-nitrosoimidazole has a good linear relationship, with r = 0.9986.

[0055] (4) Verification of system precision System precision solution: 0.9 ng / mL reference solution.

[0056] Precisely measure 1 μL of the system precision solution, inject it into an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) instrument, inject it repeatedly 6 times, record the chromatogram, and calculate the RSD% of the peak area of impurity 2-methyl-5-nitro-1-nitrosoimidazole. The results are shown in Table 7.

[0057] Table 7 Results of the system precision test

[0058] Conclusion: When the system precision solution is injected repeatedly 6 times, the RSD of the peak area of impurity 2-methyl-5-nitro-1-nitrosoimidazole is less than 10%, and the system precision meets the requirements.

[0059] (5) Verification of method precision Blank test solution: The same test solution as in the "specificity" item.

[0060] Method precision solution: Take about 500 mg of morinidazole, weigh accurately, place it in a 10 mL volumetric flask, accurately add 0.1 mL of the reference solution at 90 ng / mL, and prepare the test solution according to the method under "Example 2". Prepare 6 portions in the same way.

[0061] Accurately measure 1 μL of each of the above solutions respectively, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are shown in Table 8.

[0062] Table 8 Results of method precision test

[0063] Conclusion: The RSD of the recovery rate of the content of impurity 2-methyl-5-nitro-1-nitrosoimidazole in 6 test samples is 4.7%, and the method precision meets the requirements.

[0064] (6) Verification of method accuracy Blank test solution: The same as the test solution under "Specificity".

[0065] Method accuracy solution: Take about 500 mg of morinidazole, weigh accurately, place it in a 10 mL volumetric flask, accurately add 0.1 mL of the reference solution at 90 ng / mL, and prepare the test solution according to the method under "Example 2". Prepare 6 portions in the same way.

[0066] Accurately measure 1 μL of each of the above solutions respectively, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are shown in Table 9. Figure 7 as shown.

[0067] Table 9 Results of method accuracy test

[0068] Conclusion: The recovery rate of the content of impurity 2-methyl-5-nitro-1-nitrosoimidazole is between 91.62 and 104.52, and the method accuracy meets the requirements.

[0069] (7) Verification of solution stability Reference solution: Take the reference solution at 0.9 ng / mL.

[0070] Test solution: Take the test solution under "Method precision".

[0071] Accurately measure 1 μL of each of the above solutions respectively at 0, 2, 4, 8, 12, 16, and 24 hours, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are shown in Table 10.

[0072] Table 10 Results of solution stability test

[0073] Conclusion: The reference solution is stable when stored at room temperature for 24 hours, and the test solution is stable when stored at room temperature for 24 hours.

[0074] (8) Method durability Solution for method durability test: Take the test solution under "Method precision".

[0075] Test conditions: Column temperature (±5 °C), drying gas temperature (±10 °C), and other chromatographic conditions remain unchanged.

[0076] Respectively take 1 μL of the solution for method durability test and inject it into the ultra-high performance liquid chromatography-mass spectrometry instrument, and record the chromatogram. The results are shown in Table 11.

[0077] Table 11 Results of method durability test

[0078] Conclusion: By changing the column temperature and drying gas temperature, the detection of impurity 2-methyl-5-nitro-1-nitrosoimidazole is not interfered, and the method durability meets the requirements.

[0079] In summary, it can be seen that the ultra-high performance liquid chromatography-mass spectrometry detection method for 2-methyl-5-nitro-1-nitrosoimidazole in the morinidazole raw material of the present invention has high sensitivity, good resolution, strong specificity, good durability, and accurate method, which is of great significance for the quality control of morinidazole raw materials.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention in detail and are not restrictive. Those skilled in the art can modify or equivalently replace the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention, and all should be covered within the scope of the claims of the present invention.

Claims

1. A potential genotoxic impurity in a morpholinoazole raw material and a preparation thereof, characterized in that: The potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole, and its structural formula is as follows: 。 2. A method for separating and purifying potential genotoxic impurities in the morpholinazole raw material and its preparation as claimed in claim 1, characterized in that: The following steps are involved: (1) Take morpholiniazole raw material, add hydrogen peroxide solution, heat it, then add methanol-water to dissolve and dilute it, shake it well, and filter it; (2) using semi-preparative liquid chromatography to perform chromatographic separation, collecting the chromatographic peak eluate of 2-methyl-5-nitro-1-nitrosoimidazole, and concentrating it by nitrogen blowing to obtain a 2-methyl-5-nitro-1-nitrosoimidazole concentrated solution; (3) The collected 2-methyl-5-nitro-1-nitrosoimidazole concentrate was subjected to isocratic elution with a high-efficiency preparative liquid reverse phase C8 acetonitrile-water and isocratic elution with a reverse phase C18 methanol-water, respectively. The chromatographic peak eluates were collected and combined, and freeze-dried to obtain purified 2-methyl-5-nitro-1-nitrosoimidazole.

3. The separation and purification method according to claim 2, characterized in that In step (1), the ratio of morpholinonitrile to hydrogen peroxide solution is 10 mg:1 mL; the volume concentration of the hydrogen peroxide solution is 1% to 3%; the heating treatment is performed in a water bath at 60° C. for 24 h; and the volume ratio of methanol to water is 1:

1.

4. The separation and purification method according to claim 2, characterized in that: In step (2), the chromatographic conditions of the semi-preparative liquid chromatography are: using an Agilent 1200 semi-preparative liquid chromatograph, an Agilent Eclipse XDB C18, 9.4×250 mm, 5µm, preparative column, methanol-0.1% phosphoric acid solution as the mobile phase, gradient elution, the volume ratio of methanol to 0.1% phosphoric acid solution is 20:80 to 100:0, the column temperature is 40°C, the detection wavelength is 254nm, the flow rate is 2mL / min, and the injection volume is 400μL.

5. The separation and purification method according to claim 2, characterized in that: In step (3), the volume ratio of acetonitrile to water is 50:50; the volume ratio of methanol to water is 60:

40.

6. Use of the potential genotoxic impurity as claimed in claim 1 in the quality control of morpholino nidazole raw materials and preparations thereof, characterized in that: The ultra-high performance liquid chromatography-mass spectrometry analysis method is used, which specifically includes the following steps: (1) Preparation of standard curve solution Accurately measure the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole reference substance, dissolve it in a solvent and quantitatively dilute it to prepare solutions of different concentrations to obtain a standard curve solution; (2) Preparation of test solution Take morpholiniazole raw material, first add solvent 1 to heat and extract, then add solvent 2 to mix and dissolve, shake well, filter to obtain the test solution; (3) Determination The solvent, standard curve solution and test solution were accurately measured respectively, and morpholiniazole and potential genotoxic impurities were chromatographed and separated under gradient elution conditions by ultra performance liquid chromatography-mass spectrometry, and analyzed and detected by mass spectrometry. The content of potential genotoxic impurities in the test solution was calculated by peak area according to the standard curve method.

7. The use according to claim 6, characterized in that: In step (1), the concentration of the standard curve solution is 0.45-2.25 ng / mL; the solvent is methanol-water, and the volume ratio of methanol to water is 40:60-60:

40.

8. The use according to claim 6, characterized in that: In step (2), the solvent 1 is methanol; the solvent 2 is water; the volume ratio of methanol to water is 40:60 to 60:40; the heating treatment is carried out in a water bath at 40 to 60° C.; and the concentration of the test solution is 50 mg / mL.

9. The use according to claim 6, characterized in that: The liquid chromatography conditions described in step (3) are as follows: chromatographic column: pentafluorophenylsilane bonded silica gel as the stationary phase, specification model Agilent Poroshell 120 PFP, 3.0×100 mm, 1.9 μm; Mobile phase: A: 0.1%-0.5% formic acid aqueous solution; B: methanol; gradient elution; flow rate: 0.3-0.5 mL / min; column temperature: 35-45°C; injection volume: 1 μL; The gradient elution procedure is: ； The conditions of the mass spectrometry are: Ion source: Agilent Jet Stream electrospray ionization source; Scanning mode: positive ion scanning mode; Monitoring mode: Multiple reaction monitoring; The ion source parameters are: ； The monitoring ion pairs of the compounds are: 。 10. The use according to any one of claims 6 to 9, characterized in that: If a 2-methyl-5-nitro-1-nitrosoimidazole peak is detected in the chromatogram of the test solution, the 2-methyl-5-nitro-1-nitrosoimidazole shall not exceed 18 ppb according to the standard curve method.

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