A potential genotoxic impurity in a morpholine nitazoxanide raw material and formulations thereof, and methods of making and using the same

Abstract

The present application relates to the technical field of medicine, in particular to a potential genotoxic impurity in a morpholine nitroimidazole raw material and preparation and a preparation method and application thereof.The potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole.A new structure compound 2-methyl-5-nitro-1-nitrosoimidazole of diazoles is found in the synthesis process of the morpholine nitroimidazole raw material for the first time.The compound is identified as a potential genotoxic impurity because of containing a nitrosamine warning structure.A method for analyzing the potential genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole by using ultra-high performance liquid chromatography-mass spectrometry is established for the first time, which is of great significance to the quality control of the morpholine nitroimidazole raw material and preparation.The detection method provides a reference for the quality evaluation of other starting materials, intermediates and raw materials that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a potential genotoxic impurity in morpholinidazole raw material and its preparations, as well as its preparation method and application. Background Technology

[0002] Morpholinazole, chemically named 1-[3-(4-morpholinyl)-2-hydroxypropyl]-2-methyl-5-nitro-1H-imidazolium, has the following structural formula: Moroxydazole is China's first third-generation nitroimidazole antibacterial drug with independent intellectual property rights. Its anti-anaerobic activity is stronger than metronidazole, tinidazole, and ornidazole, and it has better safety. After entering susceptible microbial cells, the nitro group in the molecule is reduced to a cytotoxic amino group by electron transport proteins in an anaerobic or low-oxygen environment at a low redox potential. This disrupts the double helix structure of DNA, causing the degradation of synthesized DNA, inhibiting cellular DNA synthesis and transcription, and leading to pathogen cell death. Currently, the main marketed drug with moroxydazole as the active ingredient is moroxydazole sodium chloride injection, clinically used to treat gastroenteritis, pelvic inflammatory disease, cholecystitis, pneumonia, appendicitis, etc.

[0003] The synthetic route of morpholinonitrazole is as follows:

[0004] .

[0005] The starting material molecules of morpholinidazole contain secondary amine structures. Therefore, during the synthesis of morpholinidazole, there is a possibility of generating impurities containing nitrosamine genotoxicity warning structures. Potential genotoxic impurities can cause gene mutations or in vivo mutagenesis in cells at trace levels, causing direct or indirect damage to human genetic material, leading to genotoxic effects such as carcinogenesis and teratogenesis. Nitrosamines are recognized as potent carcinogens. The International Agency for Research on Cancer (IARC) lists 18 nitrosamine compounds, including 2 Group 1 substances, 5 Group 2A substances, and 11 Group 2B substances.

[0006] Currently, the European EMA, the US FDA, and regulatory agencies worldwide have set clearer requirements for potentially genotoxic impurities, and an increasing number of pharmaceutical companies are focusing on the control and detection of these impurities during new drug development. Therefore, the discovery of new potentially genotoxic impurities and the achievement of highly sensitive and selective analysis of such impurities have become challenges in the field of drug development. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a newly discovered potential genotoxic impurity in morpholinidazole raw material and its preparations. The impurity is a new diazole compound, 2-methyl-5-nitro-1-nitrosoimidazole.

[0008] The present invention further provides a method for preparing the above-mentioned potentially genotoxic impurities.

[0009] Another objective of this invention is to provide the application of the aforementioned potentially genotoxic impurities in the quality control of morpholinidazole raw materials and their preparations. An ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analytical method is employed, which is simple, rapid, highly specific, sensitive, and reproducible. This method can be applied to the quality control of morpholinidazole raw materials and their 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.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] This invention provides a potential genotoxic impurity in morpholinidazole raw material and its formulations, wherein the potential genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole, with the following structural formula:

[0012] .

[0013] This invention also provides a method for separating and purifying potentially genotoxic impurities in the above-mentioned morpholine nitroazole raw material and its preparations, comprising the following steps:

[0014] (1) Take morpholine nitroazole raw material, add hydrogen peroxide solution, heat treatment, then add methanol-water to dissolve and dilute, shake well, and filter;

[0015] (2) Chromatographic separation was performed by semi-preparative liquid chromatography. The eluent of the chromatographic peak of 2-methyl-5-nitro-1-nitrosoimidazole was collected and concentrated by nitrogen blowing to obtain concentrated 2-methyl-5-nitro-1-nitrosoimidazole.

[0016] (3) The collected 2-methyl-5-nitro-1-nitrosoimidazole concentrate was subjected to reverse phase C8 acetonitrile-water isocratic elution and reverse phase C18 methanol-water isocratic elution respectively. The chromatographic peak eluents were collected and combined, and then freeze-dried to obtain purified 2-methyl-5-nitro-1-nitrosoimidazole.

[0017] Furthermore, in step (1), the ratio of morpholinidazole to hydrogen peroxide solution is 10 mg: 1 mL; the volume concentration of hydrogen peroxide solution is 1% to 3%; the heating treatment is performed under a 60°C water bath for 24 hours; and the volume ratio of methanol to water is 1:1.

[0018] Furthermore, in step (2), the chromatographic conditions of the semi-preparative liquid chromatography method are as follows: an Agilent 1200 semi-preparative liquid chromatograph is used, with an Agilent Eclipse XDB C18 (9.4×250 mm, 5µm) preparative column, using methanol-0.1% phosphoric acid solution as the mobile phase, gradient elution, a volume ratio of methanol to 0.1% phosphoric acid solution of 20:80 to 100:0, a column temperature of 40℃, a detection wavelength of 254nm, a flow rate of 2mL / min, and an injection volume of 400μL.

[0019] Furthermore, in step (3), the volume ratio of acetonitrile to water is 50:50; the volume ratio of methanol to water is 60:40.

[0020] This invention also provides the application of the aforementioned potentially genotoxic impurities in the quality control of morpholinidazole raw materials and their preparations, employing an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analytical method, specifically including the following steps:

[0021] (1) Preparation of standard curve solution

[0022] Precisely measure the potentially genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole reference standard, dissolve it in a solvent and quantitatively dilute it to prepare solutions of different concentrations, thus obtaining a standard curve solution;

[0023] (2) Preparation of the test solution

[0024] Take morpholine nitroazole raw material, first add solvent 1 and heat to extract, then add solvent 2 to mix and dissolve, shake well, filter, and obtain the test solution;

[0025] (3) Measurement

[0026] Solvent, standard curve solution, and test solution were precisely measured separately. Morpholine nitroazole and potential genotoxic impurities were separated by chromatographic separation using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) system under gradient elution conditions. The results were analyzed by a mass spectrometer detector. The content of potential genotoxic impurities in the test solution was calculated by peak area using the standard curve method.

[0027] Furthermore, in step (1), the concentration of the standard curve solution is 0.45 to 2.25 ng / mL; the solvent is methanol-water, and the volume ratio of methanol to water is 40:60 to 60:40.

[0028] Furthermore, in step (2), solvent 1 is methanol; 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.

[0029] Furthermore, the liquid chromatography conditions described in step (3) are as follows: Column: Agilent Poroshell 120 PFP, 3.0×100mm, 1.9μm, with pentafluorophenylsilane-bonded silica gel as the stationary phase; Mobile phase: A: 0.1%~0.5% formic acid aqueous solution; B: methanol; Gradient elution; Flow rate: 0.3~0.5mL / min; Column temperature: 35~45℃; Injection volume: 1μL;

[0030] The gradient elution procedure is as follows:

[0031] .

[0032] The conditions for the mass spectrometry are as follows:

[0033] Ion source: Agilent Jet Electrospray Ionization Source (AJS ESI);

[0034] Scanning method: Positive ion scanning mode;

[0035] Monitoring mode: Multiple response monitoring (MRM);

[0036] The ion source parameters are:

[0037] .

[0038] The monitoring ion pairs of the compound are:

[0039] .

[0040] Furthermore, 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 peak shall not exceed 18 ppb according to the standard curve method.

[0041] This invention employs ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to achieve trace analysis of 2-methyl-5-nitro-1-nitrosoimidazole, a potential genotoxic impurity in morpholinidazole raw materials. The pretreatment method provided by this invention achieves high extraction and recovery rates of 2-methyl-5-nitro-1-nitrosoimidazole and improves the stability of the sample solution. Secondly, by selecting a pentafluorophenylsilane-bonded silica gel column and using methanol as the organic phase, sufficient chromatographic separation of the 2-methyl-5-nitro-1-nitrosoimidazole peak from the morpholinidazole peak is achieved. Formic acid is added to the mobile phase to enhance the mass spectrometric signal of 2-methyl-5-nitro-1-nitrosoimidazole. Through primary mass spectrometry full-scan analysis and secondary fragment ion scanning to screen for quantitative and qualitative ion pairs, the collision energy of each ion pair is optimized using MassHunter software. Furthermore, mass spectrometry parameters such as drying gas temperature, sheath gas temperature, and capillary voltage are optimized, ultimately achieving trace detection and precise quantification of 2-methyl-5-nitro-1-nitrosoimidazole.

[0042] This invention provides an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) technique for the separation and quantitative detection of 2-methyl-5-nitro-1-nitrosoimidazole, a novel diazole compound, in morpholinidazole raw materials. This effectively achieves quality control of potentially genotoxic impurities in morpholinidazole raw materials, reduces adverse drug reactions in patients, and ensures patient safety to a certain extent. It also provides a reference for the quality evaluation of other starting materials or intermediates that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

[0043] The beneficial effects of this invention are as follows:

[0044] 1. This invention is the first to discover a novel diazole compound, 2-methyl-5-nitro-1-nitrosoimidazole, in the synthesis process of morpholinidazole raw material. This compound is identified as a potential genotoxic impurity because it contains a nitrosamine warning structure.

[0045] 2. This invention establishes for the first time an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method for the analysis of 2-methyl-5-nitro-1-nitrosoimidazole, a potential genotoxic impurity, which is of great significance for the quality control of morpholinidazole raw materials and its preparations.

[0046] 3. The detection method of this invention provides a reference for the quality evaluation of other starting materials, intermediates and active pharmaceutical ingredients that can produce 2-methyl-5-nitro-1-nitrosoimidazole.

[0047] 4. The detection method of the present invention has high sensitivity and can effectively detect the impurity 2-methyl-5-nitro-1-nitrosoimidazole, with a detection limit of 2.7 ppb and a quantitation limit of 9.1 ppb.

[0048] 5. The detection method of this invention is highly specific, accurate and reliable. The recovery rate of the impurity 2-methyl-5-nitro-1-nitrosoimidazole is 96.3%; the repeatability RSD is 4.7%; and the precision RSD is 2.9%. Attached Figure Description

[0049] Figure 1 It is 2-methyl-5-nitro-1-nitrosoimidazole 1 H-NMR spectrum;

[0050] Figure 2 It is 2-methyl-5-nitro-1-nitrosoimidazole 1 C-NMR spectrum;

[0051] Figure 3 High-resolution mass spectrum of 2-methyl-5-nitro-1-nitrosoimidazole;

[0052] Figure 4 The ultraviolet spectrum of 2-methyl-5-nitro-1-nitrosoimidazole;

[0053] Figure 5 a: Blank solvent chromatogram for specificity verification experiment; a: Quantitative ion pair m / z 128.0→82.0 Extraction chromatogram, b: Qualitative ion pair m / z Extract the chromatogram from 128.0 to 42.0.

[0054] Figure 6 Chromatograms of mixed solutions from specificity verification experiments; a: Chromatogram of morpholinonitrazole-extracted ions; b: Quantitative ion pair of MNIMZ. m / z 128.0→82.0 Extraction chromatogram; c: Qualitative ion of MNIMZ m / z Extract the chromatogram from 128.0 to 42.0.

[0055] Figure 7 Chromatogram of the test solution for the method accuracy validation experiment; a: Quantitative ion pair m / z 128.0→82.0 Extraction chromatogram, b: Qualitative ion pair m / z Extract the chromatogram from 128.0 to 42.0.

[0056] Figure 8 The standard curve for impurity 2-methyl-5-nitro-1-nitrosoimidazole is shown. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments. These embodiments are provided to better illustrate the content of the invention and are not intended to limit it. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified. Therefore, non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0058] Example 1: Isolation and purification of impurities

[0059] (1) Destructive testing

[0060] Weigh approximately 50 mg of morpholine nitroazole raw material accurately and 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 in a 60°C water bath for 24 hours. Then add methanol-water (50:50) to dissolve and dilute to the mark. Shake well and filter.

[0061] (2) Separation and preparation of impurities by semi-preparative liquid chromatography:

[0062] An Agilent 1200 semi-preparative liquid chromatograph was used with an Agilent Eclipse XDB C18 (9.4 × 250 mm, 5 µm) preparative column. Methanol-0.1% phosphoric acid solution was used as the mobile phase with gradient elution. The volume ratio of methanol to 0.1% phosphoric acid solution was 20:80 to 100:0. The column temperature was 40 °C, the detection wavelength was 254 nm, the flow rate was 2 mL / min, and the injection volume was 400 μL. The eluent of the chromatographic peak of the impurity 2-methyl-5-nitro-1-nitrosoimidazole was collected and concentrated by nitrogen blowing.

[0063] (3) Purification:

[0064] The concentrated 2-methyl-5-nitro-1-nitrosoimidazole impurity obtained in step (2) was subjected to isocratic elution with reverse-phase C8 acetonitrile-water (50:50) and reverse-phase C18 methanol-water (60:40) respectively. The chromatographic peak eluents were collected and combined, and then freeze-dried to obtain the purified 2-methyl-5-nitro-1-nitrosoimidazole impurity.

[0065] The impurity 2-methyl-5-nitro-1-nitrosoimidazole obtained by separation 1 H-NMR spectrum as follows Figure 1 As shown, 1 C-NMR spectrum as shown Figure 2 As shown; the mass spectrum of 2-methyl-5-nitro-1-nitrosoimidazole is as follows. Figure 3 As shown; the UV spectrum of 2-methyl-5-nitro-1-nitrosoimidazole is as follows. Figure 4 As shown.

[0066] Example 2 Quality Control Method

[0067] (1) Preparation of standard curve solution

[0068] Accurately measure an appropriate amount of the impurity 2-methyl-5-nitro-1-nitrosoimidazole reference standard, dissolve it in the solvent [methanol-water (50:50)] and quantitatively dilute it to prepare a solution containing approximately 0.45, 0.6, 0.9, 1.5, and 2.25 ng of each impurity per ml.

[0069] (2) Preparation of the test solution

[0070] Weigh approximately 500 mg of morpholine nitroazole raw material accurately and place it in a 10 mL volumetric flask. First, add 6 mL of methanol and heat at 40 °C to extract. Then, add 4 mL of water to dissolve and dilute to the mark, shake well, and filter.

[0071] (3) Measurement

[0072] Accurately measure 1 μL of each of the solvent, standard curve solution, and test solution, and inject them into the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument, recording the chromatograms. Calculate the content of 2-methyl-5-nitro-1-nitrosoimidazole in the test solution based on the peak area using the standard curve method.

[0073] The chromatographic conditions were as follows: an Agilent Poroshell 120 PFP column (3.0 × 100 mm, 1.9 μm) with pentafluorophenylsilane-bonded silica gel as the stationary phase was used; 0.1%–0.5% formic acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B, with linear gradient elution performed according to the program in Table 1; the flow rate was 0.3–0.5 mL / min, the column temperature was 35–45 °C, and the injection volume was 1 μL.

[0074] Table 1

[0075]

[0076] The mass spectrometry conditions were as follows: electrospray ionization source (AJS ESI), positive ion scanning mode, and multiple reaction monitoring (MRM). The quantitative ion pair for 2-methyl-5-nitro-1-nitrosoimidazole was 128→82 with a collision energy of 20 V, and the qualitative ion pair was 128→42 with a collision energy of 42 V. Ion source parameters are shown in Table 2.

[0077] Table 2

[0078]

[0079] Example 3

[0080] The method provided in Example 2 was subjected to specificity verification, detection limit verification, repeatability verification, accuracy verification, and robustness verification.

[0081] (1) Specificity verification

[0082] Blank solvent: methanol-water (50:50)

[0083] Impurity 2-methyl-5-nitro-1-nitrosoimidazole localization solution: 90 ng / mL reference solution.

[0084] Blank test solution: Weigh 500 mg of the test sample accurately, place it in a 10 mL volumetric flask, add 6 mL of methanol and heat at 40 °C to extract, then add 4 mL of water to dissolve and dilute to the mark, and shake well.

[0085] Mixed solution: Weigh 500 mg of the test sample accurately and place it in a 10 mL volumetric flask. Accurately add 1 mL of 90 ng / mL impurity 2-methyl-5-nitro-1-nitrosoimidazole reference solution and 6 mL of methanol. Heat and extract at 40 °C. Add 4 mL of water to dissolve and dilute to the mark. Shake well to obtain the mixed sample solution.

[0086] Accurately measure 1 μL of each of the above solutions and inject them into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument, then record the chromatograms. The results are shown in Table 3. Figure 5 , Figure 6 As shown.

[0087] Table 3 Specificity test results

[0088]

[0089] Conclusion: Neither the blank solvent nor morpholinidazole interfered with the detection of the impurity 2-methyl-5-nitro-1-nitrosoimidazole, and the specificity verification met the requirements.

[0090] (2) Validation of limit of detection / limit of quantitation

[0091] Limit of quantitation solution: Take 0.45 ng / mL of the reference solution as the limit of quantitation solution.

[0092] Detection limit solution: Accurately measure 3 mL of the quantitation limit solution, place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0093] Accurately measure 1 μL of each of the above solutions and inject them into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument, then record the chromatograms. The results are shown in Tables 4 and 5. Figure 7 As shown.

[0094] Table 4 Results of detection limit test

[0095]

[0096] Table 5 Results of Limit of Quantitation Test

[0097]

[0098] Conclusion: The limit of detection for the impurity 2-methyl-5-nitro-1-nitrosoimidazole was 2.7 ppb, and the limit of quantification was 9.1 ppb.

[0099] (3) Linearity and range verification

[0100] 1 μL of each standard curve solution was precisely measured and injected into the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument. Chromatograms were recorded, and a linear regression equation was performed on the peak area against concentration. The correlation coefficient r ≥ 0.995. Specific results are shown in Table 6. Figure 8 .

[0101] Table 6 Results of Linear Experiments

[0102]

[0103] Conclusion: The impurity 2-methyl-5-nitro-1-nitrosoimidazole showed good linearity in the concentration range of 0.45 ng / mL to 2.27 ng / mL, with r = 0.9986.

[0104] (4) System precision verification

[0105] System precision solution: 0.9 ng / mL reference solution.

[0106] 1 μL of the system's precision solution was precisely measured and injected into the ultra-high performance liquid chromatography-mass spectrometry instrument. The injection was repeated 6 times, and the chromatograms were recorded. The RSD% of the peak area of ​​the impurity 2-methyl-5-nitro-1-nitrosoimidazole was calculated. The results are shown in Table 7.

[0107] Table 7 System precision test results

[0108]

[0109] Conclusion: The system precision solution was repeatedly injected 6 times, and the RSD of the peak area of ​​the impurity 2-methyl-5-nitro-1-nitrosoimidazole was less than 10%, indicating that the system precision met the requirements.

[0110] (5) Method precision verification

[0111] Blank test solution: Same as the test solution under "Specificity".

[0112] Method precision solution: Accurately weigh approximately 500 mg of morpholinidazole and place it in a 10 mL volumetric flask. Accurately add 0.1 mL of 90 ng / mL reference solution and prepare the test solution according to the method described in "Example 2". Prepare 6 samples using the same method.

[0113] Each of the above solutions was precisely measured to 1 μL and injected into an ultra-high performance liquid chromatography-mass spectrometry instrument. The chromatograms were recorded, and the results are shown in Table 8.

[0114] Table 8 Results of Method Precision Test

[0115]

[0116] Conclusion: The RSD of the recovery rate of the impurity 2-methyl-5-nitro-1-nitrosoimidazole in the 6 test samples was 4.7%, and the method precision met the requirements.

[0117] (6) Method accuracy verification

[0118] Blank test solution: Same as the test solution under "Specificity".

[0119] Method accuracy solution: Accurately weigh approximately 500 mg of morpholinidazole and place it in a 10 mL volumetric flask. Accurately add 0.1 mL of 90 ng / mL reference solution and prepare the test solution according to the method described in "Example 2". Prepare 6 solutions in the same manner.

[0120] Accurately measure 1 μL of each of the above solutions and inject them into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument. Record the chromatograms. The results are shown in Table 9. Figure 7 As shown.

[0121] Table 9. Results of Method Accuracy Tests

[0122]

[0123] Conclusion: The recovery rate of the impurity 2-methyl-5-nitro-1-nitrosoimidazole was between 91.62% and 104.52%, and the accuracy of the method met the requirements.

[0124] (7) Solution stability verification

[0125] Reference solution: Take 0.9 ng / mL of reference solution.

[0126] Test solution: Take the test solution from the "Method Precision" section.

[0127] At 0, 2, 4, 8, 12, 16 and 24 hours, 1 μL of the above solution was accurately measured and injected into an ultra-high performance liquid chromatography-mass spectrometry instrument. The chromatograms were recorded, and the results are shown in Table 10.

[0128] Table 10 Results of solution stability test

[0129]

[0130] Conclusion: The reference solution was stable at room temperature for 24 hours, and the test solution was stable at room temperature for 24 hours.

[0131] (8) Method durability

[0132] Method robustness solution: Take the test solution from the "Method precision" section.

[0133] Experimental conditions: column temperature (±5℃), drying gas temperature (±10℃), other chromatographic conditions remained unchanged.

[0134] Take 1 μL of the method robustness solution and inject it into the ultra-high performance liquid chromatography-mass spectrometry instrument. Record the chromatograms. The results are shown in Table 11.

[0135] Table 11 Results of Method Durability Tests

[0136]

[0137] Conclusion: Changing the column temperature and the temperature of the drying gas does not interfere with the detection of the impurity 2-methyl-5-nitro-1-nitrosoimidazole, and the method robustness meets the requirements.

[0138] 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 morpholinidazole raw material of the present invention has high sensitivity, good resolution, strong specificity, good robustness and accuracy, and is of great significance for the quality control of morpholinidazole raw material.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention in detail and are not intended to limit it. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a potentially genotoxic impurity in the quality control of morpholinidazole raw materials and its preparations, characterized in that, The potentially genotoxic impurity is 2-methyl-5-nitro-1-nitrosoimidazole, with the following structural formula: 。 2. The application according to claim 1, characterized in that, The method for separating and purifying potentially genotoxic impurities in the morpholine nitroazole raw material and its preparations includes the following steps: (1) Take morpholine nitroazole raw material, add hydrogen peroxide solution, heat treatment, then add methanol-water to dissolve and dilute, shake well, and filter; (2) Chromatographic separation was performed by semi-preparative liquid chromatography. The eluent of the chromatographic peak of 2-methyl-5-nitro-1-nitrosoimidazole was collected and concentrated by nitrogen blowing to obtain concentrated 2-methyl-5-nitro-1-nitrosoimidazole. (3) The collected 2-methyl-5-nitro-1-nitrosoimidazole concentrate was subjected to reverse phase C8 acetonitrile-water isocratic elution and reverse phase C18 methanol-water isocratic elution respectively. The chromatographic peak eluents were collected and combined, and then freeze-dried to obtain purified 2-methyl-5-nitro-1-nitrosoimidazole.

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

1.

4. The application according to claim 2, characterized in that, In step (2), the chromatographic conditions of the semi-preparative liquid chromatography method are as follows: an Agilent 1200 semi-preparative liquid chromatograph, Agilent Eclipse XDB C18, 9.4×250 mm, 5µm, 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℃, the detection wavelength is 254nm, the flow rate is 2mL / min, and the injection volume is 400μL.

5. The application 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. The application according to any one of claims 1-5, characterized in that, The analysis of potential genotoxic impurities in morpholine nitrazole raw materials and preparations using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) includes the following steps: (1) Preparation of standard curve solution Precisely measure the potentially genotoxic impurity 2-methyl-5-nitro-1-nitrosoimidazole reference standard, dissolve it in a solvent and quantitatively dilute it to prepare solutions of different concentrations, thus obtaining a standard curve solution; (2) Preparation of the test solution Take morpholine nitroazole raw material, first add solvent 1 and heat to extract, then add solvent 2 to mix and dissolve, shake well, filter, and obtain the test solution; (3) Measurement Solvent, standard curve solution, and test solution were precisely measured separately. Morpholine nitroazole and potential genotoxic impurities were separated by chromatographic separation using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) system under gradient elution conditions. The results were analyzed by a mass spectrometer detector. The content of potential genotoxic impurities in the test solution was calculated by peak area using the standard curve method.

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

40.

8. The application according to claim 6, characterized in that, In step (2), solvent 1 is methanol; solvent 2 is water; the volume ratio of methanol to water is 40:60 to 60:40; the heat 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 application 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 stationary phase, Agilent Poroshell 120 PFP, 3.0×100mm, 1.9μm; 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℃; injection volume: 1 μL; The gradient elution procedure is as follows: ； The conditions for the mass spectrometry are: Ion source: Agilent jet electrospray ionization source; Scanning method: Positive ion scanning mode; Monitoring mode: Multiple response monitoring; The ion source parameters are: ； The monitoring ion pairs of the compound are: 。 10. The application according to claim 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 peak shall not exceed 18 ppb according to the standard curve method.

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