Detection method of related substances of metronidazole and morpholine chloride injection

Abstract

This invention belongs to the field of pharmaceutical testing technology, specifically relating to a method for detecting related substances in morphonitroazole sodium chloride injection. The method includes high-performance liquid chromatography (HPLC) to detect related substances in morphonitroazole sodium chloride injection. The HPLC test conditions include: a column packed with octadecylsilane-bonded silica gel; mobile phases A and B, where mobile phase A is an aqueous ammonium acetate solution and mobile phase B is methanol; and gradient elution is employed. This application uses a less toxic acetate buffer-methanol mobile phase system, resulting in higher safety. Further optimization through gradient elution halves the gradient analysis time, increasing detection efficiency. This method has been validated, demonstrating strong specificity and good separation, and is suitable for the detection of related substances in morphonitroazole sodium chloride injection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, specifically relating to a method for detecting related substances in morpholine nitrazole sodium chloride injection. Background Technology

[0002] The chemical name of morpholinidazole is 1-[3-(4-morpholinyl)-2-hydroxypropyl]-2-methyl-5-nitro-1H-imidazolium, and its chemical formula is […].

[0003] Moroxydazole belongs to the third-generation nitroimidazole class of antibacterial drugs. Its antibacterial mechanism is mainly related to the nitro group contained in its molecule. The nitro group can be reduced to an amino group or other cytotoxic groups in an anaerobic environment, thereby affecting the metabolic process of pathogens. Currently, the main formulation sold with moroxydazole as the active ingredient is moroxydazole sodium chloride injection.

[0004] Moroxydazole sodium chloride injection is a nitroimidazole antibacterial drug mainly used to treat infectious diseases caused by anaerobic bacteria, such as lower respiratory tract infections, pelvic inflammatory disease, and sepsis. Its active ingredient, moroxydazole, exerts its bactericidal effect by inhibiting bacterial DNA synthesis and has broad-spectrum anti-anaerobic activity.

[0005] Related substances in morphonitroazole sodium chloride injection are mainly generated from the introduction and degradation of the active pharmaceutical ingredient morphonitroazole. During the production and storage of morphonitroazole, various impurities may be generated, such as degradation products and process impurities, directly affecting the safety and efficacy of the drug. Therefore, the detection of related substances in morphonitroazole sodium chloride injection is particularly crucial in drug quality control.

[0006] Chinese patent CN115561347A discloses a high-performance liquid chromatography (HPLC) method for the detection of related substances in morpholinidazole raw material. This method uses potassium dihydrogen phosphate buffer solution as mobile phase A and acetonitrile as mobile phase B. Acetonitrile is harmful to human health and the environment and is also costly. Furthermore, the elution time of this method is relatively long, exceeding 100 minutes. Summary of the Invention

[0007] Therefore, the technical problem to be solved by this invention is how to efficiently and accurately detect related substances in morpholine nitrazole sodium chloride injection, while improving the safety of the detection process and reducing harm to the human body and the environment.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, this application provides a method for detecting related substances in morpholine nitroazole sodium chloride injection, which includes detecting related substances in morpholine nitroazole sodium chloride injection using high performance liquid chromatography (HPLC). The HPLC test conditions include: the chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is methanol; and gradient elution is used.

[0009] In some embodiments of this application, the molar concentration of ammonium acetate in the aqueous solution is 40-60 mmol / L, and the pH of the aqueous solution is 7.0-8.0.

[0010] In some embodiments of this application, the ammonium acetate aqueous solution is an ammonium acetate-ammonia buffer solution. Preferably, the pH of the ammonium acetate-ammonia buffer solution is 7.5-8.0, and more preferably 7.6-7.8.

[0011] In some embodiments of this application, the gradient elution includes the following elution sequence: From 0 to 5 min, the volume percentage of mobile phase A was 97 ± 1%, and the volume percentage of mobile phase B was 3 ± 1%. Over 5-20 minutes, the volume percentage of mobile phase A decreased from 97±1% to 80±1%, while the volume percentage of mobile phase B increased from 3±1% to 20±1%. After 20-30 minutes, the volume percentage of mobile phase A was 80±1%, and the volume percentage of mobile phase B was 20±1%. Over 30-40 minutes, the volume percentage of mobile phase A decreased from 80±1% to 55±1%, while the volume percentage of mobile phase B increased from 20±1% to 45±1%. After 40-45 minutes, the volume percentage of mobile phase A was 55±1%, and the volume percentage of mobile phase B was 45±1%. Over 45-45.1 min, the volume percentage of mobile phase A increased from 55±1% to 97±1%, while the volume percentage of mobile phase B decreased from 45±1% to 3±1%.

[0012] From 45.1 to 55 min, the volume percentage of mobile phase A was 97 ± 1%, and the volume percentage of mobile phase B was 3 ± 1%. In some embodiments of this application, it further includes the step of filtering mobile phase A and mobile phase B respectively with an impurity trapping column before gradient elution.

[0013] In some embodiments of this application, the related substances of the morpholine nitroazole sodium chloride injection include impurity E and impurity F; preferably, the related substances of the morpholine nitroazole sodium chloride injection also include one or more of impurity A, impurity C, impurity D, impurity G and impurity H.

[0014] In some embodiments of this application, the detection method includes the following steps: Prepare the reference solution; Prepare the test solution; The reference solution and the test solution were analyzed by high performance liquid chromatography, respectively.

[0015] In some embodiments of this application, the reference solution is prepared using a mixed solution of ammonium acetate aqueous solution and methanol as a solvent, preferably with a concentration of 0.1-5 μg / ml; preferably, the reference solution includes morpholine nitrazole reference solution, impurity E reference solution, and impurity F reference solution; more preferably, the reference solution further includes one or more of impurity A reference solution, impurity C reference solution, impurity D reference solution, impurity G reference solution, and impurity H reference solution; and / or, The solvent of the test solution is a mixture of ammonium acetate aqueous solution and methanol, and preferably the concentration of morpholinidazole in the test solution is 0.1-1 mg / ml.

[0016] In some embodiments of this application, the detection wavelength used in the high-performance liquid chromatography is 310-330 nm, preferably 311-320 nm; and / or, The column temperature used in high-performance liquid chromatography is 28-32℃; and / or, The flow rate of the mobile phase used in high performance liquid chromatography is 0.8-1.2 ml / min, preferably 0.9-1.1 ml / min.

[0017] Secondly, this application also provides the application of the detection method described in the first aspect in the field of quality control of morpholinidazole and its preparations.

[0018] Beneficial effects: This application uses a less toxic acetate buffer-methanol mobile phase system, resulting in higher safety. Further optimization through gradient elution halve the gradient analysis time, increasing detection efficiency. The method has been validated, demonstrating strong specificity and good separation, and is suitable for the detection of related substances in morpholine nitrazole sodium chloride injection. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Figure 1 The chromatogram of the test solution in Example 1 is shown below. Figure 2 This is the chromatographic spectrum of the solution used in Example 1 to demonstrate the system suitability. Figure 3 The chromatogram for the detection of morpholine nitroazole sodium chloride injection by high performance liquid chromatography in Comparative Example 1 is shown. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The principles and features of the present invention are described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0023] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0024] In this application, the term "related substances" refers to impurities generated or introduced during the production and storage of morpholine nitrazole sodium chloride injection. These impurities may be generated or degraded, including intermediates, byproducts, and other organic impurities.

[0025] In this application, v / v% refers to volume percentage.

[0026] In one specific embodiment of this application, a method for detecting related substances in morpholine nitroazole sodium chloride injection is provided, comprising the following steps: detecting related substances in morpholine nitroazole sodium chloride injection using high performance liquid chromatography (HPLC), wherein the HPLC test conditions include: the chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is methanol; and gradient elution is used.

[0027] In some embodiments of this application, the pH of the ammonium acetate aqueous solution is 7.0-8.0, preferably 7.5-8.0, and more preferably 7.6-7.8.

[0028] In some embodiments of this application, the molar concentration of ammonium acetate in the aqueous solution of ammonium acetate is 40-60 mmol / L.

[0029] In some embodiments of this application, the ammonium acetate aqueous solution is an ammonium acetate-ammonia buffer solution, wherein the pH of the ammonium acetate-ammonia buffer solution is 7.0-8.0, preferably 7.5-8.0, and more preferably 7.6-7.8. This ammonium acetate-ammonia buffer solution is prepared by a method comprising preparing a solution of ammonium acetate and water, and then adjusting the pH of the solution with ammonia.

[0030] In some embodiments of this application, the gradient elution gradient in the above method is as follows: 0-5 min, the volume percentage of mobile phase A is 97±1%, and the volume percentage of mobile phase B is 3±1%; 5-20 min, the volume percentage of mobile phase A decreases from 97±1% to 80±1%, and the volume percentage of mobile phase B increases from 3±1% to 20±1%; 20-30 min, the volume percentage of mobile phase A is 80±1%, and the volume percentage of mobile phase B is 20±1%; 30-40 min, the volume percentage of mobile phase A decreases from 80±1% to 80±1%, and the volume percentage of mobile phase B increases from 3±1% to 20±1%; 20-30 min, the volume percentage of mobile phase A decreases from 80±1%, and the volume percentage of mobile phase B increases from 3±1% to 20±1%; 30-40 min, the volume percentage of mobile phase A decreases from 80±1%, and the volume percentage of mobile phase B increases from 3±1% to 20±1%. The volume percentage of mobile phase A decreased from 0±1% to 55±1%, while the volume percentage of mobile phase B increased from 20±1% to 45±1%; from 40 to 45 min, the volume percentage of mobile phase A was 55±1%, and the volume percentage of mobile phase B was 45±1%; from 45 to 45.1 min, the volume percentage of mobile phase A increased from 55±1% to 97±1%, while the volume percentage of mobile phase B decreased from 45±1% to 3±1%; from 45.1 to 55 min, the volume percentage of mobile phase A was 97±1%, and the volume percentage of mobile phase B was 3±1%.

[0031] In some embodiments of this application, the gradient elution gradient in the above method is as follows: 0-5 min, the volume percentage of mobile phase A is 97%, and the volume percentage of mobile phase B is 3%; 5-20 min, the volume percentage of mobile phase A decreases from 97% to 80%, and the volume percentage of mobile phase B increases from 3% to 20%; 20-30 min, the volume percentage of mobile phase A is 80%, and the volume percentage of mobile phase B is 20%; 30-40 min, the volume percentage of mobile phase A decreases from 80% to 55%, and the volume percentage of mobile phase B increases from 20% to 45%; 40-45 min, the volume percentage of mobile phase A is 55±1%, and the volume percentage of mobile phase B is 45±1%; 45-45.1 min, the volume percentage of mobile phase A increases from 55±1% to 97±1%, and the volume percentage of mobile phase B decreases from 45±1% to 3±1%; 45.1-55 min, the volume percentage of mobile phase A is 97±1%, and the volume percentage of mobile phase B is 3±1%. In some embodiments of this application, filtering mobile phase A and mobile phase B with an impurity trapping column before gradient elution can improve separation efficiency, extend column life, and reduce maintenance costs.

[0032] The detection method provided in this application solves the problems of poor specificity, long detection time, and high cost of existing high-performance liquid chromatography systems by selecting appropriate mobile phases and controlling the conditions of gradient elution programs, while ensuring effective separation of the main peak and impurities. It features high specificity, good precision, high accuracy, simplicity, high efficiency, and low cost, and can be applied to the quality control of morpholinonitrazole sodium chloride injection. At the same time, it greatly reduces the cost of chromatographic columns and liquid chromatograph consumables, making it more suitable for commercial production.

[0033] In some embodiments of this application, the related substances of the morpholine nitrazole sodium chloride injection include impurity E and impurity F. In some embodiments of this application, the related substances of the morpholine nitrazole sodium chloride injection also include one or more of impurities A, C, D, G, and H.

[0034] The chemical names, structural formulas, and sources of impurities A, C, D, E, F, G, and H are shown in the table below.

[0035] In some embodiments of this application, the high-performance liquid chromatography method includes the following steps: Prepare the reference solution; Prepare the test solution; High-performance liquid chromatography was used to analyze the reference solution and the test solution.

[0036] In some embodiments of this application, the reference solution includes a morpholinidazole reference solution, an impurity E reference solution, and an impurity F reference solution; preferably, the reference solution further includes one or more of the following: an impurity A reference solution, an impurity C reference solution, an impurity D reference solution, an impurity G reference solution, and an impurity H reference solution. In some embodiments, the solvent of the reference solution is a mixture of ammonium acetate aqueous solution and methanol. In some embodiments, the concentration of the reference is 0.1-5 μg / ml, preferably 0.5-2 μg / ml.

[0037] In some embodiments of this application, the concentration of the morpholinidazole reference solution is 0.1-1 μg / ml.

[0038] In some embodiments of this application, the concentration of the impurity A reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0039] In some embodiments of this application, the concentration of the impurity C reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0040] In some embodiments of this application, the concentration of the impurity D reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0041] In some embodiments of this application, the concentration of the impurity E reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0042] In some embodiments of this application, the concentration of the impurity F reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0043] In some embodiments of this application, the concentration of the impurity G reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0044] In some embodiments of this application, the concentration of the impurity H reference solution is 1-5 μg / ml, preferably 1-3 μg / ml.

[0045] In some embodiments of this application, the test sample is a solution containing morpholine nitrazole sodium chloride injection, wherein the solvent of the test sample solution is a mixed solution of ammonium acetate aqueous solution and methanol; preferably, the concentration of morpholine nitrazole in the test sample solution is 0.1-1 mg / ml.

[0046] In some embodiments of this application, the volume ratio of ammonium acetate aqueous solution to methanol is 75-85:15-25. In some embodiments, the pH of the ammonium acetate aqueous solution is 7.0-8.0, preferably 7.5-8.0, and more preferably 7.6-7.8. In some embodiments of this application, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 40-60 mmol / L. In some embodiments of this application, the ammonium acetate aqueous solution is an ammonium acetate-ammonia buffer solution, wherein the concentration of ammonium acetate is 40-60 mmol / L and the pH is 7.0-8.0. This ammonium acetate-ammonia buffer solution is prepared by a method comprising preparing a solution of ammonium acetate and water, and then adjusting the pH of the solution with ammonia.

[0047] In some embodiments of this application, the content of related substances in the above-mentioned method for detecting related substances in morpholine nitroazole sodium chloride injection is determined by the external standard method.

[0048] It should be noted that the external standard method described in this application refers to a method of quantification by comparing the peak area or peak height of the analyte in the reference substance and the pure sample of the analyte in the reference substance with that in the sample.

[0049] In some embodiments of this application, the detection wavelength used in the high-performance liquid chromatography is 310-330 nm, preferably 311-320 nm.

[0050] In some embodiments of this application, the column temperature of the chromatographic column used in the high-performance liquid chromatography is 28-32°C. In some embodiments of this application, the flow rate of the mobile phase used in high performance liquid chromatography is 0.8-1.2 ml / min, preferably 0.9-1.1 ml / min.

[0051] This application also provides the application of the above-mentioned detection methods in the field of quality control of morpholinidazole and its preparations.

[0052] The detection method for related substances in the morpholine nitrazole sodium chloride injection of the present invention will be specifically described below through specific embodiments. In the embodiments, all original reagent materials are commercially available, and experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0053] Table 1. Reagents used in the examples and comparative examples Impurity A 1-(3-chloro-2-hydroxypropyl)-2-methyl-nitroimidazole <![CDATA[Calculated on the basis of C7H 10 ClN3O3, 99.9%]]> China National Institutes for Food and Drug Control Impurity C 1-(2,3-epoxypropyl)-2-methyl-5-nitroimidazole 94.33 Shenzhen Moco Biochemical Technology Co., Ltd. Impurity D 3-(2-methyl-5-nitro-1H-imidazol-1-yl)-2-morpholinopropane-1-ol 90.25 Shenzhen Moco Biochemical Technology Co., Ltd. Impurity E 4-(2-hydroxy-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propyl)morpholine-4-oxide 97.27 Shenzhen Moco Biochemical Technology Co., Ltd. impurity F 1-(2-(hydroxymethyl)-5-nitro-1H-imidazol-1-yl)-3-morpholinopropane-2-ol 97.2 TLCPHARMACEUTICALSTANDARDS Impurity G 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole 96.65 Shenzhen Moco Biochemical Technology Co., Ltd. impurity H 2-Methyl-5-nitroimidazole <![CDATA[99.6% calculated as C4H5N3O2]]> China National Institutes for Food and Drug Control Moroxydazole / 99.9 Jilin Boda Pharmaceutical Co., Ltd. Moroxydine sodium chloride injection / 100ml: 0.5g morpholinone and 0.9g sodium chloride Hainan Aike Pharmaceutical Co., Ltd. Table 2. Instruments used in the examples High Performance Liquid Chromatography Vanquish Thermo Fisher Example 1

[0054] Determination of related substances in morpholine nitroazole sodium chloride injection by high performance liquid chromatography Chromatographic conditions: High Performance Liquid Chromatography (HPLC): Chromatographic column: Octadecylsilane-bonded silica gel (Waters Xbridge C18, 4.6 mm × 250 mm, 5 μm) was used as the packing material. Mobile phase A: Ammonium acetate buffer solution with pH 7.7. The ammonium acetate buffer solution with pH 7.7 is prepared according to the following steps: take 3.8g of ammonium acetate, add 1000ml of water to dissolve it, and then adjust the pH value to 7.7 with ammonia. Mobile phase B: Methanol (100wt%); Gradient elution was performed using mobile phase A and mobile phase B. The gradient elution conditions are shown in Table 3 below. All mobile phases were filtered online using a Welch Ghost-Buster Column (4.6×50mm) (the Ghost-Buster Column was installed after the pump and before the injector). The flow rate was 1.0 ml / min, the column temperature was 30℃, the detection wavelength was 319 nm, the detector was a UV-Vis detector, and the injection volume was 20 μl.

[0055] Table 3 Gradient elution conditions 0 97 3 5 97 3 20 80 20 30 80 20 40 55 45 45 55 45 45.1 97 3 55 97 3 1. Preparation of morpholinidazole reference solution Take an appropriate amount of morpholine nitrazole reference standard, accurately weigh it, dissolve it in a solvent and quantitatively dilute it to prepare a solution containing about 0.5 μg of morpholine nitrazole per 1 ml. The solvent is prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0056] 2. Preparation of the test solution Take a precise amount of morpholine nitrazole sodium chloride injection, dilute it with a solvent to prepare a solution containing approximately 0.5 mg of morpholine nitrazole per ml. The solvent is prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0057] 3. Reference solutions for each known impurity (impurity localization solutions): Take appropriate amounts of reference standards for impurities A, C, D, E, F, G, and H, respectively, dissolve and dilute them with solvents to prepare solutions containing approximately 2 μg of each known impurity per ml.

[0058] 4. Detection of related substances Take 20 μl each of the known impurity reference solution, the morpholinidazole reference solution, and the test solution, and inject them into the liquid chromatograph. Record the chromatogram for 55 minutes. The relevant peaks in the chromatogram of the test solution are located using the corresponding impurity peaks in the impurity localizing solution and the main peak in the morpholinidazole reference solution, as shown in Table 4. The chromatogram of the test solution is shown below. Figure 1 As shown.

[0059] Table 4. Chromatogram retention times of impurity localization solution and morpholinone reference solution

[0060] For impurity peaks in the chromatogram of the test solution, calculate the content of each impurity using the impurity reference standard method with correction factors. The correction factors and limits for each impurity are shown in Table 5. Chromatographic peaks in the test solution chromatogram smaller than the main peak area of ​​the sensitivity solution can be ignored. The sensitivity solution is prepared as follows: accurately measure an appropriate amount of morpholinidazole reference standard solution and dilute it with solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per ml.

[0061] Table 5 Correction factors and limits for each impurity Impurity A 0.8 ≤0.1% Impurity C 0.7 ≤0.1% Impurity D 1.2 ≤0.1% Impurity E 1.0 ≤0.1% impurity F 1.2 ≤0.1% Impurity G 0.7 ≤0.1% impurity H 0.6 ≤0.1% Other individual impurities 1.0 ≤0.1% Total impurities / ≤0.4% Depend on Figure 1 It can be seen that the test solution contains impurity E, with a content of 0.027%.

[0062] The methodological validation of the above-mentioned detection method for related substances in morpholine nitroazole sodium chloride injection was carried out.

[0063] 1. Method specificity 1) System adaptability test Solution preparation The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0064] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole and 1 μg of each known impurity per 1 ml.

[0065] Reference solution: Weigh an appropriate amount of morpholine nitroazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitroazole per 1 ml.

[0066] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0067] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis. The signal-to-noise ratio of the sensitivity solution is 33.4. The separation results of each impurity in the system suitability solution from morpholinidazole are shown in Table 6.

[0068] Table 6. Results of separation degree between various impurities and morpholinonitrazole in the system suitability solution. impurity H 0.33 3.67 Impurity E 0.37 13.39 Impurity G 0.48 26.51 Impurity C 0.69 8.90 impurity F 0.77 3.64 Impurity D 0.80 9.27 Impurity A 0.91 5.93 Moroxydazole / / System suitability requirements: In the system suitability solution chromatogram, impurities H, E, G, C, F, D, A, and morpholinidazole elute sequentially, and the resolution between each chromatographic peak should meet the requirements.

[0069] Five precise injections of the reference solution were taken, 20 µl each, and injected into the liquid chromatograph for analysis. The peak area RSD (relative standard deviation) results of the reference solution are shown in Table 7.

[0070] Table 7. Peak Area RSD of Reference Solution

[0071] The system suitability solution chromatogram results showed that impurities H, E, G, C, F, D, and A, along with morpholinazole, elute sequentially, with resolutions greater than 1.5, meeting the requirements. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak was 33.4, greater than 10. After five consecutive injections of the reference solution, the RSD of the main component peak area was 0.38%, less than 2.0%, meeting the validation requirements. This indicates that the method meets the system suitability requirements.

[0072] 2) Interference and selectivity tests Solution preparation The blank solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0073] Blank excipient solution: Take an appropriate amount of sodium chloride, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing approximately 0.9 mg of sodium chloride per 1 ml.

[0074] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with blank solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole per ml and 1 μg each of impurities A, C, D, E, F, G and H.

[0075] Locational solutions for each known impurity: Take appropriate amounts of reference standards for impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H, respectively, dissolve and dilute them with blank solvent to prepare solutions containing approximately 2 μg of each known impurity per 1 ml.

[0076] Preparation of morpholinidazole reference solution Take an appropriate amount of morpholine nitrazole reference standard, accurately weigh it, dissolve it in a solvent and quantitatively dilute it to prepare a solution containing about 0.5 μg of morpholine nitrazole per 1 ml. The solvent is prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0077] Test solution: Take an appropriate amount of morpholine nitrazole sodium chloride injection and dilute it with blank solvent to prepare a solution containing approximately 0.5 mg of morpholine nitrazole per 1 ml.

[0078] Accurately measure 20 µl of each of the above solutions and inject them separately into the liquid chromatograph for analysis. The results are as follows: Figure 1 As shown in Table 8.

[0079] Table 8 Chromatographic analysis results of positioning solutions and system suitability solutions

[0080] Depend on Figure 2 As shown in Table 8, the blank solvent and blank excipients did not interfere with the elution of any known impurities or the main peak. In the chromatogram of the system suitability solution, impurities H, E, G, C, F, D, and A eluted sequentially with morpholinazole, and the resolution between each peak was greater than 1.5. This meets the validation requirements.

[0081] 3) Forced degradation test Forced degradation tests are conducted in a liquid state and employ methods such as strong light irradiation, high temperature and oxidative decomposition, and acid / alkali hydrolysis. The amount of degradation products is adjusted by the exposure time, temperature and concentration.

[0082] Solution preparation: The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0083] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole and 1 μg of each known impurity per 1 ml.

[0084] Blank excipient stock solution: Take an appropriate amount of sodium chloride, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing approximately 9 mg of sodium chloride per 1 ml.

[0085] Blank excipient solution - Undamaged: Accurately measure 1 ml of blank excipient stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0086] Test solution - Undamaged: Accurately measure 1 ml of morpholine nitroazole sodium chloride injection, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0087] Blank excipient solution - acid destruction: Accurately measure 1 ml of blank excipient stock solution and place it in a 10 ml volumetric flask. Add 1 ml of 0.1 mol / L hydrochloric acid solution and let it stand at room temperature in the dark for 4 hours. Then add 1 ml of 0.1 mol / L sodium hydroxide solution to neutralize it. Dilute to the mark with solvent and shake well.

[0088] Test solution - acid destruction: Accurately measure 1 ml of morphonitroazole sodium chloride injection and place it in a 10 ml volumetric flask. Add 1 ml of 0.1 mol / L hydrochloric acid solution and let it stand at room temperature in the dark for 4 hours. Then add 1 ml of 0.1 mol / L sodium hydroxide solution to neutralize. Dilute to the mark with solvent and shake well.

[0089] Blank excipient solution - alkaline destruction: Accurately measure 1 ml of blank excipient stock solution and place it in a 10 ml volumetric flask. Add 1 ml of 0.1 mol / L sodium hydroxide solution and let it stand at room temperature in the dark for 4 hours. Then add 1 ml of 0.1 mol / L hydrochloric acid solution to neutralize it. Dilute to the mark with solvent and shake well.

[0090] Test solution - alkaline destruction: Accurately measure 1 ml of morpholine nitroazole sodium chloride injection, place it in a 10 ml volumetric flask, add 1 ml of 0.1 mol / L sodium hydroxide solution, let it stand at room temperature in the dark for 4 hours, then add 1 ml of 0.1 mol / L hydrochloric acid solution to neutralize, dilute to the mark with solvent, and shake well.

[0091] Blank excipient solution - oxidative degradation: Accurately measure 1 ml of blank excipient stock solution, place it in a 10 ml volumetric flask, add 1 ml of 3% hydrogen peroxide solution, let it stand at room temperature in the dark for 1 hour, dilute to the mark with solvent, and shake well.

[0092] Test solution - Oxidative destruction: Accurately measure 1 ml of morpholine nitroazole sodium chloride injection, place it in a 10 ml volumetric flask, add 1 ml of 3% hydrogen peroxide solution, let it stand at room temperature in the dark for 1 hour, dilute to the mark with solvent, and shake well.

[0093] Blank excipient solution - high temperature destruction: Accurately measure 1 ml of blank excipient stock solution, place it in a 10 ml volumetric flask, place it at 60°C for 4 h, cool to room temperature, dilute to the mark with solvent, and shake well.

[0094] Test solution - high temperature destruction: Accurately measure 1 ml of morpholine nitroazole sodium chloride injection, place it in a 10 ml volumetric flask, place it at 60℃ for 4 h, cool to room temperature, dilute to the mark with solvent, and shake well.

[0095] Blank excipient solution - light damage: Accurately measure 1 ml of blank excipient stock solution, place it in a 10 ml transparent volumetric flask, and place it in a strong light stability test chamber (5000±500 lx illuminance) for 4 h. Dilute to the mark with solvent and shake well.

[0096] Test solution - light damage: Accurately measure 1 ml of morpholine nitroazole sodium chloride injection, place it in a 10 ml transparent volumetric flask, and place it in a strong light stability test chamber (5000±500 lx illuminance) for 4 h. Dilute to the mark with solvent and shake well.

[0097] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis. The relative retention time and impurity content results are shown in Table 9. The impurity content is calculated by normalizing the peak area.

[0098] Table 9. Results of relative retention time and impurity content

[0099] The results showed that under all degradation conditions, the minimum separation degree between the main component peak and the adjacent impurity peak was 9.52, which was greater than 1.5; the material balance was within the range of 90% to 110%; and the purity factor of the morpholinonitrazole peak was the minimum of 998, which was greater than 980. These results met the validation requirements.

[0100] Therefore, this method has good specificity.

[0101] 2. Detection line and limit of quantitation Solution preparation The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0102] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole and 1 μg of each known impurity per 1 ml.

[0103] Limit of Quantification Solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholinidazole reference standard, dissolve them in solvent and gradually dilute them to make the S / N value ≥10.

[0104] Detection limit solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholinidazole reference standard, dissolve them in solvent and gradually dilute them to make the S / N value ≥ 3.

[0105] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms, and the experimental results of the limit of quantitation and limit of detection are shown in Table 10 and Table 11, respectively.

[0106] Table 10 Results of Limit of Quantitation Experiment

[0107]

[0108] Table 11 Detection Limit Experiment Results

[0109] Experimental results show that the sum of squared ratios (S / N) for the limits of quantitation (LOQ) of morpholinidazole and all known impurities is greater than 10. With six consecutive injections of the LOQ solution, the relative standard deviations (RSDs) of the peak areas for all known impurities and morpholinidazole are less than 20%, and the RSDs of the retention times are less than 2%, meeting the validation requirements. The sum of squared ratios (S / N) for the limits of detection (LOD) of morpholinidazole and all known impurities is greater than 3, meeting the validation requirements.

[0110] The above results indicate that this method has good sensitivity.

[0111] 3. Linearity and Range Within the quantitation limits of 200% for the determination of each impurity and main component, six linear solutions with different concentrations were prepared, and the peak areas of each component were measured. Linear regression analysis was performed with concentration as the abscissa (X) and peak area as the ordinate (Y).

[0112] The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0113] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole and 1 μg of each known impurity per 1 ml.

[0114] Linear stock solution: Take 1 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, add solvent and dilute to 10 ml to prepare the corresponding reference standard stock solution. Then take 2 ml of each impurity reference standard stock solution and 1 ml of morpholine nitrazole reference standard stock solution, add them to a 20 ml volumetric flask and dilute to volume with solvent to prepare a mixed solution containing approximately 5 μg of morpholine nitrazole and 10 μg each of impurities A, C, D, E, F, G and H per ml.

[0115] Six linear solutions with different impurity concentrations and morpholine nitrazole concentrations were prepared and denoted as linear solution 1, linear solution 2, linear solution 3, linear solution 4, linear solution 5, and linear solution 6, respectively.

[0116] Linear Solution 1: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitroazole reference standard, and then add solvent to prepare a mixed solution containing approximately 0.027 μg of morpholine nitroazole, 0.027 μg of impurity A, 0.016 μg of impurity C, 0.024 μg of impurity D, 0.026 μg of impurity E, 0.025 μg of impurity F, 0.011 μg of impurity G, and 0.013 μg of impurity H per 1 ml.

[0117] Linear solution 2: Measure 1 ml of the linear stock solution and place it in a 50 ml volumetric flask. Add solvent to make up to the mark and mix well to obtain linear solution 2, which is equivalent to the limit concentration of 20%. The concentration of morpholinidazole is 0.1 μg / ml and the concentration of each impurity is 0.2 μg / ml.

[0118] Linear solution 3: Measure 1 ml of the linear stock solution and place it in a 20 ml volumetric flask. Add solvent to make up to the mark and mix well to obtain linear solution 3, which is equivalent to 50% of the limit concentration. The concentration of morpholinidazole is 0.25 μg / ml and the concentration of each impurity is 0.5 μg / ml.

[0119] Linear solution 4: Measure 1 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to the mark, mix well to obtain linear solution 4, which is equivalent to the limit concentration of 100%, wherein the concentration of morpholinidazole is 0.5 μg / ml, and the concentration of each impurity is 1 μg / ml.

[0120] Linear solution 5: Measure 3 ml of the linear stock solution and place it in a 20 ml volumetric flask. Add solvent to make up to the mark and mix well to obtain linear solution 5, which is equivalent to 150% of the limit concentration. The concentration of morpholinidazole is 0.75 μg / ml and the concentration of each impurity is 1.5 μg / ml.

[0121] Linear solution 6: Measure 2 ml of the linear stock solution and place it in a 10 ml volumetric flask. Add solvent to make up to the mark and mix well to obtain linear solution 6, which is equivalent to a limit concentration of 200%. The concentration of morpholinidazole is 1 μg / ml and the concentration of each impurity is 2 μg / ml.

[0122] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis. The results are shown in Table 12. Table 12 Results of Linearity and Range Tests

[0123]

[0124]

[0125]

[0126]

[0127] The results showed that for impurity A, within the concentration range of 0.027 µg / ml to 2.168 µg / ml (equivalent to 200% of the limit of quantitation), a good linear relationship was observed between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-intercept| / 100% concentration response value ≤ 25%. For impurity C, within the concentration range of 0.016 µg / ml to 2.138 µg / ml (equivalent to 200% of the limit of quantitation), a good linear relationship was observed between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-intercept| / 100% concentration response value ≤ 25%. For impurity D, within the concentration range of 0.024 µg / ml to 1.921 µg / ml (equivalent to 200% of the limit of quantitation), a good linear relationship was observed between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-intercept| / 100% concentration response value ≤ 25%. Impurity E, within the concentration range of 0.026 µg / ml to 2.101 µg / ml (equivalent to 200% of the limit of quantitation), exhibits a good linear relationship between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a concentration response value of |Y-intercept| / 100% ≤ 25%. Impurity F, within the concentration range of 0.025 µg / ml to 1.996 µg / ml (equivalent to 200% of the limit of quantitation), also exhibits a good linear relationship between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a concentration response value of |Y-intercept| / 100% ≤ 25%. Impurity G, within the concentration range of 0.011 µg / ml to 2.173 µg / ml (equivalent to 200% of the limit of quantitation), also exhibits a good linear relationship between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a concentration response value of |Y-intercept| / 100% ≤ 25%. Impurity H, within the concentration range of 0.013 µg / ml to 2.086 µg / ml (equivalent to 200% of the limit of quantitation), exhibits a good linear relationship between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-intercept| / 100% concentration response value ≤ 25%. Moroxydine, within the concentration range of 0.027 µg / ml to 1.096 µg / ml (equivalent to 200% of the limit of quantitation), also exhibits a good linear relationship between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-intercept| / 100% concentration response value ≤ 25%.

[0128] In summary, for the above samples, within the concentration range equivalent to the limit of quantitation to 200% of the limit concentration, a good linear relationship was observed between concentration and peak area, with a correlation coefficient r ≥ 0.990 and a |Y-axis intercept| / 100% concentration response value ≤ 25%, meeting the validation requirements. This indicates that the method has a good linear relationship.

[0129] 4. Precision 4.1 Repeatability Solution preparation: The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0130] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitroazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitroazole and 1 μg of each known impurity per 1 ml.

[0131] Reference solution: Weigh an appropriate amount of morpholine nitrazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitrazole per 1 ml.

[0132] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0133] Impurity reference standard stock solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H reference standards, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 10 μg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H per ml.

[0134] Spiked test solution: Accurately measure 1 ml of morpholine nitrazole sodium chloride injection into a 10 ml volumetric flask, add 1 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, and shake well to prepare the spiked test solution. Prepare 6 parallel spiked test solutions.

[0135] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis. Calculate the content of each impurity by peak area using the external standard method, and calculate the RSD of the results for the 6 spiked test solutions. The results are shown in Table 13.

[0136] Table 13 Repeatability Test Results

[0137] The results showed that the RSD of the detection amount of each known impurity in the six spiked test solutions was less than 5%, which met the validation requirements and indicated that the method had good repeatability.

[0138] 4.2 Intermediate Precision Solution preparation The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0139] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitroazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitroazole and 1 μg of each known impurity per 1 ml.

[0140] Reference solution: Weigh an appropriate amount of morpholine nitrazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitrazole per 1 ml.

[0141] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0142] Impurity reference standard stock solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H reference standards, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 10 μg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H per ml.

[0143] Spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, and shake well to prepare the spiked test solution. Prepare 6 parallel spiked test solutions.

[0144] Another analyst conducted tests on different dates using different instruments. The spiked test solution was prepared according to the repeatability test, and six parallel solutions were prepared. The content of each impurity was calculated by peak area using the external standard method, and the results were compared with the repeatability test results. The results are shown in Table 14.

[0145] Table 14 Intermediate Precision Test Results

[0146] The results showed that the RSD of the detection amount of each known impurity in the 12 spiked test solutions was less than 10%, which met the validation requirements and indicated that the intermediate precision of the method was good.

[0147] 5. Accuracy Solution preparation The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0148] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitroazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitroazole and 1 μg of each known impurity per 1 ml.

[0149] Reference solution: Weigh an appropriate amount of morpholine nitrazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitrazole per 1 ml.

[0150] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0151] Test solution: Take an appropriate amount of morpholine nitrazole sodium chloride injection and dilute it with solvent to prepare a solution containing approximately 0.5 mg of morpholine nitrazole per 1 ml.

[0152] Impurity reference standard stock solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H reference standards, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 10 μg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H per ml.

[0153] 50% spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 0.5 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, shake well, and prepare 3 parallel portions.

[0154] 100% spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, shake well, and prepare 3 parallel solutions.

[0155] 150% spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 1.5 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, shake well, and prepare 3 parallel solutions.

[0156] Accurately measure 20 µl of each of the above solutions and inject them into the liquid chromatograph for analysis. The results are shown in Table 15.

[0157] Table 15 Accuracy Measurement Results

[0158]

[0159]

[0160]

[0161] The results showed that the recoveries of all known impurities were between 90% and 108% at nine concentration levels, with RSDs all less than 3%, meeting the validation requirements and indicating that the method had good accuracy.

[0162] 6. Solution stability Solution preparation The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0163] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitrazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitrazole and 1 μg of each known impurity per 1 ml.

[0164] Reference solution: Weigh an appropriate amount of morpholine nitroazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitroazole per 1 ml.

[0165] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0166] Test solution: Take an appropriate amount of morpholine nitrazole sodium chloride injection and dilute it with solvent to prepare a solution containing approximately 0.5 mg of morpholine nitrazole per 1 ml.

[0167] Impurity reference standard stock solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference standards respectively, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 10 μg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H per ml.

[0168] Spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, and shake well.

[0169] During the experiment, the reference solution, the test solution, and the spiked test solution were placed in an autosampler at 5℃ and room temperature, and the solutions were injected at different time points to investigate their stability. The results are shown in Tables 16-18.

[0170] Table 16 Results of stability test of reference solution

[0172] Table 17 Results of the stability test of the test sample solution

[0173] Table 18 Results of Stability Tests on Spiked Samples

[0174] The results showed that: (1) When the reference solution was placed at 5℃ for 49h, the peak area of ​​the main component at each time point was the largest change rate of 2.0% compared with 0h, which was less than 5.0%, indicating that the reference solution had good stability within 49h at 5℃; when the reference solution was placed at room temperature for 49h, the peak area of ​​the main component at each time point was the largest change rate of 0.6% compared with 0h, which was less than 5.0%, indicating that the reference solution had good stability within 49h at room temperature. (2) After the test solution was placed at 5°C for 49 hours, the absolute value of the change in the content of each known and unknown single impurity at each time point compared with 0 hours was 0.002%, less than 0.05%, and the absolute value of the change in the total impurity content was 0.002%, less than 0.1%. No new impurities exceeding the reporting limit were found, indicating that the test solution was stable within 49 hours at 5°C. After the test solution was placed at room temperature for 49 hours, the absolute value of the change in the content of each related substance and unknown single impurity at each time point compared with 0 hours was 0.002%, less than 0.05%, and the absolute value of the change in the total impurity content was 0.002%, less than 0.1%. No new impurities exceeding the reporting limit were found, indicating that the test solution was stable within 49 hours at room temperature. (3) When the spiked test solution was placed at 5°C for 49 hours, the absolute value of the change in the content of each impurity was 0.003% at each time point compared with 0 hours, which is less than 0.05%, indicating that the test solution was stable within 49 hours at 5°C. When the spiked test solution was placed at room temperature for 49 hours, the absolute value of the change in the content of each impurity was 0.050% at each time point compared with 0 hours, which is less than 0.1%, indicating that the spiked test solution was stable within 49 hours at room temperature.

[0175] 7. Durability The solvent was prepared by mixing ammonium acetate buffer (pH 7.7) and methanol (100 wt%) at a volume ratio of 80:20.

[0176] System suitability solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and morpholine nitroazole reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of morpholine nitroazole and 1 μg of each known impurity per 1 ml.

[0177] Reference solution: Weigh an appropriate amount of morpholine nitrazole reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 1 μg of morpholine nitrazole per 1 ml.

[0178] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with a solvent to prepare a solution containing approximately 0.25 µg of morpholinidazole per 1 ml.

[0179] Impurity reference standard stock solution: Take appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H reference standards, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 10 μg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H per ml.

[0180] Spiked test solution: Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of impurity reference stock solution, dissolve and dilute to the mark with solvent, and shake well.

[0181] To investigate the method's robustness against variable experimental factors, the following factors were examined: column temperature, flow rate, buffer pH, and the concentration of different batches of columns (Waters Xbridge C18 (4.6 mm × 250 mm, 5 μm)) and ammonium acetate.

[0182] The experimental designs are shown in Table 19. Table 19 Durability Test Results

[0183] Accurately pipette the above solution and perform chromatographic analysis under conditions 1-8 in Table 19. The results are shown in Tables 20 and 21. Table 20 System Suitability Test Results

[0184] Table 21 Test Results of the Sample

[0185] The results showed that: (1) In the chromatograms of the system suitability solutions under various robustness conditions, impurities H, E, G, C, F, D, and A, along with morpholinidazole, elute sequentially, with a resolution greater than 1.5 between each peak; in the chromatograms of the sensitivity solutions, the signal-to-noise ratio of the main component peaks was greater than 10. This met the validation requirements. (2) When the chromatographic conditions changed slightly (pH change ±0.1, column temperature change ±2℃, flow rate change ±0.1 ml / min, and different batches of chromatographic columns, etc.), the absolute value of the change in impurity content under each condition compared with the standard conditions was the largest at 0.032%, which was less than 0.05%, meeting the validation requirements.

[0186] Therefore, this method is durable.

[0187] Comparative Example 1 High performance liquid chromatography was used to determine related substances in morpholine nitroazole sodium chloride injection.

[0188] 1. Chromatographic conditions: High Performance Liquid Chromatography (HPLC): Chromatographic column: Octadecylsilane-bonded silica gel (Waters Xbridge C18, 4.6 mm × 250 mm, 5 μm) was used as the packing material. Isocratic elution was performed using a mobile phase consisting of a mixture of 0.05 mol / L phosphate buffer, acetonitrile, and methanol, with a volume ratio of 80:10:10. 0.05 mol / L phosphate buffer solution is prepared as follows: Dissolve 6.8g of potassium dihydrogen phosphate in approximately 900ml of water, then add 10ml of triethylamine. Adjust the pH to 7.0 with phosphoric acid, and then dilute with water to 1000ml.

[0189] The mobile phase was filtered in-line using a Welch Ghost-Buster Column (4.6 x 50 mm) (the Ghost-Buster Column was installed after the pump and before the injector). The flow rate was 1.0 ml / min, the column temperature was 30℃, the detection wavelength was 319 nm, the detector was UV-Vis, and the injection volume was 20 μl.

[0190] 2. Preparation of the test solution Accurately weigh the sodium chloride injection of morpholinedazole, dissolve it in the mobile phase, and quantitatively dilute it to prepare a solution containing approximately 0.5 mg of morpholinedazole per 1 ml.

[0191] 3. Reference solution Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with the mobile phase and shake well.

[0192] 4. Detection of related substances Inject 20 μl each of the test solution and the reference solution into the liquid chromatograph and record the chromatogram for 55 minutes. Analyze using the main component impurity reference method; the chromatogram is shown below. Figure 2 As shown.

[0193] Depend on Figure 2 It can be seen that the H peak of impurity partially overlaps with the G peak of impurity, indicating poor specificity and failing to meet the basic requirements of analysis.

[0194] In summary, this application improves detection efficiency and saves experimental time and costs by optimizing chromatographic conditions and controlling the gradient elution time to 55 minutes. This method is more sensitive, and a concentration of 0.2 μg / ml is sufficient to meet the sensitivity requirements; This method comprehensively analyzes the impurity profile of morpholine nitric acid sodium chloride injection based on the synthesis process of morpholine nitric acid raw material, which can effectively separate morpholine nitric acid from various impurities, thereby ensuring the accuracy of the detection results.

[0195] This invention provides a comprehensive methodological validation of the effectiveness and detection capability of the method. The validation results show that the detection method has high accuracy, good precision, and good robustness.

[0196] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting related substances of a morpholine nitazoxanide sodium chloride injection, characterized by, It includes the use of high performance liquid chromatography (HPLC) to detect related substances in morpholine nitroazole sodium chloride injection. The HPLC test conditions include: the chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is methanol; and gradient elution is used.

2. The detection method according to claim 1, characterized in that, The ammonium acetate aqueous solution has a molar concentration of 40-60 mmol / L and a pH of 7.0-8.

0.

3. The detection method according to claim 1 or 2, characterized in that, The ammonium acetate aqueous solution is an ammonium acetate-ammonia buffer solution. Preferably, the pH of the ammonium acetate-ammonia buffer solution is 7.5-8.0, and more preferably 7.6-7.

8.

4. The detection method according to any one of claims 1-3, characterized in that, The gradient elution includes the following elution sequence: From 0 to 5 minutes, the volume percentage of mobile phase A was 97 ± 1%, and the volume percentage of mobile phase B was 3 ± 1%. Over 5-20 minutes, the volume percentage of mobile phase A decreased from 97±1% to 80±1%, while the volume percentage of mobile phase B increased from 3±1% to 20±1%. After 20-30 minutes, the volume percentage of mobile phase A was 80±1%, and the volume percentage of mobile phase B was 20±1%. Over 30-40 minutes, the volume percentage of mobile phase A decreased from 80±1% to 55±1%, while the volume percentage of mobile phase B increased from 20±1% to 45±1%. After 40-45 minutes, the volume percentage of mobile phase A was 55±1%, and the volume percentage of mobile phase B was 45±1%. Over 45-45.1 min, the volume percentage of mobile phase A increased from 55±1% to 97±1%, while the volume percentage of mobile phase B decreased from 45±1% to 3±1%. From 45.1 to 55 min, the volume percentage of mobile phase A was 97±1%, and the volume percentage of mobile phase B was 3±1%.

5. The detection method according to any one of claims 1-4, characterized in that, It also includes the step of filtering mobile phase A and mobile phase B separately with an impurity trapping column before gradient elution.

6. The detection method according to any one of claims 1-5, characterized in that, The related substances of the morpholine nitroazole sodium chloride injection include impurity E and impurity F; preferably, the related substances of the morpholine nitroazole sodium chloride injection also include one or more of impurities A, C, D, G and H.

7. The detection method according to any one of claims 1-6, characterized in that, The detection method includes the following steps: Prepare the reference solution; Prepare the test solution; The reference solution and the test solution were analyzed by high performance liquid chromatography, respectively.

8. The detection method according to claim 7, characterized in that, The reference solution is prepared using a mixed solution of ammonium acetate aqueous solution and methanol as a solvent, preferably with a concentration of 0.1-5 μg / ml; preferably, the reference solution includes morpholine nitrazole reference solution, impurity E reference solution, and impurity F reference solution; more preferably, the reference solution further includes one or more of impurity A reference solution, impurity C reference solution, impurity D reference solution, impurity G reference solution, and impurity H reference solution; and / or, The solvent of the test solution is a mixture of ammonium acetate aqueous solution and methanol, and preferably the concentration of morpholinidazole in the test solution is 0.1-1 mg / ml.

9. The detection method according to any one of claims 1-8, characterized in that, The detection wavelength used in high-performance liquid chromatography is 310-330 nm, preferably 311-320 nm; and / or, The column temperature used in high-performance liquid chromatography is 28-32℃; and / or, The flow rate of the mobile phase used in high performance liquid chromatography is 0.8-1.2 ml / min, preferably 0.9-1.1 ml / min.

10. The application of the detection method according to any one of claims 1-9 in the field of quality control of morpholinidazole and its preparations.

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