Detection method of morphonidazole isomer
By employing a high-performance liquid chromatography method with a specific combination of packing agent and mobile phase, the isomers of morpholinidazole were successfully separated and detected, solving the problem of poor separation effect in the prior art and achieving efficient and accurate isomer detection.
Patent Information
- Application Number
- CN202511123027.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-09-05
- Filing Date
- 2025-08-12
- Publication Date
- 2025-12-09
AI Technical Summary
The lack of effective high-performance liquid chromatography (HPLC) methods in the current technology for separating and detecting isomers of morpholinidazole affects product quality control.
A chromatographic column packed with covalently bonded amylose-tris(3-chloro-5-methylphenylcarbamate) on a silica gel surface was used. Mobile phase A was adjusted to pH 2.0 ± 0.5 with 0.05 mol/L potassium dihydrogen phosphate solution, and mobile phase B was acetonitrile. Isocratic or gradient elution was performed at a specific volume ratio, and detection was performed using an ultraviolet detector or a diode array detector.
The method achieves effective separation and accurate detection of morpholinonitrazole isomers, with good peak shape, high resolution, and meets the detection requirements. It also exhibits good repeatability and linearity.
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Figure CN121090740A_ABST
Abstract
Description
[0001] The present application claims priority from Chinese patent application 202411237661.6 with the filing date of 2024 / 09 / 05. The present application incorporates the entire text of the above-mentioned Chinese patent application. TECHNICAL FIELD
[0002] The present application belongs to the technical field of pharmaceutical analysis, and specifically relates to a detection method of morpholine nitroimidazole isomers. BACKGROUND
[0003] Morpholine nitroimidazole, with the chemical name of (1-(3-(4-morpholinyl)-2-hydroxypropyl)-2-methyl-5-nitro-1H-imidazole, molecular formula C11H18N4O4, molecular weight 270.29, is a new third-generation nitroimidazole drug, mainly used for surgical infections and gynecological infections caused by anaerobic bacteria in clinical practice, and the original research manufacturer is Jiangsu Haosen Pharmaceutical. Through the observation of morpholine nitroimidazole I, II and III clinical trial cases, it is proved that morpholine nitroimidazole has strong activity against anaerobic bacteria, fast onset, good tolerance, high safety, and does not need to adjust the dosage for traditional adverse reactions of nitroimidazole drugs such as mild and moderate liver dysfunction.
[0004] Morpholine nitroimidazole contains a chiral carbon atom in its structure, and the R and S configuration isomers have the following structural formulas
[0005]
[0006] The content of each isomer of morpholine nitroimidazole directly affects the quality of morpholine nitroimidazole as a raw material drug, so the control of the isomer content in morpholine nitroimidazole is of great significance to the product quality.
[0007] At present, there are very few related reports on the detection of morpholine nitroimidazole isomers by high performance liquid chromatography (HPLC), therefore, the development of an HPLC detection method which can effectively separate and accurately detect morpholine nitroimidazole isomers plays an important role in the control of the product quality. SUMMARY
[0008] To solve the above problems, the present application provides a detection method of morpholine nitroimidazole isomers, which is a high performance liquid chromatography method, using a chromatographic column with straight-chain starch-tris(3-chloro-5-methylphenylcarbamate) covalently bonded on the surface of silica gel as the filler, mobile phase A is 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.0±0.5 with phosphoric acid, mobile phase B is acetonitrile, and the mobile phases A and B are isocratically eluted at a volume ratio of (20-30):(80-70).
[0009] In some preferred embodiments, the detection method provided by the present application uses mobile phase A and B in a volume ratio of (23-27) : (77-73) for isocratic elution; for example, the mobile phase A and B are used in a volume ratio of 23:77, 25:75, 27:73, or any sub-range or numerical point thereof for gradient elution.
[0010] In some embodiments, the detection method provided by the present application uses a CHIRALPAK IG column with a size of 250 mm x 4.6 mm and a particle size of 5 μm.
[0011] In some embodiments, the detection method provided by the present application uses mobile phase A which is a 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.0 ± 0.2 with phosphoric acid.
[0012] In some embodiments, the detection method provided by the present application uses a mobile phase flow rate of 0.3-0.5 ml / min; for example, the mobile phase flow rate is 0.35 ml / min, 0.4 ml / min, 0.45 ml / min, or any sub-range or numerical point thereof.
[0013] In some embodiments, the detection method provided by the present application uses a sample solution of the detection method which is a solution containing 0.1 mg / ml of morpholine nitroimidazole.
[0014] In some embodiments, the detection method provided by the present application uses a sample injection amount of 5 μl-10 μl; for example, the sample injection amount is 5 μl, 10 μl, or any sub-range or numerical point thereof.
[0015] In some embodiments, the detection method provided by the present application uses a column temperature of 30-45°C; for example, the column temperature is 35°C, 38°C, 40°C, 42°C, or any sub-range or numerical point thereof.
[0016] In some embodiments, the detection method provided by the present application uses an ultraviolet detector or a diode array detector.
[0017] The "diode array detector" mentioned in the present application is also known as DAD or PDA detector.
[0018] In some embodiments, the detection method provided by the present application uses a detection wavelength of 319 nm.
[0019] In some embodiments, the solvent used for preparing the sample solution in the detection method provided by the present application is an aqueous acetonitrile solution; preferably, the solvent used for preparing the sample solution is 60% to 75% acetonitrile; for example, the solvent used for preparing the sample solution is 60% acetonitrile, 75% acetonitrile, or any sub-range or numerical point thereof.
[0020] In some embodiments, the detection method provided by the present application comprises the following steps:
[0021] Step 1: Prepare a system suitability solution: dissolve the metronidazole reference substance in a solvent to prepare a solution containing metronidazole at a concentration of 0.1 mg / ml;
[0022] Step 2: Prepare a sample solution: dissolve the metronidazole sample in a solvent to prepare a solution containing metronidazole at a concentration of 0.1 mg / ml;
[0023] Step 3: Precisely take the system suitability solution and the sample solution respectively, inject them into the liquid chromatograph, and detect under the above chromatographic conditions to record the chromatogram.
[0024] In some embodiments, the solvent in step 1 and step 2 is an aqueous acetonitrile solution, preferably 60% to 75% acetonitrile; more preferably 75% acetonitrile.
[0025] In some embodiments, the detection method provided by the present application, the isomer content calculation method is area normalization method.
[0026] In some embodiments, the detection method provided by the present application, the structure of the metronidazole isomer is as follows:
[0027]
[0028] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present application. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1-1 HPLC detection chromatogram of Example 1-1;
[0030] Figure 1-2 HPLC detection chromatogram of Example 1-2;
[0031] Figure 1-3 HPLC detection chromatogram of Example 1-3;
[0032] Figure 1-4 HPLC detection chromatogram of Example 1-4;
[0033] Figure 2 HPLC detection profile of Example 2;
[0034] Figure 3 HPLC detection profile of Example 3;
[0035] Figure 4 HPLC detection profile of Example 4;
[0036] Figure 5 HPLC detection profile of Example 5;
[0037] Figure 6 HPLC detection profile of Example 6;
[0038] Figure 7 HPLC detection profile of Example 7;
[0039] Figure 8 HPLC detection profile of Example 8;
[0040] Figure 9 HPLC detection profile of Example 9;
[0041] Figure 10 HPLC detection profile of Example 10;
[0042] Figure 11 HPLC detection profile of Example 11;
[0043] Figure 12 HPLC detection profile of Example 12;
[0044] Figure 13 HPLC detection profile of Example 13;
[0045] Figure 14 HPLC detection profile of Example 14;
[0046] Figure 15 HPLC detection profile of Example 15;
[0047] Figure 16 HPLC detection profile of Comparative Example 1;
[0048] Figure 17 HPLC detection profile of Comparative Example 2;
[0049] Figure 18 HPLC detection profile of Comparative Example 3;
[0050] Figure 19 HPLC detection profile of Test Example 1; DETAILED DESCRIPTION
[0051] The above summary of the present application will be further explained in the detailed description of specific embodiments in the form of examples. This should not be understood, however, as limiting the scope of the subject matter of the present application to the examples described below, but rather as an illustration of the application based on the above summary.
[0052] High performance liquid chromatograph: Shimadzu LC-2030C
[0053] Detector: UV detector (model: Shimadzu LC-2030C)
[0054] Example 1-1 to 1-4 Chromatographic conditions 1-1 to 1-4 (mobile phase: 0.1% formic acid solution-acetonitrile)
[0055] Chromatographic column: CHIRALPAK IG 250mm x 4.6mm, 5μm;
[0056] Mobile phase: 0.1% formic acid solution-acetonitrile (40:60);
[0057] Flow rate: 0.4ml / min;
[0058] Column temperature: 30°C;
[0059] Injection volume: 10μl;
[0060] Detection wavelength: 319nm;
[0061] Test sample solution: About 5mg of the test sample of moroxydine was accurately weighed into a 50ml volumetric flask, dissolved and diluted to the mark with the solvent, and shaken to give a test sample solution containing 0.1mg / ml of moroxydine. The solvent used to prepare the test sample is shown in the following table.
[0062] Table 1 Solvents used to prepare the test sample
[0063] Example Number 1-1 1-2 1-3 1-4 Solvent 60% acetonitrile 40% acetonitrile 50% acetonitrile 70% acetonitrile Chromatogram APPENDIX Figure 1-1 APPENDIX Figure 1-2 APPENDIX Figure 1-3 APPENDIX Figure 1-4 Results Not baseline separated Not separated Not separated Not baseline separated
[0064] The detection results are shown in the following table. Figures 1-1 through 1-4 The results show that under the chromatographic conditions, the isomer peaks of Examples 1-1 and 1-4 did not reach baseline separation, and the peak areas could not be accurately integrated; the isomer peaks of Examples 1-2 and 1-3 overlapped with each other and were not separated. When different concentrations of acetonitrile solution were used as the solvent, the separation effect of the isomers of moroxydine was slightly different.
[0065] Example 2 Chromatographic condition 2 (mobile phase: 0.1% formic acid solution-acetonitrile)
[0066] Mobile phase: 0.1% formic acid solution-acetonitrile (50:50); the remaining chromatographic conditions are the same as those of Example 1-1.
[0067] The results are shown in Figure 1 and indicate that the isomer peaks are not separated. Figure 2
[0068] Example 3 Chromatographic condition 3 (mobile phase: 0.1% formic acid solution-acetonitrile)
[0069] Mobile phase: 0.1% formic acid solution-acetonitrile (70:30); the rest of the chromatographic conditions are the same as in Example 1-1.
[0070] The results are shown in Figure 1 and indicate that the isomer peaks are not separated. Figure 3 The results are shown in Figure 1 and indicate that the isomer peaks are not separated.
[0071] Example 4 Chromatographic condition 4 (mobile phase: 0.1% formic acid solution-acetonitrile)
[0072] Mobile phase: 0.1% formic acid solution-acetonitrile (75:25); the rest of the chromatographic conditions are the same as in Example 1-1.
[0073] The results are shown in Figure 1 and indicate that the isomer peaks are not separated. Figure 4 The results are shown in Figure 1 and indicate that the isomer peaks are not separated.
[0074] Example 5 Chromatographic condition 5 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0)-acetonitrile)
[0075] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0)-acetonitrile (40:60);
[0076] Test sample solution: morpholine nitazoxanide solution, containing morpholine nitazoxanide at a concentration of 0.1 mg / ml, solvent: 75% acetonitrile.
[0077] The rest of the chromatographic conditions are the same as in Example 1-1.
[0078] The results are shown in Figure 1 and indicate that the isomer peaks are not separated. Figure 5 The results are shown in Figure 1 and indicate that the isomer peaks are not separated.
[0079] Example 6 Chromatographic condition 6 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0)-acetonitrile)
[0080] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0)-acetonitrile (25:75); column temperature: 35°C; the rest of the chromatographic conditions are the same as in Example 5.
[0081] The results are shown in Figure 1 and indicate that the isomer peaks are not separated. Figure 6 The results are shown in Figure 1 and indicate that the isomer peaks are not separated.
[0082] Example 7 Chromatographic condition 7 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0) - acetonitrile)
[0083] Injection volume: 5 μl; the rest of the chromatographic conditions are the same as in Example 6.
[0084] The detection results are shown in the attached Figure 7 chromatogram. The results show that the isomer peak separation is 2.5 and the peak shape is good.
[0085] Example 8 Chromatographic condition 8 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0) - acetonitrile)
[0086] Column temperature: 40°C; injection volume: 5 μl; the rest of the chromatographic conditions are the same as in Example 6.
[0087] The detection results are shown in the attached Figure 8 chromatogram. The results show that the isomer peak separation is 3.0 and the peak shape is good.
[0088] Example 9 Chromatographic condition 9 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0) - acetonitrile)
[0089] Flow rate: 0.35 ml / min; the rest of the chromatographic conditions are the same as in Example 8.
[0090] The detection results are shown in the attached Figure 9 chromatogram. The results show that the isomer peak separation is 3.07 and the peak shape is good, but the running time is long.
[0091] Example 10 Chromatographic condition 10 (mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.0) - acetonitrile)
[0092] Flow rate: 0.45 ml / min; the rest of the chromatographic conditions are the same as in Example 8.
[0093] The detection results are shown in the attached Figure 10 chromatogram. The results show that the isomer peak separation is 2.86 and can be completely separated, and the peak shape is good.
[0094] Column temperature: 38°C; the rest of the chromatographic conditions are the same as in Example 8.
[0095] The detection results are shown in the attached Figure 11 chromatogram. The results show that the isomer peak separation is 2.8 and can be completely separated, and the peak shape is good.
[0096] Column temperature: 42°C; other chromatographic conditions are the same as in Example 8.
[0097] The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good. Figure 12 The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good.
[0098] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH value 2.0) - acetonitrile (23:77);
[0099] Other chromatographic conditions are the same as in Example 8.
[0100] The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good. Figure 13 The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good.
[0101] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH value 2.0) - acetonitrile (27:73); other chromatographic conditions are the same as in Example 8.
[0102] The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good. Figure 14 The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good.
[0103] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH value 2.2) - acetonitrile (25:75); other chromatographic conditions are the same as in Example 8.
[0104] The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good. Figure 15 The detection results are shown in Figure 8, which show that the isomer peak separation is 3.17, can be completely separated, and the peak shape is good.
[0105] Comparative Example 1
[0106] Chromatographic column: Phenomenexe Gemini C18 250 mm x 4.6 mm, 5 μm;
[0107] Mobile phase A: potassium dihydrogen phosphate solution (0.01 mol / L potassium dihydrogen phosphate solution, pH value 7.0), mobile phase B is acetonitrile, and the elution program is shown in the table below;
[0108] Flow rate: 1.0 ml / min;
[0109] Column temperature: 30°C;
[0110] Injection volume: 5 μl;
[0111] Detection wavelength: 319 nm;
[0112] Test solution: Morinamide solution, containing morinamide at a concentration of 0.1 mg / ml, solvent: water.
[0113]
[0114]
[0115] The detection results are shown in the attached Figure 16 chromatogram, and the results show that the method does not separate the isomers of morinamide.
[0116] Comparative Example 2
[0117] Column: CHIRALPAK IC 250 mm x 4.6 mm, 5 μm;
[0118] Mobile phase: n-hexane-isopropanol (85:15);
[0119] Flow rate: 1.0 ml / min;
[0120] Column temperature: 27°C;
[0121] Injection volume: 5 μl;
[0122] Detection wavelength: 319 nm;
[0123] Test solution: Morinamide solution, containing morinamide at a concentration of 0.1 mg / ml, solvent: anhydrous ethanol.
[0124] The detection results are shown in the attached Figure 17 chromatogram, and the results show that the isomer peak separation is 1.18, which cannot achieve baseline separation.
[0125] Comparative Example 3
[0126] Column: CHIRALPAK IG 250 mm x 4.6 mm, 5 μm;
[0127] Mobile phase: methanol-water (80:20);
[0128] Flow rate: 0.4 ml / min;
[0129] Column temperature: 30°C;
[0130] Injection volume: 5 μl;
[0131] Detection wavelength: 319 nm;
[0132] Test solution: moroxydine solution, containing moroxydine at a concentration of 0.1 mg / ml, solvent being 75% acetonitrile.
[0133] The detection results are shown in the attached Figure 18 chromatogram, and the results show that the isomer peaks are coincided as one peak and cannot be separated.
[0134] Specificity test of test example 1
[0135] Solution preparation:
[0136] Blank solution: 75% acetonitrile aqueous solution.
[0137] System suitability solution: moroxydine reference substance 5.09 mg was accurately weighed into a 50 ml volumetric flask, dissolved and diluted to the mark with 75% acetonitrile aqueous solution, and shaken to obtain a system suitability solution.
[0138] S-moroxydine stock solution: S-moroxydine reference substance 25.01 mg was accurately weighed into a 50 ml volumetric flask, dissolved and diluted to the mark with 75% acetonitrile aqueous solution, and shaken to obtain an S-moroxydine stock solution.
[0139] R-moroxydine stock solution: R-moroxydine reference substance 25.10 mg was accurately weighed into a 50 ml volumetric flask, dissolved and diluted to the mark with 75% acetonitrile aqueous solution, and shaken to obtain an R-moroxydine stock solution.
[0140] S-moroxydine reference substance solution: 2 ml of the S-moroxydine stock solution was transferred into a 20 ml volumetric flask, diluted to the mark with the solvent, and shaken to obtain an S-moroxydine reference substance solution.
[0141] R-moroxydine reference substance solution: 2 ml of the R-moroxydine stock solution was transferred into a 20 ml volumetric flask, diluted to the mark with the solvent, and shaken to obtain an R-moroxydine reference substance solution.
[0142] Test solution: about 5 mg of moroxydine test sample was accurately weighed into a 50 ml volumetric flask, dissolved and diluted to the mark with 75% acetonitrile aqueous solution, and shaken to obtain a test solution.
[0143] Chromatographic conditions: the chromatographic conditions of example 8 were used.
[0144] The above reference substance solution, system suitability solution and test solution were respectively taken for sample injection and detection, and the chromatogram was recorded. The results are shown in Table 2 below:
[0145] Table 2: Specificity test results
[0146] Solution Name S-moIynil nitazoxanide retention time min R-moIynil nitazoxanide retention time min Two peak separation S-moIynil nitazoxanide reference solution 7.317 / / R-moIynil nitazoxanide reference solution / 8.158 / System suitability solution 7.317 8.158 3.06 Test solution 7.300 8.139 3.05
[0147] The results show that the separation degree of S and R isomers of moroxydine nitrate in the system suitability solution reaches 3.06 (as shown in the attached Figure 19 chromatogram) under the chromatographic conditions, and the isomers can be completely separated with good peak shape, meeting the detection requirements.
[0148] Test Example 2: Investigation of the limit of quantification and detection limit
[0149] 5 ml of the S-moroxydine nitrate stock solution and 5 ml of the R-moroxydine nitrate stock solution in Test Example 1 were transferred into the same 50 ml volumetric flask, diluted to the calibration mark with 75% acetonitrile water, and shaken to prepare a mixed solution.
[0150] The mixed solution was diluted with 75% acetonitrile water in stages, and detected using the chromatographic conditions of Example 8. When the concentration at which the chromatographic peak height is about 10 times the baseline noise is the limit of quantification concentration; and the concentration at which the chromatographic peak height is about 3 times the baseline noise is the detection limit concentration.
[0151] The results show that the limit of quantification concentration of S-moroxydine nitrate is about 0.0677 μg / ml, and the limit of quantification concentration of R-moroxydine nitrate is about 0.0687 μg / ml under the chromatographic conditions.
[0152] The detection limit concentration of S-moroxydine nitrate is about 0.0203 μg / ml, and the detection limit concentration of R-moroxydine nitrate is about 0.0206 μg / ml.
[0153] Test Example 3: Investigation of linearity and range
[0154] The mixed solution prepared in Test Example 2 was precisely measured at 3 ml, 4 ml, 5 ml, 6 ml, and 7 ml, respectively, and transferred into 10 ml volumetric flasks, which were diluted to the calibration mark with 75% acetonitrile water, and shaken to prepare linear solutions.
[0155] The linear solutions were precisely measured at 5 μl, injected into the liquid chromatograph using the chromatographic conditions of Example 8, and the chromatogram was recorded. The concentration (μg / ml) was taken as the abscissa, and the peak area was taken as the ordinate. Linear regression was performed, and the linear regression equations are shown below, indicating that the linear relationship of the chromatographic conditions is good.
[0156] S-moroxydine nitrate: Y = 28538.5399x - 5567.5420 R 2 = 0.9999;
[0157] R-moroxydine nitrate: Y = 28612.4632x - 6211.1351 R 2 = 0.9999.
[0158] Test Example 4: Investigation of repeatability
[0159] About 5 mg of the test sample of metronidazole was precisely weighed into a 50-ml volumetric flask, dissolved and diluted with 75% aqueous acetonitrile to the mark, shaken well to serve as the test solution. Six samples were prepared in the same manner.
[0160] The chromatographic conditions of Example 8 were used. Five μl of each of the above solutions were precisely measured and injected into the liquid chromatograph, respectively, and the chromatograms were recorded. The results are shown in Table 3.
[0161] Table 3 Results of the reproducibility test
[0162] % Content 1 2 3 4 5 6 Average RSD% R-moIynil nitazoxanide 50.00 49.99 50.01 49.99 50.00 50.02 50.00 0.02 S-moIynil nitazoxanide 50.00 50.01 49.99 50.01 50.00 49.98 50.00 0.02
[0163] The results show that under the chromatographic conditions, the RSD of the reproducibility test of S-metronidazole and R-metronidazole is 0.02%, indicating that the chromatographic conditions are reproducible.
Claims
1. A method for detecting isomers of morpholinonitrazole, characterized in that, The method is high performance liquid chromatography, using a chromatographic column with linear starch-tris(3-chloro-5-methylphenylcarbamate) covalently bonded to the silica gel surface as the packing material. The mobile phase A is a 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.0±0.5 with phosphoric acid. The mobile phase B is acetonitrile. The mobile phases A and B are eluted isocratically at a volume ratio of (20-30):(80-70). Preferably, mobile phases A and B are eluted isocratically at a volume ratio of (23-27):(77-73).
2. The detection method as described in claim 1, characterized in that, The chromatographic column was a CHIRALPAK IG column with dimensions of 250 mm x 4.6 mm and 5 μm.
3. The detection method as described in claim 1 or 2, characterized in that, The mobile phase A is a 0.05 mol / L potassium dihydrogen phosphate solution, and the pH is adjusted to 2.0 ± 0.2 with phosphoric acid.
4. The detection method according to any one of claims 1 to 3, characterized in that, The flow rate of the mobile phase is 0.3 to 0.5 ml / min; preferably, the flow rate is 0.35 ml / min, 0.4 ml / min or 0.45 ml / min.
5. The detection method according to any one of claims 1 to 4, characterized in that, The test solution of the detection method is a solution containing morpholinidazole at a concentration of 0.1 mg / ml, and the injection volume is 5 μl to 10 μl; preferably, the injection volume is 5 μl or 10 μl.
6. The detection method according to any one of claims 1 to 5, characterized in that, The detection method employs an ultraviolet detector or a diode array detector; preferably, an ultraviolet detector is used, with a detection wavelength of 319 nm.
7. The detection method according to any one of claims 1 to 6, characterized in that, The column temperature of the detection method is 30℃~45℃; preferably, the column temperature is 35℃, 38℃, 40℃ or 42℃.
8. The detection method according to any one of claims 1 to 7, characterized in that, The solvent used in the detection method for preparing the test solution is an aqueous solution of acetonitrile; preferably, the solvent used for preparing the test solution is 60% to 75% acetonitrile; more preferably, the solvent used for preparing the test solution is 60% acetonitrile or 75% acetonitrile.
9. The detection method according to any one of claims 1 to 8, characterized in that, Includes the following steps: Step 1, prepare system suitability solution: Take morpholine nitroazole reference standard, dissolve it in solvent and prepare a solution containing morpholine nitroazole at a concentration of 0.1 mg / ml; Step 2, prepare the test solution: Take the morpholine nitroazole test sample, dissolve it in a solvent and prepare a solution containing morpholine nitroazole at a concentration of 0.1 mg / ml; Step 3: Accurately measure the system suitability solution and the test solution, inject them into the liquid chromatograph, detect them under chromatographic conditions, and record the chromatograms; In step 1 and step 2, the solvent is an aqueous solution of acetonitrile, preferably 60% to 75% acetonitrile; more preferably 75% acetonitrile.
10. The detection method according to any one of claims 1 to 9, characterized in that, The structural formula of the morpholine nitroazole isomer is as follows: