Method for determining position isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile

Abstract

The invention belongs to the technical field of tablet quality detection, and particularly relates to a method for determining a positional isomer in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile. Comprising the following steps: detecting a nitro position isomer and a methoxyl position isomer in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile by adopting a normal phase chromatography, and detecting the nitro position isomer and the methoxyl position isomer in the 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile by adopting a chromatographic column taking amylose-tri (3, 5-dimethylphenyl carbamate) coated silica gel as a filling agent, a mixed solution system of normal hexane-isopropanol-triethylamine is used as a mobile phase, and isocratic elution is carried out. The detection method is high in specificity, high in sensitivity, wide in detection range and good in accuracy, durability and linearity, meanwhile, operation is easy and convenient to implement, nitro position isomers and methoxyl position isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile can be detected at the same time, and the medication safety is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug quality detection, and more particularly to a method for determining nitro positional isomers and methoxy positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile by liquid chromatography. Background Art

[0002] The compound 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile (CAS No. 675126-26-8) is an important intermediate in the synthesis of some tinib-type anticancer drugs. During its preparation, nitro and methoxy positional isomers may be generated. Failure to use reliable detection methods to control these isomers may increase the difficulty of synthesizing the final product. The specific structural formula of this compound is as follows:

[0003]

[0004] Currently, there is no reported method for detecting the nitro positional isomers and methoxy positional isomers of this compound. In order to ensure the purity of the final product, it is necessary to develop an analytical method with strong specificity, high sensitivity, good accuracy and durability. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for detecting the content of nitro positional isomers and methoxy positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile. The method has strong specificity, good sensitivity, linearity, accuracy and durability, and can achieve quality control of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile during the production process.

[0006] In order to achieve the above object, the present invention adopts normal phase chromatography for detection, wherein,

[0007] The chromatographic column was filled with amylose-tris (3,5-diphenylcarbamate) coated silica gel and the mobile phase was a mixed solution of n-hexane-isopropanol-triethylamine for isocratic elution.

[0008] The filler of the chromatographic column in the present invention is amylose-tris(3,5-xylylcarbamate)-coated silica gel. The inventors discovered that adding triethylamine to the mobile phase and using the amylose-tris(3,5-xylylcarbamate)-coated silica gel as the filler in the chromatographic column can separate 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, its nitro positional isomers, and its methoxy positional isomers. This is likely due to the fact that triethylamine makes the mobile phase alkaline, improving the peak shapes of the components and thus enhancing the specificity of the method.

[0009] Furthermore, the normal phase chromatography conditions specifically include:

[0010] Instrument: Normal phase high performance liquid chromatography;

[0011] Chromatographic column: Chiral chromatographic column filled with amylose-tris (3,5-diphenylcarbamate) coated silica gel;

[0012] Mobile phase: n-hexane-isopropanol-triethylamine (volume ratio: 85:15:0.1 to 85:15:0.3);

[0013] Detector: PDA or UV detector;

[0014] Flow rate: 0.8-1.2 mL / min;

[0015] Detection wavelength: 245-255nm;

[0016] Column temperature: 30-40°C;

[0017] Injection volume: 10 μL.

[0018] Preferably, the chromatographic column is a CHIRALPAK AD-H chromatographic column,

[0019] 4.6mm×250mm, 5μm.

[0020] Furthermore, the structure of the positional isomer is shown below:

[0021]

[0022] Furthermore, the high performance liquid chromatography detection method of the present invention comprises at least the following steps:

[0023] (1) Prepare the test solution: Take an appropriate amount of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, add solvent and ultrasonically dissolve it, dilute it to the scale with solvent, and shake well;

[0024] (2) Preparation of reference stock solutions: Take an appropriate amount of the nitro positional isomer reference substance, accurately weigh it, add a solvent and ultrasonically dissolve it, dilute it to the mark with the solvent to obtain a nitro positional isomer stock solution; take an appropriate amount of the methoxy positional isomer reference substance, accurately weigh it, add a solvent and ultrasonically dissolve it, dilute it to the mark with the solvent to obtain a methoxy positional isomer stock solution; accurately measure the nitro positional isomer and methoxy positional isomer stock solutions into the same volumetric flask, dilute it to the mark with the solvent, and shake well;

[0025] (3) Prepare the reference solution: Accurately measure the reference stock solution, dilute to the mark with solvent, and shake well;

[0026] (4) Prepare the test sample spike solution: Take an appropriate amount of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, dilute it to the mark with solvent, and shake well;

[0027] (5) Injection: Accurately measure equal amounts of solvent, test sample spiked solution, reference solution, and test sample solution, and inject them into a high performance liquid chromatograph. Record the chromatogram and calculate the contents of nitro positional isomers and methoxy positional isomers based on the peak area using the external standard method.

[0028] Preferably, the solvent is anhydrous ethanol.

[0029] Furthermore, based on the principle of facilitating detection, the concentrations of the test solution and the reference solution are not particularly limited and may be conventional concentrations in the art or other appropriate concentration ranges.

[0030] Preferably, the concentration of the test solution is 1 mg / mL, and the concentrations of the nitro positional isomer and methoxy positional isomer reference solutions are 1 μg / mL, respectively.

[0031] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:

[0032] The present invention combines a mobile phase of n-hexane-anhydrous ethanol-triethylamine with a chromatographic column filled with amylose-tris(3,5-dimethylphenylcarbamate)-coated silica gel to detect nitro and methoxy positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile using normal phase chromatography. This method has strong specificity, high accuracy, good linearity, sensitivity, and durability, and can achieve quality control of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0034] Figure 1 : Blank solvent chromatogram among embodiment 1.

[0035] Figure 2 : Chromatogram of test sample spiked solution in Example 1.

[0036] Figure 3 : Chromatogram of reference substance mixed solution among embodiment 1.

[0037] Figure 4 : Chromatogram of need testing solution among embodiment 1.

[0038] Figure 5 : Linear chromatogram of nitro position isomers in Example 1.

[0039] Figure 6 : Linear chromatogram of methoxy position isomers in Example 1.

[0040] Figure 7 : Chromatogram of the impurity mixed positioning solution in Comparative Example 1.

[0041] Figure 8 : Chromatogram of test solution in Comparative Example 1.

[0042] Figure 9 : Overlay of the impurity mixed positioning solution and the test solution in Comparative Example 2.

[0043] Figure 10 : Overlay of the impurity mixed positioning solution and the test solution in Comparative Example 3.

[0044] Figure 11 : Overlay of the impurity mixed positioning solution and the test solution in Comparative Example 4.

[0045] Figure 12 : Chromatogram of the impurity mixed positioning solution in Comparative Example 5.

[0046] Figure 13 : Chromatogram of the test sample spiked solution in Comparative Example 5.

[0047] Note: The nitro positional isomer is abbreviated as GFN-1K, the methoxy positional isomer is abbreviated as GFN-1J, and the main component is abbreviated as GFN-1. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The following are the materials, reagents, instruments and HPLC chromatographic conditions used in the examples and experimental examples:

[0050] 1. Instruments and HPLC Conditions

[0051] High performance liquid chromatograph: American Waters e2695 liquid chromatograph, UV detector;

[0052] MSA6.6S-0CE type one-millionth balance (Sartorius, Germany); AB265-S type one-hundred-thousandth analytical balance (METTLER TOLEDO, USA).

[0053] Chromatographic conditions: chromatographic column: CHIRALPAKAD-H, 4.6 mm × 250 mm, 5 μm; mobile phase: n-hexane, isopropanol, triethylamine = 85:15:0.1 to 85:15:0.3; flow rate: 0.8 to 1.2 mL / min; column temperature: 30 to 40°C; detection wavelength: 245 to 255 nm; injection volume: 10 μL.

[0054] 2. Materials

[0055] Nitro positional isomer reference standard (source: Guangzhou Jiatu Technology Co., Ltd., batch number: 0113-RG-0072, purity 98.8%);

[0056] Methoxy positional isomer reference substance (source: Guangzhou Jiatu Technology Co., Ltd., batch number: 0115-RG-0002, purity 99.8%);

[0057] 4-Methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile (Source: Anqing Qichuang Pharmaceutical Co., Ltd., Batch No.: 22030401, 22030402, 22030403)

[0058] n-Hexane (Source: Xilong Science, Batch No.: 240805B1)

[0059] Isopropyl alcohol (Source: Xilong Science, Batch No.: 240604A1)

[0060] Triethylamine (Source: Tianjin Damao, Batch No.: 20230701)

[0061] Anhydrous ethanol (Source: Xilong Science, Batch No.: 241216A1)

[0062] Example 1: A method for determining the nitro positional isomers and methoxy positional isomers of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile by liquid chromatography

[0063] Liquid chromatography conditions

[0064] Instrument: Waters e2695 liquid chromatograph, UV detector;

[0065] Chromatographic column: CHIRALPAKAD-H, 4.6 mm × 250 mm, 5 μm;

[0066] Mobile phase: n-hexane-isopropanol:triethylamine = 85:15:0.2;

[0067] Column temperature: 35°C

[0068] Flow rate: 1.0 mL / min;

[0069] Detection wavelength: 250nm;

[0070] Injection volume: 10 μL;

[0071] Solvent: anhydrous ethanol;

[0072] Solution preparation:

[0073] Test solution: Take an appropriate amount of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, add an appropriate amount of anhydrous ethanol and sonicate to dissolve it to make a solution containing about 1 mg per 1 mL, and shake well.

[0074] Nitro positional isomer stock solution: Take about 2 mg of nitro positional isomer reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add anhydrous ethanol and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0075] Methoxy positional isomer stock solution: Take about 2 mg of methoxy positional isomer reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add anhydrous ethanol, ultrasonically dissolve it and dilute it to the scale, and shake well.

[0076] Reference substance stock solution: Accurately measure 2 mL each of the nitro positional isomer and methoxy positional isomer stock solutions, place them in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0077] Impurity reference substance mixed solution: Accurately measure 1 mL of reference substance stock solution, place it in a 10 mL volumetric flask, dilute to the scale with solvent, and shake well.

[0078] Test sample spiked solution: Take approximately 10 mg of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, place it in a 10 mL volumetric flask, add solvent and sonicate to dissolve it, measure 1 mL of the reference stock solution, dilute it to the scale with solvent, and shake well to obtain the solution.

[0079] Test results

[0080] Accurately measure 10 μL each of blank solvent, test sample spiked solution, impurity reference solution, and test sample solution and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the table below:

[0081] Table 1 Test results

[0082]

[0083] Experimental verification

[0084] 1. Exclusivity

[0085] Solvent: anhydrous ethanol;

[0086] GFN-1J stock solution: Take about 2 mg of GFN-1J impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake it well.

[0087] GFN-1K stock solution: Take about 2 mg of GFN-1K impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake it well.

[0088] Reference substance stock solution: Accurately measure 2 mL each of GFN-1J and GFN-1K impurity stock solutions, place in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0089] Reference substance solution: Accurately measure 1 mL of reference substance stock solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0090] Test sample spiked solution: Take about 10 mg of the product, accurately weigh it, place it in a 10 mL volumetric flask, add solvent and sonicate to dissolve it, measure 1 mL of the reference sample stock solution, dilute it to the scale with solvent, shake well, and the solution is ready.

[0091] Test solution: Take about 10 mg of the product, accurately weigh it, place it in a 10 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0092] Accurately measure 10 μL of each of the above solutions, inject them into the high performance liquid chromatograph, and record the chromatogram.

[0093] like Figure 2 As shown in Table 2, in the spiked solution of the test sample, the nitro positional isomers, 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, and the methyl positional isomers can be completely separated.

[0094] Table 2 Specificity test results

[0095]

[0096] The results showed that the blank solvent did not interfere with the detection of the two isomeric impurities; in the spiked solution of the test sample, the separation between the main peak of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile and the two isomeric impurities met the requirements, and accurate quantitative detection of the nitro positional isomers and methoxy positional isomers could be achieved.

[0097] 2. Sensitivity measurement results

[0098] Take appropriate amounts of the stock solutions of the nitro positional isomer and the methoxy positional isomer, dilute them step by step with solvent, and examine the limit of quantification and the limit of detection. The results are shown in the table below:

[0099] Table 3 Sensitivity measurement results

[0100]

[0101] As can be seen from the table, the nitro position isomers and methoxy position isomers have higher sensitivity.

[0102] 3. Linearity and range

[0103] Nitro positional isomer stock solution: Take about 2 mg of nitro positional isomer impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0104] Methoxy positional isomer stock solution: Take approximately 2 mg of methoxy positional isomer impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent to dissolve it and dilute it to the scale, and shake well.

[0105] Linear stock solution: Accurately measure 2 mL each of the nitro positional isomer and methoxy positional isomer impurity stock solutions, place them in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0106] Linear solution 1: quantification limit solution;

[0107] Linear solution 2 (0.05%): Accurately measure 1 mL of the linear stock solution, place it in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0108] Linear solution 3 (0.10%): Accurately measure 1 mL of the linear stock solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0109] Linear solution 4 (0.20%): Accurately measure 2 mL of the linear stock solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0110] Linear solution 5 (0.50%): Accurately measure 5 mL of the linear stock solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0111] Accurately measure 10 μL of each solution and inject them into the high performance liquid chromatograph. Record the chromatogram. The results are shown in the table below:

[0112] Table 4 Linearity results

[0113]

[0114]

[0115] 4. Accuracy and repeatability

[0116] Nitro positional isomer stock solution: Take about 2 mg of nitro positional isomer impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0117] Methoxy positional isomer stock solution: Take approximately 2 mg of methoxy positional isomer impurity reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add solvent to dissolve it and dilute it to the scale, and shake well.

[0118] Reference substance stock solution: Accurately measure 2 mL each of the nitro positional isomer and methoxy positional isomer impurity stock solutions, place them in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0119] Reference solution: Accurately measure 1 mL of reference stock solution, place in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. (Prepare 2 in parallel)

[0120] Test solution: Take about 10 mg of the product, accurately weigh it, place it in a 10 mL volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0121] 50% recovery solution: Accurately weigh approximately 10 mg of the product and place in a 10 mL volumetric flask. Dissolve by sonication with solvent. Accurately measure 0.5 mL of the reference stock solution and dilute to volume with solvent. Shake well. Prepare 3 portions in the same manner.

[0122] 100% recovery solution: Take approximately 10 mg of this product, accurately weigh it, and place it in a 10 mL volumetric flask. Add solvent and sonicate to dissolve it. Accurately measure 1 mL of the reference stock solution, dilute it to the mark with solvent, and shake well. Prepare 6 portions in the same manner (3 portions as accuracy samples and 6 portions as repeatability samples).

[0123] 150% recovery solution: Accurately weigh approximately 10 mg of the product and place in a 10 mL volumetric flask. Dissolve by sonication with solvent. Accurately measure 1.5 mL of the reference stock solution, dilute to volume with solvent, and shake well. Prepare three portions in the same manner.

[0124] Accurately measure 10 μL each of the above-mentioned reference solution, test solution, recovery solution and repeatability solution, inject them into the liquid chromatograph respectively, record the chromatogram, and the results are shown in the table below:

[0125] Table 5 Accuracy results (nitro position isomers)

[0126]

[0127]

[0128] Table 6 Accuracy results (methoxy position isomers)

[0129]

[0130] Table 7 Repeatability test results

[0131] sample Nitro position isomers (%) Methoxy position isomers (%) 100％-1 0.13 0.11 100％-2 0.13 0.10 100％-3 0.13 0.10 100％-4 0.12 0.10 100％-5 0.13 0.10 100％-6 0.13 0.10 average value 0.13 0.1 RSD (%) 2.7 4.5

[0132] Conclusion: (1) This method was used to determine the recovery rates of nitro positional isomers and methoxy positional isomers. Sample solutions of three different concentrations, 50%, 100%, and 150% of the limit concentration, were prepared to determine the recovery rates. The recovery rates of each impurity in the nine solutions were all within the range of 90% to 108%, with RSDs less than 10.0%, which met the requirements and the method had good accuracy.

[0133] (2) Six sample spiked solutions were prepared according to the 100% limit. The detection results of each impurity in the six sample spiked solutions were basically consistent, and the RSD of the detection amount was less than 30%, which met the requirements and the method had good repeatability.

[0134] (4) Solution stability

[0135] The reference sample mixed solution and the test sample solution were taken separately to examine the solution stability at room temperature for 24 hours. The changes in the peak area of ​​each impurity in the reference sample solution and the detected amount of impurities in the test sample solution were examined. The results are shown in Tables 8 and 9. According to the test results, the RSD of the peak area of ​​each impurity in the reference sample solution was less than 10.0% after being placed at room temperature for 24 hours. The detected amounts of impurities GFN-1J and GFN-1K in the test sample solution were basically the same as those at 0 hours after being placed at room temperature for 24 hours, indicating that the reference sample solution and the test sample solution were stable after being placed at room temperature for 24 hours.

[0136] Table 8 Stability test results of reference solution

[0137] Time (h) Nitro positional isomers Methoxy positional isomers 0 22846 45076 2 20831 44412 4 24570 43182 8 21802 43735 12 24525 47166 18 24138 48192 24 25065 46190 mean 23397 45422 RSD% 6.9 4.1

[0138] Table 9 Solution stability test results - test solution (detection amount %)

[0139] Time (h) Nitro position isomer% Methoxy position isomer% 0 0.029 Not detected 2 0.030 Not detected 4 0.032 Not detected 8 0.029 Not detected 12 0.030 Not detected 18 0.029 Not detected 24 0.028 Not detected mean 0.030 Not detected RSD% 4.6 Not detected

[0140] 5. Durability

[0141] The sample test results under robust conditions are shown in Table 10. Under different chromatographic conditions, the average values ​​of nitro isomers and methoxy isomers in the spiked solutions of the test samples were 0.12% and 0.09%, respectively, with RSDs of 3.6% and 4.8%, respectively. This method has good robustness.

[0142] Table 10 Durability results

[0143]

[0144] Comparative Example 1

[0145] The chromatographic conditions are as follows:

[0146] Chromatographic column: CHIRALPAK IC (4.6 mm × 250 mm, 5 μm)

[0147] Mobile phase: n-hexane-isopropanol (50:50)

[0148] Column temperature: 40°C;

[0149] Flow rate: 1.0 mL / min;

[0150] Detection wavelength: 250nm;

[0151] Injection volume: 50 μL;

[0152] Diluent: methanol;

[0153] The solution was prepared as follows:

[0154] Impurity mixed location solution: Take appropriate amount of nitro positional isomer and methoxy positional isomer reference substances, dissolve them in diluent and dilute them to make a solution containing approximately 0.5 mg of each of nitro positional isomer and methoxy positional isomer per 1 mL.

[0155] Test solution: Take an appropriate amount of this product, add diluent, ultrasonically dissolve and dilute to make a solution containing about 1 mg per 1 mL, which is used as the test solution.

[0156] See the results Figure 7 and Figure 8 Under the chromatographic conditions, the impurity peak shape was poor and the main peak appeared late. We planned to increase the proportion of isopropanol in the mobile phase and add 0.2% triethylamine to continue the experiment.

[0157] Comparative Example 2

[0158] The mobile phase was n-hexane-isopropanol (40:60) (containing 0.2% triethylamine)

[0159] The rest of the chromatographic conditions and solution preparation were the same as those in Comparative Example 1. Figure 9 After changing the mobile phase, the peak shape of the impurity improved, but the main peak still eluted late and interfered with the detection of the impurity methoxy position isomers. It is planned to continue to increase the proportion of isopropanol for testing.

[0160] Comparative Example 3

[0161] The mobile phase was n-hexane-isopropanol (30:70) (containing 0.2% triethylamine), and the other chromatographic conditions and solution preparation were the same as those in Comparative Example 1. The results were as follows: Figure 10 As shown in the figure, after increasing the proportion of isopropanol, the separation between the main peak and the impurity methoxy position isomers was not improved, and it was planned to replace the AD-H column to continue the experiment.

[0162] Comparative Example 4

[0163] The chromatographic column was CHIRALPAKAD-H (4.6 mm × 250 mm, 5 μm), the mobile phase was n-hexane-isopropanol (30:70) (containing 0.2% triethylamine), and the other conditions were the same as those in Comparative Example 1. The results were as follows: Figure 11 As shown in the figure, after replacing the AD-H column, the main peak and the impurity peaks are advanced, but the main peak still interferes with the detection of the impurity methoxy position isomer. Using this column, the ratio of isopropanol is reduced to investigate the separation degree between the main peak and the methoxy position isomer.

[0164] Comparative Example 5

[0165] Chromatographic column: CHIRALPAKAD-H (4.6 mm × 250 mm, 5 μm)

[0166] Mobile phase: n-hexane-isopropanol (85:15) (containing 0.2% triethylamine)

[0167] Impurity mixed positioning solution: Take an appropriate amount of the impurity nitro positional isomer and methoxy positional isomer reference substances, dissolve them in diluent and dilute them to make a solution containing approximately 0.1 mg of nitro positional isomer and methoxy positional isomer per 1 mL, which serves as the reference substance solution.

[0168] Spiked test solution: Take appropriate amounts of the product, impurity nitro positional isomers, and methoxy positional isomer reference substances, add diluent, ultrasonically dissolve and dilute to make a solution containing approximately 1 mg of the main component and approximately 0.1 mg of the nitro positional isomers and methoxy positional isomers per 1 mL, which is used as the spiked test solution.

[0169] The other conditions are the same as those in Comparative Example 1. Figure 12 and Figure 13 As shown, after reducing the proportion of isopropanol, the resolution between the main peak and the methoxy position isomers reached 1.3, but the peak shape of the nitro position isomers was poor. It was proposed to replace the diluent with anhydrous ethanol and reduce the injection volume for further investigation. See the examples for details.

[0170] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, characterized in that: Normal phase chromatography was used for detection, The chromatographic column was filled with amylose-tris (3,5-diphenylcarbamate) coated silica gel and the mobile phase was a mixed solution of n-hexane-isopropanol-triethylamine for isocratic elution.

2. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 1, wherein: The normal phase chromatography method at least comprises: Instrument: Normal phase high performance liquid chromatography; Chromatographic column: Chiral chromatographic column filled with amylose-tris (3,5-diphenylcarbamate) coated silica gel; Mobile phase: n-hexane-isopropanol-triethylamine (volume ratio: 85:15:0.1 to 85:15:0.3); Detector: PDA or UV detector; Flow rate: 0.8-1.2 mL / min; Detection wavelength: 245-255nm; Column temperature: 30-40°C; Injection volume: 10 μL.

3. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 2, wherein: The chromatographic column is a CHIRALPAKAD-H chromatographic column, 4.6 mm×250 mm, 5 μm.

4. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 1, wherein: The structures of the positional isomers are shown below:

5. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 4, wherein: The specific steps are as follows: (1) Prepare the test solution: Take an appropriate amount of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, add solvent and ultrasonically dissolve it, dilute it to the scale with solvent, and shake well; (2) Preparation of reference stock solutions: Take an appropriate amount of the nitro positional isomer reference substance, accurately weigh it, add a solvent and ultrasonically dissolve it, dilute it to the mark with the solvent to obtain a nitro positional isomer stock solution; take an appropriate amount of the methoxy positional isomer reference substance, accurately weigh it, add a solvent and ultrasonically dissolve it, dilute it to the mark with the solvent to obtain a methoxy positional isomer stock solution; accurately measure the nitro positional isomer and methoxy positional isomer stock solutions into the same volumetric flask, dilute it to the mark with the solvent, and shake well; (3) Prepare the reference solution: Accurately measure the reference stock solution, dilute to the mark with solvent, and shake well; (4) Prepare the test sample spike solution: Take an appropriate amount of 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile, accurately weigh it, dilute it to the mark with solvent, and shake well; (5) Sampling: Accurately measure equal amounts of solvent, test sample spiked solution, reference solution, and test sample solution, and inject them into a high performance liquid chromatograph, respectively. Detect according to the method described in any one of claims 1 to 3, and record the chromatogram.

6. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 5, wherein: The solvent is anhydrous ethanol.

7. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 5, characterized in that: In principle, for the purpose of facilitating detection, there is no particular limitation on the concentrations of the test solution and the reference solution.

8. The method for determining positional isomers in 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile according to claim 5, wherein: The concentration of the test solution is 1 mg / mL, and the concentrations of the nitro positional isomer and methoxy positional isomer reference solutions are 1 μg / mL, respectively.

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