Method for determining impurity content in OAB-14 by high performance liquid chromatography

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for determining the content of impurities in OAB-14 by using high performance liquid chromatography. The method comprises the following steps: preparing a test sample labeling solution, a test sample solution and a contrast solution of OAB-14, respectively injecting the test sample labeling solution, the blank solution, the contrast solution and the test sample solution into a liquid chromatograph, recording chromatograms, and calculating according to a main component self-contrast method to obtain the content of impurities in the test sample solution; wherein the impurities are one or more of an impurity A, an impurity B, an impurity C, an impurity D, an impurity E or an impurity F. According to the method, the impurities in the OAB-14 can be qualitatively and quantitatively measured at the same time, the stability and the reproducibility are good, the separation degree between a main peak and each impurity peak is good, the precision is high, and an analysis means is provided for quality control of the OAB-14.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and particularly relates to a method for determining impurity content in OAB-14 by using high performance liquid chromatography. BACKGROUND

[0002] OAB-14, the chemical name of which is 4-(3-(2-aminoethyl)urea)-N-(1,1,4,4,5,5,8,8-octamethyl-1,2,3,4,5,6,7,8-octahydroanthracen-9-yl)benzamide, has a molecular formula of C 32 H 46 N4O2, and a molecular weight of 518.75, and has a structural formula of .

[0003] OAB-14 is mainly used for treating Alzheimer's disease (AD).

[0004] Through previous research, it is found that OAB-14 can significantly improve various behavioral disorders of APP / PS1 double transgenic AD model mice in learning and memory and social activities. Compared with existing Alzheimer's disease treatment drugs, the action target and mechanism of OAB-14 are completely different from those of most anti-Alzheimer's disease drugs that have failed in clinical trials. The anti-Alzheimer's disease mechanism of OAB-14 is complex and diverse, and is not only related to the clearance of brain β-amyloid (Aβ) deposition, but also involves multiple targets such as central anti-inflammatory, antioxidant and inhibition of neuronal apoptosis. Specifically, OAB-14 can significantly reduce the deposition of β-amyloid in the brain, reduce the overphosphorylation of Tau protein, and protect the structure and function of cerebral cortex and hippocampal neurons and their synapses. These characteristics make OAB-14 have significant advantages and potential in the treatment of Alzheimer's disease.

[0005] During the synthesis and preparation of OAB-14, various process impurities exist, which significantly affect the quality of the product, and therefore the quality control of OAB-14 is particularly important. SUMMARY

[0006] The purpose of the present application is to provide a method for determining the impurity content in OAB-14 by using high performance liquid chromatography, which can simultaneously qualitatively and quantitatively determine the impurities in OAB-14, has good stability and reproducibility, good separation degree between the main peak and each impurity peak, and high precision, thereby providing an analysis means for the quality control of OAB-14.

[0007] The method for determining the impurity content in OAB-14 by using high performance liquid chromatography is that an OAB-14 sample is weighed, impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution are added to the sample, and then the sample is diluted with a solvent to prepare a sample solution; the sample solution, a blank solution, a control solution and the sample solution are injected into a liquid chromatograph, and a chromatogram is recorded; and the content of impurities in the sample solution is calculated according to a main component self-control method; wherein the impurities are one or more of impurity A, impurity B, impurity C, impurity D, impurity E or impurity F.

[0008] The structural formula of impurity A is as follows:

[0009] The structural formula of impurity B is as follows:

[0010] The structural formula of impurity C is as follows:

[0011] The structural formula of impurity D is as follows:

[0012] The structural formula of impurity E is as follows:

[0013] The structural formula of impurity F is as follows: .

[0014] The preparation method of the sample solution is that OAB-14 samples are weighed, impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution are mixed, and then diluted with a solvent to prepare a sample solution; the concentration of OAB-14 in the sample solution is 0.8-1.2 mg / ml, and the concentration of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F is all 4.0-6.0 μg / ml; The preparation method of the impurity A stock solution is that impurity A control samples are weighed, dissolved and diluted with a solvent to prepare an impurity A stock solution; the concentration of impurity A in the impurity A stock solution is 80-120 μg / ml; The preparation method of the impurity B stock solution is that impurity B control samples are weighed, dissolved and diluted with a solvent to prepare an impurity B stock solution; the concentration of impurity B in the impurity B stock solution is 80-120 μg / ml; The preparation method of the impurity C stock solution is that impurity C control samples are weighed, dissolved and diluted with a solvent to prepare an impurity C stock solution; the concentration of impurity C in the impurity C stock solution is 80-120 μg / ml; The preparation method of the impurity D stock solution is to weigh the impurity D reference substance, dissolve and dilute it with the solvent to prepare the impurity D stock solution; the concentration of impurity D in the impurity D stock solution is 80-120 μg / ml. The preparation method of the impurity E stock solution is to weigh the impurity E reference substance, dissolve and dilute it with the solvent to prepare the impurity E stock solution; the concentration of impurity E in the impurity E stock solution is 80-120 μg / ml. The preparation method of the impurity F stock solution is to weigh the impurity F reference substance, dissolve it with trifluoroacetic acid and dilute it with methanol to prepare the impurity F stock solution; the ratio of the impurity F reference substance to trifluoroacetic acid is 2:300-500, the impurity F reference substance is in mg, and trifluoroacetic acid is in μl; the concentration of impurity F in the impurity F stock solution is 80-120 μg / ml.

[0015] The preparation method of the test sample solution is to weigh the OAB-14 sample, dissolve and dilute it with the solvent to prepare the test sample solution; the concentration of OAB-14 in the test sample solution is 0.8-1.2 mg / ml.

[0016] The preparation method of the control solution is to accurately weigh the test sample solution, dilute it with the solvent to prepare the control solution; the concentration of OAB-14 in the control solution is 4.0-6.0 μg / ml.

[0017] The solvent is a mixed solution of trifluoroacetic acid and methanol (trifluoroacetic acid-methanol solution), and the volume concentration of trifluoroacetic acid is 0.08-0.12%.

[0018] The blank solution is a mixed solution of trifluoroacetic acid and methanol (trifluoroacetic acid-methanol solution), and the volume concentration of trifluoroacetic acid is 0.08-0.12%.

[0019] The chromatographic conditions of high performance liquid chromatography include a chromatographic column, the chromatographic column uses silica gel with alkyl amide groups covalently bound and capped as the filler, the specification of the chromatographic column is: column length is 150-250 mm, column inner diameter is 4.0-4.6 mm, and filler particle size is 3.5-5 μm, and the column temperature of the chromatographic column is 23-27℃.

[0020] The chromatographic conditions also include a mobile phase, the mobile phase is a trifluoroacetic acid aqueous solution with a volume concentration of 0.12-0.18% as mobile phase A and methanol as mobile phase B; the total flow rate of mobile phase A and mobile phase B is 0.95-1.05 ml / min.

[0021] The chromatographic conditions further include a gradient elution procedure as follows: 0-10 minutes, mobile phase A accounts for 28-32% of the total volume of the mobile phase, and mobile phase B accounts for 68-72% of the total volume of the mobile phase; 10-45 minutes, mobile phase A accounts for 23-27% of the total volume of the mobile phase, and mobile phase B accounts for 73-77% of the total volume of the mobile phase; 45-60 minutes, mobile phase A accounts for 8-12% of the total volume of the mobile phase, and mobile phase B accounts for 88-92% of the total volume of the mobile phase; 60-80 minutes, mobile phase A accounts for 8-12% of the total volume of the mobile phase, and mobile phase B accounts for 88-92% of the total volume of the mobile phase; 80-80.1 minutes, mobile phase A accounts for 28-32% of the total volume of the mobile phase, and mobile phase B accounts for 68-72% of the total volume of the mobile phase; 80.1-90 minutes, mobile phase A accounts for 28-32% of the total volume of the mobile phase, and mobile phase B accounts for 68-72% of the total volume of the mobile phase.

[0022] The chromatographic conditions further include a detector, a detection wavelength and an injection volume, the detector is an ultraviolet detector, the detection wavelength is 270-274 nm, and the injection volume is 10 μl.

[0023] The present application has the following advantages: (1) The present application can simultaneously qualitatively and quantitatively determine six impurities in OAB-14, and is convenient for effectively controlling the quality of the product OAB-14.

[0024] (2) The present application has good stability and reproducibility, good separation degree between the main peak and each impurity peak, and high precision. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a nuclear magnetic hydrogen spectrum of OAB-14.

[0026] Figure 2 It is a nuclear magnetic carbon spectrum of OAB-14.

[0027] Figure 3 It is a nuclear magnetic hydrogen spectrum of impurity A.

[0028] Figure 4 It is a nuclear magnetic carbon spectrum of impurity A.

[0029] Figure 5 It is a nuclear magnetic hydrogen spectrum of impurity B.

[0030] Figure 6 It is a nuclear magnetic carbon spectrum of impurity B.

[0031] Figure 7 It is a nuclear magnetic hydrogen spectrum of impurity C.

[0032] Figure 8 It is a nuclear magnetic carbon spectrum of impurity C.

[0033] Figure 9NMR spectrum of impurity D.

[0034] Figure 10 NMR spectrum of impurity D.

[0035] Figure 11 NMR spectrum of impurity E.

[0036] Figure 12 NMR spectrum of impurity E.

[0037] Figure 13 NMR spectrum of impurity F.

[0038] Figure 14 NMR spectrum of impurity F.

[0039] Figure 15 HPLC chromatogram of spiked sample solution in Example 1.

[0040] Figure 16 HPLC chromatogram of spiked sample solution in Example 1.

[0041] Figure 17 HPLC chromatogram of spiked sample solution in Example 1.

[0042] Figure 18 HPLC chromatogram of spiked sample solution in Example 1.

[0043] Figure 19 HPLC chromatogram of spiked sample solution in Example 1.

[0044] Figure 20 HPLC chromatogram of spiked sample solution in Example 1.

[0045] Figure 21 HPLC chromatogram of spiked sample solution in Example 1.

[0046] Figure 22 HPLC chromatogram of spiked sample solution in Example 1.

[0047] Figure 23 HPLC chromatogram of spiked sample solution in Example 1.

[0048] Figure 24 HPLC chromatogram of spiked sample solution in Example 1.

[0049] Figure 25 This is the high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness test ⑥.

[0050] Figure 26 This is the high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness test ⑦.

[0051] Figure 27 This is the high-performance liquid chromatogram of the spiked solution of the test sample in Method Robustness Test ⑧.

[0052] Figure 28 This is a high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness test ⑨.

[0053] Figure 29 This is the high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness test ⑩.

[0054] Figure 30 For method durability testing High performance liquid chromatogram of the spiked solution of the test sample.

[0055] Figure 31 The image shows the high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness comparison experiment ①.

[0056] Figure 32 This is the high-performance liquid chromatogram of the spiked solution of the test sample in the method robustness comparison experiment ②.

[0057] Figure 33 This is a high-performance liquid chromatogram of the test solution in Example 2.

[0058] Figure 34 This is a high-performance liquid chromatogram of the test solution in Example 3. Detailed Implementation

[0059] The present invention will be further described below with reference to embodiments.

[0060] All instruments and reagents used in the following examples are commercially available products. Methanol: Beijing Bailingwei Technology Co., Ltd., HPLC grade; Trifluoroacetic acid: Shanghai Maclean Biochemical Technology Co., Ltd., HPLC grade.

[0061] Example 1 (1) Solution preparation: A 0.1% (v / v) trifluoroacetic acid methanol solution was used as a blank solution. A 0.1% (v / v) trifluoroacetic acid methanol solution was used as the solvent; Weigh out the OAB-14 sample, dissolve and dilute it with a solvent to prepare a solution containing 1 mg per 1 ml, which is used as the test solution; Accurately measure 1.0 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well. Then accurately measure 1.0 ml from the 10 ml solution and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well. This is the control solution. The structural formulas of impurities A, B, C, D, E, and F are shown in Table 1.

[0062] Table 1. Structural formulas of impurities A, B, C, D, E, and F.

[0063] The 1H and 1C NMR spectra of OAB-14 are shown below. Figure 1 and Figure 2 The 1H NMR spectrum and 1C NMR spectrum of impurity A are shown below. Figure 3 and Figure 4 The 1H NMR spectrum and 1C NMR spectrum of impurity B are shown below. Figure 5 and Figure 6 The 1H NMR spectrum and 1C NMR spectrum of impurity C are shown below. Figure 7 and Figure 8 The 1H NMR spectrum and 1C NMR spectrum of impurity D are shown below. Figure 9 and Figure 10 The 1H and 1C NMR spectra of impurity E are shown below. Figure 11 and Figure 12 The 1H and 1C NMR spectra of impurity F are shown below. Figure 13 and Figure 14 .

[0064] Weigh impurity A reference standard, dissolve and dilute it with a solvent to prepare a solution containing 100 μg of impurity A per 1 ml, which is used as the impurity A stock solution. Weigh out the reference standard of impurity B, dissolve and dilute it with a solvent to prepare a solution containing 100 μg of impurity B per 1 ml, and use it as the stock solution of impurity B. Weigh out the reference standard of impurity C, dissolve and dilute it with a solvent to prepare a solution containing 100 μg of impurity C per 1 ml, which is used as the stock solution of impurity C. Weigh out the reference standard of impurity D, dissolve and dilute it with a solvent to prepare a solution containing 100 μg of impurity D per 1 ml, which is used as the stock solution of impurity D. Weigh out the reference standard of impurity E, dissolve and dilute it with a solvent to prepare a solution containing 100 μg of impurity E per 1 ml, which is used as the stock solution of impurity E. Weigh impurity F reference standard, dissolve it in trifluoroacetic acid and dilute it with methanol to prepare a solution containing 100 μg of impurity F per ml, which is used as impurity F stock solution; the ratio of impurity F reference standard to trifluoroacetic acid is 2:400, and impurity F reference standard is expressed in mg, and trifluoroacetic acid is expressed in μl; Accurately measure 1.0 ml of impurity A stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity A positioning solution. Accurately measure 1.0 ml of impurity B stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity B positioning solution. Accurately measure 1.0 ml of impurity C stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity C positioning solution. Accurately measure 1.0 ml of impurity D stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity D positioning solution. Accurately measure 1.0 ml of impurity E stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity E positioning solution. Accurately measure 1.0 ml of impurity F stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the impurity F positioning solution. Weigh 20 mg of OAB-14 sample and place it in a 20 ml volumetric flask. Add 1.0 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution. Dissolve and dilute to the mark with solvent, shake well, and use as the spiking solution for the test sample. (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT liquid chromatograph with UV detector; Column: Agilent Bonus-RP, 4.6×250mm, 5μm; Detection wavelength: 272nm; Mobile phase A: 0.15% (v / v) aqueous solution of trifluoroacetic acid; Mobile phase B: Methanol; Flow rate: 1.0 ml / min; Column temperature: 25℃; Injection volume: 10 μl; The gradient elution procedure is shown in Table 2; Table 2 Gradient elution procedure for Example 1

[0065] (3) Determination method: Take 10 μl each of the above blank solution, control solution, test solution, impurity positioning solution and test spiked solution, inject them into the liquid chromatograph, record the chromatograms, and the results are shown in the figure. Figures 15-19 As shown in Table 3-4, the content of impurities in the test solution was calculated using the principal component self-comparison method.

[0066] Depend on Figure 15 and Figure 16As can be seen, OAB-14, impurity A, impurity C, impurity D, impurity E, impurity B, and impurity F eluted in sequence, and the peak area data are shown in Table 3. The resolution between each chromatographic peak is greater than 1.5, indicating that the method of this invention has good specificity.

[0067] Depend on Figure 17 It can be seen that the blank solution does not cause any interference at any peak.

[0068] Depend on Figure 18 It can be seen that the test solution contains impurities A, C, D, E and F, and the peak area data are shown in Table 4.

[0069] Table 3 Chromatographic results of the spiked solution of the test sample

[0070] Table 4 Chromatographic results of the test solution

[0071] The methodology validation process is as follows: 1. Limits of Quantitation and Limits of Detection: Accurately weigh 4 mg of OAB-14 reference standard, dissolve and dilute to 20 ml in solvent, and shake well to prepare the OAB-14 stock solution. Accurately measure the stock solutions of each impurity and the OAB-14 stock solution, and serially dilute to different concentrations. Accurately measure 10 μl of each solution and inject it into the liquid chromatograph. The limits of quantitation and detection for each impurity are determined when the measured peak response values ​​are approximately 10 times and 3 times higher than the noise signal, respectively. The results are shown in Table 5.

[0072] Table 5. Experimental results of limit of quantitation and limit of detection

[0073] 2. Linearity: Take the stock solutions of impurity A, B, C, D, E, F, and OAB-14, and dilute them stepwise with solvent to the limit of quantitation (LOQ) to prepare linearity test solutions ①; Take 0.2 ml each of the stock solutions of impurity A, B, C, D, E, F, and OAB-14, and place them in a 20 ml volumetric flask, dilute to the mark with solvent, and mix well to prepare linearity test solutions ②; Take 0.5 ml each of the stock solutions of impurity A, B, C, D, E, F, and OAB-14, and place them in a 20 ml volumetric flask, and dilute to the mark with solvent. Take 1.0 ml each of the stock solutions of impurity A, B, C, D, E, F, and OAB-14, and place them in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well to prepare the linearity test solution ④. Take 2.0 ml each of the stock solutions of impurity A, B, C, D, E, F, and OAB-14, and place them in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well to prepare the linearity test solution ⑤. Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph. Plot a curve between peak area and concentration, calculate the regression equation and correlation coefficient, and the results are shown in Table 6.

[0074] Table 6. Results of linear experiments

[0075] 3. Repeatability: Prepare a control solution and 6 spiked solutions of the test sample for determination. The repeatability test results are shown in Table 7.

[0076] Table 7 Results of Repeatability Experiments

[0077] In the control solution, the RSD of the peak area of ​​OAB-14 chromatographic peak was less than 5.0%; in the spiked solution of the test sample, the range of the results for each impurity content was less than 0.1%, and the range of the results for the total impurity content was less than 0.2%.

[0078] 4. Intermediate precision: The control solution and 6 spiked solutions of the test sample were prepared again on different dates than the repeatability test and tested. The results of the intermediate precision test are shown in Table 8.

[0079] Table 8 Results of intermediate precision experiments

[0080] In the control solution, the RSD of the peak area of ​​OAB-14 chromatographic peak was less than 5.0%; in the spiked solution of the test sample, the range of the results for each impurity content was less than 0.1%, and the range of the results for intermediate precision and repeatability (a total of 12 tests) was less than 0.1%; the range of the results for total impurity content was less than 0.2%, and the range of the results for intermediate precision and repeatability (a total of 12 tests) was less than 0.2%.

[0081] 5. Accuracy: Take 1.0 ml each of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F stock solutions and place them in a 20 ml volumetric flask. Dilute to the mark with solvent and mix well to prepare the impurity reference solution. Weigh approximately 20 mg of OAB-14 test sample and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with solvent and mix well to prepare the test solution.

[0082] Weigh approximately 20 mg of OAB-14 test sample and add 0.2 ml each of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F stock solutions to a 20 ml volumetric flask. Dissolve and dilute to the mark with solvent, shake well, and prepare three parallel aliquots as accuracy test solution 1. Weigh approximately 20 mg of OAB-14 test sample and add 1.0 ml each of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F stock solutions to a 20 ml volumetric flask. Dissolve and dilute to the mark with solvent, shake well, and prepare three parallel aliquots as accuracy test solution 2. Weigh approximately 20 mg of OAB-14 test sample and add 1.2 ml each of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F stock solutions to a 20 ml volumetric flask. Dissolve and dilute to the mark with solvent, shake well, and prepare three parallel aliquots as accuracy test solutions 3. Inject the above impurity reference solutions, test sample solutions, and each accuracy test solution according to the chromatographic conditions of Example 1, record the chromatograms, and ensure that the peak area RSD of the impurity reference solution is less than 5.0% after five injections. The recovery rates of each accuracy test solution are between 80% and 120%. The results are shown in Tables 9-14.

[0083] Table 9. Accuracy test results for impurity A

[0084] Table 10. Accuracy Experiment Results of Impurity B

[0085] Table 11. Experimental Results of Impurity C Accuracy

[0086] Table 12 Experimental Results of Impurity D Accuracy

[0087] Table 13. Experimental Results of Impurity E Accuracy

[0088] Table 14. Experimental Results of Impurity F Accuracy

[0089] 6. Solution Stability: The control solution and the spiked test solution were measured at 0, 24, 48, 72, 96, and 120 hours. If the RSD of the average peak area of ​​OAB-14 in the control solution was less than 5.0%, and the range of known impurity content in the spiked test solution was less than 0.1%, and the range of total impurity content was less than 0.2%, then the control solution and the spiked test solution were considered stable within 120 hours. The results of the solution stability test are shown in Tables 15-16, indicating that the control solution and the spiked test solution were stable within 120 hours.

[0090] Table 15 Results of stability test of control solution

[0091] Table 16 Results of the stability test of the spiked solution of the test sample

[0092] 7. Method durability: Method durability test ① (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Detection wavelength: 274 nm; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 20 See Tables 17-18; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0093] Table 17 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ①

[0094] Table 18 Experimental results of method durability test ①

[0095] Method durability test ② (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Detection wavelength: 270 nm; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 21 See Tables 19-20; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0096] Table 19 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ②

[0097] Table 20 Experimental results of method durability test ②

[0098] Method durability test ③ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Column temperature: 27℃; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 22 See Tables 21-22; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0099] Table 21 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ③

[0100] Table 22 Experimental results of method durability test ③

[0101] Method durability test ④ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Column temperature: 23℃; other chromatographic conditions are the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 23 See Tables 23-24; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0102] Table 23 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ④

[0103] Table 24 Results of Method Durability Test ④

[0104] Method durability test ⑤ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Flow rate: 1.05 ml / min; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 24 See Tables 25-26; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0105] Table 25 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑤

[0106] Table 26 Experimental results of method durability test ⑤

[0107] Method durability test ⑥ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Flow rate: 0.95 ml / min; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 25See Tables 27-28; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0108] Table 27 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑥

[0109] Table 28 Experimental results of method durability test ⑥

[0110] Method durability test ⑦ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Mobile phase A: 0.18% trifluoroacetic acid aqueous solution; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 26 See Tables 29-30; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0111] Table 29 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑦

[0112] Table 30 Experimental results of method durability test ⑦

[0113] Method durability test ⑧ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Mobile phase A: 0.12% trifluoroacetic acid aqueous solution; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 27 See Tables 31-32; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0114] Table 31 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑧

[0115] Table 32 Experimental results of method durability test ⑧

[0116] Method durability test ⑨ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: The gradient elution procedure is shown in Table 33; other chromatographic conditions are the same as in Example 1. Table 33 Gradient elution procedure in method robustness test ⑨

[0117] (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 28 See Tables 34-35; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0118] Table 34 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑨

[0119] Table 35 Experimental results of method durability test ⑨

[0120] Method durability test ⑩ (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions:

[0121] The gradient elution procedure is shown in Table 36; other chromatographic conditions are the same as in Example 1. Table 36 Gradient elution procedure in method robustness test ⑩

[0122] (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 29See Tables 37-38; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0123] Table 37 Chromatographic results of the spiked solution of the test sample in Method Robustness Test ⑩

[0124] Table 38 Experimental Results of Method Durability Test ⑩

[0125] Method durability test

[0126] (1) Solution preparation: The preparation methods for blank solution, control solution and spiked solution of test sample are the same as in Example 1; (2) Chromatographic conditions: Chromatographic column: Agilent Bonus-RP, 4.6 × 150 mm, 3.5 μm; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl each of blank solution, control solution and spiked solution of test sample, inject them into the liquid chromatograph, record the chromatograms, and see the results of the method robustness test. Figure 30 See Tables 39-40; the results show that the resolution between OAB-14 and each impurity in the chromatogram of the spiked solution of the test sample is greater than 1.5, indicating that the method has good robustness.

[0127] Table 39 Method Durability Tests Chromatographic results of the spiked solution of the test sample

[0128] Table 40 Method Durability Tests Experimental results

[0129] Method durability comparison experiment ① (1) Solution preparation: The preparation method of the spiked solution for the test sample is the same as in Example 1; (2) Chromatographic conditions: Chromatographic column: Welch Xtimate C18, 4.6 × 150 mm, 5 μm; other chromatographic conditions were the same as in Example 1; (3) Determination method: Accurately measure 10 μl of the spiked solution of the test sample and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in the figure. Figure 31According to Table 41, under these experimental conditions, the resolution of impurity C and impurity D in the chromatogram of the spiked solution of the test sample can only reach 0.8, which does not achieve complete separation of the chromatographic peaks and is not suitable for quantitative analysis, indicating that the C18 column is not applicable.

[0130] Table 41 Chromatographic results of the spiked solution of the test sample in the method robustness comparison experiment ①

[0131] Method durability comparison experiment ② (1) Solution preparation: The preparation method of the spiked solution for the test sample is the same as in Example 1; (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT liquid chromatograph with UV detector; Column: Agilent Bonus-RP, 4.6×250mm, 5μm; Detection wavelength: 272nm; Mobile phase: 0.15% trifluoroacetic acid aqueous solution - methanol (25:75); Flow rate: 1.0 ml / min; Column temperature: 25℃; Injection volume: 10 μl; The elution process uses isocratic elution of the mobile phase; (3) Determination method: Accurately measure 10 μl of the spiked solution of the test sample and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in the figure. Figure 32 According to Table 42, under these experimental conditions, the main peak of the spiked solution of the test sample was not completely separated from the impurities preceding the main peak. Furthermore, the retention time of impurity B was approximately 58 minutes, and impurity F did not elute within 180 minutes of the run time. The elution times of impurities B and F were too late to be suitable for quantitative analysis, indicating that the isocratic elution method of the mobile phase is not applicable.

[0132] Table 42 Chromatographic results of the spiked solution of the test sample in Method Robustness Comparison Experiment ②

[0133] Example 2 (1) Solution preparation: A 0.12% (v / v) trifluoroacetic acid methanol solution was used as a blank solution. A 0.12% (v / v) trifluoroacetic acid methanol solution was used as the solvent. Weigh out the OAB-14 sample, dissolve and dilute it with a solvent to prepare a solution containing 1.2 mg per ml, which will be used as the test solution. Accurately measure 1.0 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well. Then accurately measure 1.0 ml from the 10 ml solution and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well. This is the control solution. Weigh impurity A reference standard, dissolve and dilute it with a solvent to prepare a solution containing 120 μg of impurity A per 1 ml, which is used as the impurity A stock solution. Weigh out the reference standard of impurity B, dissolve and dilute it with a solvent to prepare a solution containing 120 μg of impurity B per 1 ml, which is used as the stock solution of impurity B. Weigh the reference standard of impurity C, dissolve and dilute it with a solvent to prepare a solution containing 120 μg of impurity C per 1 ml, which is used as the stock solution of impurity C. Weigh out the reference standard of impurity D, dissolve and dilute it with a solvent to prepare a solution containing 120 μg of impurity D per 1 ml, which is used as the stock solution of impurity D. Weigh the reference standard of impurity E, dissolve and dilute it with a solvent to prepare a solution containing 120 μg of impurity E per 1 ml, which is used as the stock solution of impurity E. Weigh impurity F reference standard, dissolve it in trifluoroacetic acid and dilute it with methanol to prepare a solution containing 120 μg of impurity F per ml, which is used as impurity F stock solution; the ratio of impurity F reference standard to trifluoroacetic acid is 2:500, and impurity F reference standard is expressed in mg, and trifluoroacetic acid is expressed in μl; Weigh 24 mg of OAB-14 sample and place it in a 20 ml volumetric flask. Add 1.0 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution. Dissolve and dilute to the mark with solvent, shake well, and use as the spiking solution for the test sample. (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT liquid chromatograph with UV detector; Column: Agilent Bonus-RP, 4.6×250mm, 5μm; Detection wavelength: 274nm; Mobile phase A: 0.18% trifluoroacetic acid aqueous solution (by volume); Mobile phase B: Methanol; Flow rate: 1.05 ml / min; Column temperature: 27℃; Injection volume: 10 μl; The gradient elution procedure is shown in Table 43; Table 43 Gradient elution procedure for Example 2

[0134] (3) Determination method: Take 10 μl each of the above-mentioned spiked solution, blank solution, control solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity in the test solution according to the main component self-comparison method. The experimental results are shown in the figure. Figure 33 And Table 44.

[0135] Table 44 Experimental results of the test solution in Example 2

[0136] Example 3 (1) Solution preparation: A 0.08% (v / v) trifluoroacetic acid methanol solution was used as a blank solution. A 0.08% (v / v) trifluoroacetic acid methanol solution was used as the solvent. Weigh out the OAB-14 sample, dissolve and dilute it with a solvent to prepare a solution containing 0.8 mg per 1 ml, which will be used as the test solution. Accurately measure 1.0 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well. Then accurately measure 1.0 ml from the 10 ml solution and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well. This is the control solution. Weigh impurity A reference standard, dissolve and dilute it with a solvent to prepare a solution containing 80 μg of impurity A per 1 ml, which is used as the impurity A stock solution. Weigh out the reference standard of impurity B, dissolve and dilute it with a solvent to prepare a solution containing 80 μg of impurity B per 1 ml, which is used as the stock solution of impurity B. Weigh the reference standard of impurity C, dissolve and dilute it with a solvent to prepare a solution containing 80 μg of impurity C per 1 ml, which is used as the stock solution of impurity C. Weigh out the reference standard of impurity D, dissolve and dilute it with a solvent to prepare a solution containing 80 μg of impurity D per 1 ml, which is used as the stock solution of impurity D. Weigh the reference standard of impurity E, dissolve and dilute it with a solvent to prepare a solution containing 80 μg of impurity E per 1 ml, which is used as the stock solution of impurity E. Weigh impurity F reference standard, dissolve it in trifluoroacetic acid and dilute it with methanol to prepare a solution containing 80 μg of impurity F per 1 ml, which is used as impurity F stock solution; the ratio of impurity F reference standard to trifluoroacetic acid is 2:300, and impurity F reference standard is expressed in mg, and trifluoroacetic acid is expressed in μl; Weigh 16 mg of OAB-14 sample and place it in a 20 ml volumetric flask. Add 1.0 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution. Dissolve and dilute to the mark with solvent, shake well, and use as the spiking solution for the test sample. (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT liquid chromatograph with UV detector; Column: Agilent Bonus-RP, 4.6×250mm, 5μm; Detection wavelength: 270nm; Mobile phase A: A 0.12% (v / v) aqueous solution of trifluoroacetic acid; Mobile phase B: Methanol; Flow rate: 0.95 ml / min; Column temperature: 23℃; Injection volume: 10 μl; The gradient elution procedure is shown in Table 45; Table 45 Gradient elution procedure for Example 3

[0137] (3) Determination method: Take 10 μl each of the above-mentioned spiked solution, blank solution, control solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity in the test solution according to the main component self-comparison method. The experimental results are shown in the figure. Figure 34 And Table 46.

[0138] Table 46 Experimental results of the test solution in Example 2

Claims

1. A method for determining the content of impurities in OAB-14 using high performance liquid chromatography, characterized by The test sample spiked solution, test sample solution and control solution of OAB-14 are prepared, the test sample spiked solution, blank solution, control solution and test sample solution are respectively injected into a liquid chromatograph, a chromatogram is recorded, and the content of impurities in the test sample solution is calculated according to the self-control method of main components; wherein the impurities are one or more of impurity A, impurity B, impurity C, impurity D, impurity E or impurity F; The structural formula of impurity A is as follows: The structural formula of impurity B is as follows: The structural formula of impurity C is as follows: The structural formula of impurity D is as follows: The structural formula of impurity E is as follows: The structural formula of impurity F is as follows: 。 2. A method for determining the content of impurities in OAB-14 using high performance liquid chromatography as claimed in claim 1, wherein The preparation method of the test sample spiked solution is that OAB-14 samples are weighed, impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution and impurity F stock solution are mixed and then diluted with a solvent to prepare the test sample spiked solution; The concentration of OAB-14 in the test sample spiked solution is 0.8-1.2 mg / ml, and the concentrations of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F are all 4.0-6.0 μg / ml; The preparation method of the impurity A stock solution is that impurity A control samples are weighed, dissolved and diluted with a solvent to prepare the impurity A stock solution; the concentration of impurity A in the impurity A stock solution is 80-120 μg / ml; The preparation method of the impurity B stock solution is that impurity B control samples are weighed, dissolved and diluted with a solvent to prepare the impurity B stock solution; the concentration of impurity B in the impurity B stock solution is 80-120 μg / ml; The preparation method of the impurity C stock solution is that impurity C control samples are weighed, dissolved and diluted with a solvent to prepare the impurity C stock solution; the concentration of impurity C in the impurity C stock solution is 80-120 μg / ml; The preparation method of the impurity D stock solution is that impurity D control samples are weighed, dissolved and diluted with a solvent to prepare the impurity D stock solution; the concentration of impurity D in the impurity D stock solution is 80-120 μg / ml; The preparation method of the impurity E stock solution is that impurity E control samples are weighed, dissolved and diluted with a solvent to prepare the impurity E stock solution; the concentration of impurity E in the impurity E stock solution is 80-120 μg / ml; The preparation method of the impurity F stock solution is that impurity F control samples are weighed, dissolved with trifluoroacetic acid and diluted with methanol to prepare the impurity F stock solution; the ratio of impurity F control samples to trifluoroacetic acid is 2:300-500, the impurity F control samples are measured in mg, and the trifluoroacetic acid is measured in μl; the concentration of impurity F in the impurity F stock solution is 80-120 μg / ml.

3. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 1, characterized in that The preparation method of the test sample solution is that OAB-14 samples are weighed, dissolved and diluted with a solvent to prepare the test sample solution; the concentration of OAB-14 in the test sample solution is 0.8-1.2 mg / ml.

4. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 1, characterized in that The preparation method of the control solution is that the test sample solution is accurately measured, diluted with a solvent to prepare the control solution; the concentration of OAB-14 in the control solution is 4.0-6.0 μg / ml.

5. The method for determining the content of impurities in OAB-14 by high performance liquid chromatography according to any one of claims 2-4, characterized in that The solvent is a mixed solution of trifluoroacetic acid and methanol, and the volume concentration of trifluoroacetic acid is 0.08-0.12%.

6. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 1, characterized in that The blank solution is a mixed solution of trifluoroacetic acid and methanol, and the volume concentration of trifluoroacetic acid is 0.08-0.12%.

7. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 1, characterized in that The chromatographic conditions of the high performance liquid chromatography include a chromatographic column, and specifications of the chromatographic column are as follows: a column length of 150-250 mm, a column inner diameter of 4.0-4.6 mm, a filler particle size of 3.5-5 μm, and a column temperature of 23-27 ℃.

8. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 7, characterized in that The chromatographic conditions further include a mobile phase, and the mobile phase is a trifluoroacetic acid aqueous solution with a volume concentration of 0.12-0.18% as mobile phase A and methanol as mobile phase B; and a total flow rate of the mobile phase A and the mobile phase B is 0.95-1.05 ml / min.

9. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 8, characterized in that The chromatographic conditions further include a gradient elution program, and the gradient elution program is as follows: 0-10 minutes, the mobile phase A accounts for 28-32% of a total volume of the mobile phase, and the mobile phase B accounts for 68-72% of the total volume of the mobile phase; 10-45 minutes, the mobile phase A accounts for 23-27% of the total volume of the mobile phase, and the mobile phase B accounts for 73-77% of the total volume of the mobile phase; 45-60 minutes, the mobile phase A accounts for 8-12% of the total volume of the mobile phase, and the mobile phase B accounts for 88-92% of the total volume of the mobile phase; 60-80 minutes, the mobile phase A accounts for 8-12% of the total volume of the mobile phase, and the mobile phase B accounts for 88-92% of the total volume of the mobile phase; 80-80.1 minutes, the mobile phase A accounts for 28-32% of the total volume of the mobile phase, and the mobile phase B accounts for 68-72% of the total volume of the mobile phase; and 80.1-90 minutes, the mobile phase A accounts for 28-32% of the total volume of the mobile phase, and the mobile phase B accounts for 68-72% of the total volume of the mobile phase.

10. The method for determining the content of impurities in OAB-14 using high performance liquid chromatography according to claim 7, characterized in that The chromatographic conditions further include a detector, a detection wavelength, and a sample injection amount, the detector is an ultraviolet detector, the detection wavelength is 270-274 nm, and the sample injection amount is 10 μl.

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