Method for detecting related substances in revienacin raw material medicine or preparation of revienacin raw material medicine

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting related substances in a revivanacin raw material medicine or a preparation thereof. The detection method disclosed by the invention is used for carrying out high performance liquid chromatography detection on the revivanacin raw material medicine or the preparation thereof; the conditions of the high performance liquid chromatography detection are as follows: octadecylsilane chemically bonded silica is adopted as a filling agent of a chromatographic column; the mobile phase A is an aqueous solution of hexafluorophosphate, and the mobile phase B is acetonitrile; gradient elution is adopted; the related substances comprise at least one of an impurity B, an impurity C, an impurity F and an impurity H. The detection method disclosed by the invention can be used for more comprehensively detecting related substances in the raw material medicine or the preparation of the raw material medicine, and continuous detection can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a detection method of related substances in Revefenacin raw material or its preparation. BACKGROUND

[0002] Revefenacin is a long-acting muscarinic antagonist (LAMA) which can be administered by inhalation for the treatment of chronic obstructive pulmonary disease (COPD). The chemical name of Revefenacin is 1-(2-{4-[(4-aminocarbonyl-1-piperidyl)methyl]-N-methylbenzamido}ethyl)-4-piperidyl 2-diphenylcarbamate, and the structural formula is as follows: .

[0003] Revefenacin raw material is synthesized by amination, condensation and amidation reaction, and there are many synthetic process impurities and reaction by-products, for example, impurities G, impurities H and impurities J are process impurities of the raw material synthesis; the amide bond of Revefenacin is prone to hydrolysis reaction in a solution state, producing degradation impurities A, impurities B, impurities D, impurities F and impurities I, and under oxidation conditions, degradation impurities such as impurities C and impurities E are prone to be produced. In order to ensure the efficacy and safety of the raw material, it is necessary to develop a detection method of impurities in Revefenacin raw material or its preparation.

[0004] At present, the detection method of impurities in Revefenacin can only detect a few types of impurities. For example, CN118624734A discloses a detection method of related substances in Revefenacin, which adopts high performance liquid chromatography for detection, and only detects impurities B and impurities I; CN118624733A discloses a detection method of related substance RFC1 in Revefenacin, which adopts high performance liquid chromatography for detection, and only detects single impurity RFC1. In addition, the current detection method cannot realize continuous detection, and the column performance gradually decreases in continuous detection, affecting the column life and detection accuracy. SUMMARY

[0005] Based on the above problems, the purpose of the present application includes providing a detection method of related substances in Revefenacin raw material or its preparation, which can more comprehensively detect related substances in Revefenacin raw material or its preparation, and can realize continuous detection.

[0006] In the detection method of related substances in Revefenacin raw material or its preparation of the present application, high performance liquid chromatography is used to detect the Revefenacin raw material or its preparation. The high performance liquid chromatography detection conditions include: the chromatographic column uses octadecylsilane bonded silica gel as the filler; the mobile phase A is a hexafluorophosphate aqueous solution, and the mobile phase B is acetonitrile; gradient elution is adopted; The related substances include one or more of the following impurities: Impurity B: ; Impurity C: ; Impurity F: ; and, Impurity H: .

[0007] The application establishes a detection method capable of simultaneously separating multiple impurities by adopting suitable high performance liquid chromatography detection conditions, can simultaneously detect the above-mentioned 10 impurities, does not need to use phosphate or ion pair reagents such as sodium hydrogen phosphate, potassium hydrogen phosphate, ammonium hydrogen phosphate, sodium hexyl sulfonate and sodium heptane sulfonate, effectively enhances the retention of alkaline impurities, avoids irreversible modification of the properties of the chromatographic column filler caused by long-term use of ion pair reagents, realizes good separation between the riviprenasin peak and the 10 impurity peaks, and is exclusive, sensitive and accurate, and can be well used for quality research and control of riviprenasin bulk drug or its preparation (such as an inhalant).

[0008] In some embodiments, the related substances further include one or more of the following impurities: Impurity A: ; Impurity D: ; Impurity E: ; Impurity G: ; Impurity I: ; and, Impurity J: ; and / or, The column length of the chromatographic column is 150mm-250mm, the inner diameter is 4.4mm-4.8mm, and the particle size of the filler is 3.5um-5um.

[0009] In some embodiments, the column length of the chromatographic column is 150mm, the inner diameter is 4.6mm, and the particle size of the filler is 3.5um.

[0010] In some embodiments, the model of the chromatographic column is Agilent ZORBAX Bonus-RP.

[0011] In some embodiments, the concentration of the hexafluorophosphate aqueous solution is 0.03mol / L-0.07mol / L, and the pH value is 2.2-3.0.

[0012] In some embodiments, the aqueous solution of hexafluorophosphate salt has a composition of hexafluorophosphate salt, phosphoric acid and water, and the preparation method comprises the following steps: dissolving a suitable amount of hexafluorophosphate salt in water to obtain a solution with a concentration of 0.03 mol / L-0.07 mol / L, and then adding a suitable amount of phosphoric acid or dilute phosphoric acid to adjust the pH value to 2.2-3.0 to obtain the aqueous solution of hexafluorophosphate salt. Alternatively, the hexafluorophosphate salt can be one or more of potassium hexafluorophosphate, sodium hexafluorophosphate and the like metal salts.

[0013] In some embodiments, the gradient elution procedure comprises: 0 min→65 min, volume concentration of mobile phase A 88%→65%; 65 min→75 min, volume concentration of mobile phase A 65%; 75 min→80 min, volume concentration of mobile phase A 65%→15%; 80 min→85 min, volume concentration of mobile phase A 15%; 85 min→85.1 min, volume concentration of mobile phase A 15%→88%; 85.1 min→98 min, volume concentration of mobile phase A 88%; or comprising: 0 min→65 min, volume concentration of mobile phase A 90%→70%; 65 min→75 min, volume concentration of mobile phase A 70%; 75 min→80 min, volume concentration of mobile phase A 70%→10%; 80 min→85 min, volume concentration of mobile phase A 10%; 85 min→85.1 min, volume concentration of mobile phase A 10%→90%; 85.1 min→98 min, volume concentration of mobile phase A 90%; or comprising: 0 min→65 min, volume concentration of mobile phase A 92%→775%; 65 min→75 min, volume concentration of mobile phase A 75%; 75 min→80 min, volume concentration of mobile phase A 75%→5%; 80 min→85 min, volume concentration of mobile phase A 5%; 85 min→85.1 min, volume concentration of mobile phase A 5%→75%; 85.1 min→98 min, volume concentration of mobile phase A 75%.

[0014] In some embodiments, the high performance liquid chromatography detection employs an ultraviolet-visible spectrophotometric detector or a diode array detector; optionally, the detection wavelength is 230 nm-240 nm.

[0015] In some embodiments, the flow rate of the high performance liquid chromatography detection is 0.6 mL / min-1.4 mL / min.

[0016] In some embodiments, the column temperature of the high performance liquid chromatography detection is 25℃~45℃.

[0017] In some embodiments, the injection tray temperature of the high performance liquid chromatography detection is 5℃-25℃.

[0018] In some embodiments, the step of performing high performance liquid chromatography detection on the revidum raw material drug or its preparation comprises: taking the revidum raw material drug or its preparation, dissolving with a solvent to prepare a test solution; taking the control samples of each impurity, dissolving with a solvent to prepare a control solution; performing high performance liquid chromatography detection on the control solution and the test solution to calculate the content of each impurity.

[0019] Optionally, the composition of the solvent can be a mixture of mobile phase A and mobile phase B, and the volume ratio of mobile phase A and mobile phase B can be 85-95:5-15.

[0020] Optionally, the concentration of the solute in the test solution is 0.04 mg / mL-0.06 mg / mL; the concentration of each impurity in the control solution is 0.4 μg / mL-0.6 μg / mL.

[0021] In some embodiments, after performing high performance liquid chromatography detection on the control solution and the test solution, the peak area of each impurity in the test solution is obtained, and the content of each impurity is calculated by using the principal component external standard method with a correction factor according to the obtained peak area. Specifically, the content of each impurity can be calculated according to the following formula: ; wherein, A 杂 represents the peak area of each impurity in the test solution; C 对 represents the concentration of revidum in the control solution; A 对 represents the peak area of revidum in the control solution; C 供 represents the concentration of the test solution; F represents the correction factor of the impurity and the principal component.

[0022] The calculation method of the correction factor is as follows: F=k 主 / k 杂质 ; wherein, k 主 represents the slope of the linear equation of the principal component revidum; k 杂质: Represents the slope of the linear equation for impurities.

[0023] The detection method of the present invention can simultaneously detect multiple impurities in revinapine raw material or its preparations, including impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J. The main component revinapine can be completely separated from each impurity, the resolution between revinapine and each impurity is >2, the separation between each impurity is >1, and the retention time of each chromatographic peak is consistent with the retention time of the chromatographic peak in each positioning solution.

[0024] The detection method of the present invention has good method specificity; the concentration of each impurity and the peak area show a good linear relationship, the method has good linearity; and it has high sensitivity, good recovery rate and good accuracy.

[0025] This invention employs gradient elution, which, compared to traditional detection methods, ensures that column performance does not decrease during multiple detections, enabling continuous detection and significantly improving detection efficiency.

[0026] In some preferred embodiments, the limit of detection (LOQ) is approximately 15 ng / ml, equivalent to 0.03% of the test concentration, and the limit of quantitation (LOQ) is approximately 50 ng / ml, equivalent to 0.1% of the test concentration. The average recoveries of spiked test solutions with LOQ concentrations ranging from 150% to 94.1% are 109.4%, all within the range of 90% to 110%, and the RSDs are 2.9% to 8.2%, all less than 10%. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a chromatogram of the mixed solution obtained in Example 1 of the present invention; Figure 2 The chromatogram of the mixed solution obtained in Comparative Example 1 of the present invention is shown below. Figure 3 This is a chromatogram of the mixed solution obtained in Comparative Example 2 of the present invention; Figure 4 The chromatogram of the mixed solution obtained in Comparative Example 1 of the present invention is shown below. Figure 5 This is a chromatogram of the mixed solution obtained in Comparative Example 2 of the present invention. Detailed Implementation

[0029] The method for detecting the related substances in the raw material of reviprenall or the preparation thereof of the present application is further described below in combination with the embodiments and examples. It should be understood that the examples are only used for illustrating the present application but not for limiting the scope of the present application. In addition, it should be understood that after reading the content of the present application, those skilled in the art can make various alterations or modifications to the present application, and these equivalent forms also fall within the protection scope of the appended claims of the present application.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0031] The following are some specific examples.

[0032] The experimental parameters not written in the following specific examples are preferably referred to the guidance given in the present application document, and can also be referred to the experimental manuals in the art or other experimental methods known in the art, or the experimental conditions recommended by the manufacturers.

[0033] The raw materials and reagents involved in the following specific examples can be obtained commercially or prepared by those skilled in the art according to known means.

[0034] Example 1 This example provides the separation and exclusive positioning test of reviprenall and impurities.

[0035] 1. Chromatographic condition parameters Chromatographic column: Agilent ZORBAX Bonus-RP (4.6 mm x 150 mm, 5 μm); Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (pH 2.5); mobile phase B: acetonitrile Flow rate: 1.0 ml / min; column temperature: 40 °C; detection wavelength: 235 nm; injection volume: 80 μl.

[0036] Gradient elution was performed with mobile phase A-mobile phase B according to the following program: 0 min→65 min, volume concentration of mobile phase A 90%→70%; 65 min→75 min, volume concentration of mobile phase A 70%; 75 min→80 min, volume concentration of mobile phase A 70%→10%; 80 min→85 min, volume concentration of mobile phase A 10%; 85 min→85.1 min, volume concentration of mobile phase A 10%→90%; 85.1 min→98 min, volume concentration of mobile phase A 90%.

[0037] 2. Solution preparation Solvent: mobile phase A-acetonitrile (90:10).

[0038] Control solution: about 2.5 mg of rivastigmine control was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added, and ultrasonic dissolution and dilution to the mark were performed, and the mixture was shaken to obtain a rivastigmine control stock solution; 1 ml of the above solution was taken and added to a 50-ml volumetric flask, and dilution to the mark was performed, and the mixture was shaken to obtain the solution.

[0039] Impurity control stock solution (1): about 1 mg of impurity A, impurity C, impurity D, and impurity E controls was weighed into a 10-ml volumetric flask, 3 ml of acetonitrile was added, ultrasonic dissolution was performed, and dilution to the mark was performed with solvent, and the mixture was shaken to obtain a solution containing about 100 μg / ml of each impurity. Impurity control stock solution (2): about 1 mg of impurity B, impurity F, impurity G, impurity H, impurity I, and impurity J controls was weighed into a 10-ml volumetric flask, 3 ml of methanol was added, ultrasonic dissolution was performed, and dilution to the mark was performed with solvent, and the mixture was shaken to obtain a solution containing about 100 μg / ml of each impurity. Mixed solution: about 2.5 mg of rivastigmine raw material was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added, ultrasonic dissolution was performed, and 0.5 ml of each of the impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J control stock solutions was added, and dilution to the mark was performed with solvent, and the mixture was shaken to obtain a solution containing about 0.05 mg of rivastigmine and about 0.5 μg / ml of each impurity. Raw material test sample solution: about 25 mg of rivastigmine raw material was weighed into a 50-ml volumetric flask, 3 ml of acetonitrile was added, ultrasonic shaking was performed to dissolve the mixture, dilution to the mark was performed with solvent, and the mixture was shaken to obtain a test sample stock solution; 1 ml of the above solution was taken and added to a 10-ml volumetric flask, and dilution to the mark was performed, and the mixture was shaken to obtain the solution.

[0040] Preparation test sample solution: an appropriate amount of rivastigmine inhalation solution was taken directly into a sample vial to obtain the solution. (1) The control solution was continuously tested for 5 times to investigate the stability of the chromatographic system of the method, and the specific test results are shown in Table 1.

[0041] Table 1 Precision of sample injection results 2) The solvent, each impurity control stock solution (as impurity specific positioning), mixed solution, raw material, and preparation test sample solution were injected into the high performance liquid chromatograph, and the specific test results are shown in Table 2, and the test chromatogram is shown in Figure 1 .

[0042] Specificity results 4. Test Conclusion The blank solvent peak has no interference with the main component peak and each impurity peak, the main component and the adjacent impurity in the mixed solution are completely separated, the minimum separation degree is 2.9>1.5, the impurities are well separated, the minimum separation degree is 1.3>1.2, the retention time of each chromatographic peak is consistent with the retention time of the chromatographic peak in each positioning solution, which indicates that the method is good for the separation of each impurity and can be exclusively positioned.

[0043] The content of the impurity is calculated according to the main component with a correction factor, which can be calculated according to the following formula: ; in the formula, A 杂 represents the area of each impurity peak in the test sample solution; C 对 represents the concentration of revimaline in the reference substance solution; A 对 represents the peak area of revimaline in the reference substance solution; C 供 represents the concentration of the test sample solution; and F represents the correction factor of the impurity and the main component.

[0044] The calculation method of the correction factor is: F=k 主 / k 杂质 ; in the formula, k 主 represents the slope of the linear equation of the main component revimaline; and k 杂质 represents the slope of the linear equation of the impurity.

[0045] The impurity detection results of revimaline bulk drug and revimaline inhalation solution are shown in Table 3. Under the detection method provided in the present application, each impurity can be well detected.

[0046] Table 3 Impurity detection results Example 2 The present embodiment provides the sensitivity and linearity test of the detection method of the present application.

[0047] 1. Chromatographic condition parameters: the parameters remain unchanged as in Example 1.

[0048] 2. Solution preparation Solvent: mobile phase A-acetonitrile (90:10) Reference substance stock solution (1): about 1 mg of revimaline, impurity A, impurity C, impurity D and impurity E reference substances were weighed, 3 ml of acetonitrile was added to a 10 ml volumetric flask, ultrasonic was used for dissolution, and the solvent was diluted to the mark, and then shaken. (About 100 μg / ml of each component) Stock solution of reference substance (2): about 1 mg of each of the reference substances of impurity B, impurity F, impurity G, impurity H, impurity I, impurity J was taken into a 10-ml volumetric flask, 3 ml of methanol was added to ultrasonically dissolve the sample, and the solvent was added to dilute to the mark, and shaken to mix. (about 100 μg / ml of each component) Mixed stock solution of reference substance: 1 ml of each of the stock solution of reference substance of revimaline and each impurity was taken into the same 50-ml volumetric flask, the solvent was added to dilute to the mark, and shaken to mix. (about 2 μg / ml of each component) (1) Sensitivity Limit of quantitation solution: 0.5 ml of the mixed stock solution of reference substance was taken into a 20-ml volumetric flask, the solvent was added to dilute to the mark, and shaken to mix.

[0049] Limit of detection solution: 3 ml of the limit of quantitation solution was taken into a 10-ml volumetric flask, the solvent was added to dilute to the mark, and shaken to mix.

[0050] (2) Linearity 0.5, 2.5, 4, 5, and 7.5 ml of the mixed stock solution of reference substance was taken into different 20-ml volumetric flasks, the solvent was added to dilute to the mark, and shaken to mix, to obtain the linear solutions of 10% (equivalent to LOQ), 50%, 80%, 100%, and 150% limit.

[0051] 3, Sample analysis (1) The limit of detection solution, the limit of quantitation solution, and the linear solutions of serial concentration levels were continuously injected for 6 times to test the sensitivity and linearity of the method, and the specific test results are shown in Tables 4-1 and 4-2.

[0052] Table 4-1 Sensitivity results Table 4-2 Linearity results 4, Test conclusion The sensitivity of revimaline and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J is high, the limit of detection concentration is about 15 ng / ml, which is equivalent to 0.03% of the test concentration, the limit of quantitation concentration is about 50 ng / ml, which is equivalent to 0.1% of the test concentration; a good linear relationship is shown in the range of LOQ~150% limit concentration, the correlation coefficient r is greater than 0.999, and the linearity of the method is good.

[0053] Example 3 This example provides the accuracy test of the detection method of the present application.

[0054] 1, Chromatographic condition parameters: the parameters remain unchanged as in Example 1.

[0055] 2. Solution preparation Solvent: mobile phase A - acetonitrile (90:10) Reference stock solution (1): about 1 mg of each of impurity A, impurity C, impurity D, and impurity E reference substances were weighed into a 10-ml volumetric flask, 3 ml of acetonitrile was added to dissolve the substances by ultrasonic, and the volume was made up with solvent. (Each impurity was about 100 μg / ml) Reference stock solution (2): about 1 mg of each of impurity B, impurity F, impurity G, impurity H, impurity I, and impurity J reference substances were weighed into a 10-ml volumetric flask, 3 ml of methanol was added to dissolve the substances by ultrasonic, and the volume was made up with solvent. (Each impurity was about 100 μg / ml) Mixed reference stock solution: 1 ml of each of the reference stock solutions of the impurities was transferred into the same 50-ml volumetric flask, and the volume was made up with solvent. (Each component was about 2 μg / ml) Reference solution: about 2.5 mg of riviprenasin reference substance was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added to dissolve and dilute to the mark, and the volume was made up with solvent. (Each component was about 2 μg / ml)

[0056] API accuracy stock solution: about 12.5 mg of riviprenasin active pharmaceutical ingredient was weighed into a 25-ml volumetric flask, 1 ml of acetonitrile was added to dissolve the substances by ultrasonic, and the volume was made up with solvent. (Each component was about 2 μg / ml) Accuracy blank solution: 2 ml of the API accuracy stock solution was transferred into a 20-ml volumetric flask, and the volume was made up with blank excipient solution. (Each component was about 2 μg / ml) LOQ level accuracy solution: 2 ml of the API accuracy stock solution was transferred into a 20-ml volumetric flask, 0.5 ml of the mixed reference stock solution was added, and the volume was made up with blank excipient solution. (Each component was about 2 μg / ml) 100% level accuracy solution: 2 ml of the API accuracy stock solution was transferred into a 20-ml volumetric flask, 5 ml of the mixed reference stock solution was added, and the volume was made up with blank excipient solution. (Each component was about 2 μg / ml) 150% level accuracy solution: 2 ml of the API accuracy stock solution was transferred into a 20-ml volumetric flask, 5 ml of the mixed reference stock solution was added, and the volume was made up with blank excipient solution. (Each component was about 2 μg / ml) 3. Sample analysis The control solution, accuracy base solution, LOQ, 100%, 150% level accuracy solution were injected for testing to investigate the accuracy of the method. The specific test results are shown in Table 5.

[0057] Table 5 Accuracy results 4. Test conclusion The recoveries of impurities A, B, C, D, E, F, G, H, I and J were good. The average recoveries of the spiked test sample solutions at LOQ concentration to 150% concentration were 94.1% to 109.4%, all within the range of 90% to 110%, the RSDs were 2.9% to 8.2%, all less than 10%, and the method accuracy was good.

[0058] Example 4 This example provides the continuous detection applicability of the detection method of the present application.

[0059] 1. Chromatographic condition parameters The parameters of Example 1 were kept unchanged.

[0060] 2. Solution preparation Solvents, control stock solution (1), control stock solution (2), mixed control stock solution, control solution, API accuracy stock solution: the parameters were kept unchanged as in Example 3.

[0061] Test sample solution: 2 ml of the API accuracy stock solution was taken and placed in a 20 ml volumetric flask, 5 ml of the mixed control stock solution was added, and diluted to volume with the blank excipient solution, and shaken well. Three portions were prepared in parallel. (About 50 μg / ml of revimaline, 0.5 μg / ml of each impurity) 3. Injection analysis The control solution and test sample solution were injected for testing to investigate the continuous detection applicability of the method. The specific test results are shown in Table 6.

[0062] Table 6 Continuous detection results 4. Test conclusion After continuous detection of multiple samples for 72 h, there was no difference in the continuous detection results, no impurity residue, and the detection accuracy was high.

[0063] Example 5 This example provides the application of the detection method of the present application to sample detection.

[0064] 1. Chromatographic condition parameters (1) The parameters of Example 1 were kept unchanged; (2) Chromatographic parameters were adjusted to 0.6 ml / min, 1.4 ml / min, respectively, based on Example 1. (3) Chromatographic parameters were adjusted to 25℃, 45℃, respectively, based on Example 1. (4) Chromatographic parameters were adjusted to 0.03 mol / L, 0.07 mol / L, respectively, based on Example 1. (5) Chromatographic parameters were adjusted to 88%, 92%, respectively, for the initial proportion of mobile phase A in the elution gradient, 65% and 75% at 65 min, and 15% and 5% at 80 min, based on Example 1.

[0065] 2. Preparation of solutions Solvent: mobile phase A - acetonitrile (90:10) Reference solution: about 2.5 mg of reference substance of reviparin was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added, and ultrasonic dissolution and dilution to volume were performed, followed by shaking to obtain a reviparin reference stock solution; 1 ml of the above solution was taken and placed in a 50-ml volumetric flask, and dilution to volume was performed, followed by shaking to obtain the solution.

[0066] Impurity reference stock solution (1): about 5 mg of each of impurity A, impurity C, impurity D, and impurity E reference substances was weighed into a 10-ml volumetric flask, 3 ml of acetonitrile was added for ultrasonic dissolution, and dilution to volume was performed with solvent, followed by shaking to obtain a solution containing about 500 μg / ml of each impurity. Impurity reference stock solution (2): about 5 mg of each of impurity B, impurity F, impurity G, impurity H, impurity I, and impurity J reference substances was weighed into a 10-ml volumetric flask, 3 ml of methanol was added for ultrasonic dissolution, and dilution to volume was performed with solvent, followed by shaking to obtain a solution containing about 500 μg / ml of each impurity. Mixed impurity reference stock solution: 1 ml of each of the impurity reference stock solutions was transferred into the same 20-ml volumetric flask, and dilution to volume was performed with solvent, followed by shaking to obtain a solution containing about 25 μg / ml of each component. Mixed solution: about 2.5 mg of reviparin raw material was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added, and ultrasonic dissolution was performed, followed by the addition of 0.5 ml of each of the impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J reference stock solutions, and dilution to volume was performed with solvent, followed by shaking to obtain a solution containing about 0.05 mg of reviparin and about 0.5 μg / ml of each impurity. Raw material drug test sample solution: about 25 mg of raw material drug of revimaline was weighed into a 50 ml volumetric flask, 3 ml of acetonitrile was added to ultrasonic vibration to dissolve, 5 ml of mixed impurity control sample stock solution was added, and the sample was diluted to constant volume with solvent, and was shaken to obtain a test sample stock solution; 1 ml of the above solution was taken into a 10 ml volumetric flask, diluted to constant volume with solvent, and shaken to obtain the test sample solution (about 50 μg / ml of revimaline and 0.25 μg / ml of each impurity). Preparation test sample solution: 9 ml of revimaline inhalation solution was directly taken into a 10 ml volumetric flask, 0.1 ml of mixed impurity control sample stock solution was added, and the sample was diluted to constant volume with solvent, and was shaken to obtain the test sample solution (about 50 μg / ml of revimaline and 0.25 μg / ml of each impurity). 3. Sample analysis The control sample solution, mixed solution, and test sample solution were tested to investigate the difference in impurity detection results of revimaline raw material drug and revimaline inhalation solution by the method, and the specific test results are shown in Tables 7-8.

[0067] Table 7. Detection results of revimaline raw material drug samples Table 8. Detection results of revimaline preparation (inhaler) samples 4. Test conclusion Under the chromatographic parameters, the content of impurities was calculated by the principal component external standard method with a correction factor by using the detection method provided by the application, which can be calculated according to the following formula: ; in the formula, A 杂 represents the peak area of each impurity in the test sample solution; C 对 represents the concentration of revimaline in the control sample solution; A 对 represents the peak area of revimaline in the control sample solution; C 供 represents the concentration of the test sample solution; and F represents the correction factor of the impurity and the principal component.

[0068] The calculation method of the correction factor is: F=k 主 / k 杂质 ; in the formula, k 主 represents the slope of the linear equation of the principal component; and k 杂质 represents the slope of the linear equation of the impurity.

[0069] The impurities in revimaline raw material drug and revimaline inhalation solution can be well detected, and the detection method provided by the application can be used as an effective means for the detection of related substances of revimaline raw material drug and its preparation, and provides a detection basis for drug quality control.

[0070] Comparative Example 1 This comparative example provides another detection method, which uses a different elution procedure compared to Example 1.

[0071] 1. Chromatographic condition parameters Chromatographic column: Agilent ZORBAX Bonus-RP (4.6 mm x 150 mm, 5 μm); Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (pH 2.5); mobile phase B: acetonitrile Flow rate: 1.0 ml / min; column temperature: 40 °C; detection wavelength: 235 nm; injection volume: 80 μl.

[0072] Isocratic elution with mobile phase A-mobile phase B (70:30).

[0073] 2. Solution preparation Solvent: mobile phase A-acetonitrile (90:10) Impurity control stock solution (1): about 1 mg of impurity A, impurity C, impurity D, and impurity E control was weighed into a 10-ml volumetric flask, 3 ml of acetonitrile was added to ultrasonically dissolve, and the solvent was diluted to the mark, and shaken well. (About 100 μg / ml of each impurity was contained.) Impurity control stock solution (2): about 1 mg of impurity B, impurity F, impurity G, impurity H, impurity I, and impurity J control was weighed into a 10-ml volumetric flask, 3 ml of methanol was added to ultrasonically dissolve, and the solvent was diluted to the mark, and shaken well. (About 100 μg / ml of each impurity was contained.) Mixed solution: about 2.5 mg of revimaline raw material was weighed into a 100-ml volumetric flask, about 50 ml of solvent was added to ultrasonically dissolve, and then 0.5 ml of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J control stock solution was added, respectively, and mixed well, and then the solvent was diluted to the mark, and shaken well. (About 0.05 mg of revimaline and 0.5 μg / ml of each impurity was contained.) 3. Injection analysis The mixed solution was injected for testing, and the test chromatogram is shown in Figure 2. Figure 2 .

[0074] 4. Test conclusion Under the chromatographic conditions of mobile phase A-mobile phase B (70:30), the impurities were not completely separated, and the results showed that the main peak retention time was 24.751 min, and 6 impurities were detected, and the remaining impurities were overlapped or not eluted.

[0075] Comparative Example 2 This comparative example provides another detection method, which uses a different elution procedure compared to Example 1.

[0076] 1. Chromatographic condition parameters Column: Agilent ZORBAX Bonus-RP (4.6 mm x 150 mm, 5 μm); Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (pH 2.5); mobile phase B: acetonitrile Flow rate: 1.0 ml / min; column temperature: 40 °C; detection wavelength: 235 nm; injection volume: 80 μl.

[0077] Gradient elution was performed with mobile phase A-mobile phase B according to Table 9.

[0078] Table 9 Gradient elution program 2. Preparation of solutions Solvent: mobile phase A-acetonitrile (90:10) Impurity control stock solution (1): about 1 mg of impurity A, impurity C, impurity D, impurity E control was weighed, placed in a 10 ml volumetric flask, 3 ml of acetonitrile was added to ultrasonic dissolution, and the solvent was diluted to the mark. Shake well. (About 100 μg / ml of each impurity) Impurity control stock solution (2): about 1 mg of impurity B, impurity F, impurity G, impurity H, impurity I, impurity J control was taken, placed in a 10 ml volumetric flask, 3 ml of methanol was added to ultrasonic dissolution, and the solvent was diluted to the mark. Shake well. (About 100 μg / ml of each impurity) Mixed solution: about 2.5 mg of revinacin raw material was weighed, placed in a 100 ml volumetric flask, about 50 ml of solvent was added, ultrasonic dissolution was performed, and then 0.5 ml of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J control stock solution was added respectively. Mix well, then dilute to volume with solvent, and shake well. (About 0.05 mg of revinacin and 0.5 μg / ml of each impurity) 3. Injection analysis Take the mixed solution for injection test, and the test chromatogram is shown in the following figure: Figure 3 .

[0079] 4. Test conclusion Under the chromatographic conditions of a faster elution gradient of mobile phase A-mobile phase B, the impurities were not completely separated, the results showed that the main peak retention time was 22.065 min, 7 impurities were detected, and the rest of the impurities overlapped, among which impurity E and the main peak overlapped.

[0080] Comparative Example 3 This comparative example provides another detection method, which uses a different mobile phase pH value compared to Example 1.

[0081] 1. Chromatographic condition parameters Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (pH 5.5); mobile phase B: acetonitrile Other parameters remain unchanged as in Example 1. 2. Solution preparation Solvent: mobile phase A-acetonitrile (90:10) Impurity control stock solution (1): about 1 mg of impurity A, impurity C, impurity D, and impurity E controls were weighed into a 10 ml volumetric flask, 3 ml of acetonitrile was added to ultrasonic dissolution, and the solvent was diluted to the mark, and shaken well. (About 100 μg / ml of each impurity) Impurity control stock solution (2): about 1 mg of impurity B, impurity F, impurity G, impurity H, impurity I, and impurity J controls were taken into a 10 ml volumetric flask, 3 ml of methanol was added to ultrasonic dissolution, and the solvent was diluted to the mark, and shaken well. (About 100 μg / ml of each impurity) Mixed solution: about 2.5 mg of revumenox raw material was weighed into a 100 ml volumetric flask, about 50 ml of solvent was added, ultrasonic dissolution was performed, and 0.5 ml of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J control stock solutions were added respectively, mixed well, and then diluted to constant volume with solvent, shaken well. (About 0.05 mg of revumenox and 0.5 μg / ml of each impurity) 3. Sample injection analysis The mixed solution was taken for sample injection test, and the test chromatogram is shown in Figure Figure 4 .

[0082] 4. Test conclusion Under the same elution gradient, when the pH of the mobile phase is 5.5, impurity G and impurity D show double peaks, the results show that the main peak retention time is 73.215 min, the peak time is late, 8 impurities are detected, and the separation degree of impurity B and impurity E is only 0.9, and the rest of the impurities are not separated.

[0083] Comparative Example 4 This comparative example provides another detection method, which uses a different mobile phase compared to Example 1.

[0084] 1. Chromatographic condition parameters Chromatographic column: Agilent ZORBAX Bonus-RP (4.6 mm x 150 mm, 5 μm); Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution (pH 2.5); mobile phase B: acetonitrile Flow rate: 1.0 ml / min; column temperature: 40°C; detection wavelength: 235 nm; injection volume: 80 μl.

[0085] Eluted with mobile phase A-mobile phase B according to Table 10 by gradient.

[0086] Table 10 Gradient elution program 2. Preparation of solution Solvent: mobile phase A - acetonitrile (90:10) Impurity reference stock solution (1): about 1 mg of impurity A, impurity C, impurity D, impurity E reference substance was weighed into a 10 ml volumetric flask, 3 ml of acetonitrile was added to ultrasonic dissolution, and the solvent was diluted to the mark. (About 100 μg / ml of each impurity) Impurity reference stock solution (2): about 1 mg of impurity B, impurity F, impurity G, impurity H, impurity I, impurity J reference substance was weighed into a 10 ml volumetric flask, 3 ml of methanol was added to ultrasonic dissolution, and the solvent was diluted to the mark. (About 100 μg / ml of each impurity) Mixed solution: about 2.5 mg of revumenox raw material was weighed into a 100 ml volumetric flask, about 50 ml of solvent was added, ultrasonic dissolution was performed, and 0.5 ml of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J reference stock solution was added, respectively, mixed, and then diluted to volume with solvent, and shaken. (About 0.05 mg of revumenox and 0.5 μg / ml of each impurity) 3. Sample injection analysis The mixed solution was injected for testing, and the test chromatogram is shown in the accompanying Figure 5 .

[0087] 4. Test conclusion Under the chromatographic conditions of mobile phase A as phosphate, the impurities were not completely separated, the results showed that the main peak retention time was 18.613 min, 7 impurities were detected, impurity A and impurity H were coincident, impurity E and impurity C were coincident, and impurity J and the main peak were coincident.

[0088] Each technical feature of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combination of the technical features does not exist contradictory, it should be considered within the scope of the present disclosure.

[0089] The above embodiments only express several implementation manners of the present application, but cannot be understood as the limitation to the patent scope of the present application. It should be pointed out that, for the ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching of the present application, the skilled in the art can make various modifications or changes to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, the technical solutions obtained by the skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for detecting related substances in revinapordin active pharmaceutical ingredient or its formulations, characterized in that, High-performance liquid chromatography was used to detect the new active pharmaceutical ingredient or its formulation of revinnain. The conditions for high-performance liquid chromatography (HPLC) detection include: using an octadecylsilane-bonded silica gel column as the packing material; mobile phase A being an aqueous solution of hexafluorophosphate, and mobile phase B being acetonitrile; and gradient elution. The relevant substances include one or more of the following impurities: Impurity B: ; Impurity C: ; Impurity F: ;as well as, Impurity H: .

2. The detection method according to claim 1, characterized in that, The relevant substances also include one or more of the following impurities: Impurity A: ; Impurity D: ; Impurity E: ; Impurity G: ; Impurity I: ; as well as, Impurity J: ; and / or, The chromatographic column has a length of 150mm-250mm, an inner diameter of 4.4mm-4.8mm, and a packing particle size of 3.5μm-5μm.

3. The detection method according to claim 2, characterized in that, The chromatographic column is an Agilent ZORBAX Bonus-RP.

4. The detection method according to claim 1, characterized in that, The aqueous solution of the hexafluorophosphate has a concentration of 0.03 mol / L to 0.07 mol / L and a pH value of 2.2 to 3.

0.

5. The detection method according to any one of claims 1-4, characterized in that, The gradient elution procedure includes: From 0 min to 65 min, the volume concentration of mobile phase A decreased from 88% to 65%. The mobile phase A volume concentration was 65% during the 65-75 min interval. The volume concentration of mobile phase A decreased from 65% to 15% over a period of 75 min to 80 min. The mobile phase A was kept at a volume concentration of 15% for 80 min to 85 min. The volume concentration of mobile phase A increased from 15% to 88% over a period of 85 min to 85.1 min. 85.1 min → 98 min, the volume concentration of mobile phase A is 88%; or includes: From 0 min to 65 min, the volume concentration of mobile phase A increased from 90% to 70%. The mobile phase A volume concentration was 70% during the 65-75 minute interval. The volume concentration of mobile phase A decreased from 70% to 10% over a period of 75 min to 80 min. 80 min → 85 min, the volume concentration of mobile phase A is 10%; The volume concentration of mobile phase A increased from 10% to 90% over a period of 85 min to 85.1 min. 85.1 min → 98 min, the volume concentration of mobile phase A is 90%; or includes: From 0 min to 65 min, the volume concentration of mobile phase A increased from 92% to 775%. The mobile phase A volume concentration was 75% during the 65min→75min process. The volume concentration of mobile phase A decreased from 75% to 5% over a period of 75 min to 80 min. 80 min → 85 min, the volume concentration of mobile phase A is 5%; The volume concentration of mobile phase A increased from 5% to 75% over a period of 85 min to 85.1 min. 85.1 min → 98 min, the volume concentration of the mobile phase A is 75%.

6. The detection method according to any one of claims 1-4, characterized in that, The high-performance liquid chromatography detection uses a UV-Vis spectrophotometer or a diode array detector; optionally, the detection wavelength is 230nm-240nm.

7. The detection method according to any one of claims 1-4, characterized in that, The flow rate for the high-performance liquid chromatography detection is 0.6 mL / min to 1.4 mL / min.

8. The detection method according to any one of claims 1-4, characterized in that, The column temperature for the high-performance liquid chromatography detection is 25℃-45℃.

9. The detection method according to any one of claims 1-4, characterized in that, The temperature of the injection plate for the high-performance liquid chromatography detection is 5℃-25℃.

10. The detection method according to any one of claims 1-4, characterized in that, The steps for high-performance liquid chromatography (HPLC) detection of revinnain active pharmaceutical ingredient or its formulations include: Take Revinaporne raw material or its preparation, dissolve it in a solvent to prepare the test solution; Take the reference standards of each impurity, mix them together, add solvent to dissolve them, and prepare the reference standard solution; The reference solution and the test solution were subjected to high-performance liquid chromatography (HPLC) to detect and calculate the content of each impurity.

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