Method for detecting related substances in rafenasin aerosol inhalation solution

Abstract

The invention discloses a method for detecting related substances in a rafenasin aerosol inhalation solution, and belongs to the technical field of medicine detection. Carrying out analysis and detection on the rafenasin aerosol inhalation solution by adopting an ultra-high performance liquid chromatography; chromatographic conditions of the ultra-high performance liquid chromatography are as follows: a chromatographic column takes octadecylsilane chemically bonded silica as a filler; the mobile phase A is a mixed solution of trifluoroacetic acid and water, and the mobile phase B is a mixed solution of acetonitrile, water and trifluoroacetic acid; and gradient elution. The method provided by the invention can be used for separating and detecting four impurities in the rafenasin aerosol inhalation solution, analysis can be completed in 19 minutes, the separation effect is good, the analysis time and the analysis cost are remarkably shortened, the requirement of drug detection accuracy can be met, and a basis is provided for quality control of the rafenasin aerosol inhalation solution.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a method for detecting related substances in a refenacin aerosol inhalation solution. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Revefenacin has the following chemical structure:

[0004]

[0005] Revefenacin is a long-acting muscarinic antagonist (LAMA) that is administered by aerosolization of inhaled solution to improve lung function, reduce clinical symptoms of chronic obstructive pulmonary disease (COPD), and prevent further deterioration of the disease. It is used for maintenance treatment of COPD patients. The drug was approved for marketing by the U.S. Food and Drug Administration (FDA) on November 9, 2018, and the trade name of the preparation is Yupelri. At present, the drug is not included in domestic and foreign pharmacopoeias, and there are few reports on the analysis methods of degradation impurities and related substances. Patent 2024111009342 discloses a method for detecting refenacin and impurity components in aerosolized inhalation solution of refenacin. High performance liquid chromatography is used to detect refenacin and impurity components in aerosolized inhalation solution of refenacin. The chromatographic conditions are: the chromatographic column uses octadecylsilane bonded silica gel as a filler; the mobile phase A is a mixed solution of potassium dihydrogen phosphate solution and methanol, and the mobile phase B is acetonitrile, with gradient elution. However, this method takes a long time to detect, requiring 1 hour. Therefore, establishing a set of related substance detection and analysis methods with strong specificity, good repeatability, simplicity and rapidity can provide a reference for the imitation and quality control of the drug. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for detecting related substances in a nebulized inhalation solution of revenacin. The method provided by the present invention can complete the detection in only 19 minutes, greatly shortening the analysis time, reducing the amount of solvent used, and reducing the analysis cost.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a method for detecting related substances in a refenacin aerosol inhalation solution, comprising the following steps:

[0009] The refenacin nebulized inhalation solution was analyzed by ultra-high performance liquid chromatography, and the presence of refenacin and related impurities were identified by chromatogram;

[0010] Chromatographic conditions included:

[0011] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0012] Mobile phase A is a mixed solution of trifluoroacetic acid and water, and mobile phase B is a mixed solution of acetonitrile, water and trifluoroacetic acid;

[0013] Gradient elution was used.

[0014] The second aspect of the present invention provides a method for quantitatively detecting related substances in a nebulized inhalation solution of revenacin, comprising the following steps:

[0015] dissolving revenacin and related substances in a solvent to obtain a system suitability solution;

[0016] The system suitability solution is subjected to ultra-high performance liquid chromatography analysis using the above method to obtain a chromatogram of the system suitability solution;

[0017] The refenacin nebulized inhalation solution is subjected to ultra-high performance chromatography analysis using the above method to obtain a chromatogram of the refenacin nebulized inhalation solution;

[0018] The content of related substances is calculated using the correction factor and the peak area in the chromatogram.

[0019] The third aspect of the present invention provides a method for evaluating the quality of revenacin nebulized inhalation solution, using the above method.

[0020] In a fourth aspect, the present invention provides an application of detecting related substances in quality control of revenacin nebulized inhalation solution;

[0021] The related substances include four impurities, namely impurities A, B, C and D, whose structures are as follows:

[0022]

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a method for detecting related substances in a refenacin nebulized inhalation solution. Compared with the traditional high performance liquid chromatography (HPLC), the UPLC method shortens the analysis time, reduces the amount of solvent used, reduces the analysis cost, and can meet the requirements of drug detection accuracy, providing a basis for the quality control of the refenacin nebulized inhalation solution. The method of the present invention can separate and detect four impurities in the refenacin nebulized inhalation solution, and the analysis can be completed in 19 minutes, with good separation effect, significantly shortening the analysis time and analysis cost.

[0025] When the method of the present invention is used to detect the nebulized inhalation solution of refenacin, the related substances produced after light, high temperature, strong acid, strong alkali, and oxidation damage can be well separated, and the method has good specificity. In addition, the present invention verifies the repeatability of 6 samples, and the RSD of each impurity content is less than 5%, and the precision is good. The present invention also verifies the linearity and accuracy of each impurity in the range of 50% to 150% of the limit concentration. Each impurity has r>0.999 in the above range, and the recovery rate is between 97% and 103%, with good linearity and accuracy. The test solution of the present invention still has good stability after being placed at room temperature for 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The system applicability solution spectrum in Example 1 of the present invention;

[0028] Figure 2 is the localization map of impurity A in Example 1 of the present invention;

[0029] Figure 3 is the localization map of impurity B in Example 1 of the present invention;

[0030] Figure 4 is the localization map of impurity C in Example 1 of the present invention;

[0031] Figure 5 is the localization map of impurity D in Example 1 of the present invention;

[0032] Figure 6 The specific attribute overlay image in Example 1 of the present invention;

[0033] Figure 7 This is the spectrum of the undamaged test solution in Example 2 of the present invention;

[0034] Figure 8 The graph of the test solution destroyed by acid in Example 2 of the present invention;

[0035] Fig. 9 This is a sample solution spectrum of alkali destruction in Example 2 of the present invention;

[0036] Fig.10 The graph of the test solution destroyed by oxygen in Example 2 of the present invention;

[0037] Fig.11 This is the spectrum of the test solution destroyed by high temperature in Example 2 of the present invention;

[0038] Fig.12 This is a spectrum of the test solution destroyed by light in Example 2 of the present invention. DETAILED DESCRIPTION

[0039] In view of the problems such as the long analysis time of high performance liquid chromatography (HPLC) for the detection method of related substances in refenacin nebulized inhalation solution, the present invention proposes a method for detecting related substances in refenacin nebulized inhalation solution.

[0040] A typical embodiment of the present invention provides a method for detecting related substances in a refenacin aerosol inhalation solution, comprising the following steps:

[0041] The refenacin nebulized inhalation solution was analyzed by ultra-high performance liquid chromatography, and the presence of refenacin and related impurities were identified by chromatogram;

[0042] Chromatographic conditions included:

[0043] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0044] Mobile phase A is a mixed solution of trifluoroacetic acid and water, and mobile phase B is a mixed solution of acetonitrile, water and trifluoroacetic acid;

[0045] Gradient elution was used.

[0046] In the method provided by the present invention for detecting refenacin and impurity components in refenacin nebulized inhalation solution based on UPLC, the refenacin nebulized inhalation solution is an aqueous solution for nebulization. Except for the active ingredients and impurities, the solvent (water) has no effect in the high performance liquid chromatography detection process. Therefore, during the detection, the present invention directly uses the real refenacin nebulized inhalation solution as the sample to be tested, without the need for pre-treatment. The operation is simple and the accuracy is high. The qualitative and / or quantitative analysis of refenacin and four impurity components in the refenacin nebulized inhalation solution can be achieved, which fills the gap in the detection technology of refenacin and impurity components in the refenacin nebulized inhalation solution. The method can complete the detection in only 19 minutes, greatly shortening the analysis time, reducing the amount of solvent used, and reducing the analysis cost.

[0047] In a method for detecting related substances in a refenacin nebulized inhalation solution provided by the present invention, the impurity components are four compounds having the following structures. In order to distinguish each impurity, corresponding codes are set for them respectively. The specific information is shown in Table 1.

[0048] Table 1 Structures of four impurities in refenacin nebulized inhalation solution

[0049]

[0050]

[0051] Explanation of Table 1:

[0052] The English name of impurity A is Revefenacin Impurity 2, or 1-(4-((2-(4-hydroxypiperidin-1-yl)ethyl)(methyl)carbamoyl)benzyl)piperidine-4-carboxylic acid, and its molecular formula is C 22 H 33 N3O4, MW: 403.53, No.: L233542, purchased from ANTU Research Chemicals Ltd.

[0053] The English name of impurity B is Revefenacin Impurity 17, or 1-(4-((2-(4-(([1,1'-biphenyl]-2-ylcarbamoyl)oxy)piperidin-1-yl)ethyl)(methyl)carbamoyl)benzyl)piperidine-4-carboxylic acid, CAS: 909800-36-8, with a molecular formula of C 35 H 42 N4O5, MW:598.74228.

[0054] Impurity C is 2-aminobiphenyl, its English name is 2-Aminobiphenyl, CAS: 90-41-5.

[0055] Impurity D is called Revefenacin Impurity 17 in English, and its synonyms are Revefenacin Impurity 17, or 1-(2-(4-formyl-N-methylbenzamido)ethyl)piperidin-4-yl[1,1'-biphenyl]-2-ylcarbamate, CAS: 864760-28-1, and its molecular formula is C 29 H 31N3O4, MW:485.58284.

[0056] Impurity A, impurity B, impurity C, and impurity D used in the present invention are all existing products and can be purchased, for example, they can all be purchased from ANTU Research Chemicals Ltd.

[0057] In some examples of this embodiment, the mobile phase A is a mixed solution of 3-5v% trifluoroacetic acid and water, 3-5v% trifluoroacetic acid: water = 10:990, volume ratio;

[0058] The mobile phase B is a mixed solution of acetonitrile, water and 3-5v% trifluoroacetic acid, acetonitrile: water: 3-5v% trifluoroacetic acid = 900:90:10, volume ratio.

[0059] In some examples of this embodiment, the gradient elution is as follows, in volume percent:

[0060] From 0 to 3 min, mobile phase A was reduced from 100% to 92%, and mobile phase B was increased from 0% to 8%;

[0061] From 3 to 12 min, mobile phase A decreased from 92% to 61%, and mobile phase B increased from 8% to 39%;

[0062] From 12 to 14.5 min, mobile phase A was reduced from 61% to 0%, and mobile phase B was increased from 39% to 100%;

[0063] 14.5-15.5 min, mobile phase A 0%, mobile phase B 100%;

[0064] 15.5-15.54 min, mobile phase A increased from 0% to 100%, and mobile phase B decreased from 100% to 0%;

[0065] 15.51~19min, mobile phase A 100%, mobile phase B 0%.

[0066] In some examples of this embodiment, the chromatography column is Hypersil GOLD, 100 mm×2.1 mm, 1.9 μm or Hypersil GOLD Vanquish, 100 mm×2.1 mm, 1.9 μm.

[0067] In some examples of this embodiment, the chromatographic conditions also include: column temperature 45-55°C, preferably 50°C; flow rate 0.4-0.5mL / min, preferably 0.45mL / min; injection volume 9-11μL; detection wavelength 230-240nm, preferably 235nm.

[0068] In some examples of this embodiment, the chromatographic conditions are:

[0069] The chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0070] Mobile phase A is a mixed solution of 4% trifluoroacetic acid and water, 4% trifluoroacetic acid: water = 10:990, volume ratio; mobile phase B is a mixed solution of acetonitrile, water and 4% trifluoroacetic acid, acetonitrile: water: 4% trifluoroacetic acid = 900:90:10, volume ratio;

[0071] Gradient elution; detection wavelength was 235 nm, column temperature was 50 °C, flow rate was 0.45 mL / min, injection volume was 10 μL.

[0072] The present invention also provides a method for quantitatively detecting related substances in a refenacin aerosol inhalation solution, comprising the following steps:

[0073] dissolving revenacin and related substances in a solvent to obtain a system suitability solution;

[0074] The system suitability solution is subjected to ultra-high performance liquid chromatography analysis using the above method to obtain a chromatogram of the system suitability solution;

[0075] The refenacin nebulized inhalation solution is subjected to ultra-high performance chromatography analysis using the above method to obtain a chromatogram of the refenacin nebulized inhalation solution;

[0076] The content of related substances is calculated using the correction factor and the peak area in the chromatogram.

[0077] In some examples of this embodiment, the calculation formula is as follows:

[0078]

[0079] Where:

[0080] A U : Peak area of ​​impurities in refenacin nebulized inhalation solution;

[0081] f: relative correction factor, which is the ratio of the slope of the linear regression equation of refenacin to the slope of the linear regression equation of the relevant impurities;

[0082] A S : Peak area of ​​the main peak in the system suitability solution.

[0083] In some examples of this embodiment, the solvent is citric acid aqueous solution or methanol aqueous solution. Citric acid aqueous solution and methanol aqueous solution do not interfere with the separation and detection of the refenacin aerosol inhalation solution and various impurities, and the detection method provided by the present invention has good specificity.

[0084] Another typical embodiment of the present invention provides a quality assessment method for revenacin nebulized inhalation solution, using the above-mentioned detection method.

[0085] Another typical embodiment of the present invention provides an application of detecting related substances in the quality control of refenacin nebulized inhalation solution;

[0086] The related substances are the four impurities in Table 1 above, namely impurities A, B, C and D.

[0087] The present invention provides a method for detecting refenacin and impurity components in refenacin nebulized inhalation solution based on UPLC. Compared with the traditional high performance liquid chromatography (HPLC), the UPLC method shortens the analysis time, reduces the amount of solvent used, reduces the analysis cost, and can meet the requirements of drug detection accuracy, providing a basis for the quality control of refenacin nebulized inhalation solution. The method of the present invention can separate and detect four impurities in refenacin nebulized inhalation solution, and the analysis can be completed in 19 minutes, with good separation effect, significantly shortening the analysis time and analysis cost.

[0088] When the method of the present invention is used to detect the nebulized inhalation solution of refenacin, the related substances produced after light, high temperature, strong acid, strong alkali, and oxidation damage can be well separated, and the method has good specificity. In addition, the present invention verifies the repeatability of 6 samples, and the RSD of each impurity content is less than 5%, and the precision is good. The present invention also verifies the linearity and accuracy of each impurity in the range of 50% to 150% of the limit concentration. Each impurity has r>0.999 in the above range, and the recovery rate is between 97% and 103%, with good linearity and accuracy. The test solution of the present invention still has good stability after being placed at room temperature for 48 hours.

[0089] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0090] Unless otherwise specified, the raw materials used in the following examples are all conventional products and can be purchased.

[0091] Example 1

[0092] A method for detecting refenacin and impurity components in refenacin aerosol inhalation solution based on UPLC, comprising the following steps:

[0093] 1-1 Solution Preparation

[0094] Dilution: Take an appropriate amount of citric acid and dissolve it in water to prepare a solution containing approximately 3 mg of citric acid per 1 mL of solution.

[0095] Each impurity localization solution: Accurately weigh an appropriate amount of impurity A, B, C, and D reference substances, dissolve and dilute the four impurities with diluent, and prepare 0.6 μg / mL impurity A, B, C, and D localization solutions, respectively.

[0096] System suitability solution: Take about 6 mg of refenasin and place it in a 100 mL volumetric flask, then add appropriate amounts of impurities A, B, C, and D, add diluent to dissolve by ultrasonic and dilute to the scale, shake well, and prepare a mixed solution containing 60 μg of refenasin and 0.3 μg of each impurity per 1 mL.

[0097] Test solution: Take the product, open the ampoule, and squeeze out the liquid as the test solution, which contains about 58μg / mL of refenasin.

[0098] 1-2 Chromatographic conditions

[0099] Column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0100] Mobile Phase:

[0101] Mobile phase A was 4% trifluoroacetic acid-water (10:990);

[0102] Mobile phase B was acetonitrile-water-4% trifluoroacetic acid (900:90:10);

[0103] Elution method: gradient elution;

[0104] Elution method: gradient elution; the gradient elution program is shown in Table 2;

[0105] The detection wavelength is 235nm;

[0106] Flow rate: 0.45 mL / min;

[0107] The column temperature was 50°C;

[0108] The injection volume was 10 μL.

[0109] Table 2 Gradient elution program

[0110] Time (min) Mobile phase A (%) Mobile phase B (%) 0～3 100～92 0～8 3～12 92～61 8～39 12～14.5 61～0 39～100 14.5～15.5 0 100 15.5～15.51 0～100 100～0 15.51～19 100 0

[0111] 1-3 Experimental process and conclusion

[0112] Accurately measure the above solutions (each impurity location solution, system suitability solution), inject them into the ultra-high performance liquid chromatograph respectively, and record the chromatogram.

[0113] The results are shown in Table 3 and the system suitability solution spectrum is shown in Figure 1 , impurity localization map see Figure 2 (Impurity A), Figure 3 (Impurity B), Figure 4 (Impurity C), Figure 5 (Impurity D) and specificity overlay diagram see Figure 6 .

[0114] Table 3 System suitability and impurity location results

[0115]

[0116] Conclusion: The diluent does not interfere with the separation and detection of the refenacin nebulized inhalation solution and various impurities; the elution order of the components in the system suitability solution is impurity A, impurity C, refenacin, impurity B and impurity D, and the separation between each chromatographic peak is greater than 1.5. The specificity of the present invention is good.

[0117] Example 2: Strong degradation experiment

[0118] 2-1 Solution preparation

[0119] Multiple test samples of Example 1 were mixed and used as degradation refenacin solution, and acid, alkali, oxidation, high temperature and light destructive tests were performed respectively.

[0120] The test conditions are as follows:

[0121] 1) Acid destruction: Accurately measure 1 mL of the sample solution for degradation, add 0.05 mL of 1.0 mol / L hydrochloric acid solution, incubate at room temperature in the dark for about 50 h, and add 0.05 mL of 1.0 mol / L sodium hydroxide solution to neutralize the solution.

[0122] 2) Alkali destruction: Accurately measure 1 mL of the sample solution for degradation, add 0.05 mL of 1.0 mol / L sodium hydroxide solution, place at room temperature away from light for about 27 h, and add 0.05 mL of 1.0 mol / L hydrochloric acid solution to neutralize the solution.

[0123] 3) Oxidative destruction: Accurately measure 1 mL of the sample solution for degradation, add 0.1 mL of 30% hydrogen peroxide solution, and place at room temperature in the dark for 24 hours. Prepare the oxidative degradation blank solution in the same way.

[0124] 4) High temperature destruction: Take 10 mL of the sample solution for degradation and place it in a 100°C water bath for 5 hours.

[0125] 5) Light damage: Take several samples and place them in a light box for 10 days (total illumination not less than 1.2×106Lux·hr, near-ultraviolet light energy not less than 200W·hr / m 2 ).

[0126] 2-2 Chromatographic conditions

[0127] Same as Example 1.

[0128] 2-3 Experimental process and conclusion

[0129] Accurately measure the above solution (i.e., the refenacin solution destroyed by acid, alkali, oxidation, high temperature and light), inject it into the ultra-high performance liquid chromatograph, and record the chromatogram.

[0130] The results are shown in Table 4 and the destructive test chromatogram is shown in Figure 7 (Not destroyed), Figure 8 (acid damage), Fig. 9 (Alkali destruction), Fig.10 (oxygen destruction), Fig.11 (High temperature damage), Fig.12 (Light damage).

[0131] Table 4 Impurity detection results under different degradation conditions

[0132]

[0133]

[0134] Conclusion: The nebulized inhalation solution of revenacin is relatively stable under acid degradation and oxidative degradation conditions. It is relatively unstable under light, high temperature and alkaline degradation conditions, and more impurities grow. The main degradation impurity is impurity B, which grows significantly under alkaline degradation conditions.

[0135] Under each degradation condition, the main peak purity is qualified (purity angle is less than the purity threshold), and the materials are all conserved. Under each degradation condition, the diluent does not interfere with the determination of related substances. The related substance detection method can effectively detect the degradation products in the product. The known impurity peaks in each strong degradation test solution are well separated, and the separation degree of the main peak and the adjacent impurity peaks is greater than 1.5.

[0136] Example 3: Linearity and range experiments and determination of relative correction factors

[0137] 3-1 Solution preparation

[0138] Accurately weigh appropriate amounts of each impurity reference substance and refenacin reference substance, and prepare a series of concentration linearity test solutions with concentrations ranging from the quantitative limit to about 1.5%.

[0139] 3-2 Chromatographic conditions

[0140] Same as Example 1.

[0141] 3-3 Experimental process and conclusion

[0142] The above solution (i.e., the solution prepared in step 3-1) was accurately measured and injected into the ultra-high performance liquid chromatograph to record the chromatogram. The results are shown in Table 5. A linear regression equation was made with the concentration as the abscissa and the peak area as the ordinate. Another experimenter then performed a linear test on another instrument and calculated the correction factor. The average value was calculated with the measurement result of experimenter 1 as the correction factor for the impurity.

[0143] Table 5 Linear range and relative correction factor (f) of refenacin and impurities A, B and C

[0144]

[0145] Conclusion: Within the determination range, revenacin and its impurities have a good linear relationship and meet the methodological requirements; the relative correction factor is calculated by the ratio of the slope of the principal component regression line to the slope of the impurity regression line. The relative correction factors of impurity A, impurity B, impurity C and impurity D are 1.9, 0.9, 0.5 and 0.6, respectively. Therefore, impurity A, impurity B, impurity C and impurity D are quantitatively determined by the principal component self-control method with correction factors. The peak area of ​​each impurity on the chromatogram of the test solution is measured, multiplied by the corresponding correction factor, and compared with the peak area of ​​the main component of the control solution to calculate the content of each impurity.

[0146] Example 4: Limit of Quantitation and Limit of Detection

[0147] 4-1 Solution preparation

[0148] The linear solution in Implementation Case 3 was diluted step by step, and the concentration when the signal-to-noise ratio S / N≈3 was taken as the detection limit, and the concentration when S / N≈10 was taken as the quantification limit.

[0149] 4-2 Chromatographic conditions

[0150] Same as implementation case 1.

[0151] 4-3 Experimental process and conclusion

[0152] Accurately measure the above solution, inject it into ultra-high performance liquid chromatography, record the chromatogram, and the results are shown in Table 6.

[0153] Table 6 Quantitation limit and detection limit test results

[0154]

[0155] Conclusion: The quantitative limits of each impurity are lower than the impurity reporting limit of this product (0.1%). The detection limit and quantitative limit of the present invention both meet the requirements for quantitative detection of each impurity in the test sample and have high sensitivity.

[0156] Example 5: Accuracy Experiment

[0157] The accuracy was investigated through recovery test, and the study covered the limit concentration range of 50% to 150%.

[0158] 5-1 Solution preparation

[0159] Impurity stock solution: Take appropriate amount of impurity A, B, C, and D reference substances respectively, weigh accurately, place in different volumetric bottles, add 50% methanol water to dissolve and dilute to the scale, shake well, and use as the impurity reference substance stock solution.

[0160] Accurately measure 0.5mL, 1.0mL and 1.5mL of the mixed impurity reference stock solution into a 200mL volumetric flask, add the test solution to dilute to the scale, shake well, and prepare 3 parallel copies for each spiked concentration level.

[0161] 5-2 Chromatographic conditions

[0162] Same as Example 1.

[0163] 5-3 Experimental process and conclusion

[0164] Accurately measure the above solution, inject it into ultra-high performance liquid chromatography, record the chromatogram, and calculate the accuracy by peak area according to the external standard method. The results are shown in Table 7.

[0165] Table 7 Accuracy test results

[0166]

[0167]

[0168]

[0169] Conclusion: Under 50%, 100% and 150% accuracy solutions, the average recoveries of impurity A, impurity B, impurity C and impurity D were 100.7%, 100.1%, 99.6% and 100.1%, respectively, and the RSDs were 1.1%, 1.4%, 2.5% and 1.6%, respectively, indicating that the established self-control method with correction factor is suitable for the quantitative analysis of known impurities A, impurity B, impurity C and impurity D. The test results show that the present invention has good accuracy.

[0170] Example 6: Precision Experiment

[0171] 6-1 Solution preparation

[0172] Test solution: Accurately measure 1.0 mL of the impurity reference stock solution in Implementation Case 5 and place it in a 200 mL volumetric flask. Add the test solution to dilute to the mark, shake well, and prepare 6 spiked solutions in parallel.

[0173] Control solution: Accurately measure an appropriate amount of the above test solution and dilute it with diluent to make a solution containing approximately 0.6 μg of refenacin per 1 mL of solution. Prepare 6 spiked solutions in parallel.

[0174] 6-2 Chromatographic conditions

[0175] Same as Example 1.

[0176] 6-3 Experimental process and conclusion

[0177] Accurately measure the above solution, inject it into the ultra-high performance liquid chromatograph, record the chromatogram, calculate the impurity content, and the precision results are shown in Table 8.

[0178] Table 8 Precision test results

[0179] sample Impurity A(%) Impurity B(%) Impurity C(%) Impurity D(%) 1 0.55 0.76 0.55 0.55 2 0.54 0.77 0.55 0.54 3 0.53 0.74 0.53 0.56 4 0.55 0.77 0.54 0.55 5 0.54 0.75 0.55 0.53 6 0.56 0.74 0.54 0.55 average(%) 0.55 0.76 0.54 0.55 RSD(%) 1.92 1.83 1.50 1.89

[0180] Conclusion: The test results of 6 replicate sample solutions prepared in parallel showed that there was no unknown impurity greater than the reporting limit, and the RSD value of each impurity was less than 5%, which proved that the detection method of the present invention had good repeatability.

[0181] Example 7: Solution stability experiment

[0182] 7-1 Solution preparation

[0183] The spiked test solution and control solution in Example 6 were sampled and tested at different time points.

[0184] 7-2 Chromatographic conditions

[0185] Same as Example 1.

[0186] 7-3 Experimental process and conclusion

[0187] The above solution was accurately measured and injected into the ultra-high performance liquid chromatograph. The chromatogram was recorded. The solution stability results are shown in Table 9.

[0188] Table 9 Solution stability test results

[0189]

[0190] Conclusion: After the spiked test solution and the control solution were placed at room temperature for 48 hours, there was no obvious change in impurities A, B, C, D and their own controls, and no new impurities greater than the reporting limit were produced, indicating that the spiked test solution and the control solution were stable within 48 hours.

[0191] Example 8: Durability Test

[0192] Durability refers to the degree to which the test results are not affected by slight changes in the chromatographic conditions. The preparation of the system suitability solution and the test solution is the same as in Example 1 and Example 6. On the basis of the test conditions of Example 1, the temperature, flow rate, trifluoroacetic acid content and different chromatographic columns are changed. The durability test results of the test method are shown in Tables 10 and 11.

[0193] Table 10 System suitability solution test results

[0194]

[0195]

[0196]

[0197] Table 11 Test results of test solution

[0198]

[0199]

[0200] Conclusion: When the column temperature was changed by ±5°C, the flow rate was ±0.05 mL / min, the trifluoroacetic acid was ±1 v%, and different chromatographic columns were replaced, the system suitability parameters and the RSD values ​​of the impurity contents all met the requirements, and the durability of the present invention was good.

[0201] Based on the above experimental results, the detection method of the present invention is used to detect the refenacin nebulized inhalation solution. The main related substances and the main components can be well separated, and the main impurity peaks are completely separated. A significantly higher impurity detection rate is obtained, which can provide a basis for the quality control of the refenacin nebulized inhalation solution, thereby ensuring its clinical efficacy.

[0202] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances in refenacin aerosol inhalation solution, characterized in that: The steps include: The refenacin nebulized inhalation solution was analyzed by ultra-high performance liquid chromatography, and the presence of refenacin and related impurities were identified by chromatogram; Chromatographic conditions included: The chromatographic column is filled with octadecylsilane bonded silica gel; Mobile phase A is a mixed solution of trifluoroacetic acid and water, and mobile phase B is a mixed solution of acetonitrile, water and trifluoroacetic acid; Gradient elution was used.

2. The method according to claim 1, characterized in that The related substances include four impurities, namely impurities A, B, C and D, whose structures are as follows:

3. The method according to claim 1, characterized in that The mobile phase A is a mixed solution of 3-5v% trifluoroacetic acid and water, 3-5v% trifluoroacetic acid: water = 10:990, volume ratio; The mobile phase B is a mixed solution of acetonitrile, water and 3-5v% trifluoroacetic acid, acetonitrile: water: 3-5v% trifluoroacetic acid = 900:90:10, volume ratio.

4. The method according to claim 1, characterized in that In volume percentage, the gradient elution is as follows: From 0 to 3 min, mobile phase A was reduced from 100% to 92%, and mobile phase B was increased from 0% to 8%; From 3 to 12 min, mobile phase A decreased from 92% to 61%, and mobile phase B increased from 8% to 39%; From 12 to 14.5 min, mobile phase A was reduced from 61% to 0%, and mobile phase B was increased from 39% to 100%; 14.5-15.5 min, mobile phase A 0%, mobile phase B 100%; 15.5-15.54 min, mobile phase A increased from 0% to 100%, and mobile phase B decreased from 100% to 0%; 15.51~19min, mobile phase A 100%, mobile phase B 0%.

5. The method according to claim 1, characterized in that The chromatographic column is Hypersil GOLD, 100 mm×2.1 mm, 1.9 μm or Hypersil GOLD Vanquish, 100 mm×2.1 mm, 1.9 μm.

6. The method according to claim 1, characterized in that The chromatographic conditions also include: column temperature of 45-55°C, preferably 50°C; flow rate of 0.4-0.5 mL / min, preferably 0.45 mL / min; injection volume of 9-11 μL; detection wavelength of 230-240 nm, preferably 235 nm.

7. The method according to claim 1, characterized in that The chromatographic conditions are: The chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm; Mobile phase A is a mixed solution of 4% trifluoroacetic acid and water, 4% trifluoroacetic acid: water = 10:990, volume ratio; mobile phase B is a mixed solution of acetonitrile, water and 4% trifluoroacetic acid, acetonitrile: water: 4% trifluoroacetic acid = 900:90:10, volume ratio; Gradient elution; detection wavelength was 235 nm, column temperature was 50 °C, flow rate was 0.45 mL / min, injection volume was 10 μL.

8. A method for quantitatively detecting related substances in refenacin aerosol inhalation solution, characterized in that: The steps include: dissolving revenacin and related substances in a solvent to obtain a system suitability solution; Performing ultra-high performance liquid chromatography analysis on the system suitability solution using the method described in any one of claims 1 to 7 to obtain a chromatogram of the system suitability solution; The refenacin nebulized inhalation solution is subjected to ultra-high performance chromatography analysis using the method described in any one of claims 1 to 7 to obtain a chromatogram of the refenacin nebulized inhalation solution; Calculate the content of related substances by correction factor and peak area in chromatogram; Preferably, the solvent is an aqueous citric acid solution or an aqueous methanol solution.

9. A quality assessment method for refenacin nebulized inhalation solution, characterized in that: The method according to any one of claims 1 to 8 is adopted.

10. Application of related substances in quality control of refenacin nebulized inhalation solution; The related substances include four impurities, namely impurities A, B, C and D, whose structures are as follows:

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