Method for separating rafenasin and impurities thereof by high performance liquid chromatography

Abstract

The invention relates to the field of analytical chemistry, and particularly discloses a method for separating impurities in a rafenasin inhalation solution by high performance liquid chromatography, which adopts octadecylsilane chemically bonded silica as a filler, a dibasic hydrogen phosphate solution as a mobile phase A and methanol-acetonitrile as a mobile phase B to carry out gradient elution, according to the method, all known impurities and unknown impurities in a rafenasin inhalation solution can be effectively separated, five impurities are effectively separated in one chromatography, the separation degree between a main peak and an impurity IX reaches 2.7, the separation degrees of other impurities are all larger than 1.5, the specificity is good, interference of a blank solvent and other unknown impurities is avoided, the separation efficiency is high, the anti-interference capability is high, and the method is suitable for industrial production. The analysis speed is high, and the detection accuracy is high, so that the quality of the rafenasin inhaled solution is effectively controlled.

Description

Technical Field

[0001] The present invention relates to the field of analytical technologies, and specifically relates to a method for determining impurities in revefenacin inhalation solution by high performance liquid chromatography. Background Art

[0002] Revefenacin is a long-acting muscarinic antagonist, commonly known as an anticholinergic drug, which has similar affinities for subtypes of muscarinic receptors M1 to M5. In the airway, it exhibits pharmacological effects by inhibiting M3 receptors on smooth muscle, resulting in bronchodilation.

[0003] This product is an inhalation solution, and its material composition includes revefenacin, citric acid, sodium citrate, sodium chloride, hydrochloric acid, sodium hydroxide, and water for injection. The revefenacin inhalation solution adopts a high-temperature sterilization filtration process, and process and degradation impurities may be generated during the process and subsequent high-temperature sterilization process.

[0004] Table 1 briefly describes the possible sources of impurities in the preparation process of revefenacin inhalation solution:

[0005] Table 1 Table of Impurity Sources

[0006]

[0007] As can be seen from Table 1, during the preparation process of revefenacin inhalation solution, various process impurities and degradation impurities may be generated, and the structures of multiple impurities are similar. For example, the difference between impurity I and impurity II is only the aldehyde group and carboxyl group; the difference between impurity I and impurity IX is only the aldehyde group and hydroxyl group; the difference between impurity II and impurity IX is only the carboxyl group and hydroxyl group; the structures of each impurity and revefenacin have the same parent nucleus structure, and impurity IX has a similar polarity to the main peak. The similarity of multiple groups of impurities and the similarity of the impurity and main peak structures bring difficulties to the separation.

[0008] Only by controlling the amount of impurities in the revefenacin inhalation solution below a certain limit can the quality of the product be effectively guaranteed.

[0009] Currently, there is no method reported for the quality control of revefenacin inhalation solution in China. Summary of the Invention

[0010] The present invention provides a method for separating and determining impurities in revefenacin inhalation solution by high performance liquid chromatography, which is not interfered by blank solvents and other unknown impurities, and the resolution between the main peak and impurity IX reaches 2.7, and the resolution between other impurities is greater than 1.5. It is simple, fast, and has high accuracy.

[0011] In order to achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows:

[0012] Method for Separating Relafenacin from Its Impurities by High Performance Liquid Chromatography, using octadecylsilyl silica gel as the packing material; using a dibasic phosphate solution as mobile phase A and methanol-acetonitrile as mobile phase B for gradient elution, and the gradient elution program is as follows:

[0013] Table 2 Gradient Elution Program Table

[0014] Time (min) Mobile phase A (V / V%) Mobile phase B (V / V%) 0 70～80 20～30 30 50～60 40～50 55 20～30 70～80 70 20～30 70～80 70.1 70～80 20～30 80 70～80 20～30

[0015] The impurities are the compounds shown as impurity I, impurity II, impurity IV, impurity VII, and impurity IX:

[0016]

[0017]

[0018] Impurity IX has a similar polarity to the main peak, and the elution time of the chromatographic peaks differs by within 0.5 min, and there will be partial overlap of the chromatographic peaks, and the resolution is difficult to reach 1.5. The effective separation of impurity IX from the main peak can be achieved by the present invention.

[0019] Preferably in the method, the concentration of the dibasic phosphate solution is 0.015 - 0.025 mol / L, and the volume ratio of methanol to acetonitrile is 1:4 - 6.

[0020] Preferably, the flow rate is 0.8 - 1.2 mL / min.

[0021] Preferably, the initial volume ratio of mobile phase A to mobile phase B is 73:27 - 77:23.

[0022] Preferably, the detection wavelength is 220 ± 2 nm.

[0023] Preferably, the column temperature is 30 ± 5 °C.

[0024] During detection, take the same volume of the system suitability solution, reference solution, and test solution, and inject them into the liquid chromatograph;

[0025] System suitability solution: Take appropriate amounts of the reference substances of impurity I, impurity II, impurity IV, impurity VII, and impurity IX, dissolve them in diluent 1 to prepare the stock solutions of each impurity reference substances; take an appropriate amount of the relafenacin reference substance, dissolve it in diluent 1 to prepare the relafenacin reference substance stock solution; take appropriate amounts of the impurity reference substance stock solution and the relafenacin reference substance stock solution, dissolve them in diluent 2 to prepare the system suitability solution;

[0026] Test solution: Take the relafenacin inhalation solution as the test solution;

[0027] Reference substance solution: Weigh an appropriate amount of revefenacin reference substance, dissolve it in diluent 1 to prepare a reference substance stock solution; take an appropriate amount of the reference substance stock solution and dilute it with diluent 2 to prepare a reference substance solution.

[0028] The diluent 1 is methanol, and the diluent 2 is mobile phase A: mobile phase B with a volume ratio of 75:25.

[0029] The inventors unexpectedly found that when using conventional diluents such as methanol and water, the peak shape of the main peak in the revefenacin reference substance solution was poor, and there was a solvent effect (tailing factor greater than 1.5); at the same time, the peak area of the revefenacin peak was not proportional to the concentration, which would lead to a decrease in the accuracy of impurity quantification. After treatment with diluent 1 + diluent 2, the peak shape of the main peak can be significantly improved, the solvent effect is eliminated, and the symmetry factor reaches 1.0 (the specified range is 0.9 - 1.1); the peak area of the revefenacin main peak is linearly proportional to the solution concentration, greatly improving the accuracy of impurity quantification.

[0030] When quantitatively determining impurity I, impurity II, impurity IV, impurity VII, and impurity IX, it is calculated by the external standard method of the main component without correction factor.

[0031] When quantitatively determining impurity I, impurity II, impurity IV, impurity VII, and impurity IX, the control limit is not greater than 1.0% of the main peak area of the revefenacin reference substance.

[0032] The beneficial effect of the present invention is that the method of the present invention has strong specificity. Using an octadecylsilane-bonded silica gel column, with a disodium hydrogen phosphate solution as mobile phase A and methanol - acetonitrile as mobile phase B for gradient elution, it ensures that revefenacin can be effectively separated from impurities and between impurities in a chromatographic method, without being interfered by the blank solvent and other unknown impurities. Especially, the resolution between the main peak and impurity IX reaches 2.7, which is greater than 1.5, and the resolution between other impurities is greater than 1.5, meeting the requirements of the related substance detection method. This method has the advantages of simplicity, rapidity, and high accuracy. Description of the Drawings

[0033] Figure 1 It is a comparison diagram of the test solution and the system suitability solution;

[0034] Figure 2 It is a comparison diagram under different flow rate conditions;

[0035] Figure 3 It is a comparison diagram under different detection wavelengths;

[0036] Figure 4 It is a comparison diagram under different column temperature conditions;

[0037] Figure 5 It is a linear diagram of revefenacin;

[0038] Figure 6 It is the linear graph of impurity I;

[0039] Figure 7 It is the linear graph of impurity II;

[0040] Figure 8 It is the linear graph of impurity IV;

[0041] Figure 9 It is the linear graph of impurity VII;

[0042] Figure 10 It is the linear graph of impurity IX.

[0043] Specific implementation cases

[0044] The present invention will be further described below through various embodiments, but these embodiments do not limit the protection scope of the present invention.

[0045] Example 1: Separation of impurities in the inhaled solution of revefenacin

[0046] Chromatographic conditions: Use octadecylsilane-bonded silica gel as the filler (recommended Phenomenex Gemini C18 4.6×250mm, 5μm or a chromatographic column with equivalent efficiency); use 0.02mol / L dipotassium hydrogen phosphate solution (adjust the pH value to 7.5 with phosphoric acid) as mobile phase A, and use methanol-acetonitrile (1:5) as mobile phase B, and perform gradient elution according to Table 3; the flow rate is 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 220nm; the injection volume is 100 μl.

[0047] Table 3 Gradient elution program table for Example 1

[0048] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70～80 20～30 30 50～60 40～50 55 20～30 70～80 70 20～30 70～80 70.1 70～80 20～30 80 70～80 20～30

[0049] 1. System suitability test and specificity test

[0050] (1) Solution preparation:

[0051] Diluent 1: Methanol.

[0052] Diluent 2: Mobile phase A: Mobile phase B (75:25).

[0053] Resolution solution: Precisely measure 0.5 ml of each of the above impurity reference stock solutions, place them in the same 50 ml volumetric flask, dilute to the mark with the inhaled solution of revefenacin, and mix well to obtain the resolution solution.

[0054] System suitability solution: Weigh approximately 2.9 mg each of the reference substances for impurity I, impurity II, impurity IV, impurity VII, and impurity IX, accurately weigh them, place them in separate 100-ml volumetric flasks, dissolve and dilute to the mark with diluent 1 to prepare solutions containing approximately 29 μg / ml of impurity I, impurity II, impurity IV, impurity VII, and impurity IX respectively as the stock solutions of each impurity reference substance; then weigh approximately 29 mg of the reference substance of reboxetine, accurately weigh it, place it in a 100-ml volumetric flask, dissolve it with diluent 1 and dilute to the mark, shake well, as the stock solution of the reboxetine reference substance; accurately measure 0.5 ml of each of the above stock solutions of impurity reference substances and 10.0 ml of the stock solution of the reboxetine reference substance, place them in the same 50-ml volumetric flask, dilute to the mark with diluent 2, and mix well, as the system suitability solution. At the same time, it is used as the program control solution.

[0055] Test solution: Take 5 vials of this product, mix well, as the test solution.

[0056] Reference solution: Weigh approximately 29 mg of the reference substance of reboxetine, accurately weigh it, place it in a 100-ml volumetric flask, add an appropriate amount of diluent 1, dissolve it by ultrasonic treatment, and dilute to the mark with diluent 1, shake well; accurately measure 5.0 ml of the above solution, place it in a 50-ml volumetric flask, dilute to the mark with diluent 1, shake well, to obtain the stock solution of the reference substance; take 1.0 ml of the above stock solution of the reference substance, place it in a 100-ml volumetric flask, dilute to the mark with diluent 2, shake well, and that is it.

[0057] Localization solution for each impurity: Take 5 ml of each of the above stock solutions of impurity reference substances respectively, place them in 10-ml volumetric flasks, dilute to the mark with diluent 1 respectively, and mix well, as the localization solutions for each impurity.

[0058] (2) Injection: Take 5 μl each of the blank solution, blank excipient solution, system suitability solution, resolution solution, localization solutions for each impurity, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatograms; the specific test results are shown in Tables 4 to 6 and Figure 1 .

[0059] Table 4 Specificity test results - 1

[0060]

[0061] Table 5 Specificity test results - 2

[0062]

[0063] Table 6 Specificity test results - 3

[0064]

[0065] (3) Test results: The blank solution showed no interference; the test solution did not interfere with the detection; the resolution between each known impurity and the adjacent impurity was greater than 1.5, and the resolution between the main peak and the adjacent impurity was 2.7, greater than 1.5. The system suitability and specificity of this method are good.

[0066] Example 2 - 6

[0067] The chromatographic condition parameters remain unchanged, and the following parameters are adjusted:

[0068] (1) Based on the chromatographic parameters in Example 1, the flow rates were adjusted to 0.8 ml / min and 1.2 ml / min respectively;

[0069] (2) Based on the chromatographic parameters in Example 1, the detection wavelengths were adjusted to 218 nm and 222 nm respectively;

[0070] (3) Based on the chromatographic parameters in Example 1, the column temperatures were adjusted to 25 °C and 35 °C respectively;

[0071] Under the above chromatographic conditions respectively, inject the system suitability solution, and examine the minimum resolution between each impurity in the relafenacin inhalation solution. The specific test results are shown in Table 7 and Figure 2 、 Figure 3 、 Figure 4 。

[0072] Table 7 Durability results of the system suitability solution

[0073]

[0074] The test results show that: with the changes in each chromatographic condition (including flow rate, detection wavelength, column temperature), the resolution between each impurity and between the impurity and the main peak in the system suitability solution meets the requirements, and the data results indicate that this method has good durability.

[0075] Example 7: Quantification of each impurity in the relafenacin inhalation solution

[0076] Correction factors of impurity I, impurity II, impurity IV, impurity VII, and impurity IX

[0077] Take appropriate amounts of relafenacin and reference substances of impurity I, impurity II, impurity IV, impurity VII, and impurity IX, dissolve and dilute them with methanol to prepare a series of gradient concentration mixed solutions. Accurately measure 100 μl, inject it into the liquid chromatograph, take the peak area A of each peak as the ordinate and the corresponding concentration C as the abscissa, plot the standard curves of each component, calculate the linear regression equation, and calculate the correction factor of the impurity at the same time.

[0078] The specific results are shown in Tables 8 - 13 and Figures 5 - 10 。

[0079] Table 8 Linearity of relafenacin

[0080]

[0081]

[0082] Table 9 Impurity I Linearity

[0083]

[0084] Table 10 Impurity II Linearity

[0085]

[0086] Table 11 Impurity IV Linearity

[0087]

[0088] Table 12 Impurity VII Linearity

[0089]

[0090] Table 13 Impurity IX Linearity

[0091]

[0092] Test results: When the concentration of Impurity I is in the range of 0.012 - 0.864 μg / ml, the linear relationship is good. The linear equation is y = 298.2948x - 0.9137, and R 2 is 0.9998, and the deviation of the y-axis intercept is 0.01, meeting the requirements.

[0093] When the concentration of Impurity IV is in the range of 0.013 - 0.948 μg / ml, the linear relationship is good. The linear equation is y = 244.8169x - 0.2412, and R 2 is 0.9998, and the deviation of the y-axis intercept is 0.00, meeting the requirements.

[0094] When the concentration of Levonorgestrel is in the range of 0.012 - 0.863 μg / ml, the linear relationship is good. The linear equation is y = 257.4835x - 0.4912, and R 2 is 0.9998, and the deviation of the y-axis intercept is 0.01, meeting the requirements.

[0095] When the concentration of Impurity IX is in the range of 0.011 - 0.856 μg / ml, the linear relationship is good. The linear equation is y = 310.0681x - 0.9603, and R 2 is 0.9999, and the deviation of the y-axis intercept is 0.01, meeting the requirements.

[0096] When the concentration of impurity II is in the range of 0.011 - 0.826 μg / ml, the linear relationship is good. The linear equation is y = 300.8603x - 0.1510, and R 2 is 0.9998, and the y-axis intercept deviation is 0.00, meeting the requirements.

[0097] When the concentration of impurity VII is in the range of 0.011 - 0.820 μg / ml, the linear relationship is good. The linear equation is y = 317.4785x - 0.4686, R2 is 0.9997, and the y-axis intercept deviation is 0.01, meeting the requirements.

[0098] (1) Calculation of correction factor

[0099] Using another high-performance liquid chromatograph and the above linear preparation method, the linearity of each impurity in the relafenacin inhalation solution is obtained again. According to the slopes of the linear equations of relafenacin and the impurities, the correction factor is calculated. See Table 14 for details.

[0100] Table 14 Correction factor

[0101] Experimenter 1 Calibration factor Experimenter 2 Calibration factor Average calibration factor Impurity I 0.9 Impurity I 0.9 0.9 Impurity IV 1.1 Impurity IV 1.0 1.0 Impurity IX 0.8 Impurity IX 0.9 0.8 Impurity II 0.9 Impurity II 0.9 0.9 Impurity VII 0.8 Impurity VII 0.9 0.9

[0102] (2) Recovery rates of impurity IV, impurity V and impurity VI

[0103] Take appropriate amounts of the reference substances of impurity I, impurity II, impurity IV, impurity VII, and impurity IX, and add them to the relafenacin inhalation solution at 50% - 150% of the quantitative limit and limit. Calculate the recovery rate of the impurity by taking the ratio of the measured amount minus the amount of the impurity in the sample to the added amount. The test results are shown in Tables 15 - 19.

[0104] Table 15 Test results of the recovery rate of impurity I

[0105]

[0106] Table 16 Test results of the recovery rate of impurity II

[0107]

[0108] Table 17 Test results of the recovery rate of impurity IV

[0109]

[0110] Table 18 Test results of the recovery rate of impurity VII

[0111]

[0112] Table 19 Test results of the recovery rate of impurity IX

[0113]

[0114] Comparative Example 1:

[0115] With the chromatographic condition parameters unchanged, on the basis of Example 1, both diluents 1 and 2 were adjusted to methanol. The RSD of the peak area of the reference solution and the tailing factor of the main peak are as follows:

[0116] Table 20 RSD of the peak area of the reference substance in methanol solution and the tailing factor of the main peak

[0117] RSD of peak area of reference solution Tailing factor of main peak 3.3％ 0.8

[0118] Comparative Example 2:

[0119] With the chromatographic condition parameters unchanged, on the basis of Example 1, both diluents 1 and 2 were adjusted to blank excipient solution. The RSD of the peak area of the reference solution and the tailing factor of the main peak are as follows:

[0120] Table 21 RSD of the peak area of the reference substance in blank excipient solution and the tailing factor of the main peak

[0121] RSD of peak area of reference solution Tailing factor of main peak 2.7％ 0.8

[0122] Comparative Example 3:

[0123] With the chromatographic condition parameters unchanged, on the basis of Example 1, both diluents 1 and 2 were adjusted to methanol: water - 80:20 solution. The RSD of the peak area of the reference solution and the tailing factor of the main peak are as follows:

[0124] Table 22 RSD of the peak area of the reference substance in methanol: water solution and the tailing factor of the main peak

[0125] RSD of peak area of reference solution Tailing factor of main peak 3.5％ 0.8

[0126] The results showed that the RSD of the peak area of the reference solution in Comparative Examples 1, 2, and 3 was all > 2.0%, and the tailing factor of the main peak was not within the range of 0.9 - 1.1, which did not meet the requirements of the pharmacopoeia for specificity. Under the conditions of Comparative Examples 1, 2, and 3, the separation and detection of related impurities could not be carried out.

Claims

1. A method for separating revefenacin from its impurities by high performance liquid chromatography, characterized in that: Using octadecylsilane chemically bonded silica gel as the filler; using a disodium hydrogen phosphate solution as mobile phase A and methanol-acetonitrile as mobile phase B for gradient elution, and the gradient elution program is as follows: The impurities are the compounds shown as impurity I, impurity II, impurity IV, impurity VII, and impurity IX:

2. The method according to claim 1, wherein The concentration of the disodium hydrogen phosphate solution is 0.015 - 0.025 mol / L, and the volume ratio of methanol to acetonitrile is 1:4 - 6.

3. The method according to claim 1, wherein The flow rate is 0.8 - 1.2 mL / min.

4. The method according to claim 1, wherein The initial ratio of the volume ratio of mobile phase A to mobile phase B is 73:27 - 77:

23.

5. The method according to claim 1, wherein The detection wavelength is 220 ± 2 nm.

6. The method according to claim 1, wherein The column temperature is 30 ± 5 °C.

7. The method according to claim 1, characterized in that During detection, take the same volume of the system suitability solution, reference solution, and test solution and inject them into the liquid chromatograph; System suitability solution: Take appropriate amounts of the reference substances of impurity I, impurity II, impurity IV, impurity VII, and impurity IX, dissolve them in diluent 1 to prepare the stock solutions of each impurity reference substances; take an appropriate amount of the reference substance of reboxetine, dissolve it in diluent 1 to prepare the stock solution of the reboxetine reference substance; Take appropriate amounts of the stock solutions of the impurity reference substances and the stock solution of the reboxetine reference substance, dissolve them in diluent 2 to prepare the system suitability solution; Test solution: Take the reboxetine inhalation solution as the test solution; Reference solution: Take an appropriate amount of the reboxetine reference substance, dissolve it in diluent 1 to prepare the stock solution of the reference substance; take an appropriate amount of the stock solution of the reference substance and dilute it with diluent 2 to prepare the reference solution.

8. The method according to claim 1, characterized in that, The diluent 1 is methanol, and the diluent 2 is mobile phase A:mobile phase B with a volume ratio of 75:

25.

9. The method according to claim 1, characterized in that, When quantitatively determining impurity I, impurity II, impurity IV, impurity VII, and impurity IX, calculate by the external standard method of the main component without correction factor.

10. The method according to claim 9, characterized in that when quantitatively determining impurity I, impurity II, impurity IV, impurity VII, and impurity IX, the control limits are all not greater than 1.0% times the peak area of the reboxetine reference substance.

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