Detection method of rafenasin related substances

Abstract

The invention discloses a method for detecting rafenasin related substances, which comprises the following steps: detecting by adopting a high performance liquid chromatography under the chromatographic conditions that a stationary phase is an octadecyl silane bonded silica gel chromatographic column, a mobile phase A is a phosphate buffer aqueous solution, a mobile phase B is methanol, and gradient elution is carried out; the gradient elution procedure is as follows: within 0-10 minutes, the volume fraction of the mobile phase A is gradually changed from 88-92% to 73-77%; within 10-43 min, the volume fraction of the mobile phase A is gradually changed from 73-77% to 38-42%; within 43-45 min, the volume fraction of the mobile phase A is gradually changed from 38-42% to 88-92%; within 45-50 min, the volume fraction of the mobile phase A is kept at 88-92%. According to the chromatographic conditions disclosed by the invention, effective separation between the main peak of the rafenasin and each impurity can be realized, the specificity is good, and the sensitivity is high.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting refenacin-related substances. Background Art

[0002] The chemical name of revefenacin is: N-({1,1'-biphenyl}-2-yl)carbamic acid-1-(2-{4-[(4-carbamoylpiperidin-1-yl)methyl]-N-methylbenzamido}ethyl)piperidin-4-yl-1-ester, and its chemical formula is shown in formula (I):

[0003]

[0004] Revenacin is a long-acting muscarinic antagonist, commonly known as an anticholinergic drug. It binds to the M3 muscarinic receptors on bronchial smooth muscle to inhibit bronchial contraction caused by the release of acetylcholine from parasympathetic nerve terminals. It is mainly used clinically for the maintenance treatment of chronic obstructive pulmonary disease, including the maintenance treatment of chronic bronchitis and emphysema with dyspnea and the prevention of acute attacks. Revenacin solution for inhalation has been approved by the US FDA for marketing under the trade name Yupelri.

[0005] At present, the synthesis route of revenacin is relatively long, and it is easy to introduce multiple impurities. In order to monitor the quality and safety of the product, its impurities need to be tested. However, there are currently few detection methods for revenacin impurities, and the impurities detected are also relatively small. Summary of the invention

[0006] Based on the technical problems existing in the background technology, the present invention proposes a method for detecting refenacin-related substances. The chromatographic conditions of the present invention can effectively separate the main peak of refenacin from various impurities, and have good specificity and high sensitivity.

[0007] The present invention provides a method for detecting refenacin-related substances, comprising the following steps: using high performance liquid chromatography for detection, the chromatographic conditions are: the stationary phase is an octadecylsilane bonded silica gel chromatographic column, the mobile phase A is a phosphate buffered aqueous solution, the mobile phase B is methanol, and gradient elution is performed;

[0008] The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 88-92% to 73-77%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 73-77% to 38-42%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 38-42% to 88-92%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 88-92%.

[0009] Preferably, the gradient elution procedure is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 90% to 75%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 75% to 40%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 40% to 90%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 90%.

[0010] Preferably, mobile phase A is a sodium dihydrogen phosphate buffered aqueous solution.

[0011] Preferably, the concentration of sodium dihydrogen phosphate is 2.3-2.5 g / L, and more preferably the concentration of sodium dihydrogen phosphate is 2.4 g / L.

[0012] Preferably, the pH of the sodium dihydrogen phosphate buffer solution is 2.3-2.7, more preferably the pH of the sodium dihydrogen phosphate buffer solution is 2.5.

[0013] The pH of the sodium dihydrogen phosphate buffered aqueous solution can be adjusted with phosphoric acid.

[0014] Preferably, the specifications of the chromatographic column are 250×4.6 mm, 5 μm.

[0015] Preferably, the model of the chromatographic column is Syncronis C18.

[0016] Preferably, the temperature of the chromatographic column is 35-45°C, more preferably the temperature of the chromatographic column is 40°C.

[0017] Preferably, the detection wavelength is 233-237 nm, more preferably the detection wavelength is 235 nm.

[0018] Preferably, the flow rate is 0.8-1.2 ml / min, more preferably the flow rate is 1.0 ml / min.

[0019] Preferably, the content of revenacin-related substances is calculated according to the chromatographic peak area using the principal component self-reference method.

[0020] The principal component self-control method is a conventional method in the art, and its steps are generally as follows: prepare a blank solution, a test solution, and a control solution respectively, adopt the above-mentioned chromatographic conditions, inject and detect in sequence, record the chromatogram and calculate the content of the related substances of refenacin according to the principal component self-control method based on the chromatographic peak area.

[0021] The injection volume may be 25 μl.

[0022] The blank solution was prepared by mixing mobile phase A and mobile phase B in a volume ratio of 90:10.

[0023] The above-mentioned test solution is: the refenacin solution preparation for inhalation is used as the test solution, or the refenacin raw material blank solution is used to prepare a 0.06 mg / ml solution as the test solution.

[0024] The above-mentioned control solution is: accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with the blank solution, and shake well.

[0025] Preferably, the revenacin-related substances include: at least one of impurity RF-AC, impurity RF-AL, impurity RF-SM4-2ABP, impurity RF-BPA, impurity RF-IPBA, impurity RF-CZ20, impurity RF-CZ25, and impurity RF-CZ6, and the structural formula of each impurity is as follows:

[0026] Impurity RF-AC:

[0027] Impurity RF-AL:

[0028] Impurity RF-SM4-2ABP:

[0029] Impurity RF-BPA:

[0030] Impurity RF-IPBA:

[0031] Impurity RF-CZ20:

[0032] Impurity RF-CZ25:

[0033] Impurity RF-CZ6:

[0034] The present invention can detect one of the impurities individually, or can detect the above eight impurities simultaneously.

[0035] The present invention screens chromatographic conditions for eight impurities of refenacin, and finally screens out the chromatographic conditions of the present invention, which can achieve effective separation between the main peak of refenacin and each impurity, and the separation degree is greater than 1.5. The present invention has good specificity and high sensitivity, and can provide a simple and reliable detection method for quality monitoring of refenacin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the HPLC spectrum of the system suitability solution of Example 1.

[0037] Figure 2 This is the HPLC spectrum of the system suitability solution of Comparative Example 1. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is described in detail below through specific embodiments.

[0039] Example 1

[0040] A method for detecting revenacin-related substances comprises the following steps: using a Syncronis C18 (250×4.6 mm, 5 μm) chromatographic column for high performance liquid chromatography detection, wherein mobile phase A is a sodium dihydrogen phosphate buffer solution with a concentration of 2.4 g / L and a pH of 2.5; mobile phase B is methanol, the chromatographic column temperature is 40° C., the detection wavelength is 235 nm, the flow rate is 1.0 ml / min, and gradient elution is performed;

[0041] The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 90% to 75%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 75% to 40%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 40% to 90%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 90%.

[0042] Solution preparation:

[0043] The blank solution was prepared by mixing mobile phase A and mobile phase B in a volume ratio of 90:10.

[0044] Stock solutions of each impurity: Weigh appropriate amounts of impurity RF-AC reference substance, impurity RF-AL reference substance, impurity RF-SM4-2ABP reference substance, impurity RF-BPA reference substance, impurity RF-IPBA reference substance, impurity RF-CZ20 reference substance, impurity RF-CZ25 reference substance, and impurity RF-CZ6 reference substance, add 1 ml of methanol to each of them and shake to dissolve, then dilute with blank solution to make solutions with a concentration of 0.06 mg / ml, which are used as stock solutions of each impurity.

[0045] System suitability solution: Take an appropriate amount of refenacin, add blank solution and shake to dissolve, then add an appropriate amount of each impurity stock solution, dilute with blank solution to make a solution containing 0.06 mg of refenacin and 0.6 μg of each impurity per 1 ml.

[0046] operate:

[0047] Take blank solvent and system suitability solution, inject 25 μl of each, and record the chromatogram. Figure 1 And as shown in Table 1.

[0048] Figure 1 This is the HPLC spectrum of the system suitability solution of Example 1.

[0049] Table 1 Retention time and separation results of revenacin and its impurities

[0050] Peak name Retention time (min) Relative retention time Separation>1.5 Impurity RF-IPBA 5.750 0.37 / Impurity RF-BPA 14.280 0.92 70.60 Refenacin 15.520 1.00 6.97 Impurity RF-AC 16.463 1.06 5.08 Impurity RF-CZ20 17.002 1.10 3.57 Impurity RF-CZ25 19.877 1.28 18.42 Impurity RF-CZ6 21.817 1.41 11.87 Impurity RF-AL 24.673 1.59 15.18 Impurity RF-SM4-2ABP 31.097 2.00 23.46

[0051] From Table 1 and Figure 1 It can be seen that the separation between the eight impurity peaks and between the main peak and the impurity peak in the system suitability solution is greater than 1.5, which can be effectively separated. The detection method of the present invention has good specificity.

[0052] Comparative Example 1

[0053] Mobile phase B is a methanol aqueous solution with a volume fraction of 50%, and the other aspects are the same as in Example 1. After testing the chromatogram of the system suitability solution, only 4 impurity peaks were found, and the above 8 impurities could not be effectively separated from refenacin. Figure 2 shown. Figure 2 This is the HPLC spectrum of the system suitability solution of Comparative Example 1.

[0054] Example 2 (Sensitivity Test)

[0055] The detection method is the same as in Example 1.

[0056] Solution preparation:

[0057] The blank solution was the same as in Example 1.

[0058] Test solution: Take the raw material of refenacin and prepare a solution with a concentration of 0.06 mg / ml using blank solution.

[0059] Operation: dilute the sample solution step by step and perform sampling test until S / N≈10 and S / N≈3. The quantitative limit is calculated to be 0.009 μg / ml and the detection limit is 0.003 μg / ml, i.e., impurities equivalent to 0.005% of the content of the sample solution can be detected. The detection method of the present invention has high sensitivity.

[0060] Example 3 (Recovery Test)

[0061] The detection method is the same as in Example 1.

[0062] Solution preparation:

[0063] The preparation method of blank solution and system suitability solution is the same as that of Example 1.

[0064] 80% test solution: Take impurity RF-IPBA, impurity RF-BPA, impurity RF-AC, impurity RF-CZ20, impurity RF-CZ25, impurity RF-CZ6, impurity RF-AL, impurity RF-SM4-2ABP reference substance, and refenacin reference substance, and use blank solution to prepare a mixed solution with each impurity concentration of 0.48μg / mL and refenacin concentration of 0.06mg / ml.

[0065] 100% sampled test solution: Take impurity RF-IPBA, impurity RF-BPA, impurity RF-AC, impurity RF-CZ20, impurity RF-CZ25, impurity RF-CZ6, impurity RF-AL, impurity RF-SM4-2ABP reference substance, and refenacin reference substance, and use blank solution to prepare a mixed solution with each impurity concentration of 0.6 μg / mL and refenacin concentration of 0.06 mg / ml.

[0066] 120% sample test solution: take impurity RF-IPBA, impurity RF-BPA, impurity RF-AC, impurity RF-CZ20, impurity RF-CZ25, impurity RF-CZ6, impurity RF-AL, impurity RF-SM4-2ABP reference substance, and refenacin reference substance, and use blank solution to prepare a mixed solution with each impurity concentration of 0.72μg / mL and refenacin concentration of 0.06mg / ml.

[0067] Operation: Take each solution and inject it for detection in turn, and calculate the recovery rate according to the external standard method.

[0068] The average recoveries of impurity RF-IPBA, impurity RF-BPA, impurity RF-AC, impurity RF-CZ20, impurity RF-CZ25, impurity RF-CZ6, impurity RF-AL and impurity RF-SM4-2ABP were measured to be 96.8%, 97.4%, 94.3%, 93.8%, 94.2%, 96.1%, 95.4% and 98.3%, respectively, and the RSDs were 3.3%, 2.5%, 6.1%, 5.4%, 2.6%, 6.1%, 4.8% and 5.1%, respectively; the detection method of the present invention has high accuracy.

[0069] Example 4 (Sample Testing)

[0070] The detection method is the same as in Example 1.

[0071] Solution preparation:

[0072] The preparation method of blank solution and system suitability solution is the same as that of Example 1.

[0073] Test solution: The refenacin solution for inhalation is the test solution.

[0074] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with the blank solution, and shake well.

[0075] Operation: Take each solution and inject it for testing in turn, and calculate the content of each impurity in the test sample according to the main component self-control method. The results show that impurities RF-AC, RF-AL, RF-SM4-2ABP, RF-CZ20, and RF-CZ6 are all effectively detected, while impurities RF-BPA, RF-IPBA, and RF-CZ25 are not detected. The test results are shown in Table 2.

[0076] Table 2 Test results

[0077]

[0078]

[0079] Example 5

[0080] A method for detecting revenacin-related substances comprises the following steps: using a Syncronis C18 (250×4.6 mm, 5 μm) chromatographic column for high performance liquid chromatography detection, wherein mobile phase A is a sodium dihydrogen phosphate buffer solution with a concentration of 2.4 g / L and a pH of 2.3; mobile phase B is methanol, the chromatographic column temperature is 35° C., the detection wavelength is 235 nm, the flow rate is 1.0 ml / min, and gradient elution is performed;

[0081] The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 90% to 75%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 75% to 40%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 40% to 90%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 90%.

[0082] The solution preparation and operation are the same as in Example 4.

[0083] The results showed that the separation between the eight impurity peaks and between the main peak and the impurity peak in the system suitability solution was greater than 1.5, indicating that they could be effectively separated.

[0084] Example 6

[0085] A method for detecting revenacin-related substances comprises the following steps: using a Syncronis C18 (250×4.6 mm, 5 μm) chromatographic column for high performance liquid chromatography detection, wherein mobile phase A is a sodium dihydrogen phosphate buffer solution with a concentration of 2.4 g / L and a pH of 2.7; mobile phase B is methanol, the chromatographic column temperature is 40° C., the detection wavelength is 233 nm, the flow rate is 0.8 ml / min, and gradient elution is performed;

[0086] The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 90% to 75%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 75% to 40%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 40% to 90%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 90%.

[0087] The solution preparation and operation are the same as in Example 3.

[0088] The results showed that the separation between the eight impurity peaks and between the main peak and the impurity peak in the system suitability solution was greater than 1.5, indicating that they could be effectively separated.

[0089] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting refenacin-related substances, characterized in that: The method comprises the following steps: using high performance liquid chromatography for detection, wherein the chromatographic conditions are: the stationary phase is an octadecylsilane bonded silica gel chromatographic column, the mobile phase A is a phosphate buffered aqueous solution, the mobile phase B is methanol, and gradient elution is performed; The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 88-92% to 73-77%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 73-77% to 38-42%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 38-42% to 88-92%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 88-92%.

2. The method for detecting refenacin-related substances according to claim 1, characterized in that: The procedure of gradient elution is: within 0-10 minutes, the volume fraction of mobile phase A gradually changes from 90% to 75%; within 10-43 minutes, the volume fraction of mobile phase A gradually changes from 75% to 40%; within 43-45 minutes, the volume fraction of mobile phase A gradually changes from 40% to 90%; within 45-50 minutes, the volume fraction of mobile phase A is maintained at 90%.

3. The method for detecting refenacin-related substances according to claim 1 or 2, characterized in that: Mobile phase A is sodium dihydrogen phosphate buffered aqueous solution; preferably, the concentration of sodium dihydrogen phosphate is 2.3-2.5 g / L; preferably, the pH of the sodium dihydrogen phosphate buffered aqueous solution is 2.3-2.

7.

4. The method for detecting refenacin-related substances according to any one of claims 1 to 3, characterized in that: The specifications of the chromatographic column are 250×4.6 mm, 5 μm.

5. The method for detecting refenacin-related substances according to any one of claims 1 to 4, characterized in that: The model of the chromatographic column is Syncronis C18.

6. The method for detecting refenacin-related substances according to any one of claims 1 to 5, characterized in that: The column temperature was 35-45°C.

7. The method for detecting revenacin-related substances according to any one of claims 1 to 6, characterized in that: The detection wavelength is 233-237nm.

8. The method for detecting refenacin-related substances according to any one of claims 1 to 7, characterized in that: The flow rate is 0.8-1.2ml / min.

9. The method for detecting refenacin-related substances according to any one of claims 1 to 8, characterized in that: The content of revenacin-related substances was calculated according to the chromatographic peak area using the principal component self-reference method.

10. The method for detecting refenacin-related substances according to any one of claims 1 to 9, characterized in that: Refenacin-related substances include: at least one of impurity RF-AC, impurity RF-AL, impurity RF-SM4-2ABP, impurity RF-BPA, impurity RF-IPBA, impurity RF-CZ20, impurity RF-CZ25, and impurity RF-CZ6. The structural formula of each impurity is as follows: Impurity RF-AC: Impurity RF-AL: Impurity RF-SM4-2ABP: Impurity RF-BPA: Impurity RF-IPBA: Impurity RF-CZ20: Impurity RF-CZ25: Impurity RF-CZ6:

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