Ketotifen fumarate and impurity separation method thereof

Abstract

The invention discloses a method for separating ketotifen fumarate and impurities thereof. The method comprises the step of determining the ketotifen fumarate impurities in a test sample by using a high performance liquid chromatograph. The separation and detection method disclosed by the invention is good in separation degree, simple to operate, short in peak appearance time and short in detection time, and also has good method reproducibility and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis, in particular to a method for separating and detecting impurities of ketotifen fumarate. BACKGROUND

[0002] Ketotifen fumarate (structural formula as follows) is a second-generation histamine H1 receptor antagonist, which has strong histamine H1 receptor antagonism, was listed in 1979, and is used in many regions such as Europe, Asia and Australia. The tablet is recorded in Chinese Pharmacopoeia 2000 edition. Ketotifen fumarate is mainly used for the prevention and quality of allergic asthma and allergic rhinitis asthma syndrome, and is considered as a drug with steroid-like effect but without steroid side effects. Ketotifen fumarate is particularly effective for the treatment of childhood asthma, with a cure rate of more than 80%. At present, ketotifen fumarate is mainly used in nasal spray or tablet. Since the subjects are children, improper operation of the spray may cause discomfort, and it is not recommended to use; and the use of tablets may cause heat source to affect gram-positive bacteria.

[0003] The production process of its raw material may introduce starting materials and other related substance impurities, and during the production, storage and transportation of the raw material, by-products or other degradation products may also be produced, so it is extremely important to control the quality of the starting material. Therefore, it is necessary to study the impurities, determine their structures, and establish a detection method to ensure the quality of ketotifen fumarate and drug safety. SUMMARY

[0004] In view of the above problems existing in the prior art, in order to ensure that the quality of ketotifen fumarate can be effectively controlled, The purpose of the present application is to provide a method for separating and detecting ketotifen fumarate and its related impurities. The method has high sensitivity and strong specificity.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides a separation method of ketotifen fumarate and its impurities, which comprises using a high performance liquid chromatograph to determine the ketotifen fumarate impurities in the test sample.

[0006] Preferably, the mobile phase A of the high performance liquid chromatograph is water-triethylamine (500:0.175), and the mobile phase B is methanol-triethylamine.

[0007] Preferably, the volume ratio of water to triethylamine in the mobile phase A is 500:0.175, and the volume ratio of methanol to triethylamine in the mobile phase B is 500:0.175.

[0008] Preferably, the detection wavelength of the high performance liquid chromatograph is 290 nm.

[0009] Preferably, the impurities include: impurity D, impurity E and impurity G.

[0010] Preferably, the elution condition of the high performance liquid chromatograph is as follows: Time (min) A% B% 0 65 35 15 65 35 40 37 63 55 10 90 57 65 35 72 65 35 Preferably, the column temperature of the high performance liquid chromatograph is 20-40℃.

[0011] Preferably, the injection volume of the high performance liquid chromatograph is 5-20 μl.

[0012] Preferably, the isocratic elution time of the high performance liquid chromatograph is 60-100 min.

[0013] In another aspect, the present application provides a use of the method according to any one of the above in the preparation of ketotifen fumarate bulk drug.

[0014] Effects of the application The present application provides a separation and detection method for ketotifen fumarate and its impurities, which has good separation degree, simple operation, fast peak time, short detection time, and good method reproducibility and stability. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 HPLC chromatogram of ketotifen fumarate test solution (condition 5) Figure 2 HPLC chromatogram of the specificity experiment of Example 2 of the present application DETAILED DESCRIPTION

[0016] The following examples are only used to illustrate the present application, but not as a limitation on the scope of protection of the present application.

[0017] The impurity information involved in the present application is shown in the following table: Impurity Code Impurity Source Remarks EP Impurity A Process impurity Drug substance by-product Chp Impurity I / EP Impurity B Process impurity Drug substance intermediate EP Impurity C Process impurity Drug substance by-product EP Impurity D Degradation impurity Drug substance oxidative degradation product EP Impurity E Process impurity Drug substance by-product EP Impurity F Process impurity Drug substance by-product EP Impurity G Degradation impurity Drug substance by-product Examples

[0018] Inspection method: determined according to high performance liquid chromatography (2020 edition of Chinese Pharmacopoeia, General Test 0512) (1) Reagent test solution and control Methanol (HPLC grade) Triethylamine (AR grade) Water (pure water) Impurity D control Impurity G control Impurity E control Ketotifen fumarate control (2) Instruments and equipment High performance liquid chromatograph Balance (3) Preparation of test solution, control solution Solvent: methanol-water (50:50) solution.

[0019] Blank excipient: accurately measure 5 ml of blank excipient, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well.

[0020] Test solution: accurately measure 5 ml of the product, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well.

[0021] Control solution: accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, shake well.

[0022] (4) System suitability solution Take impurities D, impurities G, impurities E and fumaric acid ketotifen control each appropriate amount, accurately weigh and dissolve in solvent and dilute quantitatively to prepare a mixture solution containing about 1.25 μg of each impurity and 0.25 mg of ketotifen per 1 ml.

[0023] (5) Sensitivity solution Take fumaric acid ketotifen control appropriate amount, accurately weigh, dissolve in solvent and dilute quantitatively to prepare a solution containing about 0.125 μg of ketotifen per 1 ml.

[0024] Accurately measure the blank solution (i.e. solvent), blank excipient, sensitivity solution, system suitability solution, test solution and control solution, respectively, inject into the liquid chromatograph, and record the chromatogram. Examples

[0025] (1) Chromatographic condition optimization Take 3 mg / ml of raw material drug light liquid destruction solution, and optimize the proportion of mobile phase on the basis of the method of fumaric acid ketotifen raw material ChP related substances gradient elution.

[0026] Chromatographic column Chromatographic column: Waters Xbridge Rp C18 4.6*150mm, 3.5 μm Column temperature: 40℃ Mobile phase A: water-triethylamine (500:0.175) Mobile phase B: methanol-triethylamine (500:0.175) Flow rate: 1.0 ml / min Detection wavelength: 297 nm Injection volume: 20 μl Test solution: 0.3 mg / ml Elution conditions are as follows: Elution gradient condition 1: Time (min) A% B% 0 40 60 12 40 60 20 10 90 25 10 90 26 40 60 31 40 60 Elution gradient condition 2: Time (min) A% B% 0 50 50 15 50 50 35 10 90 50 10 90 51 50 50 56 50 50 Elution gradient condition 3: Time (min) A% B% 0 55 45 20 55 45 50 10 90 70 10 90 72 55 45 82 55 45 Elution gradient condition 4: Time (min) A% B% 0 65 35 15 65 35 40 37 63 55 10 90 57 10 90 72 65 35 82 65 35 Elution gradient condition 5: Time (min) A% B% 0 65 35 15 65 35 40 37 63 55 10 90 57 65 35 72 65 35 As shown in the chromatogram 2, when the elution condition 1, the sample main peak has good peak shape: tailing factor 1.132, resolution 2.737; but the impurity peaks are not separated, and need to be further optimized. When the elution condition 2, the chromatographic peak resolution is better than the Chp method, and still continues to be optimized. When the elution condition 3, the impurities in the test solution of the method are basically separated, and can be used for subsequent detection. When the elution condition 4, the impurities in the test solution of the method are basically separated, but many interference peaks appear when eluting with high organic phase. When the elution condition 5, impurity D, impurity C, impurity G, ketotifen, impurity E, impurity I and impurity A are sequentially eluted, the resolution of each impurity and the main peak meets the requirements, and there is no interference peak for the detection of known impurities.

[0027] Example 2 Specificity test Blank solvent (diluent): 50% methanol.

[0028] Ketotifen fumarate positioning solution: take ketotifen fumarate reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 0.25 mg of ketotifen per 1 ml.

[0029] Impurity A reference solution: take each impurity reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 2.5 μg of impurity A per 1 ml.

[0030] Impurity I reference solution: take each impurity reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 2.5 μg of impurity I per 1 ml.

[0031] Impurity C reference solution: take each impurity reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 2.5 μg of impurity C per 1 ml.

[0032] Impurity D reference solution: take each impurity reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 2.5 μg of impurity D per 1 ml.

[0033] Impurity E reference solution: take each impurity reference substance, accurately weigh, add diluent to dissolve and dilute to prepare a solution containing about 2.5 μg of impurity E per 1 ml.

[0034] Impurity G reference solution: Take an appropriate amount of each impurity reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing approximately 2.5 μg of impurity G per 1 ml.

[0035] Impurity Mixed Solution: Take appropriate amounts of impurity D, impurity G, impurity E and ketotifen fumarate reference standard, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing approximately 1.25 μg of each impurity and 0.25 mg of ketotifen per 1 ml.

[0036] Blank excipient solution: Accurately transfer 5 ml of blank excipient solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0037] Sensitivity solution: Take an appropriate amount of ketotifen fumarate reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing approximately 0.125 μg of ketotifen per 1 ml.

[0038] Test solution: Accurately transfer 5 ml of the test solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0039] Control solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0040] Take blank solvent, blank excipient solution, sensitivity solution, test solution, ketotifen fumarate, mixed solution of known impurities, impurity A, impurity C, impurity D, impurity E, impurity G, impurity I reference solution and reference solution and determine according to the method, and record the chromatogram.

[0041] like Figure 1 As shown, the blank solvent, blank excipient solution, sensitivity solution, test solution, known impurity mixed solution, ketotifen fumarate, impurity D, impurity C, impurity G, impurity E, impurity I and impurity A are listed in sequence.

[0042] Results of specificity test: Blank solution, blank excipient, and various impurity solutions did not interfere with the detection of ketotifen; in the sensitivity solution chromatogram, the signal-to-noise ratio of the ketotifen peak was 39 > 10; in the chromatogram of the known impurity mixed solution, except for the solvent peak, impurities D, C, G, ketotifen, E, I, and A were eluted sequentially, and the resolution between impurity E and ketotifen was 4.0 > 1.5.

[0043] Example 3 Sensitivity Test The signal-to-noise ratio is used to determine the detection limit and the quantitative limit. The control stock solution is diluted to ketotifen and impurities A, C, D, E, G, I signal-to-noise ratio S / N is about 3-10, and the concentration is the detection limit of the impurity; to ketotifen and impurities A, C, D, E, G, I signal-to-noise ratio S / N is about 10-30, and the concentration is the quantitative limit of the impurity. At the same time, the control mixed solution of the quantitative limit concentration is prepared, and the peak area and retention time are determined for 6 times in parallel, and the results are shown in the following table.

[0044] Note: The signal-to-noise ratio of the quantitative limit solution is the signal-to-noise ratio of the quantitative limit solution-1.

[0045] From the above table, the quantitative limit and detection limit of the known impurity are both lower than 0.1% of the concentration of the test solution, which meets the requirements.

Claims

1. A method for separating ketotifen fumarate and its impurities, characterized by, The method comprises determining the ketotifen fumarate impurities in the test sample by using a high performance liquid chromatograph.

2. The separation method of claim 1, wherein, The mobile phase A of the high performance liquid chromatograph is water-triethylamine (500:0.175), and the mobile phase B is methanol-triethylamine.

3. The separation method according to claim 2, wherein the volume ratio of water to triethylamine in the mobile phase A is 500:0.175, and the volume ratio of methanol to triethylamine in the mobile phase B is 500:0.

175.

4. The separation method of claim 1, wherein, The detection wavelength of the high performance liquid chromatograph is 290 nm.

5. The separation method of claim 1, wherein, The impurities comprise impurity D, impurity E and impurity G.

6. The separation method of claim 1, wherein, The elution conditions of the high performance liquid chromatograph are as follows: 0-15 minutes, A is 65% and B is 35%; 15-40 minutes, A is 37% and B is 63%; 40-55 minutes, A is 10% and B is 90%; 55-57 minutes, A is 65% and B is 35%; and 57-72 minutes, A is 65% and B is 35%.

7. The separation method of claim 1, wherein, The column temperature of the high performance liquid chromatograph is 20-40℃.

8. The separation method of claim 1, wherein, The injection volume of the high performance liquid chromatograph is 5-20 μl.

9. The separation method of claim 1, wherein, The isocratic elution time of the high performance liquid chromatograph is 60-100 min.

10. Use of the method according to any one of claims 1-9 in the preparation of ketotifen fumarate bulk drug.

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