Liquid chromatography detection method for separating rupatadine fumarate and positional isomers thereof

Abstract

The invention discloses a liquid chromatography method for separating rupatadine fumarate and a positional isomer of rupatadine fumarate. According to the method, the rupatadine fumarate and the positional isomer are effectively separated by adopting reversed-phase liquid chromatography, and compared with normal-phase liquid chromatography generally adopted by isomers, the method provided by the invention is quicker, simpler and more convenient, can effectively separate isomer impurities and accurately quantify the content of the isomer impurities, and has the advantages of high accuracy and high efficiency. And the quality of the product is effectively controlled.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biology, and relates to a liquid chromatography method for separating rupatadine fumarate and its positional isomers. Background Art

[0002] Rupatadine fumarate is an antiallergic drug indicated for allergic diseases such as seasonal and perennial allergic rhinitis. Rupatadine fumarate has positional isomers, and separation of these isomers is a necessary step in the preparation, quality research, and quality control of rupatadine fumarate's API.

[0003] The chemical name of rupatadine fumarate is: 8-chloro-11-[1-[(5-methyl-3-pyridinyl)methyl]-4-piperidylidene]-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine fumarate.

[0004] Its chemical structural formula is:

[0005]

[0006] The name of the positional isomer of rupatadine fumarate is: 8-chloro-11-[1-[(5-methyl-3-pyridyl)yl]-1,2,3,6-tetrahydropyridin-4-yl]-6,11-dihydro-5H-benzo-5,6]cyclohepta[1,2-b]pyridine.

[0007] Its chemical structure is:

[0008]

[0009] When determining rupatadine fumarate and its positional isomers, a review found no literature reporting on analytical methods for positional isomers in rupatadine fumarate. The relevant substance methods for the EP standard for rupatadine fumarate and the Indian import registration standard indicate that positional isomers and rupatadine overlap as a single chromatographic peak, making it ineffective for monitoring positional isomers.

[0010] In order to overcome the above problems, the present invention provides a liquid chromatography method for analyzing rupatadine fumarate and its positional isomers. Summary of the Invention

[0011] The object of the present invention is to provide a method for detecting rupatadine fumarate and its positional isomers.

[0012] The present invention separates rupatadine fumarate from its positional isomers, achieving quality control of the rupatadine fumarate raw material. The high-performance liquid chromatography method for separating rupatadine fumarate and its positional isomers employs a chromatographic column filled with octadecyl bonded silica gel, phosphate buffer as mobile phase A, and methanol as mobile phase B, with a ratio of mobile phase A to mobile phase B ranging from 35:65 to 0:100. Mobile phase A is prepared by diluting 6.8 g of potassium dihydrogen phosphate to 1000 mL with water and adjusting the pH to 5.0 with dilute ammonia water. Gradient elution is employed in this analytical method.

[0013] The chemical name of rupatadine fumarate is: 8-chloro-11-[1-[(5-methyl-3-pyridinyl)methyl]-4-piperidylidene]-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine fumarate.

[0014] The name of the positional isomer of rupatadine fumarate is: 8-chloro-11-[1-[(5-methyl-3-pyridyl)yl]-1,2,3,6-tetrahydropyridin-4-yl]-6,11-dihydro-5H-benzo-5,6]cyclohepta[1,2-b]pyridine.

[0015] Rupatadine fumarate and its positional isomers are both existing products and can be purchased on the market.

[0016] To this end, the present invention provides a method for detecting rupatadine fumarate and its positional isomers, comprising the following steps:

[0017] Step 1: taking rupatadine fumarate raw material and dissolving it in a solvent;

[0018] Step 2, passing through a high performance liquid chromatograph to obtain a chromatogram;

[0019] Step 3, obtaining characteristic peaks of rupatadine fumarate and its positional isomers according to the chromatogram;

[0020] Step 4, calculating the content of rupatadine fumarate and its positional isomers based on the peak area of ​​the characteristic peak.

[0021] Among them, the chromatographic conditions are:

[0022] The chromatographic columns are selected from: Shimadzu WondaSil C18, 5 μm, 4.6*250 mm column, Shimadzu Shim-pack GIST C18, 5 μm, 4.6*250 mm column.

[0023] The high performance liquid chromatograph uses a chromatographic column with octadecyl bonded silica gel as a filler, phosphate buffer as mobile phase A, and methanol as mobile phase B.

[0024] The preparation method of mobile phase A is as follows: take 6.8g of potassium dihydrogen phosphate, dilute it to 1000mL with water, and adjust the pH to 5.0 with dilute ammonia water.

[0025] This analytical method uses gradient elution.

[0026] Wherein, the mobile phase gradient elution program is:

[0027] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0028] The methanol is chromatographically pure methanol.

[0029] The solvent is: mobile phase A:mobile phase B=35:65.

[0030] The chromatographic conditions are as follows: a mobile phase flow rate of 1.0 to 1.5 mL / min, preferably 1.5 mL / min; a detection wavelength of 210 to 280 nm, preferably 267 nm; and a column temperature of 25 to 40° C., preferably 40° C.

[0031] Wherein, step 1 is to prepare the rupatadine fumarate raw material into a sample solution containing 0.65 mg / ml of rupatadine fumarate.

[0032] Wherein, step 2 is to inject 5-50 μL of the sample solution into the liquid chromatograph, preferably inject 50 μL of the sample solution into the liquid chromatograph.

[0033] Detector: UV detector.

[0034] Most preferably, the chromatographic conditions of the present invention are:

[0035] Wherein: The chromatograph used in the present invention is: Shimadzu LC-20AT

[0036] Chromatographic column: WondaSil C18 (250 mm × 4.6 mm, 5 μm)

[0037] Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 5.0 with dilute ammonia water.

[0038] Detection wavelength 267nm

[0039] Column temperature: 40°C

[0040] Injection volume: 50 μL.

[0041] The mobile phase gradient elution program was:

[0042] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0043] The octadecyl bonded silica gel column used in the present invention can effectively separate rupatadine fumarate and its positional isomers, thereby achieving the separation and detection of rupatadine fumarate raw material and its positional isomer impurities (see the attached results). Figures 1 to 10 ).

[0044] The detection method of the present invention is obtained through screening, and the screening method is as follows:

[0045] Selection of chromatographic column: C18 (WondaSil, 250 mm × 4.6 mm, 5 μm) was selected. Rupatadine fumarate and its positional isomers have similar chemical structures and are difficult to separate. Therefore, a C18 column with a high separation ability filler was selected.

[0046] Wavelength selection: UV spectrum scanning of rupatadine fumarate positional isomers is performed as follows Figure 10 The ultraviolet absorption at 267.30nm is relatively strong, so 267nm is preferred as the detection wavelength.

[0047] Selection of buffer salt pH: According to the existing chromatographic conditions, pH 4.9, pH 5.0, and pH 5.1 were selected. The results showed that the chromatogram peak shape of pH 5.0 was better, and the resolution was good.

[0048] Selection of HPLC: The Shimadzu LC-20AT and Waters 2695 HPLCs were used for analysis. The results showed that under the chromatographic analysis conditions, the blank solvent and impurity peaks did not interfere with the main component. The separation degree of rupatadine fumarate and its positional isomers was greater than 1.2, indicating good method specificity.

[0049] Selection of mobile phase: At least three mobile phases were selected, and the key screening results were as follows: Under the conditions of mobile phase 2, the resolution of rupatadine fumarate and its positional isomers was less than 1.2, indicating that effective separation was not possible; under the conditions of mobile phase 3, although the resolution of rupatadine fumarate and its positional isomers was greater than 1.2, the detection time was too long, and the requirement for simple and rapid detection could not be met; under the conditions of mobile phase 1, the blank solvent and impurity peaks did not interfere with the main component, and the resolution of rupatadine fumarate and its positional isomers was greater than 1.2, indicating good method specificity.

[0050] The present method uses reversed-phase liquid chromatography to achieve effective separation of rupatadine fumarate and its positional isomers. Compared with the normal-phase liquid chromatography commonly used for isomer separation, the method provided by the present invention is faster and simpler, can effectively separate isomeric impurities, and accurately quantify their content, thereby effectively controlling the quality of the product.

[0051] The present invention detects rupatadine fumarate and its positional isomers. The significance of this method lies in the following: the separation and determination of rupatadine fumarate and its positional isomers is crucial for the quality control of the final drug. However, no method for detecting positional isomers in rupatadine fumarate has been reported in the literature. Furthermore, rupatadine fumarate and its positional isomers share similar physicochemical properties and similar chromatographic retention behaviors, making their separation somewhat challenging. Therefore, developing a method for separating and detecting rupatadine fumarate and its positional isomers is of great significance for process selection and drug quality assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 HPLC chart of rupatadine fumarate in Example 1

[0053] Figure 2 HPLC chart of rupatadine fumarate isomers in Example 1

[0054] Figure 3 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers (mobile phase 1) in Example 1

[0055] Figure 4 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers in mobile phase 2 of Example 1

[0056] Figure 5 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers in mobile phase 3 of Example 1

[0057] Figure 6 HPLC graphs of the mixed solution of rupatadine fumarate and its positional isomers in Example 2 on different chromatographic columns

[0058] Figure 7 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers at pH 4.9 of Example 3

[0059] Figure 8 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers at pH 5.1 of Example 4

[0060] Figure 9 HPLC chart of the mixed solution of rupatadine fumarate and its positional isomers at pH 5.0 of Example 5

[0061] Figure 10 UV spectra of rupatadine fumarate positional isomers DETAILED DESCRIPTION

[0062] The present invention is further illustrated by the following examples.

[0063] The products and preparation methods described herein are further described below with reference to specific examples, but the present invention is not limited to these examples. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials described can be obtained from publicly available commercial sources.

[0064] Example 1

[0065] Instruments and conditions

[0066] High performance liquid chromatograph: Shimadzu LC-20AT

[0067] Chromatographic column: Shimadzu WondaSil C18 (250mm×4.6mm, 5um)

[0068] Mobile phase 1: Use phosphate buffer as mobile phase A and methanol as mobile phase B. The preparation method of mobile phase A is as follows: take 6.8 g of potassium dihydrogen phosphate, dilute it to 1000 mL with water, and adjust the pH to 5.0 with dilute ammonia water.

[0069] The mobile phase gradient elution program was:

[0070] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0071] Flow rate: 1.5 mL / min;

[0072] Detection wavelength: 267nm;

[0073] Column temperature: 40°C

[0074] Injection volume: 50 μL.

[0075] Mobile phase 2: Phosphate buffer was used as mobile phase A, methanol was used as mobile phase B, and acetonitrile was used as mobile phase C. Mobile phase A was prepared as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 5.0 with dilute ammonia water.

[0076] Elution method: isocratic elution.

[0077] Mobile phase: Mobile phase A-Mobile phase B-Mobile phase C (50:30:20)

[0078] Flow rate: 1.5 mL / min;

[0079] Detection wavelength: 220nm;

[0080] Column temperature: 40°C

[0081] Injection volume: 50 μL.

[0082] Mobile phase 3: Use phosphate buffer as mobile phase A and methanol as mobile phase B. The preparation method of mobile phase A is as follows: take 6.8 g of potassium dihydrogen phosphate, dilute it to 1000 mL with water, and adjust the pH to 5.0 with dilute ammonia water.

[0083] Elution method: isocratic elution.

[0084] Mobile phase: Mobile phase A-Mobile phase B (40:60)

[0085] Flow rate: 1.5 mL / min;

[0086] Detection wavelength: 220nm;

[0087] Column temperature: 40°C

[0088] Injection volume: 50 μL.

[0089] Experimental steps:

[0090] Accurately weigh an appropriate amount of rupatadine fumarate and dissolve it in a solvent to prepare a test solution of approximately 0.65 mg / mL. Accurately weigh an appropriate amount of rupatadine fumarate positional isomer and dissolve it in methanol to prepare a solution of approximately 0.1 mg / mL as a stock solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of solvent to prepare the rupatadine fumarate positional isomer solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of the test solution and mix thoroughly to prepare the mixed solution. Accurately measure 50 μL of each solution and inject it into a high-performance liquid chromatograph. Perform the experiment according to the above-mentioned liquid chromatography conditions and record the chromatogram.

[0091] After testing with three mobile phases under this chromatographic condition, the results showed that: the results of mobile phase 1 are shown in the attached Figures 1 to 3 . Figure 1 The chromatographic peak with a retention time of 29.248 min is rupatadine fumarate. Figure 2 The chromatographic peak with a retention time of 27.044 min is the positional isomer of rupatadine fumarate. Figure 3 The chromatographic peak with a retention time of 29.194 min is rupatadine fumarate, and the chromatographic peak with a retention time of 27.168 min is a positional isomer. The separation degree between rupatadine fumarate and its positional isomer is greater than 1.4. This method has good specificity and high detection efficiency. Under the condition of mobile phase 2, Figure 4 The chromatographic peak with a retention time of 35.720 min is rupatadine fumarate, and the chromatographic peak with a retention time of 32.487 min is a positional isomer. The separation degree between rupatadine fumarate and its positional isomer is less than 1.2, and they cannot be effectively separated. Under the condition of mobile phase 3, Figure 5The chromatographic peak with a retention time of 49.696 min is rupatadine fumarate, and the chromatographic peak with a retention time of 45.721 min is a positional isomer. Although the separation degree of rupatadine fumarate and its positional isomers is greater than 1.2, which can achieve effective separation, the elution time of each component is too long, which cannot meet the requirements of simple and quick detection. The separation results are shown in Table 1 and the chromatogram is shown in Figures 3-5 .

[0092] Table 1 Investigation results of different mobile phases

[0093] Mobile phase Rupatadine fumarate retention time Retention time of positional isomers Separation Mobile phase 1 29.194 27.168 1.45 Mobile phase 2 35.720 32.487 1.17 Mobile phase 3 49.696 45.721 1.81

[0094] Example 2

[0095] Instruments and conditions

[0096] High performance liquid chromatograph: Waters 2695

[0097] Chromatographic column: Shimadzu Shim-pack GIST, C18 (250 mm × 4.6 mm, 5 μm)

[0098] Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 5.0 with dilute ammonia water.

[0099] The mobile phase gradient elution program was:

[0100] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0101] Detection wavelength: 267nm;

[0102] Column temperature: 40°C

[0103] Injection volume: 50 μL.

[0104] Experimental procedures

[0105] Accurately weigh an appropriate amount of rupatadine fumarate and dissolve it in a solvent to prepare a test solution of approximately 0.65 mg / mL. Accurately weigh an appropriate amount of rupatadine fumarate positional isomer and dissolve it in methanol to prepare a solution of approximately 0.1 mg / mL as a stock solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of solvent to obtain the resulting solution as the rupatadine fumarate positional isomer solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of the test solution and mix thoroughly to prepare the mixed solution. Accurately measure 50 μL of each solution and inject it into a high-performance liquid chromatograph. Perform the experiment according to the above-mentioned liquid chromatography conditions and record the chromatogram. The results are shown in the attached figure. Figure 6 , Figure 6The peak at retention time 36.612 min is rupatadine fumarate, and the peak at 34.695 min is a positional isomer. The resolution between the peaks is greater than or equal to 1.2.

[0106] Example 3

[0107] Instruments and conditions

[0108] High performance liquid chromatograph: Waters 2695

[0109] Chromatographic column: Shimadzu WondaSil C18 (250mm×4.6mm, 5um)

[0110] Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 4.9 with dilute ammonia water.

[0111] The mobile phase gradient elution program was:

[0112] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0113] Detection wavelength: 267nm;

[0114] Column temperature: 40°C

[0115] Injection volume: 50 μL.

[0116] Experimental procedures

[0117] Accurately weigh an appropriate amount of rupatadine fumarate and dissolve it in a solvent to prepare a test solution of approximately 0.65 mg / mL. Accurately weigh an appropriate amount of rupatadine fumarate positional isomer and dissolve it in methanol to prepare a solution of approximately 0.1 mg / mL as a stock solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of solvent to obtain the resulting solution as the rupatadine fumarate positional isomer solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of the test solution and mix thoroughly to prepare the mixed solution. Accurately measure 50 μL of each solution and inject it into a high-performance liquid chromatograph. Perform the experiment according to the above-mentioned liquid chromatography conditions and record the chromatogram. The results are shown in the attached figure. Figure 7 , Figure 7 The peak at retention time 25.008 min is rupatadine fumarate, and the peak at retention time 23.162 min is a positional isomer. The resolution between the peaks is greater than or equal to 1.2.

[0118] Example 4

[0119] Instruments and conditions

[0120] High performance liquid chromatograph: Waters 2695

[0121] Chromatographic column: Shimadzu WondaSil C18 (250mm×4.6mm, 5um)

[0122] Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 5.1 with dilute ammonia water.

[0123] The mobile phase gradient elution program was:

[0124] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0125] Detection wavelength: 267nm;

[0126] Column temperature: 40°C

[0127] Injection volume: 50 μL.

[0128] Experimental procedures

[0129] Accurately weigh an appropriate amount of rupatadine fumarate and dissolve it in a solvent to prepare a test solution of approximately 0.65 mg / mL. Accurately weigh an appropriate amount of rupatadine fumarate positional isomer and dissolve it in methanol to prepare a solution of approximately 0.1 mg / mL as a stock solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of solvent to obtain the resulting solution as the rupatadine fumarate positional isomer solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of the test solution and mix thoroughly to prepare the mixed solution. Accurately measure 50 μL of each solution and inject it into a high-performance liquid chromatograph. Perform the experiment according to the above-mentioned liquid chromatography conditions and record the chromatogram. The results are shown in the attached figure. Figure 8 , Figure 8 The peak at retention time 27.083 min is rupatadine fumarate, and the peak at retention time 25.310 min is a positional isomer. The resolution between the peaks is greater than or equal to 1.2.

[0130] Example 5

[0131] Instruments and conditions

[0132] High performance liquid chromatograph: Waters 2695

[0133] Chromatographic column: Shimadzu WondaSil C18 (250mm×4.6mm, 5um)

[0134] Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 mL with water, and the pH was adjusted to 5.0 with dilute ammonia water.

[0135] The mobile phase gradient elution program was:

[0136] Time (min) Mobile phase A Mobile phase B 0 35 65 35 35 65 45 0 100 50 0 100 55 35 65 60 35 65

[0137] Detection wavelength: 267nm;

[0138] Column temperature: 40°C

[0139] Injection volume: 50 μL.

[0140] Experimental procedures

[0141] Accurately weigh an appropriate amount of rupatadine fumarate and dissolve it in a solvent to prepare a test solution of approximately 0.65 mg / mL. Accurately weigh an appropriate amount of rupatadine fumarate positional isomer and dissolve it in methanol to prepare a solution of approximately 0.1 mg / mL as a stock solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of solvent to obtain the resulting solution as the rupatadine fumarate positional isomer solution. Add 3 drops of the positional isomer stock solution to 1.5 mL of the test solution and mix thoroughly to prepare the mixed solution. Accurately measure 50 μL of each solution and inject it into a high-performance liquid chromatograph. Perform the experiment according to the above-mentioned liquid chromatography conditions and record the chromatogram. The results are shown in the attached figure. Figure 9 , Figure 9 The peak at retention time 26.410 min is rupatadine fumarate, and the peak at retention time 24.627 min is a positional isomer. The resolution between the peaks is greater than or equal to 1.2.

[0142] Example 6

[0143] Investigate the stability of sample solution:

[0144] An appropriate amount of rupatadine fumarate sample was accurately weighed and dissolved in a solvent to prepare a test solution of approximately 0.65 mg / mL. At 0, 3, 6, 12, and 24 hours, 50 μL of each solution was accurately measured and injected into a high-performance liquid chromatograph. The experiment was performed according to the chromatographic conditions in Example 1 above, and the chromatogram was recorded. The results are shown in Table 2.

[0145] Table 2 Results of stability study of sample solutions (content in %)

[0146] Time (h) Rupatadine fumarate% Positional isomer % 0 99.60 0.40 3 99.61 0.39 6 99.61 0.39 12 99.61 0.39 24 99.61 0.39

[0147] The results showed that under the present chromatographic detection conditions, no new impurities were generated when the sample solution was placed at room temperature for 24 hours, and the impurity content remained basically unchanged, indicating that the test solution was stable within 24 hours at room temperature.

[0148] Example 7

[0149] Investigation sensitivity:

[0150] An appropriate amount of rupatadine fumarate sample was accurately weighed, dissolved in a solvent, and a test solution of a certain concentration was prepared. The solution was tested according to the chromatographic conditions in Example 1. The results are shown in Table 3.

[0151] Table 3 Sensitivity investigation results

[0152] Element Concentration (μg / mL) S / N Equivalent to the percentage of main component concentration Positional isomers 0.664 30 0.1％

[0153] The results showed that under the present chromatographic detection conditions, the sensitivity was suitable for the detection of rupatadine fumarate and its isomers.

[0154] Example 8

[0155] Sample determination:

[0156] Appropriate amounts of rupatadine fumarate samples from different batches were taken, accurately weighed, dissolved in a solvent, and prepared into a test solution of about 0.65 mg / mL. The solution was tested according to the chromatographic conditions in Example 1. The results are shown in Table 4.

[0157] Table 4 Determination results of different batches of rupatadine fumarate samples

[0158] batch number Rupatadine fumarate% Positional isomer % 250701 99.84 0.16 250704 99.95 0.05 250401 99.61 0.39

[0159] The results showed that under the present chromatographic detection conditions, the contents of rupatadine fumarate and its positional isomers could be determined rapidly and accurately in samples of different batches.

[0160] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments described. Without departing from the principles of the present invention, a number of changes, modifications, substitutions, combinations, and simplifications may be made, all of which are equivalent replacement methods and should also be considered as the scope of protection of the present invention.

Claims

1. A method for detecting rupatadine fumarate and its positional isomers, comprising the following steps: Step 1: taking rupatadine fumarate raw material and dissolving it in a solvent; Step 2, passing through a high performance liquid chromatograph to obtain a chromatogram; Step 3, obtaining characteristic peaks of rupatadine fumarate and its positional isomers according to the chromatogram; Step 4, calculating the content of rupatadine fumarate and its positional isomers based on the peak area of ​​the characteristic peak; in, Chromatographic conditions: The chromatographic columns are selected from: Shimadzu WondaSil C18, 5 μm, 4.6*250 mm column, Shimadzu Shim-packGIST C18, 5 μm, 4.6*250 mm column. The high performance liquid chromatograph uses a chromatographic column with octadecyl bonded silica gel as a filler, phosphate buffer as mobile phase A, and methanol as mobile phase B.

2. The method according to claim 1, characterized in that in, The mobile phase gradient elution program was: 。 3. The method according to claim 1, characterized in that The preparation method of mobile phase A is as follows: take 6.8g of potassium dihydrogen phosphate, dilute it to 1000ml with water, and adjust the pH to 5.0 with dilute ammonia water.

4. The method according to claim 1, wherein The chromatographic conditions are as follows: the flow rate of the mobile phase is 1.0-1.5 ml / min; the detection wavelength is 210-280 nm; and the column temperature of the chromatographic column is 25-40° C.

5. The method according to claim 1, wherein in, Step 1 is to prepare the rupatadine fumarate raw material into a sample solution containing 0.65 mg / ml of rupatadine fumarate.

6. The method according to claim 1, characterized in that in, Step 2 is to inject 5-50 μl of the sample solution into the liquid chromatograph, preferably inject 50 μl of the sample solution into the liquid chromatograph.

7. The method according to claim 1, characterized in that The chromatographic conditions are: Wherein: The chromatograph used in the present invention is: Shimadzu LC-20AT Chromatographic column: WondaSil C18 (250 mm × 4.6 mm, 5 μm) Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted with water to 1000 ml, and the pH was adjusted to 5.0 with dilute ammonia water. Detection wavelength 267nm Column temperature: 40°C Injection volume: 50 μl, The mobile phase gradient elution program was: 。 8. The method according to claim 1, characterized in that The following steps are involved: Take an appropriate amount of rupatadine fumarate, accurately weigh it, and dissolve it in solvent to make a test solution of about 0.65 mg / ml; An appropriate amount of rupatadine fumarate positional isomer is taken, accurately weighed, and dissolved in methanol to prepare a solution of approximately 0.1 mg / ml as a stock solution. Three drops of the positional isomer stock solution are added to 1.5 ml of solvent to obtain a solution as the rupatadine fumarate positional isomer solution. Take 3 drops of the positional isomer stock solution and add 1.5 ml of the test solution, mix well to prepare the mixed solution; Accurately measure 50 μl of each solution and inject it into the high performance liquid chromatograph, perform the experiment according to the above liquid phase conditions, and record the chromatogram. Chromatographic conditions: High performance liquid chromatograph: Shimadzu LC-20AT Chromatographic column: Shimadzu WondaSil C18 (250mm×4.6mm, 5um) Mobile phase: Phosphate buffer was used as mobile phase A, and methanol was used as mobile phase B. The preparation method of mobile phase A was as follows: 6.8 g of potassium dihydrogen phosphate was diluted to 1000 ml with water, and the pH was adjusted to 5.0 with dilute ammonia water. The mobile phase gradient elution procedure was as follows: Flow rate: 1.5 ml / min; Detection wavelength: 267nm; Column temperature: 40°C Injection volume: 50 μl.

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