Separation and determination method of rupatadine fumarate starting material SM1 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining a rupatadine fumarate starting material SM1 and impurities thereof. According to the method, phenylsilane bonded silica gel is adopted as a chromatographic column filler, mobile phases comprise a mobile phase A and a mobile phase B, the mobile phase A is a monopotassium phosphate solution, and the mobile phase B is acetonitrile; the method comprises the following steps: sequentially separating an impurity SM1a, rupatadine fumarate SM1, an impurity SM1o, an impurity SM1j, an impurity SM1g, an impurity SM1m, an impurity SM1i, an impurity SM1n, an impurity SM1k, an impurity SM1f, an impurity SM1b, an impurity SM1l and / or an impurity SM1e through linear gradient elution; the rupatadine fumarate SM1 and 12 known impurities can be separated within 62 minutes by the high performance liquid chromatography constructed by the invention, the analysis time is short, the sensitivity is high, the specificity is strong, and the reproducibility is good.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and determining a rupatadine fumarate starting material SM1 and its impurities. Background Art

[0002] Rupatadine fumarate, chemical name is 8-chloro-6,11-dihydro-11-[1-[(5-methyl-3-pyridyl)methyl]-4-piperidinylidene]-5H-benzo[5,6]cyclohepta[1,2-b]pyridine fumarate, molecular formula is C 26 H 26 ClN3·C4H4O4. Rupatadine fumarate is a new antiallergic drug with dual antagonistic effects on histamine H1 receptors and PAF receptors. It is mainly used clinically to treat seasonal and perennial allergic rhinitis and urticaria.

[0003] Rupatadine fumarate SM1 is the starting material for synthesizing rupatadine fumarate, and its quality directly affects the purity and yield of rupatadine fumarate. The structural formula of rupatadine fumarate SM1 is shown in Formula 1. According to the synthetic process of rupatadine fumarate SM1, it may contain a total of 15 impurities: Impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1d 、Impurity SM 1e 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1h 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o In order to ensure the quality of rupatadine fumarate API and preparation, it is necessary to control and detect the impurities that may exist in the rupatadine fumarate starting material SM1.

[0004]

[0005] At present, there is a lack of research on the impurities present in the synthesis process of rupatadine fumarate starting material SM1, and existing related research is mostly focused on the related substances of rupatadine fumarate. For example, invention patents with publication numbers CN112903859B, CN108508117A, and CN102043025B disclose methods for determining rupatadine and its related substances using high performance liquid chromatography, but these methods cannot achieve the above-mentioned determination of SM1 in rupatadine fumarate. 1e , SM 1i Effective detection of various impurities. Summary of the invention

[0006] In view of this, this patent established a set of HPLC methods with high accuracy, good reproducibility and good durability by screening chromatographic columns and chromatographic conditions, which can achieve the determination of related substances (impurities SM 1e 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1o ) and other individual impurities; other potential impurities (impurities SM 1a 、Impurity SM 1b 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n ) can also be effectively separated in this method.

[0007] One of the objects of the present invention is to provide a method for separating rupatadine fumarate starting material SM1 and its related substances based on high performance liquid chromatography. The method has high sensitivity and strong specificity, can achieve effective separation of rupatadine fumarate starting material SM1 and its related substances, and provide support for subsequent content detection of rupatadine fumarate starting material SM1 and its related substances.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for separating rupatadine fumarate starting material SM1 and its related substances based on high performance liquid chromatography, wherein the related substances include impurity SM 1a 、Impurity SM 1o and its enantiomers, impurity SM 1j and its enantiomers, impurity SM 1g and its enantiomers, impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l 、Impurity SM 1e Any one or more of; in the high performance liquid chromatography, the chromatographic column uses phenylsilane bonded silica gel as a filler, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a potassium dihydrogen phosphate solution, and the mobile phase B is acetonitrile; the impurities SM are sequentially eluted by linear gradient elution 1a 、Rupatadine Fumarate SM1、Impurity SM 1o 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l and / or impurities SM 1e To separate;

[0010] The structural formula of the rupatadine fumarate SM1 is shown in Formula 1; the impurity SM 1a The structural formula of is shown in Formula 2; the impurity SM 1o The structural formula is shown in Formula 3; the impurity SM 1j The structural formula is shown in Formula 4; the impurity SM 1g The structural formula is shown in Formula 5; the impurity SM 1m The structural formula is shown in Formula 6; the impurity SM 1i The structural formula of is shown in Formula 7; the impurity SM 1n The structural formula is shown in Formula 8; the impurity SM 1k The structural formula is shown in Formula 9; the impurity SM 1f The structural formula of is shown in Formula 10; the impurity SM 1b The structural formula of is shown in Formula 11; the impurity SM 1l The structural formula of is shown in Formula 12; the impurity SM 1e The structural formula is shown in Formula 13;

[0011]

[0012]

[0013] The components can be qualitatively identified according to the elution order.

[0014] Furthermore, rupatadine fumarate SM1 and its related impurities were separated simultaneously, and the separation time was 62 min.

[0015] Further, the gradient elution program is set as follows:

[0016] At 0 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18;

[0017] At 10 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18;

[0018] At 30 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45;

[0019] At 35 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55;

[0020] At 40 minutes, the volume ratio of mobile phase A to mobile phase B was 40:60;

[0021] At 40.1 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18;

[0022] At 62 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18.

[0023] Preferably, the gradient elution program is set as follows:

[0024] At 0 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0025] At 10 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0026] At 30 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45;

[0027] At 35 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55;

[0028] At 40 minutes, the volume ratio of mobile phase A to mobile phase B was 40:60;

[0029] At 40.1 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0030] At 62 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16.

[0031] Furthermore, the column temperature of the chromatographic column is 33° C.-37° C., preferably 35° C.; the flow rate of the mobile phase is 0.8 ml / min-1.0 ml / min, preferably 0.9 ml / min.

[0032] Furthermore, the injector temperature was 10°C; and the injection volume was 30 μL.

[0033] Furthermore, an acetonitrile aqueous solution is used as a preparation solvent, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 70:30.

[0034] The second object of the present invention is to provide a method for qualitatively identifying rupatadine fumarate starting material SM1 and its related substances, which can achieve effective qualitative identification of rupatadine fumarate starting material SM1 and its related substances.

[0035] To achieve the above object, the present invention adopts the following technical solutions:

[0036] The method for qualitatively identifying the rupatadine fumarate starting material SM1 and its related substances comprises separating the rupatadine fumarate starting material SM1 and its related substances by the aforementioned method; entering the detector for detection and obtaining a chromatogram; the impurity SM 1a 、Rupatadine Fumarate SM1、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1l 、Impurity SM 1e The detection wavelength is 270nm and / or 220nm; the impurity SM 1b The detection wavelength is 220nm; the impurity SM 1o The detection wavelength is 270nm.

[0037] Furthermore, the retention time of each component from short to long is as follows: impurity SM 1a 、Rupatadine Fumarate SM1、Impurity SM 1o 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l and / or impurities SM 1e .

[0038] As a preferred technical solution, the chromatographic column specifications are 4.6mm×150mm, 3μm; mobile phase A is potassium dihydrogen phosphate solution, mobile phase B is acetonitrile; the mobile phase flow rate is 0.9ml / min; the chromatographic column temperature is 35°C; the impurity SM 1a、Rupatadine Fumarate SM1、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1l 、Impurity SM 1e The detection wavelength is 270nm and / or 220nm; the impurity SM 1b The detection wavelength is 220nm; the impurity SM 1o The detection wavelength is 270nm;

[0039] Elution was performed according to a linear gradient elution program, which was set as follows:

[0040] At 0 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0041] At 10 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0042] At 30 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45;

[0043] At 35 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55;

[0044] At 40 minutes, the volume ratio of mobile phase A to mobile phase B was 40:60;

[0045] At 40.1 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16;

[0046] At 62 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16.

[0047] Further, the impurity SM was found to have a retention time of 6.2±0.5min. 1a The retention time is 10.6±1min for rupatadine fumarate SM1, and the retention time is 16.6±0.5min for impurity SM 1j The impurity SM is the one with a retention time of 18.4±0.5min. 1g , the impurity SM is the one with retention time of 21.0±0.5min 1m The impurity SM is the one with a retention time of 22.3±0.5min. 1i The impurity SM is the one with a retention time of 25.2±0.5min. 1n The impurity SM is the one with a retention time of 27.8±0.5min. 1k, the impurity SM is the one with retention time of 28.8±0.5min 1f , the impurity SM is the one with retention time of 30.2±0.5min 1b The impurity SM is the one with a retention time of 31.2±0.5min. 1l The impurity SM is at a retention time of 37.8±0.5min. 1e ; The impurity SM is the one with retention time of 13.2±0.5min 1o .

[0048] The third object of the present invention is to provide a method for detecting the content of rupatadine fumarate starting material SM1 and its related substances. The present invention can separate and detect rupatadine fumarate starting material SM1 and its related substances within 62 minutes or more than 62 minutes, with short analysis time, good durability and high sensitivity.

[0049] To achieve the above object, the present invention adopts the following technical solutions:

[0050] The method for detecting the content of rupatadine fumarate starting material SM1 and its related substances comprises the following steps:

[0051] (1) separation: separating the rupatadine fumarate starting material SM1 and its related substances by the aforementioned separation method;

[0052] (2) Detection: The above-mentioned identification method is used to detect the starting material SM1 of rupatadine fumarate and its related substances, and a chromatogram is obtained; the impurity SM 1o 、Impurity SM 1j 、Impurity SM 1i 、Impurity SM 1e The detection wavelength is 270nm; the impurity SM 1a 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l The detection wavelength is 220nm;

[0053] (3) Content calculation: Using the chromatogram obtained in step (2), the content of each component is calculated by peak area using the principal component self-comparison method with a correction factor.

[0054] Further, the content calculation formula is:

[0055] C A =(A A ×C B ) / A B×f

[0056] f is the correction factor;

[0057] C A is the concentration of the analyte;

[0058] A A is the peak area of ​​the analyte;

[0059] C B is the concentration of the reference substance;

[0060] C B is the peak area of ​​the reference substance.

[0061] Furthermore, a standard solution is prepared and a chromatogram of the standard is obtained by detection; the chromatogram obtained by testing the sample solution is calculated based on the peak area.

[0062] The content of the components of the standard is known, while the content of the components of the test sample is unknown.

[0063] The obtained standard sample chromatogram can be stored in the database as a chromatographic material for quality control in the smart pharmaceutical factory, to help the robot determine whether the products it produces are qualified and to provide quantitative improvement analysis suggestions for unqualified products.

[0064] As a preferred technical solution, the specific steps include:

[0065] Step 1. Take an appropriate amount of this product, weigh it accurately, add about 15% of the total volume of acetonitrile to dissolve it by ultrasonic, then dilute it to the scale with water, shake well, and prepare a solution with a concentration of about 0.4 mg / mL as the sample solution to be tested.

[0066] Step 2. Accurately measure the test solution with a concentration of 0.4 mg / mL and dilute it with solvent to make a solution with a concentration of about 0.8 μg / mL as the control solution;

[0067] Step 3. Accurately measure 30 μL of the sample solution and control solution, respectively, inject them into the chromatograph, and record the chromatogram. According to the measured chromatogram, the impurity content is calculated by the peak area using the main component self-reference method with a correction factor.

[0068] In the method of the present invention, under the condition of detection wavelength of 220nm, the main component rupatadine fumarate SM1 and the impurity SM 1a 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM1b 、Impurity SM 1l 、Impurity SM 1e The qualitative analysis of any one or more of the following can achieve the impurity SM 1a 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l Quantification of any one or more of the above.

[0069] Under the condition of detection wavelength of 270nm, the main component rupatadine fumarate SM1 and impurity SM 1a 、Impurity SM 1o 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1l 、Impurity SM 1e The qualitative analysis of any one or more of the following can achieve the impurity SM 1o 、Impurity SM 1j 、Impurity SM 1i 、Impurity SM 1e Quantification of any one or more of the above.

[0070] The beneficial effects of the present invention are:

[0071] The high performance liquid chromatography method provided by the present invention can separate the main peak of rupatadine fumarate SM1 and other 12 known impurities (impurity SM 1a 、Impurity SM 1b 、Impurity SM 1e 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o ) and other single impurities. This analytical method has high sensitivity, strong specificity, good reproducibility, strong durability, simple and feasible operation, and is of great significance for the control and detection of related impurities in the starting material SM1 of rupatadine fumarate. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is the chromatogram of blank solution (detection wavelength is 220nm);

[0073] Figure 2 is the chromatogram of mixed solution 1 (detection wavelength is 220 nm);

[0074] Figure 3 is the chromatogram of the test solution (detection wavelength is 220nm);

[0075] Figure 4 is the chromatogram of the detection limit solution 1 (detection wavelength is 270 nm);

[0076] Figure 5 is the chromatogram of detection limit solution 2 (detection wavelength is 220 nm);

[0077] Figure 6 is the chromatogram of the detection limit solution 3 (detection wavelength is 220 nm);

[0078] Figure 7 Durability - Chromatogram of mixed solution 1 under normal conditions (detection wavelength is 220 nm);

[0079] Figure 8 Durability - Chromatogram of mixed solution 1 under column temperature of 33°C (detection wavelength is 220 nm);

[0080] Fig. 9 Durability - Chromatogram of mixed solution 1 under column temperature of 37°C (detection wavelength is 220 nm);

[0081] Fig.10 Durability - Chromatogram of mixed solution 1 under flow rate of 0.8 ml / min (detection wavelength is 220 nm);

[0082] Fig.11 Durability - Chromatogram of mixed solution 1 under the condition of flow rate 1.0 ml / min (detection wavelength is 220 nm);

[0083] Fig.12 Durability - Chromatogram of mixed solution 1 under the condition of initial ratio of mobile phase A:B=86:14 (detection wavelength is 220 nm);

[0084] Fig.13 Durability - Chromatogram of mixed solution 1 under the condition of initial ratio of mobile phase A:B=82:18 (detection wavelength is 220nm);

[0085] Fig.14 is the chromatogram of blank solution (detection wavelength is 270nm);

[0086] Fig.15 Chromatogram of mixed solution 2 (detection wavelength is 270 nm);

[0087] Fig.16 Chromatogram of the test solution (detection wavelength is 270nm);

[0088] Fig.17 is the chromatogram of mixed solution 2 under durability-normal conditions (detection wavelength is 270 nm);

[0089] Fig.18 Durability - Chromatogram of mixed solution 2 under flow rate of 0.8 ml / min (detection wavelength is 270 nm);

[0090] Fig.19 Durability - Chromatogram of mixed solution 2 under the condition of flow rate 1.0 ml / min (detection wavelength is 270 nm);

[0091] Fig. 20 Durability (mixed solution) - Chromatogram of mixed solution 2 under the condition of mobile phase initial ratio A:B=82:18 (detection wavelength is 270nm);

[0092] Fig.21 Durability - Chromatogram of mixed solution 2 under the condition of initial mobile phase ratio A:B=86:14 (detection wavelength is 270nm);

[0093] Fig. 22 Durability - Chromatogram of mixed solution 2 under column temperature of 33°C (detection wavelength of 270 nm);

[0094] Fig.23 This is the chromatogram of mixed solution 2 under the condition of durability-column temperature of 37°C (detection wavelength is 270nm). DETAILED DESCRIPTION

[0095] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0096] The embodiments of the present invention are determined strictly in accordance with the high performance liquid chromatography method (General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0097] In the embodiment of the present invention, the impurity SM 1c 、Impurity SM 1d 、Impurity SM 1hThe structural formulas are shown in Formulas 14, 15 and 16 respectively;

[0098]

[0099] In the embodiment of the present invention, this product is the starting material SM1 of rupatadine fumarate.

[0100] In the embodiment of the present invention, the solvent / blank solution is an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 70:30.

[0101] In the embodiment of the present invention, the preparation method of the potassium dihydrogen phosphate solution is as follows: weigh 1.36 g of potassium dihydrogen phosphate and 0.5 g of tetrabutylammonium hydrogen sulfate, add 1000 ml of water to dissolve, and obtain the solution.

[0102] In the embodiment of the present invention, the analysis method is as follows:

[0103] (1) Solution preparation:

[0104] Test solution: Take an appropriate amount of the product, weigh it accurately, add about 15% of the total volume of acetonitrile and dissolve it by ultrasonic, then dilute it to the scale with water, shake well, and prepare a solution with a concentration of about 0.4 mg / mL.

[0105] Control solution: Accurately measure the test sample solution and dilute it with solvent to make a solution with a concentration of approximately 0.8 μg / mL.

[0106] (2) Chromatographic conditions:

[0107] Phenylsilane bonded silica gel was used as the filler (Phenomenex Luna Phenyl-Hexyl, 4.6 mm × 150 mm, 3 μm or a chromatographic column with equivalent performance); potassium dihydrogen phosphate solution was used as mobile phase A, acetonitrile was used as mobile phase B, and linear gradient elution was performed according to Table 1; the flow rate was 0.9 mL / min; the column temperature was 35°C; the detection wavelength was 270 nm and / or 220 nm; the injector temperature was 10°C; and the injection volume was 30 μL.

[0108] Table 1. Chromatographic conditions

[0109] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 84 16 10 84 16 30 55 45 35 45 55 40 40 60 40.1 84 16 62 84 16

[0110] (3) Determination

[0111] Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0112] Limits: If there are impurity peaks in the chromatogram of the test solution, in addition to the blank solvent peak, the content of each impurity should meet the limit requirements in Table 2 by calculating the main component self-control method with the correction factor added, and the total impurity content should not exceed 2.0%. Impurity peaks with a content of less than 0.05% are ignored.

[0113] Table 2. Impurity limit regulations (detection wavelength is 270nm)

[0114] Impurity Name Relative retention time Correction Factor Impurity Limit <![CDATA[Impurity SM 1o > 1.30 1.3 0.3％ <![CDATA[Impurity SM 1j > 1.59 1.2 0.3％ <![CDATA[Impurity SM 1i > 2.20 2.7 0.15％ <![CDATA[Impurity SM 1e > 3.74 1.7 0.15％ Other single impurities —— 1.0 0.5％

[0115] Limit (potential impurities): Impurities SM in the test solution 1n The peak area should not be greater than the impurity SM in the reference solution. 1n Peak area (0.0058%), impurity SM in the test solution 1g The peak area should not be greater than the impurity SM in the reference solution. 1g Peak area (0.1%); impurity SM is calculated by the main component self-comparison method with correction factor added 1a 、Impurity SM 1b 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1l and impurity SM 1m Content, the limits of each impurity shall comply with the provisions of Table 3.

[0116] Table 3. Potential impurity limit table (detection wavelength is 220nm)

[0117]

[0118]

[0119] Note: In order to take into account the sensitivity of each potential impurity, the potential impurity analysis method and the chromatographic conditions of the starting material SM1 are different only in the detection wavelength; 1n The proposed control limit is lower, impurity SM 1g The ultraviolet absorption at 220nm is weak, so the limit method is used to calculate the content in the test sample.

[0120] Example 1. Specificity

[0121] Impurities that may be present in the starting material SM1 of rupatadine fumarate include: Impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1d 、Impurity SM 1e 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1h 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM1n and impurity SM 1o There are 15 impurities in total. This method is effective for the main peak and impurity SM 1a 、Impurity SM 1b 、Impurity SM 1e 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o The separation of the impurities and other single impurities in the specificity test is investigated. The details are as follows:

[0122] (1) Solution preparation:

[0123] Impurity localization solution 1: Take the impurity SM separately 1a 、Impurity SM 1b 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1e An appropriate amount, accurately weighed, first added acetonitrile to dissolve, then diluted with solvent to make a solution containing about 4 μg per 1 ml.

[0124] Impurity localization solution 2: Take the impurity SM separately 1e 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1o , accurately weighed, first dissolved in acetonitrile, then diluted with solvent to make a solution containing about 4μg per 1ml.

[0125] Mixed solution 1: Take the starting material SM1 and impurity SM of the test sample rupatadine fumarate respectively 1a 、Impurity SM 1b 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1eAn appropriate amount is accurately weighed, firstly dissolve it in acetonitrile at a total volume of 15%, and then diluted with water to make a mixed solution containing about 0.4 mg of the test sample and 1.2 μg of each impurity per 1 ml.

[0126] Mixed solution 2: Take the starting material SM1 and impurity SM of the test sample rupatadine fumarate respectively 1a 、Impurity SM 1b 、Impurity SM 1f 、Impurity SM 1g 、Impurity SM 1i 、Impurity SM 1j 、Impurity SM 1k 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1e An appropriate amount is accurately weighed, firstly dissolve it in acetonitrile at a total volume of 15%, and then diluted with water to make a mixed solution containing about 0.4 mg of the test sample and 1.2 μg of each impurity per 1 ml.

[0127] (2) Detection

[0128] 1) Take 30 μl of blank solution, each impurity location solution 1, mixed solution 1, and test solution respectively, inject according to the method, detect at a detection wavelength of 220 nm, and record the chromatogram.

[0129] 2) Take 30 μl of blank solution, each impurity location solution 2, mixed solution 2, and test solution respectively, inject according to the method, detect at a detection wavelength of 270 nm, and record the chromatogram.

[0130] Results: As shown in Tables 4-9, Figure 1-Figure 3 , Figure 14-16 As shown in the figure, the blank solution and the test solution did not interfere with the determination of the known impurities; the separation between the known impurities and between the impurities and the main component was greater than 1.5, and the method specificity met the requirements.

[0131] Table 4. Specificity test results (detection wavelength is 220nm)

[0132]

[0133] Table 5. Figure 2 The integral result table

[0134]

[0135] Table 6. Figure 3 The integral result table

[0136]

[0137] Table 7. Specificity test results (detection wavelength is 270nm)

[0138]

[0139]

[0140] Note: Other impurity mixed positioning includes impurity SM 1a 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f and impurity SM 1l .

[0141] Table 8. Fig.15 The integral result table

[0142]

[0143] Table 9. Fig.16 The integral result table

[0144]

[0145] Example 2. Detection Limit

[0146] (1) Preparation of solution

[0147] Detection limit solution 1: Take the starting material SM1 and impurity SM1 of the test sample rupatadine fumarate respectively 1o 、Impurity SM 1j 、Impurity SM 1i and impurity SM 1e An appropriate amount is accurately weighed, firstly 15% acetonitrile is added to dissolve it, and then diluted with water to make a mixed solution containing about 0.05 μg of the test sample and 0.05 μg of each impurity per 1 ml.

[0148] Detection limit solution 2: Take the starting material SM1 of the test sample rupatadine fumarate and each impurity (including impurity SM 1a , SM1, impurity SM 1m 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l 、Impurity SM 1g 、Impurity SM 1j 、Impurity SM 1i and impurity SM 1e) is accurately weighed, first dissolved in 15% acetonitrile, and then diluted with water to make a mixed solution containing approximately 0.04 μg of the test sample and 0.03 μg of each impurity per 1 ml.

[0149] Detection limit solution 3: Take impurity SM separately 1n Appropriate amount, accurately weighed, first add 15% acetonitrile to dissolve, then dilute with water to make each 1ml contain about SM impurities 1n 0.007 μg of solution.

[0150] (2) Detection

[0151] 1) Take the detection limit solution 1 and inject it continuously for 3 times, detect it at the detection wavelength of 270nm, and calculate the ratio of the main peak height to the noise (signal-to-noise ratio);

[0152] 2) Take detection limit solution 2 and detection limit solution 3 and inject them three times respectively, detect at a detection wavelength of 220 nm, and calculate the ratio of the main peak height to the noise (signal-to-noise ratio).

[0153] The test results are shown in Table 10. Figure 4-Figure 6 As shown, the detection limit concentration of raw material SM1 is 0.0476 μg / ml, which is expressed as 0.012% in the sample, and the average signal-to-noise ratio is 11.2; the impurity SM 1o The detection limit concentration is 0.0472μg / ml, which is expressed as 0.012% in the sample, and the mean signal-to-noise ratio is 10.4; impurity SM 1j The detection limit concentration is 0.0368μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 8.6; impurity SM 1i The detection limit concentration is 0.0451μg / ml, which is expressed as 0.011% in the sample, and the mean signal-to-noise ratio is 6.6; impurity SM 1e The detection limit concentration is 0.0482μg / ml, which is expressed as 0.012% in the sample, and the mean signal-to-noise ratio is 10.6; impurity SM 1a The detection limit concentration is 0.013 μg / ml, which is expressed as 0.003% in the sample, and the mean signal-to-noise ratio is 11.2; impurity SM 1m The detection limit concentration is 0.026μg / ml, which is expressed as 0.007% in the sample, and the mean signal-to-noise ratio is 10.2; impurity SM 1k The detection limit concentration is 0.026μg / ml, which is expressed as 0.007% in the sample, and the mean signal-to-noise ratio is 14.8; impurity SM 1f The detection limit concentration is 0.024μg / ml, which is expressed as 0.006% in the sample, and the mean signal-to-noise ratio is 12.8; impurity SM1b The detection limit concentration is 0.025μg / ml, which is expressed as 0.006% in the sample, and the mean signal-to-noise ratio is 8.7; impurity SM 1l The detection limit concentration is 0.026μg / ml, which is expressed as 0.007% in the sample, and the mean signal-to-noise ratio is 20.1; impurity SM 1g The detection limit concentration is 0.037μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 5.7; impurity SM 1n The detection limit concentration is 0.0067 μg / ml, which is expressed as 16.8 ppm in the sample, and the average signal-to-noise ratio is 7.3; both meet the requirements of the detection limit test.

[0154] Table 10. Detection limit determination results

[0155]

[0156]

[0157] Example 3. Chromatographic Conditions Robustness

[0158] (1) Take the mixed solution 1 under the "Specificity" item of Example 1, and use a normal mobile phase, a detection wavelength of 220 nm, and different column temperatures, column flow rates, and mobile phase organic phase-buffer salt initial ratios for the predetermined test to test. After the instrument system is stable, test them separately to examine the separation between the components.

[0159] Results: As shown in Table 11-Table 12, Figure 7-Figure 13 As shown in the figure, when there is a slight fluctuation in the chromatographic conditions, the separation between the components is greater than 1.5, and the method robustness meets the requirements.

[0160] Table 11. Chromatographic conditions test results (separation)

[0161]

[0162]

[0163] Table 12. Chromatographic conditions measurement results (retention time)

[0164]

[0165] (2) Take the mixed solution 2 under the "Specificity" item of Example 1, and use a normal mobile phase, a detection wavelength of 270 nm, and different column temperatures, column flow rates, and mobile phase organic phase-buffer salt initial ratios for the predetermined test to perform tests. After the instrument system is stable, test them separately to examine the separation between the components.

[0166] Results: As shown in Table 13-Table 14, Figure 17-Figure 23 As shown in the figure, when there is a slight fluctuation in the chromatographic conditions, the separation between the components is greater than 1.5, and the method robustness meets the requirements.

[0167] Table 13. Chromatographic conditions test results (separation)

[0168]

[0169]

[0170] Table 14. Chromatographic conditions measurement results (retention time)

[0171]

Claims

1. A method for separating rupatadine fumarate starting material SM1 and its related substances based on high performance liquid chromatography, characterized in that: The related substances include impurities SM 1a 、Impurity SM 1o and its enantiomers, impurity SM 1j and its enantiomers, impurity SM 1g and its enantiomers, impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l 、Impurity SM 1e Any one or more of; in the high performance liquid chromatography, the chromatographic column uses phenylsilane bonded silica gel as a filler, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a potassium dihydrogen phosphate solution, and the mobile phase B is acetonitrile; The impurities SM were removed in sequence by linear gradient elution. 1a 、Rupatadine Fumarate SM1、Impurity SM 1o 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l and / or impurities SM 1e To separate; The structural formula of the rupatadine fumarate SM1 is shown in Formula 1; the impurity SM 1a The structural formula is shown in Formula 2; The impurity SM 1o The structural formula is shown in Formula 3; the impurity SM 1j The structural formula is shown in Formula 4; The impurity SM 1g The structural formula is shown in Formula 5; the impurity SM 1m The structural formula is shown in Formula 6; the impurity SM 1i The structural formula of is shown in Formula 7; the impurity SM 1n The structural formula is shown in Formula 8; The impurity SM 1k The structural formula is shown in Formula 9; The impurity SM 1f The structural formula of is shown in Formula 10; The impurity SM 1b The structural formula of is shown in Formula 11; The impurity SM 1l The structural formula is shown in Formula 12; The impurity SM 1e The structural formula is shown in Formula 13; 2. The method according to claim 1, characterized in that The gradient elution program was set as follows: At 0 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18; At 10 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18; At 30 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45; At 35 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55; At 40 minutes, the volume ratio of mobile phase A to mobile phase B was 40:60; At 40.1 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18; At 62 minutes, the volume ratio of mobile phase A to mobile phase B was 86-82:14-18.

3. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 33° C.-37° C., and the flow rate of the mobile phase is 0.8 ml / min-1.0 ml / min.

4. The method according to claim 1, characterized in that: An acetonitrile aqueous solution is used as a preparation solvent, wherein the volume ratio of acetonitrile to water is 70:

30.

5. A method for qualitatively identifying rupatadine fumarate starting material SM1 and its related substances, characterized in that: The method according to any one of claims 1 to 4 is used to separate the rupatadine fumarate starting material SM1 and its related substances; the impurities SM1 are detected by a detector and a chromatogram is obtained; 1a 、Rupatadine Fumarate SM1、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1l 、Impurity SM 1e The detection wavelength is 270nm and / or 220nm; the impurity SM 1b The detection wavelength is 220nm; the impurity SM 1o The detection wavelength is 270nm.

6. The method according to claim 5, characterized in that The retention time of each component from short to long is as follows: Impurity SM 1a 、Rupatadine Fumarate SM1、Impurity SM 1o 、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l and / or impurities SM 1e .

7. The method according to claim 5, characterized in that The chromatographic column specifications are 4.6 mm × 150 mm, 3 μm; mobile phase A is potassium dihydrogen phosphate solution, mobile phase B is acetonitrile; the mobile phase flow rate is 0.9 ml / min; the column temperature is 35 ° C; the impurity SM 1a 、Rupatadine Fumarate SM1、Impurity SM 1j 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1i 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1l 、Impurity SM 1e The detection wavelength is 270nm and / or 220nm; the impurity SM 1b The detection wavelength is 220nm; the impurity SM 1o The detection wavelength is 270nm; Elution was performed according to a linear gradient elution program, which was set as follows: At 0 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16; At 10 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16; At 30 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45; At 35 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55; At 40 minutes, the volume ratio of mobile phase A to mobile phase B was 40:60; At 40.1 minutes, the volume ratio of mobile phase A to mobile phase B was 84:16; At 62 minutes, the volume ratio of mobile phase A to mobile phase B was 84:

16.

8. The method according to claim 5, characterized in that The impurity SM is retained at a time of 6.2 ± 0.5 min. 1a The retention time is 10.6±1min for rupatadine fumarate SM1, and the retention time is 16.6±0.5min for impurity SM 1j The impurity SM is the one with a retention time of 18.4±0.5min. 1g , the impurity SM is the one with retention time of 21.0±0.5min 1m The impurity SM is the one with a retention time of 22.3±0.5min. 1i The impurity SM is the one with a retention time of 25.2±0.5min. 1n The impurity SM is the one with a retention time of 27.8±0.5min. 1k , the impurity SM is the one with retention time of 28.8±0.5min 1f , the impurity SM is the one with retention time of 30.2±0.5min 1b The impurity SM is the one with a retention time of 31.2±0.5min. 1l The impurity SM is at a retention time of 37.8±0.5min. 1e ; The impurity SM is the one with retention time of 13.2±0.5min 1o .

9. A method for detecting the content of rupatadine fumarate starting material SM1 and its related substances, characterized in that: The steps include: (1) separation: separating the rupatadine fumarate starting material SM1 and its related substances by the method described in any one of claims 1 to 4; (2) Detection: Detect the rupatadine fumarate starting material SM1 and its related substances by the method described in any one of claims 5 to 8, and obtain a chromatogram; the impurity SM 1o 、Impurity SM 1j 、Impurity SM 1i 、Impurity SM 1e The detection wavelength is 270nm; the impurity SM 1a 、Impurity SM 1g 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1k 、Impurity SM 1f 、Impurity SM 1b 、Impurity SM 1l The detection wavelength is 220nm; (3) Content calculation: Using the chromatogram obtained in step (2), the content of each component is calculated by peak area using the principal component self-comparison method with a correction factor.

10. The method according to claim 9, characterized in that Prepare standard solution and detect to obtain standard chromatogram; test the chromatogram obtained from sample solution and calculate based on peak area.

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