Method for separating and determining rupatadine fumarate Z3 and impurities thereof by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and determining rupatadine fumarate Z3 and impurities thereof by an HPLC (High Performance Liquid Chromatography) method. The method comprises the following steps: preparing a system applicability solution, a contrast solution and a sample solution by taking methanol as a solvent, and carrying out gradient elution by taking octadecylsilane chemically bonded silica as a chromatographic column filler, a phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; the method comprises the following steps: sequentially separating an impurity Z2, an impurity Z2e, an impurity SM1, an impurity Z3c, an impurity Z3b, rupatadine fumarate Z3, an impurity Z3d, an impurity Z3a and / or an impurity Z1 through linear gradient elution, and detecting in a detector with the detection wavelength of 270nm. According to the HPLC method provided by the invention, rupatadine fumarate Z3 and eight related impurities can be separated within 40 minutes, and the HPLC method has the characteristics of short analysis time, high sensitivity, strong specificity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for separating and determining rupatadine fumarate Z3 and its impurities by HPLC. 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 is mainly used to treat seasonal and perennial allergic rhinitis and urticaria.

[0003] Rupatadine fumarate Z3 is an important intermediate in the synthesis of rupatadine fumarate, and its structural formula is shown in Formula I. According to the synthetic process of rupatadine fumarate Z3, 11 known impurities may be introduced into rupatadine fumarate Z3: impurity SM1, impurity Z1, impurity Z2, impurity Z 3a 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 3d 、Impurity Z 2e , impurity SM2, impurity SM 2k , impurity imidazole. Among them, impurity SM1, impurity Z1, impurity Z2, impurity Z 3a 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 3d Impurity Z and impurity SM2 are key impurities in the process and have been included in the quality standard of rupatadine fumarate Z3. 2e 、Impurity SM 2k , and the impurity imidazole need further study. In order to better guarantee the quality requirements of rupatadine fumarate Z3, it is necessary to establish an exclusive, sensitive and accurate quantitative detection method that can simultaneously separate and detect the above-mentioned multiple impurities in rupatadine fumarate Z3.

[0004]

[0005] There are many existing technologies that have studied rupatadine fumarate and its related substances. For example, invention patents with publication numbers CN112903859B, CN108508117A, CN102043025B, etc. disclose high-performance liquid chromatography methods for separating and detecting various related substances in rupatadine fumarate. Among them, patents CN112903859B and CN108508117A can achieve the separation and detection of impurity Z2, but except for impurity Z2, these methods are difficult to detect impurities such as impurity SM1 and impurity Z1. At present, there is no quantitative analysis method and literature analysis method that can be used to separate rupatadine fumarate Z3 and the known impurities and research impurities included in the quality standard of rupatadine fumarate Z3. Summary of the invention

[0006] In view of this, the present invention has established a high performance liquid chromatography method to study the 8 known impurities that may be introduced into the rupatadine fumarate intermediate Z3, including impurities SM1, impurities Z1, impurities Z2, impurities Z3, and impurities SM4 that have been included in the quality standards. 3a 、Impurity Z 3b 、Impurity Z 3c And impurity Z 3d , and study impurity Z 2e One of the purposes of the present invention is to provide a method for separating rupatadine fumarate Z3 and its impurities by high performance liquid chromatography, which can separate rupatadine fumarate Z3 and its related impurities within 40 minutes, has a short analysis time and good reproducibility, and provides technical support for the subsequent qualitative and quantitative detection of rupatadine fumarate Z3 and its related impurities.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for separating rupatadine fumarate Z3 and its impurities by high performance liquid chromatography, wherein the impurities include impurity SM1, impurity Z1, impurity Z 3a 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 3d and its enantiomers, impurity Z 2e and any one or more of its enantiomers, and impurity Z2; the method comprises: using octadecylsilane bonded silica gel as a chromatographic column filler, using a phosphate solution as a mobile phase A, and acetonitrile as a mobile phase B, and sequentially removing impurity Z2, impurity Z 2e , impurity SM1, impurity Z 3c 、Impurity Z 3b 、Rupatadine fumarate Z3、Impurity Z 3d 、Impurity Z 3aand / or impurity Z1 are separated; the structural formula of the rupatadine fumarate Z3 is shown in Formula I; the structural formula of the impurity SM1 is shown in Formula II; the structural formula of the impurity Z1 is shown in Formula III; the structural formula of the impurity Z2 is shown in Formula IV; the impurity Z 3a The structural formula of is shown in Formula V; the impurity Z 3b The structural formula is shown in Formula VI; the impurity Z 3c The structural formula of is shown in Formula VII; the impurity Z 3d The structural formula is shown in Formula VIII; the impurity Z 2e The structural formula is shown in Formula IX;

[0009]

[0010]

[0011] Furthermore, the components can be characterized based on how quickly they are separated.

[0012] Further, the gradient elution program is set as follows:

[0013] At 0 min, the volume ratio of the mobile phase A to the mobile phase B is 78-74:22-26;

[0014] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26;

[0015] At 10 min, the volume ratio of the mobile phase A to the mobile phase B was 70:30;

[0016] At 20 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0017] At 30 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0018] At 30.1 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26;

[0019] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26.

[0020] Preferably, the gradient elution program is set as follows:

[0021] At 0 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0022] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0023] At 10 min, the volume ratio of the mobile phase A to the mobile phase B was 70:30;

[0024] At 20 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0025] At 30 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0026] At 30.1 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0027] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24.

[0028] Furthermore, the pH of the mobile phase A is 3.0±0.1, preferably 3.0; the concentration of the phosphate solution is 0.015 mol / L-0.025 mol / L, preferably 0.02 mol / L.

[0029] Furthermore, the preparation method of the phosphate solution is: accurately weigh an appropriate amount of sodium dihydrogen phosphate dihydrate, add water to dissolve it, and obtain a solution with a concentration of about 0.015mol / L-0.025mol / L, preferably 0.02mol / L; adjust the pH value to 3.0±0.1 with phosphoric acid.

[0030] Furthermore, the flow rate of the mobile phase is 0.6 ml / min-0.8 ml / min, preferably 0.7 ml / min; the column temperature of the chromatographic column is 33° C.-37° C., preferably 35° C.

[0031] Preferably, the injection volume is 10 μl.

[0032] Preferably, the chromatographic column specifications are 4.6 mm×150 mm, 3 μm.

[0033] The second object of the present invention is to provide a method for identifying rupatadine fumarate Z3 and its related impurities based on the above separation method, which can achieve qualitative identification of rupatadine fumarate Z3 and its eight related impurities.

[0034] To achieve the above object, the present invention adopts the following technical solutions:

[0035] The method for identifying rupatadine fumarate Z3 and its related impurities based on the aforementioned separation method comprises separating rupatadine fumarate Z3 and its related impurities by the aforementioned separation method; and detecting by using a detector with a detection wavelength of 270 nm.

[0036] Furthermore, the retention time of each component from short to long is impurity Z2, impurity Z 2e, impurity SM1, impurity Z 3c 、Impurity Z 3b 、Rupatadine fumarate Z3、Impurity Z 3d 、Impurity Z 3a , impurity Z1.

[0037] The components can be qualitatively identified based on their retention time.

[0038] As a preferred embodiment, the chromatographic column has a specification of 4.6 mm×150 mm, 3 μm; the mobile phase A is a phosphate solution with a pH value of 3.0, and the mobile phase B is acetonitrile; the flow rate is 0.7 ml / min; the column temperature is 35° C.; and the detection wavelength is 270 nm;

[0039] Separation was performed according to a linear gradient elution program to obtain a chromatogram, and the gradient elution program was as follows:

[0040] At 0 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0041] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0042] At 10 min, the volume ratio of the mobile phase A to the mobile phase B was 70:30;

[0043] At 20 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0044] At 30 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80;

[0045] At 30.1 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24;

[0046] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24.

[0047] The impurity Z2 is at a retention time of 8.6±0.5min, and the impurity Z is at a retention time of 9.2±0.5min. 2e The impurity SM1 is at a retention time of 10.1±0.5min, and the impurity Z is at a retention time of 15.1±0.5min. 3c The impurity Z is the one with a retention time of 16.9±0.5min. 3b The retention time of 18.5±0.5min is rupatadine fumarate Z3, and the retention time of 19.4±0.5min is impurity Z 3d The impurity Z has a retention time of 22.4±0.5min. 3aThe impurity Z1 has a retention time of 23.0±0.5min.

[0048] The third object of the present invention is to provide a method for detecting the content of rupatadine fumarate Z3 and its related impurities, which can achieve the detection of rupatadine fumarate Z3 and its related impurities within 40 minutes or more.

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

[0050] The method for detecting the content of rupatadine fumarate Z3 and its related impurities comprises the following steps:

[0051] (1) separation: separating the rupatadine fumarate Z3 and its related impurities by the method described in any one of claims 1 to 4;

[0052] (2) Detection: Detecting rupatadine fumarate Z3 and its related impurities using the method described in any one of claims 5 to 8 to obtain a chromatogram;

[0053] (3) Content calculation: Based on the chromatogram obtained in step (2), the content of rupatadine fumarate Z3 and its related impurities is calculated by peak area using the principal component self-reference method with a correction factor.

[0054] The impurity content calculation formula is as follows:

[0055]

[0056] Total impurities are the sum of all impurities.

[0057] Where, Az: the peak area of ​​the main component in the reference solution;

[0058] As: impurity peak area in the test solution;

[0059] F: impurity correction factor (if there is no correction factor, F=1);

[0060] N: dilution factor for preparing control solution.

[0061] Furthermore, before separation, methanol was used as solvent to prepare the sample solution, control solution and system suitability solution.

[0062] Furthermore, the correction factor of the impurity Z2 is 1.4, the correction factor of the impurity SM1 is 1.3, and the impurity Z 3c The correction factor is 1.4, the impurity Z 3b The correction factor is 1.6, the impurity Z 3d The correction factor is 1.4, the impurity Z 3aThe correction factor of is 1.2, the correction factor of impurity Z1 is 1.5, and the correction factor of other single impurities is 1.0.

[0063] Furthermore, a standard substance is prepared and a chromatogram of the standard substance is obtained by detection; and the peak area of ​​the chromatogram obtained by the test sample is calculated according to the external standard method.

[0064] The content of the components of the standard is known, while the content of the components of the test sample is unknown.

[0065] 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.

[0066] The beneficial effects of the present invention are:

[0067] The high performance liquid chromatography method provided by the present invention can remove rupatadine fumarate Z3 and seven known impurities (impurity SM1, impurity Z1, impurity Z2, impurity Z3) that may be present in rupatadine fumarate Z3 and are included in the quality standard within only 40 minutes. 3a 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 3d ) and 1 research impurity (Impurity Z 2e ) for separation, and can ensure that the impurities SM2 and impurities SM 2k The invention provides a novel method for separating rupatadine fumarate Z3 from possible impurities, and the impurity imidazole has no interference with the above impurities; effective separation can be achieved under the condition of fine-tuning of chromatographic conditions; the problem of separation and determination of rupatadine fumarate Z3 and possible impurities that cannot be solved by the prior art is provided; and the analytical method is short in time, highly sensitive, highly durable, highly specific, highly reproducible, and simple and feasible to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 is the HPLC chromatogram of blank solvent;

[0069] Figure 2 is the HPLC chromatogram of the test solution;

[0070] Figure 3 is the HPLC chromatogram of the mixed solution;

[0071] Figure 4 Durability - HPLC chromatogram of the mixed solution under the flow rate of 0.6 ml / min;

[0072] Figure 5 Durability - HPLC chromatogram of the mixed solution under the condition of flow rate 0.8 ml / min;

[0073] Figure 6 Durability - HPLC chromatogram of the mixed solution under the condition of column temperature of 33°C;

[0074] Figure 7 Durability - HPLC chromatogram of the mixed solution under the condition of column temperature of 37°C;

[0075] Figure 8 Durability - HPLC chromatogram of the mixed solution under the condition of initial ratio of mobile phase A:B=78:22;

[0076] Fig. 9 Durability - HPLC chromatogram of the mixed solution under the condition of initial ratio of mobile phase A:B=74:26;

[0077] Fig.10 This is the HPLC chromatogram of the mixed solution under the condition of durability-mobile phase pH 2.9;

[0078] Fig.11 This is the HPLC chromatogram of the mixed solution under the condition of durability-mobile phase pH 3.1;

[0079] Fig.12 For durability - HPLC chromatograms of mixed solutions of different batches of chromatographic columns;

[0080] Fig.13 The chromatogram is the limit of detection (LOD). DETAILED DESCRIPTION

[0081] 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.

[0082] In the embodiments of the present invention, the determination was carried out strictly in accordance with the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0083] In the examples of the present invention, the product is rupatadine fumarate Z3; and the solvent / blank solution is methanol.

[0084] In the embodiment of the present invention, the structural formula of impurity SM2 is shown in Formula X, and impurity SM 2k The structural formula of is shown in Formula XI, and the structural formula of the impurity imidazole is shown in Formula XII;

[0085]

[0086] In the embodiment of the present invention, the analysis method is as follows:

[0087] (1) Solution preparation

[0088] Test solution: Take an appropriate amount of the product, weigh it accurately, dissolve it in methanol and dilute it to make a solution with a concentration of about 1 mg / mL as the test solution.

[0089] Control solution: Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0090] System suitability solution: Take system suitability reference (including RUP-Z3, impurity RUP-SM1, impurity RUP-Z1, impurity RUP-Z2, impurity RUP-Z 3a 、Impurity RUP-Z 3b 、Impurity RUP-Z 3c and impurity RUP-Z 3d ) is accurately weighed, dissolved in methanol and diluted to make a solution containing about 1 mg per 1 ml.

[0091] Phosphate solution: Take 3.12 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water to dissolve it, and adjust the pH to 3.0±0.1 with phosphoric acid.

[0092] (2) Chromatographic conditions

[0093] Use octadecylsilane bonded silica gel as filler (YMC-Triart C18 ExRs, 4.6mm×150mm, 3μm or chromatographic column with equivalent performance); use phosphate solution as mobile phase A, acetonitrile as mobile phase B, perform linear gradient elution according to Table 1, and the detection wavelength is 270nm; the flow rate is 0.7ml per minute; the column temperature is 35℃; the injection volume is 10μl.

[0094] Table 1. Gradient elution program

[0095] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 76 24 5 76 24 10 70 30 20 20 80 30 20 80 30.1 76 24 40 76 24

[0096] (3) Determination

[0097] System suitability requirements: In the system suitability solution chromatogram, the main peak and impurity RUP-Z 3d The peak separation should meet the requirements.

[0098] Determination method: Accurately measure the test solution and control solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0099] Limits: If there are impurity peaks in the chromatogram of the test solution, except for the solvent peak and the gradient elution peak, the content of each impurity should meet the limit requirements in Table 2 according to the main component self-reference method with correction factors, and the total impurity content should not exceed 7.0%. Impurity peaks with a content of less than 0.05% are ignored.

[0100] Table 2

[0101]

[0102]

[0103] Example 1. Specificity

[0104] (1) Solution preparation

[0105] Impurity SM1 stock solution: Accurately weigh 24.81 mg of impurity SM1, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well.

[0106] Impurity Z1 stock solution: Accurately weigh 25.37 mg of impurity Z1, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well.

[0107] Impurity Z2 stock solution: Accurately weigh 24.71 mg of impurity Z2, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well.

[0108] Impurity Z 3a Stock solution: Accurately weigh impurity Z 3a 24.62 mg, put in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well.

[0109] Impurity Z 3b Stock solution: Accurately weigh impurity Z 3b 24.91 mg, put in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well.

[0110] Impurity Z 3c Stock solution: Accurately weigh impurity Z 3c 24.41 mg, put in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well.

[0111] Impurity Z 3d Stock solution: Accurately weigh impurity Z 3d 25.69 mg, put in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well.

[0112] Impurity Z 2e Stock solution: Accurately weigh impurity Z 2e 20.10 mg, put in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well.

[0113] Impurity SM2 stock solution: Accurately weigh 25.57 mg of impurity SM2, place it in a 25 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0114] Impurity SM 2kStock solution: Accurately weigh the impurity SM 2k 26.63 mg, put in a 25 ml volumetric flask, add 60% methanol water to dissolve and dilute to the scale, shake well.

[0115] Impure imidazole stock solution: Accurately weigh 27.36 mg of impurity imidazole, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well.

[0116] Each impurity location solution: accurately measure the above impurity stock solution (except impurity Z 2e (External) Place 0.1 ml of each solution in different 10 ml volumetric flasks, dilute to the mark with methanol and shake well.

[0117] Impurity Z 2e Positioning solution: Precisely measure impurities Z 2e Place 2.5 ml of the stock solution in a 20 ml volumetric flask, dilute to volume with methanol, and shake well to obtain impurity Z. 2e Precisely measure 1 ml of the above solution into a 10 ml volumetric flask and shake well.

[0118] Test solution: Accurately weigh 49.35 mg of the test sample, place it in a 50 ml volumetric flask, dissolve it in methanol and dilute to the scale, and shake well.

[0119] Self-control solution: Accurately measure 0.5 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0120] Mixed solution: Accurately weigh 50.62 mg of the test sample and place it in a 50 ml volumetric flask. Add an appropriate amount of methanol to dissolve it. Accurately measure 0.25 ml of each of the impurity SM1, impurity Z1, and impurity Z2 stock solutions. 3a Stock solution 0.5ml, impurity Z 3b Stock solution 0.15ml, impurity Z 3c Stock solution 75μl, impurity Z 3d Stock solution 1ml, impurity Z 2e Place 0.25 ml of the stock solution in a 50 ml volumetric flask, add methanol to dilute to the mark, and shake well.

[0121] (2) Determination

[0122] Take 10 μl of each blank solution (methanol), each impurity location solution, test solution, and mixed solution, inject them into the liquid chromatograph, and record the chromatogram.

[0123] Results: Figure 1-Figure 3 As shown in Table 3, the blank solution does not interfere with the detection of each known impurity and the test sample; the separation between each known impurity peak and between the known impurity peak and the main peak is greater than 1.2, and the method specificity meets the requirements. 2kThe peak is at the blank solvent, and the impurity imidazole has no response at 270nm, and does not interfere with the detection of other known impurities and test samples.

[0124] Table 3. Specificity test results

[0125]

[0126] Table 4. Figure 2 The integral result table

[0127]

[0128] Table 5. Figure 3 The integral result table

[0129]

[0130] Example 2. Detection Limit

[0131] (1) Solution preparation

[0132] Quantitative limit stock solution: Accurately measure the impurities SM1, impurity Z1, impurity Z2, impurity Z3, impurity Z4, impurity Z5, impurity Z6, impurity Z7, impurity Z8, impurity Z9, impurity Z10, impurity Z11, impurity Z12, impurity Z13, impurity Z14, impurity Z15, impurity Z16, impurity 3a 、Impurity Z 3b 、Impurity Z 3c 2.5 ml of stock solution, impurity Z 3d Stock solution 2ml, impurity Z 2e 3.1ml of stock solution and 2.5ml of test solution are placed in the same 25ml volumetric flask, dilute to the mark with methanol, shake well, and the solution is obtained. Accurately measure 1.5ml of the above solution, place in a 50ml volumetric flask, dilute to the mark with methanol, shake well, and the solution is obtained.

[0133] Quantitation limit solution: Accurately measure 5 ml of the quantitation limit stock solution and place it in a 50 ml volumetric flask. Add methanol to dilute to the scale and shake well.

[0134] Detection limit solution: Accurately measure 3.0 ml of the quantitative limit solution and place it in a 10 ml volumetric flask. Add methanol to dilute to the scale and shake well.

[0135] (2) Determination

[0136] The detection limit solution was sampled three times continuously, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated.

[0137] Results: As shown in Table 6-Table 7, Fig.13 As shown, the detection limit concentration of rupatadine fumarate Z3 is 0.0918 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 25.0; the detection limit concentration of impurity Z2 is 0.089 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 11.1; impurity Z2e The detection limit concentration is 0.0803 μg / ml, which is expressed as 0.008% in the sample, and the mean signal-to-noise ratio is 6.0; the detection limit concentration of impurity SM1 is 0.0893 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 8.6; the detection limit concentration of impurity Z 3c The detection limit concentration is 0.0879 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 20.4; impurity Z 3b The detection limit concentration is 0.0897 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 16.2; impurity Z 3d The detection limit concentration is 0.0925 μg / ml, which is expressed as 0.009% in the sample, and the mean signal-to-noise ratio is 17.2; impurity Z 3a The detection limit concentration is 0.0886 μg / ml, which is expressed as 0.009% in the sample, and the average signal-to-noise ratio is 17.9; the detection limit concentration of impurity Z1 is 0.0913 μg / ml, which is expressed as 0.009% in the sample, and the average signal-to-noise ratio is 13.9; both meet the requirements of the detection limit test.

[0138] Table 6. Detection limit determination results

[0139]

[0140]

[0141] Table 7. Fig.13 The integral result table

[0142]

[0143] Example 3. Chromatographic Conditions Robustness

[0144] Take the mixed solution under the "Specificity" item of Example 1, and use normal chromatographic conditions, different column temperatures, column flow rates, mobile phase organic phase-buffer salt initial ratios, buffer salt pH, and chromatographic columns of the same model but different batches for testing. After the instrument system is stable, test them separately, record the chromatograms, and examine the changes in the separation between the peaks of each component.

[0145] Results: Figure 3-Figure 12 As shown in Tables 8 and 9, when there are slight fluctuations in the flow rate, column temperature, initial ratio of mobile phase organic phase-buffer salt, and pH of mobile phase buffer salt in the chromatographic conditions and for chromatographic columns of the same model but different batches, the separation degrees between the impurities controlled by this analytical method and the test sample and the known impurities that may exist in the test sample are all greater than 1.2, the relative retention time of each component is basically consistent with the normalized content, and the durability of the chromatographic conditions of the method meets the requirements.

[0146] Table 8. Results of the durability test under chromatographic condition changes

[0147]

[0148]

[0149] Table 9. Figure 3-Figure 12 Results of the durability test under chromatographic condition changes

[0150]

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for separating rupatadine fumarate Z3 and its impurities by high performance liquid chromatography, characterized in that: The impurities include impurities SM1, impurities Z1, impurities Z 3a 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 3d and its enantiomers, impurity Z 2e and any one or more of its enantiomers, and impurity Z2; the method comprises: using octadecylsilane bonded silica gel as a chromatographic column filler, using a phosphate solution as a mobile phase A, and acetonitrile as a mobile phase B, and sequentially removing impurity Z2, impurity Z 2e , impurity SM1, impurity Z 3c 、Impurity Z 3b 、Rupatadine fumarate Z3、Impurity Z 3d 、Impurity Z 3a and / or impurity Z1 for separation; The structural formula of the rupatadine fumarate Z3 is shown in Formula I; the structural formula of the impurity SM1 is shown in Formula II; the structural formula of the impurity Z1 is shown in Formula III; the structural formula of the impurity Z2 is shown in Formula IV; 3a The structural formula of is shown in Formula V; the impurity Z 3b The structural formula of is shown in Formula VI; the impurity Z 3c The structural formula is shown in Formula VII; the impurity Z 3d The structural formula is shown in Formula VIII; the impurity Z 2e The structural formula is shown in Formula IX; 2. The method according to claim 1, characterized in that , the gradient elution program is set as follows: At 0 min, the volume ratio of the mobile phase A to the mobile phase B is 78-74:22-26; At 5 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26; At 10 min, the volume ratio of the mobile phase A to the mobile phase B was 70:30; At 20 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80; At 30 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80; At 30.1 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26; At 40 min, the volume ratio of the mobile phase A to the mobile phase B was 78-74:22-26.

3. The method according to claim 1, characterized in that The pH of the mobile phase A is 3.0±0.1; the concentration of the phosphate solution is 0.015 mol / L-0.025 mol / L.

4. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.6 ml / min-0.8 ml / min, and the column temperature of the chromatographic column is 33° C.-37° C.

5. A method for identifying rupatadine fumarate Z3 and its related impurities based on the method described in any one of claims 1 to 4, characterized in that: Separating rupatadine fumarate Z3 and its related impurities by the method described in any one of claims 1 to 4; and The detection was carried out using a detector with a detection wavelength of 270 nm.

6. The method according to claim 5, characterized in that The retention time of each component from short to long is impurity Z2, impurity Z 2e , impurity SM1, impurity Z 3c 、Impurity Z 3b 、Rupatadine fumarate Z3、Impurity Z 3d 、Impurity Z 3a , impurity Z1.

7. The method according to claim 5, characterized in that The chromatographic column specifications are 4.6 mm × 150 mm, 3 μm; The mobile phase A is a phosphate solution with a pH value of 3.0, and the mobile phase B is acetonitrile; the flow rate is 0.7 ml / min; The column temperature was 35°C; the detection wavelength was 270 nm; Separation was performed according to a linear gradient elution program to obtain a chromatogram, and the gradient elution program was as follows: At 0 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24; At 5 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24; At 10 min, the volume ratio of the mobile phase A to the mobile phase B was 70:30; At 20 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80; At 30 min, the volume ratio of the mobile phase A to the mobile phase B was 20:80; At 30.1 min, the volume ratio of the mobile phase A to the mobile phase B was 76:24; At 40 min, the volume ratio of the mobile phase A to the mobile phase B was 76:

24.

8. The method according to claim 7, characterized in that: The impurity Z2 is at a retention time of 8.6±0.5min, and the impurity Z is at a retention time of 9.2±0.5min. 2e The impurity SM1 is at a retention time of 10.1±0.5min, and the impurity Z is at a retention time of 15.1±0.5min. 3c The impurity Z is the one with a retention time of 16.9±0.5min. 3b The retention time of 18.5±0.5min is rupatadine fumarate Z3, and the retention time of 19.4±0.5min is impurity Z 3d The impurity Z has a retention time of 22.4±0.5min. 3a The impurity Z1 has a retention time of 23.0±0.5min.

9. A method for detecting the content of rupatadine fumarate Z3 and its related impurities, characterized in that: The following steps are involved: (1) separation: separating the rupatadine fumarate Z3 and its related impurities by the method described in any one of claims 1 to 4; (2) Detection: Detecting rupatadine fumarate Z3 and its related impurities using the method described in any one of claims 5 to 8 to obtain a chromatogram; (3) Content calculation: Based on the chromatogram obtained in step (2), the content of rupatadine fumarate Z3 and its related impurities is calculated by peak area using the principal component self-reference method with a correction factor.

10. The method according to claim 9, characterized in that Before separation, methanol was used as solvent to prepare the sample solution, control solution and system suitability solution.

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