Method for analyzing and detecting bilastine intermediate and related substances thereof through high performance liquid chromatography

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for separating a bilastine intermediate and related substances thereof. The method comprises the following steps: detecting a to-be-detected sample by using a high performance liquid chromatography to obtain a chromatogram; wherein the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent of a chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A and the mobile phase B are used for gradient elution, the mobile phase A is a sodium hexanesulfonate solution, the mobile phase B is acetonitrile, and the bilastine intermediate and the related substances thereof are subjected to gradient elution and HPLC (High Performance Liquid Chromatography) detection. According to the method disclosed by the invention, the bilastine intermediate and the related substances thereof can be effectively separated, so that the peaks of the related substances and the peaks of the bilastine intermediate are not overlapped, the peak shape is good, the separation requirement is met, and the quality control of drugs taking bilastine as a raw material can be effectively realized.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography analysis, and more particularly to a method for high performance liquid chromatography analysis and detection of bilastine intermediate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride and related substances. Background Technology

[0002] Bilastin is a non-sedating, long-acting antihistamine used clinically to treat allergic rhinoconjunctivitis and urticaria. Bilastin selectively antagonizes peripheral H1 receptors, has no affinity for toxic alkaloid receptors, and low affinity for other receptors. Bilastin is characterized by its non-sedating effect, lack of cardiotoxicity, non-interaction with cytochrome P450 enzyme substrates, and potent antihistamine properties.

[0003] In the synthesis of bilastin, it is necessary to control the purity of some important intermediates to reduce side reactions and impurity formation, thereby improving the yield and purity of the final product. The names and structural formulas of bilastin intermediates and related impurities are shown in the figure.

[0004]

[0005] Currently, there are no mature reports on the analysis and detection of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzi[d]imidazolium hydrochloride and related substances by high performance liquid chromatography (HPLC).

[0006] Therefore, there is an urgent need to develop a method for analyzing and detecting bilastine intermediates and related substances using high-performance liquid chromatography (HPLC) in order to effectively monitor the quality of latfubilastine. Summary of the Invention

[0007] This invention aims to at least partially solve one of the technical problems in related technologies. To this end, this invention provides a method for high-performance liquid chromatography (HPLC) analysis and detection of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride, an intermediate of bilastine, and related substances. This method can simultaneously and effectively separate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride) and related substances, thereby achieving quality control of bilastine raw material.

[0008] Therefore, the first aspect of the present invention provides a method for high performance liquid chromatography (HPLC) analysis and detection of bilastine intermediate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride and related substances, wherein the method utilizes HPLC to detect the sample to be tested to obtain a chromatogram;

[0009] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0010] The chromatographic column was packed with octadecylsilane-bonded silica gel.

[0011] The mobile phase includes mobile phase A and mobile phase B, and gradient elution is performed using mobile phase A and mobile phase B.

[0012] The mobile phase A is an ion-pairing reagent with a concentration of >0.05% and <0.7%; and the pH of the mobile phase A is adjusted to <4; the mobile phase B is acetonitrile.

[0013] The ion-pairing reagents of the present invention include one or more of sodium methanesulfonate, sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, sodium decanesulfonate, sodium dodecyl sulfonate, and sodium dodecyl sulfate.

[0014] The gradient elution conditions of the method provided by this invention are as follows:

[0015] From 0 to 8 minutes, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 75% to 85%; the content of mobile phase B is 15% to 25%.

[0016] Within 8–35 minutes, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B decreases from 75–85% to 45–55%; the content of mobile phase B increases from 15–25% to 45–55%.

[0017] For 35.1–45 min, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 75–85%; the content of mobile phase B is 15–25%.

[0018] The method provided by this invention is based on high-performance liquid chromatography (HPLC). Its technical principle is that when components dissolved in the mobile phase pass through the stationary phase, their residence times in the stationary phase differ due to variations in the magnitude and strength of their interactions with the stationary phase (adsorption, partitioning, ion attraction, exclusion, affinity), resulting in their sequential elution. The method provided by this invention can effectively separate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride) and related substances, thereby achieving quality control of pharmaceuticals using bilastine as a raw material. Furthermore, only under the specific elution conditions of this invention can the peak shapes of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride and related substances in the chromatogram be well-defined, achieving the expected resolution standard, with the resolution of each adjacent peak ≥1.5.

[0019] According to an embodiment of the present invention, the gradient elution conditions are preferably:

[0020] From 0 to 8 minutes, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 80%; the content of mobile phase B is 20%.

[0021] Within 8–35 minutes, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B decreases from 80% to 50%; the content of mobile phase B increases from 20% to 50%.

[0022] During the 35.1–45 min time, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 80%; the content of mobile phase B is 20%.

[0023] According to an embodiment of the present invention, the gradient elution conditions are more preferably as follows:

[0024] From 0 to 8 minutes, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 80%; the content of mobile phase B is 20%.

[0025] From 8 to 25.1 min, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B decreased from 80% to 50%; the content of mobile phase B increased from 20% to 50%.

[0026] During the 25.1–35 min period, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 50%; the content of mobile phase B is 50%.

[0027] During the 35.1–45 min time, the percentage content of mobile phase A in the total volume of mobile phase A and mobile phase B is 80%; the content of mobile phase B is 20%.

[0028] According to an embodiment of the present invention, the mobile phase A is a sodium hexanesulfonate solution or a sodium octanesulfonate solution.

[0029] According to an embodiment of the present invention, the mobile phase A is more preferably a sodium hexanesulfonate solution.

[0030] According to an embodiment of the present invention, the concentration of sodium hexanesulfonate in the mobile phase A is 0.15% to 0.5%.

[0031] According to an embodiment of the present invention, the method includes adjusting the pH value of the mobile phase A using phosphoric acid.

[0032] According to an embodiment of the present invention, the pH value of the mobile phase A is 2.5 to 3.5.

[0033] According to an embodiment of the present invention, the chromatographic column used in the high performance liquid chromatography is preferably an Imptakt Unision UK-C18.

[0034] According to an embodiment of the present invention, the column temperature of the chromatographic column is 30–40°C.

[0035] According to an embodiment of the present invention, the flow rate of the mobile phase is 0.8 to 1.2 ml / min.

[0036] According to an embodiment of the present invention, the detection wavelength is 200-250 nm.

[0037] According to an embodiment of the present invention, the method further includes the following steps:

[0038] (1) Prepare a sample solution containing 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride;

[0039] (2) Take 5 μl of the sample solution and inject it into the high performance liquid chromatograph. Perform high performance liquid chromatography analysis and record the chromatogram to complete the separation and detection of the sample containing the bilastine intermediate.

[0040] The second aspect of the present invention provides the application of the method described in the first aspect in the quality control of bilastine.

[0041] The advantages of this invention over the prior art are:

[0042] The high-performance liquid chromatography (HPLC) method provided by this invention for the detection of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride and its related substances can effectively separate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride and its related substances. The peaks of the six substances are symmetrical and well separated, with a resolution greater than or equal to 1.5. Under the optimal method, the limit of quantitation is 0.027 μg / mL. Furthermore, this method is simple to operate, can be performed using a common HPLC instrument, and the mobile phase medium is readily available. It is highly feasible and applicable, and can be widely used in the quality research of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride).

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0045] Figure 1 The high-performance liquid chromatogram of the sample obtained according to Example 1 of the present invention is shown;

[0046] Figure 2The high-performance liquid chromatogram of the sample obtained according to Example 2 of the present invention is shown;

[0047] Figure 3 The high-performance liquid chromatogram of the sample obtained according to Example 3 of the present invention is shown;

[0048] Figure 4 The high-performance liquid chromatogram of the sample obtained according to Example 4 of the present invention is shown;

[0049] Figure 5 The high-performance liquid chromatogram of the sample obtained according to Example 5 of the present invention is shown;

[0050] Figure 6 The high-performance liquid chromatogram of the sample obtained according to Example 6 of the present invention is shown;

[0051] Figure 7 The high-performance liquid chromatogram of the sample obtained according to Example 7 of the present invention is shown;

[0052] Figure 8 The high-performance liquid chromatogram of the sample obtained according to Example 8 of the present invention is shown;

[0053] Figure 9 The high-performance liquid chromatogram of the sample obtained according to Example 9 of the present invention is shown;

[0054] Figure 10 The high-performance liquid chromatogram of the sample obtained according to Example 10 of the present invention is shown;

[0055] Figure 11 The high-performance liquid chromatogram of the sample obtained according to Example 11 of the present invention is shown;

[0056] Figure 12 The high-performance liquid chromatogram of the sample obtained according to Example 12 of the present invention is shown;

[0057] Figure 13 The high-performance liquid chromatogram of the sample obtained in Comparative Example 1 of this invention is shown.

[0058] Figure 14 The high-performance liquid chromatogram of the sample obtained in Comparative Example 2 of this invention is shown;

[0059] Figure 15 The high-performance liquid chromatogram of the sample obtained in Comparative Example 3 of the present invention is shown;

[0060] Figure 16 The high-performance liquid chromatogram of the sample obtained in Comparative Example 4 of this invention is shown.

[0061] Figure 17 The high-performance liquid chromatogram of the sample obtained in Comparative Example 5 of this invention is shown;

[0062] Figure 18 The high-performance liquid chromatogram of the sample obtained in Comparative Example 6 of this invention is shown;

[0063] Figure 19 The high-performance liquid chromatogram of the sample obtained in Comparative Example 7 of the present invention is shown;

[0064] Figure 20 The high-performance liquid chromatogram of the sample obtained in Comparative Example 8 of this invention is shown.

[0065] Figure 21 The high-performance liquid chromatogram of the sample obtained in Comparative Example 9 of this invention is shown.

[0066] Figure 22 The high-performance liquid chromatogram of the sample obtained in Comparative Example 10 of the present invention is shown. Detailed Implementation

[0067] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0068] Example 1

[0069] Chromatographic conditions:

[0070] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0071] Column: Impakt Unision UK-C18;

[0072] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0073] Mobile phase B: Acetonitrile

[0074] Flow rate: 1.0 mL / min;

[0075] Detection wavelength: 205nm;

[0076] Column temperature: 35℃;

[0077] Injection volume: 5 μL;

[0078] Perform gradient elution according to the table below:

[0079]

[0080] Experimental steps:

[0081] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0082] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0083] See results Figure 1 , Figure 1 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.320 min.

[0084] Example 2

[0085] Chromatographic conditions:

[0086] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0087] Column: Impakt Unision UK-C18;

[0088] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0089] Mobile phase B: Acetonitrile

[0090] Flow rate: 0.8 mL / min;

[0091] Detection wavelength: 205nm;

[0092] Column temperature: 35℃;

[0093] Injection volume: 5 μL;

[0094] Perform gradient elution according to the table below:

[0095]

[0096] Experimental steps:

[0097] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0098] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0099] See results Figure 2 , Figure 2 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.529 min.

[0100] Example 3

[0101] Chromatographic conditions:

[0102] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0103] Column: Impakt Unision UK-C18;

[0104] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0105] Mobile phase B: Acetonitrile

[0106] Flow rate: 1.2 mL / min;

[0107] Detection wavelength: 205nm;

[0108] Column temperature: 35℃;

[0109] Injection volume: 5 μL;

[0110] Perform gradient elution according to the table below:

[0111]

[0112] Experimental steps:

[0113] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0114] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0115] See results Figure 3 , Figure 3 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.163 min.

[0116] Example 4

[0117] Chromatographic conditions:

[0118] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0119] Column: Impakt Unision UK-C18;

[0120] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0121] Mobile phase B: Acetonitrile

[0122] Flow rate: 1.0 mL / min;

[0123] Detection wavelength: 205nm;

[0124] Column temperature: 30℃;

[0125] Injection volume: 5 μL;

[0126] Perform gradient elution according to the table below:

[0127]

[0128] Experimental steps:

[0129] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0130] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0131] See results Figure 4 , Figure 4 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 24.993 min.

[0132] Example 5

[0133] Chromatographic conditions:

[0134] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0135] Column: Impakt Unision UK-C18;

[0136] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0137] Mobile phase B: Acetonitrile

[0138] Flow rate: 1.0 mL / min;

[0139] Detection wavelength: 205nm;

[0140] Column temperature: 40℃;

[0141] Injection volume: 5 μL;

[0142] Perform gradient elution according to the table below:

[0143]

[0144] Experimental steps:

[0145] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0146] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0147] See results Figure 5 , Figure 5 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 21.083 min.

[0148] Example 6

[0149] Chromatographic conditions:

[0150] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0151] Column: Impakt Unision UK-C18;

[0152] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 2.5 with phosphoric acid;

[0153] Mobile phase B: Acetonitrile

[0154] Flow rate: 1.0 mL / min;

[0155] Detection wavelength: 205nm;

[0156] Column temperature: 35℃;

[0157] Injection volume: 5 μL;

[0158] Perform gradient elution according to the table below:

[0159]

[0160]

[0161] Experimental steps:

[0162] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0163] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0164] See results Figure 6 , Figure 6 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.979 min.

[0165] Example 7

[0166] Chromatographic conditions:

[0167] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0168] Column: Impakt Unision UK-C18;

[0169] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.5 with phosphoric acid;

[0170] Mobile phase B: Acetonitrile

[0171] Flow rate: 1.0 mL / min;

[0172] Detection wavelength: 205nm;

[0173] Column temperature: 35℃;

[0174] Injection volume: 5 μL;

[0175] Perform gradient elution according to the table below:

[0176]

[0177] Experimental steps:

[0178] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0179] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0180] See results Figure 7 , Figure 7 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.699 min.

[0181] Example 8

[0182] Chromatographic conditions:

[0183] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0184] Column: Impakt Unision UK-C18;

[0185] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0186] Mobile phase B: Acetonitrile

[0187] Flow rate: 1.0 mL / min;

[0188] Detection wavelength: 200nm;

[0189] Column temperature: 35℃;

[0190] Injection volume: 5 μL;

[0191] Perform gradient elution according to the table below:

[0192]

[0193] Experimental steps:

[0194] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0195] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0196] See results Figure 8 , Figure 1 Peak 8 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.320 min.

[0197] Example 9

[0198] Chromatographic conditions:

[0199] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0200] Column: Impakt Unision UK-C18;

[0201] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0202] Mobile phase B: Acetonitrile

[0203] Flow rate: 1.0 mL / min;

[0204] Detection wavelength: 250nm;

[0205] Column temperature: 35℃;

[0206] Injection volume: 5 μL;

[0207] Perform gradient elution according to the table below:

[0208]

[0209] Experimental steps:

[0210] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0211] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0212] See results Figure 9 , Figure 9 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.160 min.

[0213] Example 10

[0214] Chromatographic conditions:

[0215] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0216] Column: Impakt Unision UK-C18;

[0217] Mobile phase: Mobile phase A: Weigh 1.50 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0218] Mobile phase B: Acetonitrile

[0219] Flow rate: 1.0 mL / min;

[0220] Detection wavelength: 205nm;

[0221] Column temperature: 35℃;

[0222] Injection volume: 5 μL;

[0223] Perform gradient elution according to the table below:

[0224]

[0225] Experimental steps:

[0226] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0227] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0228] See results Figure 10 , Figure 10 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 23.868 min.

[0229] Example 11

[0230] Chromatographic conditions:

[0231] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0232] Column: Impakt Unision UK-C18;

[0233] Mobile phase: Mobile phase A: Weigh 5.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0234] Mobile phase B: Acetonitrile

[0235] Flow rate: 1.0 mL / min;

[0236] Detection wavelength: 205nm;

[0237] Column temperature: 35℃;

[0238] Injection volume: 5 μL;

[0239] Perform gradient elution according to the table below:

[0240]

[0241] Experimental steps:

[0242] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0243] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0244] See results Figure 11 , Figure 11 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from other impurity peaks. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 20.319 min.

[0245] Example 12

[0246] Chromatographic conditions:

[0247] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0248] Column: Impakt Unision UK-C18;

[0249] Mobile phase: Mobile phase A: Weigh 3.00 g sodium octane sulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0250] Mobile phase B: Acetonitrile;

[0251] Flow rate: 1.0 mL / min;

[0252] Detection wavelength: 205nm;

[0253] Column temperature: 35℃;

[0254] Injection volume: 5 μL;

[0255] Perform gradient elution according to the table below:

[0256]

[0257] Experimental steps:

[0258] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0259] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0260] See results Figure 12 , Figure 12 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride), peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride is well separated from other impurity peaks. However, the detection and analysis using sodium octane sulfonate takes too long, which is not conducive to actual production testing.

[0261] Comparative Example 1

[0262] Chromatographic conditions:

[0263] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0264] Column: Impakt Unision UK-C18;

[0265] Mobile phase: Mobile phase A: Water, pH adjusted to 3.0 with phosphoric acid;

[0266] Mobile phase B: Acetonitrile

[0267] Flow rate: 1.0 mL / min;

[0268] Detection wavelength: 205nm;

[0269] Column temperature: 35℃;

[0270] Injection volume: 5 μL;

[0271] Perform gradient elution according to the table below:

[0272]

[0273] Experimental steps:

[0274] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0275] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0276] See results Figure 13 , Figure 13 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), and peak 3 is impurity C. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride has a tailed shape, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 32.770 min, and impurities B, D, and E were not detected.

[0277] Comparative Example 2

[0278] Chromatographic conditions:

[0279] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0280] Column: Impakt Unision UK-C18;

[0281] Mobile phase: Mobile phase A: 0.1% aqueous phosphoric acid solution;

[0282] Mobile phase B: Acetonitrile

[0283] Flow rate: 1.0 mL / min;

[0284] Detection wavelength: 205nm;

[0285] Column temperature: 35℃;

[0286] Injection volume: 5 μL;

[0287] Perform gradient elution according to the table below:

[0288]

[0289]

[0290] Experimental steps:

[0291] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0292] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0293] See results Figure 14 , Figure 14 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), peak 3 is impurity C, and peak 4 is impurity D. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride has a tailed appearance, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 20.677 min, and impurities B and E were not detected.

[0294] Comparative Example 3

[0295] Chromatographic conditions:

[0296] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0297] Column: Impakt Unision UK-C18;

[0298] Mobile phase: Mobile phase A: Water;

[0299] Mobile phase B: Acetonitrile

[0300] Flow rate: 1.0 mL / min;

[0301] Detection wavelength: 205nm;

[0302] Column temperature: 35℃;

[0303] Injection volume: 5 μL;

[0304] Perform gradient elution according to the table below:

[0305]

[0306] Experimental steps:

[0307] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0308] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0309] See results Figure 15 , Figure 15 Peak 1 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride), peak 2 is impurity C, peak 3 is impurity D, and peak 4 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride has an asymmetrical peak shape and a low response value. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazolium hydrochloride is around 10.299 min. Impurities A and B were not detected.

[0310] Comparative Example 4

[0311] Chromatographic conditions:

[0312] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0313] Column: Impakt Unision UK-C18;

[0314] Mobile phase: Mobile phase A: Weigh 3.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 4.0 with phosphoric acid;

[0315] Mobile phase B: Acetonitrile

[0316] Flow rate: 1.0 mL / min;

[0317] Detection wavelength: 205nm;

[0318] Column temperature: 35℃;

[0319] Injection volume: 5 μL;

[0320] Perform gradient elution according to the table below:

[0321]

[0322] Experimental steps:

[0323] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0324] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0325] See results Figure 16 , Figure 16 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride, peak 3 is impurity C, peak 4 is impurity D, and peak 5 is impurity E. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride co-occurs with impurity B, and the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 25.913 min.

[0326] Comparative Example 5

[0327] Chromatographic conditions:

[0328] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0329] Column: Impakt Unision UK-C18;

[0330] Mobile phase: Mobile phase A: Weigh 2.72g of potassium dihydrogen phosphate into a beaker, pour in 1000mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0331] Mobile phase B: Acetonitrile

[0332] Flow rate: 1.0 mL / min;

[0333] Detection wavelength: 205nm;

[0334] Column temperature: 35℃;

[0335] Injection volume: 5 μL;

[0336] Perform gradient elution according to the table below:

[0337]

[0338] Experimental steps:

[0339] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0340] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0341] See results Figure 17 , Figure 17 Peak 1 is impurity A, and peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from impurity A. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 18.497 min. Impurities B, C, D, and E were not detected.

[0342] Comparative Example 6

[0343] Chromatographic conditions:

[0344] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0345] Column: Impakt Unision UK-C18;

[0346] Mobile phase: Mobile phase A: Weigh 2.72g potassium dihydrogen phosphate and 3.00g sodium heptanesulfonate into a beaker, pour in 1000mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0347] Mobile phase B: Acetonitrile

[0348] Flow rate: 1.0 mL / min;

[0349] Detection wavelength: 205nm;

[0350] Column temperature: 35℃;

[0351] Injection volume: 5 μL;

[0352] Perform gradient elution according to the table below:

[0353]

[0354] Experimental steps:

[0355] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0356] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0357] See results Figure 18 , Figure 18 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), and peak 3 is impurity B. It can be seen that under these conditions, the main peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is symmetrical and well separated from impurities A and B. The peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.010 min. Impurities C, D, and E were not detected.

[0358] Comparative Example 7

[0359] Chromatographic conditions:

[0360] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0361] Column: Impakt Unision UK-C18;

[0362] Mobile phase: Mobile phase A: Weigh 1.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0363] Mobile phase B: Acetonitrile

[0364] Flow rate: 1.0 mL / min;

[0365] Detection wavelength: 205nm;

[0366] Column temperature: 35℃;

[0367] Injection volume: 5 μL;

[0368] Perform gradient elution according to the table below:

[0369]

[0370] Experimental steps:

[0371] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0372] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0373] See results Figure 19 , Figure 19 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), peak 3 is impurity B, peak 4 is impurity C, peak 5 is impurity D, and peak 6 is impurity E. It can be seen that under these conditions, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.760 min, but the separation degree between impurity B and 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride does not reach 1.5.

[0374] Comparative Example 8

[0375] Chromatographic conditions:

[0376] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0377] Column: Impakt Unision UK-C18;

[0378] Mobile phase: Mobile phase A: Weigh 7.00 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0379] Mobile phase B: Acetonitrile

[0380] Flow rate: 1.0 mL / min;

[0381] Detection wavelength: 205nm;

[0382] Column temperature: 35℃;

[0383] Injection volume: 5 μL;

[0384] Perform gradient elution according to the table below:

[0385]

[0386] Experimental steps:

[0387] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0388] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0389] See results Figure 20 , Figure 20 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), and peak 3 is impurity B. It can be seen that under these conditions, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 35.007 min, but impurities C, D, and E were not detected.

[0390] Comparative Example 9

[0391] Chromatographic conditions:

[0392] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0393] Column: Impakt Unision UK-C18;

[0394] Mobile phase: Mobile phase A: Weigh 0.50 g of sodium hexanesulfonate, add 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid;

[0395] Mobile phase B: Acetonitrile

[0396] Flow rate: 1.0 mL / min;

[0397] Detection wavelength: 205nm;

[0398] Column temperature: 35℃;

[0399] Injection volume: 5 μL;

[0400] Perform gradient elution according to the table below:

[0401]

[0402] Experimental steps:

[0403] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0404] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0405] See results Figure 21 , Figure 21 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), peak 3 is impurity C, and peak 4 is impurity D. It can be seen that under these conditions, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 22.257 min, but impurities B and E were not detected.

[0406] Comparative Example 10

[0407] Chromatographic conditions:

[0408] High Performance Liquid Chromatography (HPLC): Dionex: UltiMate 3000;

[0409] Column: Impakt Unision UK-C18;

[0410] Mobile phase: Aqueous phase (3.00 g sodium hexanesulfonate, 1000 mL water, pH adjusted to 3.0 with phosphoric acid): Acetonitrile = 60:40

[0411] Flow rate: 1.0 mL / min;

[0412] Detection wavelength: 205nm;

[0413] Column temperature: 35℃;

[0414] Injection volume: 5 μL;

[0415] Experimental steps:

[0416] (1) Take appropriate amounts of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1Hbenzis[d]imidazolium hydrochloride reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity E reference standard, place them in a 100mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and make a sample solution.

[0417] (2) Take 5 μl of sample solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0418] See results Figure 22 , Figure 22 Peak 1 is impurity A, peak 2 is 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride), and peak 3 is impurity B. It can be seen that under these conditions, the peak of 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H benzo[d]imidazolium hydrochloride is around 14.517 min, but impurities C, D, and E were not detected.

[0419] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0420] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for the analysis of detection of Bilastine intermediate 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1 H benzo[d]imidazole hydrochloride and its related substances by high performance liquid chromatography, characterized by, The sample to be detected is detected by using high performance liquid chromatography to obtain a chromatogram; The chromatographic conditions of the high performance liquid chromatography are as follows: The chromatographic column uses octadecylsilane-bonded silica gel as the filler; The mobile phase includes mobile phase A and mobile phase B, and gradient elution is performed with the mobile phase A and the mobile phase B, The mobile phase A is an ion pair reagent, the concentration of the ion pair reagent is >0.05% and <0.7%, and the pH value of the mobile phase A is adjusted to be <4; and the mobile phase B is acetonitrile.

2. The method of claim 1, characterized in that, characterized in that, The mobile phase A includes one or more of sodium methane sulfonate, sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecyl sulfonate, and sodium dodecyl sulfate; preferably sodium hexane sulfonate solution or sodium heptane sulfonate solution or sodium octane sulfonate solution, and more preferably sodium hexane sulfonate solution.

3. The method of claim 1, wherein, The concentration of the ion pair reagent is 0.15% to 0.5%, and the pH value of the mobile phase A is 2.5 to 3.

5.

4. The method of claim 1, wherein, The conditions of the gradient elution are as follows: 0-8 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B is 75-85%, and the content of the mobile phase B is 15-25%; 8-35 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B decreases from 75-85% to 45-55%, and the content of the mobile phase B increases from 15-25% to 45-55%; 35.1-45 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B is 75-85%, and the content of the mobile phase B is 15-25%.

5. The method of claim 1, wherein, The conditions of the gradient elution are as follows: 0-8 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B is 80%, and the content of the mobile phase B is 20%; 8-25.1 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B decreases from 80% to 50%, and the content of the mobile phase B increases from 20% to 50%; 25.1-35 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B is 50%, and the content of the mobile phase B is 50%; 35.1-45 min, the percentage content of the mobile phase A in the total volume of the mobile phase A and the mobile phase B is 80%, and the content of the mobile phase B is 20%.

6. The method of claim 1, wherein, The chromatographic column in the high performance liquid chromatography includes one of ImtaktUnision UK-C18, Zorbax Stable Bond C18, Zorbax Extend-C18, Symmetry Shield TM RPC18, XDB-C18, and SB-C18.

7. The method of claim 1, wherein, The column temperature of the chromatographic column is 30-40°C.

8. The method of claim 1, wherein, The flow rate of the mobile phase is 0.8-1.2 ml / min, and the detection wavelength is 200-250 nm.

9. The method according to any one of claims 1 to 8, characterized in that, The method further includes the following steps: (1) preparing a sample solution of the test sample containing 1-(2-ethoxyethyl)-2-(piperidin-4-yl-1H-benzo[d]imidazole hydrochloride; (2) injecting 5 μl of the sample solution into a high performance liquid chromatograph, performing high performance liquid chromatography and recording the chromatogram, and completing the separation and detection of the sample containing the intermediate of bilastine.

10. Use of the method of any one of claims 1 to 9 in the quality control of a pharmaceutical product using bilastine as a raw material.

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