Method for detecting fluorine impurities in mosapride citrate intermediate

Abstract

The invention relates to the technical field of medicine quality detection methods, in particular to a method for detecting fluorine impurities in a mosapride citrate intermediate. The detection method comprises the following steps: a test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine is detected by liquid chromatography, and a mobile phase is a monopotassium phosphate-phosphoric acid-acetonitrile buffer solution. The detection method provided by the invention has good separation degree, high response value and good sensitivity on related substances, can realize separation of the four fluorine impurities, and effectively controls the related substances of the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine, so that the product quality of mosapride citrate can be effectively ensured, and the detection method is suitable for industrial production. Meanwhile, the subsequent impurity removal treatment process can be simplified, and the use amount of an impurity removal separation solvent is further reduced.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality detection methods, and in particular to a method for detecting fluorine impurities in a mosapride citrate intermediate. Background Art

[0002] Mosapride Citrate is a selective 5-hydroxytryptamine (5-HT4) receptor agonist that can promote the release of acetylcholine, stimulate the gastrointestinal tract and exert a prokinetic effect, thereby improving the gastrointestinal symptoms of patients with functional dyspepsia, but does not affect the secretion of gastric acid. Its chemical name is 4-amino-5-chloro-2-ethoxy-N-[[4-(4-fluorobenzyl)-2-morpholinyl]methyl]benzamide citrate dihydrate, CAS number: 156925-25-6, with the following structure:

[0003]

[0004] Regarding the synthesis process of mosapride citrate, most of the literature reports use two key side chains for condensation reaction. The synthesis process is as follows:

[0005]

[0006] Among them, the key intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine (Compound III) is an oily substance with low purity, poor stability and difficulty in impurity removal. The impurities produced by further reaction of the fluorine impurities contained therein have a high structural similarity with the finished product of mosapride citrate and are difficult to remove through a simple process. Due to the lack of control over the fluorine impurities in the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine, the final product often fails to meet the requirements when detecting related substances. The presence of these impurities not only affects the quality of the product, but also poses a major risk to the safety of medication, so it is necessary to control them.

[0007] The above intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine and its fluorine-based impurity compounds have simple molecular structures and weak ultraviolet absorption. At the same time, due to the large polarity of the -NH2 group molecules, it is difficult to obtain suitable retention and separation in reversed-phase liquid chromatography. The pka value is about 9, the alkalinity is high, and it is difficult to obtain a good peak shape. Therefore, seeking an analytical method that can effectively separate the fluorine-based impurities in the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine is of great significance for the further quality control of mosapride citrate. Summary of the invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting fluorine impurities in a mosapride citrate intermediate, which can realize the detection of fluorine impurities in a mosapride citrate intermediate.

[0009] To achieve the above object, the present invention provides a method for detecting fluorine impurities in a mosapride citrate intermediate, comprising the following steps:

[0010] The test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine was detected by liquid chromatography, wherein the mobile phase was a potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer solution.

[0011] Preferably, the elution gradient of the liquid chromatography is:

[0012]

[0013] Wherein, the mobile phase A is 0.02 mol / L potassium dihydrogen phosphate-phosphate buffer, and the mobile phase B is 0.01 mol / L potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer.

[0014] Preferably, the preparation method of the mobile phase A is:

[0015] Prepare 0.02 mol / L potassium dihydrogen phosphate aqueous solution and adjust the pH value to 4.2 with phosphoric acid aqueous solution.

[0016] In some specific embodiments of the present invention, 5.4 g of potassium dihydrogen phosphate was taken, 2000 ml of water was added to dissolve it, and the pH value was adjusted to 4.2 with 10% phosphoric acid water to obtain mobile phase A.

[0017] Preferably, the preparation method of the mobile phase B is:

[0018] Prepare 0.01 mol / L potassium dihydrogen phosphate solution and adjust the pH value to 4.2 with aqueous phosphoric acid solution.

[0019] The solvent of the 0.01 mol / L potassium dihydrogen phosphate solution is preferably water and acetonitrile.

[0020] The volume ratio of water to acetonitrile is preferably 1:1.

[0021] In some specific embodiments of the present invention, 2.7 g of potassium dihydrogen phosphate was taken, 1000 ml of water was added to dissolve it, 1000 ml of acetonitrile was added, mixed, and the pH value was adjusted to 4.2 with 10% phosphoric acid water to obtain mobile phase B.

[0022] The concentration of the test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine is preferably 2.5 mg / mL.

[0023] The chromatographic column used in the liquid chromatography method is preferably filled with octadecylsilane bonded silica gel.

[0024] The chromatographic column is preferably YMC-Pack ODS-A, 4.6×150 mm, 5 μm or other chromatographic columns with equivalent performance.

[0025] The column temperature of the liquid chromatography is preferably 40°C.

[0026] The flow rate of the liquid chromatography is preferably 1 ml / min.

[0027] The detection wavelength of the liquid chromatography is preferably 220 nm.

[0028] The injection volume for the liquid chromatography is preferably 5 μl.

[0029] Preferably, in the present invention, an impurity trapping column is provided between the gradient mixer and the injector of the liquid chromatography.

[0030] The impurity trapping column is preferably Ghost Trap DS 7.6×30 mm or other chromatographic columns with equivalent performance.

[0031] The configuration of the impurity trapping column described above helps to adsorb and remove impurities in the system (including mobile phase, piping and mixer), ensuring a stable gradient elution baseline under conditions of lower wavelengths and preventing interference with the target peak.

[0032] Preferably, the above-mentioned fluorine impurities include any one or more of the following impurities:

[0033] Related substance impurity A:

[0034]

[0035] Related substance impurity B:

[0036]

[0037] Related substance impurities C:

[0038]

[0039] Related substance impurities D:

[0040]

[0041] The fluorine impurities further react with the impurities corresponding to mosapride:

[0042] Mosapride Impurity A:

[0043]

[0044] Mosapride Impurity B:

[0045]

[0046] Mosapride Impurity C:

[0047]

[0048] Mosapride Impurity D:

[0049]

[0050] The test results show that using potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer solution as the mobile phase can detect 2-aminomethyl-4-(4-fluorobenzyl)-morpholine and the above four fluorine impurities, and the separation of each substance meets the requirements.

[0051] When other conventional mobile phases are used, such as ammonium acetate-acetic acid-acetonitrile buffer solution, the above four fluorine impurities cannot be detected, and the detection of 2-aminomethyl-4-(4-fluorobenzyl)-morpholine impurity cannot be achieved.

[0052] When the chromatographic column uses octadecylsilane bonded silica filler, the column temperature is 40°C, the flow rate is 1ml / min, and the detection wavelength is 220nm, the test solution and impurities have good peaks and can be effectively separated, the response value is high, and the detection sensitivity is good, which is the optimal detection condition.

[0053] Compared with the prior art, the present invention provides a method for detecting fluorine impurities in a mosapride citrate intermediate, comprising the following steps: using liquid chromatography to detect a test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine, wherein the mobile phase is a potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer solution.

[0054] The detection method provided by the present invention has good separation degree and high response value for related substances, good sensitivity, can realize the separation of the above-mentioned four fluorine impurities, effectively control the related substances of the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine, and thus can effectively ensure the product quality of mosapride citrate, and at the same time can simplify the subsequent impurity removal treatment process, and further reduce the use of impurity removal separation solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The HPLC chromatogram of the mixed reference solution of Example 1;

[0056] Figure 2 The HPLC chromatogram of the test solution of Example 1 is shown in FIG.

[0057] Figure 3 It is the high performance liquid chromatogram of Comparative Example 1;

[0058] Figure 4 The HPLC chromatogram of the test solution of Comparative Example 2 is shown in FIG.

[0059] Figure 5 It is the HPLC chromatogram of the mosapride mixed reference substance solution of Example 2.

[0060] Figure 6 It is the HPLC chromatogram of the mosapride test solution of Example 2. DETAILED DESCRIPTION

[0061] In order to further illustrate the present invention, the detection method of fluorine impurities in the mosapride citrate intermediate provided by the present invention is described in detail below in conjunction with the examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0062] In the following specific implementations, the drugs, reagents and instruments used are as follows:

[0063] Instruments: U 3000 high performance liquid chromatograph, UV DAD detector (Dionex Technologies, Inc., USA); MS205DU 1 / 100,000 analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); XPR2 1 / 100,000 analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); pH meter (S220 Shanghai Mettler-Toledo Instrument Co., Ltd.); 85-2WS magnetic stirrer (Shanghai Huxi Industrial Co., Ltd.), etc.

[0064] Reagents: potassium dihydrogen phosphate (AR, Zhongtian Fine), sodium citrate (AR, Tianjin Yongda), hydrochloric acid (AR, Chengdu Kelong), phosphoric acid (HPLC, Tianjin Komiou), methanol (HPLC, Anhui Tiandi), acetonitrile (HPLC, Anhui Tiandi).

[0065] Test and reference substances: 2-aminomethyl-4-(4-fluorobenzyl)-morpholine test substance (batch number: J01E-2307001, manufacturer: Chongqing Inska Chemical Co., Ltd.); mosapride test substance (batch number: 20230619, manufacturer: Jilin Xiuzhen Pharmaceutical New Drug Development Co., Ltd.); 2-aminomethyl-4-(4-fluorobenzyl)-morpholine reference substance (Shenzhen Excellence Biomedicine, batch number: 20221029); impurity A reference substance (Shenzhen Excellence Biomedicine, batch number: 20230517); impurity B reference substance (Shenzhen Excellence Biomedicine, batch number: 20230622); impurity C reference substance (STD PHARM, batch number: 2372519ML-YT-01); impurity D reference substance (STD PHARM, batch number: 2372520M-YT-01); mosapride reference substance (SINCO PHARMACHEM, batch number: 22-06-0604); Mosapride impurity A reference substance (SINCO PHARMACHEM, batch number: 21-06-0504); Mosapride impurity B reference substance (STD PHARM, batch number: 1972512M-WJ-01); Mosapride impurity C reference substance (STD PHARM, batch number: 2272513M-WJ-01); Mosapride impurity D reference substance (SINCO PHARMACHEM, batch number: 23-06-0909).

[0066] Example 1

[0067] Solvent: methanol;

[0068] Preparation of mixed reference solution: Weigh 5 mg of 2-aminomethyl-4-(4-fluorobenzyl)-morpholine and 4 kinds of fluorine-related impurities A~D reference substances respectively, place them in the same 20ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well; then measure 1ml, place it in a 100ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained. (Contains 2-aminomethyl-4-(4-fluorobenzyl)-morpholine and 4 kinds of fluorine-related impurities 2.5μg / ml).

[0069] Test solution: Weigh about 25 mg of 2-aminomethyl-4-(4-fluorobenzyl)-morpholine test sample, accurately weigh it, put it into a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain;

[0070] HPLC detection conditions: chromatographic column: YMC-Pack ODS-A, 4.6×150mm, 5μm, impurity trapping column: Ghost Trap DS 7.6×30mm, 0.02mol / L potassium dihydrogen phosphate buffer (take 5.4g of potassium dihydrogen phosphate, add 2000ml of water to dissolve, and adjust the pH value to 4.2 with 10% phosphoric acid water) as mobile phase A, 0.01mol / L potassium dihydrogen phosphate buffer (take 2.7g of potassium dihydrogen phosphate, add 1000ml of water to dissolve, add 1000ml of acetonitrile, mix well, and adjust the pH value to 4.2 with 10% phosphoric acid water) as mobile phase B, gradient elution according to Table 1; detection wavelength is 220nm; flow rate is 1.0ml per minute; column temperature is 40℃; injection volume is 5μl.

[0071] Table 1

[0072]

[0073] The HPLC test data and spectrum of the mixed reference solution are shown in Table 2 and Figure 1 , 2-aminomethyl-4-(4-fluorobenzyl)-morpholine and four fluorine impurities were detected, and the separation of each substance met the requirements.

[0074] Table 2

[0075]

[0076] The HPLC test data and spectra of the test solution are shown in Table 3 and Figure 2 It can be seen that the test solution and each impurity have good peaks and can be effectively separated. The response value is high and the detection sensitivity is good. The main peak area of ​​the test solution accounts for 98.670%, impurity A is 0.029%, impurity B is 0.033%, and impurity C and impurity D are not detected.

[0077] Table 3

[0078]

[0079] Comparative Example 1

[0080] According to the HPLC conditions of Example 1, the solvent methanol was injected to investigate the effect of the impurity trapping column on the gradient elution baseline. The results are as follows: Figure 3 shown.

[0081] An impurity trapping column is installed between the gradient mixer and the injector to ensure the stability of the gradient elution baseline under lower wavelength conditions and prevent interference with the target peak.

[0082] Comparative Example 2

[0083] Solvent: methanol;

[0084] Test solution: Weigh about 25 mg of 2-aminomethyl-4-(4-fluorobenzyl)-morpholine test sample, accurately weigh it, put it into a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain;

[0085] HPLC detection conditions: chromatographic column: YMC-Pack ODS-A, 4.6×150mm, 5μm, impurity trapping column: Ghost Trap DS 7.6×30mm, 0.02mol / L ammonium acetate buffer (take 1.54g of ammonium acetate, add 2000ml of water to dissolve, and adjust the pH value to 4.2 with 10% acetic acid water) as mobile phase A, 0.01mol / L ammonium acetate buffer (take 0.77g of ammonium acetate, add 1000ml of water to dissolve, add 1000ml of acetonitrile, mix well, and adjust the pH value to 4.2 with 10% phosphoric acid water) as mobile phase B, gradient elution according to Table 1; detection wavelength is 220nm; flow rate is 1.0ml per minute; column temperature is 40℃; injection volume is 5μl.

[0086] The test solution was prepared according to the above method and tested by HPLC. The results were as follows Figure 4 As shown, the visible wavelength is 220nm, the ammonium acetate-acetic acid buffer solution has a high base absorption, only the main peak of the test product is detected and the peak shape is poor, and the impurity peaks are not obviously detected.

[0087] Example 2

[0088] Two key side chain intermediates, (±)-4-amino-5-chloro-2-ethoxybenzoic acid (isobutylformic acid) anhydride (compound II) and 2-aminomethyl-4-(4-fluorobenzyl)-morpholine (compound III), were used for condensation reaction to generate mosapride (compound IV), and the further reaction of the corresponding fluorine impurities in mosapride was investigated.

[0089] Solvent: methanol;

[0090] Preparation of Mosapride mixed reference solution: Weigh 4 mg of Mosapride and 4 kinds of Mosapride impurity A~D reference substances respectively, place them in the same 20ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well; then measure 1ml, place it in a 100ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained (containing Mosapride and 4 kinds of Mosapride impurities 2μg / ml).

[0091] Test solution: Weigh about 100 mg of Mosapride test sample, accurately weigh it, put it into a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain;

[0092] HPLC detection conditions: Chromatographic column: YMC-Pack ODS-A, 4.6×150mm, 5μm, citrate buffer (take 8.82g of sodium citrate, add 800ml of water to dissolve, adjust the pH value to 4.0 with dilute hydrochloric acid, and then dilute to 1000ml with water) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 4; detection wavelength is 274nm; flow rate is 1.0ml per minute; column temperature is 40℃; injection volume is 5μl.

[0093] Table 4

[0094]

[0095] According to the above method, the mosapride mixed reference solution and the mosapride test solution were prepared and tested by HPLC. The HPLC test data and spectra are shown in Tables 5-6 and Figure 5-6 It can be seen that mosapride and its impurities have good peaks and can be effectively separated. The main peak area of ​​the mosapride test solution accounts for 99.918%, the mosapride impurity A is 0.023%, the mosapride impurity B is 0.035%, and the mosapride impurity C and mosapride impurity D are not detected, which is basically consistent with the detection results of the corresponding fluorine impurities in the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine, indicating that the synthetic process of preparing mosapride by two key side chain condensation reactions is not effective in removing fluorine impurities. Therefore, the control of fluorine impurities in the intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine plays a very important role in the quality and safety of mosapride products.

[0096] Table 5

[0097]

[0098] Table 6

[0099]

[0100] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting fluorine impurities in a mosapride citrate intermediate, comprising the following steps: The test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine was detected by liquid chromatography, wherein the mobile phase was a potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer solution.

2. The detection method according to claim 1, characterized in that: The elution gradient of the liquid chromatography is: Wherein, the mobile phase A is 0.02 mol / L potassium dihydrogen phosphate-phosphate buffer, and the mobile phase B is 0.01 mol / L potassium dihydrogen phosphate-phosphoric acid-acetonitrile buffer.

3. The detection method according to claim 2, characterized in that: The preparation method of the mobile phase A is: Prepare 0.02 mol / L potassium dihydrogen phosphate aqueous solution and adjust the pH value to 4.2 with phosphoric acid aqueous solution; The preparation method of the mobile phase B is: Prepare 0.01 mol / L potassium dihydrogen phosphate solution and adjust the pH to 4.2 with aqueous phosphoric acid solution; The solvent of the 0.01 mol / L potassium dihydrogen phosphate solution is water and acetonitrile; The volume ratio of water to acetonitrile is 1:

1.

4. The detection method according to claim 1, characterized in that: The concentration of the test solution containing 2-aminomethyl-4-(4-fluorobenzyl)-morpholine is 2.5 mg / mL.

5. The detection method according to claim 1, characterized in that: The chromatographic column used in the liquid chromatography method is an octadecylsilane bonded silica gel filler; The chromatographic column is YMC-Pack ODS-A, 4.6×150 mm, 5 μm.

6. The detection method according to claim 1, characterized in that: The column temperature of the liquid chromatography was 40°C.

7. The detection method according to claim 1, characterized in that: The flow rate of the liquid chromatography was 1 ml / min.

8. The detection method according to claim 1, characterized in that: The detection wavelength of the liquid chromatography is 220 nm.

9. The detection method according to claim 1, characterized in that: The injection volume for the liquid chromatography was 5 μl.

10. The detection method according to claim 1, characterized in that: An impurity trapping column is arranged between the gradient mixer and the injector of the liquid chromatography method; The impurity trapping column is Ghost Trap DS 7.6×30 mm.

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