Detection method of mosapride citrate intermediate impurities

Abstract

The invention provides a method for detecting impurities in a mosapride citrate intermediate, which comprises the following steps: a test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride is detected by liquid chromatography, and the solvent of the test solution is acetonitrile. The detection method is simple, convenient and rapid, good in separation degree, high in detection efficiency, short in operation time and simple in preparation process, the condition of the mosapride citrate intermediate 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride can be rapidly and accurately determined, a monitoring method of a conventional thin-layer chromatography is replaced, and the condition of a sample is accurately and efficiently determined.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting 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. The 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] The key intermediate 4-amino-5-chloro-2-ethoxybenzoic acid (compound I) needs to be activated by isobutyl chloroformate to activate the carboxylic acid group into anhydride. 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride (compound II) is further acylated with another side chain intermediate 2-aminomethyl-4-(4-fluorobenzyl)-morpholine (compound III) to generate mosapride (compound IV). The degree of activation reaction has a decisive influence on the subsequent process reaction and the final product quality of mosapride citrate. Therefore, it is necessary to develop an impurity detection method for the intermediate 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride, which can monitor the reaction degree of the intermediate to ensure that the reaction is fully controllable, and further carry out limited control of the intermediate-related substances to ensure product quality. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting impurities in the intermediate of mosapride citrate, which can be used to detect the intermediate 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride.

[0008] To achieve the above object, the present invention provides a method for detecting impurities in a mosapride citrate intermediate, comprising the following steps:

[0009] Liquid chromatography was used to detect a test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxybenzoic acid) anhydride, wherein the solvent of the test solution was acetonitrile.

[0010] The present invention is directed to 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxybenzoic acid) anhydride (compound II), an impurity of the mosapride citrate intermediate, and its structural formula is as follows:

[0011]

[0012] The above-mentioned anhydride impurities are extremely unstable and easily react with water and alcohol, which cannot be accurately quantitatively measured. The test results show that the use of conventional solvents such as water, alcohol solvents, and n-hexane-isopropanol mixed solvents will cause the substance to be unstable and there will be a phenomenon of unknown impurities growing.

[0013] The invention provides a normal phase HPLC analysis method, which adopts acetonitrile as a solvent and is used for detecting the anhydride impurity of 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) in a mosapride citrate intermediate, thereby avoiding the instability of the anhydride impurities. The method is also applicable to the quantitative analysis of anhydride compounds in other drugs.

[0014] Preferably, the concentration of the test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxybenzoic acid) anhydride is 2 mg / mL.

[0015] Preferably, the chromatographic column used in the liquid chromatography method is a cyanosilane bonded silica gel filler.

[0016] Preferably, the liquid chromatography method uses n-hexane-isopropanol as the mobile phase for gradient elution.

[0017] The elution gradient of the liquid chromatography is preferably:

[0018]

[0019]

[0020] The present invention adopts n-hexane-isopropanol as the mobile phase and the elution gradient, so as to achieve the best separation degree between the mosapride citrate intermediate and the impurity 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride.

[0021] Preferably, the column temperature of the liquid chromatography is 30°C.

[0022] Preferably, the flow rate of the liquid chromatography is 1 ml / min.

[0023] Preferably, the detection wavelength of the liquid chromatography is 274 nm.

[0024] Preferably, the injection volume of the liquid chromatography method is 5 μl.

[0025] Preferably, in the liquid chromatography method, the concentration of the standard is 0.002 mg / mL.

[0026] Compared with the prior art, the present invention provides a method for detecting impurities in an intermediate of mosapride citrate, comprising the following steps: using liquid chromatography to detect a test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxycarboxylic acid) anhydride, wherein the solvent of the test solution is acetonitrile. The above detection method is simple and fast, has good separation, high detection efficiency, short running time, simple preparation process, can quickly and accurately determine the situation of 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxycarboxylic acid) anhydride, an intermediate of mosapride citrate, replaces the conventional thin layer chromatography monitoring method, and accurately and efficiently determines the sample situation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC chromatogram of the localization solution solvent of compound II in Example 1, n-hexane-isopropanol (1:4 volume ratio);

[0028] Figure 2 HPLC chromatogram of acetonitrile, the solvent for locating the solution of Compound II in Example 1;

[0029] Figure 3 The HPLC chromatogram of the mosapride test sample chromatographic column 1 of Example 2;

[0030] Figure 4 The HPLC chromatogram of the mosapride test sample chromatographic column 2 of Example 2;

[0031] Figure 5 HPLC chromatogram of the mixed solution of Example 3;

[0032] Figure 6 The HPLC chromatogram of the spiked test solution of Example 3. DETAILED DESCRIPTION

[0033] In order to further illustrate the present invention, the detection method of 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.

[0034] In the following specific implementation manner, the drugs, reagents and instruments used are as follows:

[0035] Mosapride reference substance (source: SINCO PHARMACHEM, batch number: 22-06-0604), mosapride test substance (source: Jilin Xiuzhen Pharmaceutical New Drug Development Co., Ltd., batch number: 20230619), 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxyformic acid) anhydride (source: Jilin Xiuzhen Pharmaceutical New Drug Development Co., Ltd., batch number: 22110401), acetonitrile (chromatographically pure, Anhui Tiandi High Purity Solvent Co., Ltd.), n-hexane (chromatographically pure, Anhui Tiandi High Purity Solvent Co., Ltd.), isopropanol (chromatographically pure, Ron Reagent Co., Ltd.), liquid chromatograph (Dionex Technology Co., Ltd., U3000), chromatographic column (Dacelide Technology Co., Ltd., CHIRALPAK AD-H (4.6×250 mm, 5 μm), chromatographic column (Akzo Nobel, Kromasil 60-5-CN (4.6×250 mm, 5 μm), electronic balance (Mettler-Toledo, XSE205), electronic balance (Mettler-Toledo, XPR2).

[0036] Example 1: Screening of solvents

[0037] Solution preparation:

[0038] Solvent 1: n-hexane-isopropanol (1:4 volume ratio)

[0039] Solvent 2: Acetonitrile

[0040] Impurity compound II positioning solution: weigh about 10 mg of compound II, accurately weigh, place in a 10 ml volumetric flask, add the above solvents to dissolve and dilute to the scale, shake well; then measure 1 ml, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0041] HPLC detection conditions:

[0042] Chromatographic column: CHIRALPAK AD-H (4.6×250mm 5μm); mobile phase A: n-hexane, mobile phase B: isopropanol, elution was performed according to the gradient in Table 1; flow rate: 1ml / min; detection wavelength: 274nm; column temperature: 30°C, injection volume: 5μl.

[0043] Table 1

[0044]

[0045] According to the above conditions, high performance liquid chromatography was performed, and the results were as follows Figure 1-2As shown, it can be seen that the impurity compound II positioning solution has an unknown peak growth in the solvent n-hexane-isopropanol (1:4 volume ratio), and is relatively stable in the solvent acetonitrile. Further confirmation of the solubility of the mosapride test sample in acetonitrile shows that the test sample acetonitrile solution of 2 mg / ml is clear and transparent, so acetonitrile is selected as the solvent.

[0046] Example 2: Selection of chromatographic column

[0047] Solution preparation:

[0048] Solvent: Acetonitrile

[0049] Mosapride test solution: Weigh about 20 mg of mosapride test solution accurately, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and obtain;

[0050] Two different chromatographic columns were selected and the determination was carried out respectively according to the HPLC detection conditions of Example 1.

[0051] Chromatographic column 1: CHIRALPAK AD-H (4.6×250mm 5μm); Chromatographic column 2: Kromasil60-5-CN (4.6×250mm, 5μm).

[0052] The results are as follows Figure 3-4 As shown, when using chromatographic column 1, the mosapride test solution has double peaks and is not completely separated. When using chromatographic column 2, the mosapride test solution is a single peak, the retention time is appropriate, and the peak state is good. Therefore, the Kromasil 60-5-CN (4.6×250mm, 5μm) chromatographic column is selected.

[0053] Example 3: Method Validation

[0054] Solution preparation:

[0055] Solvent: Acetonitrile

[0056] Mixed solution: Weigh 4 mg of Mosapride and Compound II respectively, place in the same 20 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well; then weigh 1 ml, place in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well, and the solution is obtained (containing 2 μg / ml of Mosapride and Compound II).

[0057] Impurity compound II positioning solution: weigh about 10 mg of compound II, accurately weigh, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well; then measure 1 ml, place in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0058] Spiked test solution: Weigh about 20 mg of mosapride test sample, accurately weigh it, place it in a 10 ml volumetric flask, add part of acetonitrile to dissolve it, then add 2 ml of impurity compound II positioning solution, dilute it to the scale with acetonitrile, shake well, and obtain.

[0059] HPLC detection conditions:

[0060] Chromatographic column: Kromasil 60-5-CN (4.6×250 mm, 5 μm); mobile phase A: n-hexane, mobile phase B: isopropanol, elution was performed according to the gradient in Table 1; flow rate: 1 ml / min; detection wavelength: 274 nm; column temperature: 30°C, injection volume: 5 μl.

[0061] Table 2

[0062]

[0063] According to the above conditions, high performance liquid chromatography was performed, and the results are shown in Table 2 and Figure 5-6 As shown, impurity compound II and mosapride were detected, the separation met the requirements, the spiked test solution and impurities had good peaks, the response value was high, and the detection sensitivity was good.

[0064] Example 4: Methodological Investigation

[0065] Specificity investigation: The blank solvent does not interfere with the detection of impurity compound II and mosapride, and the separation degree of each component is not less than 1.5;

[0066] System suitability investigation: The mixed solution was injected 5 times continuously, and the RSD value of the peak area of ​​each component was no more than 10%, and the RSD value of the retention time of each component was no more than 2%;

[0067] Limit of quantitation / limit of detection investigation: accurately measure an appropriate amount of the mixed solution, dilute it step by step with acetonitrile, and use the solution with a signal-to-noise ratio of about 10 as the limit of quantitation solution, and the solution with a signal-to-noise ratio of about 3 as the limit of detection solution. The limit of quantitation solution is injected 6 times continuously, and the retention time RSD is ≤2%; the peak area RSD is ≤10%;

[0068] Linearity investigation: Take appropriate amount of impurity compound II and mosapride reference substance, weigh accurately, and quantitatively dilute with acetonitrile to prepare 7 mixed solutions with different concentrations, each containing about 0.2951μg~3.9340μg of impurity compound II and about 0.3019μg~4.0250μg of mosapride in 1ml, and make linear regression equations with concentration versus peak area;

[0069] Repeatability study: Prepare 6 spiked test solutions according to Example 3, and calculate the RSD of the content of impurity compound II in the spiked test solutions;

[0070] Accuracy investigation: Prepare 2μg / ml impurity compound II reference solution, take about 20mg of mosapride test sample, accurately weigh, put in a 10ml volumetric flask, accurately add 0.15ml (quantitative limit concentration), 0.5ml (50% limit concentration), 1ml (100% limit concentration), 1.5ml (150% limit concentration) of solution containing about 20μg of impurity compound II per 1ml, dissolve in acetonitrile and dilute to scale, as the method accuracy test solution, prepare 3 copies for each concentration. Calculate the recovery rate of impurity compound II;

[0071] Stability study: The ratio of the peak area of ​​the mixed solution and the spiked test solution to 0h was studied under room temperature conditions;

[0072] Durability test: Take the 100% limit spiked test solution under the "accuracy test" item, change the column flow rate (±0.2ml / min) and column temperature (±5℃) for analysis and detection, and keep other chromatographic conditions unchanged. The results show that the test results do not change when the chromatographic conditions change slightly, and the method has good durability.

[0073] Table 3

[0074]

[0075]

[0076] 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 impurities in a mosapride citrate intermediate, comprising the following steps: Liquid chromatography was used to detect a test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxybenzoic acid) anhydride, wherein the solvent of the test solution was acetonitrile.

2. The detection method according to claim 1, characterized in that: The concentration of the test solution containing 4-amino-5-chloro-2-ethoxybenzoic acid (isobutoxybenzoic acid) anhydride is 2 mg / mL.

3. The detection method according to claim 1, characterized in that: The chromatographic column used in the liquid chromatography method is cyanosilane bonded silica gel filler.

4. The detection method according to claim 1, characterized in that: The liquid chromatography method uses n-hexane-isopropanol as the mobile phase for gradient elution.

5. The detection method according to claim 4, characterized in that: The elution gradient of the liquid chromatography is:

6. The detection method according to claim 1, characterized in that: The column temperature of the liquid chromatography was 30°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 274 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: The chromatographic column for the liquid chromatography method is Kromasil 60-5-CN (4.6×250 mm, 5 μm).

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