Vildagliptin genotoxic impurity residual quantity detection method

Abstract

The invention relates to a vildagliptin genotoxic impurity residual quantity detection method, which comprises: mixing a reference substance solution: precisely weighing a proper amount of a reference substance, adding a solvent, dissolving, and diluting to a scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL; a test sample solution: precisely weighing a proper amount of a test sample, adding a solvent for dissolving, and diluting to a scale to obtain a test sample stock solution with the concentration of 0.20 mg / mL; the reference substance and the test sample are respectively injected into 5-25 [mu] l, and the content of each impurity is calculated by an external standard method. The method is simple and convenient to operate, relatively good in repeatability and specificity and accurate and reliable in result, and can be used for detecting the toxic impurity residual quantity of the vildagliptin gene.

Description

Technical Field

[0001] The present invention relates to the technical field related to biomedical detection, and in particular to a method for detecting the residual amount of genotoxic impurities of vildagliptin. Background Art

[0002] Vildagliptin is a dipeptidyl peptidase IV inhibitor and a new oral drug for the treatment of diabetes. Vildagliptin intermediate-5 is one of the key intermediates in the synthesis of Vildagliptin, with a chemical name of S-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile, a molecular formula of C7H9ClN2O, and a molecular weight of 172.61.

[0003] Vildagliptin is a selective, competitive, and reversible DPP-4 inhibitor that inhibits the proteolytic activity of DPP-4 and enhances the activity of glucagon-like peptide (GLP-1) and gastric inhibitory polypeptide (GIP) to lower blood sugar. Compared with other types of hypoglycemic drugs, vildagliptin has a significant hypoglycemic effect and is glucose concentration-dependent, so it has less hypoglycemia, does not increase weight, and is safer. It can simultaneously improve the function of α and β cells, promote insulin secretion, inhibit glucagon secretion, improve insulin resistance, and can solve the multiple pathological and physiological defects of type 2 diabetes.

[0004] Potential toxic impurities may be produced in the synthesis process of vildagliptin, so it is necessary to design a method for detecting the residual amount of toxic impurities in the synthesized vildagliptin.

[0005] In view of the above-mentioned defects, the designers have actively carried out research and innovation in order to create a method for detecting the residual amount of genotoxic impurities in vildagliptin, making it more valuable for industrial use. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the residual amount of genotoxic impurities in vildagliptin.

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

[0008] A method for detecting residual genotoxic impurities of vildagliptin, the detection method comprising:

[0009] Chromatographic conditions: C18 column;

[0010] Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile;

[0011] Column temperature: 20-30°C;

[0012] Injection volume: 5-25ul;

[0013] Flow rate: 0.5~1.5ml / min;

[0014] Detection wavelength: 200nm~250nm;

[0015] Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL;

[0016] Test solution: Accurately weigh an appropriate amount of the test sample, add solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL;

[0017] Wherein, the above-mentioned reference substance is isopropyl benzenesulfonate, and the above-mentioned test substance is vildagliptin;

[0018] Inject 5-25ul of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

[0019] As a further improvement of the present invention, the C18 chromatographic column is a Zorbax SB-C18 chromatographic column with specifications of 250 mm×4.6 mm, 5 μm, and a column temperature of 20°C.

[0020] As a further improvement of the present invention, the C18 chromatographic column is a Zorbax SB-C18 chromatographic column with specifications of 250 mm×4.6 mm, 5 μm, and a column temperature of 25°C.

[0021] As a further improvement of the present invention, the gradient elution conditions of the above chromatogram are as follows:

[0022] Initial: flow rate: 1.0 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0023] 5min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0024] 10min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0025] 20min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0026] 30min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0027] 40min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0028] As a further improvement of the present invention, the gradient elution conditions of the above chromatogram are as follows:

[0029] Initial: flow rate: 1.5 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0030] 5min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0031] 10min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0032] 20min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0033] 30min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0034] 40min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0035] As a further improvement of the present invention, the detection wavelength is 200 nm or 250 nm.

[0036] As a further improvement of the present invention, the solvent is acetonitrile.

[0037] By means of the above scheme, the present invention has at least the following advantages:

[0038] The invention is simple to operate, has good repeatability and specificity, and produces accurate and reliable results, and can be used for detecting the residual amount of genotoxic impurities of vildagliptin.

[0039] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. DETAILED DESCRIPTION

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0041] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. The components of the embodiment of the present invention generally shown here can be arranged and designed in various configurations. Therefore, the following detailed description of the embodiment of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiment of the present invention. Based on the embodiment of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0042] Embodiment 1:

[0043] The present invention provides a method for detecting residual genotoxic impurities of vildagliptin, which is specifically as follows:

[0044] 1. Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add acetonitrile solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL;

[0045] 2. Test solution: Accurately weigh an appropriate amount of the test sample, add acetonitrile solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL;

[0046] 3. Inject 5 to 25 μl of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

[0047] Chromatographic conditions: Zorbax SB-C18 column, 250 mm × 4.6 mm, 5 μm;

[0048] Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile;

[0049] Column temperature: 20°C;

[0050] Injection volume: 20ul;

[0051] Flow rate: 1.0ml / min;

[0052] Detection wavelength: 200nm;

[0053] The gradient elution conditions were as follows:

[0054] Initial: flow rate: 1.0 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0055] 5min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0056] 10min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0057] 20min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0058] 30min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0059] 40min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0060] Embodiment 2:

[0061] The present invention provides a method for detecting residual genotoxic impurities of vildagliptin, which is specifically as follows:

[0062] 1. Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add acetonitrile solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL;

[0063] 2. Test solution: Accurately weigh an appropriate amount of the test sample, add acetonitrile solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL;

[0064] 3. Inject 5 to 25 μl of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

[0065] Chromatographic conditions: Zorbax SB-C18 column, 250 mm × 4.6 mm, 5 μm;

[0066] Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile;

[0067] Column temperature: 20°C;

[0068] Injection volume: 20ul;

[0069] Flow rate: 1.5ml / min;

[0070] Detection wavelength: 200nm;

[0071] The gradient elution conditions were as follows:

[0072] Initial: flow rate: 1.5 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0073] 5min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0074] 10min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0075] 20min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0076] 30min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0077] 40min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0078] Embodiment three:

[0079] The present invention provides a method for detecting residual genotoxic impurities of vildagliptin, which is specifically as follows:

[0080] 1. Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add acetonitrile solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL;

[0081] 2. Test solution: Accurately weigh an appropriate amount of the test sample, add acetonitrile solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL;

[0082] 3. Inject 5 to 25 μl of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

[0083] Chromatographic conditions: Zorbax SB-C18 column, 250 mm × 4.6 mm, 5 μm;

[0084] Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile;

[0085] Column temperature: 25°C;

[0086] Injection volume: 20ul;

[0087] Flow rate: 1.0ml / min;

[0088] Detection wavelength: 250nm;

[0089] The gradient elution conditions were as follows:

[0090] Initial: flow rate: 1.0 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0091] 5min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0092] 10min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0093] 20min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0094] 30min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0095] 40min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0096] Embodiment 4:

[0097] The present invention provides a method for detecting residual genotoxic impurities of vildagliptin, which is specifically as follows:

[0098] 1. Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add acetonitrile solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL;

[0099] 2. Test solution: Accurately weigh an appropriate amount of the test sample, add acetonitrile solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL;

[0100] 3. Inject 5 to 25 μl of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

[0101] Chromatographic conditions: Zorbax SB-C18 column, 250 mm × 4.6 mm, 5 μm;

[0102] Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile;

[0103] Column temperature: 25°C;

[0104] Injection volume: 20ul;

[0105] Flow rate: 1.5ml / min;

[0106] Detection wavelength: 250nm;

[0107] The gradient elution conditions were as follows:

[0108] Initial: flow rate: 1.5 mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0109] 5min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0110] 10min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0111] 20min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%;

[0112] 30min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0113] 40min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting residual genotoxic impurities in vildagliptin, characterized in that: The detection method is: Chromatographic conditions: C18 column; Mobile phase: Mobile phase A is 0.1% acetic acid in water, mobile phase B is acetonitrile; Column temperature: 20-30°C; Injection volume: 5-25ul; Flow rate: 0.5~1.5ml / min; Detection wavelength: 200nm~250nm; Mixed reference substance solution: Accurately weigh an appropriate amount of reference substance, add solvent to dissolve and dilute to the scale to obtain a reference substance stock solution with a concentration of 0.20 mg / mL; Test solution: Accurately weigh an appropriate amount of the test sample, add solvent to dissolve and dilute to the scale to obtain a test sample stock solution with a concentration of 0.20 mg / mL; Wherein, the above-mentioned reference substance is isopropyl benzenesulfonate, and the above-mentioned test substance is vildagliptin; Inject 5-25ul of the reference substance and the test substance respectively, and calculate the content of each impurity by the external standard method.

2. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The C18 chromatographic column is a Zorbax SB-C18 chromatographic column with specifications of 250 mm×4.6 mm, 5 μm, and a column temperature of 20°C.

3. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The C18 chromatographic column is a Zorbax SB-C18 chromatographic column with specifications of 250 mm×4.6 mm, 5 μm, and a column temperature of 25°C.

4. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The gradient elution conditions of the above chromatography are as follows: Initial: flow rate: 1.0 mL / min; mobile phase A: 65%, mobile phase B: 35%; 5min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%; 10min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%; 20min: flow rate: 1.0mL / min; mobile phase A: 55%, mobile phase B: 45%; 30min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%; 40min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%.

5. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The gradient elution conditions of the above chromatography are as follows: Initial: flow rate: 1.5 mL / min; mobile phase A: 65%, mobile phase B: 35%; 5min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%; 10min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%; 20min: flow rate: 1.5mL / min; mobile phase A: 55%, mobile phase B: 45%; 30min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%; 40min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%.

6. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The detection wavelength is 200nm or 250nm.

7. A method for detecting residual genotoxic impurities of vildagliptin according to claim 1, characterized in that: The solvent is acetonitrile.