Method for determining and analyzing impurities in vildagliptin

Abstract

The invention relates to a method for determining and analyzing impurities in vildagliptin, which sequentially comprises the following steps of: S1, preparing a reference substance solution, namely weighing a proper amount of impurity reference substances, putting the impurity reference substances into a container, dissolving the impurity reference substances with a solvent, and diluting the impurity reference substances to a scale to obtain a reference substance stock solution with the concentration of 0.1 mg / mL; step S2, preparing a test sample solution: weighing a proper amount of vildagliptin test sample, placing the vildagliptin test sample in a container, dissolving the vildagliptin test sample in a solvent, and diluting the vildagliptin test sample to a scale to obtain a test sample stock solution with the concentration of 0.1 mg / mL; and S3, respectively detecting the reference substance solution and the test solution by using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference substance solution and the test solution, and calculating the amount of the genotoxic impurities in the test solution. 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 determining and analyzing impurities in 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 actively carried out research and innovation in order to create a method for determining and analyzing impurities in vildagliptin to make it more valuable for industrial use. Summary of the invention

[0006] In order to solve the above technical problems, the object of the present invention is to provide a method for determining and analyzing impurities in vildagliptin.

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

[0008] A method for determining and analyzing impurities in vildagliptin comprises the following steps in sequence:

[0009] Step S1, preparing a reference substance solution: weighing an appropriate amount of an impurity reference substance and placing it in a container, dissolving it with a solvent and diluting it to the scale to obtain a reference substance stock solution with a concentration of 0.1 mg / mL;

[0010] Step S2, preparing a test solution: weighing an appropriate amount of vildagliptin test sample into a container, dissolving it with a solvent and diluting it to the scale to obtain a test sample stock solution with a concentration of 0.1 mg / mL;

[0011] Step S3, respectively detecting the reference solution and the test solution using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference solution and the test solution, and calculating the amount of genotoxic impurities in the test sample;

[0012] The above liquid chromatography conditions are:

[0013] C18 column, 3.0×100 mm, 35 μm, column temperature 25-40 °C;

[0014] Flow rate: 0.5~1.5ml / min;

[0015] Injection volume: 10-25ul;

[0016] Detection wavelength: 200nm~250nm;

[0017] Mobile phase: Mobile phase A is ammonium acetate aqueous solution, mobile phase B is acetonitrile, gradient elution;

[0018] The mass spectrometry conditions mentioned above are:

[0019] The ion source is an electrospray ion source, the mobile phase is discharged for 0 to 2 minutes, the drying gas temperature is 250 to 350°C, the drying gas flow rate is 8 to 12 L / min, the nebulizer pressure is 40 to 60 psi, the capillary voltage is 3000 to 3500 V, the sheath gas temperature is 300 to 350°C, the nozzle voltage is 400 to 500 V (+ / -), and the detection is in positive ion mode.

[0020] As a further improvement of the present invention, the C18 chromatographic column is an ACE Excel3 C18-AR chromatographic column, and the column temperature is 25°C.

[0021] As a further improvement of the present invention, the C18 chromatographic column is an ACE Excel3 C18-AR chromatographic column, and the column temperature is 40°C.

[0022] As a further improvement of the present invention, in the above-mentioned mass spectrometry conditions, the detection ion pairs m / z of the compound are 173.1→70.1, 191.0→174.1, 192.0→174.1, 206.1→174.0 and 173.1→146.0, 191.0→146.0, 192.0→146.1, 206.1→146.0.

[0023] As a further improvement of the present invention, the gradient elution conditions in the above-mentioned liquid chromatography are as follows:

[0024] Initial: flow rate: 1.0 mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0025] 5min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0026] 10min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0027] 22min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0028] 15min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0029] 20min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%.

[0030] As a further improvement of the present invention, the gradient elution conditions in the above-mentioned liquid chromatography are as follows:

[0031] Initial: flow rate: 1.5 mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0032] 5min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0033] 10min: flow rate: 1.5mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0034] 22min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0035] 15min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0036] 20min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%.

[0037] As a further improvement of the present invention, the solvent is 10% methanol or 10% acetonitrile.

[0038] As a further improvement of the present invention, the injection volume is 10ul or 20ul or 25ul.

[0039] As a further improvement of the present invention, the detection wavelength is 200 nm or 250 nm.

[0040] By means of the above scheme, the present invention has at least the following advantages:

[0041] The present invention adopts liquid chromatography and mass spectrometry to perform detection and analysis, which is time-saving, highly sensitive and has good separation.

[0042] 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.

[0043] 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

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0045] 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.

[0046] Embodiment 1:

[0047] A method for determining and analyzing impurities in vildagliptin comprises the following steps in sequence:

[0048] Step S1, preparing a reference substance solution: weighing an appropriate amount of an impurity reference substance and placing it in a container, dissolving it in 10% acetonitrile solvent and diluting it to the scale to obtain a reference substance stock solution with a concentration of 0.1 mg / mL;

[0049] Step S2, preparing a test solution: weighing an appropriate amount of vildagliptin test sample into a container, dissolving it in 10% acetonitrile solvent and diluting it to the scale to obtain a test sample stock solution with a concentration of 0.1 mg / mL;

[0050] Step S3, respectively detecting the reference solution and the test solution using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference solution and the test solution, and calculating the amount of genotoxic impurities in the test sample.

[0051] 1) Liquid chromatography conditions:

[0052] C18 column, 3.0 × 100 mm, 35 μm, column temperature 25 °C;

[0053] Flow rate: 10.ml / min;

[0054] Injection volume: 10ul;

[0055] Detection wavelength: 200nmnm;

[0056] Mobile phase: Mobile phase A is ammonium acetate aqueous solution, mobile phase B is acetonitrile;

[0057] The gradient elution conditions were as follows:

[0058] Initial: flow rate: 1.0 mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0059] 5min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0060] 10min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0061] 22min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0062] 15min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0063] 20min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%.

[0064] 2) Mass spectrometry conditions:

[0065] The ion source was an electrospray ion source, the mobile phase was discharged for 0 to 2 min, the drying gas temperature was 250 to 350°C, the drying gas flow rate was 8 to 12 L / min, the nebulizer pressure was 40 to 60 psi, the capillary voltage was 3000 to 3500 V, the sheath gas temperature was 300 to 350°C, the nozzle voltage was 400 to 500 V (+ / -), and the detection was in positive ion mode; the detection ion pairs m / z of the compounds were 173.1→70.1, 191.0→174.1, 192.0→174.1, 206.1→174.0 and 173.1→146.0, 191.0→146.0, 192.0→146.1, 206.1→146.0.

[0066] Embodiment 2:

[0067] A method for determining and analyzing impurities in vildagliptin comprises the following steps in sequence:

[0068] Step S1, preparing a reference substance solution: weighing an appropriate amount of an impurity reference substance and placing it in a container, dissolving it in 10% acetonitrile solvent and diluting it to the scale to obtain a reference substance stock solution with a concentration of 0.1 mg / mL;

[0069] Step S2, preparing a test solution: weighing an appropriate amount of vildagliptin test sample into a container, dissolving it in 10% acetonitrile solvent and diluting it to the scale to obtain a test sample stock solution with a concentration of 0.1 mg / mL;

[0070] Step S3, respectively detecting the reference solution and the test solution using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference solution and the test solution, and calculating the amount of genotoxic impurities in the test sample.

[0071] 1) Liquid chromatography conditions:

[0072] C18 column, 3.0 × 100 mm, 35 μm, column temperature 40 °C;

[0073] Flow rate: 1.5ml / min;

[0074] Injection volume: 25ul;

[0075] Detection wavelength: 250nm;

[0076] Mobile phase: Mobile phase A is ammonium acetate aqueous solution, mobile phase B is acetonitrile;

[0077] The gradient elution conditions were as follows:

[0078] Initial: flow rate: 1.5 mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0079] 5min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0080] 10min: flow rate: 1.5mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0081] 22min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0082] 15min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%;

[0083] 20min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%.

[0084] 2) Mass spectrometry conditions:

[0085] The ion source was an electrospray ion source, the mobile phase was discharged for 0 to 2 min, the drying gas temperature was 250 to 350°C, the drying gas flow rate was 8 to 12 L / min, the nebulizer pressure was 40 to 60 psi, the capillary voltage was 3000 to 3500 V, the sheath gas temperature was 300 to 350°C, the nozzle voltage was 400 to 500 V (+ / -), and the detection was in positive ion mode; the detection ion pairs m / z of the compounds were 173.1→70.1, 191.0→174.1, 192.0→174.1, 206.1→174.0 and 173.1→146.0, 191.0→146.0, 192.0→146.1, 206.1→146.0.

[0086] Embodiment three:

[0087] A method for determining and analyzing impurities in vildagliptin comprises the following steps in sequence:

[0088] Step S1, preparing a reference substance solution: weighing an appropriate amount of an impurity reference substance into a container, dissolving it in 10% methanol solvent and diluting it to the mark to obtain a reference substance stock solution with a concentration of 0.1 mg / mL;

[0089] Step S2, preparing a test solution: weighing an appropriate amount of vildagliptin test sample into a container, dissolving it in 10% methanol solvent and diluting it to the scale to obtain a test sample stock solution with a concentration of 0.1 mg / mL;

[0090] Step S3, respectively detecting the reference solution and the test solution using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference solution and the test solution, and calculating the amount of genotoxic impurities in the test sample.

[0091] 1) Liquid chromatography conditions:

[0092] C18 column, 3.0 × 100 mm, 35 μm, column temperature 40 °C;

[0093] Flow rate: 1.0ml / min;

[0094] Injection volume: 25ul;

[0095] Detection wavelength: 250nm;

[0096] Mobile phase: Mobile phase A is ammonium acetate aqueous solution, mobile phase B is acetonitrile;

[0097] The gradient elution conditions were as follows:

[0098] Initial: flow rate: 1.0 mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0099] 5min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0100] 10min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0101] 22min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0102] 15min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%;

[0103] 20min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%.

[0104] 2) Mass spectrometry conditions:

[0105] The ion source was an electrospray ion source, the mobile phase was discharged for 0 to 2 min, the drying gas temperature was 250 to 350°C, the drying gas flow rate was 8 to 12 L / min, the nebulizer pressure was 40 to 60 psi, the capillary voltage was 3000 to 3500 V, the sheath gas temperature was 300 to 350°C, the nozzle voltage was 400 to 500 V (+ / -), and the detection was in positive ion mode; the detection ion pairs m / z of the compounds were 173.1→70.1, 191.0→174.1, 192.0→174.1, 206.1→174.0 and 173.1→146.0, 191.0→146.0, 192.0→146.1, 206.1→146.0.

[0106] 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 determining and analyzing impurities in vildagliptin, characterized in that: The following steps are included in sequence: Step S1, preparing a reference substance solution: weighing an appropriate amount of an impurity reference substance and placing it in a container, dissolving it with a solvent and diluting it to the scale to obtain a reference substance stock solution with a concentration of 0.1 mg / mL; Step S2, preparing a test solution: weighing an appropriate amount of vildagliptin test sample into a container, dissolving it with a solvent and diluting it to the scale to obtain a test sample stock solution with a concentration of 0.1 mg / mL; Step S3, respectively detecting the reference solution and the test solution using a liquid chromatography tandem mass spectrometer to obtain chromatogram data of the reference solution and the test solution, and calculating the amount of genotoxic impurities in the test sample; The above liquid chromatography conditions are: C18 column, 3.0×100 mm, 35 μm, column temperature 25-40 °C; Flow rate: 0.5~1.5ml / min; Injection volume: 10-25ul; Detection wavelength: 200nm~250nm; Mobile phase: Mobile phase A is ammonium acetate aqueous solution, mobile phase B is acetonitrile, gradient elution; The mass spectrometry conditions mentioned above are: The ion source is an electrospray ion source, the mobile phase is discharged for 0 to 2 minutes, the drying gas temperature is 250 to 350°C, the drying gas flow rate is 8 to 12 L / min, the nebulizer pressure is 40 to 60 psi, the capillary voltage is 3000 to 3500 V, the sheath gas temperature is 300 to 350°C, the nozzle voltage is 400 to 500 V (+ / -), and the detection is in positive ion mode.

2. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The C18 chromatographic column is an ACE Excel3 C18-AR chromatographic column, and the column temperature is 25°C.

3. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The C18 chromatographic column is an ACE Excel3 C18-AR chromatographic column, and the column temperature is 40°C.

4. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: In the above mass spectrometry conditions, the detection ion pairs m / z of the compound are 173.1→70.1, 191.0→174.1, 192.0→174.1, 206.1→174.0 and 173.1→146.0, 191.0→146.0, 192.0→146.1, 206.1→146.

0.

5. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The gradient elution conditions in the above-mentioned liquid chromatography are as follows: Initial: flow rate: 1.0 mL / min; mobile phase A: 85%, mobile phase B: 15%; 5min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%; 10min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%; 22min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%; 15min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%; 20min: flow rate: 1.0mL / min; mobile phase A: 85%, mobile phase B: 15%.

6. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The gradient elution conditions in the above-mentioned liquid chromatography are as follows: Initial: flow rate: 1.5 mL / min; mobile phase A: 90%, mobile phase B: 10%; 5min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%; 10min: flow rate: 1.5mL / min; mobile phase A: 75%, mobile phase B: 25%; 22min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%; 15min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%; 20min: flow rate: 1.5mL / min; mobile phase A: 90%, mobile phase B: 10%.

7. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The solvent is 10% methanol or 10% acetonitrile.

8. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The injection volume is 10ul, 20ul or 25ul.

9. The method for determining and analyzing impurities in vildagliptin according to claim 1, characterized in that: The detection wavelength is 200nm or 250nm.