Method for detecting genotoxic impurities in vildagliptin and application thereof

Abstract

The invention relates to a method for detecting genotoxic impurities in vildagliptin and application thereof, and the detection method comprises the following steps: detecting a to-be-detected sample by adopting a liquid chromatography-mass spectrometry method to obtain the content of the genotoxic impurities in the to-be-detected sample. The detection method provided by the invention can realize qualitative and quantitative analysis of genotoxic impurities in vildagliptin, has the advantages of good impurity specificity, good repeatability, high precision and sensitivity, is accurate and reliable, and can be widely applied to detection of the residual quantity of the genotoxic impurities in vildagliptin.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for detecting genotoxic impurities in vildagliptin and its application. Background Technology

[0002] Vildagliptin is a commonly used medication for type 2 diabetes. It works by inhibiting dipeptidyl peptidase-4 (DPP-4), thereby reducing the inactivation of glucagon-like peptide-1 (GLP-1) in the body and increasing endogenous GLP-1 levels. GLP-1 stimulates insulin secretion from pancreatic β-cells while inhibiting glucagon secretion, thus lowering blood glucose levels.

[0003] Vildagliptin is a white or off-white crystalline powder with the chemical name (S)-1-[2-(3-hydroxyadamantane-1-amino)acetyl]pyrrolidine-2-nitrile. Its molecular formula is C1. 17 H 25 N3O2 has the following structural formula: The synthesis process of vildagliptin may generate potential genotoxic impurities, which directly affect the quality of vildagliptin. The 2025 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 9306, emphasizes the importance of controlling genotoxic impurities; therefore, a method for detecting residual genotoxic impurities in synthesized vildagliptin needs to be designed. Due to the low limits for genotoxic impurities, developing a method that can accurately quantify them is challenging. However, vildagliptin is not included in the pharmacopoeias of various countries, and only some literature or patents report on the detection of genotoxic impurities in vildagliptin, which is still insufficient. For example, patent CN115586276A discloses a method for detecting genotoxic impurities in vildagliptin, mainly detecting (S)-1-chloroacetylpyrrolidine-2-carboxamide, (S)-1-chloroacetylpyrrolidine-2-carboxamide, (2-chloroacetyl)-L-proline, and 1-(chloroacetyl)-L-proline methyl ester. However, no methods have been reported for the simultaneous determination of genotoxic impurities in vildagliptin, including dichloroacetic acid, (2-chloroacetyl)-L-proline, 1-(chloroacetyl)-L-proline methyl ester, (2S)-N-dichloroacetyl-2-cyanotetrahydropyrrole, and (2S)-N-chloroacetyl-2-cyanopyrrolidine. Therefore, developing simple and reproducible analytical methods for multiple genotoxic impurities is crucial for ensuring the quality of the vildagliptin raw material. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting genotoxic impurities in vildagliptin and its application. The detection method provided by the present invention employs liquid chromatography-mass spectrometry (LC-MS) with simultaneous scanning in both positive and negative ion modes. LC achieves good separation of various impurities, while mass spectrometry performs qualitative and quantitative analysis of each impurity. Therefore, the detection method of the present invention can achieve qualitative and quantitative analysis of multiple genotoxic impurities in vildagliptin, possessing advantages such as high impurity separation, good specificity, high precision, good repeatability, and high sensitivity. The method is accurate and reliable and can be widely applied to the quality testing of vildagliptin.

[0005] The present invention adopts the following technical solution: In a first aspect, the present invention provides a method for detecting genotoxic impurities in vildagliptin, the method comprising: using liquid chromatography-mass spectrometry to detect the sample to be tested, thereby obtaining the content of genotoxic impurities in the sample to be tested.

[0006] The genotoxic impurities include (2S)-N-dichloroacetyl-2-cyanotetrahydropyrrole, and include any one or a combination of at least two of (2-chloroacetyl)-L-proline, 1-(chloroacetyl)-L-proline methyl ester, (2S)-N-chloroacetyl-2-cyanopyrrole, or dichloroacetic acid.

[0007] In this invention, the impurities include, but are not limited to, genotoxic impurities generated during the production or storage of vildagliptin raw materials. The detection method provided by this invention employs liquid chromatography coupled with mass spectrometry. Liquid chromatography achieves good separation of each impurity, while mass spectrometry qualitatively identifies each impurity. This detection method is accurate and reliable, possessing advantages such as good specificity, high precision, good repeatability, and high sensitivity. It can simultaneously detect multiple genotoxic impurities and is widely used in the quality testing of vildagliptin.

[0008] Preferably, the genotoxic impurities include (2S)-N-dichloroacetyl-2-cyanotetrahydropyrrole, (2-chloroacetyl)-L-proline, 1-(chloroacetyl)-L-proline methyl ester, (2S)-N-chloroacetyl-2-cyanopyrrole and dichloroacetic acid. In this invention, liquid chromatography-mass spectrometry is used to determine the structural information of each impurity.

[0009] Preferably, the mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of formic acid and mobile phase B is acetonitrile.

[0010] Preferably, the volume concentration of the mobile phase A is 0.09%-0.11%, for example, it can be 0.09%, 0.10%, 0.11%, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0011] Preferably, the liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows: From 0 to 3 minutes, the volume percentage of mobile phase A is 83-87%, and the volume percentage of mobile phase B is 13-17%. After a period of uniform change until the 10th minute, the volume percentage of mobile phase A was 27-33%, and the volume percentage of mobile phase B was 67-73%. After a period of uniform change until the 11th minute, the volume percentage of mobile phase A was 83-87%, and the volume percentage of mobile phase B was 13-17%. After 11 minutes, the volume percentage of mobile phase A was 83-87%, and the volume percentage of mobile phase B was 13-17%.

[0012] Preferably, the chromatographic column used in the liquid chromatography is an octadecylsilane-bonded chromatographic column.

[0013] Preferably, the chromatographic column used in the liquid chromatography has a particle size of 1.5-5 μm, such as 1.5 μm, 1.7 μm, 2.1 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.6 μm, 5 μm, etc., a column length of 100-300 mm, such as 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, etc., and an inner diameter of 2-4.6 mm, such as 2 mm, 2.1 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.6 mm, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0014] In this invention, when the octadecylsilane-bonded chromatographic column is used, such as the Waters ACQUITY UPLCBEH Shield RP18 column (1.7 μm, 2.1 × 150 mm), the peak shapes of each impurity are good and can be accurately detected.

[0015] Preferably, the column temperature of the liquid chromatography column is 30-40℃, more preferably 33-37℃, for example, 33℃, 34℃, 35℃, 36℃, 37℃, etc., but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0016] Preferably, the injection flow rate of the liquid chromatography is 0.1-0.5 mL / min, more preferably 0.2-0.4 mL / min, for example, it can be 0.2 mL / min, 0.3 mL / min, or 0.4 mL / min, but is not limited to the values ​​listed above. Other values ​​not listed in the above range are also applicable.

[0017] Preferably, the mass spectrometer is a triple quadrupole mass spectrometer.

[0018] Preferably, the mass spectrometer is an ESI mass spectrometer.

[0019] Preferably, the detection method is a simultaneous scanning of positive and negative ions.

[0020] Secondly, the present invention provides the application of the method for detecting genotoxic impurities in vildagliptin as described above in the quality testing of vildagliptin.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The detection method of liquid chromatography-mass spectrometry provided by the present invention can simultaneously determine the five impurities in vildagliptin qualitatively and quantitatively. The method is interference-free and has the advantages of good specificity, high impurity separation, high precision, good durability, and suitability for quality control. The method is accurate and reliable.

[0022] (2) The detection method provided by the present invention has high sensitivity, and the detection sensitivity limit of each impurity can be as low as 1.4 ng / mL and the detection limit can be as low as 0.4 ng / mL. Attached Figure Description

[0023] Figure 1 This is the EIC integral spectrum of impurity GTI-1-1 in Example 1; Figure 2 This is the EIC integral spectrum of impurity GTI-1-2 in Example 1; Figure 3 This is the EIC integral spectrum of impurity GTI-2-1 in Example 1; Figure 4 This is the EIC integral spectrum of impurity GTI-2-2 in Example 1; Figure 5 This is the EIC integral spectrum of impurity GTI-3-1 in Example 1; Figure 6 This is the EIC integral spectrum of impurity GTI-3-2 in Example 1; Figure 7 This is the EIC integral spectrum of impurity GTI-4-1 in Example 1; Figure 8This is the EIC integral spectrum of impurity GTI-4-2 in Example 1; Figure 9 This is the EIC integral spectrum of impurity GTI-5-1 in Example 1; Figure 10 This is the EIC integral spectrum of impurity GTI-5-2 in Example 1; Figure 11 This is the positive ion TIC spectrum from Example 1; Figure 12 This is the negative ion TIC spectrum from Example 1; Figure 13 This is the TIC spectrum in Comparative Example 1. Detailed Implementation

[0024] Example 1: Determination of the mobile phase ratio in liquid chromatography This embodiment compares the proportions of mobile phase in gradient elution in high-performance liquid chromatography, specifically including: (1) Solution preparation Reference solution: Take appropriate amounts of each impurity reference standard GTI-1, GTI-2, GTI-3, GTI-4, and GTI-5, accurately weigh them, dissolve them in acetonitrile, and quantitatively dilute them to prepare a solution containing approximately 0.3 mg per mL as the reference standard stock solution. Accurately measure an appropriate amount of the above solution and dilute it with water to prepare a solution containing approximately 30 ng per mL.

[0025] Spiked test solution: Accurately weigh an appropriate amount of vildagliptin and measure an appropriate amount of the reference stock solution, dissolve in water and dilute quantitatively to prepare a solution containing approximately 2 mg of vildagliptin and approximately 30 ng of each impurity per 1 mL.

[0026] (2) Chromatographic conditions Chromatographic column: Waters ACQUITY UPLC BEH Shield RP18 1.7μm, 2.1×150mm Mobile phase A: Formic acid aqueous solution Mobile phase B: Acetonitrile Elution gradient: Injection volume: 5 μL Column temperature: 35℃ Flow rate: 0.3 mL / min Detection wavelength: 210nm (3) Mass spectrometry conditions Ion source: Electrospray ionization (ESI) source Ion type: Simultaneous scanning of positive and negative ions Monitoring mode: Multiple reaction detection (MRM) positive ions negative ions Other parameters Group 1: Mobile phase A: 0.05% formic acid aqueous solution; Group 2: Mobile phase A: 0.09% formic acid aqueous solution; Group 3: Mobile phase A: 0.10% formic acid aqueous solution; Group 4: Mobile phase A: 0.11% formic acid aqueous solution; Group 5: Mobile phase A: 0.15% formic acid aqueous solution.

[0027] (4) Experimental steps and conclusions Conclusion: When the proportion of formic acid in the mobile phase is between 0.09% and 0.11%, various impurities can be effectively separated and detected. The best separation effect is achieved at a flow rate of 0.10%. Figure 1-10 The corresponding EIC spectra of each impurity are shown (the horizontal axis represents time, and the vertical axis represents intensity). Figure 11-12 The corresponding TIC spectra show that when the formic acid content is 0.05% and 0.15%, impurities GTI1 and GTI2 cannot be effectively separated.

[0028] Example 2: Determination of the column temperature range for chromatography This embodiment compares column temperatures in high-performance liquid chromatography, specifically including: (1) Based on group 3 of Example 1, the column temperature of the chromatographic column was changed to 30℃, 33℃, 35℃, 37℃ and 40℃ respectively, while other conditions remained unchanged; (2) Experimental results and conclusions Conclusion: When the column temperature is between 33℃ and 37℃, all impurities can be effectively separated and detected. When the column temperature is 30℃, impurities GTI1 and GTI2 cannot be effectively separated, and when the column temperature is 40℃, impurities GTI3 and GTI4 cannot be effectively separated.

[0029] Example 3: Determination of Column Flow Rate This embodiment compares the injection flow rate in high-performance liquid chromatography (HPLC) detection, specifically including: (1) Based on group 3 of Example 1, the injection flow rate was changed to 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min and 0.5 mL / min, while other conditions remained unchanged; (2) Experimental results and conclusions Conclusion: When the flow rate is between 0.2 mL / min and 0.4 mL / min, all impurities can be effectively separated and detected. When the flow rate is 0.1 mL / min, impurities GTI1, GTI2, and GTI3 cannot be effectively separated. When the flow rate is 0.5 mL / min, impurities GTI3 and GTI4 cannot be effectively separated.

[0030] Example 4: Determination of the elution procedure (1) Based on group 3 of Example 1, the elution gradient was changed as follows, while other conditions remained unchanged; Group 1: Group 2: Group 3: (2) Experimental results and conclusions Conclusion: The elution gradient of group 2 yields the best impurity separation.

[0031] Example 5 Specificity Test The test samples and conditions were the same as those in Example 1, Group 3.

[0032] The test results are shown in the table below: Conclusion: The blank solvent does not interfere with the detection of any impurities, and the detection of any impurities is unaffected.

[0033] Example 6 Sensitivity Test Blank solvent: water; Sensitivity test solution: Take the reference solution described in Example 1, dilute it to a signal-to-noise ratio (S / N) of 10:1 and a signal-to-noise ratio (S / N) of 3:1 and then perform the determination. This is the limit of quantitation and limit of detection of the method of the present invention. The chromatographic and mass spectrometry conditions were as described in Example 1, Group 3.

[0034] The test results are shown in the table below: Conclusion: The detection sensitivity of each impurity can reach as low as 1.4 ng / mL, and the detection limit can be as low as 0.4 ng / mL.

[0035] Example 7 Repeatability Test Blank solvent: water; Repeatability test solution: Take the spiked test solution described in Example 1 as the repeatability test solution and prepare six parallel portions; denoted as solution-1, solution-2, solution-3, solution-4, solution-5, and solution-6; The chromatographic and mass spectrometry conditions were as described in Example 1, Group 3.

[0036] The test results are shown in the table below: Conclusion: The maximum RSD of impurity content in the 6 repeatable solutions was 1.8%, all less than 3%, indicating that the method of the present invention has good repeatability.

[0037] Example 8 Stability Test Blank solvent: water; Stability test solution: The spiked test solution described in Example 1 was used as the stability test solution; The chromatographic and mass spectrometry conditions were as described in Example 1, Group 3.

[0038] The test results are shown in the table below: Conclusion: The results show that the peak area change of each impurity in the test solution within 17.5 h is less than 20.0%, indicating that the solution is stable.

[0039] Example 9 Linear Diluent: Water.

[0040] Linear stock solution: Take appropriate amounts of each of the impurity reference standards GTI-1, GTI-2, GTI-3, GTI-4, and GTI-5, dissolve them in acetonitrile, and quantitatively dilute them to prepare a solution containing approximately 0.3 mg per 1 mL; accurately measure an appropriate amount of the above solution, dilute it with water to prepare a solution containing approximately 1.5 μg per 1 mL, and use it as the linear stock solution.

[0041] Prepare linear solutions of the corresponding concentrations using the linear stock solution according to the table below, and mix thoroughly. The chromatographic and mass spectrometry conditions were as described in Example 1, Group 3.

[0042] The test results are shown in the table below: Within the concentration range of 5.7814 ng / mL to 57.8140 ng / mL, the linear equation between the peak area and concentration of impurity GTI-1 is y = 34405.5944x + 22526.2683, with a linear correlation coefficient (r) of 0.9997, which is greater than 0.999. This indicates a good linear relationship between the peak area and concentration of impurity GTI-1. Within the concentration range of 1.4177 ng / mL to 56.7087 ng / mL, the linear equation between the peak area and concentration of impurity GTI-2 is y = 117531.7637x + 24881.5302, with a linear correlation coefficient (r) of 0.9996, which is greater than 0.999, indicating a good linear relationship between the peak area and concentration of impurity GTI-2. Within the concentration range of 1.4330 ng / mL to 57.3215 ng / mL, the linear equation between the peak area and concentration of impurity GTI-3 is y = 285926.9676x + 168945.1612, with a linear correlation coefficient (r) of 0.9997, which is greater than 0.999. This indicates a good linear relationship between the peak area and concentration of impurity GTI-3. Within the concentration range of 1.4229 ng / mL to 56.9165 ng / mL, the peak area of ​​impurity GTI-4 showed a linear relationship with concentration as y = 21609.5151x + 9566.7804, with a linear correlation coefficient (r) of 0.9996, which is greater than 0.999. This indicates a good linear relationship between the peak area and concentration of impurity GTI-4. Within the concentration range of 5.5845 ng / mL to 55.8453 ng / mL, the linear equation between the peak area and concentration of impurity GTI-5 is y = 16099.9704x + 790.4390, with a correlation coefficient (r) of 0.9995, which is greater than 0.999. This indicates a good linear relationship between the peak area and concentration of impurity GTI-5. The results above indicate that, within the linear concentration range, the linear correlation coefficients (r) of the genotoxic impurities GTI-1~5 of vildagliptin are all greater than 0.999, and their peak areas have a good linear relationship with concentration. Compared with the correlation coefficient r=0.99 required by general standards, the accuracy of the determination is improved by more than two orders of magnitude. The detection method is more accurate and has a wider detection range.

[0043] Example 10 Recovery rate Diluent: Water.

[0044] Stock solution of impurities 1-5: Take appropriate amounts of each impurity reference standard GTI-1, GTI-2, GTI-3, GTI-4, and GTI-5, dissolve them in acetonitrile, and quantitatively dilute them to prepare a solution containing approximately 0.3 mg per mL; accurately measure an appropriate amount of the above solution, dilute it with water to prepare a solution containing approximately 1.5 μg per mL, and use it as the stock solution of reference standards.

[0045] 50% recovery solution: Weigh approximately 100 mg of vildagliptin accurately, place it in a 50 mL volumetric flask, add an appropriate amount of water to dissolve it, then accurately add 0.5 mL of the reference stock solution, dilute with water to the mark, and shake well.

[0046] 100% recovery solution: Weigh approximately 100 mg of vildagliptin accurately, place it in a 50 mL volumetric flask, add an appropriate amount of water to dissolve it, then accurately add 1 mL of the reference stock solution, dilute with water to the mark, and shake well.

[0047] 150% recovery solution: Weigh approximately 100 mg of vildagliptin accurately, place it in a 50 mL volumetric flask, add an appropriate amount of water to dissolve it, then accurately add 1.5 mL of the reference stock solution, dilute to the mark with diluent, and shake well.

[0048] The chromatographic and mass spectrometry conditions were as described in Example 1, Group 3.

[0049] The test results are shown in the table below: With test solutions at 50%, 100%, and 150% accuracy, the average recovery rates within and between groups of impurities GTI-1 to GTI-5 remained between 90% and 110%, with a concentrated recovery range, which can accurately meet the requirements for quantitative determination of each impurity.

[0050] The RSD of the recovery rate of the nine samples measured in this embodiment is less than 3.0%. Compared with the standard requirement of recovery rate RSD ≤ 10.0%, the RSD of the detection method disclosed in this invention is reduced to less than one-third of the standard requirement. The accuracy of the detection results is significantly improved by more than two times, further proving that the method is accurate in detecting the above impurities GTI-1~5.

[0051] Comparative Example 1 This comparative example provides a method for detecting genotoxic impurities in vildagliptin, with specific steps referenced in patent CN115586276A: (1) Solution preparation Reference solution: Take appropriate amounts of each impurity reference standard GTI-1, GTI-2, GTI-3, GTI-4, and GTI-5, accurately weigh them, dissolve them in acetonitrile, and quantitatively dilute them to prepare a solution containing approximately 0.3 mg per mL as the reference standard stock solution. Accurately measure an appropriate amount of the above solution and dilute it with water to prepare a solution containing approximately 30 ng per mL.

[0052] (2) Chromatographic conditions Column: Agilent ZORBAX SB-Aq 3.5μm 3.0×100mm Mobile phase A: 5 mM ammonium formate aqueous solution Mobile phase B: Acetonitrile Elution gradient: Injection volume: 5 μL Column temperature: 40℃ Flow rate: 0.3 mL / min Detection wavelength: 200nm (3) Mass spectrometry conditions Ion source: Electrospray ionization (ESI) source Ion type: Positive ion scan Monitoring mode: Multiple reaction detection (MRM) positive ions Other parameters (3) Conclusion: The test results are attached. Figure 13 GTI-5 did not show a peak, while the other four genotoxic impurities showed normal peaks, but the peak positions of GTI-3 and GTI-4 overlapped.

[0053] Comparative Example 2 This comparative example provides a method for detecting genotoxic impurities in vildagliptin, with specific steps referenced in patent CN114441657A: (1) Solution preparation Reference solution: Take appropriate amounts of each impurity reference standard GTI-1, GTI-2, GTI-3, GTI-4, and GTI-5, accurately weigh them, dissolve them in acetonitrile, and quantitatively dilute them to prepare a solution containing 0.3 mg per mL as the reference standard stock solution. Accurately measure an appropriate amount of the above solution and dilute it with water to prepare a solution containing 30 ng per mL.

[0054] (2) Chromatographic conditions Chromatographic column: Octadecyl bonded silica gel as the packing material Mobile phase: 0.02 mol / L ammonium dihydrogen phosphate solution, pH adjusted to 3.5 with 2.5 mol / L phosphoric acid: acetonitrile = 65:35 Column temperature: 30℃ Injection volume: 20 μL Flow rate: 1.0 mL / min Detection wavelength: 230nm (3) Conclusion: GTI1, GTI2, GTI4 and GTI5 did not show peaks, while GTI3 showed a normal peak, but the response was low.

[0055] In summary, the embodiments of the present invention can achieve the separation and detection of impurities GTI1, GTI2, GTI3, GTI4, and GTI5 with high sensitivity. The conditions described in Comparative Example 1 cannot achieve the separation of GTI3 and GTI4, and cannot detect GTI5. Comparative Example 2 uses high performance liquid chromatography, which has poor detection sensitivity and cannot accurately quantify the impurities.

[0056] The applicant declares that this invention illustrates the method for detecting genotoxic impurities in vildagliptin and its application through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0057] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for detecting genotoxic impurities in vildagliptin, characterized in that, The detection method includes: using liquid chromatography-mass spectrometry to detect the sample to be tested, and obtaining the content of genotoxic impurities in the sample to be tested; The genotoxic impurities include (2S)-N-dichloroacetyl-2-cyanotetrahydropyrrole, and include any one or a combination of at least two of (2-chloroacetyl)-L-proline, 1-(chloroacetyl)-L-proline methyl ester, (2S)-N-chloroacetyl-2-cyanopyrrole, or dichloroacetic acid.

2. The detection method according to claim 1, characterized in that, The genotoxic impurities include (2S)-N-dichloroacetyl-2-cyanotetrahydropyrrole, (2-chloroacetyl)-L-proline, 1-(chloroacetyl)-L-proline methyl ester, (2S)-N-chloroacetyl-2-cyanopyrrole and dichloroacetic acid.

3. The method for detecting genotoxic impurities in vildagliptin according to claim 1 or 2, characterized in that, The mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B; The mobile phase A is an aqueous formic acid solution; The mobile phase B is acetonitrile.

4. The method for detecting genotoxic impurities in vildagliptin according to claim 3, characterized in that, The volume concentration of formic acid in the mobile phase A is 0.09%-0.11%.

5. The method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-4, characterized in that, The liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows: From 0 to 3 minutes, the volume percentage of mobile phase A is 83-87%, and the volume percentage of mobile phase B is 13-17%. After a period of uniform change until the 10th minute, the volume percentage of mobile phase A was 27-33%, and the volume percentage of mobile phase B was 67-73%. After a period of uniform change until the 11th minute, the volume percentage of mobile phase A was 83-87%, and the volume percentage of mobile phase B was 13-17%. After 11 minutes, the volume percentage of mobile phase A was 83-87%, and the volume percentage of mobile phase B was 13-17%.

6. The method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-5, characterized in that, The liquid chromatography column used is an octadecylsilane bonded column; Preferably, the chromatographic column used in the liquid chromatography has a particle size of 1.5-5 μm, a column length of 100-300 mm, and an inner diameter of 2-4.6 mm.

7. The method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-6, characterized in that, The column temperature of the liquid chromatography column is 30-40℃, preferably 33-37℃.

8. The method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-7, characterized in that... The injection flow rate of the liquid chromatography is 0.1-0.5 mL / min, preferably 0.2-0.4 mL / min.

9. The method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-8, characterized in that, The mass spectrometer is a triple quadrupole mass spectrometer. Preferably, the mass spectrometer is an ESI mass spectrometer; Preferably, the detection method is a simultaneous scanning of positive and negative ions.

10. The application of a method for detecting genotoxic impurities in vildagliptin according to any one of claims 1-9 in the quality testing of vildagliptin.

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