Method for detecting nitrosamine impurity in metformin vildagliptin preparation and application thereof

Abstract

The invention provides a detection method of nitrosamine impurities in a metformin vildagliptin preparation and application thereof, the detection method comprises the following steps: mixing a to-be-detected sample with a solvent to obtain a test solution, then carrying out liquid chromatography-mass spectrometry detection on the test solution, and determining the content of nitrosamine impurities in the metformin vildagliptin preparation according to the content of nitrosamine impurities in the metformin vildagliptin preparation. And determining the type and / or content of the nitrosamine impurity in the to-be-detected sample according to the detection result. The detection method provided by the invention can qualitatively and quantitatively detect various nitrosamine impurities in the metformin vildagliptin preparation at the same time, has the advantages of high impurity separation degree, good specificity, high precision, good repeatability and high sensitivity, is accurate and reliable, and can be widely applied to quality control of the metformin vildagliptin preparation.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting nitrosamine impurities in metformin-vildagliptin preparations and its application. Background Technology

[0002] Metformin and vildagliptin tablets are a combination preparation, consisting of metformin hydrochloride and vildagliptin, suitable for adult patients with type 2 diabetes. This product is used in conjunction with diet and exercise therapy for patients whose blood glucose is not well controlled after reaching the maximum tolerated dose of metformin monotherapy or who are currently receiving combination therapy with vildagliptin and metformin. Vildagliptin is a selective dipeptide-peptidase-4 (DPP-4) inhibitor. After administration, vildagliptin rapidly and completely inhibits DPP-4 activity, increasing the levels of fasting and postprandial endogenous intestinal glucagon-1 (GLP-1) and GIP (glucose-dependent insulinotropic peptide). Metformin is an antiglycemic agent that improves glucose tolerance in patients with type 2 diabetes, lowering both basal and postprandial blood glucose. It inhibits hepatic gluconeogenesis, reduces small intestinal glucose absorption, and improves peripheral tissue sensitivity to insulin by increasing glucose uptake and utilization in peripheral tissues.

[0003] N-nitrosamines (also known as nitrosamines) are a class of substances, many of which are potent mutagens and are classified as possible human carcinogens. Since June 2018, nitrosamine impurities have been a concern for the pharmaceutical industry and global regulatory agencies. Global regulatory agencies collaborate to share information and knowledge on nitrosamine issues, aiming to promote convergence on technical issues and reduce and mitigate patient exposure to harmful nitrosamine impurities in pharmaceutical products. N-nitrosamine impurities are present in metformin vildagliptin tablets and are impurities that affect drug efficacy; therefore, quantitative detection of these impurities is urgently needed for quality control of metformin vildagliptin tablets.

[0004] Chinese patent CN117820188A discloses a "nitrosamine impurity compound of vildagliptin and its preparation, application and detection method", and CN117820189A discloses a "vildagliptin with low nitrosamine impurity content and its purification method". Both invention patents control N-nitrosovildagliptin (CAS No.: 3034601-47-0), with CN117820188A providing a detailed study on the detection method of N-nitrosovildagliptin. However, these existing technologies cannot effectively control various nitrosamine impurities in vildagliptin, and in compound preparations, the difficulty of controlling nitrosamine impurities is further increased due to the interference of metformin.

[0005] Therefore, there is an urgent need to develop a method that can simultaneously and accurately detect multiple nitrosamine impurities in metformin vildagliptin tablets in order to further improve the quality of metformin vildagliptin tablets and ensure the safety of medication. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting nitrosamine impurities in metformin-vildagliptin preparations and its application. The detection method provided by this invention can simultaneously qualitatively and quantitatively detect multiple nitrosamine impurities in metformin-vildagliptin preparations, 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 control of metformin-vildagliptin preparations.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] On one hand, the present invention provides a method for detecting nitrosamine impurities in metformin-vildagliptin preparations, the method comprising the following steps:

[0009] The sample to be tested is mixed with a solvent to obtain a test solution. The test solution is then subjected to liquid chromatography-mass spectrometry for detection. The type and / or content of nitrosamine impurities in the sample to be tested are determined based on the detection results.

[0010] The nitrosamine impurity includes any one or a combination of at least two of impurities A, B, C, or D. The structures of impurities AD are shown below:

[0011]

[0012]

[0013] The detection method provided by this invention uses liquid chromatography and mass spectrometry. Liquid chromatography achieves good separation and qualitative analysis of each impurity, while mass spectrometry performs qualitative and quantitative analysis of each impurity. The detection method is accurate and reliable, and has the advantages of good specificity, high precision, good repeatability and high sensitivity. It can detect four nitrosamine impurities at the same time and can be widely used in the quality control of metformin and vildagliptin preparations.

[0014] Preferably, the nitrosamine impurity includes at least two combinations of impurity A, impurity B, impurity C, or impurity D.

[0015] Preferably, the nitrosamine impurities include impurity A, impurity B, impurity C, and impurity D.

[0016] Preferably, the solvent includes any one or a combination of at least two of water, acid, or organic solvents.

[0017] Preferably, the organic solvent includes methanol and / or acetonitrile.

[0018] Preferably, the acid includes formic acid.

[0019] Preferably, the solvent includes formic acid, acetonitrile, and water.

[0020] Preferably, the mobile phase in the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A includes an aqueous solution of formic acid, and mobile phase B includes a formic acid acetonitrile solution and / or a formic acid methanol solution.

[0021] Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%, such as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable, with 0.08-0.12% being preferred.

[0022] Preferably, the mobile phase B is a formic acid acetonitrile solution.

[0023] Preferably, the liquid chromatography employs gradient elution, and the gradient elution procedure is as follows:

[0024] From 0 to 5 minutes, the volume percentage of mobile phase A is 85-95%, and the volume percentage of mobile phase B is 5-15%.

[0025] From 5 to 25 minutes, the volume percentage of mobile phase A changes uniformly to 10-20%, and the volume percentage of mobile phase B changes uniformly to 80-90%.

[0026] Between 25 and 26 minutes, the volume percentage of mobile phase A changes uniformly to 85-95%, and the volume percentage of mobile phase B changes uniformly to 5-15%.

[0027] Between 26 and 30 minutes, the volume percentage of mobile phase A was 85-95%, and the volume percentage of mobile phase B was 5-15%.

[0028] Preferably, the liquid chromatography uses an octadecylsilane bonded silica gel column, and more preferably an ACE Excel 3Super C18 column.

[0029] Preferably, the column temperature of the liquid chromatography is 30-40℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, but not limited to the values ​​listed above. Other values ​​not listed above are also applicable, but 30-35℃ is preferred.

[0030] Preferably, the injection flow rate of the liquid chromatography is 0.5-0.7 mL / min, such as 0.5 mL / min, 0.55 mL / min, 0.6 mL / min, 0.65 mL / min or 0.7 mL / min, but is not limited to the values ​​listed above. Other values ​​not listed in the above range are also applicable.

[0031] Preferably, the mass spectrometer is a triple quadrupole mass spectrometer.

[0032] Preferably, the mass spectrometer is an ESI mass spectrometer or an APCI mass spectrometer, more preferably an ESI mass spectrometer.

[0033] Preferably, the mass spectrometer scan type is SIM, SRM, or MRM, with MRM being the preferred method.

[0034] Preferably, the quantitative ion pair of impurity A is 333 / 151, and the qualitative ion pair is 333 / 180.

[0035] Preferably, the quantitative ion pair of impurity B is 351 / 70, and the qualitative ion pair is 351 / 115.

[0036] Preferably, the quantitative ion pair of the impurity C is 352 / 70, and the qualitative ion pair is 352 / 180.

[0037] Preferably, the quantitative ion pair of the impurity D is 317 / 93, and the qualitative ion pair is 317 / 164.

[0038] The selection of the specific parameters and reagents mentioned above can effectively improve the detection results, and enhance the specificity, precision, repeatability and sensitivity of the detection.

[0039] On the other hand, the present invention also provides the application of the detection method in the quality control of metformin vildagliptin preparations.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention provides a method for detecting nitrosamine impurities in metformin vildagliptin preparations. By using liquid chromatography coupled with mass spectrometry, liquid chromatography achieves good separation and qualitative identification of each impurity, while mass spectrometry performs qualitative and quantitative analysis of each impurity. The detection method is accurate and reliable, with advantages such as good specificity, high precision, good repeatability, and high sensitivity. It can simultaneously detect four nitrosamine impurities and can be widely used in the quality control of metformin vildagliptin preparations. Attached Figure Description

[0042] Figure 1 This is the mass spectrum obtained using the ACE Excel 3Super C18 column in Example 2;

[0043] Figure 2 This is the mass spectrum obtained using the ACQUITY UPLC HSS T3 column in Example 2;

[0044] Figure 3 This is the mass spectrum of the mobile phase in group 2 used in Example 3;

[0045] Figure 4 This is the HPLC chromatogram of the spiked test solution in Example 6. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0047] Example 1

[0048] This embodiment provides a method for detecting nitrosamine impurities in metformin vildagliptin tablets, including the following steps:

[0049] (1) Solution preparation

[0050] Blank solvent: volume ratio of formic acid:acetonitrile:water = 0.1:30:70;

[0051] Test solution: Take an appropriate amount of metformin vildagliptin tablet powder (equivalent to 25mg vildagliptin), accurately weigh it, place it in a 10mL volumetric flask, add blank solvent to dissolve and dilute to the mark, shake well, centrifuge, and take the supernatant to obtain the test solution;

[0052] Reference solutions: Accurately weigh approximately 3 mg each of impurity A, impurity B, impurity C, and impurity D reference standards, place them in separate 20 mL volumetric flasks, dissolve and dilute to the mark with solvent, and shake well to obtain impurity stock solutions; accurately measure 1 mL of each impurity stock solution into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well, then accurately measure another 1 mL into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the mixed reference standard stock solution; accurately measure 1 mL of the mixed reference standard stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution; prepare two parallel solutions.

[0053] (2) Liquid chromatography conditions:

[0054] Mobile phase A: 0.1% formic acid aqueous solution;

[0055] Mobile phase B: 0.1% formic acid acetonitrile solution;

[0056] Flow rate: 0.6 mL / min;

[0057] Column: ACE Excel 3Super C18, 4.6mm × 250mm, 3μm;

[0058] Injection volume: 20 μL;

[0059] Column temperature: 35℃;

[0060] Gradient elution procedure:

[0061]

[0062]

[0063] (3) Mass spectrometry conditions

[0064]

[0065] (4) Using the above liquid chromatography and mass spectrometry conditions, the solution obtained in step (1) was detected by liquid chromatography-mass spectrometry.

[0066] (5) Sample test results and conclusions

[0067] Impurity identification: The elution order of the reference solution was impurity B, impurity C, impurity A, and impurity D. Furthermore, the resolution R between each peak was controlled above 2.5, significantly higher than 1.5, indicating complete separation.

[0068] Impurity quantification: Calculated by peak area using the external standard method, the content of impurity A in the test solution was 0.15 ppm, the content of impurity B was 0.05 ppm, and impurities C and D were not detected.

[0069]

[0070] Example 2: Screening of chromatographic columns

[0071] This embodiment involves screening the chromatographic column type for liquid chromatography, specifically including:

[0072] Based on Example 1, the detection results of changing the chromatographic column type are shown in the table below:

[0073]

[0074] The retention times of the control solution in group 1 (six replicates) are shown in the table below:

[0075]

[0076] Conclusion: Using column 1, the four nitrosamine impurities were well separated, while using column 2, impurity A showed bifurcation. Therefore, the separation effect obtained using column 2 is superior to that using column 1. When continuously detecting the impurity reference solution using column 1, the maximum RSD of the retention time for each nitrosamine impurity was 0.89%, lower than 1.0%, demonstrating good method precision.

[0077] Example 3: Determination of the chromatographic mobile phase

[0078] This embodiment compares different mobile phases in gradient elution in high performance liquid chromatography, specifically including:

[0079] (1) Based on the detection conditions of Example 1, the type of mobile phase was changed according to the conditions described below, while other conditions remained unchanged, and the reference solution was detected:

[0080] Group 1: Same mobile phase as in Example 1;

[0081] Group 2: The only difference from the mobile phase in Example 1 is that the acetonitrile in mobile phase B is replaced with methanol;

[0082] (2) Experimental procedures and conclusions

[0083] The corresponding detection results using the mobile phases of Group 1 and Group 2 are shown in the table below:

[0084]

[0085] Conclusion: When using a 0.1% formic acid aqueous solution-0.1% formic acid methanol solution as the mobile phase system to detect the mixed impurity solution, the peaks of each impurity were late and the resolution was worse than in Example 1. When using a 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution as the mobile phase system, the peak elution time and resolution were both better.

[0086] Example 4: Determination of Column Temperature Range

[0087] This embodiment compares column temperatures in high-performance liquid chromatography, specifically including:

[0088] (1) Based on Example 1, the column temperature of the chromatographic column was changed to 30℃, 35℃ and 40℃ respectively, while other conditions remained unchanged, and the content of each nitrosamine impurity in the spiked test solution was detected.

[0089] (2) Experimental procedures and conclusions

[0090] The detection results at different column temperatures are shown in the table below:

[0091]

[0092] Conclusion: When the column temperature is 40℃, the RD value of impurity B is greater than 15.0% compared with that at 35℃. When the column temperature is between 30-35℃, the RD values ​​of all impurities are less than 15.0% compared with that at 35℃.

[0093] Example 5: Determination of chromatographic conditions and flow rate

[0094] This embodiment compares the injection flow rate in high-performance liquid chromatography (HPLC) detection, specifically including:

[0095] (1) Based on Example 1, the injection flow rate was changed to 0.5 mL / min, 0.6 mL / min and 0.7 mL / min, while other conditions remained unchanged, and the content of each nitrosamine impurity in the spiked test solution was detected;

[0096] (2) Experimental procedures and conclusions

[0097] The detection results for different injection flow rates are shown in the table below:

[0098]

[0099] Conclusion: When the flow rate is 0.7 mL / min, the RD value of impurity A is greater than 15.0% compared with 0.6 mL / min. When the flow rate is between 0.5 and 0.6 mL / min, the RD values ​​of all impurities are less than 15.0% compared with 0.6 mL / min.

[0100] Example 6 Specificity Test

[0101] (1) Liquid chromatography conditions:

[0102] Mobile phase A: 0.1% formic acid aqueous solution;

[0103] Mobile phase B: 0.1% formic acid acetonitrile solution;

[0104] Flow rate: 0.6 mL / min;

[0105] Column: ACE Excel 3Super C18, 4.6mm × 250mm, 3μm;

[0106] Injection volume: 20 μL;

[0107] Column temperature: 35℃;

[0108] Gradient elution procedure:

[0109] Time (min) Mobile phase B (vt%) Mobile phase A (vt%) 0 90 10 5 90 10 25 15 85 26 90 10 30 90 10

[0110] (2) Mass spectrometry conditions

[0111]

[0112] (3) Test methods

[0113] Blank solvent: volume ratio of formic acid:acetonitrile:water = 0.1:30:70;

[0114] Test solution: Take an appropriate amount of metformin vildagliptin tablet powder (equivalent to 25mg vildagliptin), accurately weigh it, place it in a 10mL volumetric flask, add blank solvent to dissolve and dilute to the mark, shake well, centrifuge, and take the supernatant to obtain the test solution;

[0115] Reference solutions: Accurately weigh approximately 3 mg each of impurity A, impurity B, impurity C, and impurity D reference standards, place them in separate 20 mL volumetric flasks, dissolve and dilute to the mark with solvent, and shake well to obtain impurity stock solutions; accurately measure 1 mL of each impurity stock solution into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well, then accurately measure another 1 mL into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the mixed reference standard stock solution; accurately measure 1 mL of the mixed reference standard stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution; prepare two parallel solutions.

[0116] Blank excipient solution: Accurately weigh approximately 57 mg of blank excipient, place it in a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and filter to obtain the solution.

[0117] Metformin hydrochloride solution: Accurately weigh approximately 425 mg of metformin hydrochloride, place it in a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and filter to obtain the solution.

[0118] Spiked test solution: Weigh an appropriate amount of the fine powder of this product (approximately equivalent to 25 mg of vildagliptin) accurately, place it in a 10 mL volumetric flask, accurately add 1 mL of the mixed stock solution of the reference standard, add solvent to dissolve and dilute to the mark, shake well, centrifuge, and take the supernatant to obtain the test solution.

[0119] The test results are shown in the table below:

[0120]

[0121] Conclusion: Blank solvent, blank excipient, and metformin hydrochloride do not interfere with the detection of various nitrosamine impurities, and other peaks in the test sample do not interfere with the detection of various nitrosamine impurities, indicating that the detection method of the present invention has good specificity.

[0122] Example 7 Sensitivity Test

[0123] Test method:

[0124] Blank solvent: volume ratio of formic acid:acetonitrile:water = 0.1:30:70;

[0125] 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 the limit of detection of the method of the present invention.

[0126] The chromatographic and mass spectrometry conditions are the same as in Example 1.

[0127] The test results are shown in the table below:

[0128] name Limit of Quantification (ppm) Detection limit (ppm) Impurity A 0.30 0.10 Impurity B 0.04 0.01 Impurity C 0.04 0.01 Impurity D 0.03 0.01

[0129] Conclusion: The limits of quantitation for impurities A, B, C, and D are 0.30, 0.04, 0.04, and 0.03 ppm, respectively, and the limits of detection are 0.10, 0.01, 0.01, and 0.01 ppm, respectively. The sensitivity of this method is higher than that of high performance liquid chromatography.

[0130] Example 8 Repeatability Test

[0131] Blank solvent: volume ratio of formic acid:acetonitrile:water = 0.1:30:70;

[0132] Reference solutions: Accurately weigh approximately 3 mg each of impurity A, impurity B, impurity C, and impurity D reference standards, place them in separate 20 mL volumetric flasks, dissolve and dilute to the mark with solvent, and shake well to obtain impurity stock solutions; accurately measure 1 mL of each impurity stock solution into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well, then accurately measure another 1 mL into a 20 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the mixed reference standard stock solution; accurately measure 1 mL of the mixed reference standard stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution; prepare two parallel solutions.

[0133] Repeatability test solution: Accurately weigh an appropriate amount of metformin-vildagliptin tablet powder (approximately equivalent to 25 mg of vildagliptin), place it in a 10 mL volumetric flask, accurately add 1 mL of the reference standard stock solution, dissolve and dilute to the mark with solvent, shake well, centrifuge, and take the supernatant. Prepare 6 parallel aliquots; label them as repeatability-1, repeatability-2, repeatability-3, repeatability-4, repeatability-5, and repeatability-6.

[0134] The chromatographic and mass spectrometry conditions are the same as in Example 1.

[0135] The test results are shown in the table below:

[0136] sample Impurities D / ppm Impurity A / ppm Impurity B / ppm Impurity C / ppm Repeatability 1 15.2 13.7 16.0 19.1 Repeatability 2 14.5 13.1 16.0 19.4 Repeatability 3 14.3 13.4 16.1 19.1 Repeatability 4 14.2 13.9 15.9 18.8 Repeatability 5 13.9 13.2 15.9 18.7 Repeatability 6 14.0 13.3 15.7 18.8 RSD% (n=6) 3.3 2.3 0.86 1.4

[0137] Conclusion: The maximum RSD of impurity content in the 6 reproducible solutions was 3.3%, all less than 5.0%, indicating that the method of the present invention has good reproducibility.

[0138] Example 9 Accuracy Test

[0139] Blank solvent: volume ratio of formic acid:acetonitrile:water = 0.1:30:70;

[0140] Background solution: Take the test sample solution described in Example 1 as the background solution;

[0141] Impurity stock solution: Accurately weigh approximately 3 mg each of impurity A, impurity B, impurity C, and impurity D reference standards, place them in different 20 mL volumetric flasks, dissolve and dilute to the mark with solvent, and shake well to obtain the impurity stock solution.

[0142] Accuracy stock solution: Accurately measure 1 mL of each impurity stock solution into a 20 mL volumetric flask, dilute with solvent to the mark, shake well, then accurately measure another 1 mL into a 20 mL volumetric flask, dilute with solvent to the mark, shake well, and the solution is ready.

[0143] 50% recovery solution: Take an appropriate amount of metformin vildagliptin tablet powder (approximately equivalent to 25 mg of vildagliptin), accurately weigh it, place it in a 10 mL volumetric flask, accurately add 0.5 mL of accuracy stock solution, dissolve and dilute to the mark with solvent, shake well, centrifuge, and take the supernatant to obtain the solution. Prepare 3 parallel portions.

[0144] 100% recovery solution: Take an appropriate amount of metformin vildagliptin tablet powder (approximately equivalent to 25 mg of vildagliptin), accurately weigh it, place it in a 10 mL volumetric flask, accurately add 1.0 mL of accuracy stock solution, dissolve and dilute to the mark with solvent, shake well, centrifuge, and take the supernatant to obtain the solution. Prepare 3 parallel portions.

[0145] 150% recovery solution: Take an appropriate amount of metformin vildagliptin tablet powder (approximately equivalent to 25 mg of vildagliptin), accurately weigh it, place it in a 10 mL volumetric flask, accurately add 1.5 mL of accuracy stock solution, dissolve and dilute to the mark with solvent, shake well, centrifuge, and take the supernatant. Prepare 3 parallel portions.

[0146] The chromatographic and mass spectrometry conditions are the same as in Example 1.

[0147] The test results are shown in the table below:

[0148] Impurity A

[0149]

[0150] Impurity B

[0151]

[0152]

[0153] Impurity C

[0154]

[0155] Impurity D

[0156]

[0157] Conclusion: At the limit concentrations of 50%, 100%, and 150%, the recoveries of impurity A, B, C, and D were calculated using the external standard method at three concentrations of 50%, 100%, and 150% for the nine samples. The recoveries were 75.7%–98.8%, 102.9%–110.6%, 117.6%–124.0%, and 81.8%–90.7%, all within the range of 70%–125%. The maximum RSD for the recoveries of each impurity in the nine samples was 10.4%, all less than 15.0%, which meets the requirements and indicates good method accuracy.

[0158] Comparative Example 1

[0159] The embodiments of the present invention were examined with reference to the detection conditions and methods described in CN117820188A. The method of Comparative Example 1 differs from that of Example 1 in that:

[0160] The chromatographic conditions of this invention are compared with those of the prior art in the table below:

[0161]

[0162] Comparative Example 1 Mass spectrometry conditions: Ion source: ESI+; Scan type: MRM; Electron multiplier voltage: 650V; Drying gas temperature: 300℃; Nebulizer gas pressure: 45psi; Sheath gas flow rate: 11L / min; Capillary voltage: 3500V; Nozzle voltage: 500V.

[0163] The tests were conducted using the conditions described in Comparative Example 1 and Example 1:

[0164] Resolution results:

[0165]

[0166] Conclusion: In the detection method of Comparative Example 1, impurities B and C could not be baseline separated, with a resolution of only 0.5, and impurity D did not show a peak. In this invention, the lowest resolution is 2.5. Compared with the detection method of Comparative Example 1, the detection capability and resolution of this method are significantly improved.

[0167] The applicant declares that this invention illustrates the method for detecting nitrosamine impurities in metformin-vildagliptin preparations and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0168] 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.

[0169] 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 nitrosamine impurities in metformin-vildagliptin preparations, characterized in that, The detection method includes the following steps: The sample to be tested is mixed with a solvent to obtain a test solution. The test solution is then subjected to liquid chromatography-mass spectrometry for detection. The type and / or content of nitrosamine impurities in the sample to be tested are determined based on the detection results. The nitrosamine impurity includes any one or a combination of at least two of impurities A, B, C, or D. The structures of impurities AD are shown below:

2. The detection method according to claim 1, characterized in that, The nitrosamine impurity includes at least two combinations of impurity A, impurity B, impurity C, or impurity D; Preferably, the nitrosamine impurities include impurity A, impurity B, impurity C, and impurity D.

3. The detection method according to claim 1 or 2, characterized in that, The solvent includes any one or a combination of at least two of water, acid, or organic solvents; Preferably, the organic solvent includes methanol and / or acetonitrile; Preferably, the acid includes formic acid; Preferably, the solvent includes formic acid, acetonitrile, and water.

4. The detection method according to any one of claims 1-3, characterized in that, The mobile phase in the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A includes an aqueous solution of formic acid and mobile phase B includes an acetonitrile solution of formic acid and / or a methanol solution of formic acid. Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%, more preferably 0.08-0.12%; Preferably, the mobile phase B is a formic acid acetonitrile solution.

5. The detection method according to claim 4, characterized in that, The liquid chromatography employs gradient elution, and the gradient elution procedure is as follows: From 0 to 5 minutes, the volume percentage of mobile phase A is 85-95%, and the volume percentage of mobile phase B is 5-15%. From 5 to 25 minutes, the volume percentage of mobile phase A changes uniformly to 10-20%, and the volume percentage of mobile phase B changes uniformly to 80-90%. Between 25 and 26 minutes, the volume percentage of mobile phase A changes uniformly to 85-95%, and the volume percentage of mobile phase B changes uniformly to 5-15%. Between 26 and 30 minutes, the volume percentage of mobile phase A was 85-95%, and the volume percentage of mobile phase B was 5-15%.

6. The detection method according to any one of claims 1-5, characterized in that, The liquid chromatography uses an octadecylsilane bonded silica gel column, preferably an ACE Excel 3Super C18 column.

7. The detection method according to any one of claims 1-6, characterized in that, The column temperature of the liquid chromatography is 30-40℃, preferably 30-35℃; Preferably, the injection flow rate of the liquid chromatography is 0.5-0.7 mL / min.

8. The detection method according to any one of claims 1-7, characterized in that, The mass spectrometer is a triple quadrupole mass spectrometer. Preferably, the mass spectrometer is an ESI mass spectrometer or an APCI mass spectrometer, more preferably an ESI mass spectrometer; Preferably, the mass spectrometer scan type is SIM, SRM, or MRM, with MRM being the preferred method.

9. The detection method according to any one of claims 1-8, characterized in that, The quantitative ion pair of impurity A is 333 / 151, and the qualitative ion pair is 333 / 180. Preferably, the quantitative ion pair of impurity B is 351 / 70, and the qualitative ion pair is 351 / 115; Preferably, the quantitative ion pair of the impurity C is 352 / 70, and the qualitative ion pair is 352 / 180; Preferably, the quantitative ion pair of the impurity D is 317 / 93, and the qualitative ion pair is 317 / 164.

10. The application of a detection method according to any one of claims 1-9 in the quality control of metformin-vildagliptin preparations.

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