A method for detecting genotoxic impurities of vildagliptin

Abstract

The invention discloses a method for detecting genotoxic impurities of vildagliptin, belongs to the field of chemical analysis, adopts a high performance liquid chromatography tandem mass spectrometer to detect the genotoxic impurities of vildagliptin, and can accurately detect the genotoxic impurities in vildagliptin through the chromatographic detection conditions and mass spectrometric detection conditions of the high performance liquid chromatography tandem mass spectrometer. The detection method has high sensitivity and good separation degree.

Description

Technical Field

[0001] The invention relates to a method for detecting chemical substances, in particular to a method for detecting genotoxic impurities in vildagliptin, and belongs to the field of chemical analysis. Background Art

[0002] Vildagliptin is a selective, competitive, and reversible DPP24 inhibitor. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like polypeptide 21 (GLP21) are important hormones for maintaining glucose concentrations in the body, and both have incretin effects. In patients with type 2 diabetes, the insulinotropic effect of GIP is impaired, and only GLP21 can exert an insulinotropic effect. It promotes insulin secretion by acting on receptors on the pancreatic β-cell membrane. GLP21 can also inhibit the secretion of glucagon and inhibit gastric emptying, thereby increasing satiety (suppressing appetite). DPP24 exists in many tissues bound to proteins, such as the kidney, liver, brush edge of the small intestinal membrane, pancreatic ducts, lymphocytes, and endothelial cells. It can rapidly inactivate GLP21 by hydrolyzing the second alanine at the N-terminus. Vildagliptin inhibits the activity of the enzyme by binding to DPP24 to form a DPP24 complex, increasing the concentration of GLP21 and prompting pancreatic beta cells to produce insulin while reducing the concentration of glucagon, thereby lowering blood sugar, with no significant effect on body weight.

[0003] The chemical formula of vildagliptin is (-)-(2S)-1-[[(3-hydroxytricyclo[3.3.1.1[3,7]]silan-1-yl)amino]acetyl]pyrrolidine-2-carbonitrile. Genotoxic impurities may be present in the synthetic production process, mainly (S)-1-chloroacetylpyrrolidine-2-carboxamide, (S)-1-chloroacetylpyrrolidine-2-carbonitrile, (2-chloroacetyl)-1-proline, (2-chloroacetyl)-1-proline methyl ester and other genotoxic impurities that may affect the quality of the product. Therefore, it is necessary to detect the genotoxic impurities in vildagliptin.

[0004] Existing patent CN114441657A discloses a method for detecting genotoxic impurities in vildagliptin, which mainly detects one or a combination of (S)-1-(2-chloroacetyl)pyrrolidine-2-carboxamide and (2S)-N-chloroacetyl-2-cyanopyrrole. At present, the detection methods for various genotoxic impurities in vildagliptin have not been able to accurately and sensitively detect the genotoxic impurities (S)-1-chloroacetylpyrrolidine-2-carboxamide, (S)-1-chloroacetylpyrrolidine-2-carbonitrile, (2-chloroacetyl)-1-proline, and (2-chloroacetyl)-1-proline methyl ester in vildagliptin. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting genotoxic impurities of vildagliptin, using a high performance liquid chromatography tandem mass spectrometer to detect genotoxic impurities of vildagliptin. The method of the present invention can accurately detect four genotoxic impurities in vildagliptin with high sensitivity and good separation.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for detecting genotoxic impurities of vildagliptin adopts a high performance liquid chromatography tandem mass spectrometer to detect the genotoxic impurities of vildagliptin.

[0008] Furthermore, genotoxic impurities include (S)-1-chloroacetylpyrrolidine-2-carboxamide (referred to as impurity J), (S)-1-chloroacetylpyrrolidine-2-carbonitrile (referred to as impurity H), (2-chloroacetyl)-1-proline (referred to as impurity I), and (2-chloroacetyl)-1-proline methyl ester (referred to as impurity K).

[0009] Furthermore, the chromatographic conditions include: the chromatographic column is Agilent ZORBAX SB-Aq 3.5μm3.0×100mm, the column temperature is 40°C, the flow rate is 0.3ml / min, the injection volume is 5μL, the mobile phase A is 5MM ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the elution is gradient.

[0010] Further, the gradient elution program was 0 min, 90% A, 10% B; 2 min, 90% A, 10% B; 6 min, 25% A, 75% B; 9 min, 25% A, 75% B; 10 min, 90% A, 10% B; 12 min, 90% A, 10% B.

[0011] Furthermore, the following mass spectrometry conditions are included: the ion source is an ESI source, the temperature is 600° C., the curtain gas is 35 psi, the capillary voltage is 5500 V, the spray gas is 50 psi, the auxiliary heating gas is 45 psi, and the scanning mode is positive ion scanning.

[0012] Further, in the positive ion scanning mode, the detection ion pairs m / z of the compound 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.

[0013] Furthermore, the following steps are included:

[0014] S1. Preparation of reference substance solution: (1) Preparation of stock solution: Accurately weigh 0.01g of reference substance and place it in a 10ml brown volumetric flask, dilute to the mark with methanol, and prepare a stock solution of about 1000mg / L for use; (2) Preparation of mixed intermediate solution: Intermediate solution 1: Take 0.1ml of the above four stock solutions and place them in a 10ml volumetric flask, dilute to the mark with solvent, and the concentration of intermediate solution 1 is 10mg / L for use; Intermediate solution 2: Take 1ml of intermediate solution 1 and place it in a 10ml volumetric flask, dilute to the mark with solvent, and the concentration of intermediate solution 2 is 1mg / L. Intermediate solution 2 is the reference substance solution;

[0015] S2. Sample solution: weigh 0.1 g of sample, dissolve it with solvent, transfer it to a 10 ml volumetric flask, dilute to the mark with solvent, and take 1 ml for testing;

[0016] S3. The reference solution and the sample solution are respectively detected by high performance liquid chromatography tandem mass spectrometry to obtain the chromatogram data of the reference solution and the sample solution, and the amount of genotoxic impurities in the sample is calculated.

[0017] Furthermore, step S1 includes the preparation of a mixed standard working solution: 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 300 μL of the intermediate solution 2 are taken respectively and the volume is fixed to 1 ml with a solvent, and diluted into standard working solutions with concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, and 300 μg / L.

[0018] Furthermore, in step S1, the stock solution and the intermediate solution 1 are stored at 4°C, and the intermediate solution 2 is prepared and used immediately.

[0019] Furthermore, the solvent is a mixture of methanol and water, and the volume ratio of methanol to water is 1:9.

[0020] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0021] The method of the present invention can quickly and accurately detect the genotoxic impurities of vildagliptin, is time-saving, highly sensitive and has good separation.

[0022] First, the present invention uses a high performance liquid chromatography tandem mass spectrometer to detect genotoxic impurities in vildagliptin with high sensitivity, baseline separation and good resolution. If high performance liquid chromatography or other methods are used for determination and analysis, the sensitivity is low and the chromatogram cannot be detected, and the method is not applicable.

[0023] Secondly, the present invention can better separate and detect compound impurities H, impurity J, impurity I and impurity K in the sample by limiting the gradient elution program in the spectral conditions, and obtain corresponding chromatograms with baseline separation and good separation degree; if the gradient elution program of other chromatographic conditions is adopted, the compounds in the sample cannot be separated well, and the compound chromatogram obtained by detection has some baselines not separated and the separation degree is poor.

[0024] Finally, the present invention can accurately detect genotoxic impurities by limiting mass spectrometry conditions, and the obtained chromatogram is clear, baseline-separated, and has good separation. If other mass spectrometry conditions are used, genotoxic impurities cannot be accurately detected, interference exists in the obtained chromatogram, and the baseline is not separated, and the separation is poor. Among them, the detection ion pairs in the mass spectrometry conditions are further limited. By using the detection ion pairs of the present invention, the corresponding compounds can be accurately detected, and the chromatograms can be obtained with baseline separation and good separation. If other detection ion pairs are used, only other compounds can be monitored, and the genotoxic impurities of the present application cannot be accurately detected, and the corresponding chromatograms can be obtained with baseline separation and good separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 When detecting impurity H, the chromatogram (impurity H) of the sample solution of the present invention;

[0027] Figure 2 When detecting impurity J, the chromatogram (impurity J) of the sample solution of the present invention;

[0028] Figure 3 When detecting impurity I, the chromatogram (impurity I) of the sample solution of the present invention;

[0029] Figure 4 When detecting impurity K, the chromatogram (impurity K) of the sample solution of the present invention;

[0030] Figure 5 It is the chromatogram of impurity H of the reference substance solution of the present invention;

[0031] Figure 6 It is the impurity J chromatogram of the reference substance solution of the present invention;

[0032] Figure 7 It is the impurity I chromatogram of the reference substance solution of the present invention;

[0033] Figure 8 It is the impurity K chromatogram of the reference substance solution of the present invention;

[0034] Fig. 9 It is the blank solution chromatogram of the impurity H process in the specific test of the present invention;

[0035] Fig.10 It is the blank solution chromatogram of the impurity J process of the specific test of the present invention;

[0036] Fig.11 It is the blank solution chromatogram of the impurity I process of the specific test of the present invention;

[0037] Fig.12 It is the blank solution chromatogram of the impurity K process in the specific test of the present invention;

[0038] Fig.13 It is a chromatogram of a spiked sample solution of impurity H in the specific test of the present invention;

[0039] Fig.14 It is a chromatogram of a spiked sample solution of impurity J in the specific test of the present invention;

[0040] Fig.15 It is a chromatogram of a spiked sample solution of impurity I in the specific test of the present invention;

[0041] Fig.16 It is a chromatogram of a spiked sample solution of impurity K in the specific test of the present invention;

[0042] Fig.17 It is a chromatogram of the detection limit test impurity H added at a concentration level of 0.35 mg / kg in the present invention;

[0043] Fig.18 This is the chromatogram of the detection limit test impurity J added at a concentration level of 0.35 mg / kg in the present invention;

[0044] Fig.19 This is a chromatogram of the detection limit test impurity I added at a concentration level of 0.35 mg / kg in the present invention;

[0045] Fig. 20 This is a chromatogram of the detection limit test impurity K added at a concentration level of 0.35 mg / kg in the present invention;

[0046] Fig.21 is a chromatogram of the impurity H added at a concentration level of 1.0 mg / kg in the quantitative limit test of the present invention;

[0047] Fig. 22 is a chromatogram of the quantitative limit test impurity J added at a concentration level of 1.0 mg / kg in the present invention;

[0048] Fig.23 is a chromatogram of the impurity I added at a concentration level of 1.0 mg / kg in the quantitative limit test of the present invention;

[0049] Fig.24 is a chromatogram of the impurity K added at a concentration level of 1.0 mg / kg in the quantitative limit test of the present invention;

[0050] Fig.25 It is the linear curve diagram of impurity H of the present invention;

[0051] Fig.26 It is the linear curve graph of impurity J of the present invention;

[0052] Fig. 27 is a linear curve graph of impurity I of the present invention;

[0053] Fig.28 It is the impurity K linear curve diagram of the present invention;

[0054] In the accompanying drawings, Figure 1-24 The vertical coordinate Y is Intensity, cps; e4 represents *10 4 , e5 represents *10 5 , e 6 Representative*10 6 ; The horizontal axis X is Time, min; Figure 25-28 The ordinate Y is the peak area response value, and the abscissa X is the concentration, ng / ml. DETAILED DESCRIPTION

[0055] Below is a combination of the embodiments and Figures 1 to 28 The present invention is described in further detail:

[0056] A method for detecting genotoxic impurities of vildagliptin is disclosed, wherein a high performance liquid chromatography tandem mass spectrometer is used to detect genotoxic impurities of vildagliptin. The genotoxic impurities of vildagliptin include (S)-1-chloroacetylpyrrolidine-2-carboxamide (referred to as impurity J), (S)-1-chloroacetylpyrrolidine-2-carbonitrile (referred to as impurity H), (2-chloroacetyl)-1-proline (referred to as impurity I), and (2-chloroacetyl)-1-proline methyl ester (referred to as impurity K).

[0057] The test conditions are as follows:

[0058] Chromatographic conditions:

[0059] Chromatographic column: Agilent ZORBAX SB-Aq 3.5 μm 3.0 × 100 mm;

[0060] Column temperature: 40°C;

[0061] Flow rate: 0.3ml / min;

[0062] Injection volume: 5 μL;

[0063] Mobile phase A: 5MM ammonium acetate aqueous solution;

[0064] Mobile phase B: acetonitrile;

[0065] Gradient elution: 0 min, 90% A, 10% B; 2 min, 90% A, 10% B; 6 min, 25% A, 75% B; 9 min, 25% A, 75% B; 10 min, 90% A, 10% B; 12 min, 90% A, 10% B;

[0066] Mass spectrometry conditions:

[0067] Ion source: ESI source;

[0068] Ion source conditions:

[0069] Temperature: 600℃;

[0070] Air curtain gas: 35psi;

[0071] Capillary voltage: 5500V;

[0072] Spray gas: 50psi;

[0073] Auxiliary heating gas: 45psi;

[0074] Scanning mode: positive ion scanning;

[0075]

[0076]

[0077] Note: * indicates quantitative ions

[0078] The steps are as follows:

[0079] S1. Preparation of reference solution and mixed standard working solution:

[0080] (1) Preparation of stock solutions: Accurately weigh 0.01 g (accurate to 0.00001) of each reference substance of Impurity H, Impurity J, Impurity I and Impurity K and place it in a 10 ml brown volumetric flask. Add methanol to the mark to prepare approximately 1000 mg / L stock solutions of Impurity H, Impurity J, Impurity I and Impurity K. Store at 4°C for future use.

[0081] (2) Preparation of mixed intermediate solution:

[0082] Intermediate solution 1: Take 0.1 ml of each of the above four stock solutions, place them in a 10 ml volumetric flask, and dilute to the mark with methanol-water (1:9). The concentration of intermediate solution 1 is 10 mg / L. Store at 4°C for later use.

[0083] Intermediate solution 2: Take 1 ml of intermediate solution 1, place it in a 10 ml volumetric flask, and dilute to the mark with methanol-water (1:9). The concentration of intermediate solution 2 is 1 mg / L. Intermediate solution 2 is the reference solution. Intermediate solution 2 should be prepared and used immediately.

[0084] (3) Preparation of mixed standard working solution: Take 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 300 μL of intermediate solution 2 respectively, dilute to 1 ml with methanol-water (1:9), and dilute to standard working solutions with concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, and 300 μg / L.

[0085] S2. Sample solution: Accurately weigh 0.1 g (accurate to 0.0001 g) of vildagliptin sample, dissolve it in methanol-water (1:9), transfer it to a 10 ml volumetric flask, make up to the mark with methanol-water (1:9), and take 1 ml for testing.

[0086] S3. The reference solution and the sample solution are respectively detected by high performance liquid chromatography tandem mass spectrometry to obtain the chromatogram data of the reference solution and the sample solution, and the amount of genotoxic impurities in the sample is calculated.

[0087] Example 1

[0088] The specific implementation process of this embodiment 1 is as follows:

[0089] 1. Instruments, equipment, reagents and reference substances

[0090] 1.1 Instruments and Equipment

[0091] (1) High performance liquid chromatography tandem mass spectrometer: AB5500: ABSciex

[0092] (2) Electronic balance: Model BT25S, sensitivity 0.00001 g, Sartorius Scientific Instruments Co., Ltd.; Model BSA224S, sensitivity 0.0001 g, Sartorius Scientific Instruments Co., Ltd.;

[0093] 1.2 Reagents: Methanol is chromatographically pure, water is ultrapure water: resistivity is 18.25MΩ*cm

[0094] 1.3 Reference substances

[0095] Table 1 Reference material information

[0096]

[0097] 2. Experimental steps

[0098] 2.1 Preparation of reference solution and mixed standard working solution

[0099] 2.1.1 Preparation of stock solutions: Use an electronic balance to accurately weigh 0.01 g (accurate to 0.00001) of reference substances of impurity H, impurity J, impurity I and impurity K and place them in a 10 ml brown volumetric flask. Add methanol to make dilution to the mark to prepare about 1000 mg / L stock solutions of impurity H, impurity J, impurity I and impurity K. Store at 4 °C for future use.

[0100] 2.1.2 Preparation of mixed intermediate solution:

[0101] (1) Intermediate solution 1: Take 0.1 ml of each of the four stock solutions mentioned above, place them in a 10 ml volumetric flask, and dilute to the mark with methanol-water (1:9). The concentration of intermediate solution 1 is 10 mg / L. Store at 4°C for later use.

[0102] (2) Intermediate solution 2: Take 1 ml of intermediate solution 1 and place it in a 10 ml volumetric flask. Add methanol-water (1:9) to make up to the mark. The concentration of intermediate solution 2 is 1 mg / L. Intermediate solution 2 is the reference solution. Intermediate solution 2 should be prepared and used immediately.

[0103] 2.1.3 Preparation of mixed standard working solution: Take 10μL, 20μL, 50μL, 100μL, 200μL, and 300μL of intermediate solution 2 respectively, make up to 1ml with methanol-water (1:9), and dilute into standard working solutions with concentrations of 10μg / L, 20μg / L, 50μg / L, 100μg / L, 200μg / L, and 300μg / L.

[0104] 2.2 Sample solution: Use an electronic balance to accurately weigh 0.1 g (accurate to 0.0001 g) of vildagliptin sample, dissolve it in methanol-water (1:9), transfer it to a 10 ml volumetric flask, make up to the mark with methanol-water (1:9), and take 1 ml for testing.

[0105] 3. Instrument conditions

[0106] 3.1 Liquid phase conditions

[0107] Chromatographic column: Agilent ZORBAX SB-Aq 3.5 μm 3.0 × 100 mm;

[0108] Column temperature: 40°C;

[0109] Flow rate: 0.3ml / min;

[0110] Injection volume: 5 μL;

[0111] Elution mode: gradient elution;

[0112] Table 2 Gradient elution program

[0113]

[0114]

[0115] 3.2 Mass spectrometry conditions

[0116] Ion source: ESI source;

[0117] Ion source conditions:

[0118] Temperature: 600℃;

[0119] Air curtain gas: 35psi;

[0120] Capillary voltage: 5500V;

[0121] Spray gas: 50psi;

[0122] Auxiliary heating gas: 45psi;

[0123] Scanning mode: positive ion scanning;

[0124] Table 3 Multiple reaction monitoring (MRM)

[0125]

[0126] Note: * indicates quantitative ions

[0127] 4. Test results

[0128] The reference solution and the sample solution were detected by high performance liquid chromatography tandem mass spectrometry to obtain the chromatogram data of the reference solution and the sample solution, see Figure 1-Figure 8 , calculate the amount of genotoxic impurities in the sample.

[0129] Depend on Figures 1 to 4 The results showed that no genotoxic impurities H, J, I and K were detected in the vildagliptin samples.

[0130] Table 4 Test results

[0131] Test items unit Test results Detection limit Impurity H mg / kg Not detected 0.35 Impurity J mg / kg Not detected 0.35 Impurity I mg / kg Not detected 0.35 Impurity K mg / kg Not detected 0.35

[0132] 5. Method Validation

[0133] 5.1 System adaptability

[0134] To ensure the validity of the final operation results, the reference substances of impurity H, impurity J, impurity I and impurity K were measured. The reference substance with the same concentration was measured 6 times and the relative standard deviation (RSD) of its retention time and peak area was calculated. Acceptance standard: relative standard deviation is less than 6%. The results are as follows:

[0135] Table 5 System suitability results

[0136]

[0137]

[0138] Conclusion: The relative standard deviation is less than 6%, the test results are all passed, and the system adaptability meets the requirements.

[0139] 5.2 Exclusivity

[0140] Using methanol-water (1:9) as a blank solution, sample solutions with added concentration levels of 15 mg / kg of impurity H, impurity J, impurity I and impurity K were prepared as spiked sample solutions.

[0141] To examine the specificity, the blank solution, standard working solution, sample solution and spiked sample solution were checked for mutual interference. Figure 9-Figure 16 , the results are shown in the following table:

[0142] Table 6 Specificity verification results

[0143]

[0144] Conclusion: There are no target peaks in the sample solution, that is, impurities H, impurity J, impurity I and impurity K are not detected. Target peaks appear in both the standard working solution and the spiked sample solution, that is, impurities H, impurity J, impurity I and impurity K are detected. There is no interference in the blank solution, that is, the chromatogram of the blank solution has no interference with the target peaks. The specificity test meets the requirements.

[0145] 5.3 Detection limit

[0146] Prepare 6 sample solutions with impurity H, impurity J, impurity I and impurity K added at a concentration level of 0.35 mg / kg (wherein the sample solution sampling amount is 0.1 g and the fixed volume is 10 ml), test on the machine, and obtain the test results, see Figure 17-Figure 20 Acceptance criteria: The signal-to-noise ratio (S / N) should be greater than 3.

[0147] When the sample size is 0.1g and the fixed volume is 10ml, the detection limits of impurities H, J, I and K can at least meet 0.35mg / kg. The verification results are as follows:

[0148] Table 7 Detection limit signal-to-noise ratio data

[0149] frequency Impurity H Impurity J Impurity I Impurity K 1 110.9 10.4 9.0 4.3 2 110.1 11.0 8.7 5.0 3 121.1 10.0 9.8 4.1 4 88.3 9.5 7.8 4.3 5 111 7.2 9.5 3.9 6 101.2 7.8 6.2 3.7

[0150] Conclusion: The signal-to-noise ratios S / N are all greater than 3, and the detection limits of impurities H, impurity J, impurity I and impurity K are 0.35 mg / kg, which is verified to meet the requirements.

[0151] 5.4 Limit of quantitation

[0152] Prepare 6 sample solutions with impurity H, impurity J, impurity I and impurity K added at a concentration level of 1.0 mg / kg (wherein the sample solution sampling amount is 0.1 g and the fixed volume is 10 ml), test on the machine, and obtain the test results, see Figure 21-24 Acceptance criteria: The signal-to-noise ratio (S / N) should be greater than 10.

[0153] When the sample size is 0.1g and the fixed volume is 10ml, the quantitative limit of impurity H, impurity J, impurity I and impurity K is 1.0mg / kg. The verification results are as follows:

[0154] Table 8 Quantitation limit signal-to-noise ratio data

[0155] frequency Impurity H Impurity J Impurity I Impurity K 1 359.7 16.6 20.8 11.2 2 294.3 16.3 15.9 10.1 3 317.1 16.5 13.6 11.0 4 311.7 15.9 17.7 10.7 5 343.8 19.6 18.2 12.1 6 392.6 13.7 17.9 14.1

[0156] Conclusion: The signal-to-noise ratios S / N were all greater than 10, and the limits of quantification of impurities H, J, I and K were 1.0 mg / kg, which was verified to meet the requirements.

[0157] 5.5 Limit of Quantitation Precision

[0158] Add the quantitative limit concentration level of 1.0 mg / kg to the sample and calculate the quantitative limit (1.0 mg / kg) sample test results. Acceptance criteria: The relative standard deviation RSD of all test results should not be greater than 10%. The results are as follows:

[0159] Table 9 Quantitation limit results

[0160]

[0161] Conclusion: The RSD of the results of 6 parallel determinations was no more than 10%, and the precision test of the limit of quantitation met the requirements.

[0162] 5.6 Linearity

[0163] Referring to the preparation of the mixed standard working solution, accurately prepare impurity H solution, impurity J solution, impurity I solution and impurity K solution with concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L and 300 μg / L, respectively, and perform determination to obtain their peak area response values ​​on LC-MS / MS, and establish a linear regression equation with the peak area response value as the ordinate and the concentration as the abscissa, see Figure 25-28 Acceptance criteria: R 2 Greater than 0.995. The verification results are as follows:

[0164] Table 10 Working curve

[0165]

[0166]

[0167] Conclusion: Impurity H linear regression equation y = 3.1 × 10 4 x+7.68×10 4 (r=1.0000), impurity J linear regression equation y=1.57×10 4 x+3.79×10 4 (r = 0.9999), impurity I linear regression equation y = 2.38 × 10 4 x+1.28×10 4 (r = 0.9999), impurity K linear regression equation y = 3.01 × 10 3 x-1.25×10 4 (r=0.9995). In the concentration range of 10-300 ng / ml, the correlation coefficient of the linear curve is ≥0.995, the linear relationship is good, and the linear test meets the requirements.

[0168] 5.7 Accuracy

[0169] The accuracy of the test results of this method is evaluated by the recovery rate of the spiked sample solution. The samples are added with impurity H, impurity J, impurity I and impurity K solutions at low, medium and high concentration levels, which are spiked sample solutions. Three spiked sample solutions at each concentration level are prepared and tested. Acceptance criteria: The recovery rate should be within the range of 80-115%. The verification results are shown in the following table:

[0170] Table 11 Impurity H Accuracy Results

[0171]

[0172]

[0173] Table 12 Impurity J Accuracy Results

[0174]

[0175] Table 13 Impurity I Accuracy Results

[0176]

[0177] Table 14 Impurity K accuracy results

[0178]

[0179] Conclusion: The recoveries in Tables 11 to 14 were all in the range of 80-115%, and the accuracy test results met the requirements.

[0180] 5.8 Repeatability

[0181] Add impurities H, J, I and K at a concentration level of 15 mg / kg to the sample to form a spiked sample. The spiked sample with the same added concentration level (15 mg / kg) is measured and the results of 6 measurements are evaluated. Acceptance criteria: The RSD of the results of 6 parallel measurements is required to be no greater than 6%. The verification results are as follows:

[0182] Table 15 Repeatability results

[0183]

[0184]

[0185] Conclusion: The relative standard deviation is no more than 6%, and the repeatability test meets the requirements.

[0186] 5.9 Durability

[0187] The flow rate in the test conditions is 0.3 ml / min, the column temperature is 40°C, and the nebulizer temperature is 600°C. The influence of changing the conditions on the accuracy is to change the flow rate (±0.02 ml / min), the column temperature (±2°C), and the nebulizer temperature (±10°C) respectively. Impurities H, impurity J, impurity I, and impurity K at a concentration level of 15 mg / kg are added to the sample for determination. Each time a condition is changed, the determination is repeated 6 times and the relative standard deviation RSD is calculated. Acceptance criteria: The RSD of retention time and peak area should be less than 6%. The results are as follows:

[0188] Table 16 Durability results

[0189]

[0190] Conclusion: The relative standard deviations (RSDs) of retention time and peak area were less than 6%, and the durability test met the requirements.

[0191] 5.10 Intermediate precision

[0192] Two experimenters were employed to measure the spiked samples at the same concentration level (15 mg / kg) 6 times at different times and calculate their relative standard deviation (RSD). Acceptance criteria: The relative standard deviation (RSD) of all measurement results is no more than 11%. The verification results are as follows:

[0193] Table 17 Precision results

[0194]

[0195] Conclusion: The relative standard deviation of the spiked samples at the added concentration level (15 mg / kg) was less than 11%, and the intermediate precision test met the requirements.

[0196] 5.11 Conclusion

[0197] Based on the above tests, it can be seen that: the system adaptability meets the requirements, so the system used in the method of the present invention is effective and applicable; the specificity test meets the requirements, indicating that the method of the present invention can accurately and selectively analyze and detect genotoxic impurities; the detection limit test meets the requirements, indicating that the detection limit of the method of the present invention is reasonably set and the detection method has high sensitivity; the quantitative limit test meets the requirements, indicating that the method of the present invention can accurately determine the amount of genotoxic impurities and has sensitive quantitative detection capabilities; the accuracy test meets the requirements, indicating that the method of the present invention has good precision and high accuracy of the analysis results; the repeatability test meets the requirements, indicating that the method of the present invention is repeatable and feasible; the durability test meets the requirements, that is, when the test conditions (flow rate, column temperature, nebulizer temperature) change, the method of the present invention can still maintain the accuracy of the measurement, indicating that the chromatographic system of the method of the present invention is suitable for this method; the intermediate precision test meets the requirements, that is, the precision obtained under the conditions between the repeatability conditions and the reproducibility conditions is high, indicating that the measurement precision of the method of the present invention is good.

[0198] All the above verification items meet the requirements, and the method of the present application can be used for the detection of genotoxic impurities H, impurity J, impurity I and impurity K in vildagliptin.

[0199] In summary, the present application embodiment confirms that the genotoxic impurities H, impurity J, impurity I and impurity K in vildagliptin can be accurately and quickly detected in only 12 minutes by using a high performance liquid chromatography tandem mass spectrometer and by limiting the chromatographic conditions and mass spectrometry conditions, and the detection method has high sensitivity and good separation. Mainly, the first, the gradient elution program used in the present application can better separate the genotoxic impurities in vildagliptin, so that it detects the corresponding impurities, thereby obtaining the chromatogram of impurities, baseline separation, and good separation. Second, the mass spectrometry conditions used in the present application can quickly detect the corresponding impurities, obtain the corresponding chromatogram, good separation, and high sensitivity. Third, the detection ion pair used in the present application can accurately detect the corresponding impurities, obtain the corresponding chromatogram, high sensitivity, and strong accuracy.

[0200] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and all of them belong to the protection scope of the present application.

Claims

1. A method for detecting genotoxic impurities of vildagliptin, characterized in that: The genotoxic impurities of vildagliptin were detected by high performance liquid chromatography tandem mass spectrometry; The genotoxic impurities include (S)-1-chloroacetylpyrrolidine-2-carboxamide referred to as impurity J, (S)-1-chloroacetylpyrrolidine-2-carbonitrile referred to as impurity H, (2-chloroacetyl)-1-proline referred to as impurity I, and (2-chloroacetyl)-1-proline methyl ester referred to as impurity K; The chromatographic conditions included the following: the chromatographic column was Agilent ZORBAX SB-Aq 3.5 μm 3.0×100 mm, the column temperature was 40°C, the flow rate was 0.3 ml / min, the injection volume was 5 μL, the mobile phase A was 5 MM ammonium acetate aqueous solution, the mobile phase B was acetonitrile, and the gradient elution was performed; The gradient elution program was as follows: 0 min, 90% A, 10% B; 2 min, 90% A, 10% B; 6 min, 25% A, 75% B; 9 min, 25% A, 75% B; 10 min, 90% A, 10% B; 12 min, 90% A, 10% B; The following mass spectrometry conditions were included: the ion source was an ESI source, the temperature was 600°C, the curtain gas was 35 psi, the capillary voltage was 5500 V, the spray gas was 50 psi, the auxiliary heating gas was 45 psi, and the scanning mode was positive ion scanning; In the positive ion scanning mode, the detection ion pairs of the compound were m / z 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.

2. The method for detecting genotoxic impurities of vildagliptin according to claim 1, characterized in that: The steps include: S1. Preparation of reference solution: (1) Preparation of stock solution: Accurately weigh 0.01g of reference solution of impurity H, impurity J, impurity I and impurity K respectively and place them in a 10ml brown volumetric flask, dilute to the mark with methanol, and prepare about 1000mg / L of impurity H, impurity J, impurity I and impurity K stock solution for use; (2) Preparation of mixed intermediate solution: Intermediate solution 1: Take 0.1ml of the above four stock solutions respectively, place them in a 10ml volumetric flask, dilute to the mark with solvent, the concentration of intermediate solution 1 is 10mg / L, and set aside; Intermediate solution 2: Take 1ml of intermediate solution 1, place them in a 10ml volumetric flask, dilute to the mark with solvent, the concentration of intermediate solution 2 is 1mg / L, and intermediate solution 2 is the reference solution; S2. Sample solution: weigh 0.1 g of sample, dissolve it with solvent, transfer it to a 10 ml volumetric flask, dilute to the mark with solvent, and take 1 ml for testing; S3. The reference solution and the sample solution are respectively detected by high performance liquid chromatography tandem mass spectrometry to obtain the chromatogram data of the reference solution and the sample solution, and the amount of genotoxic impurities in the sample is calculated.

3. The method for detecting genotoxic impurities of vildagliptin according to claim 2, characterized in that: The S1 step includes the preparation of a mixed standard working solution: 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 300 μL of the intermediate solution 2 are taken respectively and the volume is fixed to 1 ml with a solvent, and diluted to form standard working solutions with concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, and 300 μg / L.

4. A method for detecting genotoxic impurities of vildagliptin according to claim 2 or 3, characterized in that: In the step S1, the solvent is a mixture of methanol and water, and the volume ratio of methanol to water is 1:

9.

5. The method for detecting genotoxic impurities of vildagliptin according to claim 2, characterized in that: In the step S1, the stock solution and the intermediate solution 1 are stored at 4° C., and the intermediate solution 2 is prepared and used immediately.

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