A method for detecting linagliptin enantiomers and its application

Abstract

The present invention belongs to the technical field of pharmaceutical analysis and detection, and particularly relates to a method and application for detecting linagliptin enantiomers. This method can simultaneously separate and determine linagliptin and its enantiomers under one chromatographic condition. The resolution of the chromatographic peaks of both is good, and the detection limit and quantification limit also meet the requirements for qualitative and quantitative analysis of drugs. It has high sensitivity, as well as high recovery rate, precision and stability, good reproducibility and high accuracy. It can be used for monitoring the impurity linagliptin isomers during the production process of linagliptin, which is beneficial to controlling and improving the quality of linagliptin. At the same time, the operation is simple, the detection time is short, and it is very suitable for the quality control of raw materials in large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis and detection. More specifically, it relates to a method for detecting linagliptin enantiomers and its application. Background Art

[0002] Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor, which can improve the blood glucose control level of adult patients with type 2 diabetes on the basis of diet control and physical exercise. During the preparation process, the optical isomer (S)-3-aminopiperidine dihydrochloride of the reaction raw material (R)-3-aminopiperidine dihydrochloride exists and reacts with other reaction raw materials to generate linagliptin enantiomers. Linagliptin enantiomers remain as impurities in the linagliptin raw material drug. On the one hand, according to the guiding principles for the pharmaceutical research of chiral drugs, different stereoisomers of chiral drugs may have differences in pharmacodynamics, pharmacokinetics, and toxicology, etc. This isomer is very likely to have certain toxic side effects and affect the efficacy; on the other hand, the existence of linagliptin enantiomers in the raw material drug directly affects the purity of linagliptin and reduces the quality of linagliptin products. Therefore, in order to strictly control the product quality and study the enantiomers of this product, it is urgent to detect and control linagliptin enantiomers to obtain qualified finished products.

[0003]

[0004] Currently, the separation of chiral drugs mainly uses normal-phase chromatography. For example, Dong Haixia (Dong Haixia. Determination of the S-isomer of linagliptin by HPLC [J]. Shandong Chemical Industry, 2017, 46(10): 3.) disclosed a method for determining the S-isomer of linagliptin by HPLC. This method uses an AD-H normal-phase chromatographic column, the mobile phase is ethanol-ethanolamine (100:0.1), the column temperature is 35°C, the flow rate is 0.4 ml / min, and normal-phase chromatography is carried out at 220 nm; similarly, a Chinese patent application disclosed a method for determining the content of linagliptin enantiomers by high-performance liquid chromatography. It uses a Daicel AD-H normal-phase chromatographic column, the mobile phase is n-hexane-ethanol-trifluoroacetic acid (60:40:0.1), the column temperature is 30°C, the flow rate is 1.0 ml / min, and normal-phase chromatography is carried out at 230 nm. The above normal-phase chromatography methods can all measure linagliptin enantiomers well, but the chiral separation system applicability of this method is narrow, the detection instrument requires no water and inorganic salts, the mobile phase used has a high cost, and the reagents used are highly toxic; moreover, generally, the system needs to be replaced in advance, the efficiency is low, and the replacement process is complex. If care is not taken, it may cause blockage of the instrument and chromatographic column, and the operation is relatively troublesome. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing normal-phase chromatography method, such as high reagent cost, high toxicity, and cumbersome operation, and to provide a method for detecting the enantiomers of linagliptin, which method is simple in operation, strong in specificity, high in sensitivity, and stable and reliable.

[0006] The object of the present invention is to provide the application of the method for detecting the enantiomers of linagliptin in the quality control of linagliptin.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] A method for detecting the enantiomers of linagliptin, which is detected by reversed-phase high-performance liquid chromatography. The conditions of the reversed-phase high-performance liquid chromatography are as follows:

[0009] An amylose silica chiral column is used as the chromatographic column. Under the conditions of a column temperature of 25 - 35 °C and a flow rate of 0.8 - 1.2 ml / min, an ammonium acetate solution with a concentration of 0.05 - 0.15 mol / L is used as mobile phase A, and acetonitrile is used as mobile phase B. Isocratic elution is carried out under the condition that the volume ratio of A:B is (700 - 900):(100 - 300), and detection is carried out at a detection wavelength of 295 nm.

[0010] The inventors have found through research that the enantiomers of linagliptin have relatively large absorption at 224 nm and 296 nm, and the absorption at 224 nm is much greater than that at 296 nm. In the prior art, the wavelength is generally selected at 224 nm when detecting it. The present invention creatively selects 296 nm as the detection wavelength. Although the response value of the linagliptin enantiomers decreases relatively, the detector noise in the high wavelength band is smaller than the end absorption, and the sensitivity increases.

[0011] Preferably, the chromatographic column is a CHIRALPAK AD-H amylose silica chiral column with a size of 4.6 mm × 250 mm and a particle size of 5 μm.

[0012] Further, the pH of the ammonium acetate solution is 4 - 6.

[0013] Preferably, mobile phase A is a 0.1 mol / L ammonium acetate solution.

[0014] Preferably, the volume ratio of A:B is 900:100.

[0015] Preferably, the flow rate is 1.0 ml / min.

[0016] Even further, in the conditions of the reversed-phase high-performance liquid chromatography, the injection volume is 10 μl.

[0017] Further, the limit of quantitation of the method is 0.04%.

[0018] Furthermore, the detection limit of the method is 0.02-0.04%.

[0019] Further, when preparing the test sample for the method for detecting linagliptin enantiomers, the mobile phase is used as the solution to prepare the corresponding test sample. Preferably, the concentration of the test sample solution is 0.2 μg / ml - 600 μg / ml.

[0020] In addition, the present invention also provides the application of the method for detecting linagliptin enantiomers in the quality control of linagliptin.

[0021] The present invention has the following beneficial effects:

[0022] A method for detecting linagliptin enantiomers according to the present invention can simultaneously separate and determine linagliptin and linagliptin enantiomers under one chromatographic condition. The resolution of the chromatographic peaks of both is good, and the detection limit and quantitation limit also meet the requirements of qualitative and quantitative analysis of drugs. It has high sensitivity, high recovery rate, precision and stability, good reproducibility and high accuracy. It can be used for the monitoring of impurity linagliptin isomers during the production process of linagliptin, which is beneficial to controlling and improving the quality of linagliptin. At the same time, the operation is simple and the detection time is short, which is very suitable for the quality control of raw materials in large-scale industrial production. Description of the Drawings

[0023] Figure 1 It is the detection chromatogram of the system suitability solution in Example 1 of the present invention.

[0024] Figure 2 It is the detection limit detection chromatogram in Example 1 of the present invention.

[0025] Figure 3 It is the quantitation limit detection chromatogram in Example 1 of the present invention.

[0026] Figure 4 It is the detection chromatogram of the system suitability solution in Comparative Example 1 of the present invention.

[0027] Figure 5 It is the detection chromatogram of the system suitability solution in Comparative Example 2 of the present invention.

[0028] Figure 6 It is the detection chromatogram of the system suitability solution in Comparative Example 3 of the present invention.

[0029] Figure 7 It is the quantitation limit detection chromatogram in Comparative Example 4 of the present invention.

[0030] Figure 8 It is the standard curve graph in the methodological investigation of the method for detecting linagliptin enantiomers in Application Example 1 of the present invention. Detailed Embodiments

[0031] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] Example 1 A method for detecting the enantiomers of linagliptin

[0034] The method for detecting the enantiomers of linagliptin specifically includes the following steps:

[0035] 1. Chromatographic conditions:

[0036] Mobile phase: 0.1 mol / L ammonium acetate solution (adjusted to pH 5.0 with acetic acid) - acetonitrile (900:100), isocratic elution;

[0037] Detector: UV detector, detection wavelength is 295 nm;

[0038] Chromatographic column: CHIRALPAK AD-H amylose silica chiral column 4.6 mm × 250 mm, 5 μm;

[0039] Column temperature is 30 °C, flow rate is 1.0 ml per minute, injection volume is 10 μl.

[0040] 2. Sample preparation:

[0041] Stock solution of linagliptin enantiomers: Take about 15 mg of linagliptin enantiomer reference substance, accurately weigh, place it in a 100 ml volumetric flask, add an appropriate amount of mobile phase, sonicate to dissolve, dilute to the scale with mobile phase, as the mother liquor of linagliptin enantiomers. Accurately measure 5 ml of the mother liquor of linagliptin enantiomers, place it in a 20 ml volumetric flask, dilute to the scale with mobile phase, and shake well.

[0042] System suitability solution: Take 25 mg of linagliptin, accurately weigh, place it in a 50 ml volumetric flask, add an appropriate amount of mobile phase, sonicate to dissolve, add 1 ml of the stock solution of linagliptin enantiomers, and dilute with mobile phase to prepare a mixed solution containing about 0.75 μg of enantiomers and 0.5 mg of linagliptin per 1 ml.

[0043] Test solution: Take about 25 mg of linagliptin, accurately weigh, place it in a 50 ml volumetric flask, add an appropriate amount of mobile phase, sonicate to dissolve, and quantitatively dilute with mobile phase to prepare a solution containing about 0.5 mg per 1 ml.

[0044] Reference solution: Weigh accurately about 15 mg of linagliptin reference substance, place it in a 100-ml volumetric flask, add an appropriate amount of mobile phase, sonicate to dissolve, dilute to the mark with mobile phase to obtain the mother reference solution. Then accurately measure 1 ml, place it in a 200-ml volumetric flask, dilute to the mark with mobile phase, shake well to prepare a solution containing about 0.75 μg of linagliptin per 1 ml.

[0045] 3. Test results:

[0046] (1) To analyze the separation and detection ability of the linagliptin peak and the piriglitazone enantiomers in the test solution, inject the blank solvent (mobile phase), system suitability solution, and reference solution for detection. The test results are as Figure 1 shown in Table 1.

[0047] Table 1 Resolution results of the system suitability solution

[0048]

[0049]

[0050] The results show that the blank solvent does not interfere with the determination of linagliptin enantiomers, and in the system suitability solution, the resolution between linagliptin and its enantiomers meets the requirements (resolution > 1.5, Chinese Pharmacopoeia).

[0051] (2) To analyze the detection ability of the method of the present invention for linagliptin enantiomers, serially dilute the linagliptin enantiomer reference stock solution and examine the limit of detection (LOD) and limit of quantitation (LOQ) of impurities. The results are as Figures 2 - 3 shown in Table 2.

[0052] Table 2 Results of limit of quantitation and limit of detection

[0053] Name Peak area S / N Detection limit / % Linagliptin enantiomers 2825 5.0 0.02 Name Peak area S / N Quantitation limit / % Linagliptin enantiomers 5122 10.1 0.04

[0054] The results show that the detection method of the present invention has a low limit of detection and limit of quantitation for linagliptin enantiomers.

[0055] Example 2 A method for detecting linagliptin enantiomers

[0056] The difference from Example 1 is that in Example 2, the mobile phase is: 0.1 mol / L ammonium acetate solution (adjusted to pH 5.0 with acetic acid)-acetonitrile (700:300). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 3.

[0057] Table 3 Resolution results of the unified system suitability solution

[0058] Name Retention time / min Resolution Linagliptin 7.030 / Linagliptin enantiomers 9.513 2.46

[0059] As can be seen from the table, the resolution between linagliptin and its enantiomer determined by the method of the present invention meets the requirements (resolution > 1.5).

[0060] Example 3 A method for detecting linagliptin enantiomers

[0061] The difference from Example 1 is that in Example 3, the mobile phase is: 0.05 mol / L ammonium acetate solution (adjusted to pH 5.0 with acetic acid)-acetonitrile (900:100). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 4.

[0062] Table 4 Resolution results of the system suitability solution

[0063] Name Retention time / min Resolution Linagliptin 8.575 / Linagliptin enantiomers 12.809 3.47

[0064] As can be seen from the table, the resolution between linagliptin and its enantiomer determined by the method of the present invention meets the requirements (resolution > 1.5).

[0065] Example 4 A method for detecting linagliptin enantiomers

[0066] The difference from Example 1 is that in Example 4, the mobile phase is: 0.15 mol / L ammonium acetate solution (adjusted to pH 5.0 with acetic acid)-acetonitrile (900:100). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 5.

[0067] Table 5 Resolution results of the system suitability solution

[0068] Name Retention time / min Resolution Linagliptin 8.559 / Linagliptin enantiomers 12.833 3.50

[0069] As can be seen from the table, the resolution between linagliptin and its enantiomer determined by the method of the present invention meets the requirements (resolution > 1.5).

[0070] Example 5 A method for detecting linagliptin enantiomers

[0071] The difference from Example 1 is that in Example 5, the mobile phase is: 0.1 mol / L ammonium acetate solution (adjusted to pH 4.0 with acetic acid)-acetonitrile (900:100). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 6.

[0072] Table 6 Resolution results of the system suitability solution

[0073] Name Retention time / min Resolution Linagliptin 6.871 / Linagliptin enantiomers 9.665 3.14

[0074] As can be seen from the table, the resolution of linagliptin and linagliptin enantiomers determined by the method of the present invention meets the requirements (resolution > 1.5).

[0075] Example 6 A method for detecting linagliptin enantiomers

[0076] The difference from Example 1 is that in Example 6, the mobile phase is: 0.1 mol / L ammonium acetate solution (adjusted to pH 6.0 with acetic acid)-acetonitrile (900:100). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 7.

[0077] Table 7 Resolution results of the system suitability solution

[0078] Name Retention time / min Resolution Linagliptin 10.123 / Linagliptin enantiomers 15.741 3.76

[0079] As can be seen from the table, the resolution of linagliptin and linagliptin enantiomers determined by the method of the present invention meets the requirements (resolution > 1.5).

[0080] Example 7 A method for detecting linagliptin enantiomers

[0081] The difference from Example 1 is that in Example 7, the flow rate is 0.8 ml / min. For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 8.

[0082] Table 8 Resolution results of the system suitability solution

[0083] Name Retention time / min Resolution Linagliptin 9.669 / Linagliptin enantiomers 14.436 3.63

[0084] As can be seen from the table, the resolution of linagliptin and linagliptin enantiomers determined by the method of the present invention meets the requirements (resolution > 1.5).

[0085] Example 8 A method for detecting linagliptin enantiomers

[0086] The difference from Example 1 is that in Example 8, the flow rate is 1.2 ml / min. For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Table 9.

[0087] Table 9 Resolution results of the system suitability solution

[0088] Name Retention time / min Resolution Linagliptin 7.551 / Linagliptin enantiomers 11.217 3.36

[0089] As can be seen from the table, the resolution of linagliptin and linagliptin enantiomers determined by the method of the present invention meets the requirements (resolution > 1.5).

[0090] Comparative Example 1 A method for detecting linagliptin enantiomers

[0091] The difference from Example 1 is that in Comparative Example 1, the chromatographic column is CHIRALPAK IC cellulose-3,5-dichlorophenylcarbamate. For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Figure 4 and Table 10.

[0092] Table 10 Resolution Results of System Suitability Solution

[0093] Name Retention time / min Resolution Linagliptin 5.784 / Linagliptin enantiomers 7.242 No resolution shown

[0094] The results show that the resolution between linagliptin and linagliptin enantiomers in this detection method cannot be shown and does not meet the requirements (resolution > 1.5).

[0095] Comparative Example 2 A Method for Detecting Linagliptin Enantiomers

[0096] The difference from Example 1 is that in Comparative Example 2, the mobile phase is n-hexane-anhydrous ethanol-methanol (80:15:5). For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Figure 5 and Table 11.

[0097] Table 11 Resolution Results of System Suitability Solution

[0098] Name Retention time / min Resolution Linagliptin 40.435 / Linagliptin enantiomers 35.595 1.38

[0099] As can be seen from the table, the resolution between linagliptin and linagliptin enantiomers in this detection method does not meet the requirements (resolution > 1.5).

[0100] Comparative Example 3 A Method for Detecting Linagliptin Enantiomers

[0101] The difference from Example 1 is that in Comparative Example 3, the mobile phase is 0.1 mol / L ammonium acetate solution (adjusted to pH 5.0 with acetic acid)-acetonitrile with a ratio of 600:400. For other operations and parameters, refer to Example 1. Take the system suitability solution for detection, and the results are shown in Figure 6 and Table 12.

[0102] Table 12 Resolution Results of System Suitability Solution

[0103] Name Retention time / min Resolution Linagliptin 4.916 / Linagliptin enantiomers 7.415 No resolution shown

[0104] The results show that the resolution between linagliptin and linagliptin enantiomers in this detection method cannot be shown and does not meet the requirements (resolution > 1.5).

[0105] Comparative Example 4 A Method for Detecting Linagliptin Enantiomers

[0106] The difference from Example 1 is that in Comparative Example 4, the detection wavelength is 225 nm. For other operations and parameters, refer to Example 1. Take the solution for detecting the limit of quantitation in Example 1 for detection. The results are shown in Figure 7 and Table 13.

[0107] Table 13 Results of the Limit of Quantitation Detection

[0108] Name Peak area S / N Quantitation limit / % Linagliptin enantiomers 12051 7.1 0.05

[0109] As can be seen from the table, the detection sensitivity of linagliptin enantiomers in this detection method does not meet the requirements (S / N > 10, Chinese Pharmacopoeia).

[0110] Methodological Investigation of the Detection Method for Linagliptin Enantiomers in Application Example 1

[0111] 1. Linearity and Range Test

[0112] Accurately pipette appropriate amounts of the linagliptin enantiomer stock solution in Example 1 respectively to prepare a reference solution with a linagliptin enantiomer concentration of 0.2 - 3.5 μg / ml; refer to the liquid chromatography conditions in Example 1 for injection and detection. Perform linear regression with the concentration as the abscissa and the peak area as the ordinate, and calculate the regression equation and the correlation coefficient. The results are shown in Table 14 and Figure 8 .

[0113] Table 14 Linear Results of Linagliptin Enantiomers

[0114]

[0115] According to the measurement results, it can be known that for linagliptin enantiomers, there is a good linear relationship between the peak area and the concentration in the range of 0.2118 μg / ml - 3.529 μg / ml, where r is 0.9999, and the ratio of the Y-axis intercept to the 100% concentration response value is 0.1%.

[0116] 2. Precision Test

[0117] Refer to the conditions in Example 1 and inject the reference solution continuously for 5 times. The results are shown in Table 15.

[0118] Table 15 Results of the Precision Test

[0119]

[0120] As can be seen from the table, the RSD of the main peak area of the reference solution is less than 5.0%, indicating good precision.

[0121] 3. Accuracy Test

[0122] Accuracy stock solution: Accurately measure 25 ml of the linagliptin enantiomer mother liquor, transfer it to a 200-ml volumetric flask, dilute it to the mark with the mobile phase, and shake well.

[0123] Accuracy test solution: Take 25 mg of linagliptin, accurately weigh it, place it in a 50-ml volumetric flask, add an appropriate amount of the mobile phase, sonicate to dissolve it, accurately add 1 ml, 2 ml, and 3 ml of the accuracy stock solution respectively, and then dilute with the mobile phase to prepare solutions with relative enantiomer contents of 50%, 100%, and 150%. Prepare 2 parallel samples for each concentration; take the reference solution and test solution in Example 1, refer to the liquid chromatography conditions in Example 1, inject the samples for detection, calculate the content of linagliptin enantiomers by the external standard method based on the peak area, and the results are shown in Table 16.

[0124] Table 16 Results of accuracy determination

[0125]

[0126] As can be seen from the table, the average recovery rate of linagliptin enantiomers is 98.6%, the single recovery rate is between 95.64% and 101.31%, the recovery rate is relatively high, and the RSD is 2.1%, indicating high accuracy.

[0127] 4. Solution stability test

[0128] Take the reference solution and 100% accuracy test solution in the accuracy test item, place them at room temperature for 0 h, 4 h, 8 h, 12 h, and 18 h respectively. Accurately measure the reference solution and test solution, inject them into the liquid chromatograph, and perform the determination according to the chromatographic conditions in Example 1. Record the chromatogram, measure and calculate the change rate of the peak area of the reference solution and the difference in the content of the enantiomers in the test solution compared with that at 0 h. The results are shown in Table 17.

[0129] Table 17 Results of solution stability test

[0130]

[0131] As can be seen from the table, compared with that at 0 h, when the test solution is placed at room temperature for 18 h, the range of the enantiomer content is 0.00% < 0.05%; when the reference solution is placed at room temperature for 18 h, the maximum change rate of the peak area is -2.7%, within 10%. Therefore, both the test solution and the reference solution can maintain a stable state when placed at room temperature for 18 h.

[0132] Through the above verification process and results, it is proved that the invention can accurately and effectively detect the content of linagliptin enantiomers, and the method has strong specificity, high sensitivity, and is stable and reliable.

[0133] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for detecting the enantiomers of linagliptin, characterized in that, the detection is carried out by reverse-phase high performance liquid chromatography, and the conditions of the reverse-phase high performance liquid chromatography are as follows: CHIRALPAK AD-H amylose silica chiral column is used as the chromatographic column. At a column temperature of 25 - 35 °C and a flow rate of 0.8 - 1.2 ml / min, 0.05 - 0.15 mol / L ammonium acetate solution is used as mobile phase A, and acetonitrile is used as mobile phase B. Isocratic elution is carried out under the condition of A:B volume ratio of (700 - 900):(100 - 300), and detection is carried out at a detection wavelength of 295 nm.

2. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the chromatographic column is CHIRALPAK AD-H amylose silica chiral column 4.6 mm × 250 mm, 5 μm.

3. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the pH of the ammonium acetate solution is 4 - 6.

4. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the mobile phase A is 0.1 mol / L ammonium acetate solution.

5. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the A:B volume ratio is 900:

100.

6. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the flow rate is 1.0 ml / min.

7. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, in the conditions of the reverse-phase high performance liquid chromatography, the injection volume is 10 μl.

8. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the quantitation limit of the method is 0.04%.

9. The method for detecting the enantiomers of linagliptin according to claim 1, characterized in that, the detection limit of the method is 0.02 - 0.04%.

10. Application of the method for detecting the enantiomers of linagliptin according to any one of claims 1 - 9 in the quality control of linagliptin.

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