Mass analysis method for separating and determining tiagliptin hydrobromide isomer

Abstract

The invention discloses a quality analysis method for separating and measuring tigliptin hydrobromide isomers, which relates to the technical field of quality analysis, and comprises the steps of high performance liquid chromatography conditions, preparation of a system applicability solution, preparation of a reference substance solution, preparation of a test solution and measurement. According to the method, a silica gel surface is covalently bonded with a cellulose-tri (3, 5-dichlorophenyl carbamate) column, a mobile phase is n-hexane-absolute ethyl alcohol-acetonitrile-ethanolamine-trifluoroacetic acid (10: 85: 5: 0.25: 0.1), the column temperature is 20-30 DEG C, the detection wavelength is 254 nm, and the flow velocity is 0.4-0.65 ml per minute. The method can be used for completely separating the tigliptin hydrobromide and the isomer thereof, and can be used for detecting the isomer in the tigliptin hydrobromide. The problem that isomers cannot be effectively separated by the existing detection method for the related substances of the tigliptin hydrobromide is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass analysis technology, in particular to a method for separating and determining Teneligliptin Hydrobromide Hydrate isomers. BACKGROUND

[0002] Teneligliptin Hydrobromide Hydrate, chemical name: {(2S, 4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl) piperazin-1-yl] pyrrolidin-2-yl} (1, 3-thiazol-3-yl) methanone 2.5 hydrobromide hydrate, its chemical structural formula is as follows:

[0003] Teneligliptin (teneligliptin) is also known as Teneligliptin, which is a DPP-4 inhibitor anti-diabetic drug. DPP-4 inhibitors are one of the main types of drugs for treating diabetes. This kind of drug can not only inhibit the inactivation of GLP-1 and GIP, but also increase the level of endogenous GLP-1 and GIP, promote the release of insulin from pancreatic beta cells, inhibit the secretion of glucagon from pancreatic alpha cells, increase the level of insulin, reduce blood sugar, and is not easy to induce hypoglycemia and increase body weight.

[0004] Most of the drugs with chiral center, a large number of chiral center may only one of the human body disease treatment effect, other chiral drugs are mostly no therapeutic effect, or even reduce the therapeutic effect or harmful to the human body. Hydrobromide telmisartan has 2 chiral center, so there are 3 relative chiral isomers, their physical and chemical properties are similar to hydrobromide telmisartan, so it is difficult to separate, thereby affecting the purity and quality of the final product. Therefore, the separation and determination of hydrobromide telmisartan and its isomers is very important for the quality control of hydrobromide telmisartan. At present, the quality standard of hydrobromide telmisartan raw material is only collected in IP, and USP, BP, EP, JP and Chinese Pharmacopoeia are not collected, but the isomers are not controlled in IP. The mobile phase system mentioned in the existing literature "Development and Validation of Teneligliptin Stereoisomers by HPLC Using Cellulose Based Immobilized Polysaccharide Chiral Stationary Phase" is ethanol-acetonitrile-ethanolamine (90:10:0.1), which cannot realize the separation of hydrobromide telmisartan and its isomers. In order to ensure the quality of drug research and development and production, it is of great significance to develop an accurate and effective method for separating and determining the quality of hydrobromide telmisartan isomers for the quality control of drug products.

[0005] In view of this, a quality analysis method for separating and determining the isomers of hydrobromide telmisartan is provided to overcome the above problems. SUMMARY

[0006] The purpose of the present application is to provide a quality analysis method for separating and determining the isomers of hydrobromide telmisartan to solve the problems raised in the background art.

[0007] To solve the above technical problems, the present application adopts the following technical solutions: A quality analysis method for separating and determining the isomers of hydrobromide telmisartan, comprising the following steps: (1) High performance liquid chromatography conditions: Chromatographic column: silica gel surface covalently bonded with cellulose-tris (3,5-dichlorophenyl carbamate) column (4.6mmx250mm, 5.0um); Detection wavelength: 254nm; Flow rate: 0.4ml / min-0.65ml / min; Column temperature: 20-30℃; Injection volume: 20ul; Mobile phase: n-hexane-absolute ethanol-acetonitrile-ethanolamine-trifluoroacetic acid (8:87:5:0.25:0.09)~(12:83:5:0.25:0.11); (2) Preparation of a system suitability solution; (3) Preparation of a reference solution; (4) Preparation of a test solution; (5) Determination method: accurately measure the system suitability solution, the reference solution and the test solution respectively, inject into the liquid chromatograph, record the chromatogram, and calculate the content of the enantiomer, the epimer 1 and the epimer 2 in the test solution according to the peak area by the external standard method of main components.

[0008] The structural formula of the hydrobromide telmisartan and its isomers are as follows respectively:

[0009]

[0010] The method for preparing the system suitability solution, the reference solution and the test solution is as follows: take the hydrobromide telmisartan working reference, the enantiomer reference, the epimer 1 reference and the epimer 2 reference, and prepare the system suitability solution by adding the mobile phase; take the hydrobromide telmisartan working reference, and prepare the reference solution by adding the mobile phase; take the hydrobromide telmisartan test sample, and prepare the test solution by adding the mobile phase. The proportion of the mobile phase is n-hexane-absolute ethanol-acetonitrile-ethanolamine-trifluoroacetic acid (10:85:5:0.25:0.1), the flow rate is 0.6 ml / min, the column temperature is 25 DEG C, and the wavelength is 254 nm. The method can be applied to the detection of the hydrobromide telmisartan isomers.

[0011] Compared with the prior art, the beneficial effects of the present application are: The detection method of the hydrobromide telmisartan isomers is not collected in the pharmacopoeia of each country. The present application provides a detection method of the hydrobromide telmisartan isomers, and the method has strong specificity, high sensitivity, good accuracy, high precision and good repeatability, can effectively separate and detect the hydrobromide telmisartan isomers, is used for the detection and monitoring in the production process of the hydrobromide telmisartan, and is helpful to the establishment of the quality standard of the bulk drug. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is the separation chromatogram of the hydrobromide telmisartan system suitability solution; Figure 2 It is the chromatogram of the reference solution; Figure 3 It is the isomer detection chromatogram of the test solution; Figure 4 LOQ plot for Hydrobromide Sitagliptin and Isomers; Figure 5 LOD plot for Hydrobromide Sitagliptin and Isomers. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0014] Please refer to Figures 1-5 The present application provides a technical solution: Please refer to Figures 1-5 An embodiment of a method for separating and determining the mass of isomers of Hydrobromide Sitagliptin is shown in the following: Embodiment 1: Separation and determination of Hydrobromide Sitagliptin and its isomers in a test sample: 1. Instruments and reagents: Shimadzu LC-20AD high-performance liquid chromatograph, UV ultraviolet detector, and Waters Empower3 (network version) chromatographic workstation.

[0015] N-hexane (chromatographic grade), anhydrous ethanol (chromatographic grade), acetonitrile (chromatographic grade), ethanolamine (chromatographic grade), trifluoroacetic acid (chromatographic grade).

[0016] 2. Chromatographic conditions: Chromatographic column: silica gel covalently bonded with cellulose-tris (3, 5-dichlorophenyl carbamate) column (4.6 mm x 250 mm, 5.0 μm); Detection wavelength: 254 nm; Flow rate: 0.4 ml / min to 0.65 ml / min; Column temperature: 20°C to 30°C; Injection volume: 20 μl; Mobile phase: n-hexane-anhydrous ethanol-acetonitrile-ethanolamine-trifluoroacetic acid (10:85:5:0.25:0.1).

[0017] 3. Preparation of related solutions: (1) Isomer mixed stock solution ①: Take about 10 mg of each of the enantiomeric reference substance (source: self-made), epimer 1 reference substance (source: self-made), and epimer 2 reference substance (source: self-made), accurately weigh, and place them in the same 100 ml volumetric flask. Add the mobile phase to dissolve and dilute to the mark, and shake well. (The concentration is about 100 μg / ml each.) (2) Isomer mixture stock solution 2: accurately measure 5 ml of isomer mixture stock solution 1, place in a 50 ml volumetric flask, dilute to the mark with mobile phase, and shake well. (Concentration of each is about 10 μg / ml). (3) System suitability solution: accurately weigh about 20 mg of sitagliptin hydrobromide working control (source: self-made), place in a 20 ml volumetric flask, add mobile phase to dissolve, accurately add 2 ml of isomer mixture stock solution 2, dilute to the mark with mobile phase, and shake well. (Sitagliptin hydrobromide: 1 mg / ml, enantiomer: 1 μg / ml, epimer 1: 1 μg / ml, epimer 2: 1 μg / ml). (4) Control stock solution: accurately weigh about 20 mg of sitagliptin hydrobromide working control (source: self-made), place in a 20 ml volumetric flask, dissolve and dilute to the mark with mobile phase, and shake well. (Concentration is about 1 mg / ml). (5) Control solution: accurately measure 1 ml of the control stock solution, place in a 100 ml volumetric flask, dilute to the mark with mobile phase, and shake well; accurately measure 2 ml, place in a 20 ml volumetric flask, dilute to the mark with mobile phase, and shake well. (Concentration is about 1 μg / ml). (6) Test solution: accurately weigh about 20 mg of sitagliptin hydrobromide, place in a 20 ml volumetric flask, dissolve and dilute to the mark with mobile phase, and shake well.

[0018] 4. Separation and determination method: Accurately measure 20 μl of (3) system suitability solution, (5) control solution, and (6) test solution under the preparation item above, respectively inject into the liquid chromatograph, record the chromatogram, and the determination results are shown in Table 1, and the chromatogram is shown in Figure 1 , Figure 2 and Figure 3 . The content of enantiomer, epimer 1, and epimer 2 in the test solution is calculated by peak area according to the external standard method of main component.

[0019] Table 1 System suitability separation degree test results:

[0020] The above test results show that the separation degree between each chromatographic peak is greater than 1.5, indicating that the method has good specificity.

[0021] Example 2: Methodology verification: 1. Quantitative limit and detection limit: Stock solution: accurately measure 1 ml of isomer mixture stock solution 2 under the preparation item above, place in a 10 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0022] Quantitation limit solution: precisely take 1 ml of the stock solution, put it in a 20 ml volumetric flask, add 1 ml of the control solution under (5) in the preparation of the relevant solution, dilute to the mark with the mobile phase, and shake well. (Equivalent to 0.005% of the concentration of the test solution).

[0023] Detection limit solution: precisely take 5 ml of the quantitation limit solution, put it in a 10 ml volumetric flask, dilute to the mark with the mobile phase, and shake well. (Equivalent to 0.0025% of the concentration of the test solution).

[0024] Precisely take 20 μl of the above quantitation limit solution and detection limit solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram, and the test results are shown in Table 2, and the chromatogram is shown in Figure 4 and Figure 5 .

[0025] Table 2: Test results of quantitation limit and detection limit:

[0026] The above results show that the quantitation limit concentration of each isomer and hydrobromide tigilazone is lower than 50% of the report limit concentration (0.25 μg / ml), and the signal-to-noise ratio is greater than 10; the signal-to-noise ratio of the detection limit is greater than 3, and the sensitivity meets the requirements.

[0027] 2. Linearity and range: Linear stock solution: precisely take 2 ml of the control stock solution under (4) in the preparation of the relevant solution, put it in a 20 ml volumetric flask, dilute to the mark with the mobile phase, and shake well; precisely take 5 ml, put it in a 50 ml volumetric flask, precisely add 5 ml of the isomer mixed stock solution ① under (1) in the preparation of the relevant solution, dilute to the mark with the mobile phase, and shake well. (The concentration is about 10 μg / ml each).

[0028] Linear solution: precisely take the linear stock solution according to Table 3, put it in the corresponding volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0029] Table 3: Preparation of linear solution:

[0030] Test method: precisely take 20 μl of L1-L6 linear solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram, and the test results are shown in Table 4.

[0031] Table 4: Test results of linearity and range:

[0032] The above results show that: enantiomer, epimer 1, epimer 2 and trelagliptin hydrobromide have good linear relationship between peak area and concentration at the concentration of 0.005% to 0.20% of the test solution, and the correlation coefficient (r) is greater than 0.990.

[0033] 3. Accuracy: Reference solution: the reference solution in the preparation of the above related solution (5) is used as the reference solution.

[0034] Isomer mixture stock solution: the isomer mixture stock solution 2 in the preparation of the above related solution (2) is used as the isomer mixture stock solution.

[0035] Test solution: the test solution in the preparation of the above related solution (6) is used as the test solution.

[0036] Accuracy sample solution: an appropriate amount of trelagliptin hydrobromide is accurately weighed according to the following table 5, placed in the corresponding volumetric flask, added with an appropriate amount of mobile phase to dissolve, and then an appropriate amount of isomer mixture stock solution is accurately measured and placed in the corresponding volumetric flask, diluted to the mark with mobile phase, and shaken uniformly. Three sample solutions are prepared for each level.

[0037] Table 5 Preparation method of isomer accuracy sample solution:

[0038] Determination method: 20 μl of the reference solution, test solution and accuracy sample solution under the accuracy item are accurately measured and injected into the liquid chromatograph, respectively. The reference solution is continuously injected for 6 times, and the chromatogram is recorded. The determination results of trelagliptin hydrobromide isomers are shown in tables 6-8.

[0039] Table 6 Accuracy test results of enantiomer:

[0040] Table 7 Accuracy test results of epimer 1:

[0041] Table 8 Accuracy test results of epimer 2:

[0042] Results and analysis: At three levels (50% limit concentration, 100% limit concentration, 150% limit concentration), the recovery of enantiomers was between 91.3% and 96.1% by principal component external standard method, with RSD of 1.87%; the recovery of epimer 1 was between 95.1% and 103.3%, with RSD of 3.05%; the recovery of epimer 2 was between 101.3% and 106.9%, with RSD of 1.68%, which met the verification requirements, and the accuracy test results of the method met the requirements.

Claims

1. A method for the separation and determination of ticagliptin hydrobromide isomers, characterized in that, Includes the following steps: (1) Setting high performance liquid chromatography conditions: A chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel was used. The detection wavelength was 254 nm, the flow rate was 0.4 ml / min to 0.65 ml / min, the column temperature was 20℃ to 30℃, and the mobile phase consisted of n-hexane, anhydrous ethanol, acetonitrile, ethanolamine and trifluoroacetic acid. (2) Prepare system suitability solution, reference solution and test solution; (3) Inject the above three solutions into the liquid chromatograph, record the chromatograms, and calculate the content of ticagliptin hydrobromide isomers in the test solution.

2. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The volume ratio of each component in the mobile phase is n-hexane-anhydrous ethanol-acetonitrile-ethanolamine-trifluoroacetic acid (8:87:5:0.25:0.09) to (12:83:5:0.25:0.11).

3. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 2, characterized in that: The volume ratio of the mobile phase is n-hexane-anhydrous ethanol-acetonitrile-ethanolamine-trifluoroacetic acid (10:85:5:0.25:0.1).

4. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The column temperature in the high performance liquid chromatography conditions is 25℃.

5. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The flow rate in the high performance liquid chromatography conditions was 0.6 ml / min.

6. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The injection volume in the high performance liquid chromatography conditions is 20 μl.

7. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The solution is prepared as follows: Tilgliptin hydrobromide working reference standard, enantiomer reference standard, epimer 1 reference standard, and epimer 2 reference standard are added to the mobile phase to prepare a system suitability solution; Tilgliptin hydrobromide working reference standard is added to the mobile phase to prepare a reference solution; Tilgliptin hydrobromide test sample is added to the mobile phase to prepare a test sample solution.

8. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: The calculation was performed using the principal component external standard method, with peak area as the primary factor.

9. The method for separating and determining the ticagliptin hydrobromide isomers as described in claim 1, characterized in that: Tigliptin hydrobromide isomers include enantiomers, epimer 1, and epimer 2.

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