Method for analyzing sitagliptin related substances in sitagliptin metformin sustained release tablets

Abstract

The invention relates to an improved method for detecting sitagliptin related substances in sitagliptin and metformin sustained-release tablets, which is characterized in that the auto-polymerization rate of a mobile phase B is delayed by increasing the proportion of a phosphate buffer solution in the mobile phase B, and after the improved method is continuously operated for 168 hours, no system pressure abnormity shown in Figure 1 occurs. Besides, by selecting a proper mobile phase, controlling the concentration and the flow velocity of the mobile phase, the column temperature of a chromatographic column and other conditions and adjusting a gradient elution procedure, the sitagliptin related substances in the sitagliptin metformin sustained release tablet are quantitatively analyzed, and the method can effectively reduce the probability of abnormal pressure of a high performance liquid chromatography system and improve the detection accuracy of the sitagliptin related substances in the sitagliptin metformin sustained release tablet. According to the method, the sitagliptin related substances in the sitagliptin metformin sustained-release tablets can be continuously detected for a long time, meanwhile, the sitagliptin and other impurities can be effectively separated, and the method is high in separation degree, good in accuracy and good in durability.

Description

Invention Field

[0001] This invention belongs to the field of pharmaceutical analysis technology and provides an improved method for detecting sitagliptin-related substances in sitagliptin metformin extended-release tablets. Background of the Invention

[0002] Sitagliptin-metformin extended-release tablets are a combination hypoglycemic drug that effectively controls blood glucose levels in patients with type 2 diabetes, thereby achieving a hypoglycemic effect. In our company's testing method for related substances in sitagliptin of this drug, acetonitrile is used as mobile phase B. However, under these conditions, during routine testing, acetonitrile is easily subjected to light, high temperature, oxidation, etc., which can lead to self-polymerization, increased viscosity, and affect the stability of the pressure in the high-performance liquid chromatography system. Figure 1 This ultimately affects the accurate quantification of related substances. In severe cases, it can cause the ruby ​​in the inlet check valve to stick, leading to excessive pressure, affecting system operation, and greatly hindering the feasibility of our company's long-term continuous testing of the drug. Therefore, there is an urgent need for a testing method that can slow down the self-polymerization rate of the mobile phase. Summary of the Invention

[0003] This invention provides an improved method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets. The key feature is that by increasing the proportion of phosphate buffer in mobile phase B, changing mobile phase B from acetonitrile to [0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) - acetonitrile] (45:55), the rate of self-polymerization in mobile phase B is slowed down. After the improvement, the method ran continuously for 168 hours without any degradation. Figure 1 The system pressure shown is abnormal. This method employs high-performance liquid chromatography (HPLC), using a reversed-phase column with isocratic or gradient elution.

[0004] In some typical embodiments, mobile phase A and / or mobile phase B are used as eluents, wherein mobile phase A is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5); mobile phase B is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55); in some embodiments, the concentration of the phosphate buffer is 0.020 mol / L to 0.030 mol / L; preferably, the concentration of the phosphate buffer is 0.025 mol / L.

[0005] In some embodiments, the phosphate buffer solution needs to be adjusted to a pH of 2.3 to 2.7 with phosphate, preferably, the pH of the phosphate buffer solution is 2.5.

[0006] In some embodiments, the initial ratio of the mobile phase gradient of mobile phase A and mobile phase B is (74-78):(26-22), preferably 76:24, and the sum of the ratios of mobile phase A and mobile phase B is 100%; wherein the ratio of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the ratio of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

[0007] In some embodiments, the reversed-phase column uses a non-polar stationary phase as packing material; preferably, the reversed-phase column uses octadecylsilane-bonded silica gel as packing material; more preferably, the reversed-phase column is YMC-PackODS-AM with dimensions of 4.6 mm × 150 mm and 3 μm.

[0008] In some embodiments, the column temperature is 35°C to 45°C; preferably, the column temperature is 40°C.

[0009] In some embodiments, the flow rate of the eluent is 0.8 to 1.2 ml / min; preferably, the flow rate of the eluent is 1.0 ml / min.

[0010] In some embodiments, the detection method is performed on a high-performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high-performance liquid chromatograph using an ultraviolet detector with a detection wavelength of 205 nm.

[0011] On the other hand, the present invention provides a method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets, characterized in that: the method is high-performance liquid chromatography (HPLC), using a reversed-phase column, with an ultraviolet absorption detector; detection wavelength: 205 nm; flow rate: 1.0 ml / min; column temperature: 40 °C; injection volume: 20 μl; mobile phase A: [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5), mobile phase B: [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55); gradient elution is performed according to the following table:

[0012]

[0013] In some typical embodiments, the present invention provides a method for detecting sitagliptin-related substances in sitagliptin metformin extended-release tablets, characterized by the following steps:

[0014] (1) Preparation of test solution: Take an appropriate amount of sitagliptin metformin sustained-release tablets, accurately weigh them, add an appropriate amount of 50% methanol solution, shake for an appropriate time, take the supernatant, dilute it with [0.1% phosphoric acid solution-acetonitrile] (95∶5) solution to prepare a solution of appropriate concentration and filter it.

[0015] (2) Preparation of control solution: Take an appropriate amount of the test sample and dilute it with [0.1% phosphoric acid solution-acetonitrile] (95∶5) solution to prepare a solution of appropriate concentration.

[0016] (3) Test sample determination: Octadecylsilane-bonded silica gel (YMC-Pack ODS-AM) was used as the filler.

[0017] A 4.6 mm × 150 mm column (3 μm or equivalent performance) was used; mobile phase A was [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5), and mobile phase B was [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55), eluted according to the table below; the flow rate was 1.0 mL per minute; the column temperature was 40 °C; and the detection wavelength was 205 nm.

[0018]

[0019] Take an appropriate amount of the test solution and / or the control solution, inject it into the liquid chromatograph, and record the chromatogram;

[0020] (4) Calculate the related substances of sitagliptin in sitagliptin metformin extended-release tablets according to the self-comparison method.

[0021] Those skilled in the art will readily understand that some operational steps of the analytical method described in this application can be interchanged without affecting the results. For example, the steps for preparing the test solution, the control solution, and the system suitability solution can be arbitrarily interchanged.

[0022] In this application, the relevant substances are also described as impurities.

[0023] In this application, a 0.025 mol / L sodium dihydrogen phosphate solution (with pH adjusted to 2.5 using phosphoric acid) is also described as a phosphate buffer solution.

[0024] In this application, "appropriate amount" means that, according to the experimental purpose, the amount of each compound is within the detection line or quantitation limit of its high performance liquid chromatograph.

[0025] In this application, "suitable concentration" means that, according to the experimental purpose, the concentration of each compound is within the detection limit or quantitation limit of its high-performance liquid chromatograph.

[0026] The present invention provides an improved method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets. By increasing the proportion of phosphate buffer in mobile phase B, and modifying mobile phase B from acetonitrile to [0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) - acetonitrile] (45:55), the self-polymerization rate of mobile phase B is slowed down. By selecting a suitable mobile phase and controlling the concentration and flow rate of the mobile phase, as well as the chromatographic column and column temperature, various impurities can be effectively separated. This method has good specificity, accuracy, and robustness, and can quantify multiple impurities in sitagliptin-metformin extended-release tablets while effectively reducing the probability of pressure abnormalities in the high-performance liquid chromatography system, ensuring long-term continuous testing of sitagliptin-related substances in sitagliptin-metformin extended-release tablets. Attached Figure Description

[0027] Figure 1 System pressure abnormal fluctuation diagram Figure 2 Analysis method improved system pressure returns to normal fluctuation diagram Detailed Implementation

[0028] Unless otherwise specified, all reagents and active pharmaceutical ingredients used in the following embodiments of this invention are commercially available.

[0029] In the following embodiments, impurities 1 to 5 have the following structures:

[0030]

[0031] Example 1: Determining the improved mobile phase composition and calculating the gradient procedure

[0032] In the original implementation scheme, mobile phase A was [0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) - acetonitrile] (95:5), mobile phase B was acetonitrile, and the gradient program was as follows:

[0033]

[0034] Based on the above conditions, the proportions of 0.025 mol / L sodium dihydrogen phosphate solution and acetonitrile in the liquid phase system at each time point are calculated as follows:

[0035]

[0036] Using the maximum amount of 0.025 mol / L sodium dihydrogen phosphate solution in mobile phase B as a standard, the composition of mobile phase B was determined to be [0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) - acetonitrile] (45:55). Based on the above data, a quadratic equation was established, and the adjusted gradient program was calculated as follows:

[0037]

[0038] Example 2 Specificity Test

[0039] The liquid chromatography conditions and solution preparation information from Example 1 are as follows:

[0040] 1. Solution preparation:

[0041] 1) System suitability solution: Weigh appropriate amounts of sitagliptin phosphate reference standard and impurities 1-5, and dilute with [0.1% phosphoric acid solution-acetonitrile] (95:5) to prepare a solution containing approximately 640 μg of sitagliptin phosphate and 1 μg each of impurities 1-5 per ml.

[0042] 2) Test solution: Take 5 sitagliptin metformin sustained-release tablets, place them in a volumetric flask containing an appropriate amount of 50% methanol solution, shake rapidly to dissolve, dilute to the mark with 50% methanol solution, shake well, centrifuge, accurately measure 5 ml of the supernatant, place it in a 10 ml volumetric flask, dilute to the mark with [0.1% phosphoric acid solution-acetonitrile] (95:5), shake well, and filter.

[0043] 3) Blank solvent: [0.1% phosphoric acid solution - acetonitrile] (95:5).

[0044] 2. Chromatographic conditions:

[0045] Chromatographic column: YMC-Pack ODS-AM (150mm×4.6mm, 3μm);

[0046] Mobile phase A: [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5);

[0047] Mobile phase B: [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55);

[0048] Column temperature: 40℃;

[0049] Gradient elution procedure:

[0050]

[0051] Flow rate: 1.0 ml / min

[0052] Detection wavelength: 205nm

[0053] Column temperature: 40℃

[0054] Injection volume: 20 μl

[0055] 3. Inject 20 μl of blank solvent, system suitability solution, and test solution into the liquid chromatograph and record the chromatograms. The results are shown in Table 1.

[0056] Table 1. Results of specificity experiments

[0057]

[0058]

[0059] The results showed that the blank solvent did not interfere with the determination of related substances; the purity of the main component peak of the test solution was 1000; the minimum resolution between the impurities and the main peak of sitagliptin metformin sustained-release tablets was 3.58, all of which were greater than 1.5, indicating that the method has good specificity.

[0060] Example 3: Comparison of consistency of chromatographic conditions before and after improvement

[0061] Six parallel aliquots of the test solution from Example 2 were prepared, and 20 μl of each aliquot was injected into the liquid chromatograph under the same chromatographic conditions before and after adjustment. The chromatograms were recorded. The results are shown in Table 2.

[0062] Table 2. Test results before and after chromatographic condition adjustment.

[0063]

[0064] The results showed that none of the impurities were detected before and after the chromatographic conditions were improved. The method had good repeatability, and the test results were consistent before and after the improvement.

[0065] Example 4 Durability Test

[0066] 1) Impurity mixed stock solution: Weigh appropriate amounts of sitagliptin phosphate reference standard and impurities 1-5, and dilute with [0.1% phosphoric acid solution-acetonitrile] (95:5) to prepare a solution containing approximately 6.4 mg sitagliptin phosphate and 10 μg each of impurities 1-5 per ml.

[0067] 2) Spiked test solution: Take 5 sitagliptin metformin sustained-release tablets, place them in a volumetric flask containing an appropriate amount of 50% methanol solution, shake rapidly to dissolve, dilute to the mark with 50% methanol solution, shake well, centrifuge, and take the supernatant as the test solution; accurately measure 5 ml of the test solution and 1 ml of the impurity mixed solution into the same 10 ml volumetric flask, dilute to the mark with [0.1% phosphoric acid solution-acetonitrile] (95:5), and shake well.

[0068] 3) Control solution: Accurately measure 1 ml of the spiked test solution and place it in a 100 ml volumetric flask. Add [0.1% phosphoric acid solution-acetonitrile] (95:5) to dilute to the mark and shake well. Accurately measure 1 ml of the control solution and place it in a 10 ml volumetric flask. Add [0.1% phosphoric acid solution-acetonitrile] (95:5) to dilute to the mark and shake well.

[0069] Blank solution, spiked test solution, control solution, and system suitability solution from Example 2 were analyzed under standard chromatographic conditions, with flow rate variations of ±0.2 ml / min, temperature variations of ±5 °C, mobile phase pH variations of ±0.2, initial mobile phase ratio variations of ±2%, and potassium dihydrogen phosphate concentration variations of ±0.005 mol / L. The RSDs of related substances were calculated to assess robustness. The robustness chromatographic conditions are shown in Table 3, and the robustness results are shown in Table 4.

[0070] Table 3. Durability Chromatographic Conditions

[0071]

[0072] Table 4 Durability Results

[0073]

[0074]

[0075] The results showed that under the conditions of flow rate variation ±0.2 ml / min, temperature variation ±5℃, mobile phase pH variation ±0.2, initial mobile phase ratio variation ±2%, and potassium dihydrogen phosphate concentration variation ±0.005 mol / L, the RSD of each impurity content was less than 10.0%, and the minimum resolution of the system suitability solution was >1.5, indicating that the method has good robustness.

Claims

1. An improved method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets, characterized in that... By increasing the proportion of phosphate buffer in mobile phase B, and modifying mobile phase B from acetonitrile to [0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) - acetonitrile] (45:55), the rate of self-polymerization of mobile phase B is slowed down. This method employs high-performance liquid chromatography (HPLC), which uses a reversed-phase column and elutes at isocratic or gradient rates.

2. The detection method as described in claim 1, wherein mobile phase A and / or mobile phase B are used as eluents, wherein mobile phase A is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5); mobile phase B is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55); the concentration of the phosphate buffer is 0.020 mol / L to 0.030 mol / L; preferably, the concentration of the phosphate buffer is 0.025 mol / L; wherein, The phosphate buffer solution needs to be adjusted to a pH of 2.3 to 2.7 with phosphate, preferably, the pH of the phosphate buffer solution is 2.

5.

3. The detection method according to claim 1, wherein the reversed-phase chromatographic column uses a non-polar stationary phase as packing material; preferably, the reversed-phase chromatographic column uses octadecylsilane-bonded silica gel as packing material; more preferably, the reversed-phase chromatographic column is YMC-Pack ODS-AM, with dimensions of 4.6mm × 150mm and 3μm.

4. The detection method as described in claim 1, wherein the column temperature is 35℃~45℃; preferably, the column temperature is 40℃.

5. The detection method as described in claim 2, wherein the initial ratio of the mobile phases in the gradient elution of mobile phase A and mobile phase B is... The ratio is (78-74):(22-26), preferably 76:24, and the sum of the ratios of mobile phase A and mobile phase B is 100%; wherein the ratio of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the ratio of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

6. The detection method as described in claim 2, wherein the mobile phase A and mobile phase B are eluted in a gradient according to the following procedure: Furthermore, during gradient elution, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of the volume of mobile phase A to the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the volume of mobile phase B to the total volume of the eluent.

7. The detection method according to claim 1, wherein the flow rate of the eluent is 0.8 to 1.2 ml / min; preferably, the flow rate of the eluent is 1.0 ml / min.

8. The detection method as described in claim 1, wherein the detection method is performed on a high-performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high-performance liquid chromatograph using an ultraviolet detector with a detection wavelength of 205 nm.

9. A method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets, characterized in that: The method is high performance liquid chromatography, which uses a reversed-phase column, an ultraviolet absorption detector, and a detection wavelength of 205 nm. The column temperature is 40℃; Mobile phase A is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile]. (95∶5)； The flow rate was 1.0 ml / min; Mobile phase B is [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile]. (45∶55); Perform gradient elution according to the table below; 10. A method for detecting sitagliptin-related substances in sitagliptin-metformin extended-release tablets, characterized in that, Includes the following steps: (1) Preparation of test solution: Take an appropriate amount of sitagliptin metformin sustained-release tablets, accurately weigh them, add an appropriate amount of 50% methanol solution, shake for an appropriate time, take the supernatant, dilute it with [0.1% phosphoric acid solution-acetonitrile] (95:5) solution to prepare a solution of appropriate concentration and filter it; (2) Preparation of control solution: Take an appropriate amount of the test sample and dilute it with [0.1% phosphoric acid solution-acetonitrile] (95∶5) solution to prepare a solution of appropriate concentration; (3) Test sample determination: Octadecylsilane-bonded silica gel was used as the packing material (YMC-Pack ODS-AM, 4.6 mm × 150 mm, 3 μm or equivalent column); mobile phase A was [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (95:5), and mobile phase B was [0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 2.5 with phosphoric acid) - acetonitrile] (45:55), eluted according to the table below; the flow rate was 1.0 ml per minute; the column temperature was 40 ℃; and the detection wavelength was 205 nm. Take an appropriate amount of the test solution and / or the control solution, inject it into the liquid chromatograph, and record the chromatogram; (4) Quantitative analysis of sitagliptin-related substances in the test sample was performed using a self-comparison method.

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