Method for detecting related substances of dapagliflozin tablets

Abstract

The invention discloses a method for detecting related substances of dapagliflozin tablets, and belongs to the technical field of medicine detection. The related substances comprise an impurity 383-1D, an impurity 383-1E, an impurity 383-1F and an impurity 383-1G, and the detection method comprises the following detection conditions: adopting a high performance liquid chromatography method, taking octadecyl silica gel as a chromatographic column, taking potassium hydrogen phosphate as a buffer solution, taking acetonitrile as a diluted solution, taking a methanol-water solution as a mobile phase A, taking an acetonitrile-water solution as a mobile phase B, carrying out gradient elution, and carrying out gradient elution to obtain the impurity 383-1D, impurity 383-1E, impurity 383-1F and impurity 383-1G. And separating the related substances of the dapagliflozin tablet, and detecting to obtain the amount of the related substances. By detecting the amount of related substances, the production of the related substances in the production process is controlled, so that the product quality of the medicine is guaranteed.

Description

Technical Field

[0001] This invention relates to the detection of related substances in pharmaceuticals, and in particular to a method for detecting related substances in dapagliflozin tablets, belonging to the field of pharmaceutical testing technology. Background Technology

[0002] Dapagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor. It increases urinary glucose excretion by inhibiting SGLT2, reducing the reabsorption of filtered glucose, lowering the renal glucose threshold, and thus increasing urinary glucose excretion. It is indicated for adult patients with type 2 diabetes, heart failure, and chronic kidney disease. Its chemical name is (1S)-1,5-anhydride-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-D-glucanol, hydrated with (2S)-1,2-propanediol (1:1:1).

[0003] Currently, impurities are generated during the preparation of dapagliflozin tablets, which may affect the quality of the drug. It is necessary to test for these substances to control their generation during production and ensure product quality. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting related substances in dapagliflozin tablets, thereby solving the problem of detecting related substances in dapagliflozin tablets in the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for detecting related substances in dapagliflozin tablets, wherein the related substances include impurities 383-1D, 383-1E, 383-1F, and 383-1G, and the detection method includes the following detection conditions: High-performance liquid chromatography (HPLC) was used with an octadecyl silica gel column, potassium hydrogen phosphate as a buffer solution and acetonitrile as a diluent, and methanol-water solution as mobile phase A and acetonitrile-water solution as mobile phase B for gradient elution to separate and detect the related substances of dapagliflozin tablets.

[0006] Furthermore, the chromatographic column has dimensions of 250 mm × 5 mm and a diameter of 5 μm.

[0007] Furthermore, the detection wavelength was 255 nm; the flow rate was 1.5 ml / min; the column temperature was 25 °C; and the injection volume was 15 μl.

[0008] Furthermore, the preparation method of mobile phase A solution is as follows: take 2000 ml of water, add 1 ml of methanol, mix, and sonicate for 10 min to obtain the solution; the preparation method of mobile phase B solution is as follows: take 2000 ml of acetonitrile, add 1 ml of water, mix, and sonicate for 10 min to obtain the solution.

[0009] Further, the preparation method of potassium hydrogen phosphate buffer solution is as follows: Weigh 13.66g of potassium hydrogen phosphate, dissolve it in 1500ml of water, and adjust the pH to 11±0.05 with 45% potassium hydroxide aqueous solution.

[0010] Furthermore, the diluent is a potassium hydrogen phosphate buffer solution: acetonitrile with a volume ratio of 40:60.

[0011] Furthermore, the preparation method of the test solution is as follows: Take 5 dapagliflozin tablets, place them in a 100ml volumetric flask, add diluent to dissolve them, sonicate for 10min, shake for 10min to dissolve dapagliflozin, dilute to the mark with diluent, shake well, and filter to obtain the test solution.

[0012] Further, the mixed impurity reference solution: accurately weigh impurity 383-1D, impurity 383-1E, impurity 383-1F, and impurity 383-1G reference standards respectively, dissolve and dilute them with diluent to prepare a solution containing 0.1 ml of impurity 383-1D, impurity 383-1E, impurity 383-1F, and impurity 383-1G per ml.

[0013] Further, the preparation method of the system suitability solution is as follows: Accurately weigh 10 mg of 383-1F reference standard, 20 mg of 383-1D reference standard, 10 mg of 383-1E reference standard, and 10 mg of 383-1G reference standard, and place them separately in different 10 ml volumetric flasks. Add 50% acetonitrile to dissolve and dilute to the mark, and shake well to prepare the stock solutions for each reference standard. Accurately measure 1 ml of each stock solution of reference standard and place it in the same 50 ml volumetric flask. Dilute to the mark with the diluent and shake well to prepare the mixed impurity stock solution. Separately, accurately weigh 12.5 mg of dapagliflozin reference standard and place it in a 100 ml volumetric flask. Accurately add 2 ml of the mixed impurity stock solution, add the diluent, sonicate to dissolve, and dilute to the mark. Shake well.

[0014] Furthermore, the gradient elution program was as follows: 0 min, 90% mobile phase A, 10% mobile phase B; 3 min, 90% mobile phase A, 10% mobile phase B; 33 min, 5% mobile phase A, 95% mobile phase B; 36 min, 5% mobile phase A, 95% mobile phase B; 37 min, 90% mobile phase A, 10% mobile phase B; 43 min, 90% mobile phase A, 10% mobile phase B.

[0015] The technological advancements achieved by this invention due to the adoption of the above technical solutions are as follows: The detection method of the present invention uses the following limited detection conditions: high performance liquid chromatography with octadecyl silica gel as the chromatographic column, potassium hydrogen phosphate as the buffer solution and acetonitrile as the diluent, methanol-water solution as mobile phase A and acetonitrile-water solution as mobile phase B, and gradient elution is performed to separate and detect the related substances of dapagliflozin tablets. Attached Figure Description

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0017] Figure 1 This is a chromatogram of the solution for the system applicability of this invention; Figure 2 This is the chromatogram of the blank solution of this invention; Figure 3 This is the chromatogram of impurity 383-1D reference standard of the present invention; Figure 4 This is the chromatogram of impurity 383-1E reference standard of the present invention; Figure 5 This is the chromatogram of impurity 383-1F reference standard of the present invention; Figure 6 This is the chromatogram of impurity 383-1G reference standard of the present invention; Figure 7 This is the chromatogram of the mixed impurity reference solution of the present invention; Figure 8 This is the chromatogram of the test solution of the present invention; In the attached figure, the vertical axis represents AU; the horizontal axis represents Minutes. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present invention and their accompanying drawings will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] A method for detecting related substances in dapagliflozin tablets, wherein the related substances include impurities 383-1D, 383-1E, 383-1F, and 383-1G. The chemical name of impurity 383-1D is (2S,3R,4R,5S,6R)-2-(4-chloro-3-((4-ethoxyphenyl)(hydroxy)methyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-5-triol, with the molecular formula C2. 21 H 25ClO7; the chemical name of impurity 383-1E is (2-chloro-5-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)phenyl)(4-ethoxyphenyl) methyl ketone, with the molecular formula C 21 H 23 ClO7; the chemical name of impurity 383-1F is (3R,4R,5S,6R)-2-(4-chloro-3-(4-hydroxybenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, with the molecular formula C. 19 H 21 ClO6; the chemical name of impurity 383-1G is 1,5-dehydr-1-C-[4-bromo-3-[(4-ethoxyphenyl)methyl]phenyl]-(1S)-D-glucol, with the molecular formula C 21 H 25 BrO6.

[0020] The testing methods include the following testing conditions: High-performance liquid chromatography (HPLC) was employed with an octadecyl silica column (250 mm × 5 mm, 5 μm); the detection wavelength was 255 nm; the flow rate was 1.5 mL / min; the column temperature was 25 °C; and the injection volume was 15 μL. A gradient elution was performed using potassium hydrogen phosphate buffer and acetonitrile as diluent, with methanol-water solution as mobile phase A and acetonitrile-water solution as mobile phase B. The gradient elution program was as follows: 0 min, 90% mobile phase A, 10% mobile phase B; 3 min, 90% mobile phase A, 10% mobile phase B; 33 min, 5% mobile phase A, 95% mobile phase B; 36 min, 5% mobile phase A, 95% mobile phase B; 37 min, 90% mobile phase A, 10% mobile phase B; 43 min, 90% mobile phase A, 10% mobile phase B. This method enabled the separation and detection of related substances in dapagliflozin tablets.

[0021] It also includes the following steps: (1) Preparation method of mobile phase A solution: Take 2000ml of water, add 1ml of methanol, mix, and sonicate for 10min to obtain the solution; Preparation method of mobile phase B solution: Take 2000ml of acetonitrile, add 1ml of water, mix, and sonicate for 10min to obtain the solution.

[0022] (2) Preparation method of potassium hydrogen phosphate buffer solution: Weigh 13.66g of potassium hydrogen phosphate, add 1500ml of water to dissolve, and adjust the pH to 11±0.05 with 45% potassium hydroxide aqueous solution.

[0023] (3) The dilution solution is a potassium hydrogen phosphate buffer solution with a volume ratio of 40:60:acetonitrile.

[0024] (4) The preparation method of the test solution is as follows: Take 5 dapagliflozin tablets, put them in a 100ml volumetric flask, add diluent to dissolve them, sonicate for 10min, shake for 10min to dissolve the dapagliflozin tablets, dilute to the mark with diluent, shake well, filter, and the solution is obtained.

[0025] (5) Mixed impurity reference solution: Accurately weigh impurity 383-1D, impurity 383-1E, impurity 383-1F and impurity 383-1G reference standards respectively, dissolve and dilute them with diluent to prepare a solution containing 0.1 ml of impurity 383-1D, impurity 383-1E, impurity 383-1F and impurity 383-1G per ml.

[0026] (6) Preparation of system suitability solutions: Accurately weigh 10 mg of 383-1F reference standard, 20 mg of 383-1D reference standard, 10 mg of 383-1E reference standard, and 10 mg of 383-1G reference standard, and place them in separate 10 ml volumetric flasks. Add 50% acetonitrile to dissolve and dilute to the mark, and shake well to prepare stock solutions for each reference standard. Accurately measure 1 ml of each stock solution and place it in the same 50 ml volumetric flask. Dilute to the mark with diluent and shake well to prepare impurity mixed stock solution. Separately, accurately weigh 12.5 mg of dapagliflozin reference standard and place it in a 100 ml volumetric flask. Accurately add 2 ml of impurity mixed stock solution, add diluent, sonicate to dissolve, and dilute to the mark. Shake well.

[0027] By defining the detection conditions, high-performance liquid chromatography (HPLC) was used with octadecyl silica gel as the chromatographic column, potassium hydrogen phosphate as the buffer solution and acetonitrile as the diluent, and methanol-water solution as the mobile phase A and acetonitrile-water solution as the mobile phase B, and gradient elution was performed to separate and detect the related substances of dapagliflozin tablets.

[0028] 1. Instruments, equipment, reagents, and test samples

[0029]

[0030]

[0031] 2. Testing conditions

[0032] 3. Solution preparation (1) Preparation method of mobile phase A solution: Take 2000ml of water, add 1ml of methanol, mix, and sonicate for 10min to obtain the solution; Preparation method of mobile phase B solution: Take 2000ml of acetonitrile, add 1ml of water, mix, and sonicate for 10min to obtain the solution.

[0033] (2) Preparation method of potassium hydrogen phosphate buffer solution: Weigh 13.66g of potassium hydrogen phosphate, add 1500ml of water to dissolve, and adjust the pH to 11±0.05 with 45% potassium hydroxide aqueous solution.

[0034] (3) The dilution solution is a potassium hydrogen phosphate buffer solution with a volume ratio of 40:60:acetonitrile.

[0035] (4) The preparation method of the test solution is as follows: Take 5 dapagliflozin tablets, put them in a 100ml volumetric flask, add diluent to dissolve them, sonicate for 10min, shake for 10min to dissolve dapagliflozin, dilute to the mark with diluent, shake well, filter, and the solution is obtained.

[0036] (5) Mixed impurity reference solution: Accurately weigh impurity 383-1D, impurity 383-1E, impurity 383-1F and impurity 383-1G reference standards respectively, dissolve and dilute them with diluent to prepare a solution containing 0.1 ml of impurity 383-1D, impurity 383-1E, impurity 383-1F and impurity 383-1G per ml.

[0037] (6) Preparation of system suitability solutions: Accurately weigh 10 mg of 383-1F reference standard, 20 mg of 383-1D reference standard, 10 mg of 383-1E reference standard, and 10 mg of 383-1G reference standard, and place them in separate 10 ml volumetric flasks. Add 50% acetonitrile to dissolve and dilute to the mark, and shake well to prepare stock solutions for each reference standard. Accurately measure 1 ml of each stock solution and place it in the same 50 ml volumetric flask. Dilute to the mark with diluent and shake well to prepare impurity mixed stock solution. Separately, accurately weigh 12.5 mg of dapagliflozin reference standard and place it in a 100 ml volumetric flask. Accurately add 2 ml of impurity mixed stock solution, add diluent, sonicate to dissolve, and dilute to the mark. Shake well.

[0038] 4. Test Results and Calculations The control solution, reference solution, and test solution were analyzed using high-performance liquid chromatography (HPLC) to obtain corresponding chromatographic data. The amounts of related substances were calculated from the chromatographic data. In the chromatogram of the system suitability test solution, the elution order was 383-1F, 383-1D, 383-1E, and 383-1G.

[0039] 5. Method Validation 5.1 Exclusivity System suitability solutions: Accurately weigh 10 mg of 383-1F reference standard, 20 mg of 383-1D reference standard, 10 mg of 383-1E reference standard, and 10 mg of 383-1G reference standard, and place them separately in different 10 ml volumetric flasks. Add 50% acetonitrile to dissolve and dilute to the mark, then shake well to prepare the stock solutions for each reference standard. Accurately measure 1 ml of each stock solution and place it in the same 50 ml volumetric flask. Dilute to the mark with the diluent and shake well to prepare the mixed impurity stock solution. Separately, accurately weigh 12.5 mg of dapagliflozin reference standard and place it in a 100 ml volumetric flask. Accurately add 2 ml of the mixed impurity stock solution, add the diluent, sonicate to dissolve, and dilute to the mark. Shake well.

[0040] Acceptance criteria: In the system suitability solution chromatogram, the resolution between the 383-1E and 383-1G peaks and the dapagliflozin peak should be no less than 2.0, and the resolution between each known impurity peak should be no less than 1.0.

[0041] Accurately measure 15 μl and inject it into the liquid chromatograph. Record the chromatogram data. The results are shown in the table below:

[0042] Conclusion: The resolutions between peaks 383-1E and 383-1G and the dapagliflozin peak were 13.74 and 2.74, respectively, both greater than 2.0, which meets the requirements; the minimum resolution between each known impurity peak and its adjacent impurity peak was 10.92, greater than 1.0, which also meets the requirements.

[0043] 5.2 Limit of Detection and Limit of Quantification Prepare two parallel solutions of the limit of detection concentration and inject them separately. The signal-to-noise ratio (S / N) of each component should be ≥3. Prepare six parallel solutions of the limit of quantitation concentration and inject them separately. The signal-to-noise ratio (S / N) of each component should be ≥10. For the six solutions of the limit of quantitation, the RSD of the retention time of each component peak should not be greater than 1%, and the RSD of the peak area should not be greater than 10%.

[0044] Accurately measure 15 μl of each sample and inject them into the liquid chromatograph. Record the chromatograms. The results are shown in the table below:

[0046]

[0047]

[0048]

[0049] Conclusion: When the detection limit was tested twice, the signal-to-noise ratio (S / N) of each component was greater than 3. When six parallel solutions of the quantitation limit concentration were prepared, the S / N of each component was greater than 10. The RSD of the peak retention time of each component was 0.1%, which was less than 1%. The RSD of the peak area was 3.9%-9.0%, which was less than 10%. The quantitation limit concentration of each component was less than 30% of the limit concentration, which met the validation requirements.

[0050] 5.3 Linearity and Range Prepare linear solutions according to the table below, accurately measure 15 μl of each linear solution, inject them into the liquid chromatograph, and record the chromatogram data.

[0051]

[0052] Acceptance criteria: Within a certain concentration range, the injection concentration and peak area should have a linear relationship, the correlation coefficient r should be no less than 0.990, the absolute value of the Y-axis intercept / 100% response value should be no greater than 25%, and the RSD of the response factor should be no greater than 10%.

[0053]

[0054]

[0055]

[0057]

[0058]

[0059]

[0060] Conclusion: The correlation coefficients (r) of all components are greater than 0.990, the absolute values ​​of the Y-intercept / 100% response value are all less than 25%, and the RSD of the response factor is less than 10%, indicating good linearity of this method. The correction factors for 383-1F, 383-1D, 383-1E, and 383-1G are all between 0.9 and 1.1, and are calculated as 1.0.

[0061] 5.4 Accuracy Acceptance criteria: Using the self-comparison method, the average recovery rate at each concentration should be between 90% and 108%, and the RSD should not exceed 10%.

[0062]

[0063]

[0064]

[0065]

[0066] Conclusion: The average recovery rates of all impurities were between 90% and 108%, and the RSDs were all less than 10%, which met the validation requirements. The accuracy of this method is good.

[0067] 5.5 Repeatability Acceptance criteria: The RSD of known impurities in 6 labeled test solutions is not greater than 10%.

[0068]

[0069] Conclusion: The RSD of known impurities in the 6-point labeled test solution is between 1.2% and 4.8%, which is less than 10%, meeting the validation requirements. The method has good repeatability.

[0070] 5.6 Intermediate Precision Acceptance criteria: The RSD of known impurities in 12 labeled test solutions should not exceed 15%.

[0071]

[0072] Conclusion: The RSD of known impurities in the 12-labeled test solution is between 1.6% and 4.9%, less than 15%, which meets the requirements. The intermediate precision of this method is good.

[0073] All of the above validation items meet the requirements, and the method of this application can be used for the detection of related substances in dapagliflozin tablets.

[0074] In summary, this embodiment demonstrates that high-performance liquid chromatography (HPLC) can be used to detect related substances in dapagliflozin tablets. These related substances include impurities 383-1D, 383-1E, 383-1F, and 383-1G. By defining the detection conditions for HPLC, the amount of related substances in dapagliflozin tablets can be accurately detected. The detection method exhibits high sensitivity and good resolution. Furthermore, the detection time is short, improving the efficiency of the detection process.

[0075] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and all fall within the protection scope of this application.

Claims

1. A method for detecting related substances in dapagliflozin tablets, characterized in that, The related substances include impurities 383-1D, 383-1E, 383-1F, and 383-1G. The detection method includes the following detection conditions: High-performance liquid chromatography (HPLC) was used with an octadecyl silica gel column, potassium hydrogen phosphate as a buffer solution and acetonitrile as a diluent, and methanol-water solution as mobile phase A and acetonitrile-water solution as mobile phase B for gradient elution to separate and detect the related substances of dapagliflozin tablets.

2. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, The chromatographic column has a size of 250 mm × 5 mm and a diameter of 5 μm.

3. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, The detection wavelength was 255 nm; the flow rate was 1.5 ml / min; the column temperature was 25 °C; and the injection volume was 15 μl.

4. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, Preparation method of mobile phase A solution: Take 2000ml of water, add 1ml of methanol, mix, and sonicate for 10min. Preparation method of mobile phase B solution: Take 2000ml of acetonitrile, add 1ml of water, mix, and sonicate for 10min.

5. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, Preparation method of potassium hydrogen phosphate buffer solution: Weigh 13.66g of potassium hydrogen phosphate, add 1500ml of water to dissolve, and adjust the pH to 11±0.05 with 45% potassium hydroxide aqueous solution.

6. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, The dilution solution is a potassium hydrogen phosphate buffer solution: acetonitrile with a volume ratio of 40:

60.

7. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, The preparation method of the test solution is as follows: Take 5 dapagliflozin tablets, place them in a 100ml volumetric flask, add diluent to dissolve them, sonicate for 10min, shake for 10min to dissolve dapagliflozin, dilute to the mark with diluent, shake well, and filter to obtain the test solution.

8. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, Mixed impurity reference solution: Accurately weigh impurity 383-1D, impurity 383-1E, impurity 383-1F, and impurity 383-1G reference standards respectively, dissolve and dilute them with diluent to prepare a solution containing 0.1 ml of each of impurity 383-1D, impurity 383-1E, impurity 383-1F, and impurity 383-1G per ml.

9. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, Preparation of system suitability solutions: Accurately weigh 10 mg of 383-1F reference standard, 20 mg of 383-1D reference standard, 10 mg of 383-1E reference standard, and 10 mg of 383-1G reference standard, and place them separately in different 10 ml volumetric flasks. Add 50% acetonitrile to dissolve and dilute to the mark, then shake well to prepare the stock solutions for each reference standard. Accurately measure 1 ml of each stock solution and place it in the same 50 ml volumetric flask. Dilute to the mark with the diluent and shake well to prepare the mixed impurity stock solution. Separately, accurately weigh 12.5 mg of dapagliflozin reference standard and place it in a 100 ml volumetric flask. Accurately add 2 ml of the mixed impurity stock solution, add the diluent, sonicate to dissolve, and dilute to the mark. Shake well.

10. The method for detecting related substances in dapagliflozin tablets according to claim 1, characterized in that, The gradient elution program was as follows: 0 min, 90% mobile phase A, 10% mobile phase B; 3 min, 90% mobile phase A, 10% mobile phase B; 33 min, 5% mobile phase A, 95% mobile phase B; 36 min, 5% mobile phase A, 95% mobile phase B; 37 min, 90% mobile phase A, 10% mobile phase B; 43 min, 90% mobile phase A, 10% mobile phase B.

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