A method for simultaneously determining the content of dapagliflozin metformin

Abstract

The application provides a method for simultaneously determining the content of dapagliflozin and metformin, which adopts a reverse-phase high performance liquid chromatography method to separate and determine the content of dapagliflozin and metformin in dapagliflozin and metformin sustained-release tablets. The method has good separation degree, solvent does not interfere with detection, and good method specificity. The detection method is simple, rapid, accurate, high in sensitivity, good in repeatability and accuracy, can quickly and accurately perform qualitative and quantitative analysis on the content of dapagliflozin and metformin hydrochloride in dapagliflozin and metformin sustained-release tablets, and ensures the controllability of the quality of the product.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis, and more specifically to a method for simultaneously determining the content of dapagliflozin and metformin. Background Technology

[0002] Currently, commonly used drugs for treating type 2 diabetes include traditional drugs such as biguanides, sulfonylureas, α-glucosidase inhibitors, and thiazolidinediones, as well as newer hypoglycemic drugs such as sodium-glucose cotransporter-2 (SGLT-2) inhibitors and dipeptidyl peptidase-IV (DDP-IV) inhibitors. Sodium-glucose cotransporter-2 (SGLT-2) is a protein mainly expressed in the proximal tubules of the kidney, and its main function is to reabsorb glucose filtered from urine in the renal tubules.

[0003] The molecular formula of metformin hydrochloride is C4H 11 N5·HCl, with a molecular weight of 165.62, has the structural formula shown in Formula i. Metformin hydrochloride is a highly soluble, low-permeability drug that primarily acts on extrapancreatic tissues, inhibiting intestinal glucose absorption, increasing peripheral tissue glucose utilization, and reducing hepatic gluconeogenesis, thereby lowering blood glucose levels. Because metformin hydrochloride has a significant hypoglycemic effect, does not cause hypoglycemia, and can significantly reduce postprandial hyperglycemia, it is suitable for patients with type 2 diabetes and is a first-line drug for treating type 2 diabetes. It also improves insulin resistance, and therefore is widely used clinically.

[0004] The molecular formula of dapagliflozin is C 21 H 25 ClO6·C3H8O2·H2O, with a molecular weight of 502.98, has the structural formula shown in Formula ii. Dapagliflozin is an SGLT-2 inhibitor that reduces the reabsorption of filtered glucose by inhibiting SGLT-2, thereby lowering the renal glucose threshold and increasing the excretion of glucose in the urine.

[0005]

[0006] Dapagliflozin and metformin hydrochloride belong to the sodium-glucose cotransporter-2 (SGLT-2) inhibitor class and the biguanide class, respectively. Their mechanisms of action differ, and combined use has a synergistic effect and can reduce adverse reactions. Currently marketed dapagliflozin-metformin extended-release tablets, marketed under the brand name Xigduo XR, are used to treat type 2 diabetes in adults. Summary of the Invention

[0007] The purpose of this invention is to simultaneously determine the content of dapagliflozin and metformin hydrochloride in dapagliflozin-metformin extended-release tablets using the same high-performance liquid chromatography system.

[0008] To achieve the above objectives, the technical solution of the present invention is: a method for simultaneously determining the content of dapagliflozin and metformin, characterized in that the method is a high-performance liquid chromatography method, using a mixed stationary phase chromatographic column with reversed phase, anion and cation exchange functions.

[0009] In some embodiments, the chromatographic column uses high-purity porous spherical silica particles with charged nanopolymer microspheres as the stationary phase; in some typical embodiments, the chromatographic column is a Thermo Acclaim Trinity P1 with specifications of 3mm × 150mm and 3μm.

[0010] In some implementations, the method uses an ammonium dihydrogen phosphate buffer-acetonitrile mixture as the eluent and elutes isocratically.

[0011] In some embodiments, the concentration of the ammonium dihydrogen phosphate buffer solution is 16 g / L to 18 g / L, and in some typical embodiments, the concentration of the ammonium dihydrogen phosphate buffer solution is 17 g / L.

[0012] In some embodiments, the ammonium dihydrogen phosphate buffer solution is an aqueous solution of ammonium dihydrogen phosphate.

[0013] In some embodiments, the pH of the ammonium dihydrogen phosphate buffer solution is 4.7 to 5.2; in some typical embodiments, the pH of the ammonium dihydrogen phosphate buffer solution is 5.0.

[0014] In some embodiments, the pH of the ammonium dihydrogen phosphate buffer solution is adjusted by triethylamine.

[0015] In some embodiments, the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile is 75:25 to 71:29, and in some typical embodiments, the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile is 73:27.

[0016] In some embodiments, the flow rate of the eluent is 0.6 to 1.0 ml per minute, and in some typical embodiments, the flow rate of the eluent is 0.8 ml per minute.

[0017] In some embodiments, the column temperature is 32–38°C; in some typical embodiments, the column temperature is 35°C.

[0018] In some embodiments, the method is high-performance liquid chromatography with a diode array detector, wherein the detection wavelength of dapagliflozin is 218–222 nm and the detection wavelength of metformin hydrochloride is 253–257 nm; in some typical embodiments, the detection wavelength of dapagliflozin is 220 nm and the detection wavelength of metformin hydrochloride is 255 nm.

[0019] On the other hand, the present invention provides a method for simultaneously determining the content of dapagliflozin and metformin, characterized in that:

[0020] The analytical method is performed on a high-performance liquid chromatograph; it uses a mixed stationary phase column that simultaneously has reversed-phase, anion and cation exchange functions.

[0021] The analytical method employs a diode array detector, with dapagliflozin detected at a wavelength of 220 nm and metformin hydrochloride detected at a wavelength of 255 nm.

[0022] The analytical method uses a column temperature of 35°C.

[0023] The analytical method uses an ammonium dihydrogen phosphate buffer solution-acetonitrile mixed solution as the eluent, and performs isocratic elution. The concentration of the ammonium dihydrogen phosphate buffer solution is 17 g / L, the pH of the ammonium dihydrogen phosphate buffer solution is 5.0, and the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile is 73:27.

[0024] The flow rate of the eluent was 0.8 ml per minute;

[0025] Inject the test solution and the reference solution separately;

[0026] The contents of dapagliflozin and metformin in the test sample were calculated using the external standard method.

[0027] In some specific embodiments, the present invention provides a method for simultaneously determining the content of dapagliflozin metformin, characterized by comprising the following steps:

[0028] (1) Preparation of test solution: Take an appropriate amount of dapagliflozin metformin sustained-release tablets, grind them finely in an agate mortar, transfer them all to a volumetric flask, add an appropriate amount of methanol to dissolve by sonication, make up to volume with methanol, accurately measure an appropriate amount of supernatant and place it in another volumetric flask, dilute to the mark with a mixture of 17 g / L ammonium dihydrogen phosphate buffer solution (pH 5.0) and acetonitrile (70:30), shake well, filter, and take the filtrate as the test solution;

[0029] (2) Preparation of reference solution: Accurately weigh appropriate amounts of dapagliflozin and metformin hydrochloride reference standards, dissolve and dilute them with a mixture of 17 g / L ammonium dihydrogen phosphate buffer solution (pH 5.0) and acetonitrile (70:30), make up to volume, and shake well to obtain the solution.

[0030] (3) Determination of test samples: A ThermoAcclaim Trinity P1 (3mm×150mm, 3um) column was used; 17g / L ammonium dihydrogen phosphate buffer (pH5.0)-acetonitrile (73:27) was used as the mobile phase, and the eluent flow rate was 0.8ml per minute; the column temperature was 35℃; a diode array detector was used as the detector, with dapagliflozin detected at a wavelength of 220nm and metformin hydrochloride detected at a wavelength of 255nm; the injection volume was 3μl; the test sample solution and the reference solution were respectively injected into the liquid chromatograph, and isocratic elution was performed, and the chromatograms were recorded.

[0031] (4) Content calculation:

[0032]

[0033] Where: the correction factor f = C R / A R ;

[0034] C R This indicates the concentration of the dapagliflozin / metformin hydrochloride reference solution, in mg / ml.

[0035] A R This indicates the peak area of ​​the dapagliflozin / metformin hydrochloride reference solution;

[0036] A t This indicates the peak area of ​​dapagliflozin / metformin hydrochloride in the test solution;

[0037] G indicates the labeled amount of dapagliflozin / metformin hydrochloride, in mg;

[0038] N represents the dilution factor of the test sample.

[0039] Those skilled in the art will readily understand that, in implementing this invention, they can appropriately adjust the order of the above steps according to actual needs without affecting the implementation of the assay method, such as the order of preparation of the reference solution and the test solution.

[0040] In this invention, "appropriate amount" means that, according to the experimental purpose, the amount of each compound is within the detection limit or quantitation limit of its high-performance liquid chromatograph.

[0041] In this invention, the diluent refers to a mixture of 17 g / L ammonium dihydrogen phosphate buffer solution (pH 5.0) and acetonitrile (70:30).

[0042] In this invention, "mL" refers to milliliters; "mg" refers to milligrams; "μg" refers to micrograms; and "min" refers to minutes.

[0043] The beneficial effects of this invention are as follows: The method provided by this invention can accurately determine the content of dapagliflozin and metformin hydrochloride in dapagliflozin-metformin extended-release tablets. It has good separation, the solvent does not interfere with the detection, the method has good specificity, and the detection method is simple, rapid, accurate, sensitive, repeatable and accurate. It can quickly and accurately perform qualitative and quantitative analysis of the content of dapagliflozin and metformin hydrochloride in dapagliflozin-metformin extended-release tablets, ensuring the controllability of the product quality. Attached Figure Description

[0044] Figure 1 Overlay chromatogram of reference standard and blank excipient solution at detector absorption wavelength of 220 nm

[0045] Figure 2 Overlay chromatogram of reference standard and blank excipient solution at detector absorption wavelength of 255 nm

[0046] Figure 3 The chromatogram of the test solution at a detector absorption wavelength of 220 nm

[0047] Figure 4 The chromatogram of the test solution at a detector absorption wavelength of 255 nm

[0048] Figure 5 Refer to the chromatogram of the reference solution in Example 1. Detailed implementation method:

[0049] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0050] The metformin hydrochloride reference standard used in this invention was purchased from the National Institutes for Food and Drug Control, the dapagliflozin reference standard was produced by Nanjing Chia Tai Tianqing Pharmaceutical Co., Ltd., and the dapagliflozin metformin extended-release tablets were produced by Nanjing Chia Tai Tianqing Pharmaceutical Co., Ltd.

[0051] Example 1: Specificity Test

[0052] Diluent: Weigh 17g of ammonium dihydrogen phosphate, dissolve it in 1 liter of water, adjust the pH to 5.0 with triethylamine, and mix 700 ml of this buffer solution with 300 ml of acetonitrile until homogeneous.

[0053] Blank excipient solution: Take a total of 500 mg of blank excipients (hydroxypropyl methylcellulose, microcrystalline cellulose, anhydrous lactose and crospovidone), place them in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge and take 2.00 ml of the supernatant into a 20 ml volumetric flask, dilute to the mark with diluent, shake well, filter, and take the filtrate as the blank excipient solution.

[0054] Reference solution: Weigh appropriate amounts of dapagliflozin and metformin hydrochloride reference standards accurately, dissolve and dilute with diluent to prepare a solution containing approximately 10 μg of dapagliflozin and 1 mg of metformin hydrochloride per 1 ml, which is used as the reference solution.

[0055] Test solution: Take 5 tablets of dapagliflozin metformin sustained-release tablets, grind them finely in an agate mortar, transfer the whole tablet to a 500ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge and take 2.00ml of the supernatant into a 20ml volumetric flask, dilute to the mark with diluent, shake well, filter, and take the filtrate as the test solution.

[0056] Chromatographic conditions: A ThermoAcclaim Trinity P1 (3 mm × 150 mm, 3 μm) column was used; the mobile phase was 17 g / L ammonium dihydrogen phosphate buffer (pH 5.0)-acetonitrile (73:27); the eluent flow rate was 0.8 mL / min; the column temperature was 35 °C; a diode array detector was used as the detector, with dapagliflozin detected at 220 nm and metformin hydrochloride detected at 255 nm; the injection volume was 3 μl.

[0057] Test method: Accurately measure 3 μl each of the blank excipient solution and the reference solution, inject them into the liquid chromatograph, and record the chromatograms. The superimposed chromatogram of the blank excipient solution and the reference solution is shown below. Figures 1-2 .

[0058] The results showed that the excipients did not interfere with the detection of the two components and had good specificity.

[0059] Example 2: Linearity and Range

[0060] Solution preparation: Take appropriate amounts of dapagliflozin and metformin hydrochloride reference standards, dissolve and dilute with diluent to prepare linear solutions containing approximately 2 μg dapagliflozin (0.2 mg metformin hydrochloride), 5 μg dapagliflozin (0.5 mg metformin hydrochloride), 8 μg dapagliflozin (0.8 mg metformin hydrochloride), 10 μg dapagliflozin (1 mg metformin hydrochloride), 12 μg dapagliflozin (1.2 mg metformin hydrochloride), and 15 μg dapagliflozin (1.5 mg metformin hydrochloride) per milliliter (equivalent to 20%, 50%, 80%, 100%, 120%, and 150% of the test sample concentrations).

[0061] Experimental method: Under the chromatographic conditions of Example 1, accurately measure 3 μl of each of the above linear solutions, inject them into the liquid chromatograph, record the chromatogram, plot the standard curve with concentration as the abscissa and peak area as the ordinate, and calculate the regression equation.

[0062] The results showed that the linear regression equation for dapagliflozin was y = 1.0629 × 10⁻⁶. 7x – 7.7372 × 10 2 (γ=0.9999), the concentration showed a good linear relationship with the peak area in the range of 0.00203mg / ml to 0.0152mg / ml. The linear regression equation for metformin hydrochloride was y=1.3343×10 6 x + 2.0710 × 10 4 (γ=0.9999), the concentration showed a good linear relationship with the peak area in the range of 0.1992mg / ml to 1.494mg / ml.

[0063] Example 3: Accuracy

[0064] Reference solution: Weigh appropriate amounts of dapagliflozin and metformin hydrochloride reference standards accurately, dissolve and dilute with diluent to prepare a solution containing approximately 10 μg of dapagliflozin and 1 mg of metformin hydrochloride per 1 ml, which is used as the reference solution.

[0065] 80% recovery solution: Accurately weigh approximately 10 mg of dapagliflozin reference standard, approximately 0.8 g of metformin hydrochloride reference standard, and approximately 500 mg of blank excipient. Place them in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge, and transfer 2.00 ml of the supernatant to a 20 ml volumetric flask. Dilute to the mark with diluent, shake well, filter, and use the filtrate as the 80% recovery solution. Prepare three parallel aliquots.

[0066] 100% recovery solution: Accurately weigh approximately 12.5 mg of dapagliflozin reference standard, approximately 1 g of metformin hydrochloride reference standard, and approximately 500 mg of blank excipient. Place them in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge, and transfer 2.00 ml of the supernatant to a 20 ml volumetric flask. Dilute to the mark with diluent, shake well, filter, and use the filtrate as the 100% recovery solution. Prepare three parallel aliquots.

[0067] 120% recovery solution: Accurately weigh approximately 15 mg of dapagliflozin reference standard, approximately 1.2 g of metformin hydrochloride reference standard, and approximately 500 mg of blank excipient. Place them in a 100 ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge, and transfer 2.00 ml of the supernatant to a 20 ml volumetric flask. Dilute to the mark with diluent, shake well, filter, and use the filtrate as the 120% recovery solution. Prepare three parallel aliquots.

[0068] Experimental method: Under the chromatographic conditions of Example 1, 3 μl each of the reference solution and the recovery solution were injected into the liquid chromatograph, the chromatograms were recorded, and the recovery rate was calculated by the external standard method. The experimental results are shown in Tables 1 and 2.

[0069] Table 1. Results of Dapagliflozin Accuracy Test

[0070]

[0071]

[0072] Table 2. Results of the accuracy test for metformin hydrochloride.

[0073]

[0074] The results showed that the average recoveries of dapagliflozin and metformin hydrochloride were 99.1% and 100.1%, respectively, both within the range of 98.0% to 102.0%; the RSDs were 0.3% and 0.8%, respectively, both less than 2.0%, indicating good method accuracy.

[0075] Example 4: Precision

[0076] 4.1 Sample injection precision

[0077] Under the chromatographic conditions of Example 1, the reference solution from "Example 1" was injected six times consecutively, the chromatograms were recorded, and the peak areas and retention times RSDs were calculated. The results are shown in Table 3.

[0078] Table 3 Results of the injection precision test

[0079]

[0080]

[0081] The results showed that after six consecutive injections, the peak area RSD of dapagliflozin and metformin hydrochloride was less than 2.0%, and the retention time RSD was less than 1.0%, indicating good injection precision.

[0082] 4.2 Repeatability

[0083] Reference solution: Weigh appropriate amounts of dapagliflozin and metformin hydrochloride reference standards accurately, dissolve and dilute with diluent to prepare a solution containing approximately 10 μg of dapagliflozin and 1 mg of metformin hydrochloride per 1 ml, which is used as the reference solution.

[0084] Test solution: Take 5 dapagliflozin / metformin extended-release tablets, grind them finely in an agate mortar, transfer the entire mixture to a 500ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge, and take 2.00ml of the supernatant into a 20ml volumetric flask. Dilute to the mark with diluent, shake well, filter, and use the filtrate as the test solution. Prepare 6 portions using the same method.

[0085] Under the chromatographic conditions of Example 1, accurately measure 3 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the contents of dapagliflozin and metformin hydrochloride using the external standard method. The RSDs of the six content results were calculated, and the results are shown in Table 4.

[0086] Table 4 Results of Repeatability Tests

[0087]

[0088]

[0089] The results showed that the RSD of dapagliflozin and metformin hydrochloride in the six test solutions was less than 2.0%, indicating that the method had good repeatability.

[0090] 4.3 Intermediate Precision

[0091] Different operators prepared and tested each solution under the repeatability test according to the chromatographic conditions of Example 1 at different times. The results are shown in Table 5.

[0092] Table 5 Results of intermediate precision test

[0093]

[0094] The results showed that the RSD of dapagliflozin and metformin hydrochloride in the 12 test solutions was less than 2.0%, indicating that the method had good intermediate precision.

[0095] Example 5 Durability

[0096] The preparation of the reference solution and the test solution is the same as in Example 1.

[0097] The effects of single variables were investigated by individually varying the flow rate, column temperature, detection wavelength, organic phase ratio, pH of the ammonium dihydrogen phosphate buffer, and salt concentration, while keeping all other conditions consistent with those in Example 1. 3 μL each of the blank solvent, reference solution, and test solution were injected into the liquid chromatograph, and chromatograms were recorded. The contents of dapagliflozin and metformin in the test solution were investigated, and the results are shown in the table below.

[0098]

[0099]

[0100] The results showed that, under varying system parameters, the contents of dapagliflozin and metformin hydrochloride did not change significantly compared to the chromatographic conditions in Example 1, indicating that the method was robust.

[0101] Example 6: Sample Measurement

[0102] Reference solution: Accurately weigh appropriate amounts of dapagliflozin and metformin hydrochloride reference standards, dissolve and dilute with diluent to prepare a solution containing approximately 10 μg of dapagliflozin and 1 mg of metformin hydrochloride per 1 ml. Prepare two aliquots using the same method.

[0103] Test solution: Take 5 tablets of dapagliflozin / metformin extended-release tablets from different batches, grind them finely in an agate mortar, transfer the entire mixture to a 500ml volumetric flask, add an appropriate amount of methanol, sonicate for 15 minutes, dilute to volume with methanol, centrifuge, and take 2.00ml of the supernatant into a 20ml volumetric flask. Dilute to the mark with solvent, shake well, filter, and use the filtrate as the test solution. Prepare two portions of each batch using the same method.

[0104] Under the chromatographic conditions of Example 1, accurately measure 3 μl each of the blank excipient solution, reference solution, and each batch of test solution, inject them into the liquid chromatograph, and record the chromatograms. Calculate the contents of dapagliflozin and metformin hydrochloride using the external standard method. The results are shown in Table 6.

[0105] Table 6 Sample Measurement Results

[0106]

[0107] Reference Example 1

[0108] The solutions were prepared in the same manner as the control solution and test solution in Example 6. Two copies of each were prepared.

[0109] Chromatographic conditions: A Waters μBondapak C18 column (10 μm, 3.9 × 300 mm) was used, with a mobile phase of 0.5 g / L NaCl and 0.5 g / L sodium heptanesulfonate (pH adjusted to 3.85 with phosphoric acid) mixed solution-acetonitrile (90:10), and isocratic elution. The flow rate was 1.0 mL / min, the column temperature was 30 °C, the detection wavelength was 218 nm, and the injection volume was 10 μL.

[0110] Determination method: Take 15 μl of each solution and determine it under the above chromatographic conditions, and record the chromatogram.

[0111] The results are as follows Figure 5 As shown: Metformin hydrochloride has a retention time of approximately 6.6 minutes, while dapagliflozin did not show a peak within 20 minutes.

Claims

1. A method for simultaneously determining the content of dapagliflozin and metformin, characterized in that, The method is high-performance liquid chromatography, and includes the following steps: (1) Preparation of test solution: Take dapagliflozin metformin sustained-release tablets, add methanol and sonicate to dissolve, make up to volume with methanol, accurately measure the supernatant and place it in another volumetric flask, dilute to the mark with ammonium dihydrogen phosphate buffer solution-acetonitrile mixture, shake well, filter, and take the filtrate as the test solution; the blank excipients of dapagliflozin are hydroxypropyl methylcellulose, microcrystalline cellulose, anhydrous lactose and crospovidone; (2) Preparation of reference solution: Accurately weigh dapagliflozin and metformin hydrochloride reference standards, dissolve and dilute them with ammonium dihydrogen phosphate buffer solution-acetonitrile mixture, make up to volume, shake well, and the solution is ready; (3) Determination of test sample: A Thermo Acclaim Trinity P1 column was used; the mobile phase was ammonium dihydrogen phosphate buffer solution-acetonitrile; the column temperature was 32-38℃; a diode array detector was used as the detector, the detection wavelength of dapagliflozin was 218-222nm and the detection wavelength of metformin hydrochloride was 253-257nm; the test sample solution and the reference solution were respectively injected into the liquid chromatograph, and isocratic elution was performed, and the chromatograms were recorded; the ratio of the mobile phase ammonium dihydrogen phosphate buffer solution to acetonitrile was 75:25-71:29; (4) Calculate the contents of dapagliflozin and metformin hydrochloride in the test sample by external standard method.

2. The method as described in claim 1, characterized in that: The chromatographic column has the following specifications: 3mm × 150mm, 3μm.

3. The method as described in claim 1, characterized in that: The ratio of ammonium dihydrogen phosphate buffer solution to acetonitrile in the mobile phase is 73:

27.

4. The method as described in claim 1, characterized in that: The concentration of the ammonium dihydrogen phosphate buffer solution is 16 g / L to 18 g / L.

5. The method as described in claim 4, characterized in that: The concentration of the ammonium dihydrogen phosphate buffer solution is 17 g / L.

6. The method as described in claim 1, characterized in that: The pH value of the ammonium dihydrogen phosphate buffer solution is 4.7–5.

2.

7. The method as described in claim 6, characterized in that: The pH of the ammonium dihydrogen phosphate buffer solution is 5.

0.

8. The method as described in claim 6, characterized in that: The pH of the ammonium dihydrogen phosphate buffer solution is adjusted by triethylamine.

9. The method as described in claim 1, characterized in that: The flow rate of the eluent is 0.6 to 1.0 mL per minute.

10. The method as described in claim 9, characterized in that: The flow rate of the eluent is 0.8 mL per minute.

11. The method as described in claim 1, characterized in that: The column temperature of the chromatographic column is 35℃.

12. The method as described in claim 1, characterized in that: The detection wavelength for dapagliflozin is 220 nm, and the detection wavelength for metformin hydrochloride is 255 nm.

13. The method as described in claim 1, characterized in that: The injection volume was 3 μL.

14. The method as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of test solution: Take dapagliflozin metformin sustained-release tablets, add methanol and sonicate to dissolve, make up to volume with methanol, accurately measure the supernatant and place it in another volumetric flask, dilute to the mark with 17 g / L ammonium dihydrogen phosphate buffer solution-acetonitrile mixture, shake well, filter, and take the filtrate as the test solution; the blank excipients of dapagliflozin are hydroxypropyl methylcellulose, microcrystalline cellulose, anhydrous lactose and crospovidone; the pH of the ammonium dihydrogen phosphate buffer solution is 5.0, and the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile is 70:30; (2) Preparation of reference solution: Accurately weigh dapagliflozin and metformin hydrochloride reference standards, dissolve and dilute them with 17 g / L ammonium dihydrogen phosphate buffer solution-acetonitrile mixture, make up to volume, shake well, and the solution is obtained; the pH of the ammonium dihydrogen phosphate buffer solution is 5.0, and the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile is 70:

30. (3) Test sample determination: A Thermo Acclaim Trinity P1 column was used; the mobile phase was 17 g / L ammonium dihydrogen phosphate buffer solution-acetonitrile, the eluent flow rate was 0.8 mL / min; the column temperature was 35℃; a diode array detector was used as the detector, the detection wavelength of dapagliflozin was 220 nm, and the detection wavelength of metformin hydrochloride was 255 nm; the injection volume was 3 μL; the test sample solution and the reference solution were respectively injected into the liquid chromatograph, and isocratic elution was performed, and the chromatograms were recorded; the column specifications were: 3 mm × 150 mm, 3 μm; the pH of the ammonium dihydrogen phosphate buffer solution was 5.0, and the ratio of the ammonium dihydrogen phosphate buffer solution to acetonitrile in the mobile phase was 73:27; (4) Calculate the contents of dapagliflozin and metformin hydrochloride in the test sample by external standard method.

Images