A method for separating and determining related substances of key starting materials of dapagliflozin by HPLC

Abstract

The invention relates to a method for separating and determining related substances of a key starting raw material of dapagliflozin by using HPLC, comprising the following steps: using octadecylsilane bonded silica gel as a filler; using 0.002% to 0.1% (V / V) phosphoric acid aqueous solution as a mobile phase A, and using an acetonitrile-methanol mixed ratio as a mobile phase B, and performing gradient elution; the invention optimizes the detection result by comprehensively considering the comprehensive influence of an analytical column, a mobile phase, a gradient elution program, a flow rate, and a column temperature on separation and detection, and can quickly and efficiently separate impurities A, B, C, D, E, F, G, H, I, J, K, and L in the key starting raw material of dapagliflozin under the same chromatographic conditions, and the detection method has high sensitivity, strong specificity, is fast and simple, and is easy to operate, and can effectively control its quality, and is suitable for separating and determining related substances of the key starting raw material of dapagliflozin.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a method for separating and determining related substances of key starting raw materials of dapagliflozin by using HPLC. Background Art

[0002] Dapagliflozin (trade name Farxiga), a new anti-diabetic drug jointly developed by Bristol-Myers Squibb and AstraZeneca, was approved for marketing by the European Medicines Agency on November 12, 2012. It is the first SGLT2 inhibitor approved for the treatment of type 2 diabetes. It was approved for marketing by the US FDA on January 8, 2014 to improve blood sugar control in adult patients with type 2 diabetes. Dapagliflozin is a new sodium-glucose co-transporter 2 (SGLT2) inhibitor drug that inhibits the reabsorption of glucose by the kidneys, increases glucose excretion, and thus reduces the elevated blood sugar levels of diabetic patients. Its molecular formula: C 21 H 25 ClO6·C3H8O2·H2O molecular weight: 502.98, chemical structure is as follows (1):

[0003]

[0004] 5-Bromo-2-chloro-4'-ethoxydiphenylmethane is the key starting material for the synthesis of dapagliflozin, with the molecular formula: C 15 H 14 BrClO, molecular weight: 325.63, chemical structure is as follows (2):

[0005]

[0006] During the preparation process of the starting material, multiple impurities are generated due to various factors such as raw materials, synthesis process, degradation, etc. The synthesis process of 5-bromo-2-chloro-4'-ethoxydiphenylmethane:

[0007]

[0008] The impurities generated are as follows:

[0009]

[0010]

[0011] The analysis and detection of the main starting material plays an important role in reaction control and yield improvement, and also directly affects the quality of the final product dapagliflozin. Therefore, it is very necessary to establish a simple, stable and effective analysis and detection method for the analysis and detection of the main starting material of dapagliflozin.

[0012] CN114577944A discloses a method for detecting related substances in 5-bromo-2-chloro-4'-ethoxydiphenylmethane, wherein 12 impurities are detected and separated, and the structures of each impurity are as follows:

[0013]

[0014] The patent does not provide the synthesis process of 5-bromo-2-chloro-4'-ethoxydiphenylmethane. Judging from the impurity structure, the materials, intermediates and by-product impurities were examined. Among them, SM1-9-1, SM1-9-2 and SM1-9-3 are all impurities of material SM1-A. As the synthesis process of SM-1 generates corresponding by-product impurities, the risk of SM1-9-1, SM1-9-2 and SM1-9-3 existing in SM-1 is extremely low; in addition, the impurities brought in by the materials and the by-products produced in the process are far from meeting the quality control requirements in the actual production process. Referring to the import standard JX20140204 of dapagliflozin tablets, its related substances control 4 known impurities, which are all related to the impurities (E, I, L, C) of 5-bromo-2-chloro-4'-ethoxydiphenylmethane, while CN 114577944 A does not control impurities E, L, C. The impurity structure and analysis of the relevant substances controlled in the import standard JX20140204 of Dapagliflozin Tablets are as follows

[0015] Summary of the invention

[0016] In order to overcome the above-mentioned technical problems existing in the prior art, the inventors, after conducting a lot of in-depth research, provide a method for separating and determining related substances of the key starting raw materials of dapagliflozin by HPLC, which has the advantages of being rapid, simple, highly sensitive and having good separation degree.

[0017] The technical solution adopted by the present invention is:

[0018] A method for separating and determining related substances of key starting materials of dapagliflozin by HPLC comprises the following steps:

[0019] Octadecylsilane bonded silica gel is used as a filler; 0.002% to 0.1% (V / V) phosphoric acid aqueous solution is used as mobile phase A, and acetonitrile-methanol mixed ratio is used as mobile phase B, and gradient elution is performed; the flow rate is 0.8 to 1.2 ml / min; the column temperature is 30 to 40° C.; and an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 205 to 230 nm.

[0020] The filler is octadecylsilane bonded silica gel, preferably Agilent 5TC-C18 250×4.6mm and Nap ChromCore 120C18 5μm 250mm×4.6mm;

[0021] The mobile phase A is 0.002% to 0.1% (V / V) phosphoric acid aqueous solution. The acidic condition of the mobile phase mainly affects the retention time and peak shape of the chromatographic peak of impurity K, preferably 0.02% (V / V);

[0022] The mobile phase B is a mixture of acetonitrile and methanol, and the volume ratio is acetonitrile-methanol (5:95-20:80). As the proportion of acetonitrile increases, the separation degree between impurity C and impurity R, and impurity D and impurity F decreases; as the proportion of acetonitrile decreases, the separation degree between impurity E and impurity H decreases; acetonitrile-methanol (10:90) is preferred;

[0023] The conditions of the gradient elution are:

[0024] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 20～30 45～70 5 18 75 35 12 95 45 10 95

[0025] Preferred:

[0026] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0027] This results in the best separation.

[0028] The flow rate is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0029] The column temperature is 30-40°C, preferably 35°C.

[0030] The detection wavelength of the external detector is 205-230nm. After ultraviolet scanning, this product has strong absorption at 205-230nm, and the maximum absorption is about 226nm, so 226℃ is preferred.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The method for separating and determining related substances of the key starting raw material of dapagliflozin by HPLC of the present invention optimizes the detection result by comprehensively considering the comprehensive influence of the analytical column, the mobile phase, the gradient elution program, the flow rate and the column temperature on the separation and detection, and can quickly and efficiently separate the impurities A, B, C, E, F, G, H, I, J, K and L in the key starting raw material of dapagliflozin under the same chromatographic conditions, and the detection method has high sensitivity, strong specificity, is fast and simple, and is easy to operate, and can effectively control its quality, and is suitable for separating and determining related substances of the key starting raw material of dapagliflozin. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatogram is a blank solution detected under the conditions of Example 1 in the present invention;

[0034] Figure 2 The chromatogram of the system suitability solution tested under the conditions of Example 1 in the present invention;

[0035] Figure 3 The chromatogram of the test solution detected according to the conditions of Example 1 in the present invention;

[0036] Figure 4 The chromatogram is a quantitative limit solution detected according to the conditions of Example 1 in the present invention;

[0037] Figure 5 The chromatogram is a detection limit solution detected under the conditions of Example 1 in the present invention;

[0038] Figure 6 The chromatogram of the system suitability solution tested under the conditions of Example 2 in the present invention (column temperature -30°C);

[0039] Figure 7 The chromatogram of the system suitability solution tested under the conditions of Example 2 in the present invention (column temperature -40°C);

[0040] Figure 8 The chromatogram of the test solution detected according to the conditions of Example 3 in the present invention (gradient starting ratio mobile phase A-mobile phase B (55:45));

[0041] Fig. 9 The chromatogram of the test solution detected according to the conditions of Example 3 in the present invention (gradient starting ratio mobile phase A-mobile phase B (30:70));

[0042] Fig.10 The chromatogram of the test solution detected according to the conditions of Example 4 in the present invention (flow rate -0.8ml / min);

[0043] Fig.11 The chromatogram of the test solution detected according to the conditions of Example 4 in the present invention (flow rate -1.2ml / min)

[0044] Fig.12 The chromatogram of the test solution detected according to the conditions of Example 5 in the present invention (mobile phase B: acetonitrile: methanol (volume ratio 5:95));

[0045] Fig.13 The chromatogram of the test solution detected according to the conditions of Example 5 in the present invention (mobile phase B: acetonitrile: methanol (volume ratio 20:80));

[0046] Fig.14 The chromatogram of the test solution detected according to the conditions of Example 6 in the present invention (mobile phase A: 0.002% phosphoric acid aqueous solution);

[0047] Fig.15 The chromatogram of the test solution detected according to the conditions of Example 6 in the present invention (mobile phase A: 0.1% phosphoric acid aqueous solution);

[0048] Fig.16 The chromatogram of the test solution detected according to the conditions of Example 7 in the present invention (chromatographic column: Napu ChromCore120C185μm250mm×4.6mm) DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some variations and improvements can also be made without departing from the inventive concept. These all belong to the protection scope of the present invention. Reagents and instruments used that do not indicate the manufacturer, all can be obtained through commercial conventional products.

[0050] The key starting materials and impurity reference substances of dapagliflozin used in the examples of the present invention were all prepared by the inventors.

[0051] Example 1

[0052] The chromatographic conditions were as follows:

[0053] Chromatographic column: Agilent TC-C18 250×4.6mm

[0054] Mobile phase A: 0.02% phosphoric acid

[0055] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0056] Column temperature: 35°C

[0057] Flow rate: 1.0ml / min

[0058] Detection wavelength: 226nm

[0059] Injection volume: 10 μL

[0060] The gradient elution program was:

[0061] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0062] Solution preparation:

[0063] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0064] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0065] Test solution: Take about 25 mg of the key starting material of dapagliflozin, accurately weigh it, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0066] Stock solution of dapagliflozin key starting material reference substance: Take about 25 mg of dapagliflozin key starting material working reference substance, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0067] Quantitative limit solution: Accurately measure 1 ml of the impurity reference substance stock solution and the dapagliflozin key starting material reference substance stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, accurately measure 1 ml to 25 ml, add diluent to dilute to the scale, shake well, and obtain.

[0068] Detection limit solution: Accurately measure 5 ml of the quantitative limit solution and place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0069] Determination: Inject the blank solution (i.e. diluent), system suitability solution, test solution, quantitation limit solution and detection limit solution into the high performance liquid chromatograph for detection and record the chromatogram.

[0070] The chromatograms of blank solution, system suitability solution, test solution, quantitation limit solution and detection limit solution are shown in Figure 1. Figure 1 , 2 , 3, 4 and 5, it can be seen that Figure 1 It shows that the blank does not interfere with the impurity inspection; Figure 2 It shows that the separation between each impurity and the key starting material of dapagliflozin is good. The specific spectral data of the system suitability of the key starting material of dapagliflozin are shown in Table 2; Figure 3It shows that impurities A, B, D, F, G, H, I, J and K were not detected in the homemade key starting material samples of dapagliflozin, and the detected impurities C, E and L were all below 0.1wt%; the other single impurities detected were all below 0.1wt%; Figure 4 The results showed that the limits of quantification of the key starting materials of dapagliflozin and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L were 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt%, 0.04wt% and 0.04wt%, respectively; Figure 5 The results show that the detection limits of the key starting raw materials of dapagliflozin and impurities A, B, C, D, E, F, G, H, I, J, K and L are 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt%, 0.02wt% and 0.02wt%, respectively, and the detection sensitivity of the method is high.

[0071] Table 1 System suitability data of key starting materials of dapagliflozin in Example 1

[0072]

[0073] Example 2

[0074] The same chromatographic column, mobile phase, flow rate as in Example 1 were used, with only the column temperature being different, and the key starting materials of dapagliflozin and impurities A, B, C, D, E, F, G, H, I, J, K and L were separated and detected at 30° C. and 40° C., respectively.

[0075] The chromatographic conditions were as follows:

[0076] Chromatographic column: Agilent TC-C18 250×4.6mm

[0077] Mobile phase A: 0.02% phosphoric acid

[0078] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0079] Column temperature: 30℃ / 40℃

[0080] Flow rate: 1.0ml / min

[0081] Detection wavelength: 226nm

[0082] Injection volume: 10 μL

[0083] The gradient elution program was:

[0084] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0085] Solution preparation:

[0086] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0087] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0088] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Figure 6 , 7 .

[0089] The results show that under the above chromatographic conditions, the baseline is stable, the column temperature increases, the retention time of each peak is slightly advanced, the separation degree of impurity H and impurity E is slightly reduced, and there is no obvious difference in the separation degree between other peaks. The present invention can achieve baseline separation of 13 substances within 30-40°C. Considering the separation degree and laboratory temperature and other factors, the present invention is most preferably performed at a column temperature of 35°C for chromatographic separation.

[0090] Table 2 System suitability profile of key starting materials of dapagliflozin in Example 2 (different column temperatures)

[0091]

[0092]

[0093] Example 3

[0094] The same chromatographic column, column temperature, flow rate, and mobile phase as in Example 1 were used, only the starting ratio of the mobile phase was changed, and the gradient elution program was shown in Tables 3 and 4, and the key starting raw materials of dapagliflozin and impurities A, B, C, D, E, F, G, H, I, J, K, and L were separated and detected.

[0095] The chromatographic conditions were as follows:

[0096] Chromatographic column: Agilent TC-C18 250×4.6mm

[0097] Mobile phase A: 0.02% phosphoric acid

[0098] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0099] Column temperature: 35°C

[0100] Flow rate: 1.0ml / min

[0101] Detection wavelength: 226nm

[0102] Injection volume: 10 μL

[0103] The gradient elution program was:

[0104] Table 3 Gradient elution program

[0105] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 55 45 5 35 75 35 5 95 45 5 95

[0106] Table 4 Gradient elution program

[0107] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 70 5 35 75 35 5 95 45 5 95

[0108] Solution preparation:

[0109] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0110] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0111] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Figure 8 , 9 .

[0112] The results show that under the above chromatographic conditions, the baseline is stable, the initial ratio of organic phase (mobile phase B) increases, and the separation degree of impurity J and impurity K is significantly reduced; the initial ratio of organic phase (mobile phase B) decreases, the separation degree of impurity J and impurity K is significantly reduced, and the separation degree of impurity D and impurity F, impurity H and impurity E are slightly reduced. The present invention can achieve baseline separation of 13 substances within the initial ratio of mobile phase A-mobile phase B (55:45-30:70) of the gradient elution program. Considering the separation degree and other factors, the present invention most preferably uses the initial ratio of mobile phase A-mobile phase B (35:65) of the gradient elution program.

[0113] Table 5 System suitability profile of key starting materials of dapagliflozin in Example 3 (gradient starting ratio) Data table

[0114]

[0115] Example 4

[0116] The same chromatographic column, column temperature, and mobile phase ratio as in Example 1 were used, and only the flow rate was changed. The key starting materials of dapagliflozin and impurities A, B, C, D, E, F, G, H, I, J, K, and L were separated and detected at flow rates of 0.8 ml / min and 1.2 ml / min, respectively.

[0117] The chromatographic conditions were as follows:

[0118] Chromatographic column: Agilent TC-C18 250×4.6mm

[0119] Mobile phase A: 0.02% phosphoric acid

[0120] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0121] Column temperature: 35°C

[0122] Flow rate: 0.8ml / min / 1.2ml / min

[0123] Detection wavelength: 226nm

[0124] Injection volume: 10 μL

[0125] The gradient elution program was:

[0126] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0127] Solution preparation:

[0128] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0129] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0130] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.10 , 11 .

[0131] The results show that under the above chromatographic conditions, the baseline is stable, and as the flow rate decreases, the retention time of each peak increases slightly, and the separation degree of impurity H and impurity E decreases slightly. The present invention can achieve baseline separation of 13 substances within the flow rate (0.8ml / min to 1.2ml / min). Considering the separation degree and analysis time, the most preferred flow rate of the present invention is 1.0ml / min.

[0132] Table 6 System suitability profile of key starting materials of dapagliflozin in Example 4 (different flow rates)

[0133]

[0134] Example 5

[0135] The same chromatographic column, column temperature, mobile phase ratio, and flow rate as in Example 1 were used, and only the acetonitrile-methanol mixing ratio of the mobile phase B was changed. The key starting materials of dapagliflozin and impurities A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L were separated and detected under the conditions of mobile phase B: acetonitrile: methanol (volume ratio of 5:95) and acetonitrile: methanol (volume ratio of 20:80), respectively.

[0136] The chromatographic conditions were as follows:

[0137] Chromatographic column: Agilent TC-C18 250×4.6mm

[0138] Mobile phase A: 0.02% phosphoric acid

[0139] Mobile phase B: acetonitrile: methanol (volume ratio 5:95) / acetonitrile: methanol (volume ratio 20:80)

[0140] Column temperature: 35°C

[0141] Flow rate: 1.0ml / min

[0142] Detection wavelength: 226nm

[0143] Injection volume: 10 μL

[0144] The gradient elution program was:

[0145] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0146] Solution preparation:

[0147] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0148] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0149] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.12 , 13 .

[0150] The results showed that under the above chromatographic conditions, the baseline was stable, and as the ratio of acetonitrile to methanol in mobile phase B increased, the separation between impurity C and impurity R, and between impurity D and impurity F decreased slightly;

[0151] The mobile phase B is a mixture of acetonitrile and methanol. The reduction of the acetonitrile ratio slightly reduces the separation degree of impurity H and impurity E. The present invention can achieve baseline separation of 13 substances in the mobile phase B of acetonitrile and methanol, and the volume ratio of acetonitrile and methanol (5:95 to 20:80). Considering the separation degree and other factors, the most preferred mobile phase B of the present invention is a mixture of acetonitrile and methanol, and the volume ratio is acetonitrile and methanol (10:90).

[0152] Table 7 System suitability profile of key starting materials of dapagliflozin in Example 5 (different mobile phases B)

[0153]

[0154] Example 6

[0155] The same chromatographic column, column temperature, mobile phase ratio, and flow rate as in Example 1 were used, and only the concentration of the mobile phase A, phosphoric acid aqueous solution, was changed. The key starting materials of dapagliflozin and impurities A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, and impurity L were separated and detected under the conditions of mobile phase A: 0.002% phosphoric acid aqueous solution and 0.1% phosphoric acid aqueous solution, respectively.

[0156] The chromatographic conditions were as follows:

[0157] Chromatographic column: Agilent TC-C18 250×4.6mm

[0158] Mobile phase A: 0.002% phosphoric acid aqueous solution / 0.1% phosphoric acid aqueous solution

[0159] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0160] Column temperature: 35°C

[0161] Flow rate: 1.0ml / min

[0162] Detection wavelength: 226nm

[0163] Injection volume: 10 μl

[0164] The gradient elution program was:

[0165] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0166] Solution preparation:

[0167] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0168] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0169] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.14 , 15 .

[0170] The results show that under the above chromatographic conditions, the baseline is stable. The retention time of impurity K varies greatly with the different phosphoric acid concentrations of mobile phase A. When the phosphoric acid is 0%, impurity K is retained in the chromatographic column and cannot be eluted. When the phosphoric acid content is less than 0.01%, the retention time of impurity K varies greatly, the peak order changes, and the peak shape of the impurity K chromatographic peak varies greatly. The lower the phosphoric acid content, the worse the peak shape (the smaller the theoretical plate number value); when the phosphoric acid content is greater than 0.01%, the retention time of impurity K is stable and the peak shape is better. The present invention can achieve baseline separation of 13 substances within the phosphoric acid concentration of mobile phase A (0.002% to 0.1%). Considering the factors such as separation degree, stability of retention time and pH range tolerated by the chromatographic column, the present invention most preferably uses 0.02% phosphoric acid aqueous solution as mobile phase A.

[0171] Table 8 System suitability profile of key starting materials of dapagliflozin in Example 6 (different mobile phases A)

[0172]

[0173] Example 7

[0174] The same column temperature, mobile phase ratio and flow rate as in Example 1 were used, and only the brand of the chromatographic column was changed to separate and detect the key starting materials of dapagliflozin and impurities A, B, C, D, E, F, G, H, I, J, K and L.

[0175] The chromatographic conditions were as follows:

[0176] Chromatographic column: Nap ChromCore 120 C18 5μm 250mm×4.6mm

[0177] Mobile phase A: 0.02% phosphoric acid in water

[0178] Mobile phase B: acetonitrile: methanol (volume ratio 10:90)

[0179] Column temperature: 35°C

[0180] Flow rate: 1.0ml / min

[0181] Detection wavelength: 226nm

[0182] Injection volume: 10 μl

[0183] The gradient elution program was:

[0184] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 35 65 5 35 75 35 5 95 45 5 95

[0185] Solution preparation:

[0186] Impurity reference substance stock solution: Take approximately 12.5 mg each of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance, impurity G reference substance, impurity H reference substance, impurity I reference substance, impurity J reference substance, impurity K reference substance, and impurity L reference substance, accurately weigh, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: acetonitrile-water (80:20) (V / V).

[0187] System suitability solution: Take about 50 mg of the key starting material working reference substance of dapagliflozin, accurately weigh it, and place it in a 100 ml volumetric flask. Accurately add 1 ml of the above impurity reference substance stock solution into the above 100 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0188] Determination: Take the system suitability solution and inject it into the high performance liquid chromatograph and record the chromatogram. Fig.16 .

[0189] The results show that under the above chromatographic conditions, the baseline is stable, and there is no obvious difference in the chromatographic peaks of different chromatographic columns. The present invention can achieve baseline separation of 13 substances in a chromatographic column (ChromCore 120 C18 5μm 250mm×4.6mm).

[0190] Table 9 System suitability profiles of key starting materials of dapagliflozin in Example 7 (different chromatographic columns)

[0191]

[0192] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for separating and determining related substances of key starting materials of dapagliflozin by HPLC, comprising the following steps: using octadecylsilane bonded silica gel as a filler, using a phosphoric acid aqueous solution with a volume ratio of 0.01% to 0.1% as a mobile phase A, and a mixture of acetonitrile and methanol as a mobile phase B, and performing gradient elution; the flow rate is 0.8 to 1.2 ml / min; the column temperature is 30 to 40° C.; and an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 205 to 230 nm; The mobile phase B is an acetonitrile-methanol mixture with a volume ratio of 5:95 to 20:80; The conditions of the gradient elution are: The key starting material-related substances of dapagliflozin include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, and the specific structural formula is as follows:

2. The method according to claim 1, characterized in that The fillers are Agilent 5TC-C18 250×4.6 mm and Nap ChromCore 120C18 5μm 250mm×4.6mm.

3. The method according to claim 1, characterized in that The mobile phase A is a 0.02% by volume phosphoric acid aqueous solution.

4. The method according to claim 1, characterized in that The mobile phase B is an acetonitrile-methanol mixture with a volume ratio of 10:

90.

5. The method according to claim 1, characterized in that: The flow rate is 1.0 ml / min.

6. The method according to claim 1, characterized in that The column temperature was 35°C.

7. The method according to claim 1, characterized in that The detection wavelength of the ultraviolet detector is 205-230nm. After ultraviolet scanning, the product has strong absorption at 205-230nm, and the maximum absorption is at 226nm.

8. The method according to claim 1, characterized in that The degree of separation between the 13 components of dapagliflozin, including key starting materials, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L is greater than 1.5.

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