Specific quantitative detection method for 2-epimer and alpha-isomer in dapagliflozin

Abstract

The invention discloses a specific quantitative detection method for 2-epimer and alpha-isomer in dapagliflozin, which adopts high performance liquid chromatography and uses a chiral chromatographic column as a separation unit. A filling agent of the chiral chromatographic column is [N-(R)-(+)-1-(1-naphthyl) ethyl] methacrylamide bonded silica gel; the mobile phase is obtained by compounding a disodium hydrogen phosphate solution and acetonitrile; the dapagliflozin 2-epimer and the dapagliflozin alpha-isomer are quantitatively determined by isocratic elution, the flow velocity of a mobile phase is 0.5-1.5 ml / min, the column temperature is 20-40 DEG C, efficient separation and quantitative detection of the dapagliflozin 2-epimer and the dapagliflozin alpha-isomer can be realized, the separation degree is greater than 1.5, quantitative detection of the 2-epimer can be carried out, and the method is suitable for industrial production. And the method has good sensitivity, specificity, precision, linearity, accuracy, solution stability and durability.

Description

Technical Field

[0001] This application relates to dapagliflozin, and more particularly to a specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin. Background Technology

[0002] Dapagliflozin, a sodium-dependent glucose transporter inhibitor developed by Bristol-Myers Squibb and AstraZeneca, has played a significant role in the treatment of diabetes and related fields since its approval in the European Union in December 2012 and in the United States in January 2014. The structural formula of dapagliflozin is as follows: With the increasing demands for drug quality and safety in the pharmaceutical industry, the detection and control of impurities in dapagliflozin has become a key research focus. The synthesis of dapagliflozin involves various chemical substances and reaction steps, resulting in multiple isomers, including the 2-epimer and α-isomer.

[0003] The 2-epimer of dapagliflozin arises because the starting material gluconolactone in the dapagliflozin synthesis route contains a 2-epimer impurity—d-mannonico-δ-lactone. d-mannonico-δ-lactone also participates in the synthesis of dapagliflozin, generating the 2-epimer. The structural formula of the 2-epimer of dapagliflozin is as follows: The α-isomer of dapagliflozin is formed because the nucleophile cannot achieve 100% stereoselectivity in the glycosylation coupling reaction that ultimately forms the C-glycosidic bond during the synthesis of dapagliflozin. The structural formula of the α-isomer of dapagliflozin is as follows: Currently, there are methods for detecting the 2-epimeter in gluconolactone, a starting material. However, there are no publicly reported methods for detecting the 2-epimeter in dapagliflozin, a finished product.

[0004] When using existing traditional chromatographic analysis methods to detect impurities in dapagliflozin, the inventors found that existing methods have limitations for chiral isomers with different chiral carbon positions, such as dapagliflozin, dapagliflozin 2-epimer, and dapagliflozin α-isomer. The 2-epimer and α-isomer can only be detected as a single peak, and it is impossible to effectively separate and detect the content of the 2-epimer and α-isomer separately.

[0005] Therefore, it is impossible to directly and accurately quantify the 2-episode in the finished dapagliflozin product. Even if the content of the 2-episode in the finished product is reduced by controlling the introduction of d-mannonic acid-δ-lactone in the starting material, this cannot guarantee that the content of the 2-episode in the final product meets the requirements, which is not conducive to ensuring the quality of the finished dapagliflozin product and makes it difficult to study the transmission of related impurities in the production process. Summary of the Invention

[0006] To address the issue that the 2-epimer and α-epimer of dapagliflozin are technically impossible to detect separately, a quantitative detection method for the 2-epimer of dapagliflozin is provided.

[0007] The above-mentioned objective of this invention is achieved through the following technical solutions: A specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin was developed using high performance liquid chromatography with a chiral column as the separation unit. The packing material for the chiral chromatographic column is [N-(R)-(+)-1-(1-naphthyl)ethyl]methacrylamide-bonded silica gel; the mobile phase is prepared by mixing disodium hydrogen phosphate solution with acetonitrile. Isocratic elution was used to quantitatively determine the 2-epimer and α-isomer of dapagliflozin.

[0008] Optional: The mobile phase is prepared by mixing 0.05 mol / L disodium hydrogen phosphate solution with acetonitrile at a volume ratio of 75:25; the pH of the disodium hydrogen phosphate solution is adjusted to 4.2–6.2 with phosphoric acid.

[0009] Optional: mobile phase flow rate of 0.5–1.5 ml / min, column temperature of 20–40 °C.

[0010] Optionally, the chromatographic column has a length of 200-250 mm and an inner diameter of 4.6 mm.

[0011] Optional: Detection wavelength is 200-250nm.

[0012] Optional: The filler particle size is 3 to 5 μm.

[0013] Optional: Injection volume 5–20 μl.

[0014] By adopting the above technical solution, efficient separation and quantitative detection of dapagliflozin 2-epimer and dapagliflozin α-epimer can be achieved with a resolution greater than 1.5. The method can perform quantitative detection of the 2-epimer, and the method has good sensitivity, specificity, precision, linearity, accuracy, solution stability and robustness.

[0015] In summary, this application has at least the following beneficial effects: 1. High performance liquid chromatography (HPLC) is used, employing a chiral column with specific packing material and a compounded mobile phase, with isocratic elution, to achieve high efficiency separation of dapagliflozin 2-epimer and dapagliflozin α-isomer, with high resolution; 2. It can quantitatively detect dapagliflozin 2-epimer, with a limit of quantification of 0.3 μg / ml and a limit of detection of 0.1 μg / ml, and the method has good sensitivity; 3.2- The epimer showed good linearity in the range of 0.303 μg / ml to 2.019 μg / ml (R = 0.9990), with method recoveries ranging from 98.60% to 103.69% and RSD% (n = 9) not exceeding 1.75%. It exhibited strong specificity, precision, linearity, accuracy, solution stability, and robustness. Attached Figure Description

[0016] Figure 1 This is the chromatogram of the blank solution used in Example 1. Figure 2 This is the chromatogram of the system suitability solution used in Example 1. Figure 3 This is the chromatogram of the solution used in Example 1 to detect the limit of detection. Figure 4 This is the chromatogram of the solution used in Example 1 for detection at the limit of quantitation. Figure 5 This is the chromatogram of the test solution in Example 1. Detailed Implementation

[0017] The following provides a more detailed description of this application in conjunction with specific details.

[0018] raw material: Acetonitrile is of HPLC grade; The dapagliflozin sample was prepared in-house by Beijing Huikang Boyuan Pharmaceutical Technology Co., Ltd. Dapagliflozin is a self-produced product of Beijing Huikang Boyuan Pharmaceutical Technology Co., Ltd. Dapagliflozin 2-epimer was purchased from China National Standard Pharmaceutical Group Co., Ltd. Dapagliflozin α isomer was purchased from China National Standard Pharmaceutical Group Co., Ltd. [N-(R)-(+)-1-(1-naphthyl)ethyl]methacrylamide bonded silica columns can be purchased directly as finished products; Disodium hydrogen phosphate solution is of AR grade; Phosphoric acid is classified as AR grade.

[0019] Example 1 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin is as follows: The liquid chromatograph used is a high performance liquid chromatograph; Chromatographic column: 250 mm in length and 4.6 mm in inner diameter, packed with 5 μm of [N-(R)-(+)-1-(1-naphthyl)ethyl]methacrylamide bonded silica gel; Solvent: Acetonitrile-water (30:70) (v / v).

[0020] Mobile phase: 0.05 mol / L disodium hydrogen phosphate solution (pH adjusted to 5.2 with phosphoric acid) - acetonitrile (75:25) (v / v).

[0021] Elution conditions: column temperature 30℃, detection wavelength 220nm, flow rate 1.0mL / min, injection volume 10μL; Running time: 70 minutes.

[0022] Experimental steps: Blank solution: solvent System suitability solution: Dissolve dapagliflozin, dapagliflozin 2-epimer, and dapagliflozin α-isomer in solvent and dilute to prepare a mixed solution of dapagliflozin, dapagliflozin 2-epimer, and dapagliflozin α-isomer, wherein the concentrations of dapagliflozin, dapagliflozin 2-epimer, and dapagliflozin α-isomer are 1 μg / ml.

[0023] Limit of Quantification Solution: Dissolve dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer in solvent and dilute to prepare a limit of quantification solution with the following concentrations: dapagliflozin 0.3 μg / ml, dapagliflozin 2-epimer 0.3 μg / ml, and dapagliflozin α-isomer 0.3 μg / ml.

[0024] Detection limit solution: Dissolve dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer in solvent and dilute to prepare detection limit solution. The concentration of dapagliflozin is 0.1 μg / ml, the concentration of dapagliflozin 2-epimer is 0.1 μg / ml, and the concentration of dapagliflozin α-isomer is 0.1 μg / ml.

[0025] Test solution: Dissolve the dapagliflozin sample in solvent and dilute to prepare a 1 mg / ml solution.

[0026] Recovery rate detection solution group: Dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer were dissolved in solvent and diluted to prepare the recovery rate detection solution group; the specific details of the recovery rate detection solution group are as follows. Recovery test solution 1: The concentrations of dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer were all 0.303 μg / ml. Recovery test solution 2, recovery test solution 1, the concentration of dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer were all 1.161 μg / ml; Recovery test solution 3, recovery test solution 1, the concentration of dapagliflozin sample, dapagliflozin 2-epimer, and dapagliflozin α-isomer were all 2.019 μg / ml.

[0027] Take blank solution, system suitability solution, limit of quantitation solution, limit of detection solution, test solution, and recovery test solution respectively, and perform high performance liquid chromatography analysis under the above chromatographic conditions. Record the chromatograms and analysis tables. The results are shown in the appendix. Figures 1-5 See Table 1.

[0028] Table 1. Calculation Results of Recovery Rate of Recovery Solution Group Figure 1 This indicates that the blank solution did not interfere with the detection of dapagliflozin 2-epimer, dapagliflozin α-isomer, or the main peak of dapagliflozin. Figure 2 This indicates that the separation between dapagliflozin 2-epimer, dapagliflozin α-isomer, and dapagliflozin is good, effectively detecting impurities in the sample, and the quantitative detection results are accurate.

[0029] Figure 3-4 It is known that the detection limit of dapagliflozin 2-episode and dapagliflozin α-isomer in this detection method is 0.1 μg / ml, thus the detection method of this application has high sensitivity for the detection of dapagliflozin 2-episode and dapagliflozin α-isomer.

[0030] Figure 5 As shown in Table 1, neither the 2-epimer of dapagliflozin nor the α-isomer of dapagliflozin were detected by this detection method. Therefore, the impurity content of the test solution is low and meets the requirements for the preparation of dapagliflozin.

[0031] Comparative Example 1 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the mobile phase is a mixture of 0.05 mol / L disodium hydrogen phosphate solution and methanol at a volume ratio of 75:25; the pH of the disodium hydrogen phosphate solution is adjusted to 5.2 with phosphoric acid.

[0032] Comparative Example 2 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the mobile phase is a mixture of diammonium hydrogen phosphate solution and acetonitrile at a volume ratio of 75:25; and the pH of the disodium hydrogen phosphate solution is adjusted to 5.2 with phosphoric acid.

[0033] Comparative Example 3 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the chromatographic column packing material is octadecylsilane-bonded silica gel.

[0034] Example 2 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the mobile phase is a mixture of 0.05 mol / L disodium hydrogen phosphate solution and acetonitrile at a volume ratio of 60:40; the pH of the disodium hydrogen phosphate solution is adjusted to 5.2 with phosphoric acid.

[0035] Example 3 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the mobile phase is a mixture of 0.05 mol / L disodium hydrogen phosphate solution and acetonitrile at a volume ratio of 60:40; the pH of the disodium hydrogen phosphate solution is adjusted to 4.2 with phosphoric acid.

[0036] Example 4 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the mobile phase is a mixture of 0.05 mol / L disodium hydrogen phosphate solution and acetonitrile at a volume ratio of 60:40; the pH of the disodium hydrogen phosphate solution is adjusted to 6.2 with phosphoric acid.

[0037] Example 5 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in the elution conditions: column temperature 40℃, detection wavelength 220nm, flow rate 0.5mL / min, and injection volume 10μL.

[0038] Example 6 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in the elution conditions: column temperature 20℃, detection wavelength 220nm, flow rate 1.5mL / min, and injection volume 10μL.

[0039] Example 7 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the chromatographic column length is 180 mm.

[0040] Example 8 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the chromatographic column length is 200 mm.

[0041] Example 9 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the chromatographic column length is 250 mm.

[0042] Example 10 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the chromatographic column length is 300 mm.

[0043] Example 11 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the detection wavelength is 190 nm.

[0044] Example 12 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the detection wavelength is 200 nm.

[0045] Example 13 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the detection wavelength is 250 nm.

[0046] Example 14 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the detection wavelength is 260 nm.

[0047] Example 15 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the particle size of the packing material in the chromatographic column is 3 μm.

[0048] Example 16 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the particle size of the packing material in the chromatographic column is 4 μm.

[0049] Example 17 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the particle size of the packing material in the chromatographic column is 7 μm.

[0050] Example 18 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the injection volume is 5 μl.

[0051] Example 19 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the injection volume is 15 μl.

[0052] Example 20 The specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin differs from that in Example 1 in that the injection volume is 20 μl.

[0053] The results of the detection of Comparative Examples 1-3 and Examples 2-20 were calculated and statistically analyzed, including whether the blank solution produced peaks, whether the three components in the system suitability solution were effectively separated, the peak separation degree of recovery detection solution 1, and the average recovery rate and average RSD% (n=9) of the three solutions in the recovery detection solution group. The results are shown in Table 2.

[0054] Table 2. Statistical table of test results for Comparative Examples 1-3 and Examples 1-20 Referring to Table 2, comparing Example 1 and Comparative Examples 1-3, Comparative Examples 1-3 failed to effectively separate peaks in both the system adaptability solution and the recovery detection solution group, and could not distinguish between the 2-epimer and α-isomer of dapagliflozin. Therefore, the specific quantitative detection method for 2-epimer and α-isomer of dapagliflozin in this application requires a specific combination of the packing agent and mobile phase components in the chromatographic column to achieve efficient separation and quantitative detection of 2-epimer and α-isomer of dapagliflozin.

[0055] In conjunction with Examples 1 to 4, when testing the system adaptability solution and recovery rate detection solution groups, the peaks were effectively separated in Examples 1 to 4. Among them, Example 1 had the highest peak separation and the lowest average RSD%.

[0056] Therefore, the mobile phase of this application can be obtained by mixing 0.05 mol / L disodium hydrogen phosphate solution with acetonitrile; the pH of the disodium hydrogen phosphate solution is adjusted to 4.2-6.2 with phosphoric acid; wherein the volume ratio of disodium hydrogen phosphate solution to acetonitrile is 75:25, and the optimal pH of the disodium hydrogen phosphate solution is 5.2.

[0057] Comparing Examples 1 and 5-6, both Examples 1 and 5-6 effectively separated peaks when detecting the system adaptability solution and recovery rate test solution groups. Example 1 showed the highest peak resolution and the lowest average RSD%. Therefore, the elution adjustment in this application can be a mobile phase flow rate of 0.5-1.5 ml / min and a column temperature of 20-40°C; among these, 1.0 ml / min and a column temperature of 30°C are optimal.

[0058] In conjunction with Examples 1 and 7-10, both Examples 1 and 7-10 effectively separated peaks when detecting the system adaptability solution and recovery rate detection solution group. However, the resolution of Example 7 was 1.35, lower than 1.4; the resolutions of Examples 9 and 10 were greater than 1.6, far exceeding 1.5. Therefore, the column length in this application can be 180-300 nm. Considering separation effect, detection accuracy, and cost, 200-250 mm is generally selected to meet the detection needs of most situations.

[0059] In conjunction with Examples 1 and 11-14, and Examples 1 and 7-10, peaks were effectively separated when detecting the system adaptability solution and recovery rate detection solution groups. Among them, Examples 1, 12, and 13 had higher separation and lower average RSD%, therefore, a detection wavelength of 200-250 nm is preferred in this application.

[0060] In conjunction with Examples 1 and 15-17, both Examples 1 and 15-17 effectively separated peaks when detecting the system adaptability solution and recovery rate detection solution groups. Among them, the resolution of Examples 1, 15, and 16 was higher than that of Example 17, and the average RSD% of Examples 1, 15, and 16 was lower than that of Example 17. Therefore, a particle size of 3-5 μm for the chromatographic column packing material in this application is preferable.

[0061] Combining Examples 1 and 18-20, both Examples 1 and 18-20 effectively separated peaks when detecting the system adaptability solution and recovery rate detection solution group. Among them, Example 20 showed a significant improvement in other performance aspects, with an increasing average RSD% trend. Therefore, the injection volume in this application is 5-20 μl, and it is not advisable to further increase the injection volume to avoid affecting the detection accuracy.

[0062] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but as long as they are within the scope of protection claimed by the present invention, they are protected by patent law.

Claims

1. A specific quantitative detection method for the 2-epimer and α-isomer of dapagliflozin, characterized in that, High performance liquid chromatography was employed, using a chiral column as the separation unit. The packing material for the chiral chromatographic column is [N-(R)-(+)-1-(1-naphthyl)ethyl]methacrylamide bonded silica gel; The mobile phase was prepared by mixing disodium hydrogen phosphate solution with acetonitrile; Isocratic elution was used to quantitatively determine the 2-epimer and α-isomer of dapagliflozin.

2. The method for specific quantitative detection of the 2-epimer and α-isomer of dapagliflozin according to claim 1, characterized in that, The mobile phase was prepared by mixing 0.05 mol / L disodium hydrogen phosphate solution with acetonitrile at a volume ratio of 75:25; the pH of the disodium hydrogen phosphate solution was adjusted to 4.2-6.2 with phosphoric acid.

3. The quantitative detection method for dapagliflozin 2-epimer according to claim 1, characterized in that, The mobile phase flow rate is 0.5~1.5 ml / min, and the column temperature is 20~40℃.

4. The method for specific quantitative detection of the 2-epimer and α-isomer of dapagliflozin according to claim 1, characterized in that, The chromatographic column has a length of 200-250 mm and an inner diameter of 4.6 mm.

5. The method for specific quantitative detection of the 2-epimer and α-isomer of dapagliflozin according to claim 1, characterized in that, The detection wavelength is 200~250nm.

6. The method for specific quantitative detection of the 2-epimer and α-isomer of dapagliflozin according to claim 1, characterized in that, The filler particle size is 3~5μm.

7. The method for specific quantitative detection of the 2-epimer and α-isomer of dapagliflozin according to claim 1, characterized in that, Injection volume: 5~20μl.

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