Detection method of dapagliflozin peroxide genotoxic impurities
The detection of peroxidized genotoxic impurities in dapagliflozin through high performance liquid chromatography has solved the problem of insufficient detection sensitivity and specificity in the prior art, and achieved the guarantee of the safety of dapagliflozin drug use.
Patent Information
- Application Number
- CN202411776993.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to effectively detect peroxidized genotoxic impurities in dapagliflozin, especially when the control limit of 150ppm is reached, the sensitivity and specificity of the analysis method are insufficient, resulting in the threat of drug safety.
High performance liquid chromatography (HPLC) was used to detect peroxidized genotoxic impurities in dapagliflozin. By selecting appropriate mobile phase and gradient elution procedures, accurate quantity detection of the impurities was achieved, avoiding the use of high-cost LC/MS combined technology.
This method can reduce the detection cost while improving the detection sensitivity and accuracy, meet the control limit of 150ppm, and provides reliable guarantees for the drug safety of dapagliflozin.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting dapagliflozin peroxidase genotoxic impurities. Background Art
[0002] Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) selective inhibitor that improves blood sugar control by inhibiting SGLT2 to excrete excess glucose through urine. Dapagliflozin is a hypoglycemic drug jointly developed by AstraZeneca and Bristol-Myers Squibb. It was approved for marketing in my country in March 2017. Its structural formula is as follows:
[0003]
[0004] The dapagliflozin peroxide genotoxic impurity (impurity A), with the structural formula shown in Formula II, has the potential to cause mutagenesis and may directly or indirectly damage human DNA, leading to gene mutations and even tumors. It has always been a focus of attention in terms of drug safety.
[0005]
[0006] According to the medication cycle of dapagliflozin tablets and the ICH M7 guidelines, the acceptable limit of peroxide genotoxic impurities in dapagliflozin tablets is 150ppm. The detection of impurities at this concentration level has high requirements for the sensitivity and specificity of the analytical method. Currently, many literature reports that the detection of genotoxic impurities uses LC / MS or GS / MS combined technology to achieve detection at lower concentration levels. The universality of chromatography and mass spectrometry technology is not strong, and many pharmaceutical research and development and production companies do not have this detection capability. Therefore, there are certain difficulties in the detection and control of genotoxic impurities.
[0007] The carbon atom connected to the peroxide bond in the structure of the peroxide genotoxic impurity of dapagliflozin is a chiral carbon atom, which has two stereoisomers. Under normal HPLC conditions, the chromatographic peaks appear as double peaks, which is not conducive to quantitative detection. Therefore, existing reports all use LC / MS combined technology to perform quantitative detection of the impurity by mass spectrometry. Summary of the invention
[0008] The present invention detects peroxidation genotoxic impurities in dapagliflozin by high performance liquid chromatography, and does not need to use LC / MS coupling technology, and the detection cost is greatly reduced. High performance liquid chromatography is used, water or buffer is used as mobile phase A, and a mixed solution of methanol and acetonitrile is used as mobile phase B. The method has strong specificity, high sensitivity and accuracy, and provides a guarantee for the safety of dapagliflozin.
[0009] To achieve the above object, the present invention provides the following technical solutions:
[0010] A method for detecting dapagliflozin peroxidase genotoxic impurities adopts high performance liquid chromatography, wherein the detection conditions of the high performance liquid chromatography include: using an octadecyl bonded silica gel column as a chromatographic column; mobile phase A is any one of water, trifluoroacetic acid aqueous solution, and ammonium acetate buffer; mobile phase B is a mixture of methanol and acetonitrile, with the volume of methanol accounting for not less than 40%; and gradient elution is performed.
[0011] Furthermore, the peroxidation genotoxic impurity is a compound represented by Formula II:
[0012]
[0013] Furthermore, the concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.08-0.12%; preferably, the concentration of trifluoroacetic acid is 0.1%.
[0014] Furthermore, the pH of the ammonium acetate buffer is 4-5, preferably pH 4.5; in some embodiments, the acid for adjusting the pH of the ammonium acetate buffer is acetic acid.
[0015] Furthermore, the concentration of ammonium acetate in the ammonium acetate buffer is 10-30 mmol / L, preferably 20 mmol / L.
[0016] Furthermore, the detection conditions of the high performance liquid chromatography method also include: column temperature of 35-45°C, injection volume of 5-15μl, flow rate of 0.8-1.2ml / min; detection wavelength of 222-226nm; further preferably, the column temperature is 40°C, the flow rate is 1.0ml / min; the detection wavelength is 224nm; and the injection volume is preferably 10μl.
[0017] Furthermore, the chromatographic column is a Sepax HP-C18 column. Further preferably, the Sepax HP-C18 column has the following specifications: particle size×inner diameter×column length: 5 μm×4.6 mm×150 mm.
[0018] When performing the gradient elution program, the technician can adaptively adjust the appropriate elution program according to the adjustment of the mobile phase B. In some embodiments, the mobile phase A can be any one of water, trifluoroacetic acid aqueous solution, and ammonium acetate buffer, the mobile phase B is methanol, and the elution program is set as follows:
[0019]
[0020] In some embodiments, mobile phase A can be any one of water, trifluoroacetic acid aqueous solution, and ammonium acetate buffer, and mobile phase B is a mixture of methanol and acetonitrile, with methanol accounting for 40% by volume. The elution program is set as follows:
[0021]
[0022] In some embodiments, acetonitrile-water solution is used as a solvent for dissolving the sample for detection, and preferably the solvent is a solution in which acetonitrile and water have a volume ratio of 1:1.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] 1. The present invention adopts high performance liquid chromatography and selects methanol or a methanol-acetonitrile mixed solution as mobile phase B for gradient elution, so that the chromatographic peak of the peroxide genotoxic impurity can be presented as a single chromatographic peak, and the impurity can be accurately quantified without the need for LC / MS coupling technology with high detection and maintenance costs.
[0025] 2. This method is used to detect peroxide genotoxic impurities in dapagliflozin. The method is easy to operate, with high sensitivity and accuracy, and can meet the control limit of 150 ppm, providing a reliable guarantee for the control of genotoxic impurities in dapagliflozin, thereby ensuring the safety of clinical use. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is the blank solution chromatogram in Example 1;
[0027] Figure 2 It is the chromatogram of the reference substance solution in Example 1;
[0028] Figure 3 This is the chromatogram of the spiked test solution (dapagliflozin API) in Example 1;
[0029] Figure 4 This is the chromatogram of the spiked test solution (dapagliflozin tablets) in Example 1;
[0030] Figure 5 The quantitative limit solution chromatogram in Example 2;
[0031] Figure 6 This is the chromatogram of the spiked test solution (dapagliflozin API) in Example 4;
[0032] Figure 7 This is the chromatogram of the spiked test solution (dapagliflozin tablets) in Example 4;
[0033] Figure 8 This is the chromatogram of the spiked test solution (dapagliflozin API) in Example 5;
[0034] Fig. 9 This is the chromatogram of the spiked test solution (dapagliflozin tablets) in Example 5;
[0035] Fig.10 The chromatogram of the positioning solution in Example 6;
[0036] Fig.11The chromatogram of the positioning solution in Example 7;
[0037] Fig.12 The chromatogram of the positioning solution in Comparative Example 1;
[0038] Fig.13 The chromatogram of the positioning solution in Comparative Example 2;
[0039] Fig.14 The chromatogram of the positioning solution in Comparative Example 3;
[0040] Fig.15 The chromatogram of the positioning solution in Comparative Example 4;
[0041] Fig.16 The chromatogram of the positioning solution in Comparative Example 5;
[0042] Fig.17 The chromatogram of the positioning solution in Comparative Example 6;
[0043] Fig.18 This is the chromatogram of the positioning solution in Comparative Example 7. DETAILED DESCRIPTION
[0044] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below through specific embodiments, but the content and scope of the present invention are not limited by the following embodiments.
[0045] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention, and the peroxide genotoxic impurity reference substance was purchased from Shenzhen Zhenqiang Biotechnology Co., Ltd. The IMP A peak in each chromatogram is a peroxide genotoxic impurity.
[0046] Example 1 Specificity Test
[0047] (1) Chromatographic conditions
[0048] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0049] Mobile phase A: water
[0050] Mobile phase B: methanol
[0051] Column temperature: 40°C
[0052] Flow rate: 1.0ml / min
[0053] Detection wavelength: 224nm
[0054] Injection volume: 10 μl
[0055] Gradient Program:
[0056]
[0057] (2) Solution preparation
[0058] Solvent: Take 500ml of water and 500ml of acetonitrile and mix well.
[0059] Blank solution: solvent.
[0060] Mobile phase A: Take 1000 ml of water and degas by ultrasonication.
[0061] Mobile phase B: Take 1000 ml of methanol and degas by ultrasonication.
[0062] Stock solution of reference substance: Weigh about 15 mg of peroxide genotoxic impurity reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add solvent to dissolve it by ultrasonic and dilute it to the scale, shake it well; accurately measure 5 ml of the above solution, place it in a 100 ml volumetric flask, and dilute it to the scale with solvent.
[0063] Reference substance solution: Accurately measure 5 ml of the reference substance stock solution mentioned above, place it in a 50 ml volumetric flask, and dilute to the scale with solvent.
[0064] Spiked test solution (API): Weigh approximately 250 mg of dapagliflozin API, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, accurately add 5 ml of reference stock solution, dilute to the mark with solvent, and shake well.
[0065] Spiked test solution (dapagliflozin tablets): Take 25 dapagliflozin tablets, place in a 50 ml volumetric flask, add an appropriate amount of solvent, ultrasonicate for 20 minutes, accurately add 5 ml of reference stock solution, dilute to the scale with solvent, shake well, filter through a 0.22 μm PTFE filter membrane, discard 3 ml of the initial filtrate, and take the subsequent filtrate.
[0066] (3) Detection methods and test results
[0067] Inject the blank solution, reference solution, and each spiked test solution into the chromatograph and record the chromatograms. The blank solution chromatogram, reference solution chromatogram, spiked test solution chromatogram (dapagliflozin API), and spiked test solution chromatogram (dapagliflozin tablets) are shown in Figure 1. Figure 1 , Figure 2 , Figure 3 and Figure 4 shown.
[0068] Under the chromatographic conditions, the blank solution, dapagliflozin API and dapagliflozin tablet matrix did not interfere with the detection of peroxide genotoxic impurities, indicating that the method had good specificity.
[0069] Example 2 Detection limit and quantification limit test
[0070] (1) Chromatographic conditions: same as in Example 1.
[0071] (2) Solution preparation
[0072] The solvent, mobile phase A, mobile phase B, reference substance stock solution, and reference substance solution were the same as those in Example 1.
[0073] Detection limit solution (10% of the limit concentration): Accurately measure 1 ml of the reference solution, place it in a 10 ml volumetric flask, and dilute to the mark with solvent.
[0074] Quantitative limit solution (30% of the limit concentration): Accurately measure 1.5 ml of the reference stock solution, place it in a 50 ml volumetric flask, and dilute to the scale with solvent.
[0075] (3) Detection methods and test results
[0076] Inject the detection limit solution and the quantitative limit solution into the chromatograph and record the chromatogram. Figure 5 The S / N of the detection limit solution and the S / N of the quantification limit solution were 15.5 and 50.1, respectively, which were much larger than the acceptable standards (the acceptable standards were 3 and 10, respectively), indicating that the sensitivity of this method was high.
[0077] Example 3 Accuracy Test
[0078] (1) Chromatographic conditions: same as in Example 1.
[0079] (2) Solution preparation
[0080] The solvent, mobile phase A, mobile phase B, reference substance stock solution and reference substance solution were the same as those in Example 1.
[0081] Quantitative limit accuracy solution (dapagliflozin API): Weigh approximately 250 mg of dapagliflozin API, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, accurately add 1.5 ml of reference substance stock solution, and dilute to scale with solvent. Prepare 3 portions in parallel.
[0082] Quantitative limit accuracy solution (Dapagliflozin tablets): Take 25 Dapagliflozin tablets, place in a 50ml volumetric bottle, add an appropriate amount of solvent, sonicate for 20 minutes, accurately add 1.5ml of reference substance stock solution, dilute to scale with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard 3ml of the initial filtrate, and take the subsequent filtrate. Prepare 3 copies in parallel.
[0083] 50% accuracy solution (dapagliflozin API): Weigh about 250 mg of dapagliflozin API, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, accurately add 2.5 ml of reference substance stock solution, and dilute to scale with solvent. Prepare 3 portions in parallel.
[0084] 50% accuracy solution (Dapagliflozin tablets): Take 25 Dapagliflozin tablets, place in a 50ml volumetric bottle, add an appropriate amount of solvent, sonicate for 20 minutes, accurately add 2.5ml of reference substance stock solution, dilute to scale with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard 3ml of the initial filtrate, and take the subsequent filtrate. Prepare 3 copies in parallel.
[0085] 100% accuracy solution (Dapagliflozin API): Weigh approximately 250 mg of Dapagliflozin API, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, accurately add 5 ml of reference stock solution, and dilute to scale with solvent. Prepare 3 portions in parallel.
[0086] 100% accuracy solution (Dapagliflozin tablets): Take 25 Dapagliflozin tablets, place in a 50ml volumetric bottle, add an appropriate amount of solvent, sonicate for 20 minutes, accurately add 5ml of reference stock solution, dilute to scale with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard 3ml of the initial filtrate, and take the subsequent filtrate. Prepare 3 copies in parallel.
[0087] 150% accuracy solution (Dapagliflozin API): Weigh about 250 mg of Dapagliflozin API accurately, place in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, accurately add 7.5 ml of reference substance stock solution, and dilute to the mark with solvent. Prepare 3 portions in parallel.
[0088] 150% accuracy solution (Dapagliflozin tablets): Take 25 Dapagliflozin tablets, place in a 50ml volumetric bottle, add an appropriate amount of solvent, sonicate for 20 minutes, accurately add 7.5ml of reference substance stock solution, dilute to scale with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard 3ml of the initial filtrate, and take the subsequent filtrate. Prepare 3 copies in parallel.
[0089] (3) Detection methods and test results
[0090] Each accuracy solution was injected into the chromatograph and the chromatogram was recorded. Table 1 and Table 2 are the accuracy test results of the peroxide genotoxic impurities in dapagliflozin API and dapagliflozin tablets, respectively. The spike recovery rates were between 95% and 105%, and the RSDs were 2.00% and 2.23%, respectively, indicating that the method has high accuracy and good precision.
[0091] Table 1 Dapagliflozin API Accuracy Test Results
[0092]
[0093] Table 2 Dapagliflozin tablets accuracy test results
[0094]
[0095] Example 4
[0096] (1) Chromatographic conditions:
[0097] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0098] Mobile phase A: 0.1% trifluoroacetic acid solution
[0099] Mobile phase B: methanol
[0100] Column temperature: 40°C
[0101] Flow rate: 1.0ml / min
[0102] Detection wavelength: 224nm
[0103] Injection volume: 10 μl
[0104] Gradient program: same as Example 1
[0105] (2) Solution preparation
[0106] Solvent: Take 500ml of water and 500ml of acetonitrile and mix well.
[0107] Blank solution: solvent.
[0108] Mobile phase A: Take 1000 ml of water, add 1 ml of trifluoroacetic acid, and degas by ultrasonication.
[0109] Mobile phase B: Take 1000 ml of methanol and degas by ultrasonication.
[0110] The reference substance stock solution, reference substance solution, and each spiked test solution are the same as those in Example 1.
[0111] (3) Detection methods and test results
[0112] Inject the blank solution, reference solution, and each spiked test solution into the chromatograph and record the chromatograms. The chromatograms of the spiked test solution (dapagliflozin API) and the chromatograms of the spiked test solution (dapagliflozin tablets) are as follows: Figure 6 and Figure 7 When mobile phase A is trifluoroacetic acid aqueous solution, the peroxide genotoxic impurity presents a single chromatographic peak, the test sample matrix does not interfere with the detection, and the peroxide genotoxic impurity and adjacent impurity peaks are well separated.
[0113] Example 5
[0114] (1) Chromatographic conditions:
[0115] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0116] Mobile phase A: 20mmol / L ammonium acetate buffer
[0117] Mobile phase B: methanol
[0118] Column temperature: 40°C
[0119] Flow rate: 1.0ml / min
[0120] Detection wavelength: 224nm
[0121] Injection volume: 10 μl
[0122] Gradient program: same as Example 1.
[0123] (2) Solution preparation
[0124] Solvent: Take 500ml of water and 500ml of acetonitrile and mix well.
[0125] Blank solution: solvent.
[0126] Mobile phase A: Weigh 1.54 g of ammonium acetate, add 1000 ml of water, dissolve by ultrasonication, adjust the pH to 4.5 with acetic acid, and filter.
[0127] Mobile phase B: Take 1000 ml of methanol and degas by ultrasonication.
[0128] The reference substance stock solution, reference substance solution, and each spiked test solution are the same as those in Example 1.
[0129] (3) Detection methods and test results
[0130] Inject the blank solution, reference solution, and each spiked test solution into the chromatograph and record the chromatograms. The chromatograms of the spiked test solution (dapagliflozin API) and the chromatograms of the spiked test solution (dapagliflozin tablets) are as follows: Figure 8 and Fig. 9 When mobile phase A is ammonium acetate buffer, the peroxide genotoxic impurity presents a single chromatographic peak, the test sample matrix does not interfere with the detection, and the peroxide genotoxic impurity and adjacent impurity peaks are well separated.
[0131] Example 6
[0132] (1) Chromatographic conditions:
[0133] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0134] Mobile phase A: water
[0135] Mobile phase B: methanol-acetonitrile (70:30)
[0136] Column temperature: 40°C
[0137] Flow rate: 1.0ml / min
[0138] Detection wavelength: 224nm
[0139] Injection volume: 10 μl
[0140] Gradient Program:
[0141]
[0142]
[0143] (2) Solution preparation
[0144] Solvent: Take 500ml of water and 500ml of acetonitrile and mix well.
[0145] Blank solution: solvent.
[0146] Mobile phase A: Take 1000 ml of water and degas by ultrasonication.
[0147] Mobile phase B: Take 700 ml of methanol and 300 ml of acetonitrile, mix well, and degas by ultrasonication.
[0148] Peroxide genotoxic impurity localization solution: Weigh about 5 mg of peroxide genotoxic impurity reference substance, place it in a 50 ml volumetric flask, add solvent to ultrasonically dissolve and dilute to the scale.
[0149] (3) Detection methods and test results
[0150] Inject the peroxide genotoxic impurity location solution into the chromatograph and record the chromatogram. Fig.10 As shown, under the chromatographic conditions, the chromatographic peak of the peroxide genotoxic impurity also presents a single chromatographic peak, which can be used to detect the peroxide genotoxic impurity of dapagliflozin.
[0151] Example 7
[0152] (1) Chromatographic conditions:
[0153] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0154] Mobile phase A: water
[0155] Mobile phase B: methanol-acetonitrile (40:60)
[0156] Column temperature: 40°C
[0157] Flow rate: 1.0ml / min
[0158] Detection wavelength: 224nm
[0159] Injection volume: 5 μl
[0160] Gradient program: same as Example 6
[0161] (2) Solution preparation
[0162] Solvent: Take 500ml of water and 500ml of acetonitrile and mix well.
[0163] Blank solution: solvent.
[0164] Mobile phase A: Take 1000 ml of water and degas by ultrasonication.
[0165] Mobile phase B: Take 400 ml of methanol and 600 ml of acetonitrile, mix well, and degas by ultrasonication.
[0166] Peroxide genotoxic impurity localization solution: same as Example 6.
[0167] (3) Detection methods and test results
[0168] Inject the peroxide genotoxic impurity location solution into the chromatograph and record the chromatogram. Fig.11 As shown, under the chromatographic conditions, the chromatographic peak of the peroxide genotoxic impurity also presents a single chromatographic peak, which can be used to detect the peroxide genotoxic impurity of dapagliflozin.
[0169] Comparative Example 1
[0170] Refer to the chromatographic conditions of the import registration standard for dapagliflozin tablets.
[0171] (1) Chromatographic conditions
[0172] Chromatographic column: Zorbax SB C18, 4.6 mm × 150 mm, 5 μm
[0173] Mobile phase A: 0.1% trifluoroacetic acid in water
[0174] Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile
[0175] Column temperature: 35°C
[0176] Flow rate: 1.0ml / min
[0177] Detection wavelength: 224nm
[0178] Injection volume: 10 μl
[0179] Gradient Program:
[0180]
[0181] (2) Solution preparation
[0182] Solvent: Take 500ml of acetonitrile and 500ml of water and mix well.
[0183] Mobile phase A: Take 1000 ml of water, add 1 ml of trifluoroacetic acid, mix well, and degas by ultrasonication.
[0184] Mobile phase B: Take 1000 ml of acetonitrile, add 1 ml of trifluoroacetic acid, mix well, and degas by ultrasonication.
[0185] Peroxide genotoxic impurity localization solution: same as Example 6.
[0186] (3) Detection methods and test results
[0187] Inject the peroxide genotoxic impurity location solution into the chromatograph and record the chromatogram. Fig.12 As shown, under this chromatographic condition, the chromatographic peak of the peroxide genotoxic impurity presents a double peak.
[0188] Comparative Example 2
[0189] (1) Chromatographic conditions
[0190] Chromatographic column: Zorbax SB C18, 4.6 mm × 150 mm, 5 μm
[0191] Mobile phase A: water
[0192] Mobile phase B: acetonitrile
[0193] Column temperature: 35°C
[0194] Flow rate: 1.0ml / min
[0195] Detection wavelength: 224nm
[0196] Injection volume: 5 μl
[0197] Gradient program: same as Example 6.
[0198] (2) Solution preparation
[0199] Peroxide genotoxic impurity localization solution: same as Example 6.
[0200] (3) Test results
[0201] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.13 As shown in Figure 2, under a neutral mobile phase system, the chromatographic peak of peroxide genotoxic impurities still shows double peaks.
[0202] Comparative Example 3
[0203] (1) Chromatographic conditions
[0204] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0205] Mobile phase A: water
[0206] Mobile phase B: acetonitrile
[0207] Column temperature: 35°C
[0208] Flow rate: 1.0ml / min
[0209] Detection wavelength: 224nm
[0210] Injection volume: 5 μl
[0211] Gradient program: same as Example 6.
[0212] (2) Solution preparation
[0213] Peroxide genotoxic impurity localization solution: same as Example 6.
[0214] (3) Test results
[0215] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.14 When a hydrophilic and highly selective reverse phase column is used, the chromatographic peak of the peroxide genotoxic impurity still shows a double peak.
[0216] Comparative Example 4
[0217] The detection was performed using a Comixsil RP-100 column with a mixed separation mode, which combines three separation modes: ion exchange, hydrophobic and HILIC.
[0218] (1) Chromatographic conditions
[0219] Column: Comixsil RP-100, 4.6 mm × 150 mm, 5 μm
[0220] Mobile phase A: 0.5% sulfuric acid in water
[0221] Mobile phase B: acetonitrile
[0222] Column temperature: 35°C
[0223] Flow rate: 1.0ml / min
[0224] Detection wavelength: 224nm
[0225] Injection volume: 5 μl
[0226] Gradient program: same as Example 6.
[0227] (2) Solution preparation
[0228] Mobile phase A: Take 1000 ml of water, add 5 ml of sulfuric acid, mix well, and degas by ultrasonication.
[0229] Peroxide genotoxic impurity localization solution: same as Example 6.
[0230] (3) Detection methods and test results
[0231] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.15 When the chromatographic column is used in mixed separation mode, the chromatographic peak of peroxide genotoxic impurities still presents double peaks.
[0232] Comparative Example 5
[0233] (1) Chromatographic conditions
[0234] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0235] Mobile phase A: 10mmol / L potassium dihydrogen phosphate solution (5mol / L sodium hydroxide solution adjusted to pH 8.0)
[0236] Mobile phase B: acetonitrile
[0237] Column temperature: 35°C
[0238] Flow rate: 1.0ml / min
[0239] Detection wavelength: 224nm
[0240] Injection volume: 5 μl
[0241] Gradient program: same as Example 6.
[0242] (2) Solution preparation
[0243] Mobile phase A: Weigh 1.36 g of potassium dihydrogen phosphate, add 1000 ml of water, dissolve by ultrasonication, adjust the pH to 8.0 with 5 mol / L sodium hydroxide solution, and filter.
[0244] Peroxide genotoxic impurity localization solution: same as Example 6.
[0245] (3) Detection methods and test results
[0246] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.16 When using an alkaline mobile phase system, the chromatographic peak of the peroxide genotoxic impurity still presents a double peak.
[0247] Comparative Example 6
[0248] (1) Chromatographic conditions
[0249] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0250] Mobile phase A: water
[0251] Mobile phase B: methanol-acetonitrile (20:80)
[0252] Column temperature: 35°C
[0253] Flow rate: 1.0ml / min
[0254] Detection wavelength: 224nm
[0255] Injection volume: 5 μl
[0256] Gradient program: same as Example 6.
[0257] (2) Solution preparation
[0258] Mobile phase B: Take 200 ml of methanol and 800 ml of acetonitrile, mix well, and degas by ultrasonication.
[0259] Peroxide genotoxic impurity localization solution: same as Example 6.
[0260] (3) Detection methods and test results
[0261] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.17 When a methanol-acetonitrile (20:80) mixed solution was used as mobile phase B, the chromatographic peak of the peroxide genotoxic impurity showed a double peak, but the peak shape was improved compared with that of pure acetonitrile as the mobile phase.
[0262] Comparative Example 7
[0263] (1) Chromatographic conditions
[0264] Chromatographic column: Sepax HP-C18, 4.6mm×150mm, 5μm
[0265] Mobile phase A: water
[0266] Mobile phase B: methanol-acetonitrile (30:70)
[0267] Column temperature: 35°C
[0268] Flow rate: 1.0ml / min
[0269] Detection wavelength: 224nm
[0270] Injection volume: 5 μl
[0271] Gradient program: same as Example 6.
[0272] (2) Solution preparation
[0273] Mobile phase B: Take 300 ml of methanol and 700 ml of acetonitrile, mix well, and degas by ultrasonication.
[0274] Peroxide genotoxic impurity localization solution: same as Example 6.
[0275] (3) Detection methods and test results
[0276] Inject the peroxide genotoxic impurity positioning solution into the chromatograph and record the chromatogram. Fig.18 When the methanol-acetonitrile (30:70) mixed solution was used as mobile phase B, the peak shape of the chromatographic peak of the peroxide genotoxic impurity was significantly improved, but the chromatographic peak was round-headed and not sharp. The test results showed that as the amount of methanol in mobile phase B increased, the peak shape became better and better.
Claims
1. A method for detecting genotoxic impurities of dapagliflozin, characterized in that: A high performance liquid chromatography method is used, and the detection conditions of the high performance liquid chromatography method include: using an octadecyl bonded silica gel column as a chromatographic column; mobile phase A is any one of water, trifluoroacetic acid aqueous solution, and ammonium acetate buffer; mobile phase B is a mixture of methanol and acetonitrile, and the volume of methanol accounts for not less than 40%; and gradient elution is performed.
2. The detection method according to claim 1, characterized in that: The peroxidation genotoxic impurity is a compound represented by formula II:
3. The detection method according to claim 1, characterized in that: The concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.08-0.12%.
4. The detection method according to claim 1, characterized in that: The pH of the ammonium acetate buffer is 4-5.
5. The detection method according to claim 1 or 4, characterized in that: The concentration of ammonium acetate in the ammonium acetate buffer is 10-30 mmol / L.
6. The detection method according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography method also include: column temperature of 35-45°C, injection volume of 5-15 μl, flow rate of 0.8-1.2 ml / min; and detection wavelength of 222-226 nm.
7. The detection method according to claim 1, characterized in that: The chromatographic column was selected as a Sepax HP-C18 column.
8. The detection method according to claim 7, characterized in that: The specifications of the Sepax HP-C18 column are particle size×inner diameter×column length: 5 μm×4.6 mm×150 mm.
9. The detection method according to claim 1, characterized in that: The mobile phase B is methanol, and the elution program is set as follows:
10. The detection method according to claim 1, characterized in that: The mobile phase B is a mixture of methanol and acetonitrile, with methanol accounting for 40% by volume, and the elution program is set as follows: