Detection method of sitagliptin phosphate related substances

Abstract

The present invention relates to a method for detecting related substances of sitagliptin phosphate, wherein the column temperature is 30°C, the flow rate of the mobile phase is 1.0 ml / min, the detection wavelength is 205 nm, the stationary phase is cyanosilane bonded silica gel, the mobile phase is mobile phase A and B; mobile phase A is an ion pair reagent added to a phosphate buffer solution, and the pH value is adjusted with acid; mobile phase B is acetonitrile, and gradient elution is performed; diluent; mixed impurity stock solution: impurities B, C, D, E, F, and G are weighed and diluted, and 1.0 ml of the obtained mixed solution is taken for dilution; system suitability solution: dilution of sitagliptin phosphate and mixed impurity stock solution; sample solution: dilution of sitagliptin phosphate sample; sample solution is injected into a high performance liquid chromatograph, and the content of related substances in sitagliptin phosphate is calculated by the main component self-comparison method. Related substances in sitagliptin phosphate can be efficiently separated under the same chromatographic conditions, the quality of the raw material drug can be effectively controlled, the specificity is good, the accuracy is strong, the precision is high, and the operation is performed at room temperature.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting sitagliptin phosphate-related substances. Background Art

[0002] Type 2 diabetes is one of the types of diabetes and is the most common type of diabetes. It is caused by insulin resistance, insufficient insulin utilization, and relatively insufficient insulin secretion, which lead to increased blood sugar levels. It mostly occurs after adulthood and accounts for more than 90% of all diabetic people. Patients with type 2 diabetes will also have many clinical complications, including diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, as well as stroke and myocardial infarction. The treatment of type 2 diabetes is mainly to improve insulin sensitivity, mainly oral hypoglycemic drugs. When the pancreatic function of patients with type 2 diabetes is severely damaged, exogenous insulin needs to be supplemented.

[0003] Sitagliptin is a potent and highly selective dipeptidyl peptidase 4 (DPP-4) inhibitor that can increase insulin release and reduce glucagon levels in a glucose-dependent manner. It is effective and safe for the treatment of type 2 diabetes.

[0004] Sitagliptin's chemical name is (R)-3-amino-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butane-1-one, chemical structure:

[0005]

[0006] Sitagliptin phosphate is a highly selective small molecule dipeptidyl peptidase-4 (DPP-4) inhibitor developed by Merck Pharmaceuticals at Merck Research Laboratories in New Jersey, USA, by screening more than 800,000 compounds and synthesizing and improving more than 2,000 compounds. The compound was first shown in animal studies to improve blood sugar control and pancreatic islet function. After passing toxicology and safety evaluations, it soon entered clinical trials and was renamed sitagliptin.

[0007] In the clinical research phase, Merck scientists used only two years and one quarter to prove that oral sitagliptin alone or in combination with metformin and other hypoglycemic drugs can significantly improve blood sugar control. On October 17, 2006, the FDA officially approved sitagliptin (trade name, Januvia) for marketing, becoming the first DPP-4 inhibitor approved worldwide, and also ushering in a new era of using gliptins to treat diabetes.

[0008] At present, the API of sitagliptin phosphate is included in both EP and USP. In the related substance detection method, the mobile phase is acetonitrile-0.01 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.0 with phosphoric acid) (volume ratio 15:85), and the mobile phase elution is isocratic elution. The disadvantages are:

[0009] ①The retention time of each impurity is unstable;

[0010] ②The separation effect of impurities D, E and impurities F, G is poor;

[0011] ③The detection time is too long. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a method for detecting sitagliptin phosphate-related substances to overcome the deficiencies in the above-mentioned prior art.

[0013] The technical solution of the present invention to solve the above technical problem is as follows: A method for detecting sitagliptin phosphate related substances comprises the following steps:

[0014] Chromatographic conditions:

[0015] The column temperature was 30°C, the flow rate of the mobile phase was 1.0 ml / min, the detection wavelength was 205 nm, the stationary phase was cyanosilane bonded silica gel, and the mobile phases were mobile phase A and mobile phase B; mobile phase A was phosphate buffer solution with an ion pair reagent, and the pH value was adjusted to 2.0 with acid; mobile phase B was acetonitrile, with gradient elution;

[0016] Solution preparation:

[0017] Diluent: acetonitrile and water;

[0018] Mixed impurity stock solution: accurately weigh impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G respectively, dissolve them with appropriate amount of acetonitrile, dilute to scale with diluent, mix well, accurately measure 1.0 ml of the obtained mixed solution, dilute to scale with diluent, and mix well;

[0019] System suitability solution: Take sitagliptin phosphate and accurately pipette 1.0 ml of mixed impurity stock solution, mix, dissolve and dilute to scale with diluent, and mix well;

[0020] Sample solution: Take a sample of sitagliptin phosphate, dissolve it in diluent and dilute to scale, mix well;

[0021] Separation analysis:

[0022] The sample solution and system suitability solution (standard solution) were respectively injected into the high performance liquid chromatograph, and the content of related substances in sitagliptin phosphate was calculated by the main component self-reference method.

[0023] Based on the above technical solution, the present invention can also be improved as follows.

[0024] Further: the phosphate buffer solution is potassium dihydrogen phosphate solution.

[0025] Going further: The concentration of potassium dihydrogen phosphate solution is 0.01 mol / L.

[0026] Further: the ion pair reagent is sodium octane sulfonate.

[0027] Furthermore: the concentration of sodium octane sulfonate is 0.005 mol / L to 0.01 mol / L.

[0028] Going further: the concentration of sodium octane sulfonate is 0.007 mol / L.

[0029] Further: phosphoric acid is used in mobile phase A to adjust the pH value.

[0030] Further: the volume ratio of the diluent is acetonitrile: water = 5:95; or, the volume ratio of the diluent 2 is acetonitrile: water = 30:70.

[0031] Further: Gradient elution is specifically:

[0032] In the elution gradient from 0 to 5 min, the volume ratio of acetonitrile to phosphate buffer solution was 10%:90%;

[0033] 5. From 01min to 30min, the volume ratio of acetonitrile increased to 30%, and the volume ratio of phosphate buffer solution decreased to 70%;

[0034] From 30.01 min to 35 min, the volume ratio of acetonitrile to phosphate buffer solution remained unchanged at 30%:70%;

[0035] From 35.01 min to 45 min, the volume ratio of acetonitrile increased to 40%, and the volume ratio of phosphate buffer solution decreased to 60%;

[0036] From 45.01min to 48min, the volume ratio of acetonitrile to phosphate buffer solution remained unchanged at 40%:60%;

[0037] From 48.01min to 52min, the volume ratio of acetonitrile increased to 70%, and the volume ratio of phosphate buffer solution decreased to 30%;

[0038] From 52.01min to 55min, the volume ratio of acetonitrile to phosphate buffer solution remained unchanged at 30%:70%;

[0039] At 55.01min-56min, the volume ratio of acetonitrile decreased to 10%, and the volume ratio of phosphate buffer solution increased to 90%;

[0040] From 56.01min to 65min, the volume ratio of acetonitrile to phosphate buffer solution remained unchanged at 10%:90%.

[0041] Further: impurity B is 7-[(3R)-N-tert-butyloxycarbonyl-3-amino-1-oxo-4-(2,4,5-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine;

[0042] The structural formula is:

[0043]

[0044] Impurity C is (R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid hydrochloride;

[0045] The structural formula is:

[0046]

[0047] Impurity D is (E)-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)but-3-en-1-one;

[0048] The structural formula is:

[0049]

[0050] Impurity E is (E)-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)but-2-en-1-one;

[0051] The structural formula is:

[0052]

[0053] Impurity F is 7-[(3R)-3-amino-1-oxo-4-(2,5-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine hydrochloride;

[0054] The structural formula is:

[0055]

[0056] Impurity G is 7-[(3R)-3-amino-1-oxo-4-(2,4-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine hydrochloride;

[0057] The structural formula is:

[0058] .

[0059] further:

[0060] The solution preparation is as follows:

[0061] Diluent: acetonitrile and water in a volume ratio of 5:95;

[0062] Mixed impurity stock solution: accurately weigh 15 mg of each of impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, place them in a 50 ml volumetric flask, dissolve them with an appropriate amount of acetonitrile, dilute to the mark with diluent, and mix well; accurately measure 1.0 ml of the resulting mixed solution and place it in a 20 ml volumetric flask, dilute to the mark with diluent, and mix well;

[0063] System suitability solution: Take 13 mg of sitagliptin phosphate, containing about 10 mg of sitagliptin, and place it in a 10 ml volumetric flask. Accurately pipette 1.0 ml of mixed impurity stock solution into this volumetric flask, dissolve and dilute to the scale with diluent, and mix well;

[0064] Sample solution: Take 13 mg of sitagliptin phosphate sample, place it in a 10 ml volumetric flask, dissolve it with diluent and dilute to the scale, and mix well.

[0065] The beneficial effects of the present invention are:

[0066] The present invention provides a high performance liquid chromatography method for rapidly and efficiently analyzing sitagliptin and its related substances (degradation products, intermediates and starting materials in sitagliptin phosphate), thereby realizing the separation and determination of the related substances in sitagliptin phosphate under the same chromatographic conditions, the detection method has good specificity, high accuracy, high precision, accurate results, simple method operation, room temperature operation, and good solution stability and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : HPLC spectrum of the blank solution prepared according to Example 1;

[0068] Figure 2 : HPLC spectrum of the system suitability solution prepared according to Example 1;

[0069] Figure 3 : Linear spectrum of impurity C of the linear solution prepared according to Example 3;

[0070] Figure 4 : Linear spectrum of impurity F of the linear solution prepared according to Example 3;

[0071] Figure 5: Linear spectrum of impurity G of the linear solution prepared according to Example 3;

[0072] Figure 6 : Linear spectrum of sitagliptin phosphate of linear solution prepared according to Example 3;

[0073] Figure 7 : Linear spectrum of impurity D of the linear solution prepared according to Example 3;

[0074] Figure 8 : Linear spectrum of impurity E of the linear solution prepared according to Example 3;

[0075] Fig. 9 : Linear spectrum of impurity B of the linear solution prepared according to Example 3. DETAILED DESCRIPTION

[0076] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0077] Example 1

[0078] Chromatographic conditions

[0079] Instruments: Agilent 1260 high performance liquid chromatograph, G7115A UV detector;

[0080] Chromatographic column: a chromatographic column with cyanosilane bonded silica gel as the stationary phase (250*4.6mm, 5µm);

[0081] Mobile phase A: Add 0.007 mol / L sodium octane sulfonate to 0.01 mol / L potassium dihydrogen phosphate solution, and adjust the pH to 2.0 with phosphoric acid;

[0082] Mobile phase B: acetonitrile;

[0083] Gradient elution: from 0 to 5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90%;

[0084] 5. From 01min to 30min, the volume ratio of acetonitrile increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 70%;

[0085] From 30.01min to 35min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0086] From 35.01min to 45min, the volume ratio of acetonitrile increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 60%;

[0087] From 45.01min to 48min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 40%:60%;

[0088] From 48.01min to 52min, the volume ratio of acetonitrile increased to 70%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 30%;

[0089] At 52.01min to 55min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0090] At 55.01min-56min, the volume ratio of acetonitrile decreased to 10%, and the volume ratio of potassium dihydrogen phosphate solution increased to 90%;

[0091] At 56.01min to 65min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90% and remains unchanged.

[0092] Flow rate: 1.0ml / min;

[0093] Detection wavelength: 205nm;

[0094] Column temperature: 30°C;

[0095] Injection volume: 20 µl;

[0096] The diluent was acetonitrile: water = 5:95 (V:V);

[0097] Solution preparation:

[0098] Blank solution: diluent;

[0099] Mixed impurity stock solution: accurately weigh about 15 mg of each of impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, place them in a 50 ml volumetric flask, dissolve them with an appropriate amount of acetonitrile, dilute to the mark with diluent, and mix well; accurately measure 1.0 ml of the above mixed solution and place it in a 20 ml volumetric flask, dilute to the mark with diluent, and mix well;

[0100] System suitability solution: Take about 13 mg of sitagliptin phosphate (including about 10 mg of sitagliptin) and place it in a 10 ml volumetric flask. Accurately pipette 1.0 ml of the above mixed impurity stock solution into this volumetric flask, dissolve it with diluent and dilute to the scale, and mix well.

[0101] Testing steps: Take blank solution and system suitability solution respectively, perform HPLC analysis under the above-mentioned setting conditions, and record the chromatogram;

[0102] Figure 1 HPLC spectrum of the blank solution prepared in Example 1; Figure 2 The HPLC spectrum of the system suitability solution prepared in Example 1 is shown as follows;

[0103] Table 1 - Liquid chromatography analysis results of Example 1 system suitability solutions;

[0104]

[0105] Example 2

[0106] Stability testing of system suitability solutions

[0107] Chromatographic conditions

[0108] Instruments: Agilent 1260 high performance liquid chromatograph, G7115A UV detector;

[0109] Chromatographic column: a chromatographic column with cyanosilane bonded silica gel as the stationary phase (250*4.6mm, 5µm);

[0110] Mobile phase A: Add 0.007 mol / L sodium octane sulfonate to 0.01 mol / L potassium dihydrogen phosphate solution, and adjust the pH to 2.0 with phosphoric acid;

[0111] Mobile phase B: acetonitrile;

[0112] Gradient elution: from 0 to 5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90%;

[0113] 5. From 01min to 30min, the volume ratio of acetonitrile increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 70%;

[0114] From 30.01min to 35min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0115] From 35.01min to 45min, the volume ratio of acetonitrile increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 60%;

[0116] From 45.01min to 48min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 40%:60%;

[0117] From 48.01min to 52min, the volume ratio of acetonitrile increased to 70%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 30%;

[0118] At 52.01min to 55min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0119] At 55.01min-56min, the volume ratio of acetonitrile decreased to 10%, and the volume ratio of potassium dihydrogen phosphate solution increased to 90%;

[0120] At 56.01min to 65min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90% and remains unchanged.

[0121] Flow rate: 1.0ml / min;

[0122] Detection wavelength: 205nm;

[0123] Column temperature: 30°C;

[0124] Injection volume: 20 µl;

[0125] The diluent was acetonitrile: water = 5:95 (V:V);

[0126] Solution preparation:

[0127] Blank solution: diluent;

[0128] Mixed impurity stock solution: accurately weigh about 15 mg of each of impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, place them in a 50 ml volumetric flask, dissolve them with an appropriate amount of acetonitrile, dilute to the mark with diluent, and mix well; accurately measure 1.0 ml of the above mixed solution and place it in a 20 ml volumetric flask, dilute to the mark with diluent, and mix well;

[0129] System suitability solution: Take about 13 mg of sitagliptin phosphate (including about 10 mg of sitagliptin) and place it in a 10 ml volumetric flask. Accurately pipette 1.0 ml of the above mixed impurity stock solution into this volumetric flask, dissolve it with diluent and dilute to the scale, and mix well.

[0130] Test method: The system suitability solution was placed for 24 hours, and samples were taken and injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h;

[0131] Under the set chromatographic conditions, the relative standard deviations of the areas of sitagliptin phosphate and each impurity in each needle spectrum of the system suitability solution within 24 hours were calculated, as shown in Table 2. The relative standard deviations (RSDs) of the peak areas of each substance did not exceed 2%;

[0132] Table 2 - Results of stability test analysis of various substances in liquid chromatography of the system suitability solution of Example 2

[0133] name Impurity C Impurity F Impurity G Sitagliptin phosphate Impurity D Impurity E Impurity B 0 hours 44.53 83.52 88.88 47113.6 75.43 145.35 68.49 2 hours 44.31 84.04 87.39 47136.4 75.55 144.50 67.90 4 hours 44.49 84.07 87.46 47153.2 75.44 144.42 68.48 6 hours 44.55 84.18 87.35 47300.6 75.43 144.15 69.90 8 hours 44.68 84.08 87.48 47303.6 75.39 144.35 67.81 10 hours 44.76 84.23 87.52 47352.6 76.57 146.17 67.05 12 hours 44.54 84.23 87.52 47355.9 77.11 146.14 69.09 24 hours 45.24 83.88 89.23 47575.0 76.89 144.69 68.01 average value 44.64 84.03 87.85 47286.36 75.98 144.97 68.34 RSD(%) 0.62 0.28 0.85 0.32 0.98 0.56 1.27

[0134] Example 3

[0135] A method for detecting sitagliptin phosphate-related substances comprises the following steps:

[0136] Chromatographic conditions

[0137] Instruments: Agilent 1260 high performance liquid chromatograph, G7115A UV detector;

[0138] Chromatographic column: a chromatographic column with cyanosilane bonded silica gel as the stationary phase (250*4.6mm, 5µm);

[0139] Mobile phase A: Add 0.007 mol / L sodium octane sulfonate to 0.01 mol / L potassium dihydrogen phosphate solution, and adjust the pH to 2.0 with phosphoric acid;

[0140] Mobile phase B: acetonitrile;

[0141] Gradient elution: from 0 to 5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90%;

[0142] 5. From 01min to 30min, the volume ratio of acetonitrile increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 70%;

[0143] From 30.01min to 35min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0144] From 35.01min to 45min, the volume ratio of acetonitrile increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 60%;

[0145] From 45.01min to 48min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 40%:60%;

[0146] From 48.01min to 52min, the volume ratio of acetonitrile increased to 70%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 30%;

[0147] At 52.01min to 55min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%;

[0148] At 55.01min-56min, the volume ratio of acetonitrile decreased to 10%, and the volume ratio of potassium dihydrogen phosphate solution increased to 90%;

[0149] At 56.01min to 65min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90% and remains unchanged.

[0150] Flow rate: 1.0ml / min;

[0151] Detection wavelength: 205nm;

[0152] Column temperature: 30°C;

[0153] Injection volume: 20 µl;

[0154] Dilution 1: acetonitrile: water = 5:95 (V:V);

[0155] Dilution 2 is acetonitrile: water = 30:70 (V:V);

[0156] Solution preparation:

[0157] Stock solution: accurately weigh 19 mg of sitagliptin phosphate, about 15 mg of impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, respectively, place them in different 100 ml volumetric bottles, dissolve and dilute to the scale with diluent 2, mix well, and use as the stock solution of each substance;

[0158] Linear master solution a: Accurately measure 5.0 ml of each impurity stock solution and sitagliptin phosphate stock solution, place them in the same 50 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0159] Linear mother solution b: accurately measure 2.0 ml of impurity B and impurity C stock solutions, 1.0 ml of sitagliptin phosphate stock solution, impurity D, impurity E, impurity F, and impurity G impurity stock solutions, place them in the same 100 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0160] Linear solution 1: Accurately measure 1.0 ml of linear master solution b, place it in a 20 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0161] Linear solution 2: Accurately measure 1.0 ml of linear master solution a, place it in a 20 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0162] Linear solution 3: Accurately measure 1.0 ml of linear stock solution a, place it in a 10 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0163] Linear solution 4: Accurately measure 3.0 ml of linear master solution a, place in a 20 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0164] Linear solution 5: Accurately measure 2.0 ml of linear stock solution a, place in a 10 ml volumetric flask, add diluent 1 to dilute to the scale, and mix well;

[0165] Detection steps: Under the above-mentioned set conditions, perform HPLC analysis in the order of blank sample and linear solutions 1 to 5, and record the chromatogram;

[0166] Standard curve drawing:

[0167] Draw a calibration curve based on the mass concentration and peak area of ​​the standard series. Perform linear regression with the ratio of the linear solution as the horizontal axis (X) and the peak area as the vertical axis (Y) to obtain the regression equation and correlation coefficient;

[0168] Within the measurement range, each impurity and sitagliptin phosphate have a good linear relationship, and the linear correlation coefficient R2 is not less than 0.999. The data are shown in Tables 3 to 9.

[0169] Table 3 - Liquid Chromatography Impurity C Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0170]

[0171] Table 4 - Liquid Chromatography Impurity F Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0172]

[0173] Table 5 - Liquid Chromatography Impurity G Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0174]

[0175] Table 6 - Liquid chromatography peak analysis results of sitagliptin phosphate in linear solution of Example 3

[0176]

[0177] Table 7 - Liquid Chromatography Impurity D Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0178]

[0179] Table 8 - Liquid Chromatography Impurity E Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0180]

[0181] Table 9 - Liquid Chromatography Impurity B Chromatographic Peak Analysis Results of Example 3 Linear Solution

[0182]

[0183] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting sitagliptin phosphate related substances, characterized in that: The steps include: Chromatographic conditions: The column temperature was 30°C, the flow rate of the mobile phase was 1.0 ml / min, the detection wavelength was 205 nm, the mobile phases were mobile phase A and mobile phase B; gradient elution; Chromatographic column: chromatographic column with cyanosilane bonded silica gel as stationary phase, 250*4.6mm, 5μm; Mobile phase A: Add 0.007 mol / L sodium octane sulfonate to 0.01 mol / L potassium dihydrogen phosphate solution, and adjust the pH to 2.0 with phosphoric acid; Mobile phase B: acetonitrile; Gradient elution: from 0 to 5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10%:90%; 5. From 01min to 30min, the volume ratio of acetonitrile increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 70%; From 30.01min to 35min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%; From 35.01min to 45min, the volume ratio of acetonitrile increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 60%; From 45.01min to 48min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 40%:60%; From 48.01min to 52min, the volume ratio of acetonitrile increased to 70%, and the volume ratio of potassium dihydrogen phosphate solution decreased to 30%; At 52.01min-55min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution remained unchanged at 30%:70%; At 55.01min-56min, the volume ratio of acetonitrile decreased to 10%, and the volume ratio of potassium dihydrogen phosphate solution increased to 90%; At 56.01min~65min, the volume ratio of acetonitrile and potassium dihydrogen phosphate solution is 10%:90% and remains unchanged; Solution preparation: Diluent: acetonitrile and water; Mixed impurity stock solution: accurately weigh impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G respectively, dissolve them with appropriate amount of acetonitrile, dilute to scale with diluent, mix well, accurately measure 1.0 ml of the obtained mixed solution, dilute to scale with diluent, and mix well; The impurity B is 7-[(3R)-N-tert-butyloxycarbonyl-3-amino-1-oxo-4-(2,4,5-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine; The structural formula is: The impurity C is (R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid hydrochloride; The structural formula is: The impurity D is (E)-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl)-4-(2,4,5-trifluorophenyl)but-3-en-1-one; The structural formula is: The impurity E is (E)-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl)-4-(2,4,5-trifluorophenyl)but-2-en-1-one; The structural formula is: The impurity F is 7-[(3R)-3-amino-1-oxo-4-(2,5-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine hydrochloride; The structural formula is: The impurity G is 7-[(3R)-3-amino-1-oxo-4-(2,4-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-α]pyrazine hydrochloride; The structural formula is: System suitability solution: Take sitagliptin phosphate and accurately pipette 1.0 ml of mixed impurity stock solution, mix, dissolve and dilute to scale with diluent, and mix well; Sample solution: Take sitagliptin phosphate, dissolve it in diluent and dilute to scale, mix well; Separation analysis: The sample solution and system suitability solution were injected into the high performance liquid chromatograph respectively, and the contents of related substances in sitagliptin phosphate were calculated by the main component self-reference method.

2. A method for detecting related substances of sitagliptin phosphate according to claim 1, characterized in that: The solution preparation is as follows: Diluent: acetonitrile and water in a volume ratio of 5:95; Mixed impurity stock solution: accurately weigh 15 mg of each of impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, place them in a 50 ml volumetric flask, dissolve them with an appropriate amount of acetonitrile, dilute to the mark with diluent, and mix well; accurately measure 1.0 ml of the resulting mixed solution and place it in a 20 ml volumetric flask, dilute to the mark with diluent, and mix well; System suitability solution: Take 13 mg of sitagliptin phosphate and place it in a 10 ml volumetric flask. Accurately pipette 1.0 ml of the mixed impurity stock solution into the volumetric flask, dissolve it with diluent and dilute to the mark, and mix well. Sample solution: Take 13 mg of sitagliptin phosphate sample, place it in a 10 ml volumetric flask, dissolve it with diluent and dilute to the scale, and mix well.

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