CN115754078A - Method for detecting related substances of sitagliptin phosphate - Google Patents
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Abstract
The invention relates to a method for detecting related substances of sitagliptin phosphate, wherein the column temperature is 30 ℃, the flow rate of a mobile phase is 1.0ml/min, the detection wavelength is 205nm, a stationary phase is cyano silane bonded silica gel, and the mobile phase is mobile phases A and B; the mobile phase A is phosphate buffer solution, an ion pair reagent is added, and the pH value is adjusted by acid; the mobile phase B is acetonitrile, and gradient elution is carried out; diluting the solution; mixed impurity stock solution: weighing B, C, D, E, F, G as an impurity for dilution, and diluting 1.0ml of the obtained mixed solution; system applicability solution: diluting sitagliptin phosphate and a mixed impurity stock solution; sample solution: diluting a sitagliptin phosphate sample; injecting the sample solution into a high performance liquid chromatograph, and calculating the content of related substances in the sitagliptin phosphate according to a main component self-contrast method. The method can efficiently separate related substances in sitagliptin phosphate under the same chromatographic condition, effectively control the quality of the raw material medicine, and has the advantages of good specificity, strong accuracy, high precision and room-temperature operation.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a method for detecting related substances of sitagliptin phosphate.
Background
Type 2 diabetes is one of the diabetes types, and is the most common type of diabetes. The diabetes mellitus type 2 patients also have a plurality of clinical complications including diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, stroke, myocardial infarction and the like. The treatment of type 2 diabetes mainly adopts the improvement of insulin sensitivity, mainly adopts oral hypoglycemic drugs, and needs to supplement exogenous insulin when the function of the pancreatic islet of a type 2 diabetes patient is seriously damaged.
Sitagliptin is a potent and highly selective sitagliptin dipeptidyl peptidase 4 (DPP-4) inhibitor, can increase insulin release and reduce pancreatic glucagon levels in a glucose-dependent manner, and has significant efficacy and high safety for treating type 2 diabetes.
Sitagliptin is chemically known as (R) -3-amino-1- (3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7 (8H) -yl) -4- (2,4,5-trifluorophenyl) butan-1-one, chemical structural formula:
sitagliptin phosphate is a high selectivity small molecule dipeptidyl peptidase-4 (DPP-4) inhibitor small molecule which is developed by screening more than 80 ten thousand compounds in Merck research laboratory of the city of New Jersey in the Messandon, is synthesized and improved by more than 2000 compounds, is firstly shown to improve blood sugar control and islet function in animal research, and is also rapidly entered into clinical test after toxicological and safety evaluation, and is called sitagliptin.
In the clinical study period, scientists in the visadon only took 2 years and 1 quarter to confirm that sitagliptin, either orally administered alone or in combination with metformin and other hypoglycemic agents, significantly improved glycemic control. The approved approval of sitagliptin (trade name, jiranavin) by FDA in 2006, 10, 17 days is on the market, and the DPP-4 inhibitor which is approved in the world is the first, and simultaneously, a new age for treating diabetes by applying the sitagliptin (-gliptin) is created.
At present, sitagliptin phosphate bulk drugs are recorded in EP and USP, in the related substance detection method, the mobile phase is acetonitrile-0.01 mol/L potassium dihydrogen phosphate solution (pH is adjusted to 2.0 by phosphoric acid) (volume ratio is 15: 85), and the mobile phase elution is isocratic elution, which has the defects that:
(1) the retention time of each impurity is unstable;
(2) the separation effect of the impurity D, E and the impurity F, G is poor;
(3) the detection time is too long.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting related substances of sitagliptin phosphate, so as to overcome the defects in the prior art.
The technical scheme for solving the technical problems is as follows: a method for detecting related substances of sitagliptin phosphate comprises the following steps:
chromatographic conditions are as follows:
the temperature of a chromatographic column is 30 ℃, the flow rate of a mobile phase is 1.0ml/min, the detection wavelength is 205nm, the stationary phase is cyano silane bonded silica gel, and the mobile phase is a mobile phase A and a mobile phase B; the mobile phase A is phosphate buffer solution, an ion pair reagent is added, and the pH value is adjusted to 2.0 by acid; the mobile phase B is acetonitrile, and gradient elution is carried out;
solution preparation:
diluting liquid: acetonitrile and water;
mixed impurity stock solution: accurately weighing impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, dissolving with appropriate amount of acetonitrile, adding diluent to dilute to scale, mixing, accurately weighing 1.0ml of the obtained mixed solution, adding diluent to dilute to scale, and mixing;
system applicability solution: mixing sitagliptin phosphate and 1.0ml of mixed impurity stock solution obtained by precision transfer, dissolving and diluting the mixture to a scale by using a diluent, and uniformly mixing;
sample solution: taking a sitagliptin phosphate sample, dissolving the sitagliptin phosphate sample by using a diluent, diluting the sitagliptin phosphate sample to a scale, and uniformly mixing;
separation and analysis:
respectively injecting the sample solution and the system applicability solution (standard solution) into a high performance liquid chromatograph, and calculating the content of related substances in the sitagliptin phosphate according to a main component self-contrast method.
On the basis of the technical scheme, the invention can be further improved as follows.
Further: the phosphate buffer solution is potassium dihydrogen phosphate solution.
Further: the concentration of the potassium dihydrogen phosphate solution was 0.01mol/L.
Further: the ion pairing agent is sodium octane sulfonate.
Further: the concentration of the octane sodium sulfonate is 0.005 mol/L-0.01 mol/L.
Further: the concentration of sodium octane sulfonate is 0.007mol/L.
Further: and the pH value of the mobile phase A is adjusted by adopting phosphoric acid.
Further: the volume ratio of the diluent to the diluent is acetonitrile to water = 5; or, the diluent 2 is acetonitrile to water = 30.
Further: the gradient elution is specifically as follows:
when the elution gradient is 0-5 min, the volume ratio of acetonitrile to phosphate buffer solution is 10 percent to 90 percent;
when the reaction time is 5.01-30 min, the volume ratio of acetonitrile is increased to 30%, and the volume ratio of phosphate buffer solution is reduced to 70%;
when the time is 30.01 min-35 min, the volume ratio of the acetonitrile to the phosphate buffer solution is unchanged, wherein the volume ratio of the acetonitrile to the phosphate buffer solution is 30 percent to 70 percent;
when 35.01 min-45 min, the volume ratio of acetonitrile is increased to 40%, and the volume ratio of phosphate buffer solution is reduced to 60%;
45.01 min-48 min, the volume ratio of acetonitrile and phosphate buffer solution is 40% to 60% unchanged;
48.01 min-52 min, the acetonitrile volume ratio is increased to 70%, and the phosphate buffer solution volume ratio is reduced to 30%;
52.01 min-55 min, the volume ratio of the acetonitrile to the phosphate buffer solution is 30 percent to 70 percent;
55.01 min-56 min, the acetonitrile volume ratio is reduced to 10%, and the phosphate buffer solution volume ratio is increased to 90%;
5363 and 56.01 min-65 min, the volume ratio of acetonitrile to phosphate buffer solution is 10% to 90%.
Further: impurity B is 7- [ (3R) -N-tert-butoxycarbonyl-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 as follows:
impurity C is (R) -3-amino-4- (2,4,5-trifluorophenyl) butanoic acid hydrochloride;
the structural formula is as follows:
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;
the structural formula is as follows:
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;
the structural formula is as follows:
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 as follows:
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 as follows:
further:
the solution preparation specifically comprises the following steps:
diluting liquid: acetonitrile and water in a volume ratio of 5;
mixed impurity stock solution: accurately weighing 15mg of each of the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G, placing the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G in a 50ml measuring flask, dissolving the impurity D, the impurity E, the impurity F and the impurity G in proper amount of acetonitrile, adding diluent to dilute the mixture to a scale, and uniformly mixing the mixture; precisely measuring 1.0ml of the obtained mixed solution, placing the mixed solution into a 20ml volumetric flask, adding a diluent to dilute the mixed solution to a scale, and uniformly mixing the diluted solution and the volumetric flask;
system applicability solution: taking 13mg of sitagliptin phosphate containing about 10mg of sitagliptin, placing the sitagliptin phosphate into a 10ml volumetric flask, precisely transferring 1.0ml of mixed impurity stock solution into the volumetric flask, dissolving the mixed impurity stock solution by using a diluent, diluting the mixed impurity stock solution to a scale, and uniformly mixing the mixed impurity stock solution and the scale;
sample solution: taking a sitagliptin phosphate sample of 13mg, placing the sample in a 10ml volumetric flask, dissolving the sample with a diluent, diluting the sample to a scale mark, and mixing the sample and the diluent.
The invention has the beneficial effects that:
the invention provides a high performance liquid chromatography for rapidly and efficiently analyzing sitagliptin and related substances thereof (degradation products, intermediates and starting materials in sitagliptin phosphate), thereby realizing the separation and determination of the related substances in the sitagliptin phosphate under the same chromatographic condition.
Drawings
FIG. 1: HPLC profile of the blank solution prepared according to example 1;
FIG. 2: HPLC profile of the system suitability solution prepared according to example 1;
FIG. 3: linear solution prepared according to example 3, linear profile of impurity C;
FIG. 4: linear solution prepared according to example 3, linear profile of impurity F;
FIG. 5: linear solution prepared according to example 3, linear profile of impurity G;
FIG. 6: a linear solution prepared according to example 3, a sitagliptin phosphate linear profile;
FIG. 7: linear solution prepared according to example 3, linear profile of impurity D;
FIG. 8: linear solution prepared according to example 3, linear profile for impurity E;
FIG. 9: linear solution prepared according to example 3, linear profile of impurity B.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1
Chromatographic conditions
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, G7115A ultraviolet detector;
a chromatographic column: chromatography column with cyano silane bonded silica gel as stationary phase (250 x 4.6mm,5 μm);
mobile phase A: adding 0.007mol/L sodium octane sulfonate into 0.01mol/L potassium dihydrogen phosphate solution, and adjusting the pH value to 2.0 by using phosphoric acid;
mobile phase B: acetonitrile;
gradient elution: when the time is 0-5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10 percent to 90 percent;
when the reaction time is 5.01-30 min, the volume ratio of acetonitrile is increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 70%;
when the time is 30.01 min-35 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 30 percent to 70 percent;
when 35.01 min-45 min, the volume ratio of acetonitrile is increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 60%;
45.01 min-48 min, the volume ratio of acetonitrile and potassium dihydrogen phosphate solution is 40: 60%;
48.01 min-52 min, the acetonitrile volume ratio is increased to 70%, and the potassium dihydrogen phosphate solution volume ratio is reduced to 30%;
52.01 min-55 min, the volume ratio of the acetonitrile to the potassium dihydrogen phosphate solution is 30 percent to 70 percent;
55.01 min-56 min, the acetonitrile volume ratio is reduced to 10%, and the potassium dihydrogen phosphate solution volume ratio is increased to 90%;
5363 and 56.01-65 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10% to 90%
Flow rate: 1.0ml/min;
detection wavelength: 205nm;
column temperature: 30 ℃;
sample introduction volume: 20 mu l of the solution;
the diluent was acetonitrile: water =5 (V: V);
preparing a solution:
blank solution: diluting the solution;
mixed impurity stock solution: accurately weighing about 15mg of each of the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G, placing the weighed materials into a 50ml measuring flask, dissolving the materials with a proper amount of acetonitrile, adding a diluent to dilute the materials to a scale, and uniformly mixing the materials; precisely measuring 1.0ml of the mixed solution, placing the mixed solution into a 20ml volumetric flask, adding a diluent to dilute the mixed solution to a scale, and uniformly mixing the diluted solution and the scale;
system applicability solution: taking about 13mg of sitagliptin phosphate (containing about 10mg of sitagliptin), placing the sitagliptin phosphate into a 10ml volumetric flask, precisely transferring 1.0ml of the mixed impurity stock solution into the volumetric flask, dissolving and diluting the mixed impurity stock solution to a scale by using a diluent, and uniformly mixing;
a detection step: respectively taking a blank solution and a system applicability solution, carrying out high performance liquid chromatography analysis under the set conditions, and recording a chromatogram;
FIG. 1 is an HPLC profile of a blank solution prepared in example 1; FIG. 2 is an HPLC profile of a system suitability solution formulated in example 1;
table 1-results of liquid chromatography analysis of the system suitability solution of example 1;
example 2
Stability testing of System suitability solutions
Chromatographic conditions
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, G7115A ultraviolet detector;
a chromatographic column: a column (250 × 4.6mm,5 μm) using a cyano silane-bonded silica gel as a stationary phase;
mobile phase A: adding 0.007mol/L sodium octane sulfonate into 0.01mol/L potassium dihydrogen phosphate solution, and adjusting the pH value to 2.0 by using phosphoric acid;
mobile phase B: acetonitrile;
gradient elution: when the time is 0-5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10 percent to 90 percent;
when the reaction time is 5.01-30 min, the volume ratio of acetonitrile is increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 70%;
when the time is 30.01 min-35 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 30 percent to 70 percent;
when 35.01 min-45 min, the volume ratio of acetonitrile is increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 60%;
45.01 min-48 min, the volume ratio of acetonitrile and potassium dihydrogen phosphate solution is 40: 60%;
48.01 min-52 min, the acetonitrile volume ratio is increased to 70%, and the potassium dihydrogen phosphate solution volume ratio is reduced to 30%;
52.01 min-55 min, the volume ratio of the acetonitrile to the potassium dihydrogen phosphate solution is 30 percent to 70 percent;
55.01 min-56 min, the acetonitrile volume ratio is reduced to 10%, and the potassium dihydrogen phosphate solution volume ratio is increased to 90%;
5363 and 56.01-65 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10% to 90%
Flow rate: 1.0ml/min;
detection wavelength: 205nm;
column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
the dilution was acetonitrile: water =5 (V: V);
preparing a solution:
blank solution: diluting the solution;
mixed impurity stock solution: accurately weighing about 15mg of each of the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G, placing the impurities into a 50ml measuring flask, dissolving the impurities with a proper amount of acetonitrile, adding a diluent to dilute the impurities to a scale, and uniformly mixing the impurities; precisely measuring 1.0ml of the mixed solution, placing the mixed solution into a 20ml volumetric flask, adding a diluent to dilute the mixed solution to a scale, and uniformly mixing the diluted solution and the scale;
system applicability solution: taking about 13mg of sitagliptin phosphate (containing about 10mg of sitagliptin), placing the sitagliptin phosphate into a 10ml volumetric flask, precisely transferring 1.0ml of the mixed impurity stock solution into the volumetric flask, dissolving and diluting the mixed impurity stock solution to a scale by using a diluent, and uniformly mixing;
the test method comprises the following steps: the system applicability solution is placed for 24 hours and is sampled and injected at 0h,2h,4h,6h,8h,12h and 24h respectively;
under the set chromatographic conditions, respectively analyzing, and calculating the relative standard deviation of the areas of sitagliptin phosphate and each impurity in each needle map in the system applicability solution within 24h, wherein the Relative Standard Deviation (RSD) of the peak areas of all substances is not more than 2% as shown in Table 2;
TABLE 2 analysis results of stability test of each substance in liquid chromatography of system applicability solution of example 2
Example 3
A method for detecting related substances of sitagliptin phosphate comprises the following steps:
chromatographic conditions
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, G7115A ultraviolet detector;
a chromatographic column: a column (250 × 4.6mm,5 μm) using a cyano silane-bonded silica gel as a stationary phase;
mobile phase A: adding 0.007mol/L sodium octane sulfonate into 0.01mol/L potassium dihydrogen phosphate solution, and adjusting the pH value to 2.0 by using phosphoric acid;
and (3) mobile phase B: acetonitrile;
gradient elution: when the time is 0-5 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10 percent to 90 percent;
when the reaction time is 5.01-30 min, the volume ratio of acetonitrile is increased to 30%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 70%;
when the time is 30.01 min-35 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 30 percent to 70 percent;
when 35.01 min-45 min, the volume ratio of acetonitrile is increased to 40%, and the volume ratio of potassium dihydrogen phosphate solution is decreased to 60%;
45.01 min-48 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 40: 60%;
48.01 min-52 min, the acetonitrile volume ratio is increased to 70%, and the potassium dihydrogen phosphate solution volume ratio is reduced to 30%;
52.01 min-55 min, the volume ratio of the acetonitrile to the potassium dihydrogen phosphate solution is 30 percent to 70 percent;
55.01 min-56 min, the acetonitrile volume ratio is reduced to 10%, and the potassium dihydrogen phosphate solution volume ratio is increased to 90%;
5363 and 56.01-65 min, the volume ratio of acetonitrile to potassium dihydrogen phosphate solution is 10% to 90%
Flow rate: 1.0ml/min;
detection wavelength: 205nm;
column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
diluent 1 is acetonitrile: water =5 (V: V);
diluent 2 is acetonitrile: water =30 (V: V);
preparing a solution:
stock solution: respectively and precisely weighing 19mg of sitagliptin phosphate, 15mg of impurity B, 15mg of impurity C, 15mg of impurity D, 15mg of impurity E, 15mg of impurity F and 15mg of impurity G, respectively placing the weighed materials into different 100ml measuring bottles, dissolving and diluting the weighed materials to a scale by using a diluent 2, and uniformly mixing the weighed materials to obtain stock solutions of the materials;
linear mother liquor a: precisely measuring 5.0ml of each impurity stock solution and sitagliptin phosphate stock solution, placing the impurity stock solutions and the sitagliptin phosphate stock solutions into the same 50ml volumetric flask, adding the diluent 1 to dilute to a scale, and uniformly mixing;
linear mother liquor b: accurately measuring 2.0ml of impurity stock solutions of impurities B and C, 1.0ml of impurity stock solutions of sitagliptin phosphate, D, E, F and G respectively, placing the measured solutions into a same 100ml volumetric flask, adding the diluent 1 to dilute the solution to a scale, and mixing the diluted solution and the volumetric flask uniformly;
linear solution 1: precisely measuring 1.0ml of linear mother liquor b, placing the linear mother liquor b in a 20ml volumetric flask, adding the diluent 1 to dilute the linear mother liquor b to a scale, and uniformly mixing;
linear solution 2: precisely measuring 1.0ml of linear mother liquor a, placing the linear mother liquor a in a 20ml volumetric flask, adding the diluent 1 to dilute the mother liquor to a scale, and uniformly mixing;
linear solution 3: precisely measuring 1.0ml of linear mother liquor a, placing the linear mother liquor a in a 10ml volumetric flask, adding the diluent 1 to dilute the linear mother liquor a to a scale, and uniformly mixing;
linear solution 4: precisely measuring 3.0ml of linear mother liquor a, placing the linear mother liquor a in a 20ml volumetric flask, adding the diluent 1 to dilute the linear mother liquor a to a scale, and uniformly mixing;
linear solution 5: precisely measuring 2.0ml of linear mother liquor a, placing the linear mother liquor a in a 10ml volumetric flask, adding diluent 1 to dilute the linear mother liquor a to a scale, and uniformly mixing;
a detection step: under the set conditions, sequentially carrying out high performance liquid chromatography analysis on the blank sample and the linear solutions 1-5, and recording a chromatogram;
drawing a standard curve:
and (5) drawing a calibration curve according to the mass concentration and the peak area of the standard series. Performing linear regression by taking the proportion of the linear solution as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a regression equation and a correlation coefficient;
in the measuring range, each impurity and sitagliptin phosphate have good linear relation, the linear correlation coefficient R2 is not less than 0.999, and the data are shown in tables 3-9.
FIG. 3 is a linear profile of impurity C in the linear solution prepared in example 3;
FIG. 4 is a linear spectrum of impurity F in the linear solution prepared in example 3;
FIG. 5 is a linear profile of impurity G in the linear solution prepared in example 3;
FIG. 6 is a linear profile of impurity D in the linear solution prepared in example 3;
FIG. 7 is a linear profile of impurity E in the linear solution prepared in example 3;
FIG. 8 is a linear profile of impurity B in the linear solution prepared in example 3;
TABLE 3 liquid chromatography impurity C chromatographic peak analysis results of example 3 linear solution
TABLE 4 liquid chromatography impurity F chromatographic peak analysis results of the linear solution of example 3
TABLE 5 chromatographic impurity G chromatographic peak analysis results of the linear solution of example 3
TABLE 6 liquid chromatography sitagliptin phosphate chromatographic peak analysis results of example 3 linear solution
TABLE 7 liquid chromatography impurity D chromatographic peak analysis results of the linear solution of example 3
TABLE 8 liquid chromatography impurity E chromatographic peak analysis results of the linear solution of example 3
TABLE 9 chromatographic impurity B chromatographic peak analysis results of the linear solution of example 3
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A method for detecting related substances of sitagliptin phosphate is characterized by comprising the following steps:
chromatographic conditions are as follows:
the temperature of a chromatographic column is 30 ℃, the flow rate of a mobile phase is 1.0ml/min, the detection wavelength is 205nm, a stationary phase is cyano silane bonded silica gel, and the mobile phase is a mobile phase A and a mobile phase B; the mobile phase A is phosphate buffer solution, an ion pair reagent is added, and the pH value is adjusted to 2.0 by acid; the mobile phase B is acetonitrile, and gradient elution is carried out;
solution preparation:
diluting liquid: acetonitrile and water;
mixed impurity stock solution: accurately weighing impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, dissolving with appropriate amount of acetonitrile, adding diluent to dilute to scale, mixing, accurately weighing 1.0ml of the obtained mixed solution, adding diluent to dilute to scale, and mixing;
system applicability solution: mixing sitagliptin phosphate and 1.0ml of mixed impurity stock solution obtained by precision transfer, dissolving and diluting the mixture to a scale by using a diluent, and uniformly mixing;
sample solution: taking sitagliptin phosphate, dissolving the sitagliptin phosphate by using a diluent, diluting the sitagliptin phosphate to a scale, and uniformly mixing;
separation and analysis:
and respectively injecting the sample solution and the system applicability solution into a high performance liquid chromatograph, and calculating the content of related substances in the sitagliptin phosphate according to a main component self-contrast method.
2. The method for detecting sitagliptin phosphate related substances according to claim 1, which is characterized in that: the phosphate buffer solution is potassium dihydrogen phosphate solution.
3. The method for detecting sitagliptin phosphate related substances according to claim 2, characterized in that: the concentration of the potassium dihydrogen phosphate solution is 0.01mol/L.
4. The method for detecting sitagliptin phosphate related substances according to claim 1, which is characterized in that: the ion pair reagent is sodium octane sulfonate.
5. The method for detecting sitagliptin phosphate related substances according to claim 4, wherein the method comprises the following steps: the concentration of the sodium octane sulfonate is 0.005 mol/L-0.01 mol/L.
6. The method for detecting sitagliptin phosphate related substances according to claim 5, wherein the method comprises the following steps: the concentration of the sodium octane sulfonate is 0.007mol/L.
7. The method for detecting sitagliptin phosphate related substances according to claim 5, wherein the method comprises the following steps: and the pH value of the mobile phase A is adjusted by adopting phosphoric acid.
8. The method for detecting sitagliptin phosphate related substances according to claim 1, which is characterized in that: the gradient elution is specifically as follows:
when the elution gradient is 0-5 min, the volume ratio of acetonitrile to phosphate buffer solution is 10 percent to 90 percent;
when the time is 5.01 min-30 min, the volume ratio of the acetonitrile is increased to 30 percent, and the volume ratio of the phosphate buffer solution is reduced to 70 percent;
when the concentration is 30.01 min-35 min, the volume ratio of the acetonitrile to the phosphate buffer solution is unchanged from 30 percent to 70 percent;
when 35.01 min-45 min, the volume ratio of acetonitrile is increased to 40%, and the volume ratio of phosphate buffer solution is reduced to 60%;
45.01 min-48 min, the volume ratio of acetonitrile to phosphate buffer solution is 40% to 60% unchanged;
48.01 min-52 min, the acetonitrile volume ratio is increased to 70%, and the phosphate buffer solution volume ratio is reduced to 30%;
52.01 min-55 min, the volume ratio of the acetonitrile to the phosphate buffer solution is 30 percent to 70 percent;
55.01 min-56 min, the acetonitrile volume ratio is reduced to 10%, and the phosphate buffer solution volume ratio is increased to 90%;
5363 and 56.01 min-65 min, the volume ratio of acetonitrile to phosphate buffer solution is 10% to 90%.
9. The method for detecting sitagliptin phosphate related substances according to claim 1, which is characterized in that: the impurity B is 7- [ (3R) -N-tert-butoxycarbonyl-3-amino-1-oxo-4- (2,4,5-difluorophenyl) butyl ] -5,6,7,8-tetrahydro-3- (trifluoromethyl) -1,2,4-triazolo [4,3-alpha ] pyrazine;
the impurity C is (R) -3-amino-4- (2,4,5-trifluorophenyl) butyric acid hydrochloride;
the 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;
the 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;
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 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-alpha ] pyrazine hydrochloride.
10. The method for detecting sitagliptin phosphate related substances according to claim 1, which is characterized in that:
the solution preparation specifically comprises the following steps:
diluting liquid: acetonitrile and water in a volume ratio of 5;
mixed impurity stock solution: accurately weighing 15mg of each of the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G, placing the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G in a 50ml measuring flask, dissolving the impurity D, the impurity E, the impurity F and the impurity G in proper amount of acetonitrile, adding diluent to dilute the mixture to a scale, and uniformly mixing the mixture; precisely measuring 1.0ml of the obtained mixed solution, placing the mixed solution into a 20ml volumetric flask, adding a diluent to dilute the mixed solution to a scale, and uniformly mixing the diluted solution and the volumetric flask;
system applicability solution: taking 13mg of sitagliptin phosphate, placing the sitagliptin phosphate in a 10ml volumetric flask, precisely transferring 1.0ml of mixed impurity stock solution into the volumetric flask, dissolving and diluting the mixed impurity stock solution to a scale by using a diluent, and uniformly mixing;
sample solution: taking a sitagliptin phosphate sample 13mg, placing the sample in a 10ml volumetric flask, dissolving the sample with a diluent, diluting the sample to a scale mark, and mixing the sample and the diluent uniformly.
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