CN112394109A - Method for detecting impurities in ticagrelor - Google Patents
Method for detecting impurities in ticagrelor Download PDFInfo
- Publication number
- CN112394109A CN112394109A CN201910740912.5A CN201910740912A CN112394109A CN 112394109 A CN112394109 A CN 112394109A CN 201910740912 A CN201910740912 A CN 201910740912A CN 112394109 A CN112394109 A CN 112394109A
- Authority
- CN
- China
- Prior art keywords
- solution
- cyclopropylamine
- difluorophenyl
- impurity
- acetonitrile
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for detecting impurities in ticagrelor. Provided is a detection method of a chemical including (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the detection method including: treating (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate with a diluent solvent to obtain a control solution; treating the chemical with a diluent solvent to obtain a chemical solution; performing high performance liquid chromatography detection on the chemical solution, and detecting the content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the chemical by using an external standard method; wherein the diluting solvent is a mixed solution containing acetonitrile and sodium bicarbonate solution. The impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is detected by the method, and the detection limit is low and reaches 0.18 ng. And the detection peak pattern is good.
Description
Technical Field
The invention relates to the field of chemical substance analysis and detection, in particular to a method for detecting impurities in ticagrelor, and particularly relates to a method for detecting impurities (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Background
Ticagrelor (Ticagrelor, trade name: brilnta), a selective Adenosine Diphosphate (ADP) receptor antagonist developed by AstraZeneca pharmaceutical limited (AstraZeneca AB), reversibly blocks ADP-mediated platelet activation and aggregation by activating the P2Y12 receptor.
The FDA approved ticagrelor tablets for sale on day 20/7/2011 for patients with acute coronary syndrome (unstable angina, non-ST elevation myocardial infarction or ST elevation myocardial infarction), including patients receiving drug therapy and Percutaneous Coronary Intervention (PCI) therapy, to reduce the incidence of thrombotic cardiovascular events. The anti-platelet effect of the ticagrelor tablet is reversible, the ticagrelor tablet directly acts on ADP receptors, is not metabolized by the liver, is not influenced by metabolism in the receptors, and has the effect taking time of 30 minutes. The composition has outstanding advantages of rescuing ACS patients, has a non-platelet-mediated effect, can improve the concentration of blood adenosine, improve the blood supply of cardiac muscle, reduce the area of myocardial infarction, has stronger and more durable platelet inhibition effect in the maintenance phase than clopidogrel, and is suitable for long-term use of patients with acute coronary syndrome. Therefore, the ticagrelor and the clopidogrel are both platelet inhibitors, and clinical tests show that the ticagrelor and the clopidogrel are both excellent in safety and effectiveness and have the potential of replacing the clopidogrel, so that the ticagrelor and the clopidogrel have great market potential and wide application prospect.
(1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is the starting material for the synthesis of ticagrelor. Due to limitations in process technology and level of control, unreacted (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is readily present as an impurity in the ticagrelor product produced. (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine has an carcinogenicity warning structure, and the content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor needs to be controlled within 8ppm per day to ensure that the total intake of not more than 1.5 μ g per day, according to ICH Q3A (R2), M7 and reference 1 (reference 1: Romuraldo Benigi, Cecilia Bossa, Olga Tcherenskaia.development of structural impurities for the in vivo microbial assay in rodentts [ J ]. JRC Scientific and Technical Reports,24 ]), reference 2 (reference 2: ticagrelor tablets (JXHL1000319, 320) International Multi-centre clinical trial application pharmaceutical review report.) to specifically mention that the substance is a genotoxic impurity; therefore, it is necessary to monitor the content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Chinese patent application No. 201510718834.0 discloses a method for detecting (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor, but it requires the use of a special C18 column, which is weak in durability. The chinese patent application No. 201710681392.6 also discloses a method for detecting (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor, but it uses expensive liquid, and the detection cost is high.
Therefore, it is necessary to develop a method for detecting (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor, which has high sensitivity, low detection limit, good durability and low cost.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a method for detecting impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor with high sensitivity and low detection limit.
The inventor finds out in the research process that:
since (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is poor in stability, when the content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is measured by high performance liquid chromatography, (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate was used as a control, and then analyzing the high performance liquid chromatography detection result of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, comparing the high performance liquid chromatography detection result of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample to be detected, and determining the content of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample to be detected. The inventor finds that the treatment of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate with a mixture containing acetonitrile and a weak base solution, such as a weak base solution of sodium bicarbonate, a weak base solution of sodium carbonate, a weak base solution of sodium acetate and a weak base solution of disodium hydrogen phosphate, particularly with a mixture containing acetonitrile and a solution of sodium bicarbonate, can fully ionize (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, has a good peak shape in a liquid chromatography detection process, does not generate a forward lag or a tailing in a liquid chromatography detection pattern, and forms a good distinction with other substances, so that the accurate determination and detection of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine can be realized, therefore, the monitoring of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample to be detected can be realized by using a mixed solution containing acetonitrile and a weak base solution and by means of high performance liquid chromatography detection.
Specifically, the invention provides the following technical scheme:
the invention provides a detection method of a chemical, wherein the chemical comprises (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, and the detection method comprises the following steps: (1) treating (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate with a diluting solvent to obtain a control solution; (2) treating the chemical with a diluting solvent so as to obtain a chemical solution; (3) performing high performance liquid chromatography detection on the chemical solution and the reference solution, and detecting (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the chemical based on the high performance liquid chromatography detection result of the reference solution; wherein the diluting solvent is a mixed solution containing acetonitrile and sodium bicarbonate solution.
Wherein, the step (1) and the step (2) are not limited in sequence when preparing the reference substance solution and the chemical solution. A reference solution can be prepared first, and then a chemical solution can be prepared; or preparing the chemical solution first and then preparing the reference solution, or preparing the reference solution and the chemical solution simultaneously. The content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the chemicals is detected by using the detection method provided by the invention, and the detection limit is low and reaches 0.18 ng. And the detection peak has good shape, no trailing or forward delay phenomenon and high accuracy. The method provided by the invention has the advantages of low detection cost, strong specificity and good durability, and can be used for detection by common C18 chromatographic columns of various brands.
According to an embodiment of the present invention, the method for detecting a chemical described above may further include the following technical features:
in some embodiments of the present invention, the chemical contains ticagrelor, and the content of ticagrelor in the chemical is more than 99%. By applying the detection method provided by the invention, the ultra-low content (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the ticagrelor can be detected, and the method is used for monitoring the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the ticagrelor product, so that the quality of the medicine is controlled.
In some embodiments of the invention, the concentration of the sodium bicarbonate solution is between 0.01mol/L and 0.02 mol/L. The mixed solution of the sodium bicarbonate solution with the concentration and the acetonitrile is used as a diluting solvent, the prepared solution is very stable, and a stable and accurate detection map can be formed when high performance liquid chromatography detection is carried out, so that the method is used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine.
In some embodiments of the invention, the volume ratio of the acetonitrile to the sodium bicarbonate solution in the dilution solvent is 40:60 to 60: 40. By means of the detection of the dilution solvent, the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine has good peak pattern and high response value, and can realize accurate detection of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine.
In some embodiments of the invention, the volume ratio of the acetonitrile to the sodium bicarbonate solution in the dilution solvent is 45: 55. The peak pattern of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is good by means of the diluted solvent, and the impurity peak is not interfered by the ticagrelor and other impurity peaks of the ticagrelor, so that the method is used for accurately detecting the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine.
In some embodiments of the invention, the high performance liquid chromatography detection conditions are: the detection wavelength is 205-215nm, and the column temperature is 20-40 ℃. Therefore, the method can be used for accurately detecting the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine.
In some embodiments of the invention, step (3) further comprises: and carrying out gradient elution on the chemical solution and the impurity reference solution by using a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution containing acetonitrile and phosphate buffer solution, the pH value of the mobile phase A is 2.8-3.2, and the mobile phase B is acetonitrile.
In some embodiments of the invention, the concentration of the phosphate buffer is 0.01mol/L to 0.03 mol/L.
In some embodiments of the invention, the volume ratio of the acetonitrile to the phosphate buffer in the mobile phase a is 20:80 to 40: 60. In some embodiments of the invention, the gradient elution conditions are:
| time (min) | Flow rate (ml/min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 0.9～1.1 | 100 | 0 |
| 10 | 0.9～1.1 | 100 | 0 |
| 12 | 0.9～1.1 | 15 | 85 |
| 20 | 0.9～1.1 | 15 | 85 |
| 21 | 0.9～1.1 | 100 | 0 |
| 30 | 0.9～1.1 | 100 | 0 |
。
The detection method provided by the invention can realize accurate detection of the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity, has high repeatability and accuracy and low detection limit which reaches 0.18 ng. And the obtained peak has good shape and does not have trailing or forward delay phenomenon through high performance liquid chromatography detection. Meanwhile, the detection method provided by the invention depends on a mixed solution containing acetonitrile and sodium bicarbonate solution as a diluting solvent, so that the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine can be fully ionized, a good peak pattern can be obtained in the high performance liquid chromatography detection process, the dependence of the detection method on a chromatographic column is reduced, the application range of the method is wide, and the method can be suitable for various C18 columns.
Drawings
FIG. 1 is a high performance liquid chromatogram of an air-white solution in experimental group 1 of example 1 with an absorption value ranging from-50 mAU to 300 mAU;
FIG. 2 is a high performance liquid chromatogram of an empty solution in experimental group 1 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 3 is a high performance liquid chromatogram of mandelic acid control of experimental group 1 of example 1 with an absorption value ranging from-2 to 20 mAU;
FIG. 4 is a high performance liquid chromatogram of the impurity control of Experimental group 1 of example 1 at an absorbance range of-50 to 250 mAU;
FIG. 5 is a high performance liquid chromatogram of the impurity control in experimental group 1 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 6 is a high performance liquid chromatogram of the test sample in the experimental group 1 of example 1 when the absorption value range is-1000-6000 mAU;
FIG. 7 is a high performance liquid chromatogram of the test sample of experimental group 1 of example 1 with an absorption value ranging from-2 to 20 mAU;
FIG. 8 is a high performance liquid chromatogram of the sample spiked solution of experimental group 1 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 9 is a high performance liquid chromatogram of the impurity control solution of experimental group 2 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 10 is a high performance liquid chromatogram of the sample-spiked solution of experimental group 2 of example 1 at an absorption value ranging from-2 to 20 mAU.
FIG. 11 is a high performance liquid chromatogram of the impurity control solution of experimental group 3 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 12 is a high performance liquid chromatogram of the sample spiked solution of experimental group 3 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 13 is a high performance liquid chromatogram of the test solutions of test sample addition in test group 4 of example 1 with an absorbance range of-2 to 20 mAU;
FIG. 14 is a high performance liquid chromatogram of the sample spiked solution of experimental group 5 of example 1 at an absorption value ranging from-2 to 20 mAU;
FIG. 15 is a high performance liquid chromatogram of the impurity reference solution of control group 1 when the absorption value range is-2-20 mAU;
FIG. 16 is a high performance liquid chromatogram of the impurity reference stock solution of the reference group 1 when the absorption value range is-2-20 mAU;
in fig. 1 to 16, the abscissa represents Time (Time) and the ordinate represents Absorbance (Absorbance).
Fig. 17 is a linear regression plot of the concentration of the impurity control solution versus the peak area, wherein the abscissa represents the concentration of the impurity control solution and the ordinate represents the peak area of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the impurity control solution, in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Example 1 provides a method for accurately determining the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor by performing high performance liquid chromatography detection by using different dilution solvents for treatment.
Experimental group 1
Chromatographic conditions are as follows:
a chromatographic column: kromasil C18 (250X 4.6mm 5 μm)
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of sodium dihydrogen phosphate dihydrate, adding water to dissolve 1000ml, and adjusting pH to 3.0 +/-0.1 by using phosphoric acid) (wherein the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed as in table 1 below:
TABLE 1 gradient elution conditions
| Time (min) | Flow rate (ml/min) | A | B％ | |
| 0 | 1.0 | 100 | 0 | |
| 10 | 1.0 | 100 | 0 | |
| 12 | 1.0 | 15 | 85 | |
| 20 | 1.0 | 15 | 85 | |
| 21 | 1.0 | 100 | 0 | |
| 30 | 1.0 | 100 | 0 |
Column temperature: 35 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: acetonitrile-0.01 mol/L aqueous sodium bicarbonate solution (wherein the volume ratio of acetonitrile to 0.01mol/L aqueous sodium bicarbonate solution is 45:55)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponded to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the prepared impurity control solution was 0.212. mu.g/ml in terms of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: weighing Ticagrelor 0.25g precisely, placing into a 10ml measuring flask, adding a diluting solvent to dissolve, quantitatively diluting to scale, and shaking up to obtain the Ticagrelor (wherein the concentration is 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and sample addition solution 20 μ l each, injecting into liquid chromatograph, and recording chromatogram.
Wherein, FIG. 1 is a high performance liquid chromatogram of a space-time white solution with an absorption value range of-50-300 mAU; FIG. 2 is a high performance liquid chromatogram of a space-time white solution with an absorption value range of-2-20 mAU.
FIG. 3 is a high performance liquid chromatogram of mandelic acid control with absorption value ranging from-2 mAU to 20 mAU.
FIG. 4 is a high performance liquid chromatogram of an impurity reference substance with an absorption value range of-50 mAU to 250 mAU; FIG. 5 is a high performance liquid chromatogram of the impurity reference substance in the absorption value range of-2-20 mAU.
FIG. 6 is a high performance liquid chromatogram of the test sample with absorption value range of-1000 and 6000 mAU; FIG. 7 is a high performance liquid chromatogram of the sample with an absorption value ranging from-2 mAU to 20 mAU.
FIG. 8 is a high performance liquid chromatogram of the sample solution when the absorption value range is-2-20 mAU.
As can be seen from FIG. 3, the peak time of mandelic acid was about 5.126 minutes, and from FIGS. 7 and 8, the peak time of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine was about 7.300 minutes. Further, as is clear from FIGS. 7 and 8, mandelic acid was liberated by the treatment with the diluting solvent, and peaks of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine and mandelic acid were simultaneously shown in the high performance liquid chromatogram. Fig. 8 shows hplc analysis results of the test sample spiking solution, in which ticagrelor had a peak time of about 15.698 minutes, as can be seen from fig. 3 and 8, wherein the test sample spiking solution contained mandelic acid (peak time of 5.126 minutes) and (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (peak time of 7.326 minutes) due to the treatment with the diluent solvent.
Referring to fig. 6 and 7, mandelic acid and (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine were not detected in the sample solution, indicating that the sample solution did not contain (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine as an impurity.
Wherein the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample-to-be-tested solution can be calculated by referring to the following formula:
the content (%) of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is (a-like × C-pair)/(a-like × C-like) × 100%
Impurity limit: 0.0008 percent
In the above formula:
sample A-area of peak of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine when sample A is added to standard solution;
peak area of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in p-impurity control solution;
concentration of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (μ g/ml) in control solution of C p-impurity;
sample C-concentration of test sample plus standard solution (. mu.g/ml).
A sample A: 0.191; a pair: 0.190
C, pair: 0.212. mu.g/ml; and C, sample C: 2.5X 104μg/ml
The content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the formula: 0.000852 percent. The value is consistent with the theoretical value of impurities in the sample solution, which shows that the liquid chromatography condition can be used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Experimental group 2
Chromatographic conditions are as follows:
a chromatographic column: SHISEIDO C18250 x 4.6mm
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of dihydrate and sodium dihydrogen phosphate, adding water to dissolve 1000ml, adjusting pH to 3.0 +/-0.1 with phosphoric acid) (wherein, the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed according to the conditions of gradient elution as set forth in table 1 of experimental group 1;
column temperature: 40 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: acetonitrile-0.01 mol/L aqueous sodium bicarbonate solution (wherein the volume ratio of acetonitrile to 0.01mol/L aqueous sodium bicarbonate solution is 40:60)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponded to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the prepared impurity control solution was 0.212. mu.g/ml in terms of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: weighing Ticagrelor 0.25g precisely, placing into a 10ml measuring flask, adding a diluting solvent to dissolve, quantitatively diluting to scale, and shaking up to obtain the Ticagrelor (wherein the concentration is 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and sample addition solution 20 μ l each, injecting into liquid chromatograph, and recording chromatogram.
Wherein, FIG. 9 is a high performance liquid chromatogram of the impurity reference solution in the experimental group 2 when the absorption value range is-2-20 mAU; FIG. 10 is a high performance liquid chromatogram of the sample-spiked solution in experiment group 2 at an absorption value range of-2 to 20 mAU. The HPLC chromatograms of the other blank solutions, mandelic acid control solution and test solution are similar to those of test group 1 and are not shown.
Wherein the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the following formula:
the content (%) of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is (a-like × C-pair)/(a-like × C-like) × 100%
Impurity limit: 0.0008 percent
In the above formula:
sample A-area of peak of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine when sample A is added to standard solution;
peak area of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in p-impurity control solution;
concentration of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (μ g/ml) in control solution of C p-impurity;
sample C-concentration of test sample plus standard solution (. mu.g/ml).
The content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the formula: 0.000733 percent. The value is consistent with the theoretical value of impurities in the sample solution, which shows that the liquid chromatography condition can be used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Experimental group 3
Chromatographic conditions are as follows:
a chromatographic column: kromasil C18 (250X 4.6mm 5 μm)
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of dihydrate and sodium dihydrogen phosphate, adding water to dissolve 1000ml, adjusting pH to 2.8 +/-0.1 with phosphoric acid) (wherein the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed according to the following gradient elution conditions of table 2:
TABLE 2 gradient elution conditions
| Time (min) | Flow rate (ml/min) | A | B％ | |
| 0 | 1.1 | 100 | 0 | |
| 10 | 1.1 | 100 | 0 | |
| 12 | 1.1 | 15 | 85 | |
| 20 | 1.1 | 15 | 85 | |
| 21 | 1.1 | 100 | 0 | |
| 30 | 1.1 | 100 | 0 |
Column temperature: 30 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: acetonitrile-0.01 mol/L aqueous sodium bicarbonate solution (wherein the volume ratio of acetonitrile to 0.01mol/L aqueous sodium bicarbonate solution is 60:40)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponded to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the prepared impurity control solution was 0.212. mu.g/ml in terms of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: taking 0.25g of ticagrelor, precisely weighing, and placing 10ml of ticagrelorDissolving in a bottle, quantitatively diluting to scale, and shaking to obtain the final product (concentration: 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and sample addition solution 20 μ l each, injecting into liquid chromatograph, and recording chromatogram.
FIG. 11 is a high performance liquid chromatogram of the impurity control solution in the experimental group at an absorption value range of-2-20 mAU; FIG. 12 is a high performance liquid chromatogram of the sample solution in the experimental group when the absorption value range is-2-20 mAU. The HPLC chromatograms of the other blank solutions, mandelic acid control solution and test solution are similar to those of test group 1 and are not shown.
Wherein the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the following formula:
the content (%) of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is (a-like × C-pair)/(a-like × C-like) × 100%
Impurity limit: 0.0008 percent
In the above formula:
sample A-area of peak of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine when sample A is added to standard solution;
peak area of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in p-impurity control solution;
concentration of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (μ g/ml) in the control solution of C p-impurity;
sample C-concentration of test sample plus standard solution (. mu.g/ml).
The content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample standard is calculated according to the formula: 0.000912 percent. The value is consistent with the theoretical value of impurities in the sample solution, which shows that the liquid chromatography condition can be used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Experimental group 4
Chromatographic conditions are as follows:
a chromatographic column: kromasil C18 (250X 4.6mm 5 μm)
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of sodium dihydrogen phosphate dihydrate, adding water to dissolve 1000ml, and adjusting pH to 3.0 +/-0.1 by using phosphoric acid) (wherein the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed according to the gradient elution conditions shown in table 1 in experimental group 1:
column temperature: 35 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: acetonitrile-0.015 mol/L aqueous sodium bicarbonate solution (wherein the volume ratio of the acetonitrile to the 0.01mol/L aqueous sodium bicarbonate solution is 45:55)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponded to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the prepared impurity control solution was 0.212. mu.g/ml in terms of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: weighing Ticagrelor 0.25g precisely, placing into a 10ml measuring flask, adding a diluting solvent to dissolve, quantitatively diluting to scale, and shaking up to obtain the Ticagrelor (wherein the concentration is 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and sample addition solution 20 μ l each, injecting into liquid chromatograph, and recording chromatogram.
FIG. 13 is a high performance liquid chromatogram of the sample solution in the experimental group when the absorption value range is-2-20 mAU. The high performance liquid chromatograms of the other blank solutions, mandelic acid control solution, impurity control solution and test solution are similar to those of experimental group 1 and are not shown.
Wherein the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the following formula:
the content (%) of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is (a-like × C-pair)/(a-like × C-like) × 100%
Impurity limit: 0.0008 percent
In the above formula:
sample A-area of peak of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine when sample A is added to standard solution;
peak area of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in p-impurity control solution;
concentration of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (μ g/ml) in the control solution of C p-impurity;
sample C-concentration of test sample plus standard solution (. mu.g/ml).
The content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample standard is calculated according to the formula: 0.000853 percent. The value is consistent with the theoretical value of impurities in the sample solution, which shows that the liquid chromatography condition can be used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Chromatographic conditions are as follows:
a chromatographic column: kromasil C18 (250X 4.6mm 5 μm)
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of sodium dihydrogen phosphate dihydrate, adding water to dissolve 1000ml, and adjusting pH to 3.0 +/-0.1 by using phosphoric acid) (wherein the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed according to the gradient elution conditions shown in table 1 in experimental group 1:
column temperature: 35 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: acetonitrile-0.02 mol/L aqueous sodium bicarbonate solution (wherein the volume ratio of acetonitrile to 0.01mol/L aqueous sodium bicarbonate solution is 45:55)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponded to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the prepared impurity control solution was 0.212. mu.g/ml in terms of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: weighing Ticagrelor 0.25g precisely, placing into a 10ml measuring flask, adding a diluting solvent to dissolve, quantitatively diluting to scale, and shaking up to obtain the Ticagrelor (wherein the concentration is 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and sample addition solution 20 μ l each, injecting into liquid chromatograph, and recording chromatogram.
FIG. 14 is a high performance liquid chromatogram of the sample solution in the experimental group when the absorption value range is-2-20 mAU. The high performance liquid chromatograms of the other blank solutions, mandelic acid control solution, impurity control solution and test solution are similar to those of experimental group 1 and are not shown.
Wherein the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample solution is calculated according to the following formula:
the content (%) of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is (a-like × C-pair)/(a-like × C-like) × 100%
Impurity limit: 0.0008 percent
In the above formula:
sample A-area of peak of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine when sample A is added to standard solution;
peak area of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in p-impurity control solution;
concentration of impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine (μ g/ml) in the control solution of C p-impurity;
sample C-concentration of test sample plus standard solution (. mu.g/ml).
The content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the sample standard is calculated according to the formula: 0.000850 percent. The value is consistent with the theoretical value of impurities in the sample solution, which shows that the liquid chromatography condition can be used for accurately determining (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in ticagrelor.
Control group 1
Chromatographic conditions are as follows:
a chromatographic column: kromasil C18 (250X 4.6mm 5 μm)
A detector: UV detector
Detection wavelength: 210nm
Mobile phase A: acetonitrile-0.01 mol/L phosphate buffer (taking 1.56g of dihydrate and sodium dihydrogen phosphate, adding water to dissolve 1000ml, adjusting pH to 3.0 +/-0.1 with phosphoric acid) (wherein the volume ratio of acetonitrile to 0.01mol/L phosphate buffer is 20: 80);
mobile phase B: acetonitrile
Gradient elution was performed according to the gradient elution conditions of experimental group 1 table 1:
column temperature: 35 deg.C
Sample introduction amount: 20 μ l
Diluting the solvent: 60% aqueous acetonitrile solution (v/v)
Blank solution: i.e. the diluent solvent.
Mandelic acid control solution: taking 6.97mg of mandelic acid reference substance, placing in a 100ml measuring flask, adding a diluting solvent to dissolve and dilute to scale; precisely measuring 0.1ml of the solution, placing the solution into a 25ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated.
Preparing an impurity reference substance stock solution: taking 10.07mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate, placing the (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate into a 50ml measuring flask, adding 60% acetonitrile water solution (v/v), dissolving and diluting to a scale mark; precisely measuring 2ml of the solution, placing the solution into a 100ml measuring flask, and adding 60% acetonitrile aqueous solution (v/v) to dilute to scale to obtain an impurity reference substance stock solution.
Impurity control solution: and (3) taking 1ml of the impurity reference substance stock solution, putting the impurity reference substance stock solution into a 10ml measuring flask, and adding a diluting solvent to a constant volume to be calibrated. (since 1.9mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate corresponds to 1mg of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, the concentration of the impurity control solution was 0.212. mu.g/ml, as converted to (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine).
Preparing a test solution: weighing Ticagrelor 0.25g precisely, placing into a 10ml measuring flask, adding a diluting solvent to dissolve, quantitatively diluting to scale, and shaking up to obtain the Ticagrelor (wherein the concentration is 2.5 × 10)4μg/ml)。
Preparing a sample and a standard solution: taking 0.25g of ticagrelor, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of impurity reference substance stock solution into the measuring flask (equivalent to 2.12 mu g of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurity), adding a diluting solvent for dissolving, quantitatively diluting to a scale, and shaking up to obtain the ticagrelor-containing compound.
The measurement method is as follows:
precisely measuring blank solution, mandelic acid reference solution, impurity reference solution, sample solution and impurity reference stock solution by 20 μ l, respectively injecting into liquid chromatograph, and recording chromatogram.
Wherein, FIG. 13 is a high performance liquid chromatogram of the impurity reference solution when the absorption value range is-2-20 mAU; FIG. 14 is a high performance liquid chromatogram of the impurity reference stock solution with absorption value ranging from-2 mAU to-20 mAU. The HPLC chromatograms of other test sample solutions, test sample solution, mandelic acid control solution and blank solution are not specifically shown. As can be seen from the results shown in fig. 13, the mandelate salt of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine was not completely ionized, and the response value of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine was low, reaching only the detection limit, and the content thereof could not be accurately detected; as can be seen from the results shown in fig. 14, after increasing the concentration of the impurity control solution, the peak pattern of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine advances, and shoulder seams appear, and the content thereof cannot be effectively detected.
Control group 2
Control 2 differs from experiment 1 in that control 2 used acetonitrile at a volume ratio of 45:55 and 0.01mol/L aqueous sodium hydroxide as a dilution solvent. Then, high performance liquid chromatography was performed by referring to the method of experimental group 1.
The experimental result shows that: the diluted solvent is used for processing, and an unknown impurity peak which interferes with the detection of known impurities appears through high performance liquid chromatography detection, and may be generated by degradation of main components or other impurities of the main components.
The inventor finds out in the research process that: the detection of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine can also be achieved by performing high performance liquid chromatography detection using a diluted solvent comprising a mixture of acetonitrile and other weak base solutions (e.g., sodium acetate solution, disodium hydrogen phosphate solution, etc.), but the detection effect is slightly lower in stability and accuracy than the mixture of acetonitrile and sodium bicarbonate. Moreover, the inventor also finds out in the research process that: when acetonitrile and an aqueous sodium bicarbonate solution are used as the diluting solvent, the volume of the acetonitrile and the aqueous sodium bicarbonate solution is changed, for example, the acetonitrile and the aqueous sodium bicarbonate solution in a volume ratio of 35:65 and 0.01mol/L are used as the diluting solvent; or acetonitrile with the volume ratio of 65:35 and 0.01mol/L sodium bicarbonate water solution are used as a diluting solvent, and the solution treated by the diluting solvent can not be completely dissolved or is unstable when placed; the subsequent high performance liquid chromatography detection can be influenced. Similarly, when the concentration of the aqueous sodium bicarbonate solution is increased to 0.02mol/L or more, the solution treated with the diluting solvent is not completely dissolved or is unstable when left alone; the subsequent high performance liquid chromatography detection can also be influenced.
Example 2
In order to further verify the effects of the detection method provided by the invention in all aspects, the following experiments are carried out:
1. specificity
Mandelic acid control solution: taking 6.97mg of mandelic acid control, using the diluent solvent of the experimental group 1, and fixing the volume to 100ml, taking 0.1ml of the solution, and fixing the volume to 25ml using the diluent solvent of the experimental group 1.
Sampling blank solvent (diluting solvent), sample solution, mandelic acid reference solution, and impurity reference solution 20 μ l each.
The experimental results show that: under the detection method, the blank solvent and mandelic acid have no interference to the detection of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, and the detection method has strong specificity.
2. Precision of the system
20 mu l of the impurity control solution in the experimental group 1 of the example 1 is continuously injected into a sample of 6 needles, a chromatogram is recorded, and the peak area is measured, and the result is shown in Table 3.
TABLE 3 time to Peak and Peak area
| No. | 1 | 2 | 3 | 4 | 5 | 6 | Mean value | RSD％ |
| RTmin | 7.300 | 7.300 | 7.300 | 7.300 | 7.300 | 7.306 | 7.301 | 0.03 |
| Peak area | 0.191 | 0.192 | 0.193 | 0.191 | 0.193 | 0.194 | 0.192 | 0.63 |
The RSD of the retention time of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is calculated to be 0.03%, and the RSD of the peak area is calculated to be 0.63%, which indicates that the detection method of the invention has good system precision.
3. Linear experiment
0.5 ml, 0.8 ml, 1ml, 1.2ml and 1.5ml of impurity reference substance stock solutions in the experimental group 1 of the example 1 are respectively put into 510 ml measuring bottles, and the diluting solvent of the experimental group 1 is added for constant volume and is shaken up. And (5) injecting samples respectively. The peak area results are shown in table 4 below:
TABLE 4 Peak area results
| Concentration (μ g/ml) | Peak area |
| 0.106 | 0.098 |
| 0.170 | 0.155 |
| 0.212 | 0.190 |
| 0.254 | 0.231 |
| 0.318 | 0.286 |
The linear results are shown in fig. 16, in which the abscissa in fig. 16 represents the concentration of the impurity control solution and the ordinate represents the peak area of the impurity control solution. The above results show that the concentration of cyclopropylamine is in a good linear relationship with the peak area when the concentration is in the range of 0.106 to 0.318. mu.g/ml.
4. Repeatability test
Accurately weighing 6 parts of samples to be detected (the samples to be detected are the same as the samples to be detected used in the embodiment 1 and do not contain (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine impurities) by 0.25g respectively, putting the samples to be detected into a 10ml measuring flask, adding the diluting solvent of the experimental group 1 to dissolve and quantitatively dilute to a scale, and shaking up to obtain the sample solution to be detected.
And precisely measuring 20uL of each of the 6 parts of sample solution to be detected, detecting according to the detection method of the invention, and calculating the content of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine by using an external standard method according to the peak area. The results are shown in Table 5:
TABLE 5 test results
As can be seen from the above table, the detection method of the present invention has good reproducibility.
5. Recovery rate
The recovery was calculated using a linear equation.
Recovery of test solution: taking 0.25g of ticagrelor, putting the ticagrelor into a 10ml measuring flask, respectively adding 3 parts of impurity reference substance stock solutions of 0.8 ml, 1ml and 1.2ml of the experimental group 1 in the example 1, respectively, adding the diluting solvent of the experimental group 1 to dilute to the scale, and shaking up to obtain the ticagrelor. Respectively and precisely weighing 20 mu L of the 9 parts of solution and the impurity reference substance solution in the example 1, measuring by using the detection method, recording a chromatogram, and calculating the measurement amount, the addition amount and the recovery rate of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, wherein the results are shown in a table 6:
table 6: results of recovery test
The result shows that the recovery rate of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine measured by the detection method is 97-101%, and the RSD is 1.1%, which indicates that the detection method has good recovery rate.
6. Limit of quantification
An appropriate amount of the impurity control solution of experiment 1 of example 1 was precisely measured, and diluted with the diluent of experiment 1 step by step so that the peak height of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine was 10 times the baseline noise, and samples were continuously fed 6 times. The results are shown in Table 7:
TABLE 7 test results
The result shows that the quantitative limit of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is 0.54ng, and the method provided by the invention is proved to have high detection sensitivity and can fully meet the requirement of impurity content determination.
7. Detection limit
An appropriate amount of the impurity control solution of experiment 1 of example 1 was precisely measured, and diluted with the diluent of experiment 1 step by step so that the peak height of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine was 3 times the baseline noise, and the sample was continuously fed 3 times. The results are shown in Table 8:
TABLE 8 detection Limit results
8. Durability
Durability test article solution: recovery test the solutions used in the 100% recovery test were recovered.
And (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine content changes are examined by changing the detection wavelength, flow rate, column temperature, chromatographic column, pH and other conditions. The results are shown in Table 9:
TABLE 9 test results under different conditions
The result shows that when the column temperature of the detection method is 30-40 ℃, the detection wavelength is 205-215nm, the flow rate is 0.9-1.1ml/min, the pH of the mobile phase water phase is 2.8-3.2, and the content of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine can be accurately detected by using C18 columns of different brands, which indicates that the detection method has wider application range.
9. Stability of solution
The sample added with the standard solution in the experimental group 1 of example 1 is injected with 20 μ l of sample at 0h, 6h, 12h, 18h and 24h after preparation for investigation, and is continuously injected with 6 needles for recording chromatogram, and the stability of the impurity (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine is investigated, and the results are shown in Table 10.
TABLE 10 results of solution stability testing
As can be seen from the above embodiments, the detection method provided by the invention can realize accurate detection of impurities of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, and has a low detection limit. And the detection peak has good shape and no trailing or forward-extending phenomenon. Meanwhile, the requirements on the high performance liquid chromatography column are not high, the application range is wide, and the method can be suitable for various C18 columns.
The terms "first", "second" and "first" are used herein for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
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 a chemical, wherein the chemical comprises (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine, and the method comprises:
(1) treating (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine mandelate with a diluting solvent to obtain a control solution;
(2) treating the chemical with a diluting solvent so as to obtain a chemical solution;
(3) performing high performance liquid chromatography detection on the chemical solution and the reference solution, and detecting (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine in the chemical based on the high performance liquid chromatography detection result of the reference solution;
wherein the diluting solvent is a mixed solution containing acetonitrile and sodium bicarbonate solution.
2. The detection method according to claim 1, wherein the chemical contains ticagrelor, and the content of ticagrelor in the chemical is 99% or more.
3. The detection method according to claim 1, wherein the concentration of the sodium bicarbonate solution is 0.01 to 0.02 mol/L.
4. The detection method according to claim 1, wherein the volume ratio of the acetonitrile to the sodium bicarbonate solution in the dilution solvent is 40:60 to 60: 40.
5. The detection method according to claim 1, wherein the volume ratio of the acetonitrile to the sodium bicarbonate solution in the dilution solvent is 45: 55.
6. The detection method according to claim 1, wherein the high performance liquid chromatography detection conditions are as follows:
the detection wavelength is 205-215 nanometers, and the column temperature is 20-40 ℃, preferably 30-40 ℃.
7. The detection method according to claim 1, wherein the step (3) further comprises:
and carrying out gradient elution on the chemical solution and the control solution by utilizing a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution containing acetonitrile and phosphate buffer solution, the pH value of the mobile phase A is 2.8-3.2, and the mobile phase B is acetonitrile.
8. The method according to claim 7, wherein the concentration of the phosphate buffer is 0.01 to 0.03 mol/L.
9. The detection method according to claim 7, wherein the volume ratio of the acetonitrile to the phosphate buffer in the mobile phase A is 20: 80-40: 60.
10. The detection method according to claim 7, wherein the gradient elution conditions are:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910740912.5A CN112394109A (en) | 2019-08-12 | 2019-08-12 | Method for detecting impurities in ticagrelor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910740912.5A CN112394109A (en) | 2019-08-12 | 2019-08-12 | Method for detecting impurities in ticagrelor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112394109A true CN112394109A (en) | 2021-02-23 |
Family
ID=74602286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910740912.5A Pending CN112394109A (en) | 2019-08-12 | 2019-08-12 | Method for detecting impurities in ticagrelor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112394109A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014155389A2 (en) * | 2013-03-25 | 2014-10-02 | Glenmark Pharmaceuticals Limited; Glenmark Generics Limited | Process for preparation of ticagrelor |
| CN104311560A (en) * | 2014-09-16 | 2015-01-28 | 北京红太阳药业有限公司 | Ticagrelor preparation method |
| CN105092768A (en) * | 2014-05-21 | 2015-11-25 | 天津市汉康医药生物技术有限公司 | Method for analyzing and separating isomer impurity of ticagrelor intermediate |
| CN105424822A (en) * | 2015-10-29 | 2016-03-23 | 成都百裕科技制药有限公司 | Method for detecting (1R,2S)-2-(3,4-diflurophenyl) cyclopropylamine in ticagrelor |
| CN109387578A (en) * | 2017-08-11 | 2019-02-26 | 四川海思科制药有限公司 | The method for detecting (1R, 2S) -2- (3,4- difluorophenyl) cyclopropylamine in ticagrelor |
-
2019
- 2019-08-12 CN CN201910740912.5A patent/CN112394109A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014155389A2 (en) * | 2013-03-25 | 2014-10-02 | Glenmark Pharmaceuticals Limited; Glenmark Generics Limited | Process for preparation of ticagrelor |
| CN105092768A (en) * | 2014-05-21 | 2015-11-25 | 天津市汉康医药生物技术有限公司 | Method for analyzing and separating isomer impurity of ticagrelor intermediate |
| CN104311560A (en) * | 2014-09-16 | 2015-01-28 | 北京红太阳药业有限公司 | Ticagrelor preparation method |
| CN105424822A (en) * | 2015-10-29 | 2016-03-23 | 成都百裕科技制药有限公司 | Method for detecting (1R,2S)-2-(3,4-diflurophenyl) cyclopropylamine in ticagrelor |
| CN109387578A (en) * | 2017-08-11 | 2019-02-26 | 四川海思科制药有限公司 | The method for detecting (1R, 2S) -2- (3,4- difluorophenyl) cyclopropylamine in ticagrelor |
Non-Patent Citations (3)
| Title |
|---|
| LÍVIA MARONESI BUENO等: "HPLC method for simultaneous analysis of ticagrelor and its organic impurities and identification of two major photodegradation products", 《EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
| 张建水等: "(1R,2S)-2-(3,4-二氟苯基)环丙胺的合成", 《精细化工》 * |
| 张建礼等: "替格瑞洛原料药中有关物质的HPLC法测定", 《中国医药工业杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105431733B (en) | For detecting the analysis method of sulfated oligosaccharide | |
| CN107367562B (en) | Analysis and detection method and application of polymyxin B sulfate | |
| CN110596293B (en) | High performance liquid detection method for homopiperazine | |
| CN105717237B (en) | The detection method of GABA, Glu, DA, 5-HT and Capillary zone electropheresis in a kind of serum | |
| CN105424822B (en) | The method for detecting (1R, 2S) 2 (3,4 difluorophenyl) cyclopropylamine in ticagrelor | |
| CN111650288A (en) | Method for detecting (R) -3-tert-butyloxycarbonylaminopiperidine enantiomer | |
| CN105510482B (en) | The detection method of isomer impurities content in a kind of ticagrelor raw material | |
| CN109900830B (en) | Method for separating and determining sulfonamide impurities in celecoxib by adopting HPLC (high performance liquid chromatography) and application | |
| CN108152418A (en) | The HPLC detection methods of ketorolac tromethamine or/and impurity in ketorolac tromethamine or its preparation | |
| CN107271592B (en) | Liquid chromatographic purity detection method for completely separating tipiraxib hydrochloride from related impurities | |
| CN112630365B (en) | Method for determining content of dimyristoyl phosphatidylcholine by high performance liquid chromatography | |
| Tekko et al. | An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride | |
| CN108872406B (en) | HPLC analysis detection method for related substances in aspartic acid bulk drug | |
| CN112394109A (en) | Method for detecting impurities in ticagrelor | |
| CN113514588B (en) | High performance liquid chromatography analysis method of relevant substances of cysteamine for injection | |
| CN114689737A (en) | Analysis method of S-o-chlorophenyl glycine methyl ester tartrate related substances | |
| CN113514589B (en) | High performance liquid chromatography analysis method of stannous glucoheptonate relative substance for injection | |
| CN114965790B (en) | Liquid phase detection method for amino acid impurities in lysine zinc gluconate particles | |
| CN112611819B (en) | Method for measuring related substances in benfotiamine raw material and preparation thereof | |
| CN113933413B (en) | Method for detecting impurity K in ganciclovir and method for separating impurity | |
| CN114200050B (en) | HPLC detection method for content of related substances in p-bromoanisole | |
| CN117890496B (en) | Method for detecting related substances of compound preparation of novel oral solution of guaifenesin | |
| CN114280169B (en) | Method for determining isomer in monabivir | |
| CN113075306B (en) | Method for detecting content of edetate disodium in pantoprazole sodium | |
| CN111521693B (en) | Method for detecting isosorbide mononitrate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210223 |
|
| RJ01 | Rejection of invention patent application after publication |