CN113984933B - Detection method of KPT-330 intermediate - Google Patents
Detection method of KPT-330 intermediate Download PDFInfo
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- CN113984933B CN113984933B CN202111267577.5A CN202111267577A CN113984933B CN 113984933 B CN113984933 B CN 113984933B CN 202111267577 A CN202111267577 A CN 202111267577A CN 113984933 B CN113984933 B CN 113984933B
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- 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
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- 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
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- 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
Abstract
The invention belongs to the technical field of organic matter detection and analysis, and discloses a detection method of a KPT-330 intermediate. The detection method comprises the following steps: detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid by adopting a liquid chromatography method; the conditions of the liquid chromatography include: the adopted mobile phase comprises a mobile phase A and a mobile phase B; mobile phase a is phosphate buffer; mobile phase B is acetonitrile; the mobile phase is subjected to gradient elution according to certain conditions. The detection method can accurately detect the purity of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid in the test sample.
Description
Technical Field
The invention belongs to the technical field of organic matter detection and analysis, and particularly relates to a detection method of a KPT-330 intermediate.
Background
KPT-330 is an orally bioavailable selective CRM1 (chromosome maintenance protein 1) inhibitor. (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, CAS number: 1388842-44-1, which is a key intermediate for producing a Selingexor (KPT-330) bulk drug, and the purity and impurity of the compound directly influence the purity and the size of the impurity of the drug, thereby directly influencing the curative effect of the drug.
At present, related literature and reports on a purity detection method of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid are not found, and in order to strengthen quality control of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid, further control of quality of a Selinexor (KPT-330) bulk drug, and a detection method of purity and impurity content of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid is particularly important.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a detection method of the KPT-330 intermediate, which has the advantages of simplicity, convenience, accuracy, rapidness, reliability and the like.
The invention provides a detection method of a KPT-330 intermediate, which comprises the following steps:
detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid by adopting a liquid chromatography method;
the conditions of the liquid chromatography include: the adopted mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is phosphate buffer solution; the mobile phase B is acetonitrile;
the mobile phase eluted according to the following gradient:
0 min, 75-85% of mobile phase A and 15-25% of mobile phase B;
2 minutes, 75-85% of mobile phase A and 15-25% of mobile phase B;
for 12 minutes, the mobile phase A is 15-25%, and the mobile phase B is 75-85%;
for 22 minutes, the mobile phase A is 15-25%, and the mobile phase B is 75-85%;
23 minutes, 75-85% of mobile phase A and 15-25% of mobile phase B;
30 minutes, 75-85% of mobile phase A and 15-25% of mobile phase B.
Preferably, the detection method comprises the steps of:
dissolving a (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample in a diluent to obtain a test sample solution;
taking the sample solution, detecting by adopting a liquid chromatography method, and recording a chromatogram A;
calculating the purity of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid test sample according to the chromatogram A;
the conditions of the liquid chromatography include: the adopted mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is phosphate buffer solution; the mobile phase B is acetonitrile;
the mobile phase eluted according to the following gradient:
0 min, 78-82% of mobile phase A and 18-22% of mobile phase B; more preferably, mobile phase a is 80% and mobile phase B is 20%;
2 minutes, 78-82% of mobile phase A and 18-22% of mobile phase B; more preferably, mobile phase a is 80% and mobile phase B is 20%;
for 12 minutes, the mobile phase A is 18-22%, and the mobile phase B is 78-82%; more preferably, mobile phase a is 20% and mobile phase B is 80%;
for 22 minutes, the mobile phase A is 18-22%, and the mobile phase B is 78-82%; more preferably, mobile phase a is 20% and mobile phase B is 80%;
23 minutes, 78-82% of mobile phase A and 18-22% of mobile phase B; more preferably, mobile phase a is 80% and mobile phase B is 20%;
30 minutes, 78-82% of mobile phase A and 18-22% of mobile phase B; more preferably, mobile phase a is 80% and mobile phase B is 20%.
Preferably, the diluent is a mixture of tetrahydrofuran, water and acetonitrile, and the volume ratio of the tetrahydrofuran, the water and the acetonitrile in the diluent is 3 (3-4): (4-5), more preferably 3:3:4; the concentration of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid test sample in the test sample solution is 0.25-0.75mg/mL, and more preferably 0.5mg/mL.
Preferably, the detection method further comprises the step of a system suitability test, the system suitability test comprising: taking a (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance, and dissolving the reference substance with a diluent to obtain a system applicability solution; and detecting the system applicability solution by adopting a liquid chromatography method, and recording a chromatogram B. The purpose of the system suitability test is primarily to determine that the chromatographic system used for analysis is effective and suitable.
Preferably, the concentration of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic control in the system applicability solution is 0.25-0.75mg/mL, more preferably 0.5mg/mL.
Preferably, the system applicability test results: the theoretical plate number is not less than 2000 calculated by (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid, and the separation degree of a main peak and adjacent impurities is more than 1.5.
Preferably, the detection method further includes the step of a blank test, wherein the blank test is: and detecting the diluent by adopting a liquid chromatography method, recording a chromatogram C, and ensuring that blank is not interfered. After the chromatographic peak in the fading chromatogram C is subtracted from the chromatogram A, the purity of the test sample is calculated according to an area normalization method. The blank test aims to eliminate the influence caused by the diluent and can further improve the detection accuracy.
Preferably, the phosphate buffer is a potassium dihydrogen phosphate buffer; the concentration of the potassium dihydrogen phosphate buffer is 0.01-0.03mol/L, more preferably 0.01mol/L; the pH of the potassium dihydrogen phosphate buffer is 6.0-7.5, more preferably 7.0.+ -. 0.05. The pH of the phosphate buffer is selected to help improve the peak profile, especially when the pH is 7.0, which is optimal.
Preferably, the conditions of the liquid chromatography further include:
chromatographic column: wondaSil C18 Superb (5 um, 4.6X250 mm) (W) and its equivalent C18 column;
sample injection amount: 5-20. Mu.L, more preferably 10. Mu.L;
mobile phase flow rate: 0.7-1.0mL/min, more preferably 0.8mL/min;
column temperature: 20-30 ℃, more preferably 26 ℃;
detection wavelength: 260-270nm, more preferably 265nm;
a detector: an ultraviolet detector.
Another aspect of the invention is to provide the use of the above assay method for the preparation of CRM1 inhibitors.
In particular to an application of the detection method of the KPT-330 intermediate in preparing a Selingexor (KPT-330) bulk drug.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, phosphate buffer solution is adopted as a mobile phase A and acetonitrile is adopted as a mobile phase B, and gradient elution is carried out according to a certain proportion, so that effective separation of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid and impurities thereof is realized, the separation effect is improved, and the detection accuracy, detection speed and sensitivity are improved.
2. The detection method has the advantages of simplicity, convenience, accuracy, rapidness, reliability and the like, is beneficial to strengthening the quality control of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid, and further controls the quality of a Selingexor (KPT-330) bulk drug.
Drawings
FIG. 1 is a liquid chromatogram of a sample solution of example 1 of the present invention;
FIG. 2 is a liquid chromatogram of a system applicability solution of example 1 of the present invention;
FIG. 3 is a liquid chromatogram of a sample solution of example 2 of the present invention;
FIG. 4 is a liquid chromatogram of a sample solution of example 3 of the present invention;
FIG. 5 is a liquid chromatogram of a sample solution of example 4 of the present invention;
FIG. 6 is a liquid chromatogram of a sample solution of example 5 of the present invention;
FIG. 7 is a liquid chromatogram of a sample solution of example 6 of the present invention;
FIG. 8 is a liquid chromatogram of a sample solution of comparative example 1 of the present invention;
FIG. 9 is a liquid chromatogram of a test solution of comparative example 2 of the present invention;
FIG. 10 is a liquid chromatogram of a sample solution of comparative example 3 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Four batches of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid products produced by the same production specification instruction are taken as test products 1-4, and are detected according to the detection methods of examples 1-4, and the purity and impurities thereof are calculated by adopting an area normalization method.
Example 1
A method for detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, comprising the steps of:
1) Preparing a diluent: mixing tetrahydrofuran, water and acetonitrile according to a volume ratio of 3:3:4 to obtain a diluent;
2) Preparing a test solution: taking a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample, and dissolving the test sample with a diluent to obtain a test sample solution with the concentration of 0.5 mg/mL;
3) Preparing a system applicability solution: placing a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance into a volumetric flask, and dissolving the reference substance by using a diluent to prepare a solution containing 0.5mg of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance per milliliter to obtain a system applicability solution;
4) Blank test, precisely measuring 10 mu L of diluent, injecting into a liquid chromatograph, and recording a chromatogram; the blank must not have interference;
5) System applicability test: precisely measuring 10 mu L of system applicability solution, injecting into a liquid chromatograph, recording the chromatogram, wherein the theoretical plate number is not lower than 2000 according to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid calculation, and the separation degree of a main peak and adjacent impurities is larger than 1.5;
6) Test article solution test: precisely measuring 10 mu L of the sample solution, injecting into a liquid chromatograph, recording a chromatogram, deducting a chromatographic peak of a blank test from the chromatogram of the sample solution, and calculating the purity of the sample according to an area normalization method.
The test conditions of the liquid chromatograph of the present embodiment were set as follows: the chromatographic column is an island fluid Wondasil C18 Superb chromatographic column with the specification of 4.6X250 mm and 5 μm, and the column temperature is 26 ℃; gradient elution is carried out by adopting a double mobile phase, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution with the concentration of 0.01mol/L (the pH value is adjusted to 7.0 by sodium hydroxide solution), the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.8mL/min; the detector is an ultraviolet detector, and the detection wavelength is 265nm; the gradient elution conditions are shown in table 1, and the volume ratio of mobile phase a to mobile phase B is uniform and gradually changed.
TABLE 1 gradient elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 2 | 80 | 20 |
| 12 | 20 | 80 |
| 22 | 20 | 80 |
| 23 | 80 | 20 |
| 30 | 80 | 20 |
The chromatogram of the sample solution of this example is shown in FIG. 1, wherein "1" in FIG. 1 represents the peak corresponding to the maximum single impurity, "2" represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "3, 4" represents the peak corresponding to the other impurity (ordinate "absorpance" in FIG. 1 represents Absorbance in mAU). The integration results and detection results are shown in tables 2 and 3.
TABLE 2 integration results of chromatograms
TABLE 3 test results of test solutions
The chromatogram of the system applicability solution of this example is shown in fig. 2, in which "1" in fig. 2 represents the peak corresponding to the maximum single impurity, "2" represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "3, 4, 5" represents the peak corresponding to other impurities (ordinate "absorpance" in fig. 2 represents Absorbance in mAU). As can be seen from FIG. 2, the separation degree of the main peak from the adjacent impurities is more than 1.5, and the number of trays is high. The integration results and detection results are shown in tables 4 and 5.
TABLE 4 integral results of chromatograms
TABLE 5 detection results of System applicability solutions
Example 2
A method for detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, comprising the steps of:
1) Preparing a diluent: mixing tetrahydrofuran, water and acetonitrile according to a volume ratio of 3:3:4 to obtain a diluent;
2) Preparing a test solution: taking a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample, and dissolving the test sample with a diluent to obtain a test sample solution with the concentration of 0.5 mg/mL;
3) Preparing a system applicability solution: placing a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance into a volumetric flask, and dissolving the reference substance by using a diluent to prepare a solution containing 0.5mg of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance per milliliter to obtain a system applicability solution;
4) Blank test, precisely measuring 10 mu L of diluent, injecting into a liquid chromatograph, and recording a chromatogram; the blank must not have interference;
5) System applicability test: precisely measuring 10 mu L of system applicability solution, injecting into a liquid chromatograph, recording the chromatogram, wherein the theoretical plate number is not lower than 2000 according to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid calculation, and the separation degree of a main peak and adjacent impurities is larger than 1.5;
6) Test article solution test: precisely measuring 10 mu L of the sample solution, injecting into a liquid chromatograph, recording a chromatogram, deducting a chromatographic peak of a blank test from the chromatogram of the sample solution, and calculating the purity of the sample according to an area normalization method.
The test conditions of the liquid chromatograph of the present embodiment were set as follows: the chromatographic column is an island fluid Wondasil C18 Superb chromatographic column with the specification of 4.6X250 mm and 5 μm, and the column temperature is 26 ℃; gradient elution is carried out by adopting a double mobile phase, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution with the concentration of 0.01mol/L (the pH value is adjusted to 7.0 by sodium hydroxide solution), the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.8mL/min; the detector is an ultraviolet detector, and the detection wavelength is 265nm; the conditions for gradient elution are shown in Table 6.
TABLE 6 gradient elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 2 | 80 | 20 |
| 12 | 20 | 80 |
| 22 | 20 | 80 |
| 23 | 80 | 20 |
| 30 | 80 | 20 |
The chromatogram of the sample solution of this example is shown in FIG. 3, wherein "1" in FIG. 3 represents the peak corresponding to the maximum single impurity, "2" represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "3, 4" represents the peak corresponding to the other impurity (ordinate "absorpance" in FIG. 3 represents Absorbance in mAU). The integration results and detection results are shown in tables 7 and 8.
TABLE 7 integral results of chromatograms
TABLE 8 test results of test solutions
Example 3
A method for detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, comprising the steps of:
1) Preparing a diluent: mixing tetrahydrofuran, water and acetonitrile according to a volume ratio of 3:3:4 to obtain a diluent;
2) Preparing a test solution: taking a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample, and dissolving the test sample with a diluent to obtain a test sample solution with the concentration of 0.5 mg/mL;
3) Preparing a system applicability solution: placing a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance into a volumetric flask, and dissolving the reference substance by using a diluent to prepare a solution containing 0.5mg of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance per milliliter to obtain a system applicability solution;
4) Blank test, precisely measuring 10 mu L of diluent, injecting into a liquid chromatograph, and recording a chromatogram; the blank must not have interference;
5) System applicability test: precisely measuring 10 mu L of system applicability solution, injecting into a liquid chromatograph, recording the chromatogram, wherein the theoretical plate number is not lower than 2000 according to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid calculation, and the separation degree of a main peak and adjacent impurities is larger than 1.5;
6) Test article solution test: precisely measuring 10 mu L of the sample solution, injecting into a liquid chromatograph, recording a chromatogram, deducting a chromatographic peak of a blank test from the chromatogram of the sample solution, and calculating the purity of the sample according to an area normalization method.
The test conditions of the liquid chromatograph of the present embodiment were set as follows: the chromatographic column is an island fluid Wondasil C18 Superb chromatographic column with the specification of 4.6X250 mm and 5 μm, and the column temperature is 26 ℃; gradient elution is carried out by adopting a double mobile phase, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution with the concentration of 0.01mol/L (the pH value is adjusted to 7.0 by sodium hydroxide solution), the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.8mL/min; the detector is an ultraviolet detector, and the detection wavelength is 265nm; the conditions for gradient elution are shown in Table 9.
TABLE 9 gradient elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 2 | 80 | 20 |
| 12 | 20 | 80 |
| 22 | 20 | 80 |
| 23 | 80 | 20 |
| 30 | 80 | 20 |
The chromatogram of the sample solution of this example is shown in FIG. 4, in which "1" in FIG. 4 represents the peak corresponding to the maximum single impurity, "2" represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "3, 4" represents the peak corresponding to the other impurity (ordinate "absorpance" in FIG. 3 represents Absorbance in mAU). The integration results and detection results are shown in tables 10 and 11.
Table 10 integration results of chromatograms
TABLE 11 test results of test solutions
Example 4
A method for detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, comprising the steps of:
1) Preparing a diluent: mixing tetrahydrofuran, water and acetonitrile according to a volume ratio of 3:3:4 to obtain a diluent;
2) Preparing a test solution: taking a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample, and dissolving the test sample with a diluent to obtain a test sample solution with the concentration of 0.5 mg/mL;
3) Preparing a system applicability solution: placing a proper amount of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance into a volumetric flask, and dissolving the reference substance by using a diluent to prepare a solution containing 0.5mg of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance per milliliter to obtain a system applicability solution;
4) Blank test, precisely measuring 10 mu L of diluent, injecting into a liquid chromatograph, and recording a chromatogram; the blank must not have interference;
5) System applicability test: precisely measuring 10 mu L of system applicability solution, injecting into a liquid chromatograph, recording the chromatogram, wherein the theoretical plate number is not lower than 2000 according to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid calculation, and the separation degree of a main peak and adjacent impurities is larger than 1.5;
6) Test article solution test: precisely measuring 10 mu L of the sample solution, injecting into a liquid chromatograph, recording a chromatogram, deducting a chromatographic peak of a blank test from the chromatogram of the sample solution, and calculating the purity of the sample according to an area normalization method.
The test conditions of the liquid chromatograph of the present embodiment were set as follows: the chromatographic column is an island fluid Wondasil C18 Superb chromatographic column with the specification of 4.6X250 mm and 5 μm, and the column temperature is 26 ℃; gradient elution is carried out by adopting a double mobile phase, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution with the concentration of 0.01mol/L (the pH value is adjusted to 7.0 by sodium hydroxide solution), the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.8mL/min; the detector is an ultraviolet detector, and the detection wavelength is 265nm; the conditions for gradient elution are shown in Table 12.
TABLE 12 gradient elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 2 | 80 | 20 |
| 12 | 20 | 80 |
| 22 | 20 | 80 |
| 23 | 80 | 20 |
| 30 | 80 | 20 |
The chromatogram of the sample solution of this example is shown in FIG. 5, in which "1" in FIG. 5 represents the peak corresponding to the maximum single impurity, "2" represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "3, 4, 5, 6" represents the peaks corresponding to other impurities (ordinate "absorpance" in FIG. 5 represents Absorbance in mAU). The integration results and detection results are shown in tables 13 and 14.
TABLE 13 integration results of chromatograms
TABLE 14 test results of test solutions
From the results of the tests of examples 1 to 4, it can be seen that the retention time of the control and the test sample are almost identical by comparing the control sample with the 4 test sample. Examples 1-4 have different detection results for different samples, and can effectively separate (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid and impurities thereof, so that the method provided by the invention is accurate and stable in detection purity result.
Within the mobile phase gradient elution ratio range defined in the present invention, mobile phase a is 75% and mobile phase B is 25% as per 0 min; 2 minutes, 75% mobile phase A and 25% mobile phase B; for 12 minutes, mobile phase A was 25% and mobile phase B was 75%;22 minutes, 25% mobile phase A and 75% mobile phase B; 23 minutes, 75% mobile phase A and 25% mobile phase B; 30 minutes, 75% mobile phase A, 25% mobile phase B gradient elution ratio, or according to 0 minutes, 85% mobile phase A, 15% mobile phase B; 2 minutes, 85% mobile phase A and 15% mobile phase B; for 12 minutes, mobile phase A was 15% and mobile phase B was 85%;22 minutes, mobile phase A15%, mobile phase B85%; 23 minutes, 85% mobile phase A and 15% mobile phase B; 30 minutes, 85% mobile phase A and 15% mobile phase B, can effectively separate (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid and impurities thereof.
Example 5
Example 5 differs from example 1 in that the pH of the mobile phase A potassium dihydrogen phosphate buffer was 8.0, with the other conditions being the same as in example 1. The chromatogram of the sample solution of example 5 is shown in FIG. 6, in which "3" in FIG. 6 represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "1, 2, 4" represents the peak corresponding to impurities (in mAU on the ordinate "absorpance" in FIG. 6). As can be seen from FIG. 6, when the pH of the potassium dihydrogen phosphate buffer is not within the limit of the present invention, the chromatographic peak pattern is poor. Does not meet the chromatographic requirements. The integration results are shown in Table 15.
TABLE 15 integral results of chromatograms
Example 6
Example 6 differs from example 1 in that the pH of the mobile phase A potassium dihydrogen phosphate buffer was 5.5, with the other conditions being the same as in example 1. The chromatogram of the sample solution of example 6 is shown in FIG. 7, in which "1, 2, 3" in FIG. 7 represents peaks after branching of (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "4, 5, 6, 7, 8" represents peaks corresponding to unknown impurities (ordinate "absorpance" in FIG. 7 represents Absorbance in mAU). As can be seen from FIG. 7, when the pH of the potassium dihydrogen phosphate buffer is not within the limit of the present invention, the chromatographic peak is extremely poor, the peak is branched, and the accuracy of purity is seriously affected. The integration results are shown in Table 16.
Table 16 integration results of chromatograms
Comparative example 1
Comparative example 1 differs from example 1 in that comparative example 1 employs isocratic elution, and the elution conditions are shown in table 17.
TABLE 17 isocratic elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 20 | 50 | 50 |
The chromatogram of the sample solution of this comparative example is shown in FIG. 8, in which "5" in FIG. 8 represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "1, 2, 3, 4, 6, 7, 8" represents the peak corresponding to impurities (in mAU, the ordinate "Absorbance" in FIG. 7 represents Absorbance). As can be seen from FIG. 8, comparative example 1 has a low tray number of 1355 and less than 2000, which results in poor separation of main peaks from adjacent impurities due to the use of isocratic elution. The integration results are shown in Table 18.
Integration results of Table 18 chromatograms
Comparative example 2
Comparative example 2 is different from example 1 in that the gradient elution ratio of comparative example 2 is not within the range defined by the present invention, and the gradient elution conditions are shown in table 19.
TABLE 19 gradient elution conditions
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 2 | 70 | 30 |
| 12 | 30 | 70 |
| 22 | 30 | 70 |
| 23 | 70 | 30 |
| 30 | 70 | 30 |
The chromatogram of the sample solution of this comparative example is shown in FIG. 9, in which "3" in FIG. 9 represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "1, 2,4, 5" represents the peak corresponding to impurities (in mAU, the ordinate "Absorbance" in FIG. 9 represents Absorbance). As can be seen from fig. 9, the gradient elution ratio of comparative example 2 is not within the range defined by the present invention, resulting in a difference of less than 1.5 between the main peak and the impurity, and some of them are not separated, affecting the accuracy of purity, and not meeting the chromatographic requirements. The integration results are shown in Table 20.
Integration results of Table 20 chromatograms
Comparative example 3
Comparative example 3 differs from example 1 in that comparative example 3 mobile phase B uses methanol instead of acetonitrile, and other conditions and detection methods are the same as example 1.
The chromatogram of the sample solution of this comparative example is shown in FIG. 10, in which "2" in FIG. 10 represents the peak corresponding to (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid, and "1, 3, 4, 5, 6" represents the peak corresponding to an unknown impurity (in mAU, the ordinate "Absorbance" in FIG. 10 represents Absorbance). As can be seen from fig. 10, comparative example 3, which uses methanol instead of acetonitrile, resulted in poor peak pattern of the main peak, diverged, affected the accuracy of purity, and did not meet the chromatographic requirements. The integration results are shown in Table 21.
Table 21 integration results of chromatograms
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (9)
1. The KPT-330 intermediate detection method is characterized by comprising the following steps:
detecting (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid by adopting a liquid chromatography method;
the conditions of the liquid chromatography include: using a C18 chromatographic column, and using mobile phases including mobile phase a and mobile phase B; the mobile phase A is potassium dihydrogen phosphate buffer solution with the pH value of 7.0; the mobile phase B is acetonitrile;
the mobile phase eluted according to the following gradient:
0 min, mobile phase A80% and mobile phase B20%;
2 minutes, mobile phase A80%, mobile phase B20%;
for 12 minutes, mobile phase A was 20% and mobile phase B was 80%;
22 minutes, 20% mobile phase A and 80% mobile phase B;
23 minutes, mobile phase A80%, mobile phase B20%;
30 minutes, mobile phase A was 80% and mobile phase B was 20%.
2. The method of detection according to claim 1, comprising the steps of:
dissolving a (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample in a diluent to obtain a test sample solution;
taking the sample solution, detecting by adopting a liquid chromatography method, and recording a chromatogram A;
calculating the purity of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic acid test sample according to the chromatogram A;
the conditions of the liquid chromatography include: using a C18 chromatographic column, and using mobile phases including mobile phase a and mobile phase B; the mobile phase A is potassium dihydrogen phosphate buffer solution with the pH value of 7.0; the mobile phase B is acetonitrile;
the mobile phase eluted according to the following gradient:
0 min, mobile phase A80% and mobile phase B20%;
2 minutes, mobile phase A80%, mobile phase B20%;
for 12 minutes, mobile phase A was 20% and mobile phase B was 80%;
22 minutes, 20% mobile phase A and 80% mobile phase B;
23 minutes, mobile phase A80%, mobile phase B20%;
30 minutes, mobile phase A was 80% and mobile phase B was 20%.
3. The detection method according to claim 2, wherein the diluent is a mixture of tetrahydrofuran, water and acetonitrile, and the volume ratio of tetrahydrofuran, water and acetonitrile in the diluent is 3 (3-4): 4-5; the concentration of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid test sample in the test sample solution is 0.25-0.75mg/mL.
4. The method of claim 2, further comprising the step of testing for system suitability, the system suitability test comprising: taking a (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid reference substance, and dissolving the reference substance with a diluent to obtain a system applicability solution; and detecting the system applicability solution by adopting a liquid chromatography method, and recording a chromatogram B.
5. The method according to claim 4, wherein the concentration of the (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazol-1-yl) acrylic control in the system applicability solution is 0.25-0.75mg/mL.
6. The method according to claim 4, wherein the result of the detection: the theoretical plate number is not less than 2000 calculated by (Z) -3- (3, 5-bis (trifluoromethyl) phenyl) -1H-1,2, 4-triazole-1-yl) acrylic acid, and the separation degree of a main peak and adjacent impurities is more than 1.5.
7. The method of claim 2, further comprising the step of testing for a blank test comprising: and detecting the diluted solution by adopting a liquid chromatography method, and recording a chromatogram C.
8. The method according to claim 1, wherein the conditions of the liquid chromatography further comprise:
sample injection amount: 5-20 mu L;
mobile phase flow rate: 0.7-1.0mL/min;
column temperature: 20-30 ℃;
detection wavelength: 260-270nm.
9. Use of the assay of any one of claims 1-8 in the preparation of a CRM1 inhibitor.
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