CN113759052B - Method for detecting related substances in fluorine Lei Lana initiator - Google Patents
Method for detecting related substances in fluorine Lei Lana initiator Download PDFInfo
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- 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
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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
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Abstract
The invention discloses a detection method of related substances in a fluorine Lei Lana initiator, wherein the fluorine Lei Lana initiator is 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, the related substances are 3, 5-dichloro bromobenzene and 3, 5-dichloro-2, 2-trifluoro acetophenone, a reference substance solution and a test substance solution are prepared, chromatograms of the reference substance solution and the test substance solution are measured by using reverse-phase high performance liquid chromatography, and the impurity content is determined according to the chromatograms of the reference substance solution and the test substance solution. The method can realize complete separation between the starting material and each related substance chromatographic peak, and has the advantages of strong specificity, good recovery rate, excellent linear relation, good repeatability and good durability. The related substances are quantitatively detected by adopting a main component self-comparison method added with correction factors, so that the accurate control of the quality of the initial substances is realized, and the quality of the fluororanaftate raw material medicine product is further effectively controlled.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analytical chemistry, and particularly relates to a detection method of related substances in a fluorine Lei Lana initiator.
Background
Fluorine Lei Lana is used as a novel isoxazoline pesticide capable of rapidly and effectively killing lice and fleas, and is sold and popularized in europe, usa and other countries in 2013 so far and is used for killing lice and fleas of the parasites of dogs and the like. Starting material 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene
As a key starting material of the fluororalston crude drug, the research on the fluoro Lei Lana starting material is particularly important, and the production cost of the crude drug can be reduced so as to promote the domestic commercial production of the fluoro Lei Lana.
The related substances in the 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene mainly comprise 3, 5-dichloro bromobenzene
(impurity A), 3, 5-dichloro-2, 2-trifluoroacetophenone +.>
(impurity B). 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene is similar to impurity B in structure, and the two are not uniform in distribution of double bond electron clouds, so that the quality of fluorine Lei Lana starting material is difficult to effectively control by adopting a common alkyl column.
At present, related patent documents of the compound and related substance detection methods are not reported, and as research on fluorine Lei Lana is in depth in China, a simple and efficient method suitable for detecting related substances in a fluorine Lei Lana initiator is developed, and is a new problem to be solved urgently.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a method for detecting related substances in a fluorine Lei Lana initiator.
The technical scheme adopted by the invention is as follows:
a method for detecting related substances in a fluorine Lei Lana initiator, wherein the fluorine Lei Lana initiator is 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, and the related substances are 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoroacetophenone, comprising the following steps:
preparing a reference substance solution of a fluorine Lei Lana starting material and related substances;
preparing a fluorine Lei Lana starter test solution;
determining chromatograms of a reference substance solution and a test substance solution by using reversed-phase high performance liquid chromatography, wherein a mobile phase A of the reversed-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reversed-phase high performance liquid chromatography is diammine hydrogen phosphate solution, and an elution program is gradient elution;
according to chromatograms of the reference substance solution and the test substance solution, the content of 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoro-acetophenone in the test substance solution is determined by a main component self-comparison method added with a correction factor.
In some examples, the mobile phase B is at a concentration of 8 to 12mM.
In some examples, the gradient elution time and the volume ratio sequence of mobile phase acetonitrile are: the volume fraction is 0 min-12 min, 30-50% is 13 min-20 min, 60-68% is 21 min-30 min,30% is 30%.
In some examples, the flow rate of the mobile phase is 0.3 to 0.8 mL/min.
In some examples, the column temperature in the reverse phase high performance liquid chromatography is 25 ℃ to 35 ℃.
In some examples, the sample loading in the reverse phase high performance liquid chromatography is 5 μl to 15 μl.
In some examples, the detection wavelength in the reverse phase high performance liquid chromatography is 240nm to 260nm.
In some examples, the mass concentration of the sample solution is 400-600 μg/mL.
In some examples, the control solution and the test solution are both formulated with an aqueous acetonitrile solution having a volume fraction of 40% to 80%.
In some examples, the detection method comprises the steps of:
s1) preparing a fluorine Lei Lana starter and a reference substance solution of related substances by using an acetonitrile aqueous solution with the volume fraction of 40-80%;
s2) preparing a fluorine Lei Lana initiator sample solution with the mass concentration of 400-600 mug/mL by using an acetonitrile water solution with the volume fraction of 40-80%;
s3) determining chromatograms of the reference substance solution and the test substance solution by using a reversed-phase high performance liquid chromatography, wherein a mobile phase A of the reversed-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reversed-phase high performance liquid chromatography is a diammonium hydrogen phosphate solution with the concentration of 8-12 mM, and an elution program is gradient elution, wherein: the gradient elution time and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: 0 to 12 minutes, 30 to 50 percent of operation, 13 to 20 minutes, 60 to 68 percent of operation, 21 to 30 minutes, 30 percent of operation, and the flow rate of the mobile phase is 0.3 to 0.8 mL/min; the sample injection amount is 5 mu L-15 mu L, the chromatographic column temperature is 25-35 ℃, and the detection wavelength is 240-260 nm;
s4) determining the content of 3, 5-dichloro bromobenzene and 3, 5-dichloro-2, 2-trifluoro acetophenone in the test solution by using a main component self-contrast method added with correction factors according to chromatograms of the control solution and the test solution.
In some examples, the chromatographic column used is a YMC-Triart Phenyl chromatographic column: 4.6mm x 250mm,5 μm.
The beneficial effects of the invention are as follows:
in some examples of the invention, the reverse-phase high performance liquid chromatography is used for detecting related substances in the fluorine Lei Lana starting material, and complete separation between the starting material and chromatographic peaks of the related substances can be realized through simple mobile phase components; the detection method has the advantages of strong specificity, good recovery rate, excellent linear relation, good repeatability and good durability.
According to some examples of the invention, related substances are quantitatively detected by adopting a main component self-comparison method added with correction factors, so that the quality of the initial material 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene is accurately controlled, and the quality of the fluororalston bulk drug product is further effectively controlled.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a liquid chromatogram of a sample solution in example 1 of the present invention.
FIG. 2 is a liquid chromatogram of the impurity A localization solution in example 2 of the present invention.
FIG. 3 is a liquid chromatogram of the impurity B localization solution in example 2 of the present invention.
FIG. 4 is a liquid chromatogram showing the applicability of the system in example 2 of the present invention.
FIG. 5 is a linear diagram of impurity A in example 4 of the present invention.
FIG. 6 is a linear diagram of impurity B in example 4 of the present invention.
FIG. 7 is a liquid chromatogram of the system applicability solution in comparative example 1.
FIG. 8 is a liquid chromatogram of the system applicability solution in comparative example 2.
Detailed Description
A method for detecting related substances in a fluorine Lei Lana initiator, wherein the fluorine Lei Lana initiator is 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, and the related substances are 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoroacetophenone, comprising the following steps:
preparing a reference substance solution of a fluorine Lei Lana starting material and related substances;
preparing a fluorine Lei Lana starter test solution;
determining chromatograms of a reference substance solution and a test substance solution by using reversed-phase high performance liquid chromatography, wherein a mobile phase A of the reversed-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reversed-phase high performance liquid chromatography is diammine hydrogen phosphate solution, and an elution program is gradient elution;
according to chromatograms of the reference substance solution and the test substance solution, the content of 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoro-acetophenone in the test substance solution is determined by a main component self-comparison method added with a correction factor.
In some examples, the mobile phase B is at a concentration of 8 to 12mM.
In some examples, the gradient elution time and the volume ratio sequence of mobile phase acetonitrile are: the volume fraction is 0 min-12 min, 30-50% is 13 min-20 min, 60-68% is 21 min-30 min,30% is 30%.
In some examples, the flow rate of the mobile phase is 0.3 to 0.8 mL/min. At this flow rate, elution and separation are facilitated.
In some examples, the column temperature in the reverse phase high performance liquid chromatography is 25 ℃ to 35 ℃. The column temperature can obtain more stable data at 25-35 ℃.
In some examples, the sample loading in the reverse phase high performance liquid chromatography is 5 μl to 15 μl.
In some examples, the detection wavelength in the reverse phase high performance liquid chromatography is 240nm to 260nm.
In some examples, the mass concentration of the sample solution is 400-600 μg/mL.
In some examples, the control solution and the test solution are both formulated with an aqueous acetonitrile solution having a volume fraction of 40% to 80%.
In some examples, the detection method comprises the steps of:
preparing a fluorine Lei Lana starter and a reference substance solution of related substances by using an acetonitrile water solution with the volume fraction of 40-80%;
preparing a fluorine Lei Lana initiator solution with the mass concentration of 400-600 mug/mL by using an acetonitrile aqueous solution with the volume fraction of 40-80%;
determining chromatograms of a reference solution and a test solution by using reverse-phase high performance liquid chromatography, wherein a mobile phase A of the reverse-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reverse-phase high performance liquid chromatography is diammine hydrogen phosphate solution with the concentration of 8-12 mM, and an elution program is gradient elution, wherein: the gradient elution time and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: 0 to 12 minutes, 30 to 50 percent of operation, 13 to 20 minutes, 60 to 68 percent of operation, 21 to 30 minutes, 30 percent of operation, and the flow rate of the mobile phase is 0.3 to 0.8 mL/min; the sample injection amount is 5 mu L-15 mu L, the chromatographic column temperature is 25-35 ℃, and the detection wavelength is 240-260 nm;
according to chromatograms of the reference substance solution and the test substance solution, the content of 3, 5-dichloro bromobenzene and 3, 5-dichloro-2, 2-trifluoro acetophenone in the test substance solution is determined by using a main component self-comparison method added with a correction factor.
In some examples, the chromatographic column used is a YMC-Triart Phenyl chromatographic column: 4.6mm x 250mm,5 μm.
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
For convenience of comparison, in the following examples and comparative examples, the solvent used was an aqueous acetonitrile solution with a volume fraction of 60%; the starting material is 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, and related substances comprise 3, 5-dichloro bromobenzene (impurity A) and 3, 5-dichloro-2, 2-trifluoroacetophenone (impurity B).
Example 1: purity test:
s1: sample solution preparation: accurately weighing 10mg of fluorine Lei Lana initial material to be tested, dissolving and diluting to scale marks by using a solvent in a 20mL volumetric flask, and uniformly mixing;
s2: high performance liquid chromatography assay: YMC-Triart Phenyl chromatography column: 4.6mm×250mm,5 μm; the mobile phase is acetonitrile: 10mM diammonium phosphate solution= (30-68): (70-32) (v/v) performing gradient elution, wherein the gradient elution time and the volume ratio of the acetonitrile in the mobile phase are as follows: 0 min-12 min, 30-50% of running, 13 min-20 min, 60-68% of running, 21 min-30 min and 30% of running. Flow rate: 0.5mL/min; column temperature: 35 ℃; ultraviolet detector detection wavelength: 251nm; sample injection amount: 10. Mu.L; the sample solution was injected into the liquid chromatograph.
The detection results are shown in FIG. 1. As can be seen from fig. 1: the retention time of the initiator is 18.912min, the retention time of the impurity A is 17.465min, the retention time of the impurity B is 11.841min, the minimum separation degree is 4.98, which is far greater than 1.5, and the initiator and the impurity can be well separated. And the peak type is good, and the peak purity is high (more than or equal to 98.0%).
Example 2: specificity and system applicability test:
s1: preparation of impurity stock solution: respectively taking impurities A and B, adding a solvent to make the concentration of the impurities A and B be 0.10mg/mL, and taking the impurities A and B as stock solutions for standby;
s2: preparing an impurity positioning solution: taking 0.5mL of the impurity stock solution in a 20mL volumetric flask to obtain impurity A and impurity B positioning solutions with the concentration of 2.50 mu g/mL;
s3: system applicability solution preparation: weighing a volumetric flask with 10mg of an initial material reference substance being 20mL, adding 0.5mL of each of the impurity A and impurity B stock solutions, dissolving and diluting to a scale mark by using a solvent, and uniformly mixing to obtain a system applicability solution, wherein the concentration of the initial material is 500 mug/mL, and the concentration of the impurity A and the impurity B is 2.50 mug/mL;
s4: the measurement was performed according to the conditions of high performance liquid chromatography in example 1, and impurity a, impurity B localization solution and system applicability solution were injected into a liquid chromatograph, wherein the system applicability solution was measured 6 times in succession, and chromatograms were recorded, and the results are shown in fig. 2, 3, 4 and table 1:
as can be seen from table 1: the separation degree of each component of the system applicability solution is more than or equal to 4.98, no interference exists between the components, and the peak time of the impurity positioning solution is basically consistent with that of the system applicability solution. The main peak theoretical plate number of the 6-needle system applicability solution is 116142 ～ 116516, and is more than 2000, so that the requirement of the theoretical plate number is met; the tailing factor is between 0.97 and 0.98 and is within the range (0.8 to 2.0) required by the tailing factor; the peak area RSD value of the starting material of the 6-needle system is 0.13%, and the peak time RSD value is 0.02%.
Example 3: limit of quantification (LOQ) and limit of detection (LOD) assays:
according to the liquid chromatogram of the system applicability solution in the embodiment 2, the signal to noise ratio of each component can be preliminarily obtained, the system applicability solution is taken, the corresponding concentration with the signal to noise ratio of 2-4 is found out by adopting a gradual dilution method, and the corresponding detection limit concentration is preliminarily determined; finding out the corresponding concentration with the signal-to-noise ratio of 8-12, and primarily determining the corresponding quantitative limit concentration. The LOQ and LOD of each component were confirmed, and the final LOD and LOQ values of the compound were determined by measuring under the conditions of high performance liquid chromatography in example 1, 5-needle injection was performed for each concentration, and the response value RSD was 10% or less in accordance with the criterion of methodological verification. According to the detection limit and the quantitative limit confirmation test, the respective components LOD and LOQ are shown in table 2, respectively:
as can be seen from Table 2, the method for detecting the related substances in the fluorine Lei Lana starter has higher sensitivity to the starter, the impurity A and the impurity B, and has higher response to each substance.
Example 4: linear range relation test:
s1:200% limit mixed solution preparation: respectively sucking 1.0mL of stock solution of the impurity A and 1.0mL of stock solution of the impurity B into the same 20mL volumetric flask, and diluting the stock solutions of the impurity A and the impurity B by using a solvent to fix the volume to the scale;
s2: preparing mixed solution with each limit: 2.5mL, 4.0mL, 5.0mL, 6.0mL and 7.5mL of 200% limit mixed solution are respectively weighed into 5 10mL volumetric flasks, added with solvent to be dissolved and diluted to scale marks, and uniformly mixed to obtain the limit mixed solution with the sample concentration of 50%, 80%, 100%, 120% and 150%. Wherein, LOQ mixed solution is prepared according to the quantitative limit test result of the example 3;
s3: peak areas were measured for 5 concentration levels of the limited mixed solution according to the conditions of high performance liquid chromatography in example 1, and each sample was examined 3 times. The measured response signal and the measured object concentration are plotted as a function, and whether the measured response signal and the measured object concentration are linear or not is observed, and linear regression is performed by a least square method. The linear patterns of the impurity A and the impurity B are shown in fig. 5 and 6 respectively.
As can be seen from FIGS. 5-6, the peak area and concentration of each impurity show good linear relationship, and the calculated correlation coefficient r is greater than the standard requirement of 0.995.
Example 5: accuracy test:
s1: preparing a test solution: weighing 10mg of an initial material to be tested, placing the initial material to be tested in a 20mL volumetric flask, dissolving the initial material in a solvent, diluting the initial material to a scale mark, and uniformly mixing the initial material and the solvent;
s2: adding mixed sample solution: 5.0mL, 10mL and 15 mL of 200% limit mixed solution are respectively weighed into three 20mL volumetric flasks weighed with 10mg of starter reference, added with solvent for dissolution and dilution to scale marks, and uniformly mixed. Obtaining mixed sample solutions with the limit of 50%, 100% and 150%;
s3: the peak area of the sample solution was measured according to the high performance liquid chromatography conditions in example 1, and the recovery rate of the relevant substances in the sample solution was calculated according to the main component self-control method, and the results were shown in the following table 3:
from Table 3, the recovery rates of impurity A and impurity B at three different concentrations are 82% -97%, which meets the methodological verification requirement (80.0% -120.0%).
Example 6: repeatability test:
s1: preparing a reference substance solution: precisely weighing 10mg of initial reference substance, placing into a 20mL volumetric flask, dissolving with solvent, diluting to scale mark, and mixing to obtain reference substance solution.
S2: preparing a test solution: 6 parts of initial substances are respectively weighed, and the mass of the initial substances is respectively 10.16mg, 10.35mg, 10.22mg, 10.30mg, 10.12mg and 10.27mg, and the initial substances are added with solvents to be dissolved and diluted to scale marks, and are uniformly mixed. Solutions with the concentration of 508 mug/mL, 517.5 mug/mL, 511 mug/mL, 515 mug/mL, 506 mug/mL and 513.5 mug/mL are obtained;
s3: the peak area of the sample solution was measured according to the high performance liquid chromatography conditions in example 1, and the recovery rate of the starting material in the sample solution was calculated from the main component self-control method, and the results were shown in the following table 4:
as can be seen from Table 4, the recovery rates of the starting materials are all between 99%, which shows that the determination of the content of the starting materials by the method is accurate and reliable.
Example 7: durability and stability test:
s1: preparing a reference substance solution: precisely weighing 10mg of initial reference substance, placing into a 20mL volumetric flask, dissolving with solvent, diluting to scale mark, and mixing.
S2: preparing a test solution: respectively precisely weighing 2 parts of the initial materials and 10mg of the test materials, respectively placing in 20mL volumetric flasks, dissolving with solvent, diluting to scale mark, and mixing.
S3: the peak area of the sample solution was measured according to the high performance liquid chromatography conditions in example 1, and the influence of different column temperatures on the detection results of the starting material and the impurity was examined, and the detection was performed at the column temperature=35±5 ℃. Sample solutions were measured after 24 hours of sun exposure and compared to durability results to calculate RSD values for 3 sets of data. The results are shown in Table 5:
as can be seen from table 5, RSD values for a total of 6 data from the 3 groups all meet the durability decision criteria in the validation methodology validation. Under the condition that the fluctuation of the column temperature is not more than 5 ℃, the influence of the column temperature on each component of the fluorine Lei Lana starter test sample is within an acceptable range.
Comparative example 1:
the detection method of comparative example 1 and example 1 are the same, except that the chromatographic conditions of S2 are specifically:
s1: preparing a system applicability solution according to example 2;
s2: high performance liquid chromatography assay: YMC-Triart Phenyl chromatography column: 4.6mm×250mm,5 μm; methanol: 10mM diammonium phosphate solution= (30-68): (70-32) (v/v) performing gradient elution, wherein the gradient elution time and the volume ratio of the acetonitrile in the mobile phase are as follows: 0 min-12 min, 30-50% of running, 13 min-20 min, 60-68% of running, 21 min-30 min and 30% of running. Flow rate: 0.5mL/min; column temperature: 35 ℃; ultraviolet detector detection wavelength: 251nm; sample injection amount: 10. Mu.L; the sample solution was injected into the liquid chromatograph, and the detection result is shown in fig. 7.
As can be seen from fig. 7, neither the starter nor the impurities could be detected, nor the response.
Comparative example 2
The detection method of comparative example 2 is the same as that of example 1, except that the chromatographic conditions of S2 are specifically:
s1: preparing a system applicability solution according to example 2;
s2: high performance liquid chromatography assay: YMC-Triart Phenyl chromatography column: 4.6mm×250mm,5 μm; acetonitrile: water= (30-68): (70-32) (v/v) performing gradient elution, wherein the gradient elution time and the volume ratio of the acetonitrile in the mobile phase are as follows: 0 min-12 min, 30-50% of running, 13 min-20 min, 60-68% of running, 21 min-30 min and 30% of running. Flow rate: 0.5mL/min; column temperature: 35 ℃; ultraviolet detector detection wavelength: 251nm; sample injection amount: 10. Mu.L; the sample solution was injected into the liquid chromatograph, and the detection result is shown in fig. 8.
As can be seen from fig. 8, the starter retention time was excessive and the baseline separation from nearby impurities was not achieved.
Compared with the chromatographic conditions defined by the invention, the complete separation between the starting material and each related substance chromatographic peak can be well realized, and the method has the advantages of strong specificity, good recovery rate, excellent linear relation, good repeatability and good durability. The change of chromatographic conditions has an unpredictable effect on the detection effect.
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.
Claims (9)
1. A method for detecting related substances in a fluorine Lei Lana initiator, wherein the fluorine Lei Lana initiator is 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, and the related substances are 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoroacetophenone, comprising the following steps:
preparing a reference substance solution of a fluorine Lei Lana starting material and related substances;
preparing a fluorine Lei Lana starter test solution;
determining chromatograms of a reference substance solution and a test substance solution by using reversed-phase high performance liquid chromatography, wherein a mobile phase A of the reversed-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reversed-phase high performance liquid chromatography is diammine hydrogen phosphate solution, and an elution program is gradient elution; wherein the chromatographic column used is a YMC-Triart Phenyl chromatographic column: 4.6mm multiplied by 250mm,5 mu m, gradient elution time and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: 0 to 12 minutes, 30 to 50 percent of the operation, 13 to 20 minutes, 60 to 68 percent of the operation, 21 to 30 minutes and 30 percent of the operation according to the volume fraction;
according to chromatograms of the reference substance solution and the test substance solution, the content of 1, 3-dichloro-5- (1-trifluoromethyl-vinyl) benzene, 3, 5-dichloro-bromobenzene and 3, 5-dichloro-2, 2-trifluoro-acetophenone in the test substance solution is determined by a main component self-comparison method added with a correction factor.
2. The method of claim 1, wherein: the concentration of the mobile phase B is 8-12 mM.
3. The method of claim 1, wherein: the flow rate of the mobile phase is 0.3-0.8 mL/min.
4. The method of claim 1, wherein: the chromatographic column temperature in the reversed phase high performance liquid chromatography is 25-35 ℃.
5. The method of claim 1, wherein: the sample injection amount in the reversed phase high performance liquid chromatography is 5-15 mu L.
6. The method of claim 1, wherein: the detection wavelength in the reversed phase high performance liquid chromatography is 240 nm-260 nm.
7. The method of claim 1, wherein: the mass concentration of the sample solution is 400-600 mug/mL.
8. The method of claim 1, wherein: the reference substance solution and the sample solution are prepared by adopting acetonitrile water solution with the volume fraction of 40% -80%.
9. The method of claim 1, wherein: the method comprises the following steps:
preparing a fluorine Lei Lana starter and a reference substance solution of related substances by using an acetonitrile water solution with the volume fraction of 40-80%;
preparing a fluorine Lei Lana initiator solution with the mass concentration of 400-600 mug/mL by using an acetonitrile aqueous solution with the volume fraction of 40-80%;
determining chromatograms of a reference solution and a test solution by using reverse-phase high performance liquid chromatography, wherein a mobile phase A of the reverse-phase high performance liquid chromatography is acetonitrile, a mobile phase B of the reverse-phase high performance liquid chromatography is diammine hydrogen phosphate solution with the concentration of 8-12 mM, and an elution program is gradient elution, wherein: the gradient elution time and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: 0 to 12 minutes, 30 to 50 percent of operation, 13 to 20 minutes, 60 to 68 percent of operation, 21 to 30 minutes, 30 percent of operation, and the flow rate of the mobile phase is 0.3 to 0.8 mL/min; the sample injection amount is 5 mu L-15 mu L, the chromatographic column temperature is 25-35 ℃, and the detection wavelength is 240-260 nm;
according to chromatograms of the reference substance solution and the test substance solution, the content of 3, 5-dichloro bromobenzene and 3, 5-dichloro-2, 2-trifluoro acetophenone in the test substance solution is determined by using a main component self-comparison method added with a correction factor.
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| WO2017201134A1 (en) * | 2016-05-20 | 2017-11-23 | Avista Pharma Solutions, Inc. | Synthetic process and novel intermediates |
| CN107501199A (en) * | 2017-08-16 | 2017-12-22 | 南京农业大学 | The extraction of fluorine thunder Rana monomeric compound and purification process |
| CN113121383A (en) * | 2021-04-22 | 2021-07-16 | 江苏慧聚药业有限公司 | Related substances of Aforana and frailana synthesized building blocks and synthesis method thereof |
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