CN114113390B - Florarana intermediate and detection method of impurities thereof - Google Patents

Florarana intermediate and detection method of impurities thereof Download PDF

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CN114113390B
CN114113390B CN202111405378.6A CN202111405378A CN114113390B CN 114113390 B CN114113390 B CN 114113390B CN 202111405378 A CN202111405378 A CN 202111405378A CN 114113390 B CN114113390 B CN 114113390B
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冯绮晴
李冰冰
姜桥
龚俊
王婉盈
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Livzon New North River Pharmaceutical Co ltd
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Abstract

The invention belongs to the field of detection, and discloses a method for detecting a fluororalreceived intermediate and impurities thereof. The intermediate of fluorine Lei Lana is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, a reference substance and a sample solution are prepared by dissolving the intermediate of fluorine Lei Lana, impurities thereof, a sample to be detected and the like in acetonitrile solution, then a chromatogram is determined by using specific reversed-phase high performance liquid chromatography conditions, and the content of the intermediate of fluorine Lei Lana and the impurities thereof in the sample solution is determined by using an external standard method according to the chromatogram. The method can effectively separate the fluororalrana intermediate from impurities thereof, accurately measure the content of the fluoro Lei Lana intermediate and each impurity, is not interfered by blank and other impurities, has simple and convenient operation, high speed, high repeatability, high accuracy and high stability, can be used for controlling the quality of starting materials used in the synthesis process of the fluoro Lei Lana, and realizes the control of the preparation process of the fluoro Lei Lana.

Description

Florarana intermediate and detection method of impurities thereof
Technical Field
The invention relates to the field of medicine analysis, in particular to a method for detecting a fluororalreceived intermediate and impurities thereof.
Background
Fluoro Lei Lana (Fluralaner), CAS number 864731-61-3, molecular formula C 22 H 17 Cl 2 F 6 N 3 O 3 The broad-spectrum insecticide has good insecticidal activity on pests such as tick order, flea order, louse order, hemiptera order, diptera order and the like, and the toxicity of the broad-spectrum insecticide is higher than or equal to that of common insecticide. Fluorine Lei Lana mainly acts by interfering chloride ion channels of gamma-aminobutyric acid receptors (GABA receptors for short) and glutamic acid receptors of invertebrate nervous system, is similar to the acting targets of pesticides such as cyclopentadiene, phenylpyrazole, macrolide and the like, has no cross resistance with the existing GABA receptor type pesticides, and is safe to non-target organisms such as mammals, zebra fish, birds and the like.
Main synthesis method of fluororalanaThe intermediate is (4- (hydroxyimino) methyl) -2-methylbenzoic acid with a molecular formula of C 9 H 9 NO 3 Molecular weight is 179.18, and structural formula is
Figure BDA0003372033530000011
For related impurities of the intermediate of the fluorine Lei Lana, the content of the related impurities needs to be controlled in the process of directionally synthesizing the target final product of the fluororanaftate so as to ensure the quality of the final product.
Currently, few manufacturers of fluorine Lei Lana intermediates are available on the market, and no related patent literature report on Guan Fulei Laner intermediates and impurity analysis methods thereof exists. Therefore, the development of the analysis method for detecting the fluorine Lei Lana starting material and the impurity content thereof, which has high sensitivity and good separation degree of various impurities, has extremely important significance for realizing the key intermediate of the fluororalston and the quality control of the fluororalston.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provide a method for detecting a fluororalreceived intermediate and impurities thereof.
The technical scheme adopted by the invention is as follows:
a method for detecting a fluororalfinade intermediate, which is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and impurities thereof, wherein the impurities are at least one selected from the group consisting of 4- ((hydroxyimino) methyl) -2-methylbenzamide, 4- (hydroxymethyl) -2-methylbenzoic acid and 4-cyano-3-methylbenzaldehyde oxime, comprising the following steps:
1) Dissolving the intermediate of fluorine Lei Lana and impurities thereof by using acetonitrile solution to obtain a reference substance solution;
2) Dissolving a fluorine Lei Lana intermediate to-be-detected product by using an acetonitrile solution to prepare a sample solution;
3) Using a reversed-phase high-performance liquid chromatograph to measure fluorine Lei Lana intermediates and impurities thereof in a reference substance solution and a sample solution, wherein the mobile phase of the reversed-phase high-performance liquid chromatograph is a mixed solution of acetonitrile and perchloric acid solution, and the elution procedure is gradient elution;
4) And determining the content of the fluorine Lei Lana intermediate and impurities thereof in the sample solution by using an external standard method according to the chromatograms of the reference solution and the sample solution.
In some examples, the gradient elution and mobile phase acetonitrile in the reverse phase high performance liquid chromatography are in the following order in volume fraction: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
In some examples, the volume fraction of perchloric acid in the perchloric acid solution is from 0.05 to 0.15%.
In some examples, the mobile phase has a flow rate of 0.8 to 1.5mL/min, preferably 0.8 to 1.2mL/min.
In some examples, the column temperature of the reverse phase high performance liquid chromatography is 25-35 ℃; preferably, the column temperature is 30 ℃.
In some examples, the sample loading in the reverse phase high performance liquid chromatography is 8-15 μl.
In some examples, the solvent of the control solution and the sample solution is an aqueous acetonitrile solution with a volume fraction of 60-70%.
In some examples, the reverse phase high performance liquid chromatography column is a C18 column: 4.6 mm. Times.250 mm,5 μm.
In some examples, the detection wavelength of the reverse phase high performance liquid chromatography is 215-225 nm; preferably, the detection wavelength is 220nm.
In some examples, the method comprises the steps of:
1) Dissolving a fluorine Lei Lana intermediate and impurities thereof by using an acetonitrile water solution with the volume fraction of 60-70% to obtain a reference substance solution;
2) Dissolving a fluorine Lei Lana intermediate to-be-detected product by using an acetonitrile water solution with the volume fraction of 60-70% to prepare a sample solution;
3) Using a reversed-phase high-performance liquid chromatograph to measure fluorine Lei Lana intermediates and impurities thereof in the reference substance solution and the sample solution, wherein the mobile phase of the reversed-phase high-performance liquid chromatograph is a mixed solution of acetonitrile and perchloric acid solution with the volume fraction of 0.05-0.15%, and the elution procedure is gradient elution; the volume ratio sequence of the gradient elution and the mobile phase acetonitrile in the reversed phase high performance liquid chromatography is as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% running; the flow rate of the mobile phase is selected to be 0.8-1.2 mL/min; the temperature of the chromatographic column is 25-35 ℃;
4) And determining the content of the fluorine Lei Lana intermediate and impurities thereof in the sample solution by using an external standard method according to the chromatograms of the reference solution and the sample solution.
The beneficial effects of the invention are as follows:
the method adopts the high performance liquid chromatography to detect the intermediate of fluorine Lei Lana and the impurities thereof, can realize the effective separation between the intermediate of fluorine ralrana and the impurities thereof, accurately measure the content of the intermediate of fluorine Lei Lana and the impurities thereof, is not interfered by blank and other impurities, has simple and convenient operation, high speed, repeatability, accuracy and stability, can be used for controlling the quality of the starting materials used in the synthesis process of fluorine Lei Lana, and realizes the control of the preparation process of fluorine Lei Lana.
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FIG. 1 is a chromatogram of the control solution of example 1;
FIG. 2 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in example 1;
FIG. 3 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in example 2;
FIG. 4 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in example 3;
FIG. 5 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in example 4;
FIG. 6 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in example 5;
FIG. 7 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in comparative example 1;
FIG. 8 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in comparative example 2;
FIG. 9 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in comparative example 3;
FIG. 10 is a chromatogram of a sample solution of the intermediate fluorine Lei Lana in comparative example 4.
Detailed Description
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.
Impurity a, impurity B and impurity C in each of the following examples, comparative examples and experiments are 4- ((hydroxyimino) methyl) -2-methylbenzamide, 4- ((hydroxyimino) methyl) -2-methylbenzamide and 4-cyano-3-methylbenzaldehyde oxime, respectively.
Example 1
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.1% perchloric acid solution, and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; and (3) injecting the reference substance solution and the sample solution into a chromatograph for measurement respectively at 36-45 min and 15%.
The test results are shown in Table 1. The chromatograms obtained by detecting the reference substance solution and the sample solution are shown in fig. 1 and fig. 2, respectively.
And (3) comparing the peak area of each substance in the reference substance solution with the peak area of each substance in the sample solution (external standard method), and determining the content of the fluorine Lei Lana intermediate and impurities thereof in the sample.
Table 1, test results of example 1
Component name Retention time (min) Degree of separation Content (%)
Impurity A 11.205 13.31 0.18
Impurity B 14.255 18.35 0.16
Impurity C 26.528 / 0.12
Fluorine Lei Lana intermediate 18.617 34.24 98.48
As can be seen from table 1: the separation degree between any adjacent peaks is far greater than 1.5, which indicates that the fluorine Lei Lana intermediate and impurities are well separated, and a favorable precondition is provided for accurately testing the contents of the fluorine Lei Lana intermediate and the impurities; the content of the fluorine Lei Lana intermediate is 98.48%, the content of the impurity A is 0.18%, the content of the impurity B is 0.16%, the content of the impurity C is 0.12% and the content of each impurity is less than 0.2% by calculation of an external standard method.
Example 2
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.1% perchloric acid solution= (90-20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 0.9mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
Table 2, test results of example 2
Figure BDA0003372033530000051
Under the chromatographic conditions of example 2, the detection results of the sample solutions are shown in Table 2, and the detection chromatograms are shown in FIG. 3. As can be seen from table 2: the separation degree between any adjacent peaks is far greater than 1.5, which indicates that the fluorine Lei Lana intermediate and impurities are well separated, and a favorable precondition is provided for accurately testing the contents of the fluorine Lei Lana intermediate and the impurities; the content of the fluorine Lei Lana intermediate is 98.45%, the content of the impurity A is 0.17%, the content of the impurity B is 0.17%, the content of the impurity C is 0.11%, and the content of each impurity is less than 0.2% by calculation of an external standard method.
Example 3
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.1% perchloric acid solution= (90-20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 215nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
Table 3, test results of example 3
Figure BDA0003372033530000061
Under the chromatographic conditions of example 3, the detection results of the sample solutions are shown in Table 3, and the detection chromatograms are shown in FIG. 4. As can be seen from table 3: the separation degree between any adjacent peaks is far greater than 1.5, which indicates that the fluorine Lei Lana intermediate and impurities are well separated, and a favorable precondition is provided for accurately testing the contents of the fluorine Lei Lana intermediate and the impurities; the content of the fluorine Lei Lana intermediate is 98.42%, the content of the impurity A is 0.16%, the content of the impurity B is 0.15%, the content of the impurity C is 0.14% and the content of each impurity is less than 0.2% by calculation of an external standard method.
Example 4
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.1% perchloric acid= (90 to 20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 25 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
Table 4, test results of example 4
Figure BDA0003372033530000071
Under the chromatographic conditions of example 4, the detection results of the sample solutions are shown in Table 4, and the detection chromatograms are shown in FIG. 5. As can be seen from table 4: the separation degree between any adjacent peaks is far greater than 1.5, which indicates that the fluorine Lei Lana intermediate and impurities are well separated, and a favorable precondition is provided for accurately testing the contents of the fluorine Lei Lana intermediate and the impurities; the content of the intermediate of the fluorine Lei Lana is 98.41%, the content of the impurity A is 0.14%, the content of the impurity B is 0.15%, the content of the impurity C is 0.12% and the content of each impurity is less than 0.2% by calculation of an external standard method.
Example 5
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.05% perchloric acid= (90 to 20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
Table 5, test results of example 5
Figure BDA0003372033530000081
Under the chromatographic conditions of example 5, the detection results of the sample solutions are shown in table 5, the detection chromatograms are shown in fig. 6, and the results are shown in table 4: the separation degree between any adjacent peaks is far greater than 1.5, which indicates that the fluorine Lei Lana intermediate and impurities are well separated, and a favorable precondition is provided for accurately testing the contents of the fluorine Lei Lana intermediate and the impurities; the content of the fluorine Lei Lana intermediate is 98.46%, the content of the impurity A is 0.16%, the content of the impurity B is 0.18%, the content of the impurity C is 0.13% and the content of each impurity is less than 0.2% by calculation of an external standard method.
Comparative example 1
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase methanol: 0.1% perchloric acid solution= (90-20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
As shown in FIG. 7, the detection result of comparative example 1 shows that the baseline noise is large, and impurity C cannot be detected.
Comparative example 2
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.1% formic acid solution= (90-20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min and 15%.
The detection result of comparative example 2 is that, as shown in fig. 8, the baseline fluctuation is large, and impurity a does not appear to be peak.
Comparative example 3
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: 0.01mol/L KH 2 PO 4 Solution= (90-20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
As shown in FIG. 9, the detection result of comparative example 3 shows that the main peak shape is poor, part of impurities cannot be detected, baseline drift is obvious, and baseline separation cannot be achieved.
Comparative example 4
1) Preparing a reference substance solution: accurately weighing a fluororalrana intermediate standard substance, an impurity A, an impurity B and an impurity C, adding a mixed solution of acetonitrile and water (the volume ratio is 6:4, and the acetonitrile water solution is abbreviated as acetonitrile water solution), and dissolving to prepare a solution containing 1mg of each of the fluoro Lei Lana intermediate, 0.1mg of each of the impurity A, the impurity B and the impurity C per mL, and taking the solution as a reference substance solution;
2) Preparing a sample solution: accurately weighing 20mg of fluorine Lei Lana intermediate in a 20mL volumetric flask, adding acetonitrile aqueous solution to dissolve and dilute to scale marks to obtain a sample solution;
3) High performance liquid chromatography assay: the chromatographic test conditions were as follows: c18 chromatography column: 4.6mm by 250mm,5 μm; mobile phase acetonitrile: purified water= (90 to 20): (10-80) (v/v), and performing isocratic elution for 45min; flow rate: 1.0mL/min; column temperature: 30 ℃; ultraviolet detector detection wavelength: 220nm; sample injection amount: 10. Mu.L; the gradient elution program and the volume ratio sequence of the acetonitrile in the mobile phase are as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of operation.
As shown in FIG. 10, the result of comparative example 4 shows that the main peak and part of the impurity peaks have poor peak shape, the impurities cannot be completely separated, the baseline drift is obvious, and the standard deviation is not in accordance with the regulations.
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 (12)

1. A method for detecting a fluororalfinade intermediate, which is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and impurities thereof, wherein the impurities are at least one selected from the group consisting of 4- ((hydroxyimino) methyl) -2-methylbenzamide, 4- (hydroxymethyl) -2-methylbenzoic acid and 4-cyano-3-methylbenzaldehyde oxime, and the method is characterized in that: the method comprises the following steps:
1) Dissolving the intermediate of fluorine Lei Lana and impurities thereof by using acetonitrile solution to obtain a reference substance solution;
2) Dissolving a fluorine Lei Lana intermediate to-be-detected product by using an acetonitrile solution to prepare a sample solution;
3) Using a reversed-phase high-performance liquid chromatograph to measure fluorine Lei Lana intermediates and impurities thereof in a reference substance solution and a sample solution, wherein the mobile phase of the reversed-phase high-performance liquid chromatograph is a mixed solution of acetonitrile and perchloric acid solution, and the elution procedure is gradient elution; the volume ratio sequence of the gradient elution and the mobile phase acetonitrile in the reversed phase high performance liquid chromatography is as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% of the operation, wherein the chromatographic column of the reversed-phase high-performance liquid chromatography is a C18 chromatographic column;
4) And determining the content of the fluorine Lei Lana intermediate and impurities thereof in the sample solution by using an external standard method according to the chromatograms of the reference solution and the sample solution.
2. The method of claim 1, wherein: in the perchloric acid solution, the volume fraction of perchloric acid is 0.05-0.15%.
3. The method of claim 1, wherein: the flow rate of the mobile phase is 0.8-1.5 mL/min.
4. A detection method according to claim 3, wherein: the flow rate of the mobile phase is 0.8-1.2 mL/min.
5. The method of claim 1, wherein: the chromatographic column temperature of the reversed-phase high-performance liquid chromatography is 25-35 ℃.
6. The method of claim 5, wherein: the column temperature was 30 ℃.
7. The method of claim 1, wherein: the sample injection amount in the reversed phase high performance liquid chromatography is 8-15 mu L.
8. The detection method according to any one of claims 1 to 7, wherein: the solvent of the reference substance solution and the sample solution is acetonitrile water solution with the volume fraction of 60-70%.
9. The detection method according to any one of claims 1 to 7, wherein: the chromatographic column of the reversed-phase high-performance liquid chromatography is a C18 chromatographic column: 4.6mm×250mm,5 μm.
10. The detection method according to any one of claims 1 to 7, wherein: the detection wavelength of the reversed-phase high-performance liquid chromatography is 215-225 nm.
11. The method of claim 10, wherein: the detection wavelength was 220nm.
12. The method of claim 1, wherein: the method comprises the following steps:
1) Dissolving a fluorine Lei Lana intermediate and impurities thereof by using an acetonitrile water solution with the volume fraction of 60-70% to obtain a reference substance solution;
2) Dissolving a fluorine Lei Lana intermediate to-be-detected product by using an acetonitrile water solution with the volume fraction of 60-70% to prepare a sample solution;
3) Using a reversed-phase high-performance liquid chromatograph to measure fluorine Lei Lana intermediates and impurities thereof in the reference substance solution and the sample solution, wherein the mobile phase of the reversed-phase high-performance liquid chromatograph is a mixed solution of acetonitrile and perchloric acid solution with the volume fraction of 0.05-0.15%, and the elution procedure is gradient elution; the volume ratio sequence of the gradient elution and the mobile phase acetonitrile in the reversed phase high performance liquid chromatography is as follows: running for 0-30 min and 15-60%; 30-35 min, 60-90% of the operation; 36-45 min,15% running; the flow rate of the mobile phase is 0.8-1.2 mL/min; the temperature of the chromatographic column is 25-35 ℃;
4) And determining the content of the fluorine Lei Lana intermediate and impurities thereof in the sample solution by using an external standard method according to the chromatograms of the reference solution and the sample solution.
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