CN114137111B - Reversed-phase high performance liquid chromatography analysis method for fluororalrana intermediate oxime acid - Google Patents
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Abstract
The invention belongs to the field of analysis, and discloses a reversed-phase high-performance liquid chromatography analysis method of a fluororalrana intermediate oxime acid. The high-efficiency liquid phase analysis method of the fluoro Lei Lana intermediate oxime acid provided by the invention has the advantages of high detection sensitivity, high stability, high reliability, high theoretical plate number and relatively simple operation, can effectively measure the content of the fluororalrana intermediate oxime acid material oxime acid, and is beneficial to ensuring the quality of the fluoro Lei Lana intermediate oxime acid bulk drug and preparation.
Description
Technical Field
The invention belongs to the technical field of medicine analysis and detection, and particularly relates to a reversed-phase high performance liquid chromatography analysis method of a fluororalreceived intermediate oxime acid.
Background
The fluorine Lei Lana is chemically named as 4- [5- (3, 5-dichlorophenyl) ] -4, 5-dihydro-5-trifluoromethyl-3-isoxazolyl ] -2-methyl-N- [ 2-oxo-2- [ 2.2.2-trifluoroethyl) amino ] ] benzamide, is a novel broad-spectrum pesticide, is an effective inhibitor of ligand-gated chloride ion channels in neurons, and has remarkable selectivity on mammalian neurons and arthropod neurons. The successful research and development of the fluorine Lei Lana creates a brand-new research direction of GABA-gated chloride ion channel disruptors, is similar to the action targets of pesticides such as cyclopentadiene, phenylpyrazole, macrolides and the like, has good insecticidal activity on pests such as ticks, fleas, lice, hemiptera, diptera and the like, has higher toxicity than or equal to that of common pesticides, has obvious differences in molecular structure, action sites, selectivity, interaction resistance and the like compared with other similar pesticides, and has been applied to clinical treatment of canine and feline ectoparasitic diseases in recent years.
The chemical name of the fluoro Lei Lana intermediate oxime acid is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, CAS number: 1437051-93-8 molecular formula C 9 H 9 NO 3 The molecular weight is 179.17.
At present, related patent documents of the compound and the impurity detection method thereof are not reported, and impurities generated in the production process of the compound are gradually discovered along with the intensive research of the product in China, so that in order to strengthen the quality control of the fluorine Lei Lana intermediate oxime acid, the intermediate and the impurities, a sensitive, stable and reliable detection method is necessary to be provided.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a reversed-phase high performance liquid chromatography analysis method of a fluororalreceived intermediate oxime acid.
The technical scheme adopted by the invention is as follows:
a reverse phase high performance liquid chromatography analysis method of a fluororalfina intermediate oxime acid, wherein the fluoro Lei Lana intermediate oxime acid is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the method comprises the following steps:
preparing a standard substance solution: dissolving the fluoro Lei Lana intermediate oxime acid standard with an aqueous acetonitrile solution;
preparing a sample solution to be tested: dissolving a fluorine Lei Lana intermediate oxime acid sample to be tested by using an acetonitrile aqueous solution;
determining chromatograms of a standard substance solution and a sample to be detected by using reverse-phase high performance liquid chromatography, wherein the conditions of the reverse-phase high performance liquid chromatography are as follows: the mobile phase consists of a mobile phase A and a mobile phase B, wherein acetonitrile is the mobile phase A, a phosphoric acid solution is the mobile phase B, the mass concentration of phosphoric acid is 0.05% -0.15%, and the mobile phase A is as follows: carrying out gradient elution on the mobile phase B;
and calculating the content of the sample to be measured according to chromatograms of the standard substance and the sample to be measured.
In some examples, the aqueous acetonitrile solution has a volume concentration of 40% to 80%.
In some examples, the mobile phase in the reverse phase high performance liquid chromatography has a flow rate of 0.6 to 1mL/min.
In some examples, the column temperature of the reverse phase high performance liquid chromatography is 20 ℃ to 35 ℃.
In some examples, the reverse phase high performance liquid chromatography column is a C18 column: 4.6X1250 mm,5 μm.
In some examples, the phosphoric acid solution has a mass concentration of 0.1%.
In some examples, the detection wavelength of the reverse phase high performance liquid chromatography is 200nm to 210nm.
In some examples, the sample injection amount of the reverse phase high performance liquid chromatography is 10-20 μl.
In some examples, the concentration of the fluoro Lei Lana intermediate oxime is from 0.3 to 1mg/mL.
In some examples, the gradient elution time and the volume ratio of acetonitrile in the mobile phase are in the order:
0-30 min, 10-20 v/v% running; preferably 20v/v%;
30-31 min, 90-95 v/v% running; preferably 95v/v%;
31-40 min, 10-20 v/v% running; preferably 20v/v%.
In some examples, the high performance liquid chromatography method comprises the steps of:
preparing a standard substance solution: dissolving the fluoro Lei Lana intermediate oxime acid standard with a 60v/v% acetonitrile in water;
preparing a sample solution to be tested: dissolving a fluorine Lei Lana intermediate oxime acid sample to be detected by using a 60v/v% acetonitrile aqueous solution, wherein the concentration of the sample to be detected is 0.4mg/mL;
determining chromatograms of a standard substance solution and a sample to be detected by using reverse-phase high performance liquid chromatography, wherein the conditions of the reverse-phase high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column: 4.6X1250 mm,5 μm, column temperature: 25 ℃, sample injection amount: 15 μl, detection wavelength 205nm, mobile phase composed of mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, mobile phase B is 0.1wt% phosphoric acid solution, mobile phase A: mobile phase B was gradient eluted, flow rate: the volume ratio of acetonitrile in the gradient elution time and the mobile phase is 0.8mL/min in sequence:
0-30 min,20 v/v% running;
30-31 min,95 v/v% running;
31-40 min,20 v/v% running;
and calculating the content of the sample to be measured according to chromatograms of the standard substance and the sample to be measured.
The beneficial effects of the invention are as follows:
the high-efficiency liquid phase analysis method of the fluoro Lei Lana intermediate oxime acid provided by the invention has the advantages of high detection sensitivity, high stability, high reliability, high theoretical plate number and relatively simple operation, can effectively measure the content of the fluororalrana intermediate oxime acid material oxime acid, and is beneficial to ensuring the quality of the fluoro Lei Lana intermediate oxime acid bulk drug and preparation.
Drawings
FIG. 1 is a chromatogram of a standard solution of the intermediate oxime acid of Flirana in example 1;
FIG. 2 is a chromatogram of a standard solution of fluoro Lei Lana intermediate oxime acid in example 2;
FIG. 3 is a linear relationship diagram of example 4;
FIG. 4 is a chromatogram of a standard solution of the fluororalreceived intermediate oxime acid in comparative example 1;
FIG. 5 is a chromatogram of a standard solution of fluoro Lei Lana intermediate oxime acid in comparative example 2;
FIG. 6 is a chromatogram of a standard solution of fluoro Lei Lana intermediate oxime acid in comparative example 3;
FIG. 7 is a chromatogram of a standard solution of the intermediate oxime acid of Flirana in comparative example 4.
Detailed Description
A reverse phase high performance liquid chromatography analysis method of a fluororalfina intermediate oxime acid, wherein the fluoro Lei Lana intermediate oxime acid is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the method comprises the following steps:
preparing a standard substance solution: dissolving the fluoro Lei Lana intermediate oxime acid standard with an aqueous acetonitrile solution;
preparing a sample solution to be tested: dissolving a fluorine Lei Lana intermediate oxime acid sample to be tested by using an acetonitrile aqueous solution;
determining chromatograms of a standard substance solution and a sample to be detected by using reverse-phase high performance liquid chromatography, wherein the conditions of the reverse-phase high performance liquid chromatography are as follows: the mobile phase consists of a mobile phase A and a mobile phase B, wherein acetonitrile is the mobile phase A, a phosphoric acid solution is the mobile phase B, the mass concentration of phosphoric acid is 0.05% -0.15%, and the mobile phase A is as follows: carrying out gradient elution on the mobile phase B;
and calculating the content of the sample to be measured according to chromatograms of the standard substance and the sample to be measured.
In some examples, the aqueous acetonitrile solution has a volume concentration of 40% to 80%. The data show that the acetonitrile aqueous solution with the concentration has good dissolving capacity on the sample and does not have adverse effect on the subsequent liquid chromatography.
In some examples, the mobile phase in the reverse phase high performance liquid chromatography has a flow rate of 0.6 to 1mL/min. The elution can be better performed at this flow rate, with more accurate results.
In some examples, the mobile phase in the reverse phase high performance liquid chromatography has a flow rate of 0.8mL/min. The data indicate better results at this flow rate.
In some examples, the column temperature of the reverse phase high performance liquid chromatography is 20 ℃ to 35 ℃. In this range, a relatively accurate detection result can be obtained. In particular, the control of the column temperature at 25℃is more advantageous in obtaining accurate results.
In some examples, the reverse phase high performance liquid chromatography column is a C18 column: 4.6X1250 mm,5 μm.
In some examples, the phosphoric acid solution has a mass concentration of 0.1%.
In some examples, the detection wavelength of the reverse phase high performance liquid chromatography is 200nm to 210nm.
In some examples, the sample injection amount of the reverse phase high performance liquid chromatography is 10-20 μl.
In some examples, the concentration of the fluoro Lei Lana intermediate oxime is from 0.3 to 1mg/mL.
In some examples, the gradient elution time and the volume ratio of acetonitrile in the mobile phase are in the order:
0-30 min, 10-20 v/v% running; preferably 20v/v%;
30-31 min, 90-95 v/v% running; preferably 95v/v%;
31-40 min, 10-20 v/v% running; preferably 20v/v%.
In some examples, the high performance liquid chromatography method comprises the steps of:
preparing a standard substance solution: dissolving the fluoro Lei Lana intermediate oxime acid standard with a 60v/v% acetonitrile in water;
preparing a sample solution to be tested: dissolving a fluorine Lei Lana intermediate oxime acid sample to be detected by using a 60v/v% acetonitrile aqueous solution, wherein the concentration of the sample to be detected is 0.4mg/mL;
determining chromatograms of a standard substance solution and a sample to be detected by using reverse-phase high performance liquid chromatography, wherein the conditions of the reverse-phase high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column: 4.6X1250 mm,5 μm, column temperature: 25 ℃, sample injection amount: 15 mu L, the detection wavelength is 200 nm-210 nm, the mobile phase is composed of a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile, the mobile phase B is 0.1wt% phosphoric acid solution, and the mobile phase A is: mobile phase B was gradient eluted, flow rate: the volume ratio of acetonitrile in the gradient elution time and the mobile phase is 0.8mL/min in sequence:
0-30 min,20 v/v% running;
30-31 min,95 v/v% running;
31-40 min,20 v/v% running;
and calculating the content of the sample to be measured according to chromatograms of the standard substance and the sample to be measured.
The present invention will be described in more detail by way of examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, since various modifications and adaptations may be made by those skilled in the art in light of the teachings herein. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a selection within the suitable ranges by the description herein and are not intended to be limited to the specific data described below.
Among the reagents used in the following examples, comparative examples and experiments, acetonitrile was of foreign import HPLC grade, the brand was Fisher, and water was purified water to reduce baseline noise, and a high performance liquid chromatograph was Agilent 1260-II.
Example 1: reverse phase high performance liquid chromatography analysis method of fluorine Lei Lana intermediate oxime acid:
preparation of standard solution: about 10mg of the fluororalrana intermediate oxime acid is weighed, precisely weighed in a 25mL volumetric flask, added with a proper amount of acetonitrile water solution with volume concentration of 60% to be dissolved and diluted to a scale, and uniformly mixed to obtain the fluoro Lei Lana intermediate oxime acid with concentration of 0.4mg/mL.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% phosphoric acid solution;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
the detection chromatogram of the standard solution under the chromatographic conditions of example 1 is shown in figure 1.
As can be seen from FIG. 1, the retention time of the oxime acid of the intermediate oxime acid of the fluorine Lei Lana is 12.56min, the peak shape is good, and the peak purity is high (more than or equal to 98.0%).
Example 2: system adaptability test for measuring content of oxime acid in fluorine Lei Lana intermediate
Preparation of standard solution: weighing 10.2mg of fluororalrana intermediate oxime acid, precisely weighing, adding a proper amount of acetonitrile aqueous solution with volume concentration of 60% into a 25mL volumetric flask, dissolving and diluting to a scale, and uniformly mixing to obtain the fluoro Lei Lana intermediate oxime acid with concentration of 0.408mg/mL.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% phosphoric acid solution;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
sample solutions were taken and repeated 6 times, and the test results are shown in table 1:
TABLE 1 System suitability test results
As can be seen from Table 1, the same sample solution of fluoro Lei Lana intermediate oxime acid is repeated 6 times, the peak area RSD of fluororalfina intermediate oxime acid is 0.12%, and meets the rule that RSD is less than or equal to 2.0%, which shows that the method disclosed by the invention has good system adaptability and high test reliability.
Example 3
Repetition investigation of fluoro Lei Lana intermediate oxime acid
(1) Preparing a standard reference substance: 10mg of the fluororalrana intermediate oxime acid is weighed, precisely weighed and put into a 25mL volumetric flask, added with a diluent to be dissolved and diluted to a scale, and uniformly mixed to be used as a standard reference substance solution.
(2) Preparing a test article: weighing 6 parts of a fluororalrana intermediate oxime acid bulk drug sample: (10.2 mg,10.3mg,10.1mg,10.33mg,10.21mg,10.36 mg), each precisely weighed into a 25mL volumetric flask, dissolved and diluted to a scale with an aqueous solution of 60% acetonitrile by volume, and uniformly mixed to obtain a fluorine Lei Lana intermediate oxime acid solution with different mass concentrations, which respectively correspond to standard substance solutions No. 1-6 in sequence: 0.408mg/mL,0.412mg/mL,0.404mg/mL,0.413mg/mL,0.408mg/mL,0.414mg/mL.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% phosphoric acid solution;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
taking standard substance solutions with different mass concentrations of 1 to 6, respectively injecting samples, and measuring the content of each standard substance solution as shown in table 2:
TABLE 2 repeatability test results
As can be seen from Table 2, the average content of the fluororalrana intermediate oxime acid in the sample is 98.39%, the content of the RSD% of the fluoro Lei Lana intermediate oxime acid is 0.26%, and the method meets the rule that the content is 98.0% -102.0%, and the RSD% is not more than 2.0%, which shows that the method of the invention has good reproducibility.
Example 4
Investigation of the Linear relationship of the fluoro Lei Lana intermediate oxime acid
Preparation of standard solution: weighing 5 parts of the fluororalston intermediate oxime acid sample according to the volume concentration of the sample of 20%,50%,80%,100%,120% and five concentrations respectively: 21.48mg,54.39mg,86.68mg,108.72mg and 130.25mg are precisely weighed into a 100mL volumetric flask, a proper amount of acetonitrile water solution with volume concentration of 60% is added and diluted to a scale, and uniformly mixed to obtain fluorine Lei Lana intermediate oxime acid solutions with different mass concentrations, wherein the fluorine Lei Lana intermediate oxime acid solutions respectively correspond to standard substance solutions with serial numbers of 7-11 in sequence: 0.2148mg/mL,0.5439mg/mL,0.8668mg/mL,1.0872mg/mL,1.3025mg/mL.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% phosphoric acid water;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
sample solutions of No. 7-11 standard substances with different mass concentrations are respectively sampled, and the peak areas and standard concentrations of the solutions of the standard substances are shown in Table 3:
TABLE 3 results of Linear experiments
| Numbering device | Peak area | Standard concentration (mg/ml) |
| 7 | 4292.434 | 0.0804 |
| 8 | 10564.055 | 0.2010 |
| 9 | 16569.269 | 0.3216 |
| 10 | 20499.789 | 0.4020 |
| 11 | 24217.088 | 0.4824 |
As can be seen from table 3, linear regression was performed by the least square method with the concentration of the intermediate oxime acid of fluoro Lei Lana on the abscissa and the peak area on the ordinate. The fluorine Lei Lana intermediate oxime acid has a linear regression equation of y=49671x+426.86 and a linear regression coefficient r within the range of 0.0804-0.4824 mg/mL sample injection concentration 2 0.9996, in accordance with the rule r 2 And not less than 0.998, the method of the invention has good linearity, and the linear relation diagram is shown in figure 3.
In comparative examples 1 to 5, the standard solutions were identical to the standard solutions in example 1.
Comparative example 1
The detection procedure of comparative example 1 was the same as in example 1, except that the chromatographic assay conditions were:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% phosphoric acid solution;
flow rate: 1.2mL/min;
column temperature: 25 ℃;
sample injection amount: 15 mu L
Detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
the test results of comparative example 1 were: as shown in fig. 4, the separation degree of the adjacent peaks is less than 1.5, so that the intermediate oxime acid of fluorine Lei Lana and impurities cannot be effectively separated, and the content of the intermediate oxime acid of fluorine Lei Lana cannot be accurately detected.
Comparative example 2
The detection procedure of comparative example 2 was the same as in example 1, except that the chromatographic assay conditions were:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B: water;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
the test results of comparative example 2 were: as shown in FIG. 5, the main peak and part of impurity peaks have poor peak shape, the baseline drift is obvious, and the content of the oxime acid in the intermediate of fluorine Lei Lana cannot be accurately detected.
Comparative example 3
The detection procedure of comparative example 3 was the same as in example 1, except that the chromatographic assay conditions were:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% formic acid;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
the test results of comparative example 3 were: as shown in fig. 6, the main peak and part of impurity peaks have poor peak shape, the baseline drift is obvious, the main peak is split, and the content of the oxime acid in the intermediate of fluorine Lei Lana cannot be accurately detected.
Comparative example 4
The detection procedure of comparative example 4 was the same as in example 1, except that the chromatographic assay conditions were:
chromatographic column: c18, specification 4.6X1250 mm,5 μm;
mobile phase: a: acetonitrile, B:0.1% triethylamine;
flow rate: 0.8mL/min;
column temperature: 25 ℃;
sample injection amount: 15. Mu.L;
detection wavelength: 205nm;
gradient elution procedure:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
the test results of comparative example 4 were: as shown in fig. 7, the baseline drift was significant, and the main peak and impurity peak of the fluororalreceived intermediate oxime acid were not detected, and the content of the fluoro Lei Lana intermediate oxime acid could not be accurately detected.
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 (6)
1. The reverse-phase high performance liquid chromatography analysis method of the fluororalfina intermediate oxime acid is characterized in that the fluoro Lei Lana intermediate oxime acid is (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and is characterized in that: the method comprises the following steps:
preparing a standard substance solution: dissolving a fluorine Lei Lana intermediate oxime acid standard by using an acetonitrile aqueous solution;
preparing a sample solution to be tested: dissolving a fluorine Lei Lana intermediate oxime acid sample to be tested by using an acetonitrile aqueous solution;
determining chromatograms of a standard substance solution and a sample solution to be detected by using a reversed-phase high performance liquid chromatography, wherein a chromatographic column of the reversed-phase high performance liquid chromatography is a C18 chromatographic column: 4.6X1250 mm,5 μm, the conditions of the reverse phase high performance liquid chromatography are: the mobile phase consists of a mobile phase A and a mobile phase B, wherein acetonitrile is the mobile phase A, a phosphoric acid solution is the mobile phase B, the mass concentration of phosphoric acid is 0.05% -0.15%, the flow rate of the mobile phase in the reversed-phase high-performance liquid chromatography is 0.6-1 mL/min, and the mobile phase A: the mobile phase B is subjected to gradient elution, and the gradient elution program is as follows:
when the time is 0.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 30.0min, the volume fraction of A in the mobile phase is 95%, and the volume fraction of B is 5%;
when the time is 31.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
when the time is 40.0min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;
and calculating the content of the sample to be detected according to chromatograms of the standard substance solution and the sample to be detected.
2. The reverse-phase high performance liquid chromatography method according to claim 1, wherein: the volume concentration of the acetonitrile aqueous solution is 40-80%.
3. The reverse-phase high performance liquid chromatography method according to claim 1, wherein: the chromatographic column temperature of the reversed-phase high-performance liquid chromatography is 20-35 ℃.
4. The reverse-phase high performance liquid chromatography method according to claim 1, wherein: in the phosphoric acid solution, the mass concentration of phosphoric acid is 0.1%.
5. The reverse-phase high performance liquid chromatography method according to any one of claims 1 to 4, wherein: the detection wavelength of the reversed-phase high-performance liquid chromatography is 200 nm-210 nm.
6. The reverse-phase high performance liquid chromatography method according to any one of claims 1 to 4, wherein: the sample injection amount of the reversed-phase high-performance liquid chromatography is 10-20 mu L.
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