CN114280185B - Florarana intermediate and detection method of isomer thereof - Google Patents
Florarana intermediate and detection method of isomer thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting a fluororalreceived intermediate and an isomer thereof. In some examples of the invention, detection conditions such as a chromatographic column and a corresponding mobile phase, flow rate, column temperature and the like are screened to obtain a separation detection method applicable to a fluorine Lei Lana intermediate and a corresponding E/Z isomer thereof, wherein the method adopts Ultimate XB-C8,4.6mm multiplied by 250mm and 5 mu m, and the chromatographic column is a separation chromatographic column; acetonitrile and trifluoroacetic acid (c=0.03%v/v) mixed solution is used as a mobile phase; the intermediate of fluorine Lei Lana and the corresponding E/Z isomer impurity thereof can be effectively separated and detected.
Description
Technical Field
The invention belongs to the field of medicine analysis, and relates to a method for detecting a fluororalreceived intermediate and an isomer thereof.
Background
The successful research and development of fluorine Lei Lana opens up a new research direction of GABA-gated chloride ion channel disruptors, and draws attention and favor of animal medicine and pesticide scientists.
Fluorine Lei Lana acts mainly by interfering with GABA-gated chloride channels, similar to the target of action of pesticides such as cyclopentadienes, phenylpyrazoles, and macrolides. Fluorine Lei Lana is a broad-spectrum insecticide, has good insecticidal activity on pests of the order tick, the order flea, the order lice chemical book, the order hemiptera, the order diptera and the like, and has higher toxicity or equal toxicity to common insecticides. The fluorine Lei Lana has no obvious cross resistance with the existing pesticide, and has better insecticidal activity even on partial resistant pests.
The benzaldehyde oxime structure of the fluororalfinade intermediate-5 (E type (4- (hydroxyimino) methyl) -2-methylbenzoic acid) enables the fluororalfinade intermediate to exist in an isomer with Z configuration, the structures of the fluororalfinade intermediate-5 are highly similar, and separation detection analysis is difficult. The structural isomer of E/Z configuration of the fluororalston intermediate-5 has the following structural formula:
at present, related patent documents of a fluorine Lei Lana intermediate and an isomer analysis method thereof are not reported, and with the intensive research of the product in China, in order to enhance the quality control of the fluorine Lei Lana intermediate, a sensitive and stable detection method is necessary to be provided.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting a fluororalreceived intermediate and an isomer thereof.
The technical scheme adopted by the invention is as follows:
a method for detecting a fluororalfinade intermediate and an isomer thereof, wherein the fluoro Lei Lana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of fluoro Lei Lana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, which comprises the following steps:
s1) preparing a standard solution and a test solution of E/Z configuration isomer of a fluorine Lei Lana intermediate;
s2) performing gradient elution detection on a standard solution and a sample solution by using high performance liquid chromatography to obtain chromatograms of the standard solution and the sample solution, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is aqueous solution of trifluoroacetic acid, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is Ultimate XB-C8 chromatographic column;
s3) determining the retention time of each component in the fluorine Lei Lana intermediate sample solution according to chromatograms of the standard sample solution and the sample solution.
In some examples, the gradient elution and mobile phase acetonitrile in the high performance liquid chromatography are in the following order in volume fraction: 0 min-30 min,25% -80% of running; 30 min-40 min,80% -20% of the operation.
In some examples, the aqueous solution of trifluoroacetic acid is a 0.03% (v/v) solution of trifluoroacetic acid.
In some examples, acetonitrile is used: water=80: 20 Preparing a standard solution and a test solution of E/Z configuration isomer of the intermediate of the fluorine Lei Lana by the mixed solution of (v/v).
In some examples, the flow rate is 0.7mL/min.
In some examples, the column temperature of the high performance liquid chromatography is 30-40 ℃.
In some examples, the column temperature is 35 ℃.
In some examples, the high performance liquid chromatography sample loading is 8-15 μl.
In some examples, the high performance liquid chromatography is performed at a sample size of 10 μl.
In some examples, the detector of the high performance liquid chromatograph is an ultraviolet detector with a detection wavelength of 205nm.
In some examples, the conditions of the high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is 0.03% (v/v) trifluoroacetic acid solution, and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0 min-30 min,25% -80% of running; 30-40 min, 80-20% running, flow rate of 0.7mL/min, chromatographic column temperature of 35 ℃, sample injection amount of 10 mu L, and chromatographic column of Ultimate XB-C8: 4.6 mm. Times.250 mm,5 μm.
The beneficial effects of the invention are as follows:
in some examples of the invention, detection conditions such as a chromatographic column and a corresponding mobile phase, flow rate, column temperature and the like are screened to obtain a separation detection method applicable to a fluorine Lei Lana intermediate and a corresponding E/Z isomer thereof, wherein the method adopts Ultimate XB-C8,4.6mm multiplied by 250mm and 5 mu m, and the chromatographic column is a separation chromatographic column; acetonitrile and trifluoroacetic acid (c=0.03%v/v) mixed solution is used as a mobile phase; the intermediate of fluorine Lei Lana and the corresponding E/Z isomer impurity thereof can be effectively separated and detected.
Drawings
FIG. 1 is a chromatogram of a hollow white solution of example 1;
FIG. 2 is a chromatogram of a standard mixed solution in example 1;
FIG. 3 is a chromatogram of a standard solution in example 2;
FIG. 4 is a chromatogram of a standard solution in example 3;
FIG. 5 is a chromatogram of a standard mixed solution in a comparative example.
Detailed Description
A method for detecting a fluororalfinade intermediate and an isomer thereof, wherein the fluoro Lei Lana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of fluoro Lei Lana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, which comprises the following steps:
s1) preparing a standard solution and a test solution of E/Z configuration isomer of a fluorine Lei Lana intermediate;
s2) performing gradient elution detection on a standard solution and a sample solution by using high performance liquid chromatography to obtain chromatograms of the standard solution and the sample solution, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is aqueous solution of trifluoroacetic acid, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is Ultimate XB-C8 chromatographic column;
s3) determining the retention time of each component in the fluorine Lei Lana intermediate sample solution according to chromatograms of the standard sample solution and the sample solution.
In some examples, the gradient elution and mobile phase acetonitrile in the high performance liquid chromatography are in the following order in volume fraction: 0 min-30 min,25% -80% of running; 30 min-40 min,80% -20% of the operation.
In some examples, the aqueous solution of trifluoroacetic acid is a 0.03% (v/v) solution of trifluoroacetic acid. Studies have shown that aqueous solutions of trifluoroacetic acid at this concentration can achieve better resolution.
In some examples, acetonitrile is used: water=80: 20 Preparing a standard solution and a test solution of E/Z configuration isomer of the intermediate of the fluorine Lei Lana by the mixed solution of (v/v). The experimental result shows that the mixed solution with the ratio can obtain better separation degree and is beneficial to the detection of E/Z configuration isomers.
In some examples, the flow rate is 0.7mL/min.
In some examples, the column temperature of the high performance liquid chromatography is 30-40 ℃.
In some examples, the column temperature is 35 ℃.
In some examples, the high performance liquid chromatography sample loading is 8-15 μl.
In some examples, the high performance liquid chromatography is performed at a sample size of 10 μl.
In some examples, the detector of the high performance liquid chromatograph is an ultraviolet detector with a detection wavelength of 205nm.
In some examples, the conditions of the high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is 0.03% (v/v) trifluoroacetic acid solution, and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0 min-30 min,25% -80% of running; 30-40 min, 80-20% running, flow rate of 0.7mL/min, chromatographic column temperature of 35 ℃, sample injection amount of 10 mu L, and chromatographic column of Ultimate XB-C8: 4.6 mm. Times.250 mm,5 μm.
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.
In order to better understand the technical solution of the present invention, the following non-limiting examples are further disclosed for further details of the present invention.
The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.
In the present invention, g represents g, mL represents milliliter, nm represents nanometer, μm represents micrometer, mm represents millimeter, and min represents minute.
Example 1
Detection conditions:
instrument: the detection wavelength of the Siemens flight high performance liquid chromatograph, siemens flight DAD ultraviolet detector: 205nm;
chromatographic column: ultimate XB-C8, 4.6mm.times.250 mm,5 μm
A diluent: acetonitrile: water=80: 20 (v/v)
Mobile phase: a: acetonitrile; b:0.03% trifluoroacetic acid aqueous solution (0.3 mL of trifluoroacetic acid in 1000mL of water, mixing, suction filtration, ultrasonic wave)
Flow rate: 0.7mL/min;
column temperature: 35 ℃;
sample injection amount: 10. Mu.L;
elution time: for 40min;
the detection step comprises:
preparing a mixed standard solution: accurately weighing 5mg of the fluororalston intermediate-5 and 5mg of the fluororalston intermediate-5 isomer, putting into a 20mL volumetric flask, adding a diluent (acetonitrile: water=80:20 (v/v)) to dissolve and dilute to scale marks, and taking the mixed standard solution;
and (3) respectively taking a blank solution (diluent) and a mixed standard solution, performing detection analysis according to the conditions, and recording a chromatogram, wherein the results are shown in fig. 1 and 2. The chromatographic peak remaining for 12.41 minutes in FIG. 2 is that of the fluororalreceived intermediate-5; the 11.68 min chromatographic peak was the peak of the isomer of the fluororalreceived intermediate-5 with a resolution of 3.2.
Example 2
Detection conditions:
instrument: the detection wavelength of the Siemens flight high performance liquid chromatograph, siemens flight DAD ultraviolet detector: 205nm;
chromatographic column: ultimate XB-C8, 4.6mm.times.250 mm,5 μm
A diluent: acetonitrile: water=80: 20 (v/v)
Mobile phase: a: acetonitrile; b:0.03% trifluoroacetic acid aqueous solution (0.3 ml of trifluoroacetic acid in 1000ml of water, mixing, suction filtration, ultrasonic treatment)
Flow rate: 0.7mL/min;
column temperature: 35 ℃;
sample injection amount: 10. Mu.L;
elution time: for 40min;
the detection step comprises:
preparing a standard solution: weighing about 5mg of fluorine Lei Lana intermediate-5 sample in a 20mL volumetric flask, adding a diluent to dissolve and dilute to a scale mark to serve as a standard solution;
taking standard solution, performing high performance liquid chromatography according to detection conditions, and recording a chromatogram, wherein the result is shown in figure 3. FIG. 3 shows that the optical purity of the fluororalrana intermediate-5 is higher, the detection requirement of the intermediate is met, and the method can be used for the quality detection of the fluoro Lei Lana intermediate-5.
Example 3
Detection conditions:
instrument: the detection wavelength of the Siemens flight high performance liquid chromatograph, siemens flight DAD ultraviolet detector: 205nm;
chromatographic column: ultimate XB-C8, 4.6mm.times.250 mm,5 μm
A diluent: acetonitrile: water=80: 20 (v/v)
Mobile phase: a: acetonitrile; b:0.03% trifluoroacetic acid aqueous solution (0.3 mL of trifluoroacetic acid in 1000mL of water, mixing, suction filtration, ultrasonic wave)
Flow rate: 0.7mL/min;
column temperature: 35 ℃;
sample injection amount: 10. Mu.L;
elution time: for 40min;
the detection step comprises:
preparing a standard solution: weighing 5mg of the fluororalrana intermediate-5 isomer in a 20mL volumetric flask, adding a diluent to dissolve and dilute to a scale mark to serve as a standard solution;
taking standard solution, performing high performance liquid chromatography according to detection conditions, and recording a chromatogram, wherein the result is shown in fig. 4.
Comparative example 1
Detection conditions:
instrument: the detection wavelength of the Siemens flight high performance liquid chromatograph, siemens flight DAD ultraviolet detector: 205nm;
chromatographic column: ultimate XB-C8, 4.6mm.times.250 mm,5 μm
A diluent: acetonitrile: water = 60:40 (v/v)
Mobile phase: a: acetonitrile; b:0.03% trifluoroacetic acid aqueous solution (0.3 mL of trifluoroacetic acid in 1000mL of water, mixing, suction filtration, ultrasonic wave)
Flow rate: 0.6mL/min;
column temperature: 20 ℃;
sample injection amount: 10. Mu.L;
elution time: for 40min;
the detection step comprises:
preparing a mixed standard solution: accurately weighing 5mg of the fluororalston intermediate-5 and 5mg of the fluororalston intermediate-5 isomer, putting into a 20mL volumetric flask, adding a diluent (acetonitrile: water=60:40 (v/v)) to dissolve and dilute to scale marks, and taking the mixed standard solution;
and (3) respectively taking a blank solution (diluent) and a mixed standard solution, performing detection analysis according to the conditions, and recording a chromatogram.
The results are shown in FIG. 5. The chromatographic peak with a retention time of 13.86 minutes in FIG. 5 is a mixed chromatographic peak of the intermediate-5 of Florarana and the intermediate-5 isomer of F Lei Lana, which can be seen to be indistinguishable from each other.
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 (8)
1. A method for detecting a fluororalfinade intermediate and an isomer thereof, wherein the fluoro Lei Lana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of fluoro Lei Lana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, which is characterized in that: the method comprises the following steps:
acetonitrile was used: water=80: 20 to prepare a standard substance solution and a test substance solution of E/Z configuration isomer of the fluorine Lei Lana intermediate;
performing gradient elution detection on a standard solution and a sample solution by using high performance liquid chromatography to obtain chromatograms of the standard solution and the sample solution, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is aqueous solution of trifluoroacetic acid, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is an Ultimate XB-C8 chromatographic column, and the volume ratio sequence of gradient elution and mobile phase acetonitrile in the high performance liquid chromatography is as follows: 0 min-30 min,25% -80% of running; 30-40 min, 80-20% of the operation;
the retention time of each component in the fluorine Lei Lana intermediate sample solution was determined from the chromatograms of the standard and sample solutions.
2. The method of claim 1, wherein: the aqueous solution of trifluoroacetic acid is 0.03% trifluoroacetic acid solution.
3. The detection method according to claim 1 or 2, characterized in that: the column temperature of the high performance liquid chromatography is 30-40 ℃.
4. A detection method according to claim 3, wherein: the column temperature of the chromatographic column was 35 ℃.
5. The detection method according to claim 1 or 2, characterized in that: the sample injection amount of the high performance liquid chromatography is 8-15 mu L.
6. The method of claim 5, wherein: the sample injection amount of the high performance liquid chromatography is 10 mu L.
7. The detection method according to claim 1 or 2, characterized in that: the detector of the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength is 205nm.
8. The method of claim 1, wherein: the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is 0.03% trifluoroacetic acid solution, and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0 min-30 min,25% -80% of running; 30-40 min, 80-20% running, flow rate of 0.7mL/min, chromatographic column temperature of 35 ℃, sample injection amount of 10 mu L, and chromatographic column of Ultimate XB-C8: 4.6mm×250mm,5 μm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3643705A1 (en) * | 2018-10-24 | 2020-04-29 | Basf Se | Pesticidal compounds |
| CN113009048A (en) * | 2021-04-16 | 2021-06-22 | 成都海关技术中心 | Method for detecting content of flurarana by using dispersed solid phase extraction and liquid chromatography tandem mass spectrometry |
| CN113759044A (en) * | 2021-09-07 | 2021-12-07 | 丽珠集团新北江制药股份有限公司 | Method for detecting frainer initiator by reversed-phase HPLC |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3643705A1 (en) * | 2018-10-24 | 2020-04-29 | Basf Se | Pesticidal compounds |
| CN113009048A (en) * | 2021-04-16 | 2021-06-22 | 成都海关技术中心 | Method for detecting content of flurarana by using dispersed solid phase extraction and liquid chromatography tandem mass spectrometry |
| CN113759044A (en) * | 2021-09-07 | 2021-12-07 | 丽珠集团新北江制药股份有限公司 | Method for detecting frainer initiator by reversed-phase HPLC |
Non-Patent Citations (2)
| Title |
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| Kilp et al..Pharmacokinetics of fluralaner in dogs following a single oral or intravenous administration.《Parasites & Vectors》.2014,第7卷(第85期),1-5. * |
| 景成德;栾长福.氟雷拉纳在小动物临床的应用.《新农业》.2020,(09),59-60. * |
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