CN114280185A - Method for detecting flurarana intermediate and isomer thereof - Google Patents
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Abstract
The invention discloses a method for detecting a flurarana intermediate and an isomer thereof. Some embodiments of the present invention provide a separation and detection method suitable for frainer intermediate and its corresponding E/Z isomer by screening chromatographic column and detection conditions of adaptive mobile phase, flow rate, column temperature, etc., wherein said method employs Ultimate XB-C8, 4.6mm × 250mm, 5 μm, chromatographic column, which is a separation chromatographic column; a mixed solution of acetonitrile and trifluoroacetic acid (c =0.03% v/v) is used as a mobile phase; the intermediate of the fraserpine and the E/Z isomer impurity corresponding to the intermediate can be effectively separated and detected.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and relates to a method for detecting a flurandrine intermediate and an isomer thereof.
Background
The successful research and development of the loratadine creates a brand-new research direction of GABA (gamma-aminobutyric acid) gated chloride channel interfering agents, and draws the attention and favor of animal medicine and pesticide science workers.
The loratadine mainly plays a role by interfering GABA gated chloride ion channels, and is similar to the action targets of pesticides such as cyclopentadiene, phenylpyrazole and macrolide. The fraxidin is a broad-spectrum pesticide, has good insecticidal activity on pests of the orders of Acarina, Siphonaptera, louse chemical book, Hemiptera and Diptera, and has higher toxicity or equivalent toxicity to common pesticides. The fraxidin has no obvious cross resistance with the existing pesticide, and even has better insecticidal activity to partially resistant pests.
The structure of the benzaldoxime of the intermediate of the fraserai-5 (E type (4- (hydroxyimino) methyl) -2-methylbenzoic acid) enables the fraserai-5 to exist in an isomer with Z configuration, the structure is highly similar, and separation, detection and analysis are difficult. The structural formula of the isomer of the E/Z configuration of the frataxin intermediate-5 is as follows:
at present, no patent literature report related to the analysis method of the fradoramene intermediate and the isomer thereof exists, and with the deep research on the product in China, a sensitive and stable detection method is needed to be provided for strengthening the quality control of the fradoramene intermediate.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting a flurandrine intermediate and an isomer thereof.
The technical scheme adopted by the invention is as follows:
a detection method of a flurarana intermediate and an isomer thereof, wherein the flurarana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of the flurarana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, comprises the following steps:
s1) preparing a standard solution and a test solution of the E/Z isomer of the frataxin intermediate;
s2) carrying out gradient elution detection on the standard solution and the test solution by using high performance liquid chromatography to obtain chromatograms, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is trifluoroacetic acid water solution, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is an Ultimate XB-C8 chromatographic column;
s3) determining the retention time of each component in the frasnarina intermediate sample solution according to the 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 order of volume ratio, in volume fraction: 0min to 30min, and 25 percent to 80 percent of operation; 30-40 min, and 80-20% of 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: 80 parts of water: preparing a standard solution and a test solution of the E/Z isomer of the frataxin intermediate by using the mixed solution of 20 (v/v).
In some examples, the flow rate is 0.7 mL/min.
In some examples, the column temperature of the high performance liquid chromatography is 30 to 40 ℃.
In some examples, the column temperature is 35 ℃.
In some examples, the high performance liquid chromatography is carried out in an amount of 8-15 μ L.
In some examples, the high performance liquid chromatography is run in 10 μ L.
In some examples, the detector of the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength is 205 nm.
In some examples, the conditions of the high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is a trifluoroacetic acid solution of 0.03% (v/v), and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0min to 30min, and 25 percent to 80 percent of operation; 30 min-40 min, running at 80% -20%, the flow rate is 0.7mL/min, the chromatographic column temperature is 35 ℃, the sample injection amount is 10 mu L, and the chromatographic column is an Ultimate XB-C8 chromatographic column: 4.6mm by 250mm, 5 μm.
The invention has the beneficial effects that:
some embodiments of the present invention provide a separation and detection method suitable for frainer intermediate and its corresponding E/Z isomer by screening chromatographic column and detection conditions of adaptive mobile phase, flow rate, column temperature, etc., wherein said method employs Ultimate XB-C8, 4.6mm × 250mm, 5 μm, chromatographic column, which is a separation chromatographic column; a mixed solution of acetonitrile and trifluoroacetic acid (c is 0.03% v/v) is used as a mobile phase; the intermediate of the fraserpine and the E/Z isomer impurity corresponding to the intermediate can be effectively separated and detected.
Drawings
FIG. 1 is a chromatogram of an empty 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 of example 2;
FIG. 4 is a chromatogram of a standard solution of example 3;
FIG. 5 is a chromatogram of a standard mixed solution in the comparative example.
Detailed Description
A detection method of a flurarana intermediate and an isomer thereof, wherein the flurarana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of the flurarana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, comprises the following steps:
s1) preparing a standard solution and a test solution of the E/Z isomer of the frataxin intermediate;
s2) carrying out gradient elution detection on the standard solution and the test solution by using high performance liquid chromatography to obtain chromatograms, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is trifluoroacetic acid water solution, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is an Ultimate XB-C8 chromatographic column;
s3) determining the retention time of each component in the frasnarina intermediate sample solution according to the 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 order of volume ratio, in volume fraction: 0min to 30min, and 25 percent to 80 percent of operation; 30-40 min, and 80-20% of 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: 80 parts of water: preparing a standard solution and a test solution of the E/Z isomer of the frataxin intermediate by using the mixed solution of 20 (v/v). Experimental results show that the mixed solution with the proportion can obtain better separation degree and is beneficial to the detection of E/Z configurational isomers.
In some examples, the flow rate is 0.7 mL/min.
In some examples, the column temperature of the high performance liquid chromatography is 30 to 40 ℃.
In some examples, the column temperature is 35 ℃.
In some examples, the high performance liquid chromatography is carried out in an amount of 8-15 μ L.
In some examples, the high performance liquid chromatography is run in 10 μ L.
In some examples, the detector of the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength is 205 nm.
In some examples, the conditions of the high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is a trifluoroacetic acid solution of 0.03% (v/v), and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0min to 30min, and 25 percent to 80 percent of operation; 30 min-40 min, running at 80% -20%, the flow rate is 0.7mL/min, the chromatographic column temperature is 35 ℃, the sample injection amount is 10 mu L, and the chromatographic column is an Ultimate XB-C8 chromatographic column: 4.6mm by 250mm, 5 μm.
The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.
In order to make the technical solutions of the present invention better understood by those skilled in the art, the following further discloses some non-limiting examples to further explain the present invention in detail.
The reagents used in the present invention are either commercially available or can be prepared by the methods described herein.
In the present invention, g represents g, mL represents mL, nm represents nm, μm represents μm, mm represents mm, and min represents min.
Example 1
Detection conditions are as follows:
the instrument comprises the following steps: the high-performance liquid chromatograph of the Saimeifen, the DAD ultraviolet detector of the Saimeifen, the detection wavelength: 205 nm;
a chromatographic column: ultimate XB-C8, 4.6mm × 250mm, 5 μm
Diluent agent: acetonitrile: 80 parts of water: 20(v/v)
Mobile phase: a: acetonitrile; b: 0.03% trifluoroacetic acid in water (0.3 mL trifluoroacetic acid in 1000mL water, mixing, suction filtration, sonication)
Flow rate: 0.7 mL/min;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
elution time: 40 min;
a detection step:
preparing a mixed standard solution: accurately weighing 5mg of frataxin intermediate-5 and 5mg of frataxin intermediate-5 isomer in a 20mL volumetric flask, adding a diluent (acetonitrile: 80: 20(v/v)) to dissolve and dilute to a scale mark to obtain a mixed standard solution;
taking blank solution (diluent) and mixed standard solution respectively, performing detection analysis according to the above conditions, and recording chromatogram, the results are shown in FIG. 1 and FIG. 2. The chromatographic peak with retention time of 12.41 minutes in FIG. 2 is the chromatographic peak of frataxin intermediate-5; the peak of the chromatogram at 11.68 minutes was the peak of the isomer of frataxin intermediate-5, and the degree of separation was 3.2.
Example 2
Detection conditions are as follows:
the instrument comprises the following steps: the high-performance liquid chromatograph of the Saimeifen, the DAD ultraviolet detector of the Saimeifen, the detection wavelength: 205 nm;
a chromatographic column: ultimate XB-C8, 4.6mm × 250mm, 5 μm
Diluent agent: acetonitrile: 80 parts of water: 20(v/v)
Mobile phase: a: acetonitrile; b: 0.03% trifluoroacetic acid in water (0.3 ml trifluoroacetic acid in 1000ml water, mixing, suction filtering, ultrasonic)
Flow rate: 0.7 mL/min;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
elution time: 40 min;
a detection step:
preparation of a standard solution: weighing about 5mg of a frataxin intermediate-5 sample in a 20mL volumetric flask, adding a diluent to dissolve and dilute the sample to a scale mark to be used as a standard solution;
taking the standard solution, performing high performance liquid chromatography according to the detection conditions, and recording the chromatogram, wherein the result is shown in figure 3. FIG. 3 shows that the optical purity of the frataxin intermediate-5 is high, the detection requirement of the intermediate is met, and the method can be used for quality detection of the frataxin intermediate-5.
Example 3
Detection conditions are as follows:
the instrument comprises the following steps: the high-performance liquid chromatograph of the Saimeifen, the DAD ultraviolet detector of the Saimeifen, the detection wavelength: 205 nm;
a chromatographic column: ultimate XB-C8, 4.6mm × 250mm, 5 μm
Diluent agent: acetonitrile: 80 parts of water: 20(v/v)
Mobile phase: a: acetonitrile; b: 0.03% trifluoroacetic acid in water (0.3 mL trifluoroacetic acid in 1000mL water, mixing, suction filtration, sonication)
Flow rate: 0.7 mL/min;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
elution time: 40 min;
a detection step:
preparation of a standard solution: weighing 5mg of the intermediate-5 isomer of the frataxin in a 20mL volumetric flask, adding a diluent to dissolve the isomer and diluting the isomer to a scale mark to obtain a standard solution;
taking the standard solution, performing high performance liquid chromatography according to the detection conditions, and recording the chromatogram, wherein the result is shown in FIG. 4.
Comparative example 1
Detection conditions are as follows:
the instrument comprises the following steps: the high-performance liquid chromatograph of the Saimeifen, the DAD ultraviolet detector of the Saimeifen, the detection wavelength: 205 nm;
a chromatographic column: ultimate XB-C8, 4.6mm × 250mm, 5 μm
Diluent agent: acetonitrile: 60 parts of water: 40(v/v)
Mobile phase: a: acetonitrile; b: 0.03% trifluoroacetic acid in water (0.3 mL trifluoroacetic acid in 1000mL water, mixing, suction filtration, sonication)
Flow rate: 0.6 mL/min;
column temperature: 20 ℃;
sample introduction amount: 10 mu L of the solution;
elution time: 40 min;
a detection step:
preparing a mixed standard solution: accurately weighing 5mg of frataxin intermediate-5 and 5mg of frataxin intermediate-5 isomer in a 20mL volumetric flask, adding a diluent (acetonitrile: water: 60: 40(v/v)) to dissolve and dilute to a scale mark to obtain a mixed standard solution;
and (4) respectively taking the blank solution (diluent) and the mixed standard solution, carrying out detection analysis according to the conditions, and recording a chromatogram.
The results are shown in FIG. 5. The chromatographic peak at retention time of 13.86 minutes in FIG. 5 is a mixed chromatographic peak of the isomers of frataxin intermediate-5 and frataxin intermediate-5, and it can be seen that the two peaks are contained together and cannot be separated.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
Claims (10)
1. A method for detecting a flurarana intermediate and an isomer thereof, wherein the flurarana intermediate is E-type (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the isomer is Z-type isomer of the flurarana intermediate (4- (hydroxyimino) methyl) -2-methylbenzoic acid, and the method is characterized by comprising the following steps of: the method comprises the following steps:
preparing a standard solution, a test solution and a test solution of the E/Z isomer of the frataxin intermediate;
performing gradient elution detection on the standard solution and the test solution by using high performance liquid chromatography to obtain chromatograms of the standard solution and the test solution, wherein the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is trifluoroacetic acid water solution, and the flow rate is 0.5-1.0 mL/min; the chromatographic column is an Ultimate XB-C8 chromatographic column;
and determining the retention time of each component in the intermediate sample solution of the frailamide according to the chromatograms of the standard sample solution and the sample solution.
2. The detection method according to claim 1, characterized in that: the volume ratio sequence of gradient elution and mobile phase acetonitrile in the high performance liquid chromatography is as follows by volume fraction: 0min to 30min, and 25 percent to 80 percent of operation; 30-40 min, and 80-20% of operation.
3. The detection method according to claim 1, characterized in that: the aqueous trifluoroacetic acid solution is a 0.03% (v/v) trifluoroacetic acid solution.
4. The detection method according to claim 1, characterized in that: using acetonitrile: water = 80: preparing a standard solution and a test solution of the E/Z isomer of the frataxin intermediate by using the mixed solution of 20 (v/v).
5. The detection method according to any one of claims 1 to 4, characterized in that: the column temperature of the high performance liquid chromatography is 30-40 ℃.
6. The detection method according to claim 5, characterized in that: the column temperature was 35 ℃.
7. The detection method according to any one of claims 1 to 4, characterized in that: the sample injection amount of the high performance liquid chromatography is 8-15 mu L.
8. The detection method according to claim 7, characterized in that: the sample size of the high performance liquid chromatography is 10 mu L.
9. The detection method according to any one of claims 1 to 4, characterized in that: the detector of the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength is 205 nm.
10. The detection method according to claim 1, characterized in that: the conditions of the high performance liquid chromatography are as follows: the mobile phase A is acetonitrile, the mobile phase B is a trifluoroacetic acid solution of 0.03% (v/v), and the volume ratio sequence of the gradient elution and the mobile phase acetonitrile is as follows: 0min to 30min, and 25 percent to 80 percent of operation; 30 min-40 min, running at 80% -20%, the flow rate is 0.7mL/min, the chromatographic column temperature is 35 ℃, the sample injection amount is 10 mu L, and the chromatographic column is an Ultimate XB-C8 chromatographic column: 4.6mm by 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 |
|---|
| KILP ET AL.: "Pharmacokinetics of fluralaner in dogs following a single oral or intravenous administration" * |
| 景成德;栾长福;: "氟雷拉纳在小动物临床的应用" * |
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