CN112763586A - Chiral chromatographic separation analysis method of closantel enantiomer - Google Patents
Chiral chromatographic separation analysis method of closantel enantiomer Download PDFInfo
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- CN112763586A CN112763586A CN202011345292.4A CN202011345292A CN112763586A CN 112763586 A CN112763586 A CN 112763586A CN 202011345292 A CN202011345292 A CN 202011345292A CN 112763586 A CN112763586 A CN 112763586A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
Abstract
The invention belongs to the technical field of chiral chromatographic separation, and discloses a chiral chromatographic separation analysis method of closantel enantiomer. The method comprises the steps of taking alkane, lower alcohol and organic acid as mobile phases, balancing bonded isopropyl cyclofructan-6 at a flow rate of 0.2-1.0 mL/min, adjusting the volume ratio of the alkane and the lower alcohol in the mobile phases to be (90-99): 1-10, and taking the organic acid as 0.05-0.5% of the total volume of the alkane and the lower alcohol; injecting the closantel solution into a liquid chromatography system, wherein the injection volume is 0.1-10 mu L, the ultraviolet wavelength is 210-254 nm, the column temperature of a chromatographic column is 5-40 ℃, and performing liquid chromatography separation analysis to realize the separation of closantel enantiomers. The chiral chromatographic separation and analysis method of the invention realizes the complete separation of the two enantiomers of closantel, and has simple and stable detection process and strong practicability.
Description
Technical Field
The invention belongs to the technical field of chiral chromatographic separation, and particularly relates to a chiral chromatographic separation analysis method of closantel enantiomer.
Background
Chiral enantiomers generally have the same physical and chemical properties, but exhibit different metabolic, activity, and toxicity properties in vivo. Differences in biological activity of chiral enantiomers are often observed with one isomer being active and the other being inactive but toxic. Enantiomers of thalidomide, praziquantel, ofloxacin and the like in the chiral drugs respectively have different biological activities. R-thalidomide has a sedative effect, while the S configuration has a teratogenic effect; r-praziquantel has good activity against Schistosoma mansoni, while S-praziquantel has no therapeutic effect; the antibacterial activity of the levofloxacin is 8-128 times that of the levofloxacin and 2 times that of the ofloxacin. According to incomplete statistics, 40% of chemically synthesized drugs are chiral drugs, with only 12% of chiral drugs administered as a single enantiomer. Most of the chemical synthetic drugs used in the market are racemic compounds, and the number of the corresponding isomers with ineffectiveness or toxicity accounts for half of the total number of the chemical synthetic drugs, so that the chemical synthetic drugs not only have no treatment effect, but also cause resource waste and environmental pollution. Therefore, the research on the resolution of chiral drugs has been a focus in the pharmaceutical field.
Closantel (Closantel), also known as Closantel and Closantel, belongs to the salicylanilide group of antiparasitic drugs and contains a chiral carbon center and a pair of chiral enantiomers (see formula 1). Its chemical name is N- [ 5-chloro-4- (. alpha. - (4-chlorophenyl) -. alpha. -cyanomethyl) -2-tolyl ] -2-hydroxy-3, 5-diiodo-phenamide. Cyhaloiodosalamide is a strong uncoupler of oxidative phosphorylation, which increases the permeability of the parasite mitochondria. The action mechanism is to inhibit the phosphorylation process of the worm body, thereby preventing the generation of Adenosine Triphosphate (ATP) in the worm body and finally leading the worm body to exhaust energy and die. The closantel is a broad-spectrum antiparasitic drug and mainly shows good anti-insect effect on liver fluke infection diseases of cattle and sheep.
Closantel is widely used in veterinary clinics due to its high efficiency and broad spectrum, however, administration of racemic compounds increases the residual amount of the drug in animal tissues, wasting resources. On one hand, after the drug is metabolized and distributed in vivo, the ineffective and toxic enantiomer is discharged to the environment in the form of feces and urine, thereby polluting the environment; on the other hand, animal edible tissues that retain the ineffective or toxic enantiomer for a long period of time pose a health threat to the consumer. At present, liquid chromatography and liquid-mass spectrometry are mainly adopted to determine closantel and the residual quantity thereof in animal-derived samples, but no report is found about the separation analysis of two enantiomers of the closantel. Therefore, the development of chiral chromatographic separation analysis technology has positive significance for chiral resolution and enantiomer analysis and detection of closantel.
Disclosure of Invention
In order to solve the defects and shortcomings of the chiral analysis and separation method of the closantel enantiomer in the prior art, the invention aims to provide a chiral chromatographic separation and analysis method of the closantel enantiomer. The method takes alkane, lower alcohol and organic acid as a mobile phase mixture as a mobile phase for the first time, and realizes the complete separation of the enantiomers of closantel at the column temperature of 5-40 ℃.
The purpose of the invention is realized by the following technical scheme:
a method for chiral chromatographic separation and analysis of an enantiomer of closantel comprises the following steps:
s1, dissolving a standard substance of closantel in chromatographic grade methanol to prepare a solution of closantel;
s2, taking alkane, lower alcohol and organic acid as mobile phases, balancing the bonded isopropyl cyclofructan-6 at a flow rate of 0.2-1.0 mL/min, adjusting the volume ratio of the alkane and the lower alcohol in the mobile phases to be (90-99): 1-10, and taking the organic acid as 0.05-0.5% of the total volume of the alkane and the lower alcohol;
s3, injecting the prepared closantel solution obtained in the step S1 into a liquid chromatography system, wherein the sample injection volume is 0.1-10 mu L, the ultraviolet wavelength is 210-254 nm, the column temperature of a chromatographic column is 5-40 ℃, and liquid chromatography separation analysis is carried out, so that the separation of closantel enantiomers can be realized.
Preferably, the concentration of the closantel solution in the step S1 is 1-10 mg/mL.
Preferably, the alkane in step S2 is n-hexane, heptane or octane.
Preferably, the lower alcohol in step S2 is ethanol, propanol, isopropanol or butanol.
Preferably, the organic acid in step S2 is formic acid, acetic acid or trifluoroacetic acid.
Preferably, the balancing time in the step S2 is 0.5-2 h.
Preferably, the volume ratio of the alkane to the lower alcohol in step S2 is 97: 3; the volume content of the organic acid is 0.1%.
Preferably, the column temperature of the chromatographic column in the step S2 is 20 ℃, and the flow rate is 0.5 mL/min.
Preferably, the separation degree of the closantel enantiomer in the step S3 is 0.84-2.56; the capacity factor is 1.91-2.56.
Preferably, the time for realizing the separation of the closantel enantiomer in the step S3 is 6-30 min.
Compared with the prior art, the invention has the following beneficial effects:
1. the method takes the mixture of alkane, lower alcohol and organic acid as the mobile phase for the first time, and realizes the complete separation of the enantiomer of closantel at the column temperature of 5-40 ℃.
2. The chiral chromatographic separation and analysis method provided by the invention realizes the complete separation of the two enantiomers of closantel, and has the advantages of simple and stable detection process and strong practicability.
3. Under the chromatographic condition, the separation degree of two enantiomers of closantel is 0.84-2.56; the capacity factor is 1.91-2.56. In the chiral chromatographic separation and analysis method, the closantel enantiomer has good separation effect and high analysis speed (6-30 min), and can be applied to the analysis and detection of the closantel enantiomer.
Drawings
FIG. 1 is an HPLC chromatogram of an enantiomer of closantel obtained in example 1.
FIG. 2 is an HPLC chromatogram of an enantiomer of closantel obtained in examples 1 to 4.
FIG. 3 is an HPLC chromatogram of the enantiomers of closantel obtained in examples 5 to 6 and comparative example 1.
FIG. 4 is an HPLC chromatogram of an enantiomer of closantel obtained in comparative examples 2 to 3 and example 7.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the embodiments of the present invention are not limited thereto. The reagent materials in the examples are all commercially available; reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The instruments and reagents used in the examples of the invention were as follows: the Chiral chromatographic column is an InfinityLab Poroshell 120Chiral-CF chromatographic column produced by Agilent, the specification is 4.6mm multiplied by 100mm, the particle size is 2.7 mu m, a high performance liquid chromatograph e2695 series (Waters corporation, USA), an ultraviolet detector 2487 type (Waters corporation, America), an electronic analytical balance 92SM-202A type, the sensitive quantity is 0.0001g (Precisa company, Switzerland), an ultrasonic cleaner KQ5200B type (ultrasonic instruments, Inc. of Kunshan, Jiangsu), and a micropipetman Pipetman type (Gillson company, Germany).
The stationary phase is bonding type isopropyl cyclofructan-6; the chromatographic grade n-hexane, isopropanol, methanol, ethanol, acetonitrile and the like are all purchased from Thermo Fisher company of America; chromatographic grade trifluoroacetic acid was purchased from Sigma-Aldrich, germany; the closantel reference was purchased from dr. ehrenstorfer GmbH, germany at a content of 98.1%.
Example 1
1. Weighing a proper amount of closantel standard substance, placing the standard substance into a 10mL volumetric flask, adding chromatographic grade methanol for dissolving, fixing the volume, shaking up, and preparing into a 1mg/mL closantel solution;
2. using n-hexane, isopropanol and trifluoroacetic acid as mobile phases, balancing the bonded isopropyl cyclofructan-6 for 0.5h at the flow rate of 0.5mL/min, and adjusting the volume ratio of the n-hexane, the isopropanol and the trifluoroacetic acid to 97:3: 0.1;
3. injecting the closantel solution prepared in the step S1 into a liquid chromatography system, wherein the sample injection volume is 2 mu L, the ultraviolet wavelength is 230nm, the column temperature of a chromatographic column is 20 ℃, performing liquid chromatography separation analysis, and separating the closantel enantiomer within 15 min.
Example 2
The difference from example 1 is that: the column temperature was 30 ℃.
Example 3
The difference from example 1 is that: the column temperature was 35 ℃.
Example 4
The difference from example 1 is that: the column temperature was 25 ℃.
Example 5
The difference from example 1 is that: n-hexane, isopropanol, trifluoroacetic acid 95:5: 0.1; the column temperature was 25 ℃.
Example 6
The difference from example 1 is that: n-hexane: isopropyl alcohol: trifluoroacetic acid 90:10: 0.1; the column temperature was 25 ℃.
Comparative example 1
The difference from example 1 is that: n-hexane: isopropyl alcohol: trifluoroacetic acid 80:20: 0.1; the column temperature was 25 ℃.
FIG. 1 is an HPLC chromatogram of an enantiomer of closantel obtained in example 1. 2 chromatographic peaks are observed in 11.8min and 13.4min of closantel, and baseline separation of two enantiomers of closantel is realized. FIG. 2 is an HPLC chromatogram of an enantiomer of closantel obtained in examples 1 to 4. As can be seen from fig. 2 and table 1, the degree of separation of closantel gradually increases as the column temperature decreases. The temperature of the column is gradually reduced from 35 ℃ to 20 ℃, and the separation degree is increased from 0.40 to 2.68; the capacity factor increased from 1.91 to 2.56, with a significant change; but the selection factor does not vary much. FIG. 3 is an HPLC chromatogram of the enantiomers of closantel obtained in examples 5 to 6 and comparative example 1. As can be seen from fig. 2, in the n-hexane-isopropanol mobile phase with different volume ratios, the separation degree of closantel gradually increases and the retention time increases with the decrease of the isopropanol content. When the mobile phase composition was n-hexane, isopropanol, trifluoroacetic acid 95:5:0.1(v/v), the resolution reached 0.85.
Table 1 shows the separation results of the enantiomers of closantel obtained in examples 1 to 6 and comparative example 1, with different column temperatures and n-hexane: isopropyl alcohol: results of the effect of different flow phase ratios of trifluoroacetic acid on chiral chromatographic separation of closantel. Chiral chromatographic separation of enantiomers of closantel was performed in examples 5-6 and comparative example 1 in table 1 with a fixed mobile phase ratio of n-hexane to isopropanol to trifluoroacetic acid of 97:3:0.1(v/v) and adjusting the column temperature. The contrast separation degree is known, under the condition of the column temperature of 20 ℃, the separation degree of closantel is 2.68, and the baseline separation of enantiomers is realized.
Table 1 shows the results of the separation of the enantiomers of closantel obtained in examples 1 to 6 and comparative example 1
Example 7
The difference from example 1 is that: n-hexane: ethanol: trifluoroacetic acid 95:5: 0.1; the column temperature was 25 ℃.
Comparative example 2
The difference from example 1 is that: n-hexane: ethanol: trifluoroacetic acid 80:20: 0.1; the column temperature was 25 ℃.
Comparative example 3
The difference from example 1 is that: n-hexane: ethanol: trifluoroacetic acid 90:10: 0.1; the column temperature was 25 ℃.
Table 2 results of separation of enantiomers of closantel obtained in comparative examples 2 to 3 and example 7
Table 2 shows the results of the separation of the enantiomers of closantel obtained in comparative examples 2-3 and example 7. The result of the influence of the condition of the mobile phase of mixed n-hexane, ethanol and trifluoroacetic acid with different volume ratios on the separation of the chlorocyaniodosalicylamine chiral chromatography is obtained at the constant column temperature of 25 ℃. Column InfinityLab Poroshell 120Chiral-CF (4.6 mm. times.100 mm, 2.7 μm). As is clear from tables 1 and 2, n-hexane-isopropyl alcohol generally has a better separation effect on closantel than the mobile phase condition of n-hexane-ethyl alcohol, and the degree of separation is better when the mixed solution of n-hexane-isopropyl alcohol is 95:5, and the degree of separation is 0.85. Therefore, the normal hexane, the isopropanol and the trifluoroacetic acid are selected as main mobile phases for chiral chromatographic separation, and the volume ratio of the normal hexane to the isopropanol in the mobile phases is adjusted to 97:3 to continuously optimize the separation degree.
FIG. 4 is an HPLC chromatogram of the enantiomers of closantel obtained in comparative examples 2-3 and example 7. As can be seen from fig. 4, when the chiral chromatographic separation of the closantel enantiomer was performed using n-hexane-ethanol as the mobile phase, the closantel could not be separated by adjusting the ratio of the mobile phase. The separation degree of the closantel enantiomer is 0.84-2.56; the capacity factor is 1.91-2.56, and baseline separation of two enantiomers of closantel is realized.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for chiral chromatographic separation and analysis of an enantiomer of closantel is characterized by comprising the following steps:
s1, dissolving a standard substance of closantel in chromatographic grade methanol to prepare a solution of closantel;
s2, using alkane, lower alcohol and organic acid as mobile phases, balancing bonded isopropyl cyclofructan-6 at a flow rate of 0.2-1.0 mL/min, adjusting the volume ratio of the alkane and the lower alcohol in the mobile phases to be (90-99): 1-10, and using the organic acid as 0.05-0.5% of the total volume of the alkane and the lower alcohol;
s3, injecting the prepared closantel solution obtained in the step S1 into a liquid chromatography system, wherein the sample injection volume is 0.1-10 mu L, the ultraviolet wavelength is 210-254 nm, the column temperature of a chromatographic column is 5-40 ℃, and performing liquid chromatography separation analysis to realize the separation of the closantel enantiomer.
2. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the concentration of the solution of closantel in step S1 is 1-10 mg/mL.
3. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the alkane in step S2 is n-hexane, heptane or octane.
4. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the lower alcohol in step S2 is ethanol, propanol, isopropanol or butanol.
5. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the organic acid in step S2 is formic acid, acetic acid or trifluoroacetic acid.
6. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the equilibration time in step S2 is 0.5-2 h.
7. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the volume ratio of the alkane to the lower alcohol in step S2 is 97: 3; the volume content of the organic acid is 0.1%.
8. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the column temperature of the column in step S2 is 20 ℃ and the flow rate is 0.5 mL/min.
9. The chiral chromatographic separation and analysis method of an enantiomer of closantel as claimed in claim 1, wherein the separation degree of the enantiomer of closantel in step S3 is 0.84-2.56; the capacity factor is 1.91-2.56.
10. The method for chiral chromatographic separation and analysis of an enantiomer of closantel as claimed in claim 1, wherein the time for achieving separation of the enantiomer of closantel in step S3 is 6-30 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102778525A (en) * | 2011-04-18 | 2012-11-14 | 林维宣 | Detection method for residual quantity of salicylanilide veterinary drugs in animal-derived food |
| US20170326103A1 (en) * | 2014-08-12 | 2017-11-16 | Monash University | Lymph directing prodrugs |
| CN110187021A (en) * | 2019-05-17 | 2019-08-30 | 河北远征药业有限公司 | Method that is a kind of while measuring two kinds of drug contents in closantel sodium ivermectin injection |
| CN111107745A (en) * | 2017-09-13 | 2020-05-05 | 先正达参股股份有限公司 | Fungicidal compositions |
-
2020
- 2020-11-25 CN CN202011345292.4A patent/CN112763586A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102778525A (en) * | 2011-04-18 | 2012-11-14 | 林维宣 | Detection method for residual quantity of salicylanilide veterinary drugs in animal-derived food |
| US20170326103A1 (en) * | 2014-08-12 | 2017-11-16 | Monash University | Lymph directing prodrugs |
| CN111107745A (en) * | 2017-09-13 | 2020-05-05 | 先正达参股股份有限公司 | Fungicidal compositions |
| CN110187021A (en) * | 2019-05-17 | 2019-08-30 | 河北远征药业有限公司 | Method that is a kind of while measuring two kinds of drug contents in closantel sodium ivermectin injection |
Non-Patent Citations (3)
| Title |
|---|
| GARRETT HELLINGHAUSEN 等: "Mass Spectrometry-Compatible Enantiomeric Separations of 100 Pesticides Using Core-Shell Chiral Stationary Phases and Evaluation of Iterative Curve Fitting Models for Overlapping Peaks", 《CHROMATOGRAPHIA》 * |
| 李哲 等: "HPLC同时测定羊组织中氯氰碘柳胺和雷复尼特的残留量", 《食品研究与开发》 * |
| 赵英 等: "高效液相色谱法测定制剂中氯氰碘柳胺钠含量", 《中国兽药杂质》 * |
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