CN110596268B - Separation and detection method for isomers in loratadine racemate - Google Patents

Abstract

The invention provides a separation and detection method of isomers in a flurandran racemate, which is carried out by adopting a high performance liquid chromatography, taking amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel as a stationary phase and taking normal hexane and ethanol as a mobile phase. The method can effectively separate and measure the isomers in the fraserpine racemate, and has the advantages of high efficiency, simplicity, high sensitivity, good separation effect and separation degree of 6.681. Provides a detection method for the subsequent development of the S isomer of the fradoramene and the examination of the R isomer of the fradoramene.

Description

Separation and detection method for isomers in loratadine racemate

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a separation and detection method for isomers in a loratadine racemate.

Background

Flurarana, english name: fluralaner, CAS number 864731-61-3, molecular formula C₂₂H₁₇Cl₂F₆N₃O₃Molecular weight 556.9, white to pale yellow solid. The structural formula is shown in figure 1. Fluranide belongs to the isoxazoline class of insecticides and acaricides. The medicine composition is used for preventing and treating ectoparasites of animals such as dogs and cats, and the marketed medicine composition is a mixture of R and S configuration, but later further research results show that the S configuration is the main insecticidal active ingredient. At present, no report of isomer determination methods for fradoramene exists in related documents, enterprise standards and pharmacopoeia by inquiring, and the establishment of a separation detection method for isomers in fradoramene racemates is necessary, so that a foundation is established for the subsequent development of S isomers to be marketed and the inspection of R isomers.

Disclosure of Invention

In view of the above, the present invention is directed to a method for separating and detecting isomers of a loratadine racemate, so as to solve the above problems.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a separation and detection method of isomers in a flurandran racemate is carried out by adopting a high performance liquid chromatography, taking amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel as a stationary phase, and forming a mobile phase by using normal hexane and ethanol.

Further, the volume ratio of the n-hexane to the ethanol is (58-62): (38-42).

Preferably, the volume ratio of the n-hexane to the ethanol is 60: 40.

further, the flow rate of the mobile phase is 0.8-1.2 ml/min.

Preferably, the flow rate of the mobile phase is 1.0 ml/min.

Further, the detection wavelength of the liquid chromatogram is 200nm-400 nm.

Preferably, the detection wavelength of the liquid chromatography is 265 nm.

Further, the column length of the chromatographic column is 250mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the packing is 5 microns.

Further, the sample amount of the liquid chromatography is 5 to 50. mu.l, preferably 20. mu.l.

A separation and detection method for isomers in a flurandrine racemate comprises the following steps:

(1) taking a proper amount of the fraserpine racemate, adding ethanol to dissolve and dilute the mixture to prepare a sample solution containing about 0.5mg in each 1 ml;

(2) amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel is used as a filler; taking a mixed solution of n-hexane and ethanol with a volume ratio of 60:40 as a mobile phase; setting the flow rate to 1.0ml per minute; the detection wavelength is 265 nm; the sample amount is 20 mul; the column temperature is room temperature;

(3) and (2) injecting 5-20 mu L of the sample solution obtained in the step (1) into a high performance liquid chromatograph (4.6mm multiplied by 250mm, 5 mu m) to finish the separation and detection of isomers in the fraralanine racemate.

Compared with the prior art, the method for separating and detecting the isomers in the fraserpine racemate has the following advantages:

the method can effectively separate and measure the isomers in the fraserpine racemate, and has the advantages of high efficiency, simplicity, high sensitivity, good separation effect and separation degree of 6.681. Provides a detection method for the subsequent development of the S isomer and the subsequent examination of the R isomer.

Drawings

FIG. 1 is a structural formula of frataxin;

FIG. 2 is an HPLC chart of a fraserpine racemate;

FIG. 3 is a graph showing the effect of HPLC separation of a sample solution in the examples;

FIG. 4 is an HPLC chart of Y1 in example;

FIG. 5 is an HPLC chart of Y2 in example.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to the following examples and accompanying drawings.

Instrument and reagent

Instruments and reagents	Type and specification	Manufacturer of the product
			High performance liquid chromatograph	LC-20AT,SPD-20A,SIL-20A	SHIMADZU
Electronic balance	ES 225SM-DR	Precisa
			Chiral chromatographic column	AD-H(4.6mm×250mm，5μm)	Daicel
Fluralanane racemate	99.21％	McIdein Biotech Ltd
			N-hexane	HPLC	Tianjin Chuanpu Wei science and technology Limited
Isopropanol (I-propanol)	HPLC	XILONG SCIENTIFIC Co.,Ltd.
			Ethanol	HPLC	Tianjin Chuanpu Wei science and technology Limited

Second, method and results

(1) Preparing a sample solution:

taking a proper amount of the fraserpine racemate, preparing a solution with a certain concentration, and carrying out ultraviolet scanning within a wavelength range of 200-400 nm. The absorption of the fraralanin racemate at the wavelength of 265nm is found to be the absorption wavelength of liquid phase detection. The spectrum is shown in FIG. 2.

Taking a proper amount of the fraserpine racemate, adding ethanol to dissolve and dilute the mixture to prepare a sample solution containing about 0.5mg of the fraserpine racemate in 1 ml.

(2) Chromatographic conditions are as follows:

the chromatographic column takes amylose-tris (3, 5-dimethylphenyl carbamate) silica gel as a stationary phase, the detection wavelength is 265nm, a normal hexane-isopropanol system and a normal hexane-ethanol system are respectively used for screening to obtain a chromatogram peak shape obtained by the normal hexane-ethanol system, the chromatogram peak shape is sharp and symmetrical, the ratio of the normal hexane to the ethanol is adjusted to be (60:40), so that the peaks of two enantiomers are respectively 5.157min and 7.811min, the separation degree is 6.681, the result is shown in the following table, the chromatogram is shown in fig. 3, and the left peak in the chromatogram represents R-configuration frauran; the right peak represents the S configuration of frainer.

The chromatographic conditions are as follows: amylose-tris (3, 5-dimethylphenylcarbamate) silica gel was used as stationary phase (parameters 4.6mm × 250mm, 5 μm); n-hexane-ethanol (60:40) is used as a mobile phase; the flow rate was 1.0ml per minute; the detection wavelength is 265 nm; the sample amount is 20 mul; the solvent is ethanol; the column temperature was room temperature.

(3) The experimental steps are as follows:

(1) taking a proper amount of the fraserpine racemate, adding ethanol to dissolve and dilute the mixture to prepare a sample solution containing about 0.5mg in each 1 ml;

(2) amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel is used as a filler; taking a mixed solution of n-hexane and ethanol with a volume ratio of 60:40 as a mobile phase; setting the flow rate to 1.0ml per minute; the detection wavelength is 265 nm; the sample amount is 20 mul; the column temperature is room temperature;

(3) and (2) injecting 5-20 mu L of the sample solution obtained in the step (1) into a high performance liquid chromatograph (4.6mm multiplied by 250mm, 5 mu m) to finish the separation and detection of isomers in the fraralanine racemate.

Thirdly, result and analysis:

(1) and (3) durability test:

the flow rates are respectively changed to be 0.8ml/min and 1.2ml/min, the flow phase ratio of n-hexane-ethanol (62:38) and n-hexane-ethanol (58:42) has the wavelength of 263nm and 267nm, the column temperature is 20 ℃ and 30 ℃, the change of the chromatographic conditions only slightly influences the retention time and the peak area of the isomers, the separation degree between the isomers is still more than 2.0, and the durability is good. The durability test can show that the method has small change of the chromatographic conditions determined by the method and small influence on the detection of isomers, and the method has good separation degree and durability on R-configuration frataxin and S-configuration frataxin.

(2) Enantiomers were separated and determined:

the medium-pressure preparation liquid chromatograph is adopted, and the chromatographic column conditions are as follows:

stationary phase: amylose-tris (3, 5-dimethylphenylcarbamate) silica gel;

mobile phase: the volume ratio of the n-hexane to the ethanol is 60: 40;

flow rate: 3-7-ml/min;

sample introduction amount: 5-10 ml;

column temperature: room temperature;

concentration of fluranine racemate: 10-20mg/ml, and ethanol as solvent.

Collecting the fraxidin by stages under the conditions, concentrating and drying to obtain two products Y1 and Y2.

Respectively weighing appropriate amount of Y1 and Y2, dissolving in ethanol, diluting to obtain solution containing 5mg of each 1ml, and measuring optical rotation to obtain YI

Is +61.89, i.e. of R configuration, Y2

It is-59.03, namely S configuration.

Taking appropriate amount of Y1 and Y2, precisely weighing, dissolving in ethanol, and diluting to obtain solution containing 0.5mg per 1 ml. And (3) performing positioning according to the chromatographic conditions in the step (2). The peak-off time of Y1 and Y2 was 5.288min and 7.910min, respectively. The chromatogram is shown in FIGS. 4-5.

Therefore, the separation and detection method can separate two enantiomers in the fraserpine racemate, and the method establishes a detection method for subsequently developing S isomer and checking R isomer.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (9)

1. A separation and detection method of isomers in a flurandrine racemate is characterized by comprising the following steps: the method is carried out by adopting a high performance liquid chromatography, taking amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel as a stationary phase, and forming a mobile phase by using normal hexane and ethanol;

the volume ratio of the n-hexane to the ethanol is (58-62): (38-42).

2. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the volume ratio of the n-hexane to the ethanol is 60: 40.

3. the method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the flow rate of the mobile phase is 0.8-1.2 ml/min.

4. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the flow rate of the mobile phase was 1.0 ml/min.

5. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the detection wavelength of the liquid chromatogram is 200nm-400 nm.

6. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the detection wavelength of the liquid chromatogram is 265 nm.

7. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the sample amount of the liquid chromatogram is 5-50 μ l.

8. The method for separating and detecting isomers of a frainer racemate according to claim 1, comprising the steps of: the sample size of the liquid chromatography was 20. mu.l.

9. The method for separating and detecting an isomer in a fraserane racemate according to any one of claims 1 to 8, comprising the steps of:

(1) taking a proper amount of the fraserpine racemate, adding ethanol to dissolve and dilute the mixture to prepare a sample solution containing about 0.5mg in each 1 ml;

(2) amylose-tri (3, 5-dimethylphenyl carbamate) bonded silica gel is used as a filler; taking a mixed solution of n-hexane and ethanol with a volume ratio of 60:40 as a mobile phase; setting the flow rate to 1.0ml per minute; the detection wavelength is 265 nm; the sample amount is 20 mul; the column temperature is room temperature;

(3) injecting 5-20 mu L of the sample solution obtained in the step (1) into a high performance liquid chromatograph, wherein the type of a chromatographic column is 4.6mm multiplied by 250mm and 5 mu m;

and completing the separation and detection of isomers in the fraserpine racemate.

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