阅读说明：本技术 一种分离表断马钱子苷半缩醛内酯和断马钱子苷半缩醛内酯的液相色谱方法 (Liquid chromatography method for separating epiloganin hemiacetal lactone and secologanin hemiacetal lactone ) 是由 吴海靖 其他发明人请求不公开姓名 于 2019-12-05 设计创作，主要内容包括：本发明公开了一种分离表断马钱子苷半缩醛内酯和断马钱子苷半缩醛内酯的液相色谱方法。本领域技术人员知道,手性化合物常需要借助于手性色谱柱分离,但是手性色谱柱的成本较高,使用寿命短,重复性也不如常规的C18色谱柱,这时,基于常规C18色谱柱的手性流动相分析法也不失为一种选择,成本低,分析性能稳定。本发明使用基于常规C18色谱柱的手性流动相分析法实现了表断马钱子苷半缩醛内酯与断马钱子苷半缩醛内酯、8-表马钱子苷与马钱子苷的色谱分离,该方法可以用于检测这些化合物样品是单品还是混合品。(The invention discloses a liquid chromatography method for separating epiloganin hemiacetal lactone and secologanin hemiacetal lactone. As known to those skilled in the art, chiral compounds often need to be separated by means of a chiral chromatographic column, but the chiral chromatographic column has high cost, short service life and poor repeatability as compared with a conventional C18 chromatographic column, and in this case, a chiral mobile phase analysis method based on a conventional C18 chromatographic column is not lost as an option, so that the cost is low and the analysis performance is stable. The invention realizes the chromatographic separation of secoisolaricoside hemiacetal lactone and 8-epiloglycoside and loglycoside by using a chiral mobile phase analysis method based on a conventional C18 chromatographic column, and the method can be used for detecting whether the compound samples are single products or mixed products.)

1. A liquid chromatography method for separating epiloganin hemiacetal lactone and loganin hemiacetal lactone is characterized by comprising the following steps:

preparation of a mobile phase: precisely weighing a proper amount of D-cycloserine and blue copperas, dissolving with deionized water to prepare aqueous solutions of D-cycloserine and copper ion with the concentrations of 4mM and 1mM respectively, mixing the aqueous solutions with methanol and acetonitrile according to the volume ratio of 5:3:2, performing ultrasonic treatment, and filtering.

Setting chromatographic parameters and analyzing sample injection: the chromatographic column is C18 chromatographic column, the column temperature is set at 30 deg.C, the flow rate is 1.0mL/min, the detection wavelength is 240nm, the sample amount is 10 μ L, the mobile phase isocratic elution is performed, and the sample is injected and collected.

2. The liquid chromatography method of claim 1, wherein the high performance liquid chromatograph is an LC-20AT high performance liquid chromatograph.

3. The liquid chromatography method of claim 1, wherein the chromatography column is a Kromasil 100-5C18 chromatography column.

4. The liquid chromatography method of claim 3, wherein the column size is 250mm x 4.6mm, 5 μm.

Technical Field

The invention belongs to the field of pharmaceutical analysis, relates to separation and detection of optical isomers, and particularly relates to a liquid chromatography method for separating epiloglycoside hemiacetal lactone and secologlycoside hemiacetal lactone.

Background

The epiloganin hemiacetal lactone and the secologanin hemiacetal lactone, and the 8-epiloganin and the loganin are two pairs of isomers respectively, are chiral isomers, and have the following chemical structural formulas.

The secoisolaricoside hemiacetal lactone and secoisolaricoside hemiacetal lactone, and the 8-epistrychnine and the strychnine are chiral compounds, so that the epistrychnine and the strychnine are extremely difficult to separate on a conventional C18 chromatographic column. In this case, the secoisolaricoside hemiacetal lactone and the secoisolaricoside hemiacetal lactone may be mixed with each other, or the 8-epiloglycoside and the strychnine may be mixed with each other, and it is not possible to determine whether the compound sample is a monomer or a mixture, which affects the reliability of the pharmacodynamic test result.

Disclosure of Invention

The invention provides a liquid chromatography method for separating secoisolaricoside hemiacetal lactone and secoisolaricoside hemiacetal lactone, aiming at overcoming the defects of the prior art.

The technical scheme of the invention is as follows:

a liquid chromatography method for separating secoisolaricoside hemiacetal lactone and secoisolaricoside hemiacetal lactone comprises the following steps:

preparation of a mobile phase: precisely weighing a proper amount of D-cycloserine and blue copperas, dissolving with deionized water to prepare aqueous solutions of D-cycloserine and copper ion with the concentrations of 4mM and 1mM respectively, mixing the aqueous solutions with methanol and acetonitrile according to the volume ratio of 5:3:2, performing ultrasonic treatment, and filtering.

Setting chromatographic parameters and analyzing sample injection: the chromatographic column is C18 chromatographic column, the column temperature is set at 30 deg.C, the flow rate is 1.0mL/min, the detection wavelength is 240nm, the sample amount is 10 μ L, the mobile phase isocratic elution is performed, and the sample is injected and collected.

In a specific embodiment, the HPLC is an LC-20AT HPLC.

In a specific embodiment, the chromatography column is a Kromasil 100-5C18 chromatography column.

In a specific embodiment, the column size is 250mm × 4.6mm, 5 μm.

A liquid chromatography method for separating 8-epiloglycoside and loglycoside comprises the following steps:

preparation of a mobile phase: precisely weighing a proper amount of D-cycloserine and blue copperas, dissolving with deionized water to prepare aqueous solutions of D-cycloserine and copper ion with the concentrations of 4.5mM and 1.5mM respectively, mixing the aqueous solutions with methanol and acetonitrile according to the volume ratio of 5:3:2, performing ultrasonic treatment, and filtering.

Setting chromatographic parameters and analyzing sample injection: the chromatographic column is C18 chromatographic column, the column temperature is set at 30 deg.C, the flow rate is 1.0mL/min, the detection wavelength is 240nm, the sample amount is 10 μ L, the mobile phase isocratic elution is performed, and the sample is injected and collected.

In a specific embodiment, the HPLC is an LC-20AT HPLC.

In a specific embodiment, the chromatography column is a Kromasil 100-5C18 chromatography column.

In a specific embodiment, the column size is 250mm × 4.6mm, 5 μm.

The beneficial technical effects are as follows:

as known to those skilled in the art, chiral compounds often need to be separated by means of a chiral chromatographic column, but the chiral chromatographic column has high cost, short service life and poor repeatability as compared with a conventional C18 chromatographic column, and in this case, a chiral mobile phase analysis method based on a conventional C18 chromatographic column is not lost as an option, so that the cost is low and the analysis performance is stable. The invention realizes the chromatographic separation of secoisolaricoside hemiacetal lactone and 8-epiloglycoside and loglycoside by using a chiral mobile phase analysis method based on a conventional C18 chromatographic column, and the method can be used for detecting whether the compound samples are single products or mixed products.

Drawings

FIG. 1 is a chromatogram obtained by separating secoisolaricoside hemiacetal lactone from secoisolaricoside hemiacetal lactone, wherein the mobile phase is as follows: precisely weighing a proper amount of D-cycloserine and blue copperas, dissolving with deionized water to prepare aqueous solutions of D-cycloserine and copper ion with the concentrations of 4mM and 1mM respectively, mixing the aqueous solutions with methanol and acetonitrile according to the volume ratio of 5:3:2, performing ultrasonic treatment, and filtering; baseline separation of secoisolaricoside hemiacetal lactone from secoisolaricoside hemiacetal lactone under the chromatographic conditions is achieved;

FIG. 2 is a chromatogram obtained by separating secoisolaricoside hemiacetal lactone from secoisolaricoside hemiacetal lactone, with the mobile phase being: precisely weighing a proper amount of D-cycloserine, dissolving with deionized water to prepare an aqueous solution with the concentration of the D-cycloserine of 4mM, mixing the aqueous solution with methanol and acetonitrile according to the volume ratio of 5:3:2, performing ultrasonic treatment, and filtering; coelution semiacetal lactone and secoisolaricoside semiacetal lactone are co-eluted under the chromatographic conditions and cannot be separated;

FIG. 3 is a chromatogram obtained by separating secoisolaricoside hemiacetal lactone from secoisolaricoside hemiacetal lactone, with the mobile phase being: precisely weighing a proper amount of blue vitriol, dissolving with deionized water to prepare an aqueous solution with copper ion concentration of 1mM, mixing the aqueous solution with methanol and acetonitrile according to a volume ratio of 5:3:2, performing ultrasonic treatment, and filtering; coelution semiacetal lactone and secoisolaricoside semiacetal lactone are co-eluted under the chromatographic conditions and cannot be separated;

FIG. 4 is a chromatogram for separating loganin from loganin, wherein the mobile phase is: precisely weighing a proper amount of D-cycloserine and blue copperas, dissolving with deionized water to prepare aqueous solutions of D-cycloserine and copper ion concentrations of 4.5mM and 1.5mM respectively, mixing the aqueous solutions with methanol and acetonitrile according to a volume ratio of 5:3:2, performing ultrasonic treatment, and filtering; 8-epiloglycoside is separated from loglycoside at baseline under the chromatographic conditions;

FIG. 5 is a chromatogram for separating loganin from loganin, wherein the mobile phase is: precisely weighing a proper amount of D-cycloserine, dissolving the D-cycloserine with deionized water to prepare an aqueous solution with the concentration of the D-cycloserine being 4.5mM, mixing the aqueous solution with methanol and acetonitrile according to the volume ratio of 5:3:2, carrying out ultrasonic treatment, and filtering; co-eluting 8-epiloganin and loganin under the chromatographic condition, and separating;

FIG. 6 is a chromatogram for separating loganin from loganin, wherein the mobile phase is: precisely weighing a proper amount of blue vitriol, dissolving with deionized water to prepare an aqueous solution with copper ion concentration of 1.5mM, mixing the aqueous solution with methanol and acetonitrile according to a volume ratio of 5:3:2, performing ultrasonic treatment, and filtering; the 8-epiloganin and loganin co-elute under the chromatographic conditions and cannot be separated.

Detailed Description

The following detailed description of the invention refers to the accompanying drawings, which are included to provide a further understanding of the invention, and are incorporated by reference herein.