CN106996964A - Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer - Google Patents
Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer Download PDFInfo
- Publication number
- CN106996964A CN106996964A CN201710262396.0A CN201710262396A CN106996964A CN 106996964 A CN106996964 A CN 106996964A CN 201710262396 A CN201710262396 A CN 201710262396A CN 106996964 A CN106996964 A CN 106996964A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- hydroxypropyl
- cyclodextrin
- phase
- diene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Steroid Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a kind of method of use reversed-phased high performace liquid chromatographic separating chiral enantiomer, including：It is added to hydroxypropyl beta cyclodextrin as additive in original mobile phase, obtains required reverse-phase chromatography mobile phase；Use the reverse-phase chromatography mobile phase and reverse-phase chromatography stationary phase, the diketone of 4,9 (11) diene of chiral 16 α methyl androstanes of enantiomer 3,17 and 16 β methyl androstanes 4, the diketone of 9 (11) diene 3,17 carries out RPLC separation.Present invention selection hydroxypropyl beta cyclodextrin is used as Mobile Phase Additives, due to hydroxypropyl beta cyclodextrin, solubility is larger in water, therefore do not result in chromatographic column as Mobile Phase Additives to block, it is possible to prevente effectively from traditional beta cyclodextrin easily causes the defect of chromatographic column blocking.
Description
Technical field
The invention belongs to the separation technology field of chiral enantiomer, more particularly to one kind uses RPLC
The method of method separating chiral enantiomer, and a kind of application of hydroxypropyl-β-cyclodextrin.
Background technology
Chiral molecules is also known as chiral enantiomer, is specular and the molecule that can not be completely superposed in structure in chemistry,
With two kinds of molecular structures.Two kinds of chiral molecules with different molecular structures are in addition to optical activity has differences, physical property phase
Together, chemical property may be similar, therefore can not realize the separation of two kinds of chiral molecules using conventional high performance liquid chromatography.But
It is due to that two kinds of chiral molecules with different molecular structures often have larger difference in terms of bioactivity and pharmacological action
Not, or even effect it is therefore desirable to chiral molecule on the contrary, separated, its related activity is investigated respectively and is rationally made
With.
The oligosaccharide that beta-schardinger dextrin is made up of 7 glucopyranoses, with wide at the top and narrow at the bottom, both ends open hollow
Shape structure, its cavity inside relative hydrophobic is outside to be surrounded and relative hydropathic by polar hydroxyl groups, and this special construction causes β-ring paste
Essence has polarity and nonpolar peculiar property.Multiple chiral centers, its complexing tool are had based on beta-schardinger dextrin intramolecular
There is High level of stereoselectivity selective and cheap, therefore prior art uses beta cyclodextrin as Mobile Phase Additives, using efficient liquid
The chiral molecule of phase chromatography is split.
But, due to beta-schardinger dextrin, solubility is small in water, is used as Mobile Phase Additives and easily causes chromatographic column blocking,
Therefore the above-mentioned property of beta-schardinger dextrin largely limits it in using high performance liquid chromatography separating chiral molecule
Using.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of using reversed-phased high performace liquid chromatographic separating chiral mapping
The method of body, above-mentioned technical problem is solved with all or part of.
In order to solve the above problems, reversed-phased high performace liquid chromatographic separating chiral enantiomer is used the invention discloses one kind
Method, including：
It is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, the reverse-phase chromatography flowing needed for obtaining
Phase；
Use the reverse-phase chromatography mobile phase and reverse-phase chromatography stationary phase, the chiral Alpha-Methyl androstane -4,9 of enantiomer 16
(11)-diene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone carry out RPLC
Separation.
Alternatively, it is described by hydroxypropyl-β-cyclodextrin
Include as additive added to original mobile phase：
The hydroxypropyl-β-cyclodextrin aqueous solution is added in the original mobile phase.
Alternatively, the original mobile phase and the volume ratio of the hydroxypropyl-β-cyclodextrin aqueous solution are 40:60~30:
70；
The concentration of the hydroxypropyl-β-cyclodextrin aqueous solution is 0.02~0.04g/ml.
Alternatively, to 16 Alpha-Methyl androstane -4,9 (11)-diene -3,17- diketone and the 16 Beta-methyl androstane -
When 4,9 (11)-diene -3,17- diketone carries out RPLC separation, the flow velocity of the reverse-phase chromatography mobile phase is 1
~1.2ml/min.
Alternatively, the original mobile phase is acetonitrile or methanol.
Alternatively, the reverse-phase chromatography stationary phase is Eclipse Plus-C8 chromatographic columns or ZORBAX SB-C18 chromatograms
Post.
Alternatively, the column temperature of the Eclipse Plus-C8 chromatographic columns or the ZORBAX SB-C18 chromatographic columns be 35~
40℃。
Alternatively, the size of the Eclipse Plus-C8 chromatographic columns or the ZORBAX SB-C18 chromatographic columns is
250mm×4.6mm。
The embodiment of the present invention additionally provides a kind of application of hydroxypropyl-β-cyclodextrin, using reversed-phased high performace liquid chromatographic
The Alpha-Methyl androstane-4,9 (11) of separating chiral enantiomer 16-diene-3,17- diketone and 16 Beta-methyl androstane-4,9 (11)-diene-
During 3,17- diketone, it is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, the hydroxypropyl-β-cyclodextrin
It is combined into reverse-phase chromatography mobile phase with the original flowing.
Alternatively, the original mobile phase is acetonitrile or methanol.
Compared with prior art, the present invention includes advantages below：
The invention provides a kind of method of use reversed-phased high performace liquid chromatographic separating chiral enantiomer, hydroxypropyl is selected
Group-beta-cyclodextrin is as Mobile Phase Additives, and due to hydroxypropyl-β-cyclodextrin, solubility is larger in water, therefore as
Mobile Phase Additives do not result in chromatographic column blocking, it is possible to prevente effectively from traditional beta-schardinger dextrin easily causes lacking for chromatographic column blocking
Fall into.
The use of conventional chromatogram post is that can be achieved when being separated according to the chiral enantiomer of methods described of the embodiment of the present invention
Efficiently separating for chiral enantiomer, it is simple to operate.Compared with traditional Chiral stationary phase liquid chromatography method, the present invention is implemented
Example methods described is with low cost, more economical to be applicable, and is with a wide range of applications.
Brief description of the drawings
Fig. 1 is a kind of method of use reversed-phased high performace liquid chromatographic separating chiral enantiomer described in the embodiment of the present invention
Flow chart；
Fig. 2 is that 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers of the embodiment of the present invention exist
High-efficient liquid phase chromatogram under the conditions of different Chiral Mobile Phase Additives；
Fig. 3 is that 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers exist in the embodiment of the present invention
High-efficient liquid phase chromatogram under the conditions of different mobile phase ratios；
Fig. 4 is that 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers of the embodiment of the present invention exist
High-efficient liquid phase chromatogram under different chromatographic columns.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, it is below in conjunction with the accompanying drawings and specific real
Applying mode, the present invention is further detailed explanation.
The implementation process of the method for the invention is described in detail below by embodiment.
The embodiments of the invention provide a kind of method of use reversed-phased high performace liquid chromatographic separating chiral enantiomer.
Reference picture 1, it illustrates a kind of using reversed-phased high performace liquid chromatographic separating chiral described in the embodiment of the present invention
The flow chart of the method for enantiomer, the described method comprises the following steps：
Step 101, it is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, it is anti-phase needed for obtaining
Chromatogram flow phase.
16 Alpha-Methyl androstanes -4,9 (11)-diene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17-
Diketone is chiral enantiomer, wherein 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone is a kind of important steroid drugs
Intermediate, synthesizes significant to steroidal drug, therefore to 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone
Separating-purifying is carried out to be very important.The two structure difference is as follows：
Based on hydroxypropyl-β-cyclodextrin intramolecular there are multiple chiral centers, its complexing to have High level of stereoselectivity selection
Property, there is the performance such as larger solubility in water, the embodiment of the present invention uses the chiral molecule 16 of reversed-phased high performace liquid chromatographic
Alpha-Methyl androstane -4,9 (11)-diene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone are divided
From when, hydroxypropyl-β-cyclodextrin is added in original mobile phase, needed for original mobile phase and hydroxypropyl-β-cyclodextrin are combined into
Reverse-phase chromatography mobile phase.
In the embodiment of the present invention, original mobile phase can be acetonitrile or methanol.
When adding hydroxypropyl-β-cyclodextrin into original mobile phase, the hydroxypropyl-β-cyclodextrin of addition can be with the aqueous solution
Form addition, i.e., by the hydroxypropyl-β-cyclodextrin aqueous solution added in original mobile phase.Wherein, original mobile phase and hydroxypropyl
The volume ratio of the group-beta-cyclodextrin aqueous solution can be 40:60~30:70, the concentration control of the hydroxypropyl-β-cyclodextrin aqueous solution exists
0.02~0.04g/ml.
Step 102, using reverse-phase chromatography mobile phase and reverse-phase chromatography stationary phase, the chiral Alpha-Methyl androstane of enantiomer 16-
4,9 (11)-diene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone carry out RP-HPLC color
Spectrum separation.
Obtain after the reverse-phase chromatography mobile phase that is made up of original mobile phase hydroxypropyl-β-cyclodextrin, utilize high-efficient liquid phase color
Spectrometer, uses reverse-phase chromatography mobile phase and reverse-phase chromatography stationary phase, the chiral Alpha-Methyl androstane -4,9 (11)-two of enantiomer 16
Alkene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone are separated.Based on hydroxypropyl in mobile phase-
The addition of beta-schardinger dextrin so that 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone is while obtaining efficiently separating, no
Chromatographic column can be blocked, it is ensured that chromatography experiment is normally carried out.
In the embodiment of the present invention, reverse-phase chromatography stationary phase can be Eclipse Plus-C8 chromatographic columns or ZORBAX SB-
The column temperature of C18 chromatographic columns, Eclipse Plus-C8 chromatographic columns or ZORBAX SB-C18 chromatographic columns can be controlled at 35~40 DEG C,
The size of Eclipse Plus-C8 chromatographic columns or ZORBAX SB-C18 chromatographic columns can be 250mm × 4.6mm.
The embodiment of the present invention additionally provides a kind of application of hydroxypropyl-β-cyclodextrin, using reversed-phased high performace liquid chromatographic
The Alpha-Methyl androstane-4,9 (11) of separating chiral enantiomer 16-diene-3,17- diketone and 16 Beta-methyl androstane-4,9 (11)-diene-
During 3,17- diketone, it is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, hydroxypropyl-β-cyclodextrin and original
Beginning mobile phase is combined into reverse-phase chromatography mobile phase.Specific separation process can refer to use reversed phase high efficiency provided in an embodiment of the present invention
The method of liquid chromatography separating chiral enantiomer.The original mobile phase can be acetonitrile or methanol.
The embodiment of the present invention selects hydroxypropyl-β-cyclodextrin as Mobile Phase Additives, because hydroxypropyl-β-cyclodextrin exists
Solubility is larger in water, therefore does not result in chromatographic column blocking as Mobile Phase Additives, it is possible to prevente effectively from conventional beta-
Cyclodextrin easily causes the defect of chromatographic column blocking.
Relative to traditional enantiomers separation method, such as Chiral stationary phase liquid chromatography method, this method is used
Chromatographic column be chiral chromatographic column, because chiral chromatographic column is by special due to a class such as polymer with optically active monomer solid
The chromatographic column very made, it is expensive and selective stronger, the separation of a certain class material is only applicable to, therefore use this method pair
Chiral enantiomer separated spent by cost it is higher, and complex operation.It is efficient compared to above-mentioned traditional chiral stationary phase
Liquid chromatography, when being separated according to the chiral enantiomer of methods described of the embodiment of the present invention, uses conventional chromatogram post
Efficiently separating for chiral enantiomer is realized, it is simple to operate, it is with low cost, it is more economical to be applicable, it is with a wide range of applications.
To make those skilled in the art more fully understand the present invention, illustrate this hair below by way of multiple specific embodiments
The method of the use reversed-phased high performace liquid chromatographic separating chiral enantiomer of bright embodiment.
Embodiment 1
(1) α of about 5mg 16 (β)-methyl androstane -4,9 (11)-diene -3,17- diketone sample is weighed, using acetonitrile as solvent
Carry out sample dissolving.
(2) by step (1) sample solution after 0.2 μm of membrane filtration, respectively with acetonitrile/water, acetonitrile/0.02g/ml
The hydroxypropyl-β-cyclodextrin aqueous solution, acetonitrile/0.03g/ml hydroxypropyl-β-cyclodextrins aqueous solution, acetonitrile/0.04g/ml hydroxypropyls-
The beta-schardinger dextrin aqueous solution and acetonitrile/0.06g/mL hydroxypropyl-β-cyclodextrins aqueous solution are mobile phase, inject high performance liquid chromatograph
It is measured.
Instrument and reagent
The high performance liquid chromatographs of Agilent Agilent 1260
Chromatographic column：Eclipse Plus-C8 250mm*4.6mm, 5 μm
Reagent：Acetonitrile (chromatographically pure), distilled water (UP), hydroxypropyl-β-cyclodextrin
Mobile phase ratio：Acetonitrile:The hydroxypropyl-β-cyclodextrin aqueous solution=40:60
The flow velocity of mobile phase：1.0ml/min
The sample size of chiral enantiomer：20μL
The embodiment of the present invention to 16 Alpha-Methyl androstanes -4,9 (11)-diene -3 under various concentrations hydroxypropyl-β-cyclodextrin,
The height of 17- diketone (α compounds) and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone (beta compounds) chiral enantiomer
Effect liquid phase chromatogram figure, that is, obtain Fig. 1.
In Fig. 2, curve 1 is the high-efficient liquid phase chromatogram of α compounds, and curve 2 is the high-efficient liquid phase chromatogram of beta compounds,
Curve 3, curve 4, curve 5, curve 6 and curve 7 are the high-efficient liquid phase chromatogram of α compounds and beta compounds mixture, curve 1,
The mobile phase of curve 2 and curve 3 is acetonitrile and water, and the mobile phase of curve 4 is acetonitrile and 0.02mg/ml hydroxypropyl-β-cyclodextrins,
The mobile phase of curve 5 is acetonitrile and 0.03mg/ml hydroxypropyl-β-cyclodextrins, and the mobile phase of curve 6 is acetonitrile and 0.04mg/ml
Hydroxypropyl-β-cyclodextrin, the mobile phase of curve 7 is acetonitrile and 0.06mg/ml hydroxypropyl-β-cyclodextrins.
The embodiment of the present invention has also measured 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers
Separation parameter under the conditions of different Chiral Mobile Phase Additives, separation parameter is as shown in table 1.
16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers exist in table 1, the embodiment of the present invention
Separation parameter under the conditions of different Chiral Mobile Phase Additives
Understood based on Fig. 2 and the data of table 1, under the conditions of using Eclipse Plus-C8 chromatographic columns as reverse-phase chromatography stationary phase,
During using the mixture of acetonitrile and water as mobile phase, the retention time of α compounds is 25.9min, and the retention time of beta compounds is
25.2min, when both mix, separating degree is 0.9, it is impossible to preferably split the chiral enantiomer.
It is mobile phase to above-mentioned chiral enantiomer using the mixture of acetonitrile and the 0.02g/ml hydroxypropyl-β-cyclodextrin aqueous solution
When being split, the separating degree of two kinds of chiral enantiomers is 1.2.With acetonitrile and the 0.03g/ml hydroxypropyl-β-cyclodextrin aqueous solution
Mixture when being split for mobile phase to above-mentioned chiral enantiomer, the separating degrees of two kinds of chiral enantiomers is 1.3.With acetonitrile
When being split with the mixture of the 0.04g/ml hydroxypropyl-β-cyclodextrin aqueous solution for mobile phase to above-mentioned chiral enantiomer, two
The separating degree for planting chiral enantiomer is 1.5, and two kinds of chiral enantiomers can reach preferable separating effect.By hydroxy propyl-Beta-ring
When the concentration of dextrin in aqueous solution brings up to 0.06g/ml, the separating degree of above-mentioned chiral enantiomer is not obviously improved.
Embodiment 2
(1) the Alpha-Methyl androstanes-4,9 (11) of about 5mg 16-diene-3,17- diketone and 16 Beta-methyl androstanes-4,9 are weighed
(11) biased sample of-diene -3,17- diketone, dissolves by solvent of acetonitrile to above-mentioned biased sample.
(2) by the biased sample solution obtained by step (1) after 0.2 μm of membrane filtration, respectively with acetonitrile and 0.04g/ml
The mixture of the mixture of the hydroxypropyl-β-cyclodextrin aqueous solution, methanol and the 0.04g/ml hydroxypropyl-β-cyclodextrin aqueous solution is stream
Dynamic phase, injection high performance liquid chromatograph is measured.
Instrument and reagent
High performance liquid chromatograph：The high performance liquid chromatographs of Agilent Agilent 1260.
Chromatographic column：Eclipse Plus-C8 (250mm*4.6mm, 5 μm).
Reagent：Methanol (chromatographically pure), distilled water (UP), hydroxypropyl-β-cyclodextrin.
Mobile phase ratio：VMethanol:The hydroxypropyl-β-cyclodextrin aqueous solution=40:60.
The flow velocity of mobile phase：1.0ml/min.
The sample size of chiral enantiomer：20μL.
When other chromatographic conditions are identical, using acetonitrile/0.04g/ml hydroxypropyl-β-cyclodextrins aqueous solution as the mobile phase chirality
Enantiomer retention time is respectively 18.9 and 20.2min, and separating degree is 1.5；With methanol/0.04g/ml hydroxypropyl-β-cyclodextrins
For mobile phase, the chiral enantiomer is remained in chromatographic column the aqueous solution in 60min, and required time is longer.
Embodiment 3
(1) the Alpha-Methyl androstanes-4,9 (11) of about 5mg 16-diene-3,17- diketone and 16 Beta-methyl androstanes-4,9 are weighed
(11) biased sample of-diene -3,17- diketone, dissolves by solvent of acetonitrile to above-mentioned biased sample.
(2) by the biased sample solution obtained by step (1) through 0.2 μm of membrane filtration, afterwards with acetonitrile and 0.04g/ml hydroxyls
The mixture of the propyl-beta-cyclodextrin aqueous solution is mobile phase, and injection high performance liquid chromatograph is measured, mobile phase ratio difference
For：VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=45:55、VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=40:60、VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=35:65、
VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=30:70.
Instrument and reagent
High performance liquid chromatograph：The high performance liquid chromatographs of Agilent Agilent 1260.
Chromatographic column：Eclipse Plus-C8 (250mm*4.6mm, 5 μm).
Reagent：Acetonitrile (chromatographically pure), distilled water (UP), hydroxy-beta-cyclodextrin.
The flow velocity of mobile phase：1.0ml/min.
The sample size of chiral enantiomer：20μL.
The embodiment of the present invention is to 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers in difference
Chromatogram under the conditions of mobile phase is tested, and test result is as shown in Figure 3.In Fig. 3, curve 1 is mobile phase ratio VAcetonitrile:
VThe hydroxypropyl-β-cyclodextrin aqueous solution=30:90 high-efficient liquid phase chromatogram, curve 2 is mobile phase ratio VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=30:70
High-efficient liquid phase chromatogram, curve 3 be mobile phase ratio VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=35:65 high-efficient liquid phase chromatogram, it is bent
Line 4 is mobile phase ratio VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=40:60 high-efficient liquid phase chromatogram, curve 5 is mobile phase ratio VAcetonitrile:
VThe hydroxypropyl-β-cyclodextrin aqueous solution=45:55 high-efficient liquid phase chromatogram.
The embodiment of the present invention has also measured 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers
Separation parameter under the conditions of different mobile phases, separation parameter is as shown in table 2.
16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers exist in table 2, the embodiment of the present invention
Separation parameter under the conditions of different mobile phases
From Fig. 3 and table 2, as mobile phase ratio VAcetonitrile:V- hydroxypropyl-β-cyclodextrin the aqueous solutionFor 30:70、35:65 and 40:, should when 60
Chiral enantiomer can obtain good fractionation, and work as VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solution=40:There is appropriate retention time when 60, and
As mobile phase ratio VAcetonitrile:V- hydroxypropyl-β-cyclodextrin the aqueous solutionFor 45:When 55, the chiral enantiomer separating degree is only 0.9, it is impossible to meet its good
Good separation, as mobile phase ratio VAcetonitrile:VThe hydroxypropyl-β-cyclodextrin aqueous solutionFor 30:When 90, retention time is longer, causes operation time-consuming longer.Cause
This, original mobile phase and the volume ratio of the hydroxypropyl-β-cyclodextrin aqueous solution are preferably 40:60~30:70.
Embodiment 4
(1) the Alpha-Methyl androstanes-4,9 (11) of about 5mg 16-diene-3,17- diketone and 16 Beta-methyl androstanes-4,9 are weighed
(11) biased sample of-diene -3,17- diketone, dissolves by solvent of acetonitrile to above-mentioned biased sample.
(2) by the biased sample solution obtained by step (1) through 0.2 μm of membrane filtration, afterwards with acetonitrile and 0.04g/ml hydroxyls
The mixture of the propyl-beta-cyclodextrin aqueous solution is mobile phase, respectively using ZORBAX SB-C18 and Eclipse Plus-C8 as color
Stationary phase is composed, injection high performance liquid chromatograph is measured.
Instrument and reagent
High performance liquid chromatograph：The high performance liquid chromatographs of Agilent Agilent 1260.
Chromatographic column：250mm*4.6mm, 5 μm.
Reagent：Acetonitrile (chromatographically pure), distilled water (UP), hydroxypropyl-β-cyclodextrin.
Mobile phase ratio：Acetonitrile:The hydroxypropyl-β-cyclodextrin aqueous solution=40:60
The flow velocity of mobile phase：1.0ml/min.
The sample size of chiral enantiomer：20μL.
The embodiment of the present invention is to 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers in difference
Chromatogram under chromatographic column is tested, and test result is as shown in figure 4, in Fig. 4, curve A is that ZORBAX SB-C18 are corresponding
High-efficient liquid phase chromatogram, curve B is the corresponding high-efficient liquid phase chromatograms of Eclipse Plus-C8,
And 16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomer has been measured in different chromatograms
Separation parameter under post, separation parameter is as shown in table 3.
16 α (β)-methyl androstane -4,9 (11)-diene -3,17- diketone chiral enantiomers exist in table 3, the embodiment of the present invention
Separation parameter under different chromatographic columns
From Fig. 4 and the data of table 3, under other chromatographic condition the same terms, Eclipse Plus-C8 chromatographic columns are utilized
When carrying out the enantiomers separation, its retention time is respectively 18.9 and 20.2min, and separating degree is 1.5, the time it is appropriate and
The good fractionation of chiral enantiomer can be achieved, and its retention time is 32.0 Hes when utilizing the fractionation of ZORBAX SB-C18 chromatographic columns
33.9min, the time is longer.
Above to a kind of method for using reversed-phased high performace liquid chromatographic separating chiral enantiomer provided by the present invention with
And a kind of application of hydroxypropyl-β-cyclodextrin is described in detail, principle of the specific case used herein to the present invention
And embodiment is set forth, the explanation of above example is only intended to help the method and its core that understand the present invention to think
Think；Simultaneously for those of ordinary skill in the art, according to the thought of the present invention, in specific embodiments and applications
It will change, in summary, this specification content should not be construed as limiting the invention.
Claims (10)
1. a kind of method of use reversed-phased high performace liquid chromatographic separating chiral enantiomer, it is characterised in that including：
It is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, obtains required reverse-phase chromatography mobile phase；
Using the reverse-phase chromatography mobile phase and reverse-phase chromatography stationary phase, the chiral Alpha-Methyl androstane-4,9 (11) of enantiomer 16-
Diene -3,17- diketone and 16 Beta-methyl androstanes -4,9 (11)-diene -3,17- diketone carry out RPLC separation.
2. according to the method described in claim 1, it is characterised in that described by hydroxypropyl-β-cyclodextrin
Include as additive added to original mobile phase：
The hydroxypropyl-β-cyclodextrin aqueous solution is added in the original mobile phase.
3. method according to claim 2, it is characterised in that the original mobile phase and the hydroxypropyl-β-cyclodextrin
The volume ratio of the aqueous solution is 40:60~30:70；
The concentration of the hydroxypropyl-β-cyclodextrin aqueous solution is 0.02~0.04g/ml.
4. according to the method described in claim 1, it is characterised in that to 16 Alpha-Methyl androstane -4,9 (11)-diene -3,
When 17- diketone and the 16 Beta-methyl androstane -4,9 (11)-diene -3,17- diketone carry out RPLC separation, institute
The flow velocity for stating reverse-phase chromatography mobile phase is 1~1.2ml/min.
5. according to the method described in claim 1, it is characterised in that the original mobile phase is acetonitrile or methanol.
6. according to the method described in claim 1, it is characterised in that the reverse-phase chromatography stationary phase is Eclipse Plus-C8
Chromatographic column or ZORBAX SB-C18 chromatographic columns.
7. method according to claim 6, it is characterised in that the Eclipse Plus-C8 chromatographic columns or described
The column temperature of ZORBAX SB-C18 chromatographic columns is 35~40 DEG C.
8. the method stated according to claim 6, it is characterised in that the Eclipse Plus-C8 chromatographic columns or the ZORBAX
The size of SB-C18 chromatographic columns is 250mm × 4.6mm.
9. a kind of application of hydroxypropyl-β-cyclodextrin, it is characterised in that use reversed-phased high performace liquid chromatographic separating chiral mapping
When the Alpha-Methyl androstane-4,9 (11) of body 16-diene-3,17- diketone and 16 Beta-methyl androstanes-4,9 (11)-diene-3,17- diketone,
It is added to hydroxypropyl-β-cyclodextrin as additive in original mobile phase, the hydroxypropyl-β-cyclodextrin and the primary flow
It is dynamic combined into reverse-phase chromatography mobile phase.
10. the application stated according to claim 9, it is characterised in that the original mobile phase is acetonitrile or methanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710262396.0A CN106996964A (en) | 2017-04-20 | 2017-04-20 | Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710262396.0A CN106996964A (en) | 2017-04-20 | 2017-04-20 | Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106996964A true CN106996964A (en) | 2017-08-01 |
Family
ID=59435503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710262396.0A Pending CN106996964A (en) | 2017-04-20 | 2017-04-20 | Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106996964A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164464A (en) * | 2018-02-06 | 2018-06-15 | 美药星（南京）制药有限公司 | A kind of analysis method that can be kept completely separate (-)-huperzine and (+)-huperzine |
| CN108535398A (en) * | 2018-04-04 | 2018-09-14 | 福州海王福药制药有限公司 | A method of Tiagabine Hydrochloride chiral enantiomer is detached using reversed-phased high performace liquid chromatographic |
| WO2019041762A1 (en) * | 2017-08-31 | 2019-03-07 | 深圳创维－Rgb电子有限公司 | Advertisement delivery method, device and apparatus, and storage medium |
| CN110736798A (en) * | 2019-09-29 | 2020-01-31 | 方达医药技术（苏州）有限公司 | Separation method of chiral isomers by electrochemically modified liquid chromatography |
| CN111426769A (en) * | 2020-04-22 | 2020-07-17 | 江西省药品检验检测研究院 | Method for resolving chiral mobile phase additive HP L C of nadifloxacin enantiomer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113211A (en) * | 2013-02-04 | 2013-05-22 | 浙江工业大学 | Splitting method for racemic 2-benzene propanoic acid |
| CN104458945A (en) * | 2014-01-24 | 2015-03-25 | 广东东阳光药业有限公司 | Separation and measurement method of besifloxacin hydrochloride and isomer of besifloxacin hydrochloride |
| CN105954419A (en) * | 2016-06-01 | 2016-09-21 | 重庆华邦胜凯制药有限公司 | Method for separating and determining levocetirizine hydrochloride and enantiomer thereof through HPLC method |
-
2017
- 2017-04-20 CN CN201710262396.0A patent/CN106996964A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113211A (en) * | 2013-02-04 | 2013-05-22 | 浙江工业大学 | Splitting method for racemic 2-benzene propanoic acid |
| CN104458945A (en) * | 2014-01-24 | 2015-03-25 | 广东东阳光药业有限公司 | Separation and measurement method of besifloxacin hydrochloride and isomer of besifloxacin hydrochloride |
| CN105954419A (en) * | 2016-06-01 | 2016-09-21 | 重庆华邦胜凯制药有限公司 | Method for separating and determining levocetirizine hydrochloride and enantiomer thereof through HPLC method |
Non-Patent Citations (3)
| Title |
|---|
| P. K. ZARZYCKI等: "Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| 姚彤炜: "《体内药物分析》", 30 June 2012, 浙江大学出版社 * |
| 徐其进等: "疏水条件下甾体化合物的分离及环糊精的作用", 《分析化学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019041762A1 (en) * | 2017-08-31 | 2019-03-07 | 深圳创维－Rgb电子有限公司 | Advertisement delivery method, device and apparatus, and storage medium |
| CN108164464A (en) * | 2018-02-06 | 2018-06-15 | 美药星（南京）制药有限公司 | A kind of analysis method that can be kept completely separate (-)-huperzine and (+)-huperzine |
| CN108164464B (en) * | 2018-02-06 | 2019-09-17 | 美药星（南京）制药有限公司 | A kind of analysis method that can be kept completely separate (-)-huperzine Yu (+)-huperzine |
| CN108535398A (en) * | 2018-04-04 | 2018-09-14 | 福州海王福药制药有限公司 | A method of Tiagabine Hydrochloride chiral enantiomer is detached using reversed-phased high performace liquid chromatographic |
| CN110736798A (en) * | 2019-09-29 | 2020-01-31 | 方达医药技术（苏州）有限公司 | Separation method of chiral isomers by electrochemically modified liquid chromatography |
| CN111426769A (en) * | 2020-04-22 | 2020-07-17 | 江西省药品检验检测研究院 | Method for resolving chiral mobile phase additive HP L C of nadifloxacin enantiomer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106996964A (en) | Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer | |
| West | Current trends in supercritical fluid chromatography | |
| Sun et al. | Simultaneous rapid quantification of ginsenoside Rg1 and its secondary glycoside Rh1 and aglycone protopanaxatriol in rat plasma by liquid chromatography–mass spectrometry after solid-phase extraction | |
| Hu et al. | Determination of estrogens in pork and chicken samples by stir bar sorptive extraction combined with high-performance liquid chromatography–ultraviolet detection | |
| Li et al. | Fabrication and evaluation of chiral monolithic column modified by β-cyclodextrin derivatives | |
| GÖrÖg | Recent advances in the analysis of steroid hormones and related drugs | |
| Franco et al. | Enantiomer separation by countercurrent chromatography using cinchona alkaloid derivatives as chiral selectors | |
| Yin et al. | Determination of hyperoside and isoquercitrin in rat plasma by membrane‐protected micro‐solid‐phase extraction with high‐performance liquid chromatography | |
| Geryk et al. | Enantioselective separation of biologically active basic compounds in ultra-performance supercritical fluid chromatography | |
| Lü et al. | Rapid separation and determination of structurally related anthraquinones in Rhubarb by pressurized capillary electrochromatography | |
| Inoue et al. | An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation | |
| Zhang et al. | Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector | |
| Hofstetter et al. | Supercritical fluid chromatography: From science fiction to scientific fact | |
| Wang et al. | Sodium hyaluronate-functionalized urea-formaldehyde monolithic column for hydrophilic in-tube solid-phase microextraction of melamine | |
| D’Orazio et al. | Enantiomeric separation of some chiral analytes using amylose 3, 5-dimethylphenylcarbamate covalently immobilized on silica by nano-liquid chromatography and capillary electrochromatography | |
| Zhang et al. | Investigation of fusidic acid as a chiral selector in capillary electrophoresis | |
| Roy et al. | Effect of water addition to super/sub-critical fluid mobile-phases for achiral and chiral separations | |
| Yang et al. | Porous capillary monolithic column coupled with ultrahigh performance liquid chromatography-tandem mass spectrometry for fast and effective separation and determination of estrogens | |
| Shehzad et al. | Rational development of a selection model for solvent gradients in single‐step separation of ginsenosides from P anax ginseng using high‐speed counter‐current chromatography | |
| Dias et al. | Chromatography of oxysterols | |
| Guo et al. | Comparison of vancomycin-immobilized chiral stationary phase with its derivative for enantioseparation of drugs in high-performance liquid chromatography | |
| Guo et al. | Dummy-template Pickering emulsion imprinted microspheres online pretreatment and analysis for the estrogens in cosmetics | |
| Patel et al. | Evolution of strategies to achieve baseline separation of ten anionic, water-soluble sulfated estrogens via achiral packed column supercritical fluid chromatography | |
| Bradow et al. | Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds | |
| Joseph et al. | Application of SFC for bioanalysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170801 |