CN102323368B - Method for analyzing chiral drug enantiomers in biological body fluid through column-switching liquid chromatography - Google Patents
Method for analyzing chiral drug enantiomers in biological body fluid through column-switching liquid chromatography Download PDFInfo
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Abstract
The invention relates to a method for analyzing chiral drug enantiomers in biological body fluid through column-switching liquid chromatography, which comprises the following steps: (1) preparing a chiral drug racemate standard product into a standard solution having a certain concentration; and diluting the standard solution into a plasma sample having a certain concentration; (2) centrifuging the plasma sample, and taking the supernate as an injected sample for later use; (3) preparing a restricted-access material column; (4) preparing a chiral fixed phase; (5) putting the restricted-access material column used as a pretreatment column and the chiral fixed phase used as an analytical column into a column-switching restricted-access material-chiral fixed phase high performance liquid chromatography system; and (6) injecting the supernate of the plasma sample into a sampler in the column-switching restricted-access material-chiral fixed phase high performance liquid chromatography system; and by using borate buffer-methanol or water-methanol as a mobile phase of the pretreatment column, separating with borate buffer-isopropanol-anhydrous alcohol to obtain a chromatogram of different single enantiomers having different appearance times. The invention has the characteristics of high speed, high convenience and high sensitivity.
Description
Technical field
The present invention relates to column switching technique application and enantiomers of chiral drugs field, relate in particular to the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid.
Background technology
From world-shaking Adverse drug events last century is several, people recognize gradually: the small difference between enantiomers of chiral drugs on spatial configuration causes its aspect such as pharmacologically active, metabolic process and toxicity in human body all to have significant difference.Along with scientific and technical development, the chiral drug single enantiomer is carried out studying in more deep body, for estimating the biologic activity between each enantiomorph of chiral drug, difference between the effects and the exploitation single enantiomer new drug of illustrating between the chirality enantiomorph all have very great meaning.
The physicochemical property of each enantiomorph of chiral drug are similar, optical activity difference only, analyze the mensuration difficulty large, mainly utilize at present modern chromatographic technique (as: high performance liquid chromatography, capillary electrophoresis, supercritical fluid chromatography, high-speed countercurrent chromatography, simulated moving bed chromatography method etc.) to realize the fractionation of each enantiomorph is measured; But the bulk concentration assay method of single enantiomer be take high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry as main.
Biological fluid mesostroma complicated component, the concentration of single enantiomer is low, it analyze to be measured and all to have that sample pre-treatments step complexity is loaded down with trivial details, the sample loss large, the method recovery and accuracy low, expend time in, expend the series of problems such as solvent, therefore set up quick in a kind of biological fluid, sensitive, the single enantiomer analysis determining method extremely is necessary accurately.
Summary of the invention
Technical matters to be solved by this invention be to provide a kind of fast, the method for enantiomers of chiral drugs in convenient, sensitive post switching-liquid-phase chromatographic analysis biological fluid.
For addressing the above problem, the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid of the present invention comprises the following steps:
(1) chiral drug raceme standard items are used to anhydrous alcohol solution at the temperature of 5～30 ℃, make the standard solution that concentration is 4mg/mL～5mg/mL; This standard solution with the blank plasma dilution, is made the plasma sample that concentration is 0.5mg/mL～1.5mg/mL at the temperature of 5～30 ℃;
(2) described plasma sample is put in 1mL～5mL tool plug centrifuge tube, with the centrifugal 3～10min of the rotating speed of 1000～8000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby;
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm;
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that chirality AE.Lichrom CE 4 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that chirality AE.Lichrom CE 7 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase II;
(5) described chiral stationary phase I is connected with described the limit into filled column respectively with described chiral stationary phase II, and using describedly limitting into filled column as pretreatment column, described chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between described pretreatment column and described analytical column, by polyetheretherketone (PEEK) pitch tube, be connected, this polyetheretherketone (PEEK) pitch tube is provided with transfer valve; The external UV-detector of described analytical column;
(6) the plasma sample supernatant of described step (2) gained being injected into to the switching of described post limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol or water-methanol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 230～350nm, the mobile phase flow velocity of pre-service is 0.5mL/min～2.0mL/min, the analysis flow rate of mobile phase is 0.5mL/min～2.0mL/min, under the condition that temperature is 15～40 ℃, separated, obtain the chromatogram of the different single enantiomers of different appearance times.
Chiral drug raceme standard items in described step (1) refer to Propranolol Hydrochloride raceme standard items.
Be 1min～5min the switching time of the transfer valve in described step (5).
Borate buffer solution in described step (6) in the pretreatment column mobile phase and the volume ratio of methyl alcohol or water and methyl alcohol are 30～98: 2～70.
The volume ratio of the borate buffer solution in described step (6) in the analytical column mobile phase and isopropyl alcohol, ethanol is 10～70: 10～40: 20～50.
Described borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L according to 1～8: the solution that the pH value of 2～9 volume ratio preparation is 7.0～9.5.
The present invention compared with prior art has the following advantages:
1, limitting into filler is a new chromatograph packing material family, their separation mechanism is mainly by the filler outside surface is carried out to suitable Hydrophilic modification, making large molecule in biological sample solution can not enter in the endoporus of filler goes, biomacromolecule is in the situation that be close to dead volume and removed by wash-out, and the bore area of such filler is still reversed phase extraction agent character, analyte is retained by hydrophobic effect and electrostatic interaction.Column switching technique is by changing the position of transfer valve, realize that the conversion between injector, different chromatographic column, detecting device connects, thereby realize separating of analyte and other interference component, reach the purpose of purifying and enrichment, and then complete the concentration determination to analyte.
The present invention has set up new post switching and has limit into filler-chiral stationary phase liquid chromatography pattern, and this pattern is utilized column switching technique, realizes that in biological fluid, the quick, online of chiral drug single enantiomer concentration detects.This pattern can realize the direct injection analysis of complex sample, realize the ON-LINE SEPARATION of other invalid components in analyte and biological fluid, reduced loaded down with trivial details step in traditional off-line Bio-specimen Preparation method, the loss of having avoided analyte to cause due to pre-treatment, shortened analysis time, improve automaticity, realized the online detection of quick, convenient, sensitive enantiomers of chiral drugs concentration.Experimental results show that: in the analyzing and testing of this pattern chiral drug single enantiomer in biological fluid, there is huge superiority.
2, the present invention utilizes the PEEK pipe to connect the interface of transfer valve, form transfer valve when diverse location, limit into the change between filled column, chiral stationary phase, detecting device stream, by changing the position of transfer valve, thus realize chiral drug single enantiomer concentration in biological fluid fast, on-line determination.
3, the present invention has the characteristics such as specificity is high, favorable reproducibility, highly sensitive, analysis speed is fast, simple to operate, and applicable to the pharmacokinetic of chiral drug single enantiomer.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
The chromatogram that Fig. 1 is the embodiment of the present invention 1 blank plasma.
Fig. 2 be embodiment of the present invention 1R (+)-, the chromatogram of S (-)-Propranolol Enantiomers.
The chromatogram that Fig. 3 is the embodiment of the present invention 2 blank plasmas.
Fig. 4 be embodiment of the present invention 2R (+)-, the chromatogram of S (-)-Propranolol Enantiomers.
Fig. 5 be embodiment of the present invention 3R (+)-, the chromatogram of S (-)-Propranolol Enantiomers.
Fig. 6 be embodiment of the present invention 4R (+)-, the chromatogram of S (-)-Propranolol Enantiomers.
Embodiment
The method of enantiomers of chiral drugs in embodiment 1 post switching-liquid-phase chromatographic analysis biological fluid comprises the following steps:
(1) by chiral drug raceme standard items---Propranolol Hydrochloride raceme standard items are used anhydrous alcohol solution at the temperature of 20 ℃, make the standard solution that concentration is 5mg/mL; This standard solution with blank plasma (referring to Fig. 1) dilution, is made the plasma sample that concentration is 0.5mg/mL at the temperature of 20 ℃.
(2) plasma sample is put in 1.5mL tool plug centrifuge tube, with the centrifugal 5min of the rotating speed of 4000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby.
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm.
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that chirality AE.Lichrom CE 4 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that chirality AE.Lichrom CE 7 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase II.
(5) chiral stationary phase I and chiral stationary phase II are connected with limitting into filled column respectively, and using limitting into filled column as pretreatment column, chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between pretreatment column and analytical column, by polyetheretherketone (PEEK) pitch tube, be connected, this polyetheretherketone (PEEK) pitch tube is provided with transfer valve; The external UV-detector of analytical column.Be 1min～5min the switching time of transfer valve.
Utilize PEEK pipe to connect transfer valve, the change of Realization analysis stream, by changing the position of transfer valve, realize the mensuration of removing Propranolol Hydrochloride enantiomorph concentration in interfering material in blood plasma and blood plasma of pretreatment column.
(6) the plasma sample supernatant of step (2) gained being injected into to post switching limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 293nm, the mobile phase flow velocity of pre-service is 1.0mL/min, the analysis flow rate of mobile phase is 0.8mL/min, under the condition that temperature is 25 ℃, separated, obtain the appearance time of R (+)-Propranolol Enantiomers in 16.5～18.9min scope, the appearance time of S (-)-Propranolol Enantiomers is chromatogram (referring to Fig. 2) in 19.0～22.2min scope.
Wherein: the borate buffer solution in the pretreatment column mobile phase and the volume ratio of methyl alcohol (mL/mL) are 95: 5.
The volume ratio of the borate buffer solution in the analytical column mobile phase and isopropyl alcohol, ethanol (mL/mL) is 40: 30: 30.
The solution that it is 8.5 according to the pH value of the volume ratio (mL/mL) of 1: 9 preparation that borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L.
The method of enantiomers of chiral drugs in embodiment 2 post switching-liquid-phase chromatographic analysis biological fluids comprises the following steps:
(1) by chiral drug raceme standard items---Propranolol Hydrochloride raceme standard items are used anhydrous alcohol solution at the temperature of 30 ℃, make the standard solution that concentration is 4mg/mL; This standard solution with blank plasma (referring to Fig. 3) dilution, is made the plasma sample that concentration is 1.5mg/mL at the temperature of 30 ℃.
(2) plasma sample is put in 2.0mL tool plug centrifuge tube, with the centrifugal 3min of the rotating speed of 2000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby.
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm.
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that chirality AE.Lichrom CE 4 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that chirality AE.Lichrom CE 7 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase II.
(5) chiral stationary phase I and chiral stationary phase II are connected with limitting into filled column respectively, and using limitting into filled column as pretreatment column, chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between pretreatment column and analytical column, by polyetheretherketone (PEEK) pitch tube, be connected, this polyetheretherketone (PEEK) pitch tube is provided with transfer valve; The external UV-detector of analytical column.Be 1min～5min the switching time of transfer valve.
Utilize PEEK pipe to connect transfer valve, the change of Realization analysis stream, by changing the position of transfer valve, realize the mensuration of removing Propranolol Hydrochloride enantiomorph concentration in interfering material in blood plasma and blood plasma of pretreatment column.
(6) the plasma sample supernatant of step (2) gained being injected into to post switching limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 230nm, the mobile phase flow velocity of pre-service is 1.2mL/min, the analysis flow rate of mobile phase is 1.0mL/min, under the condition that temperature is 30 ℃, separated, obtain the appearance time of R (+)-Propranolol Enantiomers in 25.0～28.2min scope, the appearance time of S (-)-Propranolol Enantiomers is chromatogram (referring to Fig. 4) in 28.2～31.3min scope.
Wherein: the volume ratio of the borate buffer solution in the pretreatment column mobile phase and methyl alcohol or water and methyl alcohol is 90: 10.
The volume ratio of the borate buffer solution in the analytical column mobile phase and isopropyl alcohol, ethanol is 50: 20: 30.
The solution that it is 9.0 according to the pH value of the volume ratio of 8: 2 preparation that borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L.
The method of enantiomers of chiral drugs in embodiment 3 post switching-liquid-phase chromatographic analysis biological fluids comprises the following steps:
(1) by chiral drug raceme standard items---Propranolol Hydrochloride raceme standard items are used anhydrous alcohol solution at the temperature of 5 ℃, make the standard solution that concentration is 4.5mg/mL; This standard solution with the blank plasma dilution, is made the plasma sample that concentration is 1.0mg/mL at the temperature of 5 ℃.
(2) plasma sample is put in 1mL tool plug centrifuge tube, with the centrifugal 10min of the rotating speed of 1000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby.
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm.
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that chirality AE.Lichrom CE 4 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that chirality AE.Lichrom CE 7 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase II.
(5) chiral stationary phase I and chiral stationary phase II are connected with limitting into filled column respectively, and using limitting into filled column as pretreatment column, chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between pretreatment column and analytical column, by polyetheretherketone (PEEK) pitch tube, be connected, this polyetheretherketone (PEEK) pitch tube is provided with transfer valve; The external UV-detector of analytical column.Be 1min～5min the switching time of transfer valve.
Utilize PEEK pipe to connect transfer valve, the change of Realization analysis stream, by changing the position of transfer valve, realize the mensuration of removing Propranolol Hydrochloride enantiomorph concentration in interfering material in blood plasma and blood plasma of pretreatment column.
(6) the plasma sample supernatant of step (2) gained being injected into to post switching limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol or water-methanol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 350nm, the mobile phase flow velocity of pre-service is 0.5mL/min, the analysis flow rate of mobile phase is 0.5mL/min, under the condition that temperature is 15 ℃, separated, obtain the appearance time of R (+)-Propranolol Enantiomers in 33.7～37.4min scope, the appearance time of S (-)-Propranolol Enantiomers is chromatogram (referring to Fig. 5) in 37.4～42.1min scope.
Wherein: the water in the pretreatment column mobile phase and the volume ratio of methyl alcohol are 98: 2.
The volume ratio of the borate buffer solution in the analytical column mobile phase and isopropyl alcohol, ethanol is 70: 10: 20.
The solution that it is 7.0 according to the pH value of the volume ratio of 5: 5 preparation that borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L.
The method of enantiomers of chiral drugs in embodiment 4 post switching-liquid-phase chromatographic analysis biological fluids comprises the following steps:
(1) by chiral drug raceme standard items---Propranolol Hydrochloride raceme standard items are used anhydrous alcohol solution at the temperature of 25 ℃, make the standard solution that concentration is 5mg/mL; This standard solution with the blank plasma dilution, is made the plasma sample that concentration is 0.5mg/mL at the temperature of 25 ℃.
(2) plasma sample is put in 5mL tool plug centrifuge tube, with the centrifugal 4min of the rotating speed of 8000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby.
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm.
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that chirality AE.Lichrom CE 4 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that chirality AE.Lichrom CE 7 chromatographic columns that 4.6mm, length are 250mm form the chiral stationary phase II.
(5) chiral stationary phase I and chiral stationary phase II are connected with limitting into filled column respectively, and using limitting into filled column as pretreatment column, chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between pretreatment column and analytical column, by polyetheretherketone (PEEK) pitch tube, be connected, this polyetheretherketone (PEEK) pitch tube is provided with transfer valve; The external UV-detector of analytical column.Be 1min～5min the switching time of transfer valve.
Utilize PEEK pipe to connect transfer valve, the change of Realization analysis stream, by changing the position of transfer valve, realize the mensuration of removing Propranolol Hydrochloride enantiomorph concentration in interfering material in blood plasma and blood plasma of pretreatment column.
(6) the plasma sample supernatant of step (2) gained being injected into to post switching limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol or water-methanol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 300nm, the mobile phase flow velocity of pre-service is 2.0mL/min, the analysis flow rate of mobile phase is 2.0mL/min, under the condition that temperature is 40 ℃, separated, obtain the appearance time of R (+)-Propranolol Enantiomers in 15.5～17.3min scope, the appearance time of S (-)-Propranolol Enantiomers is chromatogram (referring to Fig. 6) in 17.3～20.8min scope.
Wherein: the volume ratio of the borate buffer solution in the pretreatment column mobile phase and methyl alcohol or water and methyl alcohol is 30: 70.
The volume ratio of the borate buffer solution in the analytical column mobile phase and isopropyl alcohol, ethanol is 10: 40: 50.
The solution that it is 9.5 according to the pH value of the volume ratio of 7: 3 preparation that described borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L.
Claims (5)
1. the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid comprises the following steps:
(1) chiral drug raceme standard items are used to anhydrous alcohol solution at the temperature of 5～30 ℃, make the standard solution that concentration is 4mg/mL～5mg/mL; This standard solution with the blank plasma dilution, is made the plasma sample that concentration is 0.5mg/mL～1.5mg/mL at the temperature of 5～30 ℃; Described chiral drug raceme standard items refer to Propranolol Hydrochloride raceme standard items;
(2) described plasma sample is put in 1mL～5mL tool plug centrifuge tube, with the centrifugal 3～10min of the rotating speed of 1000～8000r/min, the accurate supernatant of drawing is placed in a centrifuge tube to make sample introduction standby;
(3) preparation is limit into filled column: the hydrophilic polyvinyl alcohol (PVA) of silica gel outside surface bonding that is 5 μ m by particle diameter, and the nonpolar hexylamine of its inside surface bonding, the formation internal diameter is anti-phase the limit into filled column of inside surface that 4.6mm, length are 45mm;
(4) prepare chiral stationary phase: the internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 3,5-dimethylaminobenzoic acid ester is that the chirality AE.Lichrom CE4 chromatographic column that 4.6mm, length are 250mm forms chiral stationary phase I; The internal diameter that the Silica Surface that is 5 μ m by particle diameter is coated with 4-methylamino benzoic ether is that the chirality AE.Lichrom CE7 chromatographic column that 4.6mm, length are 250mm forms chiral stationary phase II;
(5) described chiral stationary phase I is connected with described the limit into filled column respectively with described chiral stationary phase II, and using describedly limitting into filled column as pretreatment column, described chiral stationary phase puts into the post switching as analytical column and limits into filler-Chiral stationary phase liquid chromatography system; Between described pretreatment column and described analytical column, by the polyether-ether-ketone resin pipe, be connected, this polyether-ether-ketone resin pipe is provided with transfer valve; The external UV-detector of described analytical column;
(6) the plasma sample supernatant of described step (2) gained being injected into to the switching of described post limits into the injector in filler-Chiral stationary phase liquid chromatography system, take borate buffer solution-methyl alcohol or water-methanol as the pretreatment column mobile phase, take borate buffer solution-isopropyl alcohol-absolute ethyl alcohol as the analytical column mobile phase, detecting wavelength in ultraviolet is 230～350nm, the mobile phase flow velocity of pre-service is 0.5mL/min～2.0mL/min, the analysis flow rate of mobile phase is 0.5mL/min～2.0mL/min, under the condition that temperature is 15～40 ℃, separated, obtain the chromatogram of the different single enantiomers of different appearance times.
2. the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid as claimed in claim 1, it is characterized in that: be 1min～5min the switching time of the transfer valve in described step (5).
3. the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid as claimed in claim 1 is characterized in that: the borate buffer solution in described step (6) in the pretreatment column mobile phase and the volume ratio of methyl alcohol or water and methyl alcohol are 30～98: 2～70.
4. the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid as claimed in claim 1 is characterized in that: the volume ratio of the borate buffer solution in described step (6) in the analytical column mobile phase and isopropyl alcohol, absolute ethyl alcohol is 10～70: 10～40: 20～50.
5. the method for enantiomers of chiral drugs in post switching-liquid-phase chromatographic analysis biological fluid as described as claim 3 or 4 is characterized in that: described borate buffer solution refers to the BAS of the borax soln of 0.05mol/L and 0.2mol/L according to 1～8: the solution that the pH value of 2～9 volume ratio preparation is 7.0～9.5.
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