Study Of Lamivudine With Novel Chiral Separation System

Using chiral selectors to construct chiral separation environment,it is a conventional research method to achieve enantiomeric separation.The recent research progress of chiral separation,especially single separation system and multiple separation system are reviewed in this paper.This paper also briefly overview recent researches about lamivudine.Due to the current situation of Lamivudine chiral separation system rarely reported,this paper focuses on the new methods to achieve chiral separation for lamivudine.Lamivudine is currently widely used as cytosine nucleoside antiviral drug,since it has been applied to the treatment of Hepatitis B Virus(HBV)and Human Immunodeficiency Virus(HIV).Because the clinical administration is levorotatory form,there is cell toxicity in the dextrorotatory form of lamivudine.Therefore,establishing effective chiral separation systems of lamivudine has a wide range of applications and academic significance.In this research,three kinds of own intellectual property rights ?-cyclodextrin derivatives(one is authorized patent)are chosen as chiral selectors,by means of high performance capillary electrophoresis(HPCE)and high performance liquid chromatography(HPLC),to construct chiral separation environment for Lamivudine.Chiral selectors of bis-(6-oxogen-m-carboxyl benzene sulfonyl)-?-cyclodextrin(?-CD-M1),bis-(6-oxygen-m-nitro benzene sulfonyl)-?-cyclodextrin(?-CD-N2)and bis-[6-oxygen-(3-deoxy citric acid monoester-4)]-?-cyclodextrin(?-CD-B2)are respectively used in this thesis.The HPCE chiral monolithic column is prepared within 75?m capillary column.In order to verify its inner column condition,field emission scanning electron microscopy(SEM)is used to inspect inner wall of monolithic column.In HPCE,?-CD-M1 and ?-CD-N2 chiral column are used to separate lamivudine enantiomers respectively.Through discussing separation principles and optimizing the separation conditions,the enantioseparation methods within two chiral selectors have been established.The HPCE separation conditions of ?-CD-M1 chiral column are as follows: Tris-phosphate buffer's pH is 5.1,Tris' s concentration is 50mmol/L,separation voltage 18 kV,detection wavelength 270 nm.Under this conditions,lamivudine enantiomers have separated in 10 minutes,Rs reaches 12.87.The optimum separation conditions in ?-CD-N2 HPCE chiral column are as follows: Tris-phosphate buffer's pH is 4.1,Tris' s concentration is 50mmol/L,separation voltage-23 kV,detection wavelength 270 nm,the analysis time is 20 min,Rs reaches 55.95.In ?-CD-B2 HPCE chiral column,when the Tris-phosphate buffer's pH is 2.8,Tris' s concentration is 50mmol/L,detection wavelength 270 nm,separation voltage-23 kV,Rs reaches 48.35.Comparing these experimental results,lamivudine enantiomers have been separated in three kinds of chiral selector respectively.Acid performance of ?-CD-B2 material is stronger.Lamivudine enantiomers' s linearity range in ?-CD-M1 chiral HPCE monolithic column is narrower than in ?-CD-N2 chiral HPCE monolithic column.However,it is showed that there exists mesh structure inside column by SEM,the ?-CD-N2 chiral HPCE monolithic column material bonded not dense enough,and the stability lower than ?-CD-M1 chiral HPCE monolithic column.In order to overcome their limitation,two kinds of chiral selector are mixed in a ratio to prepare chiral HPCE monolithic column.The mixed ratio materials used as a main functional monomer have been mixed with the other functional monomer,crosslinking agent,pore-foaming agent and initiator agent,and so on.Filling in the empty capillary column,the mixed material chiral monolithic column has been produced by situ polymerization under heating-up conditions in the column.Its characterization of inner column by SEM,?-CD-M1: ?-CD-N2=1:2 is best mixing ratio.Then lamivudine and bupivacaine are chosen as analyte to verify the mixed monolithic column separation ability.Preliminary results show that under the condition of operating voltage 18 kV,50mmol/L Tris-phosphate buffer pH4.8~5.2,lamivudine diastereomer has a signs of separation,RS reaches 13.20.In the range of pH4.0~5.0,the bupivacaine enantiomers are separated.To further constructing chiral separation system of lamivudine enantiomer or diastereomer,?-CD-B2 is used as chiral stationary phase to construct chiral separation environment in HPLC,and lamivudine enantiomers obtain chiral separation.The optimal separation conditions are as follows: citric acid-triethylamine buffer is pH4.0,the concentration of citric acid is 5mmol/L,acetonitrile as the organic additive,V buffer: Vacetonitrile=45%:55%,flow velocity is 0.80mL/min,detection wavelength is 270 nm,column temperature is 20?,RS reaches 3.97.To verify the separation capabilities of ?-CD-M1 and ?-CD-N2 for lamivudine enantiomers in HPLC,lamivudine enantiomers are separated under the same mobile phase system respectively in different chiral stationary phase.The results are no separation.The experiment results show that ?-CD-B2 as chiral selector in HPLC,carboxyl content of ?-CD-B2 strengthens the acting forces difference between lamivudine enantiomers and stationary phase.Even though there is no ? bond of benzene ring in ?-CD-B2,its ability to separate does not been affected.The separation performance of ?-CD-M1 and ?-CD-N2 for Lamivudine enantiomer in HPLC is significantly weaker than ?-CD-B2.The optimum separation conditions of propafenone enantiomer in ?-CD-N2 HPCE chiral monolithic column are investigated in this thesis,as followes: Trisphosphate buffer is pH4.0,Tris' s concentration is 40mmol/L,operating voltage is-25 kV,detection wavelength is 254 nm,analysis time is 15 min,RS reaches 52.82.The separation mechanism is discussed preliminarily.There is ?-? conjugation between propafenone benzene and ?-CD-N2.The synergy between nitro group,sulfonyl group and propafenone enantiomer,enhances the chiral isomer selectivity differences between ?-CD-N2 and propafenone enantiomers.Propafenone enantiomers are separated under suitable conditions.