Preparation Of Novel Bridged Bis(?-Cyclodextrin)-bonded Stationary Phases And Their Application In Chiral Separation

At present,chiral compounds are increasingly used in medicine,chemicals,foods,and pesticides.Modern scientific data reveal that many enantiomers have certain differences in drug activity,biological toxicity,metabolic pathways,and environmental migration.The use of chiral compounds will bring new problems to drug safety,food safety and environmental safety,and will directly affect people's health.Therefore,the development of a new method for the separation and analysis of enantiomers has important research significance for monitoring the quality of chiral drugs,assessing the residues of chiral pesticides in foods,and tracking the chiral pollutants in the environment.The similarities in the structure and properties of chiral enantiomers present unprecedented challenges to the selectivity of chromatographic separation materials.In this paper,the chiral separation selectivity was improved based on the co-inclusive effect between multi-cavities of bridged cyclodextrins.Two novel bridged bicyclodextrin chiral bonding stationary phases with two different bridging groups were prepared,respectively.After the characterization of chemical structure,the chiral chromatographic properties of the two new stationary phases were evaluated by using different structural solutes as probes in reversed-phase chromatography and polar organic mode,respectively.It has been found that the separation selectivity of bridged cyclodextrin stationary phase was generally better than that of mono-cyclodextrin stationary phase,which should be related to the cooperative inclusion of the adjacent cavity of bridged bicyclodextrin.Based on the optimization of chromatographic conditions,new methods for the determination of the enantiomeric content of drugs in tablets,blood,feed and meat foods were developed using the newly prepared bridged cyclodextrin columns,which service for drug safety and food safety analysis.The main work of this dissertation is related to the preparation,evaluation and separation mechanism of novel bridged bis-??-cyclodextrin?-bonded stationary phase,as well as the separation and application of relevant research work,including the following aspects:The first part briefly summarizes the differences in enantiomers of chiral compounds,leading to the importance of chiral enantiomer resolution.The commonly used chiral resolution methods,basic principles and applications were systematically summarized.The progress of high performance liquid chromatography with chiral stationary phases was outlined.The derivatized cyclodextrin type chiral stationary phases and their bonding methods were briefly introduced.The latest applications of today's click chemistry reaction and ordered mesoporous chromatography matrix materials.This is used as the theoretical basis for the topic selection and starting point of work.In the second part,a bridged ethylenediamino double?-cyclodextrin was synthesized for the first time.It was used as a chiral ligand and bonded to the surface of silica gel with active isocyanatopropyl triethoxysilane.A novel bridged ethylenediamino bis??-cyclodextrin?-bonded stationary phase?EBCDP?was obtained.The chemical structures and morphologies of the ligands and the stationary phase were characterized by mass spectrometry?MS?,elemental analysis?EA?,infrared spectroscopy?IR?,and scanning electron microscopy?SEM?.The 21 chiral drugs including acidic,basic,neutral and amphoteric drugs were used as probes to systematically evaluate the chiral chromatographic performance of the new stationary phase.The results show that the prepared bridging cyclodextrin stationary phase is suitable for a variety of separation modes and successfully and systematically resolved most enantiomers ofdifferent structural drugs,most of which can be completely separated.The resolution of 2'-hydroxyflavanone was 4.35 in reversed-phase chromatography.In the polar organic solvent mode,all 8?--blocker drugs were resolved,among them the resolution of arotinolol enantiomers was 2.05.In the ligand-exchange mode,the underivatised DL-proline,DL-tert-leucine and DL-methionine were successfully resolved by using 0.1 mmol/L CuSO₄ and 0.05mol/L KH₂PO₄ aqueous solution/methanol as mobile phases within 15 min.The good chromatographic performance of bridged cyclodextrin stationary phase was benefited from the synergistic inclusion between cavities and the auxiliary coordination of EDTA bridging groups,so that the chiral selectivity and resolution range of new stationary phases can be improved.The third part,The resolution of the enantiomers of arotinolol hydrochloride and atenolol can reach 1.71 and 1.52,and the analysis time is about 30 minuses by using ethylenediaminetetraacetic acid bridged bis-?-cyclodextrin chiral stationary phase?EBCDP?via further optimize the mobile phase composition,flow rate,column temperature and other chromatographic parameters in a polar organic mode.A HPLC-FLD method for the determination of arotinolol hydrochloride and atenolol in tablets and in plasma were established.Aroraol hydrochloride and atenolol have a good linear relationship in the concentration range of 0.05¹.0 mg/L and 0.05⁰.25mg/L,respectively,and the correlation coefficient R were about 0.9987.The detection limit were 0.034 mg·L^-1for both enantiomers.This method is simple and rapid,with good selectivity and high sensitivity.It can provide a new method for monitoring the quality and pharmacokinetics of this class of chiral drugs.In the fourth part,a‘Click chemistry'reaction was used to synthesize a triazole bridged bis??-cyclodextrin?-boned stationary phase.The 6-azido-?-cyclodextrin and6-propynylamino?-cyclodextrin were respectively synthesized,and dissolved in DMF to produce the bridged cyclodextrin in a higher yield by triphenylphosphine cuprous complex as as a catalyst.The bridged cyclodextrin was bonded to the surface of ordered mesoporous SBA-15 to obtain a novel triazole bridged double?-cyclodextrin stationary phase?TBCDP?by a reactive 3-isocyanatopropyl triethoxysilane coupling agent..The chemical structures and morphologies of the ligand and the stationary phase were charactrized byMS,IR,EA and SEM.The performance of TBCDP was systematically evaluated by different structural drugs as probes,including common triazole pesticides,flavanones,?-blockers,dansyl amino acids and aryl alcohols.The TBCDP successfully resolved 9 triazole fungicides,8flavanones,10 dansyl amino acids and 5 aryl alcohols in the reversed phase modewithin 30 min.The resolutions of hexaconazole,2'-hydroxyflavanone,dansyl-DL-tyrosine and hydroxyethylphenol enantiomers were 2.49,5.40,3.25and3.08,respectively.In polarity organic solvent mode,all 10?-blockers were resolved,among them the resolution of arotinolol hydrochloride enantiomers was 1.71.The preliminary chiral separation mechanism was proposed and provided as a theoretical basis for further practical application.The results indicated that the bridged cyclodextrin-bonded stationary phase had a relatively strong chiral separation capability without being derivatized.In the fifth part,the resolution of ractopamine enantiomers on TBCDP can reach1.61within 25 min via further optimizing the chromatographic parameters such as the composition of the mobile phase,flow rate,and column temperature in the reversed mode.A quantitative HPLC-FLD method for the determination of ractopamine enantiomers was developed in four food and feed samples with different matrices by a simple sample pretreatment.The ractopamine enantiomers had good linear relationship in the range of 0.005⁰.5?g/mLwith the correlation coefficient 0.9998.The LOD of ractopamine enantiomers were less than 5 ng/mL for both enantiomers.This method has simple,sensitive and fast for monitoring chiral ractopamine residue in food safety analysis.