Preparation Of Novel Derivated β-cyclodextrin-bonded Stationary Phases And Their Applications In Chiral Separations By HPLC

Chirality is one of the fundamental properties of nature. Many protein, enzyme,nucleic acid and polysaccharides in human body are chiral. They often have highly stereoselective interactions with chiral drugs, which make chiral enantiomers exhibit different pharmacological activity, insecticidal activity and toxicity. We must attach great importance to the research of chiral drugs and pesticides. Therefore, to develop new chiral separation materials and to establish fast, easy, efficient method for monitoring enantiomer content at low cost has important research significance to address the problem of chiral drug safety and food safety.High-performance liquid chromatography(HPLC) due to the separation of mild conditions, easy operation, high instrument commercialization degree, has become one of the preferred method for the separation and determination of enantiomers. The direct separation of chiral drugs by chiral stationary phase(CSP) with high selectivity,good reproducibility, convenient operation etc, has become the most effective method for the monitoring of drugs enantiomers. Derivatized β-cyclodextrin-based chiral stationary phases are a kind of chiral separation materials with stable chromatographic property and low cost. Due to the synergistic effect of the cavity and port,β-cyclodextrin-based phases show excellent chiral recognition abilities and have good application prospects in the separations and analysis of chiral drugs and pesticides enantiomers.In this paper, a series of β-cyclodextrin bonded chiral stationary phases with ordered mesoporous SBA-15 as matrix, were prepared and characterized, including the 6-benzyl phenethylamine β-cyclodextrin-bonded phase(BZCDP),nitrophenylamino β-cyclodextrin-bonded phase(NCDSP), 2,4-dinitrobenzene etherβ-cyclodextrin-bonded phase(NESP), ureido β-cyclodextrin bonded phase(UCDSP)and β-cyclodextrin bonded phase(β-CDSP). The CD-based stationary phases were employed for the enantioseparations common cardiovascular chiral drugs. The new quantitative method for chiral drugs enantiomers was also established. Moreover, the new stationary phase was successfully used for the enantioseparation of common triazole chiral pesticides. Based on chromatographic data, the chiral separation mechanism of new stationary phases were discussed. The main research work includes the following five aspects.In the first chapter, briefly describes all kinds of chiral separation technologies,systematically summarizes several kinds of the development situation of high performance liquid chromatography(HPLC) chiral stationary phase in recent years,among the development of cyclodextrin-based chiral stationary phases is reviewed in detail. While the mesoporous material SBA-15 as a development chromatography matrix has also carried on the related comments. All is used as the foundation and starting point in this research work.In the second chapter, a novel 6-benzyl phenethylamine-β-cyclodextrin-bonded ordered mesoporous SBA-15 stationary phase(BZCDP) for HPLC was first prepared.The chemical structure and morphology of ligand and stationary phase were characterized by mass spectrometry, elemental analysis, infrared spectroscopy,transmission electron microscopy and thermogravimetric analysis, respectively. The enantioseparation properties of the stationary phases were evaluated by using differient solute probes, such as 9 kinds of β-blockers, 8 kinds of dansyl amino acids and 6 kinds of flavanones in polar organic and reversed-phase chromatographic modes, respectively. The results show that the BZCDP is suitable for differient chromatographic separation modes with a good enantioseparation performance.BZCDP can effectively separated the β-blockers enantiomers, among the chiral selectivity factor and resolution of carvedilol were 1.30 and 1.97 in polar organic mode, respectively. The new phase exhibited excellent chiral recognation ability for dansyl amino acids, especially the dansyl tyrosine with resolution of 3.29 in reversed-phase mode. By using common acidic methanol or acetonitrile as mobile phase, 4’-hydroxy flavanone with resolution of 3.65 on BZCDP was achieved.Compared with the reported native β-cyclodextrin and γ-cyclodextrin stationary phases, the new cyclodextrin stationary phases had faster and better separation effect for the above analytes within relatively short time. Experimental data show that, in polar organic mode, inclusion and hydrogen bonding is the main force of the chiral separation; in reversed-phase mode, inclusion and hydrophobic interaction is the main driving force of the chiral separation.In the third chapter, a novel nitrophenylamino-β-cyclodextrin-bonded ordered mesoporous SBA-15 stationary phase(NCDSP) for HPLC was prepared. The fast enantioseparation of metoprolol enantiomers on the new stationary phase was achieved in polar organic solvent mode. Some chromatographic conditions, such as the methanol content, the proportion and content of glcial acetic acid /triethylamine inmobile phase and column temperature were optimized in order to maxmize the resolution. The resolution of metoprolol enantiomers reached up to 1.80. The analysis time was rather short within 15 min under the above condition. Based on the optimization, a fast quantitative method of metoprolol tartrate enantiomers in tablets was established. The good linear relationships were observed in the range of 2.2~140μg/mL for both enantiomers. The detection limts of metoprolol enantiomers were 0.2μg/mL. The proposed method has high chiral selectivity, relative sensitive, high analysis speed and lower cost by using home-made cyclodextrin-based stationary phase. It can provide a convenient and fast method for quality controlling of chiral drug metoprolol and related pharmacokinetic research.In the fourth chapter, a novel of dinitrophenyl ether β-cyclodextrin-bonded SBA-15 chiral stationary phase(NESP) for HPLC was prepared by using ordered mesoporous matrix via a “Click Chemistry” reaction. The fast enantioseparation of carvedilol enantiomers on NESP was successfully achieved in ploar organ solvent mode. The carvedilol enantiomers were completely separated with resolution 1.62 within 15 min. A new LC-MS method for the determination of carvedilol enantiomers in human plasma was established by selected positive ion monitoring of [M+H]+407.30 for the enantiomers and [M+H]+ 317.20 for S-timolol interal standard,respectively. The good linear relationships for two carvedilol enantiomers were observed in the range of 10~200 ng/mL. The low detection limits were 2.5 ng/mL for both enantiomers. The total content of carvedilol and S-/R- enantiomeric ratio in healthy human plasma after oral administration racemic carvedilol existence certain difference, which closely related with individual differences and bioavailability of the enantiomers. The results showed that the established method was simple, fast,sensitive, extract and low cost by using homemade cyclodextrin-based chiral column.It is suitable for fast quantification of carvedilol enantiomers and its pharmacokinetics study.In the fifth chapter, two kinds of new β-cyclodextrin-bonded chiral stationary phases(CDSP and UCDSP) with carbamate and urea linkages were prepared by using ordered mesoporous materials SBA-15 as matrix, β-cyclodextrin and 6-aminoβ-cyclodextrin as ligands via the addition reactions of the OH and NH2 groups ofβ-cyclodextrin with active 3-isocyanatopropyltriethoxy silane, respectively. The enantioseparation properties of two new stationary phases were evaluated and compared by adopting 10 kinds of common chiral triazole pesticides as probesincluding triticonazole, diniconazole, hexaconazole, tebuconazole, etc. in reversed-phase mode. The results show that two homemade β-cyclodextrin-based stationary phases exhibited excellent and fast chiral separation ability for triazole pesticides within 30 min. The triticonazole and diniconazole with resolutions of 3.84 and 3.23 could be achieved in this experiment, respectively. We found that triazole pesticides with appropriate size and OH groups on CDSP and UCDSP had better separation effect, indicating that the inclusion, hydrogen bonding, steric hindance and other synergic forces between β-cyclodextrin ligand and solutes could contribute enantioseparations. Compared with the reported commodity coated cellulose column,the new cyclodextrin stationary phases with urea and carbamate linkages were more stable, more practical, easy preparation and low cost. The prepared β-cyclodextrin stationary phases have good prospects in the enantiomeric monitoring of triazole pesticides residues.The above CD-based stationary phases combine with the excellent chiral recognition of cyclodextrin and the low eddy diffusion, rapid mass transfer of mesoporous SBA-15. They have simple preparation at low cost, high selectivity, fast separation and stable chromatographic property.