Studies On Preparation And Applications Of Novel Polymeric Monolithic Columns In Pharmaceutical Analysis

With the rapid development of subjects(including drug discovery, proteomics, traditional Chinese medicine, etc.), the demands for high sensitive and throughput analytical method become urgent. Chromatographic analysis technology confronts a lot of new challenges. Traditional packing columns hold a series of inadequacies, such as high cost, low stability, difficult preparation process, high backpressure etc. Rencently, monolithic columns have proved to be an effective alternative to packed columns and have attracted considerable interest owing to their facile preparation methodology and good column characteristics, such as permeability and efficiency. As we know, drug-membrane interactions play an important role in drug transport, distribution, accumulation, efficacy and resistances. Furthermore, chiral separations of active pharmactuical canditates are very important in drug discovery. Therefore, the main goal of the present research is to preparae two types(phospholipids and quinidine) of organic polymer based monolithic columns and to evaluate their applications in drug discovery. The optimized monoliths will be applied to the prediction of drug-membrane interactions or the enantioseparation of small molecules, respectively.In the first chapter, the classifications, functionalities, and applications of monolith were comprehensively introduced. In particular, the developments of phospholipid or quinidine functionlized materials were reviewed in details. On this basis, the innovative points and significances of current study were finally highlighted.In the second chapter, a novel methodology for preparing double chain PC functionalized monolithic column poly(1-dodecanoyl-2-(12-methacryloyloxy dodecanoyl)-sn-glycero-3-phosphocholine-co-ethylene dimethacrylate)(poly(MDCPC-co-EDMA)) was reported. The developed synthetic approach not only shortens the preparation progress and improves the stability of double chain PC functionalized IAM columns but also completely excludes the influence of residual silanol and amine groups. The resulting monolith exhibited excellent permeability and column efficiency. A significant correlation between CHI IAM7.4 PC. DD2 and CHI IAM7.4 MDCPC demonstrates the potential of the poly(MDCPC-co-EDMA) monolith for studying the drug-membrane interaction as a replacement of the commercial IAM columns. Furthermore, the optimized monolithic column exhibited good separation abilities for protein mixture and a set of small molecule drugs in the micro-LC mode.In the third chapter, a phospholipid analogous functionalized monolithic column poly(2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate-co-EDMA)(poly(MBP-co-EDMA)) was prepared with one step in situ co-polymerization with MBP and EDMA. The effects of the content of functional monomer, the composition of porogenic solvent on the morphologies of the monoliths were investigated. The resulting phospholipid analogous monolith exhibited excellent permeability and mechanical stability. A tapical HILIC retention mechanism and electrostatic mechanism were observed at higher ACN contents(> 75%). The MBP monoliths were further evaluated for the separation of various polar analytes. It is worth noting that the result monolith exhibits much higher efficiency and selectivity than the previously developed poly(2-methacryloyloxyethyl phosphorylcholine-co-EDMA)(poly(MPC-co-EDMA)) monolith for all polar analytes. Satisfactory separtions of small peptides was obtained on this hydrophilic phospholipid analogous functionalized monolith.In the fourth chapter, a chiral monolithic column poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)(poly(MQD-co-HEMA-co-EDMA))was prepared through one step in situ co-polymerization of MQD, HEMA and EDMA. The effects of the content of monomer, the composition of porogenic solvent on the morphologies of the monoliths were investigated. The resulting monolith exhibited excellent efficieny and enantioselectivity for N-derivatized amino acids. The chemoselectivity of the monolithic column for a multicomponent mixture of N-derivatized amino acids was also investigated. A mixture of three pairs of 3,5-dichlorobenzoyl-amino acids could be fully enantioresolved within 22.5 min.In the fifth chapter, baseline resolutions of the stereoisomers of 24 out of 53 N-derivatized di- and tri-peptides were obtained on the poly(MQD-co-HEMA-co-EDMA) monolith. The monolithic column was then applied to the quantitative analysis of L-carnosine and its enantiomeric impurity in three different commercial dietary supplements. Method validation demonstrated satisfactory results in terms of linearity, precision, selectivity, accuracy and limits of detection and quantification. The determined amounts of L-carnosine in commercial formulations were in agreement with the labeled content for all analyzed samples, and the enantiomeric impurity was found to be below the limit of detection(LOD), showing the potential of the poly(MQD-co-HEMA-co-EDMA) monolithic column as a reliable tool for the quality control of L-carnosine in dietary supplements by micro-LC.In the sixth chapter, an O-9-(tert-butylcarbamoyl) quinidine(t-Bu CQD) functionalized polymeric monolithic capillary column was prepared by the in situ copolymerization method. The chiral recognition ability of the resulting monolith was also evaluated using 47 N-derivatized amino acids, eight N-derivatized dipeptides, and two herbicides. Under the selected conditions, the enantiomers of all chiral analytes were baseline separated with exceptionally high selectivity and resolution using micro-LC. It is worth noting that this new chiral stationary phase(CSP) containing quinidine with a tert-butyl carbamate residue as chiral selector exhibits much higher enantioselectivity and diastereoselectivity than the previously developed MQD based CSP for N-derivatized amino acids and dipeptides.All contents were summarized in the seventh chapter. The future developments of phospholipid and quinidine functionalized stationary phases were also discussed.