Development And Applications Of Monolith-based Two-dimensional Liquid Chromatography System In Pharmaceutical Analysis

Liquid chromatography(LC)is an important analytical tool in the pharmaceutical field,from the screening of active components in TCM to the pharmacokinetics study of treated drugs.The fast-developing and flourishing pharmaceutical industry keep sending demands for novel LC methods with better selectivity,higher separation power and faster analytical speed.The thesis aimed at developing novel monolithic columns with a larger inner diameter or different selectivity and then setting up micro/nano two-dimensional LC(2D-LC)systems based on these monolithic columns.Further applications of these columns and 2D-LC in the pharmaceutical analysis were also investigated.The major content of each chapter as follows:In Chapter Ⅰ,the development of 2D-LC and its applications in pharmaceutical analysis,especially the analysis of traditional Chinese medicine(TCM)and natural products,were first reviewed;Researches on organic polymer-based monolithic columns were also outlined,with a particular emphasis on the size and housing materials of monolithic columns.The development of 2D-LC based on the organic polymer-based monoliths was then summarized.Finally,the research outline and innovation points of the thesis were described.In Chapter Ⅱ,to better meet the requirements of the monolith-based 2D-LC system,we focused on the preparation of organic polymer-based monoliths within housed materials with a larger ID.A novel organic polymer-based zwitterionic HILIC monolithic column SPP@EDMA-400 was developed and prepared by using N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine(SPP)as the functional monomer,ethylene glycol dimethacrylate(EDMA)as the crosslinker in the presence of the porogen methanol and the initiator azobisisobutyronitrile(AIBN).The polymerization conditions including preparation process,housing materials,polymerization time and monomer/crosslinker content ratio were carefully optimized.It was found that 400μm ID×800μm OD fused-silica capillary is a good housing material in terms of column efficiency and preparation process.Good permeability,mechanical stability,column efficiency,long-term stability and reproducibility were observed on the SPP@EDMA-400.The SPP@EDMA-400 also exhibited good separation power for various polar compounds.In Chapter Ⅲ,to further improve the hydrophilicity and column efficiency of the zwitterionic monolith,a more hydrophilic crosslinker,N,N’-methylenebisacrylamide(MBA),was employed to replace EDMA as the crosslinker.After a careful optimization of the polymerization conditions,the resultant monolith SPP@MBA-400 exhibited very high column efficiency(up to 144,000 plates/m)and good selectivity towards a wide range of small polar compounds,including organic acids,nucleobases and nucleosides,benzoic acids and acrylamides.Compared to the SPP@EDMA-400 monolith and commercial column ZIC-HIILIC(300 μm ID×150 mm,3.5 μm),the SPP@MBA-400 monolith has a higher column efficiency and better hydrophilic selectivity.In Chapter Ⅳ,according to the preparation method in Chapter Ⅱ,a novel large size hydrophobic RP monolith AOD@EDMA-400 was designed and prepared by using 3-methylacryloyl-3-oxapropyl-3-(N N-dioctadecylcarbamoyl)-propionate(AOD)as the functional monomer,EDMA as the crosslinker in the presence of 2-methyl-1-propanol and 1,4-butanediol as the dual porogen,AIBN as the initiator.Good permeability,stability and column efficiency were observed on the optimal AOD@EDMA-400 monolith.The AOD@EDMA-400 also showed good hydrophobic selectivity towards highly hydrophobic compounds such as β-TOH and γ-TOH.In Chapter Ⅴ,a fast separation for nucleotides was developed based on the SPP@MBA-400 monolith.The chemical stability of the SPP@MBA-400 under the high pH was evaluated firstly and the results showed that the coefficient of variation(RSD)of retention time on the SPP@MB A-400 monolith is lower than 3%after over 4000 consecutive injections which are equal to over 30,000 column volume.Afterward,the separation conditions influencing the retention of nucleotides were systematically optimized,including the type and concentration of buffer,buffer pH,the content of organic solvent,column temperature,etc.Under the optimized conditions,all series of nucleotides(mono-,di-,tri-phosphates nucleotides)were fully separated on the SPP@MBA-400 within 3.0 minutes.In Chapter Ⅵ,thanks to the dual retention mechanism of the zwitterionic monolith SPP@MBA-400,two modes of comprehensive two-dimensional liquid chromatography(LC×LC)based on the SPP@MBA-400 were developed,i.e.μHILIC×RPLC and μRPLC×RPLC.Several parameters including peak capacity,orthogonality,separation power of these two LC×LC system were evaluated by separating 46 phenolic acids and flavonoids(glycosides)compounds.The interface of LC×LC was also investigated.The result indicated that when using SPP@MBA-400 as the RP column,higher peak capacity and better separation power towards the separation of standards can be achieved in the presence of passive solvent modulation in the interface.The developed μRPLC×RPLC was successfully applied to monitor the content of phenolic acids and flavonoids/glycosides.In Chapter Ⅶ,to further verify the applicability of organic polymer-based monolith for 2D-LC.a series of RP monoliths with different alkyl chain length and various mixed-mode phospholipid-functionalized monoliths were first prepared.These columns were used in the second dimension for the separation of targeted analytes,while a C18 packed short capillary column was used in the first dimension for capturing samples.A systematic column selection was first performed to obtain a baseline separation of the deamidation-sensitive signature peptide of trastuzumab and its deamidated product isomers.The results showed that a cation-exchange capability of the stationary phase might favor the separation.A MDPC60PA40@EDMA-100 monolith which presents a certain amount of phosphatic acid groups(anion ion),as well as phosphocholine groups,was finally chose.Separation conditions including the type and concentration of buffer,buffer pH and column temperature were also carefully optimized.Under optimal condition,the triad peptides were fully separated using this monolith based 2D-LC.In Chapter Ⅷ,according to a summary of the whole thesis,prospects of the monoliths based two-dimensional liquid chromatography in the pharmaceutical analysis were proposed.