Study On The Preparation Of Restricted Access Monolithic Column With Large And Well Aligned Channels And Its Application In Biopharmaceutical Analysis

Currently,biopharmaceutical analysis still faces the challenges from the complex matrix components.With the popularization of ultra-high pressure liquid chromatography and high-sensitivity mass spectrometry detection technology,separation and detection technology have gotten great progress,however,the pretreatment of biological samples is still in a state of bottleneck.Solid phase extraction(SPE)has become the current mainstream technology for rapid pretreatment of biological samples.However,most of the extraction media used in most SPE technologiesare hydrophobic material.When pretreating biological samples(such as plasma or serum),it is often necessary to remove plasma proteins in advance to prevent the permanent adsorption of protein molecules on the surface of the extraction medium.This makes the sample-preparing process cumbersome and tediously long,and also causes the loss of target analytes and deteriorate detections sensitivity.Therefore,it has become one of the hot research topics that developing new SPE extraction media with the function of protein molecule restricted access for directly processing plasma(serum or whole blood).Focusing on the goal of rapidly,efficiently and directly extracting and enriching the(hydrophobic and ionic)active ingredients of traditional Chinese medicine from plasma,we carried out research on the preparation of restricted access monolithic in pipette tip and its application to directly pretreat rat plasma samples with satisfactory results.The main research contents of this paper are as follows: 1.IntroductionThe research background and purpose of this paper are introduced.The following issues are focused on: the current development status of SPE and restricted access SPE pretreatment technologies,the technical characteristics of electron transfer for atom transfer radical polymerization(ARGET-ATRP),and the status of preparing macroporous polymer materials by unidirectional freezing polymerization method.Finally,the design idea of the thesis research is proposed,confirming to prepare a polymer monolithic column with a restricted access surface in a pipette and apply it to the SPE pretreatment of rat plasma samples in a convenient and efficient manner.2.Preparation of aligned-porous restricted access monolithic tipBy using styrene(ST)as monomer,ethylene glycol dimethacrylate(EGDMA)as cross-linking agent,and dimethyl sulfoxide(DMSO)/N,N-dimethylformamide(DMF)as solvent,a well aligned macroporous hydrophobic monolithic column was prepared in a 1000μL pipette by ARGET-ATRP reaction under unidirectional freezing.Further,ARGET-ATRP was used again to graft hydroxyethyl methacrylate(HEMA)onto the surface of the hydrophobic monolithic column to form a layer of linear poly-hydroxyethyl methacrylate(p-HEMA)chain.Inside the restricted access monolithic column,long microtubes with the diameter of tens of micrometers formed along the freezing direction.These microtubes provide the possibility for the smooth passage of plasma protein molecules.The p-HEMA chain on the surface of the inner wall of the microtubule provides the function of protein molecule restricted access.Only small-molecule drugs can penetrate into the hydrophilic layer and get to the inner hydrophobic surface to be retained because of the interaction with phenyl functional groups.The restricted access monolithic column tip can be connected to a 2mL disposable syringe through a conical adapter tube to assemble a convenient SPE device,through which,quick and direct SPE treatment of rat plasma samplesis performed by manually pushing the plunger.The monolithic column cross-sections were characterized by scanning electron microscopy,and the column chemical composition was characterized by infrared spectroscopy and X-ray energy spectroscopy.Using magnolol and honokiol as the target components and HPLC/UV analysis,the adsorption capacity of hydrophobic analytes and the protein exclusion efficiency of the monolithic column were examined.3.Aligned-porous restricted access monolithic tip was used to directly extract magnolol and honokiol from rat plasmaA new RA-SPE-HPLC/UV method was established to quickly and directly analyze magnolol and honokiol in rat plasma by using the self-made convenient restricted-access tip SPE device to directly extract target molecules from rat plasma samples,followed with HPLC/UV analysis.In the experiment,the SPE conditions were optimized and the constructed RA-SPE-HPLC/UV method was also validated with quality control samples at four concentration levels,in the meantime,the method selectivity and the conditions for the plasma sample stability were also investigated.The method was applied to the detection of real plasma samples from the Magnolia Bark extracts intragastric administration rats as well as the standard-spiked test,and satisfactory results were obtained.The good linearity relation for magnolol(62.94～5035.00 ng/mL,r=0.9988)and honokiol(62.62～5010.00 ng/mL,r=0.9996),the RSDs were detected as 3.39%～11.16%,and the relative recovery rates were detected as 89.52%～108.42%.Experimental results showed that the method has good reproducibility and recovery rate,and can be applied to the biopharmaceutical analysis of other hydrophobic drugs in the future.4.Aligned-porous restricted access monolithic tip was used to directly extract fangchinoline and tetrandrine from rat plasmaBased on the self-made convenient restricted access monolithic tip SPE device and HPLC/UV,a new RA-SPE-HPLC/UV method for simultaneous determination of fangchinoline and tetrandrine in rat plasma samples was established.Under optimized SPE conditions,the method validation was carried out with rat plasma quality control samples at 4 concentration levels,and the method selectivity and sample stability conditions were investigated.The detection of fangchinoline and tetrandrine showed good linearity in the concentration ranges of 80.48～8048.00ng/mL and 80.96～8096.00 ng/mL,respectively,and the correlation coefficients(r)were 0.9988(fangchinoline)and 0.9991(tetrandrine).The RSDs were in the ranges of 3.97%～8.06%,and the relative recovery rates were 87.52%～99.15%.This analytical method has been successfully applied to the detection of actual rat plasma samples with good results.5.Preparation of a replaceable weak cation exchange aligned-porous restricted access monolithic column and its direct simultaneous extraction of coptisine and berberine in rat plasmaThe weak cation exchange agent benzoic acid was adsorbed on the hydrophobic restricting monolithic column through the π-π interaction force,so that the aligned-porous restricted access monolithic column with benzene ring as a functional group was converted into a weak cation exchange aligned-porous restricted access monolithic column.Using this self-made weak cation exchange restricted access monolithic column to directly extract polar alkaloids(coptisine and berberine)from rat plasma,a new restricted access ion exchange SPE-HPLC/UV method was constructed to analyze coptisine and berberine from rat plasma.Under optimized SPE conditions,method validation was carried out with the rat plasma quality control samples at 4 concentration levels,and the method selectivity,precision and accuracy conditions were investigated.Coptisine and berberine showed good linearity in the concentration range of 50.70～10140.00ng/mL(r=0.9995)and 50.90～10180.00 ng/mL(r=0.9997),respectively.The RSDs were in the range of 3.62%～10.14%,and the relative recovery rates were 87.10%～104.24%.Finally the constructed method was successfully applied to the detection of real plasma samples from the drug administered rats with good results.