| The pretreatment of biological samples is a crucial step in the overall bioanalysis process.It is very important to develop the convenient and fast pretreatment methods with high efficiency for determining analyte concentrations quickly and accurately with respect to those plasma(serum)samples in great quantity and small incipient volumes.As a kind of micro solid phase extraction device,pipette tip filled with monolithic polymer column exhibits many advantages in coping with plasma(serum)samples.Nowadays,there are several shortcomings in all the reported monolithic polymer tips,for example,not enough enriching capacity for HPLC/UV analysis caused from the too small volumes of column bodies to which ultraviolet-ray-initiated polymerization was applied,too bad flow permeability of column body to be connected to a pipettor for accomplishing the SPE process manually,etc.Although several documents presented the improving measurements,there are still the issues of tedious steps and repeatability in preparation.So,some improvements have been made in this master’s thesis research:(1)cellulose diacetate with ultra-high molecular weight in N,N-dimethylformamide was used as the porogen to create macropores at micrometer level to improve the flow permeability;(2)thermal initiated polymerization was used to synthesize monolithic column with larger volume to enhance the adsorptive capacity;(3)Atomic transfer radical polymerization coupled with actor regenerated by electron transfer(ARGET-ATRP)was applied to preparing the surface restricted access monolithic column in pipettor tip.The obtained restricted access monolithic tip can be connected to a hand-held pipettor to perform SPE quickly in manual manner.The thesis contents are as follows:1.Introduction In this part,the research background and purpose of this paper were introduced,including the sample pretreatment technology,solid phase extraction principle and extraction materials,the classification of monolithic column and the principle of ATPR method as well as its application in synthesizing extraction materials.Based on the existing shortcoming,the main research target of this paper is illustrated that to develop a new type of restricted access monolithic column tip for the pretreatment of biological samples,as well as the significance of the research.2.The preparation and application of microporous poly(styrene-ethylene glycol dimethacrylate)monolithic columnA macroporous monolithic column was synthesized in a 1000μL pipettor tip by in situ thermal polymerization by using styrene as functional monomer,ethylene glycol dimethacrylate as crosslinking agent and CDA/DMF as porogen.The macropores provide good permeability to the polymer column,and so that it can be attached to a hand-held pipettor for rapid solid phase extraction manually.Based on the optimal SPE conditions,a tip-SPE-HPLC/UV method was established for simultaneously determining liquiritin,liquiritigenin and isoliquiritigenin.The accuracy and precision of the method were verified by quality control samples.The recoveries were 92.4~103.6%,and the RSDs were 0.55~ 7.3%.Under the enrichment mode of SPE,the limit of quantitation of the three licorice flavonoids can reach 10.5(LQ),3.50(LQG)and 2.34(ILQG)ng/m L,respectively.The analysis method has been successfully applied to the detection of real rat plasma samples.3.The preparation and application of poly(methacrylic acid-divinylbenzene)monolithic columnA macro-porous monolithic column with partly hydrophilic property at surface was synthesized in 1000μL pipettor tip with α-methacrylic acid as functional monomer,divinylbenzene as crosslinking agent and CDA/DMF as porogen.The polymer monolithic column showed uniform structure and good permeability.The partly hydrophilic surface of column allowed the monolithic tip to be used directly for the SPE treatment of urine samples.With the optimized SPE conditions applied,good recoveries and enrichment ability were obtained.The constructed tip-SPE-HPLC/UV analysis method was applied to simultaneously determining coptisine and berberine in human urine,with the good linearity relations for coptisine at 0.100-60.0μg/m L and berberine at 0.0980-58.4μg/m L,respectively,and a correlation coefficient R2 greater than 0.9997.The RSDs of intra-day and inter-precision were detected as 0.31%~3.7% and 0.62%~6.1% respectively.The analysis method is simple and sensitive,meeting the requirement for detecting the trace analytes in real human samples.4.The preparation and application of restricted access poly(styrene-ethylene glycol dimethacrylate-hydroxyethyl methacrylate)monolithic columnBy using styrene as functional monomer,ethylene glycol dimethacrylate as crosslinking agent,and CDA/DMF as a porogen,a surface-restricted-access monolithic column was prepared in a 1000μL pipettor by ARGET-ARTP method.After synthesizing the hydrophobic monolithic column,the hydrophobic surface was grafted with hydrophilic polymer chain,poly(hydroxyethyl methacrylate),by ARGET-ARTP.The hydrophilic poly(hydroxyethyl methacrylate)chain can keep out the macromolecules in plasma(serum),for instance albumin molecules,and prevent them from being un-reversibly adsorbed onto the surface of column,in the meanwhile,the polystyrene chain at the underpart can grasp the analytes with small molecular weight by hydrophobic interaction.Therefore,the monolithic tip can be directly used to extract analyte from plasma(serum)sample without prior protein precipitation procedure.Based on the optimized SPE conditions,a tip-SPE-HPLC/UV method was established for simultaneously determining four kinds of indole alkaloids in the rat plasma.The detection results of quality control samples showed that the accuracy,precision,repeatability and stability of the method meet the analytical requirements.Moreover,the method has also been successfully applied to the simultaneous detection of rhynchophylline,isorhynchophylline,corynoxine and corynoxine B in real rat plasma samples. |
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