| As all we know,biopharmaceutical analysis plays an indispensable role in pharmaceutical research.The tested samples for biopharmaceutical analysis mainly include blood,urine,saliva,and tissue samples etc.,among which blood samples are tested the most.The rapid and accurate analysis of blood samples acts as an effective tool for researches in the fields of(clinical)pharmacology,pharmacokinetics,and clinical therapeutic drug detection.In view of the fact that there are a large number of complex matrix components in blood samples,which can easily interfere with subsequent instrument detection,direct、efficient and convenient pretreatment technique of blood sample has become a key node in biopharmaceutical analysis.At present,as the sensitivity of blood sample detection equipment(chromatographic analysis)continues to increase,and the demand for the original volume of blood samples has also been reduced accordingly.In response to this,blood sample pre-processing devices have been becoming increasingly miniaturized(small),such as solid-phase micro-extraction or micro-solid-phase extraction devices have come into being.The research and application of blood sample pretreatment technology based on micro-extraction device have also become one of the frontier hotspots in the field of biological analysis.Based on the goal of realizing the direct,convenient and efficient pretreatment of rat plasma samples,the research of this thesis carried out the preparation of new surface restricted access(RA)solid phase extraction(solid phase extraction,SPE)film roll micro column materials.In the study,the prepared film roll micro column can be conveniently rolled and placed in a 1m L medical syringe,and assembled into a convenient and restricted micro-solid phase extraction device.With the help of manually pulling and pushing operation,without prior blood sample protein precipitation operation is required,the direct solid-phase extraction pretreatment of rat blood samples can be quickly performed to achieve direct extraction and enrichment of licorice flavonoids and strychnine alkaloids from plasma samples.Combined with high performance liquid chromatographic analysis,new methods have been constructed for simultaneous detection of multiple active ingredients of traditional chinese medicine in plasma.The experimental results obtained are satisfactory.1.IntroductionIn this part,it is introduced that the research purpose and significance,focusing on the traditional SPE technique,the current development status of SPE technique,the research status of RA-SPE materials and its current status in the field of biological sample pretreatment.At the same time,the technical features of activators regenerated by electron transfer coupled with atom transfer radical polymerization(ARGET-ATRP)and its application in the preparation of restricted-access solid phase extraction materials are also introduced.Finally,the research ideas and methods of this thesis are proposed,i.e.macro porous polypropylene(PP)film is used as the support material,and the ARGET-ATRP technology is used to initiate multiple synthesis reactions on the surface to consecutively graft functional polymer materials and restricted access material on the surface of PP film.The finally prepared film material is rolled into the shape of film roll micro column and inserted into a 1m L disposable medical syringe to form a convenient restricted-access SPE device for rapid SPE pretreatment of rat plasma samples.2.Applying ARGET-ATRP method to preparing restricted access solid-phase extraction film rolls micro-column for the direct and simultaneous extraction of multiple active components of licorice from rat plasma.The restricted access solid phase extraction film roll micro-column was prepared based on the porous(pore size 20μm)polypropylene(PP)film with inert surface,soft texture and porous structure:(1)PP film saturated with polyvinyl alcohol casting liquid(composed of polyvinyl alcohol(PVA),polyethylene glycol(PEG600)and glutaraldehyde(Glu)),was clamped between two glass plates and place in the environment at room temperature over night to obtain a composite PP film with a large number of hydroxyl(-OH)groups in and on the film;(2)Through the acylation reaction,grafting 2-bromoisobutyryl bromide onto the surface of the composite polypropylene film to form a polymer film initiator;(3)Using the dormant seeds on the surface of composite PP film to initiate ARGET-ATRP reaction,grafting net-like hydrophobic functional layer(poly-benzyl methacrylate(BZMA)and ethylene glycol dimethacrylate(EGDMA),poly-BZMA-co-EDMA)on the surface of the film;(4)Using the dormant sites on the outer surface of the net-like hydrophobic functional layer,the hydrophobic poly-BZMA chain brush layer was grafted onto the outer surface by the second surface initiated ARGET-ATRP reaction to form a superimposed double-hydrophobic functional layer composed of network-like and comb-like polymer layers;(5)The dormant seeds on the outer surface of the comb-shaped functional layer were continuedly used to initiate the third ARGET-ATRP reaction,for grafting linear polyhydroxyethyl methacrylate(HEMA)hydrophilic chain brush on the outermost surface,which serves as the restricted access functional layer by blocking the penetration of macromolecular matrix components,such as proteins in the blood sample,to the hydrophobic functional layer.The final film was rolled into a micro-column rolls shape and then was put into a 1m L disposable medical syringe with a porous hydrophilic polypropylene sieve plate(with a pore size of Ф20μm)set at the bottom,to construct a portable SPE device with a manual operation plunger,pulling and pushing the plunger to perform the SPE steps of sample loading,rinsing,and elution.The surface topography and element constituent characterization were performed separately by SEM)and XPS for the film prepared at each reaction step.Using HPLC/UV as the detection method,the ability of restricted-access film roll micro-column exclusion protein was investigated,and the conditions for direct SPE treatment of rat plasma were optimized.Based on this,a new RA-SPE-HPLC/UV method was established for the direct extraction and analysis of four active(metabolic)components of licorice(glycyrrhizin,glycyrrhizin,isoliquiritin and glycyrrhetinic acid)in rat plasma samples.Methodological verification was carried out based on the plasma quality control samples at 4concentration levels.The linear ranges for the detection of four licorice components were separately detected as: glycyrrhizin(LQ)60.00～4000.00ng/m L,glycyrrhizin(LQG)19.40～4850.00ng/m L,isoliquiritin(ILQG)9.70～4850.00ng/m L,glycyrrhetinic acid(GTA)98.00～7840.00ng/m L,the RSD(%)and relative recovery rates of the measured values of the 4 quality control samples were separately detected as: 0.95%～5.70% and 92.79%～108.14% range.This method has been applied to the detection of actual plasma samples of rats fed with licorice extract,and the experimental results are satisfactory.3.Preparation of restricted-access film rolls micro-column of surface-modified nanoparticles and their use for direct simultaneous extraction of brucine and strychnine in rat plasma.Nanoparticles have a large specific surface area and good dispersibility,and they have the advantage of large loading capacity when used as SPE extraction media.This experiment attempts to graft hydrophobic functional nanoparticles on the surface of the film to prepare restricted access film roll micro-column with surface-modified nanoparticles.In order to obtain better solid-phase extraction performance,the preparation process of the film roll micro-column is listed as follows:(1)Preparing the composite polypropylene film with2-bromoisobutyryl bromide grafted on the surface by the same method as that in the previous chapter;(2)Via amination and acylation reactions,the surface of silica particles were grafted with 2-bromoisobutyryl bromide to form a silica nanoparticle initiator;(3)Using ARGET-ATRP method to graft the hydrophobic functional layer that formed by copolymerization of benzyl methacrylate(BZMA)and ethylene glycol di-methacrylate(EGDMA)on the surface of silica particles to prepare hydrophobic functional silica Nanoparticle(4)Rolling the composite polypropylene film prepared in step(1)into a roll-shaped micro-column,which was inserted into the bottom of a1 m L syringe,and then pouring a suspension liquid containing hydroxyethyl methacrylate(HEMA)and hydrophobic functional silica nanoparticles.Finally ARGET-ATRP reaction was carried out in situ in a syringe to prepare a restricted access nanoparticle-thin-film-roll micro-column solid phase extraction device.In the experiment,in addition to the SEM characterization of the surface morphology and the XPS element analysis of the film,the silica nanoparticles were also characterized by FT-IR、TEM、XPS and so on.At the same time,using HPLC/UV as a detection tool,it was investigated that the ability of the prepared nanoparticle-thin-film-roll micro-column to exclude proteins.Based on the optimal plasma SPE conditions,a new SPE-HPLC/UV analysis coupled with restricted SPE was established.The method is used to directly extract and analyze the two main alkaloid components(Brucine(BRU)and strychnine(STR))in rat blood samples.The plasma quality control samples at 4 concentration levels were detected for the methodology validation.The linear ranges were detected as separately: BRU(27.25-5450.00ng/m L),STR(27.50-5500.00ng/m L),RSD(%)and relative recovery rate of the determined concentration values of four quality control samples were calculated as separately:3.08 %～9.31%,91.64%～100.82%.This method has been applied to the detection of actual blood samples of rats of intragastric administration of the extract of Strychnia serrata,and the experimental results showed that the method is effective. |
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