Application Of Sample Preparation Technique Combined With LC-MS To The Analysis Of Biological Samples

Biological samples are characterized by the complex compositions and larger interference of substrate, meanwhile, by the subtle amount of sample and more difficulty in obtaining, as well as the lower concentration of analyte. With the development of bioscience, requirements for biological seperation and analysis techniques meet much higher standards. In order to establish a higher sensitive and selective analysis method of biological samples, the pre-treatment of samples and detection techniques are being taken into consideration, among these two aspects, pretreatment of samples is the most feasible and challenging method.Polymer monolith microextraction (PMME) which uses polymer monolithic material as the extraction medium, is a new-type of solid phase microextraction technique for samples pre-treatment with the advantages of simple preparation methods, well-distributed internal structure, long service life, good reproducibility and higher extraction efficiency. Via manufacturing different types of extraction materials, polymer monolith has been widely used in seperation and analysis of some bioactive substances such as drugs, metabolites, biological macromolecules, proteomics and other substances. The author’s research group has done lots of work in basic research and application study of polymer monolithic materials, including creating a variety of polymer monoliths especially as the representative of poly (methacrylic acid-ethylene glycol dimethacrylate) which has been applied to the detection of biological samples and achieved preferable effectiveness. At the same time, in the analysis of biological samples, other pre-treatment techniques such as extraction combined with chemical derivatization, as well as PMME combined with chromatography online method, have further improved purification and enrichment effect of biological samples and enhance the detection sensitivity and reproducibility.This thesis is mainly focused on the analysis of some biological samples using pre-treatment methods such as PMME, liquid-liquid extraction, pre-column derivatization combined with HPLC, LC-MS techniques. The main research contents are as follows: 1. Introduce some biological sample pre-treatment methods and the updated development direction, giving a brief introduction to the research of the brain cytochrome P450.2. Established a method, which use chlorzoxazone as substrate and combined liquid-liquid extraction with LC-MS method to detect CYP2E1enzymatic activity of microsomal in different brain regions of rats. The method is simple and much higher sensitivity than HPLC method as reported previously, which requires a small size of samples and meets individualized detection of different brain regions of rats.3. Established two methods to detect of CYP2D6enzymatic activity in different brain regions of rats, which uses dextromethorphan as substrate, one combined PMME with LC-MS online and another combined liquid-liquid extraction with LC-MS/MS.4. To establish an analysis method which uses2,4-dinitrophenylhydrazine as derivative reagent and combined Poly (MAA-co-EGDMA) monolith microextraction with HPLC to detect the concentration of5-Hydroxymethylfurfural in coffee, honey, beer, cola and urine. The method with high sensitivity and superior precision and accuracy is especially suitable for the trace detection of5-Hydroxymethylfurfural among some complex samples such as food, medicine, biological samples and so on.