Based On Column Before Fluorescent Tags Combined With The New Method Research Of High Performance Liquid Chromatography (hplc) Technique And Its Application In Food, Medicine And Serum Sample Analysis

With the continuous development of society, HPLC has been widely used in biochemical analysis, medicine detection, food safety, environmental protection detection etc because of its analysis separation ability. But sometimes, because the nature and low content of the sample, we can not using this method for effective detection analysis, such as fatty acid, biogenic amine, amino acids, etc. They all have very low ultraviolet absorption, so the general spectrophotometry is hard to realize accurate determination. At present column before fluorescent markers set HPLC technique has been widely used in analytical chemistry. Because of using fluorescence detection technology can make the lightless compound derived for emission fluorescence compounds, this technology in sensitivity choice has a strong advantage. This paper established based on several new fluorescent marker reagents, including (2-(11h-benzene [a] carbazole) ethyl p-toluene sulfonic acid ester (BCETS),2.-(2-(7h-double benzo [a, g] carbazole-7-ethoxy chloride ethyl formate (DBCEC) and benzene and acridone-5-acetic acid hydrazide (BAAH)of fatty acid and amino acid of high sensitive HPLC analysis method, making derivative reaction and chromatographic separation conditions are optimized, and used in other actual sample. The detailed work was given as follows:Chapter One:HPLC and chemical derivatization application method.Chapter Two:The seed oil extraction from Chinese star anise was carried out by supercritical CO2.A three factor, three level Box-Behnken factorial design (BBD) and RSM were applied to optimize the extracting parameters including pressure, temperature and amount of modifier (ethanol), and to study the interaction effect of the parameter. As results, the optimum conditions were as follows:extraction pressure:27.72MPa; extraction temperature:46.22℃; and amount of modifier:8.58%, and the maximum oil yield of25.31±0.22%was obtained under the these extractions. The composition of Chinese star anise seed oil (CSASO) was determined and quantified by HPLC/APCI/MS with fluorescence detection using BCETS as a pre-column labeling regent, and CSASO was found to be rich in unsaturated fatty acids, which accounted for71%in high to low concentrations as follows:C18:2, C18:1, C18:3and C20:4. Chapter Three:In present study, we developed a selective and sensitive method for trace AA determination in biological samples using DBCEC as labeling reagent by HPLC-FLD-MS/MS. Response surface methodology (RSM) was first employed to optimize the derivatization reaction between DBCEC with AA. In conjunction with a gradient elution, a baseline resolution of20amino acids containing acidic, neutral, and basic amino acids was achieved on a reversed-phase Hypersil BDS C18column. This method showed excellent reproducibility and correlation coefficient, and offered the exciting detection limits of0.19-1.17fmol/μL. The developed method was successfully applied to determinate AA in human serums, and shows great potential for trace AA determination in other complex physiological fluids, human or plant tissue and biological samples..Chapter Four:Using2-(12-benzo[b]acridin-5-(12H)-yl)-acetohydrazide(BAAH) as derivatization reagent, in Thermo Hypersil Gold (5μm,6mm x250mm) to realize a variety of fatty acid of the baseline separation, simultaneously mass spectrometry was operating after column, determine the derivatives of mass spectrum cracking model, and the method is applied in lotus lotus leaf and the fatty acid content analysis.