Fluorescence Derivatization Reagent Preparation And Reversed-phase High Performance Liquid Chromatography - Mass Spectrometry Analysis

For the powerful separation and analysis capacities,reversed-phase high performance liquid chromatographic method has been widely used in many fields of scientific research and application,such as analytical chemistry,biochemistry,clinical medicine,pharmaceutical chemistry,food hygiene and environmental monitoring.It is enthusiastically and initiatively adopted in many scientific fields for ultra strong separation ability,and tumed into one of the fastest developing and widely used research fields.Lacking highly sensitive all-purpose detector in reversed-phase high-performance liquid chromatography,chemical derivatization is adopted by researchers to improve detection sensitivity and achieve baseline separation. Developing new derivatization reagents and its application in HPLC is one of the active research areas for liquid chromatography.In this paper,several novel derivatization reagents were designed,prepared and characterized.The spectral properties,optimal derivatization conditions and chromatographic separation conditions were investigated.Pre-column derivatization reversed-phase high-performance liquid chromatographic methods were established and evaluated for the analysis of realistic samples.Chapter one:The development and basic theory about high performance liquid chromatography and high performance liquid chromatography coupled with mass spectrometry were simply presented.Chemical derivatization,fluorescence derivatization and their research progress were systematically introduced.At last,applications of pre-column derivatization high performance liquid chromatography were summarized.And,the research progress of computerassisted liquid chromatography method establishment in reversed-phase high performance liquid chromatographic method development was introduced.Chapter two:The design idea for the synthesis of fluorescence reagent was summaried. The design and synthesis of fluorescence reagents,10-ethyl acridine-2- sulfonohydrazide (EASH),N-(2-aminoethyl)-10- ethylacridine -2-sulfonamide,5-(2- hydroxyethyl) benzo [b]acridone(HEBA),benzo[f]chromene -2- carboxylic acid are described,and followed with their structural characterization.Chapter three:Pre-column derivatization methods for the sensitive determination of estrogens with 10-ethyl acridine -2-sulfonyl chloride(EASC) as derivatization reagent followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using EASC as labeling reagents were evaluated.The established method for the determination of trace free estradiol and estriol from real samples such as feces sample labeled with this reagent show higher sensitivity in UV,FL and MS detection than that with Dansyl-Cl.Chapter four:Pre-column derivatization methods for the sensitive determination of aliphatic amines with 10-ethyl acridine -2-sulfonyl chloride(EASC) as derivatization reagent followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using EASC as labeling reagents were evaluated.The established method for the determination of aliphatic amines from real samples such as environmental water samples was satisfactory.The mechanism of MS enhancement was proposed and the enhancement was evaluated with ACD/pKa DB software.Chapter five:Pre-column derivatization methods for the sensitive determination of aliphatic aldehydes with 10-ethyl acridine-2-sulfonohydrazide(EASH) as derivatization reagent followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using EASC as labeling reagent were evaluated.The contents of aliphatic aldehydes from real samples such as alcoholic beverage and environmental water were determined,Chapter six:A novel derivative reaction for the determination of aliphatic aldehydes with higher sensitivity has been developed.The optimal derivatization and chromatographic separation conditions with this new derivative reaction were investigated,and a pre-column derivatization RP-HPLC method was established.Chapter seven:Pre-eolumn derivatization methods for the sensitive determination of aliphatic acids with 5-(2-hydroxyethyl) benzo[b]acridone(HEBA) as derivatization reagent followed by HPLC-MS analysis have been developed.The optimal derivatizations and chromatographic separation conditions for HEBA derivatives were investigated and a pre-column derivatization RP-HPLC method using HEBA as labeling reagent were established and evaluated.The concentrations of fatty acids in real sample auricularia auricula were abtained.reversed-phase high performance liquid chromatography(RP-HPLC);high performance liquid chromatography-mass spectrometry(HPLC-MS);pre-column derivatization; fluorescent derivatization reagent;computer-assisted chromatography method development; estrogens;fatty acids(aliphatic acids);aliphatic amines;aliphatic aldehydes;aliphatic acids;...