Preparation Of Novel Fluorescent Derivatization Reagents And Their Application In Food Analysi

High performance liquid chromatography(HPLC),was widely used for the analysis of some target compounds due to its good selectivity,high separation efficiency,good sensitivity and short analysis time.Some natural compounds,such as carbazole and acridone molecules,have highly conjugated system,so they exhibited excellent fluorescence characteristics,and can be directly used for the UV or fluorescence detection using HPLC.Most of the measured target compounds showed neither natural UV absorption nor fluorescence.Therefore,chemical derivatization was necessary in order to increase detection sensitivity and improve selectivity by pre-column or post-column HPLC derivatization methods.Through the combination of derivatization technology and ultrasound-assisted dispersed liquid-liquid microextraction(UADDLM)technology,the sensitivity and selectivity of HPLC could be greatly improved.To further enhance detection effect,various factors influencing the experiment were optimized by response surface methodology(RSM),this method can effectively simplify the experimental steps and reduce sample loss,and efficiently detect and analyze different samples.The specific research contents were as follows:Chapter one: The classification,history and characteristics of HPLC were introduced.The sample pretreatment methods were compared reasonably.Fluorescence labeling reagents were summarized and enumerated.Finally,the significance of the topic selection and the research content of this paper were expounded.Chapter two: The new labeling reagent of N-(9-ethylcarbazole-3-amino)-1-H-imidazolyl formamide(ECIC)was synthesized.With the combination of ECIC labeling and UADDLM technology,a set of safe,reliable,high sensitivity and simple pretreatment method by HPLC detection was established.At the same time,the derivatization conditions(derivative temperature,time,added DMAP amount and the ratio of reagent/BAs)and the parameters of UADDLM were systematically optimized.The established methods effectively improved the extraction efficiency of the actual sample pretreatment,and improved the sensitivity and accuracy of the test.It also effectively expanded the application range of the dispersed liquid microextraction technology,and could be applied to the accurate determination of biogenic amines in different beer samples.Chapter three: The new labeling reagent of N-(9-ethyl-9H-carbazol-3-yl)-carbamic acid-2,5-dioxo-1-pyrrolidinyl ester(ECCPE)was synthesized.The derivatization conditions(the extraction agent selection,derivatization time,derivatization temperature,and the ratio of labeling reagent/BAs)were reasonably optimized by response surface methodology,the detection sensitivity,accuracy and precision of the established method have been significantly improved.The proposed method could obtain accurate detection of biogenic amines in swordfish samples.The measured results also confirmed that low temperature storage could effectively inhibit the formation of biogenic amines in swordfish samples.Chapter four: The fluorescence labeling reagent of 2-(9-acridone)-ethylchloroformate(AEC-Cl)was prepared by an improved synthesis method.It was successfully applied to the determination of amino acids in silkworm pupae by HPLC analysis.The determination of amino acid derivatives was accomplished by reverse-phase HPLC with fluorescence detection coupled with post-column online ESI-MS identification.The RSM technology was used to optimize the derivatization conditions.The developed separation technology was relatively simple and showed high accuracy and precision with little matrix interference.The amino acid derivatives exhibited high ionizable capacity in positive-ion detection mode(ESI-MS).This method provided an accurate,reliable assessment for the compositional analysis of amino acids from silkworm chrysalis samples.This method is also suitable for the composition analysis of amino acids in other samples and as well as quality-monitoring for products.