Development and Application of Fluorescent Reagent on Analysis Biogenic Amines with High Performance Liquid Chromatography

Abstract: Polyamines are small molecules alkaline aliphatic compounds, which almost presenting in every cell. Representative materials are putrescine (PUT), spermidine (SPD) and spermine (SPM). Polyamines play important physiological roles in creatures and have been implicated in many physiological functions, including cell division and differentiation, nucleic acids replication, transcription, translation, synthesis of proteins and membrane stability. More attention has been paid to explore analytical method of polyamines for high selectivity, sensitivity, repeatability and minimum running cost. Reversed phase high performance liquid chromatography fluorescence detection with pre-column derivatization is one of the most widely used method in analytical of polyamine. However there are some disadvantages in most derivative regents. In this paper we synthesized some fluorescent labeling reagents with low molecular weight and low hydrophobicity, studied the chemical reactivity and HPLC method about these regents and polyamine.The content of the research are summarized as follows:1. The syntheses of four fluorescent labeling reagents Isatoic anhydride, N-methylisatoic anhydride, Succinimido 2-aminonicotinate, Succinimido salicylate were described. The reactivity about the four reagents with primary amines、secondary amines were researched. The result was that Isatoic anhydride showed the best reactivity in the four labeling reagent. 2. Pre-column derivatization methods for the reaction of polyamine with Isatoic anhydride have been development. The optimal derivatization conditions using Isatoic anhydride as labeling reagent was that the reaction temperature is 100℃and the reaction time is 5.5h. The separation was on Capcell Pak C18 SG120(4.6 mm×250 mm, 5um)and eluted with 50 mmol?L?1 sodium dihydrogen phosphate and acetonitrile (3 : 2, v : v) using isocratic elution, The flow rate was 1.0 ml·min-1 and the temperature was 37℃. The column eluate was monitored by fluorescence detection (excitation, 310 nm; emission, 410 nm).3. The syntheses of eight Isatoic anhydride derivatives, and followed by their structural characterization and spectral properties. We choose high fluorescence quantum yield derivatives react with hexane diamine, the result showed that the fluorescence intensity of the objective product has been greatly improvement. So they may be used as derivatization reagent for high performance liquid chromatography fluorescence detection…
Key words: Polyamines; High Performance Liquid Chromatographic (HPLC); Derivative reaction; Fluorescent reagent

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