Sensitive analysis of polyamines by micellar-electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) in mineral media samples

Abstract

A micellar electrokinetic chromatography (MEKC) method coupled with laser-induced fluorescence (LIF) detection was developed and validated for the determination of five aliphatic polyamines in mineral media samples. Since polyamines lack inherent UV chromophores or fluorophores, fluorescence detection was enhanced using precapillary derivatization with fluorescein isothiocyanate isomer I (FITC). Key parameters affecting the derivatization, including reagent concentration, reaction time, and temperature, were optimized to improve sensitivity. Under optimal conditions, with a background electrolyte (BGE) of 20 mM borax and 20 mM SDS, polyamines were successfully separated, achieving limits of detection as low as 0.03 μM for cadaverine and 0.09 μM for putrescine, spermine, spermidine, and norspermidine. The method demonstrated the importance of borate complexation and the use of anionic surfactants for improving both sweeping and fluorescence signal intensity. The MEKC-LIF method was applied to analyze polyamines in mineral media from in vitro cultures of Arabidopsis thaliana and Pectobacterium betavasculorum, grown individually and in co-culture. Results showed that bacteria can synthesize or degrade polyamines, suggesting their importance in plant-bacteria interactions. The method enables sensitive analysis of polyamine metabolism, and its use provides insights into inter-kingdom communication.