Visible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatization of Amines

Abstract

To fully exploit the attractive visible diode laser-induced fluorescence (LIF) detection technique in column liquid chromatography (LC), covalent labeling procedures need to be developed which are compatible with near-infrared fluorescence. For this purpose, several red-absorbing labels containing a single succinimidyl ester functionality were synthesized and used for the derivatization of primary and secondary amines. Oxazines/thiazines, squaraines, and dicarbocyanines were examined as redabsorbing fluorophores. The quality of the LC-diode LIF system for the analytes concerned is illustrated by the concentration detection limit of the labeled n-octylamine (a test compound), which was as low as 2×10 -12 M. As expected in view of the limited reactivity of the succinimidyl ester group, the analyte concentrations required to obtain quantitative reaction with the label were equal to or higher than 2×10 -8 M. To show the applicability of the method to biological samples, urine was spiked with 5×10 -7 M 1-adamantanamine, extracted, derivatized with a dicarbocyanine-based label, and analyzed by LC-diode LIF