VISIBLE DIODE LASER-INDUCED FLUORESCENCE DETECTION IN LIQUID-CHROMATOGRAPHY AFTER PRECOLUMN DERIVATIZATION OF AMINES

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 red-absorbing 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 x 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 x 10(-8) M. To show the applicability of the method to biological samples, urine was spiked with 5 x 10(-7) M. 1-adamantanamine, extracted, derivatized with a dicarbocyanine-based label, and analyzed by LC-diode LIF.