Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o-phthalaldehyde

Abstract

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high-throughput analysis with flow-gated CE. Here, we report a fast fluorescein-labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow-gated CE. This scheme was based on the reaction between primary amines and o-phthalaldehyde in the presence of a fluorescent thiol, 2-((5-fluoresceinyl) aminocarbonyl) ethyl mercaptan (FACE-SH). The short reaction time (<30 s) was suited for on-line mixing and derivatization that was directly coupled with flow-gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE-SH, Tris(2- carboxyethyl) phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow-gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on-chip labeling of proteins retained on support in SPE.