Highly selective determination of bioactive compounds based on intra molecular fluorescence resonance energy transfer detection.

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I have developed highly selective HPLC method for the determination of tyrosine, tryptophan and their related compounds (L-DOPA, catechdlamines, 5-hydroxytryptamine, etc.). The compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. Each derivative afforded intramolecular fluorescence resonance energy transfer (FRET) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor) ; the acceptor fluorescence was observed with the excitation of the donor at 280 run. The derivatives were separated on a reversed-phase column and then effectively detected by monitoring their FRET. Through the screening study of 15 fluorogenic reagents, ο-phthalaldehyde (with 2-mercaptoethanol), Dansyl chloride, 4, 5-dimetholy-ο-phthalaldehyde and DAMTC afforded efficient FRET ; ο-phthalaldehyde was selected as a highy selective, sensitive and practical reagent. The FRET detection method was highly selective and sensitive (limits of detection ; 20-70 fmol on clumn) by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. This method could be successfully applied to human urine sample without any tedious pretre atment.