A selective fluorescence reaction for peptides and chromatographic analysis

A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 degrees C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N=3) of the detection for the tested peptides were 0.5-1.0 pmol per an injection volume (40 microl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.