Liquid Chromatographic Determination of Ornithine and Lysine Based on Intramolecular Excimer-forming Fluorescence Derivatization

Abstract

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.