Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization

A highly sensitive and selective fluorometric determination method for orni thine and lysine has been developed. This method is based on an intramolecu lar 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 moiet ies in a molecule, were converted to the corresponding dipyrene-labeled der ivatives by reaction with PSE. The derivatives afforded intramolecular exci mer fluorescence (450 - 550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled d erivatives of monoamines. The structures of the derivatives and the emissio n of excimer fluorescence were confirmed by LC with mass spectrometry and w ith 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-nois e ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-mul injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.