Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization.
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.
Yoshida
H
H
Faculty of Pharmaceutical Sciences, Fukuoka University, Johnan, Japan.
Nakano
Y
Y
Koiso
K
K
Nohta
H
H
Ishida
J
J
Yamaguchi
M
M
D002138:Calibration; D002853:Chromatography, Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008239:Lysine / Q000032:analysis* / Q000652:urine; D013058:Mass Spectrometry; D009952:Ornithine / Q000032:analysis* / Q000652:urine; D011721:Pyrenes / Q000737:chemistry; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
C458721:4-(1-pyrene)butyric acid N-hydroxysuccinimide ester; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011721:Pyrenes; D009952:Ornithine; D008239:Lysine