NEW REAGENTS FOR DETERMINATION OF AMINO-ACIDS BY LIQUID-CHROMATOGRAPHY WITH PRECOLUMN FLUORESCENCE DERIVATIZATION

A liquid-chromatography method for the determination of amino acids wi th fluorescence detection has been developed. The reactions of three f luorescent tagging reagents, namely acridone-hi-acetyl chloride (ARC-C l), carbazole-9-acetyl chloride (CRA-Cl) and carbazole-9-propionyl chl oride (CRP-CI), with amino acids are reported. Emission maxima for the ARC, CRA and CRP derivatives of amino acids were at 430 nm (lambda(ex )=404 nm), 360 nm (lambda(ex)=335 nm) and 365 nm (lambda(ex)=340 nm), respectively. In all cases, the derivatives exhibited strong fluoresce nce, whereas the reagents themselves also exhibited fluorescence. The labeled derivatives are very stable; <4% decomposition occurs after he ating at 40 degrees C for 24 h. The fluorescence intensities of the am ino-acid derivatives were higher in neutral and alkaline than in acidi c solutions. This method, combining a multi-gradient program, offers b aseline resolution of the common ARC, CRA and CRP amino-acid derivativ es than a linear acetonitrile gradient. Separation of amino-acid deriv atives was carried out on a reversed-phase C-18 column. Derivatization and chromatographic conditions were optimized. The separation of amin o-acid derivatives has good reproducibility and the relative standard deviations (n=6) for 5 pmol of each amino acid are <4%, The detection limit is at the fmol level. (C) 1998 Elsevier Science B.V.