New reagents for determination of amino acids by liquid chromatography with pre-column fluorescence derivatization Original Research Article

A liquid-chromatography method for the determination of amino acids with fluorescence detection has been developed. The reactions of three fluorescent tagging reagents, namely acridone-N-acetyl chloride (ARC-Cl), carbazole-9-acetyl chloride (CRA-Cl) and carbazole-9-propionyl chloride (CRP-Cl), with amino acids are reported. Emission maxima for the ARC, CRA and CRP derivatives of amino acids were at 430 nm (λex=404 nm), 360 nm (λex=335 nm) and 365 nm (λex=340 nm), respectively. In all cases, the derivatives exhibited strong fluorescence, whereas the reagents themselves also exhibited fluorescence. The labeled derivatives are very stable; <4% decomposition occurs after heating at 40°C for 24 h. The fluorescence intensities of the amino-acid derivatives were higher in neutral and alkaline than in acidic solutions. This method, combining a multi-gradient program, offers baseline resolution of the common ARC, CRA and CRP amino-acid derivatives than a linear acetonitrile gradient. Separation of amino-acid derivatives was carried out on a reversed-phase C18 column. Derivatization and chromatographic conditions were optimized. The separation of amino-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.