High-performance liquid chromatographic determination of amino acids by pre-column fluorescence derivatization with three new fluorescent agents

A high-performance liquid chromatographic method for the determination of amino acids with fluorescence detection has been developed. The reactions of three fluorescent tagging reagents, 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, CRC and CRP derivatives of amino acids were 430 nm (λ_ex = 404 nm), 368 nm (λ_em, = 335 nm) and 365 nm (λ_ex = 340 nm), respectively. In all cases, the derivatives exhibited strong fluorescence whereas the reagents themselves also exhibited fluorescence. The labelled derivatives are very stable, less than 4% decomposition occurs after heating at 40°C for 24 h. fluorescence intensities of amino acid derivatives were higher in neutral and alkline than in acidic solutions. This method, combining a muti-gradient program, offers baseline resolution of the common ARC, CRA and CRP amino acid derivatives from a linear acetonitrile gradient. Separations of amino acid dervatives were carried out on a reversed-phase C₁₈ column. Derivatization and chromatographic conditions were optimized: Amino acid derivatives were eluted with a good reproducibility. The relative standard deviation (n = 6) at an analytical concentration of 5 pmol of each amino acid is <4%. The detection limit is around fmol level.