Identification of 4-N, N-Dimethylamino-1-naphthylthiohydantoin Amino Acids by High Performance Liquid Chromatography with Enhanced Fluorescence Detection

Abstract

The simultaneous separation of fluorescent 4-N, N-dimethylamino-1-naphthylthiohydantoin (DNTH) amino acids was attained by high performance liquid chromatography (HPLC); they were separated in 35min on a reversedphase TSK-gel 80 TM (C₁₈) column with a linear gradient elution using 35mM of sodium acetate buffer (pH 5.0)-acetonitrile (65:35, v/v) and acetonitrile. An enhanced detection capability of DNTH-amino acids (E×345nm, Em 425nm) was achieved by the postcolumn addition of 7.5% tetraethylammonium hydroxide in dimethylsulfoxide. The established HPLC system permitted the detection of DNTH-amino acids in quanitities of 0.5pmol. The application of this method to the sequence analysis of human GRF is also described.