Abstract: A reversed-phase high performance liquid chromatographic method with pre-column derivatization for the determination of amino acids, which were known as the characteristic constituents of Isatis indigotica Fort was established. The amino acids in alkalescence were derivatized with 2,4-dinitro-fluorobenzene (DNFB). A reproducible method for simultaneous qualitative analysis of glutamic acid (Glu), argirine (Arg) and proline (Pro) and quantitative analysis of Arg and Pro in Isatis indigotica Fort has been established. NaAc buffer (pH 6.4)-acetonitrile (85∶15, v/v) as mobile phase and a Sinochrom ODS-BP column were used. The detector was operated at 360 nm. The linear regressions of the standard curves were determined for Arg and for Pro. The method was carried out over the range of 0.627-5.016 μg for Arg and 0.874-7.000 μg for Pro. The recoveries were 98.5% and 98.4% with the relative standard deviations of 2.5% and 2.3% for Arg and Pro respectively. The results indicate that among the three amino acids in Isatis indigotica Fort, Arg content was the highest, Pro the second and Glu the lowest. The method has good accuracy and repeatability and it can be used for the quality control of Isatis indigotica Fort.

Key words: 2,4-dinitro-fluorobenzene, argirine, glutamic acid, Isatis indigotica Fort , pre-column derivatization, proline, reversed-phase high performance liquid chromatography