Determination of proline by reversed-phase high-performance liquid chromatography with automated pre-column o-phthaldialdehyde derivatization Academic Article uri icon


  • A simple and sensitive fluorometric HPLC method was developed for the analysis of proline in biological samples. The HPLC apparatus consisted of an autosampler, a binary solvent delivery system, a 3-μm reversed-phase C18 column (150 × 4.6 mm I.D.) guarded by a 40-μm reversed-phase C18 column (50 × 4.6 mm I.D.), a fluorescence detector, and a computer workstation. Proline was oxidized to 4-amino-1-butanol in the presence of chloramine-T and NaBH4 in a 60°C water bath, which took 11 min. 4-Amino-1-butanol was automatically derivatized with o-phthaldialdehyde in the presence of 2-mercaptoethanol. The proline derivative was separated on a 25-min gradient program, employing solvent A (0.1 M sodium acetate-0.5% tetrahydrofuran-9% methanol; pH 7.2) and solvent B (methanol). Fluorescence was monitored with excitation at 340 nm and emission at 450 nm. The conversion of proline to 4-amino-1-butanol was quantitative, reproducible, and linear with proline concentrations ranging from 25 to 500 μM commonly present in biological samples. The described method can be readily used for quantifying proline in biomedical research. © 1993.

altmetric score

  • 3

author list (cited authors)

  • Wu, G.

citation count

  • 19
  • 20

publication date

  • July 1993