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

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-m reversed-phase C18 column (150 4.6 mm I.D.) guarded by a 40-m 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 60C 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 M commonly present in biological samples. The described method can be readily used for quantifying proline in biomedical research. 1993.

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