N-hydroxysuccinimidyl-fluorescein-O-acetate for precolumn fluorescence derivatization of amino acids and oligopeptides in liquid chromatography

A new fluorescent derivatizing reagent, N-hydroxysuccinimidyl-fluorescein-O-acetate, is used for the high-performance liquid chromatographic analysis of amino acids and oligopeptides. This reagent has the advantages of high-detection sensitivity in the visible region, specifically with amino groups, mild derivatization conditions, and little interference induced. The fluorescence properties of the reagent and its derivatives with amino acids and oligopeptides are studied. The conditions of the derivatization are investigated in detail. In the mobile phase of methanol-water (42:58, v/v) containing a 10 mM pH 5.0 citric acid-Na2HPO4 buffer, six amino acids and oligopeptides are separated in 20 min with fluorescence detection at excitation and emission wavelengths of 492 and 513 nm, respectively, with the detection limits for injected standards ranging from 0.64 to 12 fmol.