N-hydroxysuccinimidyl fluorescein-O-acetate as a highly fluorescent derivatizing reagent for aliphatic amines in liquid chromatography

A sensitive fluorescent reagent, N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA), has been developed as a precolumn derivatizing reagent for aliphatic amines in liquid chromatography (LC). SIFA was shown to be a successful reagent with advantages including high fluorescent quantum yield in the visible region, specificity to aliphatic amines, mild derivatization conditions and few by-products. Methylamine, ethylamine, ethylene diamine, isopropylamine, isobutylamine, n-butylamine, 1,4-butylene diamine and 1,5-amylene diamine were used to investigate the separation and derivatization conditions. In a pH 8.5 H3BO3-Na2BO7 buffer solution, SIFA reacted with amines at 45 degrees C for 30 min to form stable derivatives. The derivatives could be separated on a C-18 column in 18 min with methanol-water (46/54, v/v) containing 10 mmol l(-1) pH 5.40 citric acid-Na2HPO4 buffer as a mobile phase. The detection limits of these amines were in the range 85-520 amol (signal-to-noise ratio = 3), which were better than those using the other derivatizing reagents for amine analysis by LC. (C) 2000 Elsevier Science B.V. All rights reserved.