N-hydroxysuccinimidyl fluorescein-O-acetate as a fluorescent derivatizing reagent for catecholamines in liquid chromatography

A new amine-reactive derivatizing reagent, N-hydroxysuccinimidyl fluorescei n-O-acetate (SIFA), was developed for catecholamine (CA) analysis in liquid chromatography, The reactivity of this reagent with the CAs norepinephrine (NE), epinephrine (E), and dopamine (DA) was investigated in detail. In aq ueous methanol containing 32 mmol/L pH 9.0 H3BO3-Na2B4O7 buffer, SIFA react ed with NE, E, and DA under mild conditions. The derivatives were separated in 20 min on a C-18 column with a mobile phase of methanol/water (38:62, v /v) containing 10 mmol/L pH 5.0 H(3)cit-Na2HPO4 buffer. At lambda(ex)/lambd a(em) = 490/516 nm, the detection limits were 3.2, 12, and 56 fmol, respect ively, with a signal-to-noise ratio of 3, which were comparable to those us ing 1,2-diphenylethylenediamine as the derivatizing reagent for CA analysis . Amino acids, aliphatic amines, and alcohols had no obvious interference w ith the determination. The proposed method has been applied to the determin ation of CAs in human urine, with recoveries of 95.3-103.9%. (C) 2000 Acade mic Press.