N-Hydroxysuccinimidyl Fluorescein-O-acetate as a Fluorescent Derivatizing Reagent for Catecholamines in Liquid Chromatography

Abstract

A new amine-reactive derivatizing reagent, N-hydroxysuccinimidyl fluorescein-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 aqueous methanol containing 32 mmol/L pH 9.0 H3BO3–Na2B4O7 buffer, SIFA reacted with NE, E, and DA under mild conditions. The derivatives were separated in 20 min on a C18 column with a mobile phase of methanol/water (38:62, v/v) containing 10 mmol/L pH 5.0 H3cit–Na2HPO4 buffer. At λex/λem = 490/516 nm, the detection limits were 3.2, 12, and 56 fmol, respectively, with a signal-to-noise ratio of 3, which were comparable to those using 1,2-diphenylethylenediamine as the derivatizing reagent for CA analysis. Amino acids, aliphatic amines, and alcohols had no obvious interference with the determination. The proposed method has been applied to the determination of CAs in human urine, with recoveries of 95.3–103.9%.