3-Iodoacetylaminobenzanthrone as a fluorescent derivatizing reagent for thiols in high-performance liquid chromatography

Abstract

A new thiol-reactive derivatizing reagent, 3-iodoacetylaminobenzanthrone (IAB) has been developed for thiol analysis in liquid chromatography. In aqueous methanol containing 15 mM pH 8.3 H 3BO 3–KCl–Na 2CO 3 buffer, IAB reacted with thiols at 35 °C for 15 min. The derivatives of IAB with glutathione (GSH), cysteine (Cys), homocysteine (Hcy) and N-acetylcysteine (Nac) were well separated on a C 18 column with the mobile phase of methanol–water (50:50, v/v) containing 15 mM pH 2.7 H 3cit–Na 2HPO 4 buffer. At λ ex/ λ em=420/540 nm, the detection limits were 20, 20, 55 and 40 fmol (1, 1, 2.3 and 2 nM), respectively, with a signal-to-noise ratio of 3. Owing to the preferential selectivity of iodoacetamidyl moiety to SH group, amino acids, aliphatic amines, phenol and alcohols had no obvious interference with the determination. The proposed method has been applied to the determination of thiols in human blood with recoveries of 98.5–105.3%.