2-(4-N-MALEIMIDOPHENYL)-6-METHOXYBENZOFURAN - A SUPERIOR DERIVATIZING AGENT FOR FLUOROMETRIC-DETERMINATION OF ALIPHATIC THIOLS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The use of 2-(4-N-maleimidophenyl)-6-methoxybenzofuran as a fluorogenic pre-column derivatizing agent for sensitive high-performance liquid chromatographic (HPLC) determination of aliphatic thiols is described. The maleamic acid, its methyl ester and the N-maleimide derivatives of the common amine precursor were easily synthesized and characterized according to conventional methods. These derivatives lack fluorescent properties in their native form but display strong fluorescence upon reaction with aliphatic thiols. The reaction is rapid and highly selective for thiols over a wide pH range (7.1-8.8). Following derivatization, the thiol adducts were separated on a reversed-phase column (Ultrasphere-ODS) using 0.1% hexanesulfonic acid in 10 mM potassium hydrogen phosphate-acetonitrile (65:35, pH 4.5) and were detected fluorimetrically (excitation at 310 nm; emission a; 390 nm). The method is highly sensitive (femtomole range) and is easily applied to the determination of SH-containing drugs and endogenous thiols in biological samples.