Analysis of biogenic thiols in natural water samples by high-performance liquid chromatographic separation and fluorescence detection with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F)

Abstract

A sensitive and reliable reversed-phase high-performance liquid chromatography (HPLC) method has been developed to analyze naturally occurring low-molecular weight thiols using pre-column derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) and fluorescence detection in a gradient elution. The derivatization procedure and separation conditions were optimized by careful control of the reaction conditions in terms of time, temperature, pH and reducing agent. A linear calibration in the nM range was obtained for all the standard compounds, allowing measurement of thiols in natural water samples. Results show that the reducing agent tri-n-butylphosphine (TBP) not only acts as an anti-oxidant/disulfide cleaving agent, but also enables the SBD-F to react with thiols complexed by soft metals. This is the first study reported using the SBD-F labeling technique to measure phytochelatin concentrations. The methodology has been successfully applied to estuarine water samples from Galveston Bay. (C) 2000 Elsevier Science B.V. All rights reserved