Bromide–sulfur interchange: Ion chromatographic determination of total reduced thiol levels in plasma

Abstract

Plasma thiol concentration has long been recognised as a potential indicator for assessing the severity of oxidative stress processes within physiological systems. While such measurements are normally restricted to research studies, this communication has sought to develop and characterise a novel approach through which this parameter could be exploited within routine clinical settings. The protocol is based on the rapid derivatisation of reduced thiol functionalities (protein and monomolecular moieties) through the homogenous reaction of a naphthoquinone bromide derivative. Bromide released in the reaction can be easily quantified through ion chromatography (Isocractic Dionex DX-120 incorporating an IonPac® AS14 anion exchange column and a 25 μL sample loop with conductivity detector. Mobile phase consisted sodium carbonate/bicarbonate (3.5 mM/1 mM) at a flow rate of 1.5 mL/min). Method selectivity and sensitivity has been critically evaluated. The technique covers the range 15 μM–3.5 mM PSH with a detection limit of 9 μM PSH and analysis time of 5 min. The efficacy of the approach for the analysis of human plasma from five volunteers was assessed (ranging from 49 to 72 μM with an intra assay variation of less than 5% in all cases). The responses were validated through comparison with the standard Ellman colorimetric technique.