Fluorescence detection of glutathione S conjugate with aldehyde by high-performance liquid chromatography with post-column derivatization

Abstract

A high-performance liquid chromatographic method with post-column derivatization was developed for the detection of giutathione (GSH) S conjugates with aliphatic aldehydes produced during lipid peroxidation. The GSH S conjugates were separated on a reversed-phase C₁₈ column coated with synthetic polymer, using a mobile phase consisting of a methanol–phosphate buffer gradient system, and were then treated with o-phthalaldehyde (OPA) in the reaction coil. The detection of the OPA derivatives was effected by fluorimetry (λ_ex= 340 nm; λ_em= 450 nm). The detection limits calculated for some aldehydes added to the reaction system were approximately 12–111 pmol (signal-to-noise ratio = 3). This is a simple and sensitive method for the detection of GSH S conjugates with aldehydes in biological samples. By using this method, it was found that the conjugates were readily produced in rat red blood cells incubated with some alkenals and hydroxyalkenals at 37 °C for 60 min.