Simultaneous and trace determination of reduced and oxidized glutathione in minute plasma samples using dual mode fluorescence detection and column switching high performance liquid chromatography

A robust method for the simultaneous quantification of endogenous reduced glutathione (GSH) and oxidized glutathione (GSSG) in as little as 5 μl human plasma employing two-dimensional chromatographic system with parallel Hypercarb™ columns coupled with dual fluorescence detectors (FLD) has been developed. After sample preparation, 10 μl of supernatant was injected into the chromatographic system. The limits of detection (LOD) of GSH and GSSG were 0.5 and 0.040 pmol on column, respectively. Derivatization of GSH and GSSG with monobromobimane (MBB) and ortho-phthalaldehyde (OPA) provides a sensitivity and specificity that allows analysis after fingertip sampling, blood sampling from infants or multiple blood sampling from mice or other small experimental animals without sacrificing the animal.