Development and validation of a direct HPLC method for the determination of salivary glutathione disulphide using a core shell column and post column derivatization with o-phthalaldehyde

Abstract

Glutathione disulfide (GSSG) has been monitored in human saliva samples by an optimized and validated method that is based on liquid chromatography coupled to on-line post column derivatization. The analyte was separated from the sample matrix using a 100% aqueous mobile phase through a core-shell reversed phase column. Following optimization of the reaction using Box- Behnken experimental design and validation, GSSG was quantified accurately and selectively in the range of 100-2000 nmol L^-1 with a LOD of 20 nmol L^-1. GSSG was quantified in 15 out of 20 human saliva samples (75%) with a mean value of 860 nmol L^-1 (150-4600 nmol L^-1). Blocking of reduced Glutathione with N-ethylmaleimide ensured stability of the samples for at least 72 h at all temperatures examined.

Keywords: Core shell reversed phase column; Glutathione disulfide; Human saliva; Liquid chromatography; Post column derivatization.