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

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.

Automated summary

In this study, an optimized and validated method was used to monitor glutathione disulfide (GSSG) in human saliva samples. The results showed accurate quantification of GSSG in the samples, with a concentration range of 100-2000 nmol L-1 and a LOD of 20 nmol L-1. The stability of the samples was ensured by blocking reduced glutathione with N-ethylmaleimide.