Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry

A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX® extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm × 2.1 mm, 5 μm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1–100 μM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 μM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.