Quantification of reduced and oxidized thiols in mouse serum by column-switching hydrophilic interaction chromatography coupled with mass spectrometry.

An automated online solid-phase extraction method for the determination of the reduced and oxidized forms of thiols in mouse serum was developed and validated. Analysis was performed with column-switching hydrophilic interaction chromatography coupled with mass spectrometry (CS-HILIC–MS). The proposed CS-HILIC–MS method enabled the simultaneous determination of reduced and oxidized thiols in mouse serum samples. In addition, interference from endogenous compounds was removed by means of the column-switching technique. We also compared the effects of derivatization before and after preparing serum from blood samples and found that it was necessary to perform the derivatization immediately before preparing serum from blood samples. We investigated the role of thiol compounds in lipopolysaccharide (LPS)-induced acute inflammation in vivo. Serum glutathione disulfide and cystine levels were significantly decreased at 4h after LPS treatment. Our method is expected to be useful for the assessment of the roles of reduced and oxidized glutathione in the oxidative state.