Characterization, biological interactions and in-vivo detection of selenotrisulfide derivatives of glutathion, cysteine and homocysteine by HPLC-ICP-MS

Abstract

Thiol-containing compounds such as glutathione, cysteine and homocysteine react with selenite under specific conditions to form selenotrisulfides of significance in cellular systems. This study describes the synthesis and mass spectrometric characterization of selenotrisulfides of glutathione (GSH), cysteine (Cys) and homocysteine (HCys) by electrospray-MS (ESI-MS). The synthesized species are separated by reversed phase-ion pairing HPLC and detected by selective monitoring of Se (m/z 77, 78 and 80) using a collision/reaction cell-inductively coupled plasma-mass spectrometer (CRC-ICP-MS). The cell is pressurized using H₂ as reaction gas at a flow of 3.5 ml min⁻¹ for minimizing Ar polyatomic ions affecting m/z 78 and 80. Separation of the species is performed in a C₈ column with a mobile phase containing 12% methanol and 0.05% heptafluorobutyric acid (HFBA). Using this hyphenated system (HPLC-ICP-MS) LDs below 1 ppb were attained for all the species under study. HPLC-ICP-MS was used also as a tool to study the presence of an excess of GSH and the pH influence on the stability of the Se–glutathion conjugate (GS–Se–SG) as model. Similarly, GS-Se-SG is evaluated as a possible substrate for the enzyme glutathione reductase (GR) in the presence of NADPH. The isolation and HPLC-ICP-MS detection of the synthesized selenotrisulfides in rat liver cytosols of exposed animals will be also discussed.