Analysis of biothiols and ascorbate by HPLC-ESI-MS(TOF)
Ascorbate, biothiols and Hg–biothiol complexes were analysed by HPLC-electro spray ionisation (ESI)-time-of-flight mass spectrometry [MS(TOF)], using an HPLC system (Alliance 2795, Waters, Milford, MA, USA), equipped with a reverse-phase monolithic UPLC column (Mediterranea SEA18 3 µm 15 x 0.21 cm, Teknokroma), and coupled to a MS-TOF spectrometer (MicrOTOF, Bruker Daltonics, Bremen, Germany) equipped with an ESI source (Carrasco-Gil et al. , 2011), with a mobile phase made with solvents A (0.1% formic acid in miliQ water), and B (0.1% formic acid in acetonitrile)(see Extended Materials and Methods details in Supplementary online material). After chromatographic separation, sample was directed with a flow rate of 200 µL min-1to the ESI interface. The MS(TOF) operated in negative/positive ion mode at -500/4500 V endplate and spray tip voltages, respectively. The orifice voltage was set at 100 V and full scan data acquisition was carried out from m/z 50 to1000. The mass axis was calibrated externally using Li-formate adducts (10 mm LiOH, 0.2% (v/v) formic acid and 50% (v/v) 2-propanol). The HPLC-ESI-MS(TOF) system was controlled with MicrOTOF Control v.2.2 and HyStar v.3.2, and data were managed with Data Analysis v.3.4 (software packages from Bruker Daltonics). Ion chromatograms were extracted with a precision of 0.05 m/z units.