*2.2. Instrumentation*

LC–MS analysis chromatographic separation was achieved on a reversed phase pentafluorophenyl column (Supelco 2.1 μm F5, 100 × 2.1 mm) from Sigma Aldrich (Poole, Dorset, UK). Thermo Surveyor LC (Thermo Scientific, Hemel Hempsted, UK) consisted of a quaternary MS pump, vacuum degasser, thermostated autosampler (set to 5 ◦C) and a thermostated column oven (set to 25 ◦C). Mass spectrometry was performed using an LTQ XL ion trap mass spectrometer (Thermo Scientific, Hemel Hempsted, UK) equipped with a heated electrospray ionization (HESI) source maintained at 200 ◦C. The solvent evaporation was aided with auxiliary gas, sheath gas and sweep gas set to an arbitrary flow rate of 15, 60 and 1, respectively. The mass spectrometer was operated in selected reaction monitoring (SRM) MS/MS in negative mode, with collision energies of 28 eV for T4 and 27 eV for T3. The monitored transitions were 776 → 604 and 650 → 633 for T4 and T3, respectively. In SRM MS/MS mode the precursor ion is isolated and subjected to a specified amount of collision energy to induce fragmentation. The MS method is then set to monitor the precursor and a minimum of two stable product ions. Sample aliquots of 10 μL were introduced onto the column at a flow rate of 200 μL/min. The analytes were separated using an isocratic method using water +0.2% formic acid (A) and methanol +0.2% formic acid (B) as the mobile phase.
