Phytochelatin Determination by UPLC-ESI-QTOF-MS

Phytochelatins (PCs) were determined by UPLC-ESI-QTOF-MS as described earlier^{53 (link)}. Briefly, frozen homogenized plant material was extracted with 0.1% (v/v) trifluoroacetic acid containing 6.3 mM diethylene triamine pentaacetic acid and 40.04 µM N-acetylcysteine as internal standard. Prior to derivatization with monobromobimane at 45 °C for 30 min, thiol groups were reduced with Tris-(2-carboxyethyl)-phosphine. Labelled thiols were separated on a HSS T3 column (1.8 μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) by a Waters Aquity UPLC system applying a linear binary gradient of water (A) and acetonitrile (B), both acidified with 0.1% (v/v) formic acid, at a flow of 0.5 mL min^–1: 99.5% A, 0.5% B for 1 min, a linear gradient to 60.5% B at 10 min, gradient to 99.5% B at 12 min, flushing with 99.5% B for 1 min, a gradient back to initial conditions in 1 min and an additional re-equilibration for 1 min. The column temperature was set to 40 °C. Thiols were detected with a Q-TOF Premier mass spectrometer equipped with an ESI-source (Waters Corporation) operated in the V + mode. For quantification the QuanLynx module of the MarkerLynx software was used.