Benzoxazinoid Purity Analysis by UHPLC-MS

To determine the purity of the extracted benzoxazinoids, we analyzed them by UHPLC-MS and adapting a previous protocol (62 (link)). Briefly, an Acquity UHPLC system coupled to a G2-XS QTOF mass spectrometer equipped with an electrospray source and piloted by the software MassLynx 4.1 (Waters AG, Bade-Dättwil, Switzerland) was used. Gradient elution was performed on an Acquity BEH C18 column (2.1 × 50 mm i.d., 1.7 mm particle size) at 90 to 70% A over 3 min, 70 to 60% A over 1 min, 40 to 100% B over 1 min, holding at 100% B for 2.5 min, holding at 90% A for 1.5 min where A = 0.1% formic acid (FA)/water and B = 0.1% FA/acetonitrile (ACN). The flow rate was 0.4 mL/min. The temperature of the column was maintained at 40 °C, and the injection volume was 1 μL. The QTOF MS was operated in positive mode. The data were acquired over an m/z range of 50 to 1,200 with scans of 0.15 s at a collision energy of 4 V and 0.2 s with a collision energy ramp from 10 to 40 V. The capillary and cone voltages were set to 2 kV and 20 V, respectively. The source temperature was maintained at 140 °C, the desolvation was 400 °C at 1,000 L/h and cone gas flow was 50 L/h. Accurate mass measurements (<2 mg kg⁻¹) were obtained by infusing a solution of leucin encephalin at 200 ng/mL and a flow rate of 10 mL/min through the Lock Spray probe.