2.7. Benzoxazinoid analysis

To evaluate the influence of volatile exposure on benzoxazinoid defence metabolites, maize leaves were measured 5 hr after simulated herbivory (n = 5). Seventy milligrams of frozen maize leaf powder was extracted in 700 μl of acidified H₂O/MeOH (50:50 v/v; 0.1% formic acid) and then analysed with an Acquity UHPLC–MS system equipped with an electrospray source (Waters i‐Class UHPLC‐QDA, USA) using a previously established method (Robert et al., 2017). Compounds were separated on an Acquity BEH C18 column (2.1 × 100 mm i.d., 1.7‐μm particle size). Water (0.1% formic acid) and acetonitrile (0.1% formic acid) were employed as mobile phases A and B. The elution profile was 0–9.65 min, 97–83.6% A in B; 9.65–13 min, 100% B; 13.1–15 min 97% A in B. The mobile phase flow rate was 0.4 ml/min. The column temperature was maintained at 40°C, and the injection volume was 5 μl; 2‐(2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose (HDMBOA‐Glc), 2‐(2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose (DIMBOA‐Glc), and 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) were quantified in positive mode using single ion monitoring (SIM) at m/z 194 with cone voltage of 20 V; 2‐(2,4‐dihydroxy‐6,7‐dimethoxy‐l,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose (DIM₂BOA‐Glc), 2‐(2,4‐dihydroxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose (DIBOA‐Glc), 2‐(2‐hydroxy‐4,7,8‐trimethoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose (HDM₂BOA‐Glc), and 6‐methoxy‐benzoxazolin‐2‐one (MBOA) were acquired in negative scan mode (m/z 150–650) using a cone voltage of 10 V. The ESI capillary voltage was set to 0.8 kV. The probe temperature was maintained at 600°C. The detector gain was set to 1 and the sampling frequency was 5 Hz. Absolute quantities were determined using standard curves obtained from purified or synthetic DIMBOA, DIMBOA‐Glc, HDMBOA‐Glc, and MBOA as described (Maag et al., 2015).