Xevo tandem quadrupole mass spectrometer

The ABA extraction and detection method is as described in He et al. (2014) . In brief, 10 mg of dry seeds were ground and extracted with methanol/water/ acetic acid (80 : 19 : 1) and [ 2 H 6 ]ABA as internal standard. The tubes were vortexed and sonicated for 10 min. Samples were centrifuged for 10 min and the liquid phase was transferred to a glass vial. The samples were re-extracted with methanol/water/acetic acid (80 : 19 : 1). Both fractions were combined in the vial and dried in a speedvac. The residue was dissolved in 100 ml of methanol/acetic acid 99 : 1 (v/v) and 900 ml of 1% acetic acid in water. The samples were loaded on HLB columns (Oasis Õ , Waters, 30 mg 1 ml) which were previously equilibrated. The columns/samples were washed with 1 ml of methanol/ water/acetic acid (10 : 89 : 1). After washing, 1 ml of methanol/water/acetic acid (80 : 19 : 1) was added to elute the samples. The samples were dried in a vacuum concentrator and re-suspended in 100 ml of water/acetonitrile/formic acid (94.9 : 5 : 0.1). ABA analysis was performed with a Waters Xevo tandem quadrupole mass spectrometer equipped with an electrospray ionization source and coupled to an Acquity UPLC system (Waters).