Quantifying Abscisic Acid by UHPLC-MS/MS

Abscisic acid levels were determined by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) as described previously (Müller and Munné-Bosch, 2011 (link)). In short, 100 mg per sample was extracted with 200 µl methanol/isopropanol/acetic acid 50:49:1 (v/v/v) using ultrasonication and vortexing (Branson 2510 ultrasonic cleaner, Bransonic, Danbury, CT, USA) for 30 min. Deuterium-labeled ABA was then added, and after centrifugation at 600×g for 15 min at 4°C, the pellet was re-extracted using the same procedure. Supernatants were pooled and filtered through a 0.22-µm polytetrafluoroethylene (PTFE) filter (Waters, Milford, MA, USA) before analyses. ABA levels were analyzed using UHPLC-ESI-MS/MS as described in Müller and Munné-Bosch (2011) (link). Quantification was performed considering recovery rates for each sample by using a deuterium-labeled internal standard. Three replicates were conducted for each treatment.