0.22 μm filter

Plant hormones, including different jasmonates (OPDA, JA and the conjugated form JA‐Ile), ABA and the cytokinin t‐Z were extracted and quantified by ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Ground leaves (50 mg) and freeze‐dried roots (30 mg) were extracted with 250 μl of a methanol:2‐propanol:glacial acetic acid (50:49:1) mix and deuterium‐labeled standards (d5‐JA, d6‐ABA, and d5‐tZ). The extracts were subjected to ultrasonication (Branson 2510 ultrasonic cleaner, Bransonic) and vortexing for 30 min, followed by a 10 min centrifugation at 15,000 g (PrismR, Labnet International Inc.). The supernatant was collected, and the pellet was reextracted. Both supernatants were merged and filtered through a hydrophobic 0.22 μm filter (Phenomenex). Hormone levels were analyzed by UHPLC‐ESI‐MS/MS as described by Müller and Munné‐Bosch (2011 (link)). The UHPLC was coupled to a triple quadrupole mass spectrometer (QTRAP 4000, AB Sciex). A LUNA C18 column (Phenomenex, 1.6 μm, 100 × 2.1 mm) was used. Solvent A was water with 0.05% acetic acid and solvent B was acetonitrile with 0.05% acetic acid. Flow rate was set at 0.6 ml min⁻¹. Quantification was made considering recovery rates for each sample by using the deuterium‐labeled internal standards. Calibration curves for each analyte were generated using MultiQuantTM 3.0.1 software.

For bread analysis the method described by Saladino et al. [5 (link)] was followed. Briefly, 5 g of bread were accurately weighed (precision 0.1 mg), transferred to centrifuge tubes (50 mL), and 25 mL of methanol were added. Samples were crushed in Ultraturrax (T 18 digital ULTRA-TURRAX^®, Staufen, Germany) for 5 min and centrifuged at 4500 rpm for 10 min (Centrifuge 5810R, Eppendorff, Germany). Supernatant was collected in new centrifuge tubes and evaporated until complete dryness using a rotavapor (BUCHI Rotavapor R-200, Postfach, Switzerland) and turbovap (TurboVap LV Evaporator, Zymark, Hopkinton, MA, USA). Samples were then reconstituted in 1 mL of methanol, filtered with a 0.22-μm filter (Phenomenex, Madrid, Spain) and injected for HPLC–MS/qTOF analysis. All the analyses were performed by triplicate (n = 3).