Quantitative Analysis of Plant Hormones and Phenolics

For the hormonal analysis, fresh material was frozen in liquid N, ground, and freeze-dried. Fresh tissue (0.5 g) was immediately homogenized in 2.5 mL of ultrapure water, and 100 ng mL⁻¹ of a mixture of internal standards ((2H6-ABA (to quantify ABA), 2H4-SA (to quantify SA and propylparaben (to quantify phenolic compounds like ferulic acid (FA) and chlorogenic acid (CGA)), (Sigma–Aldrich, St. Louis, MO, USA)) were added prior to extraction. The samples were centrifuged at 5000 rpm for 45 min at 4 °C. The supernatant was partitioned against diethylether, dried in a speed vacuum and resuspended in 90:10 H₂O:MeOH. [78 (link)]. After extraction, a 20 µL aliquot was injected directly into an ultra-high performance liquid chromatography (UPLC) system with an ACQUITY UPLC BEH C18 column (1.7 μm 2.1 × 50 mm) (Waters, Mildford, MA, USA), which was interfaced with a triple quadrupole mass spectrometer (TQD, Waters, Manchester, United Kingdom). Version 4.1 of the MASSLYNX NT software (Micromass) was used to process the quantitative data from the calibration standards and plant samples. The concentrations of hormones and phenolic compounds were determined in each sample by normalizing the chromatographic area for each compound with the fresh weight of the corresponding sample.