Uhplc hrms

Hormone extraction of two independent assays was performed. In the first experiment, whole hypocotyl explants were transferred to SGM keeping their apical-basal polarity. Three replicates including eight hypocotyl explants were collected at 0, 6, 10, 24, 96, and 192 hae; before collection, each hypocotyl explant was sectioned into three fragments (D, BC, and A fragments). In the second experiment, hypocotyls were half-sectioned (DC and BA fragments), and each fragment was transferred to SGM keeping its apical-basal polarity. Three replicates including eight DC or BA fragments were collected at 0, 24, and 72 hae; before collection, each fragment was divided in half and hormone measurements were analyzed in D, C, B, and A fragments. Phytohormones were extracted from frozen tissues and were analyzed as described previously [80 (link),81 (link),82 (link)]. Auxin metabolites were identified according to their exact molecular masses and retention times obtained from the total ion chromatograms generated by U-HPLC-HRMS (ThermoFisher Scientific, Waltham, MA, USA) analysis, and they were quantified using analog compounds as standards.

The chemicals for the analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA), and water for ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC/HRMS) was purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA) [16 (link)]. The measurement of plasma levels of polyamines was performed using an UPLC Ultimate 3000 (Thermo Fisher Scientific) system equipped with a HESI-II electrospray source to a Q-Exactive-Orbitrap™-based mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was carried out on a C18 column of the Gemini C18 (Phenomenex, Torrance, CA, USA), 100 mm × 2 mm, particle size 3 µm, and the column was held at 37 °C. Chromatographic separation was achieved with gradient elution using a mobile phase composed of 0.05% heptafluorobutyric acid (HFBA) in water and 0.05% HFBA in methanol, as described in Ran Liu et al. and modified by Coradduzza et al. [54 (link),55 (link)].

For the extraction of polar phenols, rat serum samples underwent enzymatic hydrolysis prior to liquid-liquid extraction (LLE) with ethyl acetate (EtOAc), as reported by Vasilakopoulou et al. [31 (link)]. 100 μL of sodium acetate (CH₃COONa, 0.1 M, pH 5, Thermo Fisher Scientific, MA, USA ) were added to 100 μL of serum samples. Several 20-μL aliquots of β-glucuronidase/ sulfatase (2000/40 U) were injected into the serum samples; the samples were vortexed and incubated for 45 min in a heating bath that was set at 37 °C. To end the enzymatic hydrolysis, 100 μL of phosphoric acid (H₃PO₄, 4% v/v, Thermo Fisher Scientific, MA, USA) were added to the incubated samples. The samples then underwent further treatment with LLE () with the addition of 700 μL EtOAc (Thermo Fisher Scientific, Pittsburgh, PA, USA) to the serum samples. The samples were then vortexed, centrifuged for 5 min at 5000× g, and their supernatants were collected. The extraction process was carried out three times in total, with the supernatants being pooled, evaporated, and reconstituted in 100 μL methanol-water (MeOH-H₂O, 1:1, v/v, Thermo Fisher Scientific, Pittsburgh, PA, USA), which were analyzed with UHPLC–HRMS (Thermo Fisher Scientific, Bremen, Germany).