4.4. Methanol Extraction of Root Tissue and HPLC-UV, LC-MS/MS Analysis of Methanol Extracts

Metabolites were extracted from 40 mg fresh plant material by adding 1 mL 100% methanol (MeOH) containing 0.8 mg/mL phenyl-β-D-glucopyranoside (Sigma Aldrich, St. Louis, MO, USA) and 40 ng/mL D₆-abscisic acid (D₆-ABA) (Santa Cruz Biotechnology, Dallas, TX, USA) as internal standards. Samples were shaken for 30 sec in a paint shaker (Scandex, Büdelsdorf, Germany) and afterwards for 30 min at 200 rpm on a horizontal shaker (IKA Labortechnik, Staufen, Germany). After centrifugation, the supernatants were split for high performance liquid chromatography (HPLC)-UV and liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements.

Salicinoid analysis and quantification was performed by HPLC-UV (200 nm) as described previously in Böckler et al. [37] for the compounds salicin, salicortin, tremulacin, and homaloside D, and for 6′-O-benzoylsalicortin as described in Lackner et al. [38]. Chromatographic separation was achieved on an Agilent 1100 Series LC system (Agilent Technologies), using an EC 250/4.6 Nucleodur Sphinx column (RP 5 μm, Macherey-Nagel, Düren, Germany), with water and acetonitrile as mobile phases A and B, respectively. The mobile phase flow rate was 1 mL/min. The elution profile is listed in Supplemental Table S4 as gradient A. Salicinoids were quantified relative to the signal of the internal standard phenyl-β-D-glucopyranoside, by applying experimentally determined response factors [37,38].

The compounds salirepin, salicin-7-sulfate, and salirepin-7-sulfate were analyzed and quantified by LC-MS/MS as follows and as previously described in Lackus et al. [39]. Chromatographic separation was achieved using an Agilent 1260 infinity II LC system (Agilent Technologies) equipped with a Zorbax Eclipse XDB-C18 column (50 × 4.6 mm, 1.8 μm, Agilent Technologies), using aqueous formic acid (0.05% (v/v)) and acetonitrile as mobile phases A and B, respectively. The mobile phase flow rate was 1.1 mL/min. The elution profile is listed in Supplemental Table S4 as gradient B. The column temperature was maintained at 20 °C. The LC system was coupled to a QTRAP 6500^® tandem mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with a turbospray ion source, operated in negative ionization mode. The ion spray voltage was maintained at −4500 eV and the turbo gas temperature was set at 700 °C. Nebulizing gas was set at 60 psi, curtain gas at 40 psi, heating gas at 60 psi and collision gas at medium level. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion → product ion formation, and respective parameters are listed in Supplemental Table S5. Sulfated salicinoids and salirepin were quantified relative to the signal of the internal standard D₆-ABA, by applying experimentally determined response factors [39]. Analyst 1.6.3 software (Applied Biosystems, Darmstadt, Germany) was used for data acquisition and processing.