Flavonoids and Stilbenes Quantification by UHPLC

Flavonoids and stilbenes were identified and quantified on the samples used for RNAseq by Ultra High Performance Liquid Chromatography (UHPLC), according to^{68 (link)}. Samples were prepared as described in^{69 (link)}. Briefly, 0.4 mL of chloroform and 0.6 mL of methanol:water (2:1 v/v) were added to 0.1 g of fresh sample. The extraction was repeated by adding 0.6 mL of methanol and water (2:1 v/v) and 0.2 mL of chloroform. The aqueous-methanolic phases were combined and evaporated to dryness under N₂. Samples were re-suspended in 500 µL of methanol and water (1:1 v/v), centrifuged and transferred into an HPLC vial. Chromatographic analysis was performed using a Waters Acquity UPLC system (Milford) equipped with a Waters Acquity HSS T3 column using the solvents B (acetonitrile containing 0.1% formic acid) and A (water containing 0.1% formic acid). Mass spectrometry detection was performed on a Waters Xevo triple-quadrupole mass spectrometer detector (Milford) with an electrospray (ESI) source^{68 (link)}. Compounds were identified based on their reference standards, retention time and qualifier and quantifier ion, and were quantified using their calibration curves. Data were finally expressed as µg g⁻¹ of fresh weight (FW). Data processing was performed using the Waters MassLynx V4.1 software.