UHPLC-PDA-Orbitrap Metabolites Characterization

Liquid chromatography was performed using an UHPLC C18 column (Accucore, 150 mm × 4.6 mm ID, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) operated at 25°C. The detection wavelengths were 254, 280, 330 and 354 nm, and PDA was recorded from 200 to 800 nm for peak characterization. Mobile phases were 1% formic aqueous solution (A) and 1% formic acid in acetonitrile (B). The gradient program (time (min), % B) was: (0.00, 12); (5.00, 12); (10.00, 20); (15.00, 40); (20.00, 40); (25.00, 70); (35.00, 12); and 15 min for column equilibration before each injection. The flow rate was 1.00 mL min⁻¹, and the injection volume was 10 μL. The standards, and the extracts dissolved in ethanol, were kept at 10°C during storage in the autosampler. The HESI II and Orbitrap spectrometer parameters were optimized as previously reported (Salgado et al., 2017 (link); Torres-Benitez et al., 2017 (link)).