Milli q purification apparatus

All the employed solvents were of analytical grade and purchased from Carlo Erba (Milan, Italy). Chlorogenic acid (5-CQA) and neochlorogenic acid (3-CQA) standards were purchased from Sigma Aldrich (Milan, Italy), while cryptochlorogenic acid (4-CQA) and caffeic acid (CA) standards were purchased from PhytoLab (Vestenbergsgreuth, Germany). Acetonitrile (ACN) and methanol, both LC-MS grade, were from Merck KGaA (Darmstadt, Germany). Formic acid for LC-MS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocalteu reagent, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) diammonium salt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride (FeCl₃), and gallic acid (GA) were purchased from Sigma-Aldrich (Milan, Italy). Ultra-pure deionized water was generated by a Milli-Q purification apparatus (Millipore, Bedford, MA, USA).

All solvents for LC-HRMS were of high-purity (UPLC-MS grade). Methanol (MeOH) and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany), whereas sodium hydroxide monohydrate (≥99.9%), ammonium formate (A.F.) ≥99.0%, ammonium acetate (A.A.) and formic acid (F.A.) 99%, were provided from Fluka (Buchs, Switzerland). Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA) was used for distilled water. Analytical standards utilized in the study were of highest available purity grade. Most of them were purchased from Sigma-Aldrich (Steinheim, Germany). Additional standard solutions used for the in-house database development were prepared according to Mass Spectrometry Metabolite Library of Standards (MSMLS) protocols (more information in SM-2). A working mix solution of 5 mg L⁻¹ was prepared for analysis by gradient dilution of the stock solutions in methanol, in order to prepare the matrix-matched calibration curves. Lysine-d4, syringaldehyde and hesperetin were used as Internal Standards (IS) at a level of 10 mg L⁻¹.