Hydroxytyrosol

Ultrapure water was obtained through a purification system (Millipore Direct-Q UV, Bedford, MA, USA). Formic acid, of LC-MS grade, and ammonium acetate ≥99% were obtained from Fluka (Buchs, Switzerland). Sodium hydroxide >99% and methanol, of LC-MS grade, were acquired from Merck (Darmstadt, Germany). 2-propanol was acquired from Fisher Scientific (Geel, Belgium).
All standards had an analytical grade ≥95%. Epicatechin, ferulic acid, gallic acid, homovanillic acid, oleuropein, pinoresinol, p-coumaric acid, and syringaldehyde were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin, caffeic acid, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruhe, Germany). Luteolin and hydroxytyrosol were purchased from Santa Cruz Biotechnologies (Heidelberg, Germany). Syringic acid was obtained from Extrasynthèse (Genay, France). Stock solutions for each analyte (1000 mg/L) were dissolved in methanol and stored at −20 °C. Mixed standard working solutions were prepared every laboratory day diluting the stock solutions with water:methanol (20:80, v/v).

Methanol (LC-MS grade) and sodium hydroxide (>99%) were purchased from Merck (Darmstadt, Germany). Ammonium acetate (≥99% for HPLC) and formic acid (LC-MS Ultra) were purchased from Fluka (Buchs, Switzerland). Isopropanol was obtained from Fisher Scientific (Geel, Belgium). Ultrapure water was provided by a Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). Regarding the standards that were used, syringic acid 95% was purchased from Extrasynthèse (Genay, France). Gallic acid 98%, ferulic acid 98%, epicatechin 97%, p-coumaric (4-hydroxycinnamic acid) 98%, homovanillic acid 97%, quercetin 98%, oleuropein 98%, pinoresinol 95%, caffeic acid 99%, taxifolin 98%, vanillic acid 97% and syringaldehyde 98% (internal standard) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydroxytyrosol 98% and luteolin 98% were acquired from Santa Cruz Biotechnologies. Vanillin 99%, apigenin (4,5,7-trihydroxyflavone) 97% and tyrosol (2-(4-hydroxyphenyl) ethanol) 98% were purchased from Alfa Aesar (Karlsruhe, Germany). Stock standard solutions of individual compounds (1000 mg/L) were solubilized in methanol and stored at −20 °C in dark brown glass bottles. Intermediate standard working solutions containing the analytes were prepared by appropriate dilution of the stock solutions with methanol:water (80:20, v/v) over the concentration range 0.1–8 mg/L.

All standards and reagents were LC-MS grade and were used without any further treatments. Authentic standards of 2,5-Dihydroxybenzoic acid (gentisic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid), 4-hydroxybenzoic acid, cinnamic acid, epicatechin, eriodictyol, ferulic acid, gallic acid, myricetin, p-coumaric acid, pinoresinol, quercetin, resveratrol, rosmarinic acid, salicylic acid, syringic acid, taxifolin, and vanillic acid were purchased from Sigma-Aldrich (Stenheim, Germany). Apigenin, caffeic acid, catechin, ethyl vanillin (internal standard, IS), galangin, genistein, naringenin, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruche, Germany), whereas hydroxytyrosol and luteolin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2-Propanol (LC–MS grade) and methanol (MeOH) (LC–MS grade) were purchased from Fisher Scientific (Geel, Belgium) and Merck (Darmstadt, Germany), respectively. Formic acid 99%, ammonium acetate and sodium hydroxide monohydrate for trace analysis ≥ 99.9995% were all obtained from Fluka (Buchs, Switzerland). Ultrapure water (18.2 MΩ resistivity) was provided by a Millipore Direct-Q UV purification System (Millipore, Bedford, MA, USA). The wine samples were filtered using regenerated cellulose syringe filters (RC filters, pore size 0.2 μm, diameter 15 mm) purchased from Phenomenex (Torrance, CA, USA).