Pinoresinol

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.

The analytical standards of hydroxytyrosol, tyrosol, vanillic acid, oleuropein, luteolin, and apigenin were purchased from Extrasynthese (Genay, France). p-Coumaric acid, ferulic acid, pinoresinol, syringic acid, α-tocopherol, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and Trolox were purchased from Sigma-Aldrich (Milan, Italy). High performance liquid chromatography (HPLC) -grade methanol, hexane, and isopropanol (IPA) were purchased from Sigma-Aldrich (Milan, Italy). Water (resistivity above 18 MΩ cm) was obtained from a Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA). Solvents and solutions were filtered through a 0.45-µm polytetrafluoroethylene (PTFE) filter from Supelco (Bellefonte, PA, USA).

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).

Standards of volatile and phenolic compounds were commercially obtained as follows: (Z)-3-hexen-1-ol (Toronto research chemicals Inc., Toronto, CA, USA); linalool, nonanal, phenylethyl alcohol, decanal, eugenol, 1,3,5-trimethoxybenzene, 3-hydroxytyrosol, caffeic acid, vanillin, trans-p-coumaric acid, trans-o-coumaric acid, apigenin-7-glucoside, oleuropein, pinoresinol, quercetin, luteolin, apigenin, diosmetin tyrosol, rutin, luteolin-7-glucoside, oleacein, and ligstroside from Sigma-Aldrich (St. Louis, MO, USA); verbascoside (HWI group, Rülzheim, Germany); and oleuroside (Phytolab, Vestenbergsgreuth, Germany). Alkane standard solutions C₈-C₂₀ and C₁₀, C₂₀-C_40, and derivatization reagent N,O-Bis(trimethylsilyl)trifluoroacet-amide (BSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); pentane, diethyl-ether, acetonitrile, and methanol were from VWR (Radnor, PA, USA); and anhydrous sodium sulfate was from Kemika (Zagreb, Croatia).

Standard compounds such as hydroxytyrosol, tyrosol, luteolin, apigenin, and pinoresinol were purchased from Sigma-Aldrich (St. Louis, MO, USA), and oleuropein from Extrasynthèse (Lyon, France). Methanol reagent was from Panreac (Barcelona, Spain). HPLC-grade acetonitrile and acetic acid (assayed at >99.5%) used for preparing mobile phases were from Labscan (Dublin, Ireland) and Fluka (Switzerland), respectively. Distilled water with a resistance of 18.2 MΩ was deionized in a Milli-Q system (Millipore, Bedford, MA, USA). The stock solutions containing these analytes were prepared in methanol. All chemicals were of analytical reagent grade and used as received. All the solutions were stored in dark flasks at −20 °C until used.

2,5-Dihydroxybenzoic acid, pyrocatechol, chlorogenic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, gallic acid, (+)-catechin, vanillin, syringic acid, taxifolin, p-coumaric acid, sinapic acid, ferulic acid, 2-hydroxycinnamic acid, rosmarinic acid, pinoresinol, luteolin, quercetin, apigenin and HPLC grade of methanol and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-Hydroxybenzoic acid, vanillic acid, apigenin 7-glucoside, 3,4-dihydroxyphenylacetic acid, luteolin 7-glucoside, eriodictyol, hesperidin, and kaempferol were obtained from Fluka (St. Louis, MO, USA). The hyperoside protocatechuic acid and verbascoside were purchased from HWI Analytik (Ruelzheim, Germany). Ultra-pure water (18.2 mΩ/cm) was prepared by using a Milli-Q water purification system (Milli-Q Millipore Merck KGaA Darmstadt, Germany). All the chemicals and reagents used in the biological assay were obtained from Sigma-Aldrich.

The chemicals employed during the development of this research were of analytical grade reagent. Milli-Q water for PLE and mobile phase preparation was obtained with a Millipore system (Bedford, MA, USA). Ethanol was provided by VWR Chemicals (Radnor, PA, USA), while Fisher Chemicals (Waltham, MA, USA) supplied cellulose filters and sand for PLE experiments. The mobile phase composition was obtained with acetic acid supplied by Sigma–Aldrich (Steinheim, Germany) and LC-MS-grade acetonitrile from Fisher Chemicals (Waltham, MA, USA). The commercial standards for quantitation purposes (quinic acid, hydroxytyrosol, pinoresinol, and oleuropein) were acquired from Sigma–Aldrich (St. Louis, MO, USA), Arbo Nova (Turku, Finland) and Extrasynthese (Lyon, France).