4 o caffeoylquinic acid

Cyanidin-3-O-rutinoside (aka keracyanin), cyanidin-3-O-glucoside (aka kuromanin) and luteolin were purchased from Extrasynthese (Genay Cedex, France). 3-O-caffeoylquinic acid (aka neochlorogenic acid), 4-O-caffeoylquinic acid (aka cryptochlorogenic acid), 5-O-caffeoylquinic acid (aka chlorogenic acid), kaempferol, quercetin-3-O-rutinoside hydrate (aka rutin hydrate), quercetin dihydrate, DL-3-phenyllactic acid, 3,4-dihydroxybenzoic acid (aka protocatechuic acid), caffeic acid and toluene were purchased from Sigma-Aldrich (St. Louis, MO, USA). DL-p-hydroxyphenyllactic acid was from Santa Cruz Biotechnology (Dallas, TX, USA). HPLC-grade acetonitrile was from Sigma-Aldrich, while HPLC-grade water and LC-MS grade formic acid were purchased from VWR International (Milan, Italy).

Analytical standards of 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, protocatechuic acid, quercetin-3-rutinoside hydrate, quercetin dihydrate, kaempferol, phenyllactic acid, caffeic acid, naringenin, (+)-catechin, (−)-epicatechin, p-coumaric acid and toluene were from Sigma-Aldrich (St. Louis, MO, USA). Dihydrocaffeic acid, p-hydroxyphenyllactic acid were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while luteolin was from Extrasynthese (Genay, France). HPLC-grade acetonitrile was from Sigma-Aldrich (St. Louis, MO, USA), while HPLC-grade water and LC-MS grade formic acid were purchased from VWR International (Milan, Italy).

Analytical and HPLC grade solvents and reagents were used for chemical analyses. Acetonitrile (99.9%) and methanol (99.9%)as well as the references as quercetin, quercetin-3-O-glucoside, luteolin-7-O-glucoside, luteolin-7-O-rutinoside, rutin trihydrate, apigenin-7-O-glucoside, santin, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, were obtained from Sigma-Aldrich (Steinheim, Germany); trifluoroacetic acid (≥99%) and apigenin were supplied from Fluka Chemika (Buchs, Switzerland). The purified deionized water (18.2 mΩ/cm) was produced using the Millipore Simpak1 Synergy 185 ultra-pure (Bedford, MA, USA) water system.

Analytical-grade methanol and ethanol for extractions and solvent-partition were obtained from Sigma-Aldrich (Milan, Italy). Ultrapure water (18 MΩ) was prepared by a Milli-Q purification system (Millipore, Bedford, MA, USA). LCMS-grade acetonitrile (ACN), water, and formic acid were supplied by Romil (Cambridge, UK). Reference standards (>98% HPLC grade) apigenin, luteolin, chlorogenic acid (5-caffeoylquinc acid, 5-CQA), 4-O-caffeoylquinic acid, cynarin (1,3-dicaffeoylquinc acid, 1,3-diCQA), 1,5-dicaffeoylquinc acid, 3,5-dicaffeoylquinc acid, 4,5-dicaffeoylquinc acid, luteolin-7-O-glucoside, apigenin-7-O-rutinoside, 1,1-diphenyl-2-picrylhydrazyl (DPPH^•), and Folin–Ciocalteu’s phenol reagent were purchased from Sigma-Aldrich (Milan, Italy), 1-O-caffeoylquinic acid and 1,4-dicaffeoylquinc acid were purchased from Extrasynthase (Lyon, France). Standard stock solutions (1 mg mL⁻¹) of each compound were prepared in methanol and stored at 4 °C. Diluted solutions and standard mixtures were prepared in MeOH/H₂O 2:8, v/v.

For the determination of sugars and organic acids, we used the following standards: sucrose, glucose, fructose, tartaric, quinic, malic, citric, oxalic, shikimic and fumaric acid from Sigma-Aldrich (Steinheim, Germany). For phenolic compounds’ identification and calculation, the following standards were purchased: procyanidin B1, p-coumaric acid, epicatechin ferulic acid, catechin, quercetin-3-O-rutinoside, naringenin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-xyloside, kaempferol-3-O-glucoside and phloridzin from Fluka Chemie (Seelze, Germany), quercetin-3-O-arabinofuranoside from Apin Chemicals LTD (Compton, UK), isorhamnetin-3-O-glucoside from Extrasynthèse (Genay, France), and chlorogenic and caffeic acid, quercetin-3-O-rhamnoside, 3-O-caffeoylquinic acid, cyanidin-3-O-glucoside, pelargonidin-3-glucoside, cyanidin-3-O-rutinoside and 4-O-caffeoylquinic acid from Sigma-Aldrich. For the extraction of phenolic compounds, methanol from Sigma-Aldrich, double-distilled water purified with a Milli-Q system (Millipore, Bedford, MA, USA) and 3% formic acid from Fluka Chemie were used. The chemicals for the mobile phases were acetonitrile (HPLC-MS), formic acid, sulphuric acid and double-distilled water.

Methanol, acetonitrile, and formic acid of MS-grade were supplied from Fisher Scientific (Hampton, New Hampshire, USA). Ammonium formate of MS grade, 4-O-caffeoylquinic acid, quinic acid, shikimic acid, 3-hydroxycoumarin, 7-hydroxycoumarin, trans-caffeic, caffeic acid, trans-ferulic acid, chlorogenic acid, neochlorogenic acid 1-aminocyclohexanecarboxylic acid, methyl benzoate, betaine, norvaline, valine, proline, and choline were purchased from Sigma (St. Louis, MO, USA). Ultrapure water was generated with a Milli-Q system (Millipore, Madrid, Spain).
Green coffee beans (GCB) of the Arabica variety were roasted to light (LRC), medium (MRC), and dark (DRC) levels at 175, 185, and 195 °C during 12.36, 14.11, and 17.06 min, respectively. In order to control the roasting process, the weight loss of each sample was checked, being 13% in light coffee, 15% in medium coffee, and 17% in dark coffee. All these coffee samples were kindly donated, roasted, and grounded by “Café Fortaleza” (Vitoria, Spain) and were identical to the samples analyzed in our previous works using RPLC-MS [25 (link)] and CE-MS [26 (link)] platforms.

Methyl jasmonate (MeJA); sulfatase (from Helix pomatia); diethylaminoethyl (DEAE)-sephadex A-25; sinigrin hydrate; sodium acetate; ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate); iron (II) sulfate heptahydrate (FeSO₄·7H₂O); DPPH (1,1-diphenyl-2-picrylhydrazyl); sodium nitroprusside; acetonitrile (HPLC grade); methanol (HPLC grade); methyl tert-butyl ether (MTBE; HPLC grade); phosphoric acid; 4-O-caffeoylquinic acid; ferulic acid; sinapic acid; quercetin; kaempferol and lutein standards were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sulfanilamide and N-(1-Naphthyl)ethylenediamine dihydrochloride (NED) solutions were acquired from Promega (G2930, Madison, WI, USA). Ethanol (HPLC grade), sodium selenite (Se; Na₂SeO₃), and potassium sulfate (S; K₂SO₄) were obtained from Desarrollo de Especialidades Quimicas, S.A. de C.V. (Monterrey, NL, Mexico). Desulfoglucoraphanin was acquired from Santa Cruz Biotechnology (Dallas, TX, USA).