Gallocatechin

The following standards were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany): catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, gallocatechin gallate, procyanidin B1 and B2, procyanidin A2, procyanidin C1, kaempferol, quercetin, rutin, salicylic acid, catechin-2,3,4–¹³C₃ 99 atom % ¹³C (98 % CP), gallocatechin-2,3,4–¹³C₃ ≥ 99 atom % ¹³C (≥97 % CP), catechin-2,3,4–¹³C₃ gallate ≥99 atom % ¹³C (≥97 % CP), salicylic acid-D₄ certified reference material. Naringenin was purchased from Thermo Scientific (Waltham, MA, U.S.A).
The following chemicals were purchased from Sigma-Aldrich (Merck-Millipore, Merck KGaA, Darmstadt, Germany): dimethyl sulfoxide (DMSO), hydrochloric acid (HCl, 37 %), formic acid (FA, LiChropur 98–100 % LCMS grade), 2 M Folin-Ciocalteu′s phenol reagent (47641-500 ML-F), gallic acid (G7384-100G) and β-glucuronidase type H-1 from Helix pomatia. Sodium bicarbonate (S6014-500G, Fluka), methanol (85681.320, VWR), and acetonitrile (ACN, HiPerSolv Chromanorm) were purchased from VWR Chemicals (Radnor, PA, U.S.A.). Sodium acetate was obtained from Merck (Darmstadt, Germany) and glacial acetic acid from Fluka/Sigma-Aldrich (Thermo Fisher Scientific, Waltham, MA, U.S.A; Merck KGaA, Darmstadt, Germany).

Samples
were also diluted 1/2 with acidified water prior to the analysis of
ellagitannins. Then, following the methodology developed and validated
in our laboratory,^{33 (link)} (−)-gallocatechin
(Sigma-Aldrich, St. Louis, MO, U.S.A.) was added as an internal standard
(final concentration of 0.015 mg/mL) and the samples were filtered
(0.45 μm Millex syringe-driven filter unit) before the injection
in the HPLC–MS system. A Hewlett-Packard 1200 series LC equipped
with an AQUA C18 reversed-phase, 5 μm, 150 × 4.6 mm column
(Phenomenex, Torrance, CA, U.S.A.) thermostated at 35 °C was
employed, using an aqueous solution (2.5%) of acetic acid (AnalaR,
Normapur, VWR International, Fontenay-sous-Bois, France) (solvent
A), 100% HPLC-grade isopropanol (HiPerSolv Chromanorm, BDH Prolabo,
VWR International, Fontenay-sous-Bois, France) (solvent B), and 100%
HPLC-grade methanol (Macron Fine Chemicals, Avantor, Gliwice, Poland)
(solvent C) as eluents. HPLC conditions were previously developed
in our laboratory for the analysis of oak wood ellagitannins.^{29 (link)} The API 3200 QTrap mass spectrometer was connected
to the LC via the cell outlet, and MSⁿ–MRM analyses were performed in negative ion mode following
the conditions previously optimized for quantification of these compounds.^{33 (link)}

All of the solvents, salts, acids and bases were of analytical grade and were purchased from Sigma-Aldrich-Merck (Darmstadt, Germany) or Carlo Erba (Milan, Italy). (Poly)phenol standards used for identification and quantification purposes with HPLC-MS/MS were as follows: protocatechuic acid, gallic acid, (+)-gallocatechin, (−)-epigallocatechin, caftaric acid, (+)-catechin, (−)-epicatechin, trans-coutaric acid, astringin, trans-fertaric acid, 2,6-dihydroxybenzoic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, vanillic acid, rutin, piceid, coumaric acid, sinapic acid, ferulic acid, luteolin, quercetin, apigenin, kaempferol, procyanidin B1 (Sigma-Aldrich-Merck, Darmstadt, Germany) and procyanidin B2 (Extrasynthese, Genay Cedex, France).