2.5.1. Identification and Quantification of Individual Polyphenolic Compounds

An LC-MS method previously described [21,22] was used for the identification of individual polyphenols in the HI extracts. The 18 external standards were: apigenin, caffeic acid, caftaric acid, chlorogenic acid, ferulic acid, fisetin, gentisic acid, hyperoside, isoquercitrin, kaempferol, luteolin, myricetin, patuletin, *p*-coumaric acid, quercetin, quercitrin, rutoside, and sinapic acid (Table S1). In brief, the chromatographic separation was performed on a reverse-phase analytical column (Zorbax SB-C18, 100 mm × 3.0 mm i.d., 3.5 μm particles) with a mixture of methanol/acetic acid 0.1% (*v*/*v*) as mobile phase and a binary gradient. The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol at 35 min, isocratic elution followed with 42% methanol for the next 3 min, rebalancing with 5% methanol in the next 7 min. The flow rate was 1 mL/min, the injection volume was 5 μL, and the column temperature was 48 ◦C. The detection procedure was performed on both UV and MS mode. The UV detector was set at 330 nm until 17 min (for the detection of polyphenolic acids), then at 370 nm until the end of analysis time (for the detection of flavonoids and their aglycones). The MS system operated using an electrospray ion (ESI) source in negative mode (capillary 3000 V, nebulizer 60 psi (nitrogen), dry gas temperature 360 ◦C, and dry nitrogen gas at 12 L/min).

Another LC-MS method (LC-MS method II) previously described [23] was used to detect the other six polyphenols (epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, and vanillic acid) in HI extracts (Table S2). The chromatographic separation was accomplished on the same analytical column and in the same chromatographic conditions as mentioned before but with a slightly different binary gradient (start: 3% methanol; at 3 min: 8% methanol; at 8.5 min: 20% methanol; at 10 min: rebalance column with 3% methanol). The detection of the compounds was performed on MS mode. All identified polyphenols were measured both in the HI non-hydrolyzed and hydrolyzed extracts (equal amounts of extract and 4 M HCl kept 30 min on 100 ◦C water bath) on the basis of their peak areas and comparison with a calibration curve of their corresponding standards. The results were expressed as micrograms of phenolic compounds per dw of involucre (μg/g dw).
