Thermostatted column compartment

HPLC-grade solvents (water and acetonitrile) were purchased from VWR (Milan, Italy) and high-purity commercial analytical standards were obtained from Sigma-Aldrich (Milan, Italy). Chromatographic analyses were carried out on an Ultimate3000 UHPLC-focused instrument equipped with a binary high-pressure pump, a Photodiode Array Detector, a Thermostatted Column Compartment, and an Automated Sample Injector (Thermo Fisher Scientific, Inc., Milan, Italy). Analytical runs were performed using the same chromatographic conditions (column elution program and solvents) reported by Russo et al. (2021) [26 ]. Quantification was carried out at 280 nm for gallic acid and its derivatives (including HHDP derivatives, pedunculagins, and granatins) using gallic acid as a reference (r² = 0.9999). Similarly, quantification of ellagitannins was made at 360 nm using punicalagin (r² = 0.9998) as a reference, while ellagic acid and its derivatives were quantified at the same wavelength using ellagic acid (r² = 0.9997) as an external standard. In order to unambiguously identify the chromatographic signals, HPLC/ESI-MS analyses were also performed. The HPLC apparatus used was as described above, whilst ESI mass spectra were acquired using the same instrumentation, operating conditions, and acquisition software as previously described [18 (link)]. All analyses were carried out in triplicate.