) and ACN/HCOOH (99.9:0.1, v/v; phase B). The elution gradient and) and ACN/HCOOH (99.9:0.1, v/v;

) and ACN/HCOOH (99.9:0.1, v/v; phase B). The elution gradient and
) and ACN/HCOOH (99.9:0.1, v/v; phase B). The elution gradient and ESI supply parameters were optimized in our prior studies [25,38]. The chromatographic program was coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Samples have been analyzed in Top rated five datadependent acquisition (DDA) mode with an exclusion list containing the most intense ions detected within the blank sample (H2 O/MeOH, 90:10, v/v). For low- and high-molecularweight phenolic compound evaluation, MS information had been acquired in the variety 150000 m/z and 300000 m/z, respectively, using a resolution (complete width at half maximum, FWHM, at m/z 200) of 70,000. In full-scan mode, the automatic get manage (AGC) target worth was 200,000, the maximum ion injection time was 100 ms, and also the isolation window width was two m/z. Tandem MS (MS/MS) fragmentation was performed using a resolution (FWHM, at m/z 200) of 35,000 with the AGC target worth set at 100,000, and dynamic exclusion set to three s. Fragmentation was achieved within the higher-collision dissociation (HCD) cell at three values of normalized collision power (NCE), namely, 20-50-80 NCE inside the optimistic ion mode and 20-40-60 NCE in the damaging ion mode based on the outcomes of a previous study [25]. All samples were run in triplicate. 3.7. Data Evaluation and Phenolic AAPK-25 site compounds Validation For phenolic compound annotation, a customized data-processing workflow on Compound Discoverer three.1 (Thermo Fisher Scientific, Waltham, MA, USA) was employed [25,39]. A metabolomics-based approach was chosen, aided by a customized phenolic compound database, which was generated by combining totally free phenolic compounds (aglycones) using a series of sugars and aliphatic and aromatic acids. The database, full with IDs, accurate masses, and molecular formulas, was implemented in the mass list function to IQP-0528 Autophagy automatically match extracted m/z ratios (45,567 combinations). For simplifying MS/MS spectra manual annotation, detailed HCD fragmentation spectra for flavonoids and phenolic acids have been implemented in the compound class scoring node. Furthermore, the parameters for the predict composition tool were adapted to phenolic compounds. Extracted m/z in the raw chromatograms have been grouped, aligned, and filtered to take away background compounds and capabilities not linked with compounds present within the databases or with MS/MS spectra. Filtered compounds have been manually validated by matching fragmentation spectra to out there standards or spectra reported inside the literature. When information have been lacking, phenolic compounds have been tentatively identified as outlined by the characteristic fragmentation spectra. three.8. Caco-2 Cell Culture and Differentiation Caco-2 cells have been kindly obtained from Institut National de la Santet de la Recherche M icale (INSERM, Paris). For differentiation, Caco-2 cells were seeded on polycarbonate filters, 12 mm diameter, 0.4 pore diameter (Transwell, Corning Inc., Lowell, MA, USA) at a density of three.five 105 cells/cm2 in complete medium supplemented with ten FBS in each apical (AP) and basolateral (BL) compartments for two d to allow the formation of a confluent cell monolayer. Beginning from day 3 soon after seeding, cells had been transferred to a FBS-free medium in both compartments, supplemented with ITS (final concentration 10 mg/L insulin (I), five.5 mg/L transferrin (T), 6.7 /L sodium selenite (S); GIBCO-Invitrogen, San Giuliano Milanese, Italy) only in the BL compartment, and permitted to differentiate for 21 days with frequent medium.