) 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 source parameters were optimized in our preceding studies [25,38]. The chromatographic technique was coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Samples have been analyzed in Prime 5 datadependent acquisition (DDA) mode with an exclusion list GYKI 52466 supplier containing one of 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 within the range 150000 m/z and 300000 m/z, respectively, with a resolution (complete width at half maximum, FWHM, at m/z 200) of 70,000. In full-scan mode, the automatic get control (AGC) target worth was 200,000, the maximum ion injection time was 100 ms, along with the isolation window width was 2 m/z. Tandem MS (MS/MS) fragmentation was performed having a resolution (FWHM, at m/z 200) of 35,000 with all the AGC target value set at 100,000, and dynamic exclusion set to 3 s. Fragmentation was accomplished within the higher-collision dissociation (HCD) cell at three values of normalized collision power (NCE), namely, 20-50-80 NCE inside the positive ion mode and 20-40-60 NCE inside the negative ion mode depending on the outcomes of a preceding study [25]. All samples have been run in triplicate. 3.7. Information Analysis and Phenolic 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 selected, aided by a customized phenolic compound database, which was generated by combining no cost phenolic compounds (aglycones) with a series of sugars and aliphatic and aromatic acids. The database, total with IDs, accurate masses, and molecular formulas, was implemented inside the mass list function to 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 inside the compound class scoring node. Additionally, the parameters for the predict composition tool had been adapted to phenolic compounds. Extracted m/z from the raw chromatograms had been grouped, aligned, and filtered to remove background compounds and characteristics not related with compounds present within the databases or with MS/MS spectra. Filtered compounds have been manually validated by matching fragmentation spectra to readily available requirements or spectra reported in the literature. When information were 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 had been seeded on polycarbonate filters, 12 mm diameter, 0.four pore diameter (Transwell, Corning Inc., Lowell, MA, USA) at a density of 3.five 105 cells/cm2 in comprehensive medium supplemented with ten FBS in both apical (AP) and basolateral (BL) compartments for two d to allow the formation of a confluent cell monolayer. Starting from day 3 right after RP101988 In stock seeding, cells have been transferred to a FBS-free medium in both compartments, supplemented with ITS (final concentration ten mg/L insulin (I), 5.five mg/L transferrin (T), six.7 /L sodium selenite (S); GIBCO-Invitrogen, San Giuliano Milanese, Italy) only within the BL compartment, and permitted to differentiate for 21 days with frequent medium.
