Extraction and Analysis of Quercetin Metabolites

Fecal metabolites produced during the in vitro fecal incubation of unformulated and phytosome-formulated quercetin were extracted adopting the method reported by Bresciani et al. [18 (link)], with slight modifications. Briefly, 300 µL of each fermented sample was extracted with 0.1% (v/v) formic acid in ethyl acetate, vortexed for 30 s, sonicated for 10 min in an ultrasonic bath, vortexed for 30 s, and re-sonicated for 5 min. Finally, samples were centrifuged at 14,460× g for 10 min and the upper organic layer was transferred to a clean microfuge tube. After the first extraction, the residual pellet of the fermented samples was re-extracted following the same procedure, using 500 μL of the same solvent. Finally, supernatants were pooled and brought to dryness for about 2 h at room temperature through a centrifugal concentrator (SpeedVac Savant SPD121P, Thermo Fisher Scientific Inc., San Jose, CA, USA). Both dried residues were reconstituted in 50% (v/v) aqueous methanol acidified with 0.1% (v/v) formic acid (dilution factors of 1:10 and 1:2 for the analyses of native quercetin and its fecal metabolites, respectively), vortexed, and centrifuged at 14,460× g for 10 min before uHPLC-MSⁿ analyses.