Extraction of Polar Phenols from Rat Serum

For the extraction of polar phenols, rat serum samples underwent enzymatic hydrolysis prior to liquid-liquid extraction (LLE) with ethyl acetate (EtOAc), as reported by Vasilakopoulou et al. [31 (link)]. 100 μL of sodium acetate (CH₃COONa, 0.1 M, pH 5, Thermo Fisher Scientific, MA, USA ) were added to 100 μL of serum samples. Several 20-μL aliquots of β-glucuronidase/ sulfatase (2000/40 U) were injected into the serum samples; the samples were vortexed and incubated for 45 min in a heating bath that was set at 37 °C. To end the enzymatic hydrolysis, 100 μL of phosphoric acid (H₃PO₄, 4% v/v, Thermo Fisher Scientific, MA, USA) were added to the incubated samples. The samples then underwent further treatment with LLE () with the addition of 700 μL EtOAc (Thermo Fisher Scientific, Pittsburgh, PA, USA) to the serum samples. The samples were then vortexed, centrifuged for 5 min at 5000× g, and their supernatants were collected. The extraction process was carried out three times in total, with the supernatants being pooled, evaporated, and reconstituted in 100 μL methanol-water (MeOH-H₂O, 1:1, v/v, Thermo Fisher Scientific, Pittsburgh, PA, USA), which were analyzed with UHPLC–HRMS (Thermo Fisher Scientific, Bremen, Germany).