2.5. Plasma and Urine Prenylflavonoids and Their Metabolites 
6-PN
All prenylflavonoids (X, IX, 6-PN, and 8-PN) were measured both in plasma and urine in their unconjugated, glucuronide, and sulfated forms; each were expressed as a sum of the 3 different forms (i.e., total). Standard of X (purity: 99.6%), IX (purity: 99.6%), 8-PN (purity: 100%), and 6-PN (purity: 97%) were purchased from Phytolab (Vestenbergsgreuth, Germany). β-Glucuronidase enzyme from Urine and plasma samples were stored at −80 °C until analysis; they were then prepared with an automated workstation (Beckman Coulter, Biomek NX, Brea, CA, USA) in random order. For the plasma samples, 100 µL of plasma or enzymatic hydrolyzed plasma were loaded on Captiva EMR-lipid plate (Agilent Technologies, Santa Clara, CA, USA). An amount of 300 µL of MeOH/ACN (50:50) were added and mixed at 700 rpm for 3 min. Samples were then eluted by positive pressure. An amount of 100 µL of MeOH/ACN (50:50) were loaded on the cartridge once again and eluted by positive pressure. For the urine samples, 300 µL of water were loaded on Captiva ND plate, and 100 µL of urine or enzymatic hydrolyzed urine were added and mixed at 700 rpm for 1 min. Samples were then eluted by positive pressure and analyzed by LC-HRMS. Enzymatic hydrolyzed urine and plasma samples were prepared with 10 mg of enzyme diluted in 10 mL sodium phosphate buffer (100 mM, pH = 6.8) for β-Glucuronidase and in 10 mL sodium acetate buffer (100 mM, pH = 5) for sulfatase enzyme. An amount of 60 µL of this solution were mixed with 60 µL of plasma or urine and incubated during 1 h at 37 °C. Noting the difference between the concentrations measured before and after enzymatic hydrolysis provides the ability to calculate the concentrations of the glucuronide/sulfated forms. Liquid chromatography was performed on a UHPLC Thermo Vanquish (Thermo Scientific, Karlsruhe, Germany) in reverse phase mode with an Accucore RP-MS column (150 × 2.1 mm, 2.6 µm, Thermo Scientific) using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN). The elution gradient started at 5% B for 1 min, followed by a linear gradient rising to 90% B during 9 min. The mobile phase remained at 90% B for 5 min and then returned to initial condition after 1 min. The column was equilibrated for 4 min in initial conditions (5% B) prior to the next injection, for a total run time of 20 min. The flow rate was 0.5 mL/min, and the injection volume was 2 µL. The column was heated at 30 °C to ensure a stable column temperature and a better repeatability between runs, and the autosampler temperature was maintained at 6°C. The UHPLC system was coupled to an Orbitrap Q-Exactive Focus mass spectrometer (Thermo Scientific, Germany), and analyses were performed using an electrospray interface in negative mode, in full scan with a resolution of 35,000 FWHM in the scan range of This analytical method was validated according to internal guidelines and specificity, linearity, repeatability, and limit of quantification (LOQ) are listed in 