Plasma Curcuminoids Extraction and Quantification

Curcuminoids were extracted from plasma samples according to the method of Cuomo et al. [3 (link)] Briefly, the plasma samples were thawed for 30 min at room temperature. In the meantime, 50 µL of internal standard solution (250 ng/mL) composed of Curcumin-d6 (ISTD: CND Isotope, Pointe-Claire, QC, Canada) in methanol was added in an empty Eppendorf tube of 1.5 mL to which 0.2 mL of plasma and 100 µL of β-Glucuronidase/Arylsulfatase 10,000 U/mL from Helix pomatia (Sigma-Aldrich, Saint-Louis, MO, USA) diluted in phosphate buffer 0.1 M, pH = 6.86 were added. The tubes were vortexed for 1 min and incubated at 37 °C for 1 h to hydrolyze the phase-2 conjugates of curcuminoids [20 (link),21 (link)]. Then, curcuminoids were extracted two times from the mixture by adding 1 mL of ethyl acetate, then vortexed 1 min and sonicated for 15 min. After sonification, tubes were centrifugated at 15,000 g at room temperature for 6 min. The superior organic phase was then transferred, and the process was repeated a second time. The combined organic phase was evaporated under nitrogen stream. The curcuminoids extract was reconstituted in 100 µL of methanol, centrifuge at 5000 g for 5 min to remove any particles, and 50 µL of the supernatant was then transferred to HPLC vials.