Acacetin Protein Binding Assay

The fractions of unbound acacetin in plasma (fu_P), microsomes (fu_MIC), SGF (fu_SGF), and SIF(fu_SIF) were measured using a rapid equilibrium dialysis device (Thermo Fisher Scientific, Inc., Waltham, MA, USA) as previously described with slight modifications [25 (link)]. Briefly, 0.2 mL of the matrix spiked with acacetin (10 μM) was placed into the “sample” chamber, and 0.4 mL of PBS was placed into the adjacent “buffer” chamber. The fractions of unbound acacetin were calculated as the concentration ratio between the two chambers. The blood-to-plasma concentration ratio (R_B) of acacetin was determined as described previously [25 (link)]. Briefly, 1-mL blood spiked with acacetin (1 μM) was incubated at 37 °C for 30 min. The blood sample was centrifuged at 2000× g for 5 min to prepare the plasma sample. The concentrations of acacetin in the aforementioned plasma and buffer samples were measured using the LC-MS/MS method.