2.6. In vitro human plasma rapid equilibrium dialysis (RED) protein binding assay

Assays were performed according to guidance by the manufacturer. Gefitinib or erlotinib was mixed with 1 mL pooled human plasma at a 10 µM concentration. This mixture (300 µL) was dispensed into the sample chamber (red ring) and 500 µL dialysis buffer was put in the buffer chamber. The plate was sealed, then incubated at 37 °C on an up and-down shaker shaking at 20 rpm for 4.0 h. Samples were collected by removing 100 µL from each buffer and plasma chamber. The collected buffer samples were mixed with 100 µL plasma, and the collected plasma samples were mixed with 100 µL dialysis buffer. Then, 200 µL of acetonitrile containing internal standard was added to all samples. The samples were vortexed for 30 s, then centrifuged at 1,300 × g at 4 °C for 15 min. Supernatants were stored at 4 °C until analysis. Samples were run after all samples were collected within 5 h.