solution (Sigma-Aldrich, Co., MO, USA) were added to each sample. After the resultant mixture was centrifuged at 13,000 G for 3 min, the propofol concentration of the supernatant was measured with a high-performance liquid chromatography (HPLC) system (Shimadzu Co., Tokyo, Japan). The peak area ratio of propofol to the IS was obtained from the HPLC chromatogram at each control concentration, and calibration curves for the blood propofol level (0.156, 0.312, 0.625, 1.25, and 2.5 μg/mL) and the protein-unbound free propofol level (0.005, 0.01, 0.02, and 0.04 μg/mL) were constructed.