Sufentanil

The cells were inoculated in 96-well plates at a density of 5 × 10⁴/mL. 100 μL cell suspension was added to each well. Blank pores are free of cells. Incubate overnight in a 5% CO₂ incubator. After the cells were completely attached to the wall, the supernatant was carefully sucked from the 96-well plate. In the experimental group, the RPMI-1640 culture medium containing different concentrations of sufentanil (1, 10 μmol/L) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well, 200 μL. The negative control group and the blank control group only added the culture medium 200 μL. Six parallel control holes were set for each group. After incubation for 48 h, 5 mg/mL MTT 20 μL was added to each well for 4 h. Carefully suck off the supernatant. DMSO 150 μL (Sigma-Aldrich, St. Louis, MO, USA) is added per well. Shake the shaker gently for 10 min. After the purple particle was fully dissolved, the wavelength of the purple particle was determined at 570 nm by a microplate reader (MultiskanEX, Lab systems, Helsinki, Finland). Determine the absorbance (OD) value of each well.

1.5-2.0 mg/kg propofol (Shanghai Yuanye Biotech, B33792-100 mg), 0.4 μg/mL sufentanil (Sigma-Aldrich (Shanghai), S-008-1ML), 0.15-0.2 mg/kg cis-atracurium (Shenzhen Jianzhu Biotech, 30119630), and 30-50 μg/kg midazolam (Nhwa Pharmaceutical Co. Ltd, China) were used for anesthesia induction. Then, endotracheal intubation and mechanical ventilation were performed. After successful anesthesia induction, continuous pumping of 15-75 μg/kg·min propofol and 0.05-0.15 μg/kg·min remifentanil (Sigma-Aldrich (Shanghai), R-024-1ML) was administered intraoperatively, accompanied by auxiliary inhalation of 1-2% sevoflurane (Shanghai Fuyu Biotech, FY23996-25) and intermittent addition of atracurium (Shanghai Yuanye Biotech, Y54583).

Fentanyl, Fentanyl‐d5 and sufentanil were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). Cefazolin was obtained from Santa Cruz Biotechnology (Heidelberg, Germany), doxapram from Selleckchem (Munich, Germany) and keto‐doxapram from Tractus (London, UK). Ammonium acetate was obtained from Sigma Aldrich (Zwijndrecht, the Netherlands). Methanol, acetonitrile and formic acid were purchased from Biosolve BV (Valkenswaard, the Netherlands). All reagents were LC–MS grade, which means at least 99% purity. Water was purified by using a MilliPore Advantage A10 system. Human drug‐free plasma was obtained from the blood donation center (Sanquin, Rotterdam, the Netherlands).