Plasma Extraction and Quantification Protocol

To extract irinotecan, SN-38, and SN38G from the plasma samples, 100 μL of the plasma sample was mixed with 200 µL of acetonitrile for 3 min by using a multitube vortexer to extract analytes. After 6000-rpm centrifugation for 10 min at 4 °C, 0.1 mL of the supernatant was transferred to another 1.5-mL microtube and stored at 4 °C (analyte A). Subsequently, to collect rapamycin, 200 μL of the extracted solution (methanol/0.1 M zinc sulfate solution = 7/3) was added to 100 µL of the blood sample and vortexed for 1 min. The mixture was centrifuged at 6000 rpm at 4 °C for 10 min, and 100 μL of the supernatant was mixed with analyte A and vortexed for 10 s (analyte B). Subsequently, 10 μL of camptothecin (1 µg/mL) and 10 μL of ascomycin (3 µg/mL) were added to analyte B and then diluted with the mobile phase and mixed thoroughly. The final sample solution was injected into the UPLC/MS/MS system for analysis.