Plasma sample preparation

Blood samples were collected in tubes containing sodium heparin before and after IY5511HCL administration. Plasma samples were immediately separated from the blood in a refrigerated centrifuge (3,000 rpm, 10 min, 4℃) and stored at −70℃ until subsequent analysis. For plasma processing, the plasma samples were allowed to thaw gradually to room temperature. An aliquot (100 µL) of the plasma sample was transferred to a polypropylene tube which contains 50 µL of the IS working solution (6 µg/mL). After the tube was briefly vortexed, 1.5 mL of MTBE was added. This mixture was vortexed for 5 min and then centrifuged at 14,000 rpm for 10 min at 4℃. The supernatant was transferred to a clean tube and evaporated to dryness in a SpeedVac concentrator (SPD2010, Thermo Fisher Scientific, Waltham, MA, USA) for approximately 50 min. The dry residue was reconstituted in 500 µL of acetonitrile/water/formic acid (4:6:0.01 v/v/v), and vortexed for 3 min, and centrifuged at 14,000 rpm for 10 min at 4℃. The supernatant (200 µL) was then transferred to an HPLC vial for further HPLC-MS/MS analysis.