The current study utilized the 96-well equilibrium dialysis device purchased from HTDialysis to dialyze the test compound and the control compound at 2 μM in plasma at 37±1°C for 4 hrs. Plasma prepared from Sprague-Dawley rats, beagle dog, cynomolgus monkey and humans were previously frozen at below -60°C. The dialysis membrane strips were soaked in ultra-pure water at room temperature for approximately 1 hr. After that, each membrane strip that contains 2 membranes was separated and soaked in ethanol:water (20,80 v,v) for approximately 20 mins, after which it was ready for use or was stored in the solution at 2–8°C for up to a month. Prior to the experiment, the membrane was rinsed and soaked for 20 mins in ultra-pure water. On the day of experiment, the plasma from Sprague-Dawley rats, beagle dogs, cynomolgus monkey and humans was thawed under running cold tap water and centrifuged at 3220×g for 5 mins to remove any clots and the pH value of the resulting plasma will be checked. if required, adjusted to 7.4 ± 0.1. All samples were further processed by protein precipitation for LC/MS/MS analysis (Wuxi AppTec, China).

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