In vitro experiments for determination offm
To prepare for the experiment, 33 compounds, positive controls, and inhibitors were dissolved in DMSO to create 10 mM stock solutions, which were then stored at -40 ℃. Prior to the experiment, NADPH was dissolved in PBS to create a 3 mM working solution. HLM stock solution was diluted with PBS, and a certain volume of organic solvent, compound, positive control, or inhibitor was added to create HLM working solution, HLM inhibitor (15 μM) working solution, HLM compound (3 μM) working solution, and HLM positive control (3 μM) working solution. All these working solutions contained 0.75 mg mL-1 HLM.
To create the non-reaction control group, HLM working solution was mixed with a 3 μM compound or positive control working solution in equal volume. The non-inhibition group was created by mixing HLM working solution with a 3 μM compound or positive control working solution in equal volume. The inhibition group was created by mixing the HLM inhibitor working solution with a 3 μM compound or positive control working solution in equal volume. These reaction groups were preheated at 37 ℃ for 5 minutes, and then the same volume of NADPH working solution was added for the reaction. Prior to starting the reaction, 3 times the volume of ACN termination solution containing 20 ng mL-1 internal standard was added to the non-reaction control group to terminate the reaction, and it was then stored at -40 ℃. The other two groups were incubated at 37 ℃ at 150 rpm for 60 minutes. At the end of the reaction, 3 times the volume of ACN termination solution containing 20 ng mL-1 internal standard was added to terminate the reaction.
After the reaction, the reaction plate was shaken at 600 rpm for 10 minutes and then centrifuged at 6000 rpm for 15 minutes. The supernatant was collected for LC-MS/MS analysis.