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.