Time Dependent Inhibition, LC-MS/MS Detection (IC50 Shift)

Assay System: 

  • Human liver microsomes, pooled from 50 donors.
  • Inhibition potential against CYP isoforms (IC50) measured after pre-incubation with and without NADPH in the presence of different concentrations of test compound by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
  • Time-dependent inhibition (IC50 shift) is determined by comparing IC50 values determined after pre-incubation with and without NADPH.

Assay Conditions: 

Test Compound Concentration Eight concentrations ranging from 0.05 µM to 100 µM
Microsome Concentration 0.1 mg/mL
Percent Organic Solvent in Incubation 2% acetonitrile
CYP Isoforms Available for Testing CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP 3A4
Pre-Incubation Time 30 minutes
Incubation Time 10-20 minutes (depending on CYP isoform)
pH 7.4
Temperature 37 °C
Analysis LC-MS/MS
Number of Repeats 2


  • IC50 of test compound and positive control compound both with and without NADPH pre-incubation.
  • IC50 shift (IC50 -NADPH/ IC50 +NADPH)

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.

CYP Isoform Probe Substrate Monitored Metabolite Positive Control Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
2B6 Bupropion OH Bupropion Ticlopidine
2C8 Amodiaquine Desethylamodiaquine Gemfibrozil Glucuronide
2C9 Tolbutamide 4-OH Tolbutamide Tienilic Acid
2C19 S-Mephenytoin 4’-OH Mephenytoin (S)-Fluoxetine
2D6 Dextromethorphan Dextrorphan Paroxetine
3A4 Testosterone 6β-OH Testosterone Mibefradil
3A4 Midazolam 1’-OH Midazolam Mibefradil
*If you choose "Other", please use the comments section below to request your desired system.