CYP Reaction Phenotyping Using Chemical Inhibitors

This assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of human liver microsomes and CYP-specific chemical inhibitors.

Required from Customer

  • The test compound in powder form—a minimum of 10 mg
  • Exact molecular mass of the test compound and its salt form
  • Relevant solubility of test compound
  • The MSDS or handling and storage information, e.g., light sensitive, store at -20ºC, etc.


  • In vitro intrinsic clearance of test compound determined for each CYP isoform
  • Rank order of CYP isoform(s) responsible for test
  • compound’s metabolism


  • Test compound at a single concentration (based on Km; if not available, 1µM will be used) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

  • Pooled, mixed-donor, human liver microsomes (minimum 50 donors) with and without NADPH
  • CYP-specific chemical inhibitor(s)
  • LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the test compound divided by that of an analytical internal standard), without running a standard curve

Assay Conditions

  • This assay is run with a single incubation (N=1) per treatment
  • Incubate test compound at 37°C in the presence of human liver microsomes (0.5 mg protein/mL) and:
    • No NADPH (negative control)
    • NADPH (no-inhibitor control)
    • NADPH + a CYP-specific inhibitor (single concentration)
  • Sample the reaction mixture at 0, 10, 20, 30 and 60 minutes

Batch QC

  • Batch certification of human liver microsomes with CYP-specific probe substrates and CYP-specific chemical inhibitors
  • Use fluorimetry to confirm the addition of NADPH to the reaction mixture


  1. The customer will be asked to specify: • the concentration(s) of the test compound • the CYP(s) to be screened
  2. The customer can request: • additional replicates • additional or alternative time points • the use of a standard curve • that a positive control be run for each CYP