Metabolic Stability – Liver S9 Fractions

This assay is used to determine the percent remaining of a test compound incubated with liver S9 fraction (mouse, rat, dog, monkey, or human) in the presence of cofactors.

Required from Customer

  • Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
  • Exact molecular mass of test compound and its salt form
  • MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)


  • Table of % remaining of test compound at each time point
  • Calculated t1/2 of test compound and controls
  • Calculated in vitro intrinsic clearance of test compound


  • Test compound at 1 µM dissolved in DMSO, methanol, or acetonitrile

Assay System

  • Pooled liver S9 fraction (≥3 donors), with 1 mM NADPH, UDPGA, PAPS and GSH
    • Human: mixed gender
    • Rat, dog, primate or mouse: male only
  • LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

  • The assay is run with a single incubation (N=1)
  • Incubate test compound (1 µM) at 37°C in buffer containing 1.0 mg/mL S9 protein
  • Initiate the reaction by adding cofactors, sampling at 0, 10, 20, 30, and 60 minutes
  • Incubate positive controls (testosterone and 7-hydroxycoumarin) in parallel, sampling at 0, 10, 30, and 60 minutes

Assay QC

  • The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the S9 fraction
  • After the final time point, fluorimetry is used to confirm the addition of NADPH to the reaction mixture
  • T1/2 of control compounds must meet internal acceptance criteria


  1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
  2. This assay does not provide stability information of test compound in liver S9 incubations without cofactors.
  3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample.
  4. In vitro intrinsic clearance (CLint in-vitro) = k/P (mL/min·mg protein), where k is the elimination rate constant (min-1) and P is the protein concentration (mg/mL).