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S9 metabolic stability assay is very suitable for obtaining an early knowledge about the metabolism of a new chemical entity. This is valuable in the drug development process, because it provides essential information for selecting leads from many compounds with similar therapeutic potential.

S9 represents the supernatant obtained by differential centrifugation at 9,000 g of liver homogenate. Liver S9 is rich in both microsomal and cytosolic fractions. Optimum metabolic information about a compound is obtained from the liver S9 fractions than microsomes. The main components of S9 fractions are cytochrome P450, uridine 5′-diphospho-glucuronosyltransferase, aldehyde oxidase, xanthine oxidase, sulfotransferase, methyltransferase, N-acetyltransferase, glutathione transferase, which are considered to be major contributors for the metabolism of certain chemotypes. The S9 data set is therefore richer in content and provides an opportunity for medicinal chemists to stabilize compounds against both Phase I and II simultaneously.

Similar to microsomes, liver S9 fractions need cofactors such as β-Nicotinamide adenine dinucleotide phosphate-regenerating system (NADPH; Phase I oxidation), uridine 5’-diphospho-α-D-glucuronic acid (UDPGA; Phase II glucuronidation), 3’-phosphoadenosine-5’-phosphosulphate (PAPS; Phase II sulfation), and glutathione (GSH; Phase II) for activity, however they have a significant price benefit, are easy to store and use, and are much more amenable to high-throughput screening.

For about 10 years, Creative Bioarray has been using the liver S9 system as our primary in vitro screen to eliminate compounds with poor metabolic stability and poor probability of success. Our standard S9 stability assay measures the disappearance of test compounds over time, both in the presence and absence of cofactors needed for CYP-mediated drug oxidation and glucuronidation. S9 fractions from a variety of animal species can be used to predict interspecies differences in the rate of metabolic elimination of the studied compound.

S9 Metabolic Stability Assay

Test compound concentration
1 µM (0.1% DMSO) or custom

S9 concentration
1 mg/mL or custom

NADPH, UDPGA, PAPS or custom

Time points
0, 5, 15, 30, 45, 60 minutes

Assay controls
Positive control: compounds with known activity (Verapamil)
Negative control without cofactor
Blank control with vehicle (0.1% DMSO)

Analysis method
LC-MS/MS with internal standard

Data delivery
Intrinsic clearance (CLint)
Half-life (t1/2)
%Remaining of test compound

Compound requirements
200 µL 10 mM stock in DMSO

Features and Benefits of S9 Metabolic Stability Assay

  • Matching microsomes, S9 and cytosol for most donor pools
  • Large donor pools minimize lot-to-lot variation and increase the long-term availability of each lot.
  • Extensively characterized using validated LC-MS/MS methods


  1. Samantha J Richardson, et al.; Efficiency in drug discovery: liver S9 fraction assay as a screen for metabolic stability. Drug Metabolism Letters, 2016, 10(2): 83-90.

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