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Microsomal Stability Assay (mouse, rat, dog, monkey, human)



Readout: % remaining at time point, t1/2 determination, and intrinsic clearance (Clint) estimation

Controls:  w/o NADPH; Dextromethorphan (CYP2D6), Midazolam (CYP3A4), Phenacetin (CYP1A2), Diclofenac (CYP2C9), Omeprazole (CYP2C19)


Assay Description:

Working solutions of each compound are prepared from 10 mM stock solution in DMSO diluted to a final concentration of 100 μM in 0.05 M phosphate buffer (pH 7.4).

Aliquots of Liver Microsome working solution are transferred into 1.1 mL tubes using a multichannel pipette. Positive control (5 mixed) and test compound working solutions are transferred into the tubes. The mixtures are vortexed gently and then pre-incubated at 37 °C.  5 mM NADPH or LM buffer (no NADPH buffer) is aliquoted into the tubes using a multichannel pipette and vortexed gently.

At each time point of 0 min, 5 min, 15 min, 30 min and 60 min with NADPH or 0, 30 min and 60 min without NADPH, an aliquot is removed from each tube. Terfenadine/tolbutamide in ACN/MeOH (1:1, v/v) is added to quench and precipitate the microsomal incubations.  Samples are capped and vigorously vortexed and then centrifuged at 4 °C.

An aliquot of each supernatant is transferred for LC-MS/MSC analysis.

The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid,  solvent B: ACN with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area) for each time point.


Data Analysis:

The estimation of Clint (in uL/min/mg protein) is calculated using the following equation:

CLint (uL/min/mg protein) = ln(2) * 1000 / t1/2 / protein conc.

Unit of t1/2= min

Unit of Protein Conc. = mg/mL

Final DMSO concentration: 1%




ACN                         Acetonitrile

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

LC                             Liquid chromatography

MS                           Mass spectrometry

NADPH                   Nicotinamide adenine dinucleotide phosphate




  1. Obach, R. S.; “Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes”, Drug Metab. Dispos. 27, 1350, (1999).
  2. McGinnity, D. F.; Riley, R. J.; “Predicting drug pharmacokinetics in humans from in vitro metabolism studies”, Biochem. Soc. Trans. 29, 135, (2001).

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