Equilibrium dialysis is used in this assay to determine the percentage of test compound that binds to human, rat, dog, primate or mouse plasma proteins

Catalog number EA701

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.

Deliverables

  • Percent bound of test compound and QC compound

Substrate

  • The test compound at 5 μM in human, rat, dog, primate or mouse blood plasma at pH 7.4 with final DMSO concentration less than 1%

Assay System

  • Pooled plasma (≥3 donors)
    • Human: mixed gender
    • Rat, dog, primate or mouse plasma: male only
  • Dialysis: Rapid Equilibrium Dialysis (RED) device at 37°C

Assay Conditions

  • Use two separate wells (N=2), with one side of each dialysis well containing phosphate-buffered saline (PBS) at pH 7.4 and the other side of the well containing plasma
  • Use appropriate anticoagulant (typically sodium heparin)
  • Adjust pH of PBS and plasma to 7.4 prior to initiation of experiment
  • Sample both the plasma side and the buffer side of each well 4 hours after the system reaches 37°C
  • Use LC-MS/MS 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 calibration curve

Assay QC

  • Warfarin is co-dosed with the test compound in each well

Dynamic Range

  • 5 to 99.9% bound

Notes

  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 uses frozen / thawed pooled blood plasma.
  3. This assay can yield ambiguous results with test compounds that are unstable in plasma. Absorption Systems can determine the plasma stability of a test compound at an additional cost.
  4. The following equation is used to calculate the percentage of the test compound bound to plasma proteins based on the peak area response ratio (PARR):
    % bound =  ([PARR (donor)] – [PARR (receiver)]  x 100)/[PARR (donor)]

Options

  1. The customer can request that the experiment be performed using a Teflon chamber microdialysis apparatus with an equilibration time of >22 hours.