EA713-C

Blood to Plasma Partitioning Whole Blood

This assay is used to determine the blood-to-plasma partition coefficient of a test compound in mice, rats, and humans in vitro

Required from Customer

  • Either a minimum of 300 μL of test compound at 10 mM in DMSO or 5 mg of powder
  • The exact molecular mass of the test compound and its salt form
  • MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, solubility, etc.
  • Stability of test compound in whole blood and/or plasma from the test species, if known

Deliverables

  • The concentrations of test compound and control compound in whole blood and plasma
  • The calculated blood-to-plasma partition coefficient (KRBC/P) for the test compound and control compound
  • Hematocrit of the whole blood lot used in the study

Substrate

  • Test compound at a single concentration (typically 5 μM) in  heparinized blood and plasma with final DMSO concentration < %

Assay System

  • Fresh, whole heparinized blood and plasma from mice, rats ( ≥3 donors pooled; male only) or humans ( ≥3 donors pooled; mixed gender
  • LC-MS/MS is used to determine the peak area response ratio (PARR; peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

  • Adjust pH of blood and plasma to 7.4 prior to addition of test compound
  • Spike whole blood and reference plasma (from the same lot of whole blood) with test compound, mix, and incubate at 37°C
  • Co-incubate control compound with test compound
  • Duplicate incubation (N=2 separate incubations) in whole blood and reference plasma
  • Sample blood and prepare plasma  (test plasma) by centrifugation at 60 minutes
  • Sample reference plasma at 60 minutes

Assay QC

  • Results are accepted only when the blood-to-plasma partition coefficient of the reference compound meets internal acceptance criteria

Notes

  1. The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.

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