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

Catalog number EA711

Required from Customer

  • A study design—the customer must specify the value of a number of assay variables listed in the Options section
  • Either a minimum of 300 μL of test compound at 10 mM in DMSO, or 5 mg of powder
  • 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


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


  • Test compound at a single concentration (typically 5 μM) in  heparinized blood and plasma with final DMSO concentration <1%

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 compounds, one low- and one high-partitioning, with test compound
  • Triplicate incubation (N=3 separate incubations) in whole blood and reference plasma
  • Sample blood and prepare plasma (test plasma) by centrifugation at 0, 30, and 60 minutes
  • Sample reference plasma at the same time points
  • Assess the plasma stability of the test compound in parallel

Assay QC

  • Results are accepted only when the correct rank order and relative magnitude of blood-to-plasma partition coefficients are obtained for the control compounds


  1. Determination of the concentrations of test and control compounds in test and reference plasma, using a 5- to 8-point calibration standard curve, is available at an additional cost.
  2. The blood-to-plasma partition coefficient (KRBC/P) = 1/H x (
    [PARRref/PARRtest]-1) + 1, where H = hematocrit, PARRref is the peak area response ratio of the analyte in reference plasma, and PARRtest is the peak area response ratio of the analyte in test plasma (prepared following incubation with whole blood).
  3. Analytical method development includes:
  4. Optimization of MS: Initial conditions will be established by infusing a solution containing the test compound into an appropriate mass spectrometer. The goals in MS optimization are to maximize detector response for the test compound and determine the approximate lower limit of quantitation (LLOQ) of the test compound.
  5. Optimization of HPLC: HPLC parameters will be optimized using an appropriate selection of LC column(s) and mobile phase. The goal in HPLC optimization is to develop appropriate chromatographic run-times while maintaining adequate levels of sensitivity.
  6. Optimization of Sample Preparation: Various means of sample preparation, e.g., direct injection, dilution with appropriate solution, protein precipitation, solid phase extraction etc., will be evaluated for their efficiency in extracting the test compound from the biological matrix. The goal in extraction optimization is to achieve the required lower limit of quantitation (LLOQ) while alleviating matrix suppression effects.


  1. The customer must specify:
    • either the standard or custom report format
    • species: mouse, rat, or humanthe concentration of the test compound—analytical sensitivity will influence the range of acceptable concentrations
  2. The customer can request:
    • different sampling time points
    • determination of the concentrations of test and control compound (additional cost)