Ultracentrifugation is used in this assay to determine the percentage of test compound that binds to plasma proteins

Catalog number EA704

Required from Customer

  • A study design—the customer must specify the value of the 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
  • Molecular mass (exact mass) of test compound and its salt form
  • MSDS, handling and storage information, e.g., light sensitive, store at -20°C, etc.
  • The solubility of test compound in PBS buffer at pH 7.4


  • The concentrations of test compound and control compounds in plasma and buffer
  • The calculated percent bound of the test compound and control compounds


  • Test compound dissolved in DMSO and plasma from a source specified by the customer

Assay System

  • Rapid Equilibrium Dialysis (RED) device at 37°C
  • Blood plasma collected with an addition of anticoagulant
  • Phosphate buffered saline at pH 7.4

Assay Conditions

  • Three separate chambers are used for each test compound (N=3)
  • Dialyse the test compound in blood plasma against phosphate buffer saline
  • Incubate the dialysis chambers at 37°C
  • Sample both plasma and buffer in each chamber at 4 hours
  • Samples diluted to achieve the same analytical matrix
  • Unless the optional full method validation is ordered the concentrations of test compound are determined using a generic LC/MS/MS method with a minimum 4-point calibration curve

Assay QC

  • The results for the test compound are accepted only when the correct rank order is obtained for the percent binding of the control compounds.


  1. This assay is also available as an ExpressPlus Assay.
  2. It is required that 30-40% of test compound remain after 4 hours at 37°C in phosphate buffer saline pH 7.4 or plasma for equilibrium dialysis assays to give reliable results. If the stability of the test compound is not known, then a stability assay should be run prior to starting the dialysis experiments.
  3. This assay can yield ambiguous results on test compounds with low solubility. Absorption Systems can determine solubility of a test compound at an additional cost. See assay EA101.
  4. The control compounds are run to verify that there was no severe leakage of the chambers during the experiment and that the chambers have reached equilibrium.
  5. Sample dilution for analysis:
    • Buffer chamber is diluted 5:1 with plasma
    • Plasma chamber is diluted 1:5 with buffer
  6. Calculation of % protein binding of test compound(s) (average of triplicate studies):
    1. % bound =

      [donor] – [receiver]  x 100[donor]
    2. Calculation of % recovery of test compound(s)

    3. % recovery = [donor] + [receiver] x 100[nominal]

  7. We also offer protein binding assay analysis using ultrafiltration and ultracentrifugation technique. This assay is the most reliable (but more expensive) among the three provided that the test compound has a relatively high stability.
  8. An optional complete analytical method development includes:
    1. 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.
    2. 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.
    3. 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.
  9. An optional validation is performed using calibration standards and quality control (QC) samples containing the test compound at known concentrations.  In order for the analytical method to be considered acceptable the results from at least one method validation run must meet the following criteria:
    1. The standard curve must include a minimum of five standards with their concentration back calculated to ±15% (± 20% at the LLOQ) of their theoretical concentration when the correlation coefficient of the regression model is ≥ 0.990.
    2. The calculated concentration of at least two-thirds of all QC samples and at least one-third of the QC replicates must fall with ±15% of their respective theoretical concentration.
    3. The intra-assay coefficient of variation (CV) of the replicate determinations of the low, medium and high QC samples must not exceed ±15%, and the average concentration for each set of QC replicates as calculated using the standard curve must be within ± 15% of their theoretical concentration.
    4. The lowest standard is accepted as the lower limit of quantitation if the typical response at this concentration is at least 5 times greater than any interference in the blank matrix at the retention time of the analyte(s).
    5. The response ratio of any interfering peak(s) in the matrix blanks at the retention time of the analyte(s) must be <20% of the response to the LLOQ standard.


  1. The customer must specify:
    • either the basic, standard or custom report format
    • the source of the plasma. Sources include, but are not limited to:
    • human of mixed gender
    • male Cynomolgus primate
    • male beagle dog
    • male or female Sprague-Dawley rat
    • male or female mouse
    • the concentration of the test compound—the analytical sensitivity will influence the range of acceptable concentrations
  2. The customer can request:
    • complete analytical method development and pre-study validation
    • different sampling time points
    • the use of different contol compounds—default control compounds are Atropine and Warfarin
    • that the experiment be performed using a Teflon chamber microdialysis apparatus with an equilibration time of >22 hours.