Determination of the fractional binding of test compound to plasma proteins using an ultrafiltration technique

Catalog number EA705

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 concentration of test compound and control compound in the ultrafiltrate
  • The calculated percent bound of the test compound and QC compounds


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

Assay System

  • Amicon Micropartition device (the filter system)
  • Blood plasma collected with an addition of anticoagulant
  • Phosphate buffered saline at pH 7.4

Assay Conditions

  • Run assay in triplicate for the test compound
  • Test compound mixed with phosphate buffer saline, pH 7.4, and filter to test the non-specific binding of the test compound
  • Warm all matrices for circa 3 minutes at 37°C
  • Incubate  the mixture of test compound and control compounds for 15 minutes at 37°C
  • Centrifuge the filter at ~ 3,000 RPM then collect samples from receiver (ultrafiltrate) and retentate chamber
  • 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 can yield erroneous results on test compounds with solubilities of less than 5 μM. Absorption System can determine solubility of a test compound at an additional charge.
  2. If test compound shows high non-specific binding to Amicon device (>25%), the assay may produce invalid results due to low recovery of the test compound.
  3. Calculation of % of protein binding:
    1. Percent Bound = 1 – (Cp,f /C0)(1 + NSB) x 100
      • Where: Non Specific Binding (NSB) = 1 – (Cb, f /Cb, uf)
      • Cb, f  is the average concentration in the filtered PBS (ultrafiltrate).
      • Cb,uf  is the average concentration in the unfiltered PBS (retentate).
      • Cp, f  is the concentration in the filtered matrix.
      • C0  is the dosing concentration of the compound.
  4. We also offer protein binding assay analysis using equilibrium dialysis and ultracentrifugation technique. The equilibrium dialysis assay is less likely to be confined by non-specific binding and low solubility but the test compound has to be stable in the protein matrix for 8 to 24 hours. 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.
    4. 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:
    5. 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.
    6. 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.
    7. 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.
    8. 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).
    9. 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