This screening assay is used to determine the bioavailability of test compounds relative to a reference compound after oral administration to rats

Catalog number EA801

Required from Customer

  • Test article in powder form or preformulated
  • Dose of test article and reference compound—typically 10 mg/kg
  • Molecular mass (exact mass) of each test article and its salt form
  • MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.


  • In-life observation on each rat
  • A table containing test compound concentration in plasma samples at each time point
  • A table listing relative exposure (bioavailability) based on the AUC for each compound,  Cmax and Tmax of each compound
  • Half-life, volume of distribution, clearance after IV dosing
  • An appendix containing the bioanalytical data


  • Test compound dissolved in an appropriate dosing vehicle

Assay System

  • Conscious, fasted, Sprague-Dawley rats weighing between 200 and 400 grams. Water is offered ad libitum to the rats

Assay Conditions

  • Three rats (N=3) per dosing route group
  • Sample blood from the jugular vein at timepoints specified by the customer
  • Prepare plasma from each sample and freeze
  • Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve
  • Non-compartmental analysis is used to determine PK parameters for each test article

Assay QC

  • Adverse reactions, if any, are reported to the customer
  • Analytical data are accepted only when at least two-thirds of the analytical control samples and 60% of the standards used to create the calibration curve have a back calculated accuracy of ±15% (at LLOQ, ±20%)


  1. The amount of test compound required depends on the dose, i.e., mg of test compound per gram of rat, and the number of rats.
  2. The standard strain of rats is Sprague-Dawley. The customer can specify the use of other commercially available strains.
  3. Absorption Systems can develop the dose vehicle for this study.
  4. The customer can elect to perform the bioanalysis while Absorption System only does the dosing and sampling.
  5. The standard anticoagulant is sodium heparin. The customer can specify that another anticoagulant be used.
  6. A 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.
  7. A pre-study 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 nominal 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 nominal 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 nominal 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:
    • the use of either the standard or a custom report format
    • the dose vehicle(s)
    • the strain of rats
    • the sampling time points the concentration for IV dosing
    • the concentration for OG dosing
  2. The customer can request:
    • a different anticoagulant be used
    • that Absorption Systems determine a suitable dosing vehicle
    • that Absorption Systems prepare the dosing vehicle using the customer’s instructions
    • a more complete bioanalytical method validation, e.g., inter-day variability, and assessment of the stability of the samples
    • that Absorption Systems only dose and sample the rats (in-life only)
    • that Absorption Systems analyze the plasma collected by others
    • that Absorption Systems perform the PK analysis on data collected by others