This assay uses in vivo carotid infusion to determine the extent of brain penetration of test compound in a physiological setting, including plasma protein binding

Catalog number EA811

Required from Customer

  • A study design—the customer must specify the value of a number of assay variables listed in the Options section below
  • At least 20 mg of test compound in powder form
  • Molecular mass (exact mass) of the test compound and its salt form
  • MSDS or handling and storage information, e.g., light sensitive, store at 20°C, stability, solubility and percent purity
  • If applicable, instruction for dosing vehicle preparation

Deliverables

  • Concentrations of test compound and control compounds in brain extracts

Substrate

  • Test compound at a concentration specified by the customer

Assay System

  • 200-400 gram Sprague-Dawley rats with cannulated left carotid artery; can also be done in mice

Assay Conditions

  • Four rats are used per test compound (N=4); data from three rats that meet the acceptance criteria for the internal control compounds are used
  • Rapidly infuse test compound plus negative control, atenolol, into left carotid artery for 3 minutes
  • Remove the brain at the end of the infusion, excise and homogenize the left hemisphere and extract the tissue with acetonitrile
  • Perform pre-study work with  the test compound in brain matrix  to establish calibration range and method reproducility
  • Determine concentrations of test compound using a validated LC-MS/MS method with a minimum 8 point calibration curve and quality control samples

Assay QC

  • Analytical data are accepted only when at least two-thirds of the control samples and 5 of the 8 standards used to create the calibration curve have a back-calculated accuracy of ±15%  (at LLOQ, ±20%)
  • The negative control, atenolol, will be codosed with test compound

Notes

  1. 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.
  2. 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

Options

  1. The customer must specify:
    • either the standard or custom report format
    • the concentration of the test compound—the lower limit is circa 1 μM, and this limit  depends primarily on the sensitivity of the analytical method—there is no upper limit except as defined by solubility of the compound
  2. The customer can request:
    • additional rats used per test compound
    • plama samples to be collected and analyzed for determination of brain to plasma ratio
    • animals to be sacrificed at a time point other than the end of the infusion