
EA713-C
Blood to Plasma Partitioning Whole Blood
This assay is used to determine the blood-to-plasma partition coefficient of a test compound in mice, rats, and humans in vitro
Required from Customer
- Either a minimum of 300 μL of test compound at 10 mM in DMSO or 5 mg of powder
- The 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
Deliverables
- The concentrations of test compound and control compound in whole blood and plasma
- The calculated blood-to-plasma partition coefficient (KRBC/P) for the test compound and control compound
- Hematocrit of the whole blood lot used in the study
Substrate
- Test compound at a single concentration (typically 5 μM) in heparinized blood and plasma with final DMSO concentration < %
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 compound with test compound
- Duplicate incubation (N=2 separate incubations) in whole blood and reference plasma
- Sample blood and prepare plasma (test plasma) by centrifugation at 60 minutes
- Sample reference plasma at 60 minutes
Assay QC
- Results are accepted only when the blood-to-plasma partition coefficient of the reference compound meets internal acceptance criteria
Notes
- The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.