Final ProgramCPSA 2011
Science and Technology Coming Together to Make a Difference
October 3 - 6, 2011
Bucks County Sheraton Hotel
Langhorne, PA
Poster Abstract #11
A Novel High Throughput Method Using Full Scan HRAM and Online Extraction for Plasma Protein Binding Determination
Thermo Fisher Scientific, San Jose, California, USA
The ability of a drug to reach its intended therapeutic target as well as its ability to be eliminated from the body is affected by the reversible binding to plasma or serum proteins. Equilibrium dialysis is the accepted method for estimating protein binding. Rapid equilibrium dialysis speeds throughput when compared to the traditional method through its automation friendly footprint and because of high surface area to volume ratio of the membrane compartment which reaches equilibrium within four hours. To further increase throughput, the need to optimize each compound for QQQ analysis is eliminated by taking advantage of full scan HRAM analysis.
Plasma containing drug was added to one chamber while buffer was added to the second chamber separated by a semi-permeable membrane (8K MWCO). The samples were incubated at ~37 °C while shaking at 100 rpm for 4 hours. Afterwards, an aliquot from each chamber (200 µL plasma, 300 µL buffer) was removed and equal amounts of fresh plasma and buffer were added to respective incubated aliquots. The protein/buffer mixtures were precipitated using an acidified organic ISTD cocktail solution, thoroughly mixed, centrifuged, and the supernatant was transferred to a 96-well plate for subsequent analysis.
Protein binding determination was conducted in both human and rat plasma for a wide spectrum of low to high binding compounds such as Gabapentin (literature value: <3%), Levofloxacin (24-38%) and Warfarin (98-100%). In an effort to create a generic workflow, incubations were performed at a single concentration for the compound library analyzed. The total time to analyze the entire 16 compound sample set (n=4), for both human and rat, was approximately 5 hours. Overall, the data acquired agreed with literature values with acceptable reproducibility using either a simple mean or well-to-well calculation. The time savings provided with this workflow is useful in both a discovery and development environment due to the elimination of off-line sample preparation, chromatographic multiplexing, and removing compound optimization required for triple-stage quadrupole instruments.