Description:
Semi-Automated Plasma Protein Binding Assay Utilizing a 96-well Equilibrium Dialyzer and a Mixed Matrix Approach
Emile G. Plise, Daniel Tran and Laurent Salphati
Drug Metabolism and Pharmacokinetic Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA.
Purpose. To automate a 96-well equilibrium dialysis plasma protein binding (PPB) assay using a novel mixed-matrix approach. Methods. This method employs the HTdialysis HTD 96 equilibrium dialysis plate, the Bio-Tek Precision XS, and Zymark Rapid Plate® 96/384 liquid handling systems. A master plate containing the compounds is manually prepared. The spiking and mixing of the plasma with the compounds, as well as the transfer of spiked plasma and blank buffer to the dialysis plate are performed by the Precision XS. Following dialysis, the Precision XS transfers the dialysate buffer and plasma to a clean 96-deep well plate. The transfer is made such that the buffer containing a test article from a species is combined with the plasma of the same species which contains a different test article, in contrast to the more “traditional” method that requires fresh buffer and plasma for the preparation of the mixed-matrix. After addition of the analytical internal standard and protein precipitation with acetonitrile, the Rapid Plate® transfers the supernatant to a new plate. All samples are analyzed by LC/MS/MS.
Results. The PPB values measured in mouse, rat, and human plasma with this new method were comparable with those obtained with the established manual mixed-matrix method for four Genentech compounds and tolbutamide, used as a control (r2 was 0.96). PPB ranged from 62% to 99%. Values determined for tolbutamide were similar to published results (96.7%-98.7%, Banker et al. 2002).
Conclusions: This new automated mixed-matrix PPB method is easy to use and does not require expensive automation or standard curves. PPB values determined were similar to those obtained using the “traditional” manual method. In contrast to the “traditional” mixed-matrix method, it uses the plasma and buffer from the dialyzed samples to prepare the mixed-matrix. Consequently, this method generates 50% fewer samples and thus requires half the mass spectrometer time. It also has increased throughput. A total of 16 compounds in 3 species can be run in duplicate in a single HTdialysis plate.
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