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Rapid Equilibrium Dialysis (RED): an In-vitro High-Throughput Screening Technique for Plasma Protein Binding using Human and Rat Plasma

Jitendra Kumar Singh, Anant Solanki, Reema C Maniyar, Debarupa Banerjee and Vikas S Shirsath

Determining the extent to which a molecule binds to plasma proteins is a critical phase of drug development because the amount of plasma-bound drug influences compound dosing, efficacy, clearance rate and potential for drug interactions. Determination of the free (%Fu) and bound (%Bound) fractions of a test article in plasma is therefore a critical parameter, which is routinely determined in the process of drug discovery and development. This determination is enabled by equilibrium dialysis, an accepted and standard method for reliable estimation of the non-bound drug fraction in plasma. Although it is the preferred method, equilibrium dialysis has historically been labor-intensive, time-consuming, cost-prohibitive and difficult to automate. A Rapid Equilibrium Dialysis (RED) drug-protein binding assay using LC-MS/MS was developed using a novel technique that resulted in significantly improved assay precision and offers a speed advantage. A panel of compounds covering a range of expected protein binding was tested in plasma of human and rat species. A sensitive and selective method using quadruple tandem mass spectrophotometer interfaced with electro spray ionization was developed for the quantification of unbound drug in pretreated plasma. The mobile phases used were 0.1% Formic acid in Acetonitrile: 0.1% Formic acid in Water with gradient HPLC method. The unbound fraction of drugs was detected by mass spectrometer operated in ESI mode. In addition, data for a set of ten compounds was compared with literature values. With the described method, it is possible to screen a relatively large number of compounds for PPB in a drug discovery environment.