Semi-automated protein binding methodology using equilibrium dialysis and a novel mixed-matrix cassette approach

A semi-automated protein binding assay using equilibrium dialysis (ED) and a novel mixed-matrix methodology has been developed. This method decreases mass spectrometer run time and reduces the likelihood of experimental artifacts. In this cassette-based approach, a single matrix is prepared following dialysis by mixing dialyzed plasma and buffer containing different test compounds from the same dialysis plate. This approach differs from the traditional mixed-matrix method where fresh plasma and fresh buffer are mixed with opposing dialyzed samples. This new mixed-matrix methodology is compatible with various high-throughput ED and ultrafiltration devices, many liquid handling systems, and can be used for plasma, serum, albumin, alpha-1 acid glycoprotein, microsomal, and fine tissue homogenate binding studies. The utility of the method can be further enhanced by varying the number of replicates, concentrations, and matrices with simple modifications. Using 29 structurally diverse marketed drugs with a wide range of protein binding values reported in the literature, we have shown the new procedure reduces the total number of samples by nearly half compared to traditional methods, eliminates the need for standard curves, and increases the uniformity of the sample matrix for LC/MS/MS analysis.