Modification of the ultrafiltration technique to overcome solubility and non-specific binding challenges associated with the measurement of plasma protein binding of corticosteroids

Plasma protein binding (PPB) methodology suitable for application in the lead optimisation of a corticosteroid series known to demonstrate non-specific binding (NSB) and poorsolubility has been established. The method involved a modification to standard ultrafiltration (UF) techniques. In parallel with each experimental plasma sample, a control plasma sample was also processed by ultrafiltration. The retentate from experimental and control plasma samples were mixed back into the filtrate of the partner sample. The resulting regenerated plasma samples, one representing the experimental filtrate and one representing the experimental retentate, were then analysed by LC/MS/MS. Varying degrees of NSB were demonstrated with a number of corticosteroids, and this effect was eliminated using the modified method. Validation using a panel of established corticosteroids showed good agreement with published PPB figures. The published PPB figuffe for fluticasone propionate (FP) was, however, found to be an underesti mate, and this was subsequently confirmed, at clinically relevant plasma concentrations, to be 99.3%. The modified method was particularly suited to lead optirnisation because it provided samples in a consistent matrix compatible with standard high throughput LC/MS/MS analysis. (c) 2005 Elsevier B.V. All rights reserved.