[Fast liquid chromatographic method to determine compounds binding to human serum albumin]

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The 2- propanol concentration is increased from 0 to 25% in 3 minutes, thus allowing strongly bond compounds to elute within the 15 minutes. The HSA binding values were derived from the logarithmic values of the gradient retention times that were converted to logarithm of the equilibrium constants (logK HSA) using a calibration set of molecules. The logK HSA values for the calibration set of molecules have been derived from literature values of the % plasma protein binding. The method is fully automated and it has been used for lead optimization in more than 20 projects. The obtained HSA binding data on more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in the early drug discovery settings. The method has been validated using literature plasma protein binding data of 70 known drug molecules. It was shown that compound lipophilicity dominates the HSA binding, however compounds binding to a specific binding site show stronger HSA binding that can be expected from their lipophilicity values. The solvation equation approach has been used to characterize the non-specific lipophilic binding ability of HSA. Based on our approach we can identify compounds with specific and non-specific binding.