Determination of partitioning coefficients and protein binding using high performance liquid chromatography: Towards in vitro pharmacology in radiotracer development

Abstract

We present RP-HPLC methods for the determination of the distribution coefficient at pH 7.4, as log D, and plasma protein binding. Although there are several methods available to determine distribution coefficient (log D) and protein binding, none of them involves a general and user-friendly approach that gives the opportunity to screen large libraries of compounds for both log D and plasma protein binding, with low cost and less time-consuming assays. The log D methods are amenable to rapid determinations and offer an excellent reproducibility. Standard deviation from literature was only 0.1 as an average for method A and 0.2 in method B, though method B eluted compounds earlier and worked for a broader range of complex compounds. The plasma protein binding determination made use of Microcon centrifugal filters whereas incubated test compound in porcine plasma where ultrafiltrated and the supernatant were measured by RP-HPLC. The high-throughput protocols described herein, for determination of log D and plasma protein binding, are straightforward to set up and require very small quantities of sample (< 1 mg for both lipophilicity and protein binding). In addition to the optimized methods, trends between chemical properties and lipophilicity/plasma protein binding were investigated, with no clear correlation useful for screening purposes.