Correlation between reversed-phase high-performance liquid chromatography and plasma protein binding.

As hydrophobic interactions are involved in both reversed-phase liquid chromatography and plasma protein binding, the relationship between retention time and binding was investigated experimentally in two series of compounds. For betaxolol and its O-alkyl analogues, the nature of the O-alkyl group strongly influences the retention time on a Spherisorb CN 5-microns column. With 0.03 M acetate buffer (pH 5.6)-acetonitrile (60:40) as the mobile phase, k' values increase from 1.8 to 8.3 with a concomitant increase in plasma protein binding from 0.5% (R = H) to 88.2% (R = cyclopentylmethyl). The relationship between the free fraction and log k' is sigmoidal. In the second example, structural changes in the propyl side-chain of alpidem (a new anxiolytic) lead to minor variations in the protein binding: 98.9 to 84.9%. This slight decrease with the more polar metabolite is correlated with a sharp decrease in the k' values from 14.7 to 0.94 on a Supelcosil LC 18 DB column. Based on retention times, it should be feasible to predict qualitatively, if not quantitatively in some instances, plasma protein binding in a series of structurally similar compounds.