Prostaglandins are biological lipid mediators that control a variety of physiological and pathophysiological processes. They function as locally acting hormones and are continuously synthesized and inactivated in target tissues by respective biosynthetic and metabolic enzymes. The separation of prostaglandins and their metabolites is crucial for study of their roles in biological systems. In this article, we describe systematic studies of the retention and separation of prostaglandin E₂ (PGE₂) from its inactive metabolites 15-keto-PGE₂ and 13,14-dihydro-15-keto-PGE₂. We also describe the separation of a second prostaglandin species, PGF₂α , from its metabolites 15-keto-PGF₂α and 13,14-dihydro-15-keto-PGF₂α . Chromatography was performed on RP-18W high-performance thin-layer chromatographic plates with binary mobile phases comprising water and an organic modifier (methanol, ethanol, acetonitrile, acetone, or tetrahydrofuran). The experimental data revealed that at constant temperature, plots of the retention, RM , of the prostaglandins against the mole fraction of organic modifier in the water (XS) were nonlinear. By use of mobile phases of similar eluent strength the effect of temperature on the retention and separation of the solutes was examined. Thermodynamic data, calculated from linear Van’t Hoff plots, indicated that the mechanism of retention for each prostaglandin compound was consistent for the different mobile phases. The separation of PGE₂ and PGF₂α from each other and from their respective metabolites was rapid and robust in acetonitrile and in acetonitrile-water mobile phases. Interestingly, with 100% acetonitrile as the mobile phase the reversed-phase system apparently performed as a normal-phase system. The chromatographic system described here is applicable to the rapid processing of a large number of samples, which is a requirement for determining the biological metabolism of PGE₂ and PGF₂α and for studying the kinetics of prostaglandin-inactivating enzymes. The system is also suitable for the prepurification of complex biological samples for subsequent quantification of individual prostaglandins.
Journal Of Planar Chromatography: Modern TLC Vol. 16, Issue 2, p. 95-101