Direct isomeric separation of a 3-hydroxyproline-containing prodrug, L-693 989, by high-performance liquid chromatography with a porous graphitic carbon column

Abstract

The use of high-performance liquid chromatography (HPLC) with a porous graphitic carbon (Hypercarb) column for the direct separation of a positional isomer of L-693 989, a 3-hydroxyproline-containing prodrug, is described. The isomer peak was isolated by HPLC and analyzed by mass spectrometry and proton nuclear magnetic resonance spectroscopy. An authentic sample of the isomer was also synthesized for chromatographic comparison. The results confirm that the peak in question is a 4-hydroxyproline isomer which is difficult to separate from L-693 989 compound with the silica-based reversed-phase columns. The observed chromatographic elution supports the retention mechanism based on the unique electronic donor-acceptor interaction between the lone-pair electrons of the analyte (donor) and the delocalized electron conduction bands on the graphitized carbon stationary phase (acceptor).