High-performance liquid chromatographic determination of monohydroxy compounds by a combination of pre-column derivatization and post-column reaction detection

Abstract

A novel high-performance liquid chromatographic method was developed for the determination of monohydroxy compounds using the combined approach of pre-column derivatization and post-column reaction with fluorescence detection. Monohydroxy-containing drugs were modified by pre-column derivatization with propyl isocyanate (in pyridine, 50°C, 1 h) to form the corresponding n-propyl carbamate esters. Excess of reagent, reagent impurities and solvent were then removed by evaporation. Following chromatographic separation on a reversed-phase octadecylsilica column, the n-propyl carbamate ester derivative was subjected to post-column reaction and detection using alkaline hydrolysis to generate free propylamine, which was subsequently derivatized in-line using o-phthaladehyde and 3-mercaptopropionic acid to form a fluorescent isoindole. The fluorescence emission of the isoindole product was measured at 455 nm following excitation at 340 nm. As propyl isocyanate is highly volatile and physico-chemically different to many drug molecules, problems associated with reagent impurities and reaction by-prodycts were substantially minimized. The method was highly sensitive, allowing detection of sub-nanogram amount of monohydroxy-containing drugs, hydroxy steroids and hydroxamic acids. The utility of this derivatization scheme was demonstrated by the highly sensitive measurement in human plasma at oxiracetam (4-hydroxy-2-oxo-1-pyrrolidineacetamide), an investigational drug inteded for use in dementia and other memory disorders.