Isolation of circulating metabolites in drug discovery using high-performance liquid chromatography, and their identification by liquid chromatography coupled with tandem mass spectrometry and nuclear magnetic resonance spectroscopy

Abstract

Isolation of circulating metabolites in drug discovery using high-performance liquid chromatography, and their identification by liquid chromatography coupled with tandem mass spectrometry and nuclear magnetic resonance spectroscopy One of the major components of modern drug discovery is the structural determination and the assessment of biological activity of plasma metabolites. LC-MS/MS has played a prominent role in the identification of metabolites; however, fragmentation patterns alone may not be sufficient for identification. Consequently, it may be necessary to isolate the metabolites for NMR or LC-NMR analysis. This report describes the isolation and identification of the major plasma metabolites of two lead compounds (SCH X and SCH Y). The major metabolite of SCH X in monkey plasma constituted 65% of total compound-derived materials. Incubation of rat liver microsomes with SCH X gave the mono-hydroxylated metabolite found in monkey plasma; however, the yield was low. Incubations with microsomes from rats pre-treated with various cytochrome P450 inducers showed that the highest yield was obtained from pregnenolone 16a-carbonitrile (PCN)-induced animals. For SCH Y, two metabolites were found in bile and plasma of both rats and monkeys. Various in vitro systems did not produce amounts sufficient for isolation. Therefore, the metabolites of SCH X and SCH Y were isolated from PCN-induced rat liver microsomal incubation and rat bile, respectively. The chemical structures of the metabolites were unambiguously determined using LC-NMR analyses.

Use of quadrupole time‐of‐flight mass spectrometry in the elucidation of unknown compounds present in environmental water

“…Since the commercial introduction of liquid chromatography/nuclear magnetic resonance (LC/NMR) systems, this hyphenated technique has been mainly applied to the structural elucidation of metabolites in drug development studies12 and to the identification of natural compounds in plant extracts 13. However, recent studies have demonstrated its applicability to the analysis of unknown compounds in environmental samples as 1 H NMR spectra in general give more detailed structural information than mass spectra 14. Optimum structural information is obtained when LC/MS and LC/NMR are used together to identify unknowns in complex mixtures 15.…”