750 MHz HPLC-NMR spectroscopic identification of rat microsomal metabolites of phenoxypyridines

Directly coupled 750 MHz HPLC-H-1 NMR spectroscopy has been applied to the characterisation of low level metabolites of 3-amino-2-(2-fluorophenoxy)pyridine (AP) and 3-nitro-2-(2-fluorophenoxy)pyridine (NP) in rat microsomes. In stop-flow HPLC-NMR mode, the direct injection of microsomal extracts enabled the separation and characterisation of minor metabolites. NP is converted into AP to an extent of 93.3% and this is further metabolised to 4- and 6-hydroxy-AP (6 and 0.6% respectively). Unequivocal identification of these metabolites was achieved without the use of a radiolabel or synthetic standards and thus demonstrates the applicability of directly coupled HPLC-NMR to metabolite identification in in vitro systems. The potential exists for HPLC-NMR and HPLC-NMR-MS to provide rapid metabolic information within the timescale of high throughput lead optimisation exercises in drug discovery. (C) 1997 Elsevier Science B.V.