HPLC/1H NMR Spectroscopic Studies of the Reactive α-1-O-acyl Isomer Formed during Acyl Migration of S-Naproxen β-1-O-acyl Glucuronide

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Abstract
A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that α-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen β-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 °C. We report the first direct observation of the α-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure β-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, “dynamic” stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated α-1-O-acyl isomer to a mixture of α/β-2-O-acyl isomers; the reverse reaction from the isolated α/β-2-O-acyl isomers to the α-1-O-acyl isomer was also clearly demonstrated. This application of “dynamic” stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the α-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of α-1-O-acyl isomers from zomepirac and diflunisal β-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile α-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.