High-performance liquid chromatography directly coupled to 19F and 1H NMR for the analysis of mixtures of isomeric ester glucuronide conjugates of trifluoromethylbenzoic acids

Abstract

Acyl migration reactions of drug 1-O-acyl glucuronides are of interest because of their possible role in covalent binding to serum proteins and consequent allergic reactions. This paper describes a new way of investigating the kinetics of acyl migration reactions in a buffer system at pH 7.4, using synthetic 1-O-acyl glucuronides of model compounds (2- and 3-trifluoromethylbenzoic acids) by HPLC-NMR. HPLC directly coupled to 19F and 1H NMR were used in both stop-flow and continuous-flow modes to separate and rapidly identify a mixture of ester glucuronide isomers formed spontaneously by internal acyl migration and mutarotation of 2-, and 3-trifluoromethylbenzoic acid glucuronides [1-O-(2-trifluoromethylbenzoyl]- d-glucopyranuronic acid and 1-O-(3-trifluoromethylbenzoyl)- d-glucopyranuronic acid). The mixtures of isomers were obtained by incubation of the synthetic 2-, and 3-trifluoromethylbenzoic acid glucuronides in buffer solution (pH 7.4) at 25°C for 48 h. The β-anomer of the 1-O-acyl-glucuronide, as well as the 2-, 3-, and 4-positional glucuronide isomers (all three in both α- and β-anomeric forms) present in the isomeric mixture, were all characterised directly by NMR after separation in an isocratic chromatographic system containing phosphate buffer at pH 7.4 and acetonitrile in the mobile phase. The time-course of individual acyl migration of positional glucuronide isomers was monitored in the mobile phase in a novel stop-flow ‘dynamic’ HPLC- 19F NMR experiment. This approach to monitoring metabolite reactivity will be of great value in furthering the understanding of glucuronide rearrangement kinetics and may be of wider importance in monitoring the reactivity of other types of analytes that have been separated in an HPLC-NMR system.