HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY DIRECTLY COUPLED TO F-19 AND H-1-NMR FOR THE ANALYSIS OF MIXTURES OF ISOMERIC ESTER GLUCURONIDE CONJUGATES OF TRIFLUOROMETHYLBENZOIC ACIDS

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 i nvestigating the kinetics of acyl migration reactions in a buffer syst em at pH 7.4, using synthetic 1-O-acyl glucuronides of model compounds (2- and 3-trifluoromethylbenzoic acids) by HPLC-NMR. HPLC directly co upled to F-19 and H-1 NMR were used in both stop-flow and continuous-f low modes to separate and rapidly identify a mixture of ester glucuron ide isomers formed spontaneously by internal acyl migration and mutaro tation of 2-, and 3-trifluoromethylbenzoic acid glucuronides [1-O-(2-t rifluoromethylbenzoyl]-D-glucopyranuronic acid and 1-O-(3-trifluoromet hylbenzoyl)-D-glucopyranuronic acid). The mixtures of isomers were obt ained by incubation of the synthetic 2-, and 3-trifluoromethylbenzoic acid glucuronides in buffer solution (pH 7.4) at 25 degrees C for 48 h . The beta-anomer of the 1-O-acyl-glucuronide, as well as the 2-, 3-, and 4-positional glucuronide isomers (all three in both alpha- and bet a-anomeric forms) present in the isomeric mixture, were all characteri sed directly by NMR after separation in an isocratic chromatographic s ystem containing phosphate buffer at pH 7.4 and acetonitrile in the mo bile 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-F-19 NMR experiment. This approach to monitoring metabolite reactivity will be of great value in furthering the underst anding of glucuronide rearrangement kinetics and may be of wider impor tance in monitoring the reactivity of other types of analytes that hav e been separated in an HPLC-NMR system.