750-MHz directly coupled HPLC-NMR: Application for the sequential characterization of the positional isomers and anomers of 2-, 3-, and 4-fluorobenzoic acid glucuronides in equilibrium mixtures

Abstract

This paper presents the first demonstration of the use of directly coupled HPLC. 1 H NMR spectroscopy using 750-MHz NMR detection. We show the application of the technique in the continuous-flow mode to separate and characterize mixtures of ester glucuronide isomers formed by internal acyl migration and mutarotation of 2-, 3-, and 4-fluorobenzoic acid β-1-O-acyl glucuronides [1-O-(2-fluorobenzoyl)-D-glucopyranuronic acid, 1-O-(3-fluorobenzoyl)-D-glucopyranuronic acid, and 1-O-(4-fluorobenzoyl)-D-glucopyranuronic acid]. The equilibrated mixtures of isomers were obtained by incubation of 10 mM synthetic β-1-O-acyl glucuronides in buffer solution (pH 7.4) at 25 °C. The β-anomer of the residual 1-O-acyl-glucuronide, as well as the 2-, 3-, and 4-positional glucuronide isomers (all three as both α- and β-anomers) present in the equilibrium mixture, was separated in the chromatographic system, and the 2-, 3- and 4-positional glucuronide isomers could be characterized directly by HPLC-NMR in the continuous-flow mode. The amount of each isomer injected on column was <0.14 μmol (35 μg), showing the practicality of continuous-flow 750-MHz HPLC-NMR in analytical problems requiring the highest sensitivity and signal dispersion.