IDENTIFICATION OF THE POSITIONAL ISOMERS OF 2-FLUOROBENZOIC ACID 1-O-ACYL GLUCURONIDE BY DIRECTLY COUPLED HPLC-NMR

Directly coupled HPLC-IH NMR was used in the ''stop-flow'' mode to separate and rapidly identify an equilibrated mixture of ester glucuronide isomers formed spontaneously by intramolecular rearrangement reactions (internal acyl migration and mutarotation) of 2-fluorobenzoic acid beta-1-glucuronide (1-O-(2-fluorobenzoyl)-D-glucopyranuronic acid). The equilibrated mixture of isomers was obtained by incubation of the synthetic 2-fluorobenzoic acid glucuronide in buffer solution (pH 7.4) at 25 degrees C for 24 h. The beta-anomer of the 1-O-acyl glucuronide, and the 2-, 3-, and 4-positional glucuronide isomers (all three as both alpha- and beta-anomers) present in the equilibrium mixture, were all characterized after separation in an isocratic chromatographic system containing phosphate buffer at pH 7.4 and 1% acetonitrile in the mobile phase. The HPLC-NMR investigations also elucidated the mutarotation of the positional glucuronide isomers as well as showing the benefits of the HPLC-NMR technique as a primary analytical tool. This HPLC-NMR method will be of particular value in studies on the acyl migration reactions of nonsteroidal antiinflammatory drug glucuronides which may be related to their toxicological properties.