DEVELOPMENT OF A SIMPLE LIQUID-CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF MIXTURES OF POSITIONAL ISOMERS AND ANOMERS OF SYNTHETIC 2-FLUOROBENZOIC, 3-FLUOROBENZOIC AND 4-FLUOROBENZOIC ACID GLUCURONIDES FORMEDVIA ACYL MIGRATION REACTIONS

Many drugs containing carboxylate groups form beta-1-O-acyl glucuronid es as their major phase II metabolites in vivo. These eater glucuronid es are potentially reactive due to the susceptibility of the acyl grou p to nucleophilic reactions resulting in hydrolysis, acyl migration or covalent adduct formation. In the present study, a number of syntheti c fluorobenzoic acid glucuronide conjugates were chosen as models for chromatographic studies. A high-performance liquid chromatography meth od is presented for the simultaneous determination of the 1-, 2-, 3- a nd 4-positional isomers of the acyl glucuronides, and their alpha- and beta-anomers for the 2-, 3- and 4-fluorobenzoic acids as well as each aglycone formed as a result of hydrolysis. The same elution order was found for the acyl migrated glucuronide isomers of the three fluorobe nzoic acids in their equilibrium mixtures. The alpha-4-O-acyl isomer e luted first followed by the beta-4-O-acyl isomer, then the beta-1-O-ac yl, the beta-3-O-acyl, the alpha-3-O-acyl, the alpha-2-O-acyl and fina lly the beta-2-O-acyl isomer eluted. The method was used to determine the overall degradation rates, the acyl migration rates and the hydrol ysis rates of 1-O-(2-fluorobenzoyl)-beta-D-glucopyranuronic acid, 1-O- (3-fluorobenzoyl)-beta-D-glucopyranuronic acid and 1-O-(4-fluorobenzoy l)-beta-D-glucopyranuronic acid in a buffer system pH 7.3 at 25 degree s C. It was found that the order of beta-1-glucuronide acyl migration rates was 2-fluorobenzoyl>3-fluorobenzoyl>4-fluorobenzoyl. Both the ac yl migration rates and the elution order were interpreted in terms of electronic effect of the fluorine substituent on the carbonyl carbon.