Reactivity considerations in the analysis of glucuronide and sulfate conjugates of diflunisal

Reactivity of glucuronide and sulfate conjugates was taken into account in development of a simple isocratic HPLC method for direct assay of diflunisal (DF) and its acyl glucuronide (DAG), phenolic glucuronide (DPG), and sulfate (DS) conjugates. Whereas DPG was stable over the pH range 0-9, DAG was highly labile at neutral to slightly alkaline pH, undergoing rearrangement (isomerisation via acyl migration), hydrolysis, and in the presence of methanol, transesterification to DF methyl ester. The 2-, 3-, and 4-O-acyl positional isomers of DAG appeared as three pairs of peaks. Interconversion between partners of each pair occurred even under acidic conditions inhibitory to acyl migration, implicating mutarotation. DS was stable at neural to slightly alkaline pH, but underwent hydrolysis under relatively strongly acidic conditions. However, this hydrolysis was remarkably catalyzed (e.g., by 1,000-fold) in the presence of solvents (i.e., solvolysis) such as diethyl ether and ethyl acetate. DS (an acid) could not be extracted from aqueous solution because of this acidic solvolysis. Suitable conditions for simultaneous direct analysis (nonextractive, nonconcentrative) of DF and its reactive (DAG and DS) and unreactive (DPG) conjugates were achieved by working at pH of approximately 4.5. The procedure thus developed is suitable for plasma, urine, and bile samples, and has revealed the presence of new, as yet unidentified, metabolites of DF.