High-performance liquid chromatographic method for the simultaneous quantitation of diflunisal and its glucuronide and sulfate conjugates in human urine.

A direct high-performance liquid chromatographic (HPLC) assay was developed to simultaneously quantitate diflunisal and its three known metabolites (i.e., the phenolic and acyl glucuronides and the sulfate conjugate) in human urine. Chromatographically pure standards of the diflunisal conjugates were isolated from urine of volunteers following ingestion of multiple doses of diflunisal (500 mg twice daily). Diflunisal, its three conjugates, and an internal standard (naproxen) were separated on a reversed-phase column using gradient elution. The column eluate was monitored fluorometrically (excitation: 258 nm; emission: 428 nm). Urine samples were diluted with phosphate buffer (pH 5.75) and injected onto the column. The limit of detection was approximately 1 microgram/mL for each conjugate and 0.1 microgram/mL for diflunisal. Due to the presence in most urine samples of significant concentrations of rearrangement products of the biosynthetic 1-O-acyl glucuronide of diflunisal, the acyl glucuronide could not be reliably quantitated by direct injection of diluted urine samples. Instead, diflunisal acyl glucuronide was quantitated indirectly following alkaline hydrolysis of the urine samples. The method has been successfully used to investigate the dose-dependent glucuronidation and sulfation of diflunisal in humans.