Determination of benzarone in human plasma and urine by high-performance liquid chromatography and gas chromatography—mass spectrometry : Identification of the conjugates

Abstract Benzarone (the debrominated metabolite of the uricosuric drug benzbromarone) has been proposed for treatment of vascular disorders. An assay was developed for the quantitation of total benzarone (conjugated and unconjugated) in plasma and urine, following oral intake of benzarone. Enzymatic hydrolysis of the samples with β-glucuronidase/arylsulphatase, extraction, gradient elution high-performance liquid chromatography with reversed-phase columns and UV detection were used for the assay. The concentration ranges, precision and sensitivities were: 0.01–2 μg/ml, 3–5% and 0.01 μg/ml, respectively, for both plasma and urine. These results were validated by gas chromatography—mass spectrometry after methylated derivatives were prepared. Enzymatic hydrolysis of plasma with pure β-glucuronidase or arylsulphatase showed that the relative amounts of unconjugated, glucuronidated, and sulphated benzarone were 6, 12 and 82% respectively, for both plasma and urine.