Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans

The metabolism of diclofenac has been reported to produce reactive benzoqui none Imine intermediates. We describe the identification of mercapturic aci d derivatives of diclofenac in rats and humans. Three male Sprague-Dawley r ats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine sp ecimens were obtained, and samples were pooled from 50 individuals. Urine s amples were analyzed by liquid chromatography-tandem mass spectrometry (LC/ MS/MS). Two metabolites with MH+ ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydi clofenac. Based upon collision-induced fragmentation of the MH+ ions, accur ate mass measurements of product Ions, and comparison of LC/MS/MS propertie s of the metabolites with those of synthetic reference compounds, one metab olite was assigned as 5-hydroxy-4-(N-acetyleystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjuga te also was detected in the pooled human urine sample by multiple reaction- monitoring LC/MS/MS analysis. It is likely that these mercapturic acid deri vatives represent degradation products of the corresponding glutathione add ucts derived from diclofenac-2,5-quinone imine and 1',4'-quinone Imine, res pectively. Our data are consistent with previous findings, which suggest th at oxidative bioactivation of diclofenac In humans proceeds via benzoquinon e imine intermediates.