Nonenzymatic formation of a novel hydroxylated sulfamethoxazole derivative in human liver microsomes: implications for bioanalysis of sulfamethoxazole metabolites

Sulfamethoxazole is metabolized by microsomal CYP2C9 to a hydroxylamine that is thought to be responsible for the relatively high incidence of hypersensitivity reactions associated with the drug. Accurate quantification of the hydroxylamine requires the loss of metabolite through autoxidation to be blocked with ascorbate. In this study, a partly nonenzymatically generated arylhydroxylated derivative of sulfamethoxazole was identified by liquid chromatography/mass spectrometry in incubations of human liver microsomes, and it was found to coelute with the isomeric hydroxylamine under the conditions of three published high-performance liquid chromatography (HPLC) assays. Partial inhibition of the aryl hydroxylation by 1-aminobenzotriazole suggested some involvement of cytochrome P450. However, the formation of this compound was ascorbate-dependent, and it was enhanced by the addition of Fe2+/EDTA and inhibited by desferrioxamine but not by mannitol. These findings are consistent with the phenol being generated via an Fe2+/ascorbate/O2-oxygenating system that does not involve hydroxyl radicals. It was also produced by H2O2/ascorbate. Because the compound shares close chromatographic similarities with the hydroxylamine metabolite, it is possible that previous studies may have inaccurately characterized or quantified sulfamethoxazole metabolism.