Figure 1.

Characterization of P450-dependent NVP and 12-D₃NVP metabolite formation in primary mouse hepatocytes. Fresh primary mouse hepatocytes from male C57BL/6J mice were cultured and treated with 10 μM NVP or 12-D₃NVP for 24 h. Metabolites were extracted from the medium from these treatments and subjected to uHPLC-MS (Orbitrap) analysis. The following high resolution ions were observed to assay for the presence of P450 metabolites: 283.1190 ± 5 ppm for monooxygenated, undeuterated NVP, 286.1378 ± 5 ppm for monooxygenated, trideuterated NVP, and 285.1315 ± 5 ppm for monooxygenated, dideuterated NVP. Representative extracted ion chromatograms (XlCs) from this analysis are shown for a mixture of authentic standards for undeuterated 2-, 3-, and 12-OHNVP (A, each at 50 nM), monooxygenated, undeuterated metabolites formed during incubations with NVP (B), monooxygenated, trideuterated metabolites formed during incubations with 12-D₃NVP (C), and monooxygenated, dideuterated metabolites formed during incubations with 12-D₃NVP (D). Results are representations of four experimental replicates.