Identification of stable and reactive metabolite(s) of nelfinavir in human liver microsomes and rCYP3A4

•Structures of major and trace level stable and reactive metabolites of nelfinavir were characterized using LC–MS tools.•A total of thirty nine stable metabolites were detected.•ortho-Diquinone and N-oxide metabolites were also detected, which are known to be reactive in nature. But these metabolites did not show any interaction with nucleophiles, possibly due to steric hindrance at the site of interface.

The present study was performed to detect trace level stable and reactive metabolites of nelfinavir in human liver microsomes and rCYP3A4. Initially, chromatographic and MS parameters were optimized and fragmentation pattern of the drug was delineated. The structures of metabolites were then elucidated by comparison of their MS/MS fragmentation patterns with the drug. A total of thirty nine stable metabolites were formed, of which twelve were established to be monohydroxylated, eighteen dihydroxy, two dehydrogenated, and one each a diquinone, keto, carboxylic, N-deacylated, dealkylated, oxo and dehydro monohydroxyl metabolite. Previously, a biotransformation product with hydroxylation at tert-butyl group of nelfinavir is reported as an active metabolite of the drug. In our case, ortho-diquinone and N-oxide metabolites were detected, which are known to be reactive in nature. However, these metabolites did not show any interaction with nucleophiles, possibly due to steric hindrance at the site of interface.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide