ent-16-Oxobeyeran-19-N-methylureido (NC-8) is a recently synthesized derivative of isosteviol that showed anti-hepatitis B virus (HBV) activity by disturbing replication and gene expression of the HBV and by inhibiting the host toll-like receptor 2/nuclear factor-κB signaling pathway. To study its pharmacokinetics as a part of the drug development process, a highly sensitive, rapid, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for determining NC-8 in rat plasma. After protein precipitation extraction, the chromatographic separation of the analyte and internal standard (IS; diclofenac sodium) was performed on a reverse-phase Luna C18 column coupled with a Quattro Ultima triple quadruple mass spectrometer in the multiple-reaction monitoring mode using the transitions, m/z 347.31 → 75.09 for NC-8 and m/z 295.89 → 214.06 for the IS. The lower limit of quantitation was 0.5 ng/mL. The linear scope of the standard curve was between 0.5 and 500 ng/mL. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of < 15%. Recoveries ranged from 104% to 113.4%, and the matrix effects (absolute) were non-significant (CV ≤ 6%). The validated method was successfully applied to investigate the pharmacokinetics of NC-8 in male Sprague–Dawley rats. The present methodology provides an analytical means to better understand the preliminary pharmacokinetics of NC-8 for investigations on further drug development.

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