Simultaneous quantification of naproxcinod and its active metabolite naproxen in rat plasma using LC–MS/MS: Application to a pharmacokinetic study

• Naproxcinod and naproxen were determined simultaneously for the first time by LC–MS/MS.
• Simple one-step extraction with methanol for protein precipitation using only 50 μL plasma.
• The LLOQs of naproxcinod and naproxen are 1.00 ng/mL and 20.0 ng/mL, respectively.
• The method was successfully used in a pharmacokinetic study of naproxcinod in rats.

In this study, a liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine naproxcinod and naproxen concentrations in rat plasma for the first time. Plasma samples were prepared by simple one-step extraction with methanol for protein precipitation using only 50 μL plasma. Separation was performed on a Synergi Fusion-RP C18 column with a run time of 4 min. Naproxcinod, naproxen and internal standard concentrations were detected in the positive ion mode using multiple reaction monitoring (MRM) of the transitions at m/z 348.2 → 302.2, 231.1 → 185.1 and 271.2 → 203.1, respectively. The calibration curves were linear, with all correlation coefficients being ≥0.9952, in the range of 1.00–400 ng/mL for naproxcinod and 20.0–8000 ng/mL for naproxen. Their accuracy was in the range of −8.1% to 8.7%, and the intra- and inter-day variations were ≤4.53%. The mean extraction recovery of all analytes was more than 93.1% efficient. Stability testing showed that naproxcinod and naproxen remained stable during the whole analytical procedure. After validation, the method was successfully applied to a pharmacokinetic study of naproxcinod and naproxen in rats. The AUC0–∞ of naproxen was 74.6 times larger than that of naproxcinod, which indicated that naproxcinod was rapidly metabolized into naproxen in rats.