Simultaneous determination of nimesulide and its four possible metabolites in human plasma by LC–MS/MS and its application in a study of pharmacokinetics

• A LC–MS/MS method for determination of nimesulide and its metabolites in plasma was developed.
• The method was fully validated and successfully applied in clinical practice.
• A new conjugated metabolite of nimesulide in plasma was determined and discussed firstly.

In this study, it was the first time that we simultaneously quantified nimesulide and its possible metabolites M1, M2, M3 and M4 by employing liquid chromatography-tandem mass spectrometry (LC–MS/MS). Nimesulide-d5 was used as internal standard (IS) for validation. Analytes and IS were recovered from human plasma by protein precipitation with acetonitrile. Prepared plasma samples were analyzed under the same LC–MS/MS conditions, and chromatographic separation was realized by using an Ultimate C18 column, with run time being 5 min for each sample. Our results showed that various analytes within their concentration ranges could be quantified accurately by using the method. Mean intra- and inter-day accuracies ranged from −4.8% to 4.8% (RE), and intra- and inter-assay precision ≤6.2% (RSD). The following parameters were validated: specificity, recovery, matrix effects, dilution integrity, carry-over, sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw and post-preparative) and stock solution stability. Pharmacokinetics of nimesulide and its metabolites were calculated based on the analysis of samples collected from twelve Chinese healthy volunteers after single oral dose of 100 mg nimesulide tablets. By applying the pharmacokinetic determination into human samples, we preliminarily detected a new metabolite of nimesulide (M4*), and the concentration of M4* was relatively higher in plasma. Furthermore, we predicted part of conceivable metabolism pathway in plasma of after oral administration of 100 mg nimesulide tablets. This research provided an experimental basis for further studies on metabolic activation and biotransformation of nimesulide, and for more comprehensive conjecture of its metabolic pathways.