Determination of nimesulide and hydroxynimesulide in human plasma by high performance liquid chromatography

Two specific methods for the simultaneous determination of nimesulide, a non steroidal anti-inflammatory drug, and its hydroxylated metabolite in human plasma are described. Adopting a high performance liquid chromatographic (HPLC) system with UV detection (230 nm), the compounds, extracted from plasma in acidic medium, were separated on ODS columns under gradient conditions, using a phosphate buffer solution and methanol as mobile phase. For each method column length, gradient rate and composition were appropriately selected. The limit of quantitation was 25 ng/mL for both compounds. The two methods were validated by intra day assays at three concentration levels and applied in kinetic studies in healthy volunteers, during which inter-day assays were carried out confirming their feasibility.