Determination of nimesulide and its active metabolite in plasma samples based on solvent deproteinization and HPLC-DAD analysis

A selective, sensitive and accurate HPLC method for the assay of nimesulide and its major metabolite (4’-hydroxynimesulide) in human plasma samples is presented. The sample preparation procedure was based on a simple protein precipitation with acetonitrile. The reversed phase liquid chromatography was used as separation technique followed by UV detection of analytes at 300 nm. Separation was achieved on a Chromolith Performance RP 18e column using a mobile phase consisting of aqueous 0.2% triethylamine at pH 3.0 with H3PO4 and methanol (1:1, v/v). A small volume of plasma is required for the sample preparation procedure (200 μL), nitrazepam being used as internal standard. The limits of quantification for nimesulide and 4’-hydroxy-nimesulide are 64 ng/mL and 41 ng/mL, respectively. Relative standard deviations characterizing intra-day and inter-day precision were less than 5%. The validated method was applied during a bioavailability study for a pharmaceutical formulation containing nimesulide (uncoated tablet) available on the Romanian market. Both nimesulide and 4’-hydroxy-nimesulide were determined from the real human plasma samples obtained from 22 volunteers, included in this study, after the administration of a single oral dose.