Improved high-performance liquid chromatographic assay for nimesulide

An improved, validated HPLC assay was developed for the non-steroidal anti-inflammatory agent, nimesulide. In contrast to previous methods, the present assay requires smaller plasma volumes (0.2 ml) and utilizes a commercially available, structurally similar analogue of nimesulide, NS-398. The method involves a liquid-liquid extraction procedure that can be completed within 4 h, followed by reversed-phase HPLC analysis. Briefly, the extraction protocol required toluene extraction of acidified plasma samples, followed by back-extraction of the retained toluene phase with aqueous base. The retained aqueous alkaline phase was concentrated by toluene re-extraction. The retained toluene phase was evaporated to dryness and reconstituted with 100 microl of mobile phase. Extracted samples were injected (50 microl) onto a Shandon Hypersil BDS C18 column (5 microm particle size; 250x4.6 mm) equilibrated with 1.0 ml/min of 68:32 (v/v) methanol-citrate (0.08 M)-phosphate (0.04 M) buffer (pH 3.0) at room temperature, with detection at 240 nm. The chromatographic run time was 12 min with retention times of 5.9 min and 9.1 min for nimesulide and NS-398, respectively. The analytical method was successfully utilized for a pilot pharmacokinetic study.