Rapid determination of nimesulide in rabbit aqueous humor by liquid chromatography

A rapid method was developed for quantification of nimesulide (methanesulfonamide, N-[4-nitro-2-phenoxyphenil]) in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C 18 reversed-phase column (Ultracarb ODS) with UV detection at 300nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine (TEA) adjusted to pH 3.2 with orthophosphoric acid. The retention time was 4.5min. A simple pre-treatment with acetonitrile was used to deproteinize aqueous humor samples. The limit of quantitation was 50ng/ml. The recovery was over 90%. The relationship between peak areas and concentration was linear over the range between 0.05 and 2.5μg/ml, with r2 values over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of nimesulide