Reversed‐phase high‐performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors. Issue 12 (29th April 2014) - Maccallini, Cristina
Abstract Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of l‐[3H]‐citrulline produced from l ‐[3H]‐arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed‐phase high‐performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of l‐citrulline with o‐phthaldialdehyde/N‐acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N‐[3‐(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well‐established radiometric assay.