Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors

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-[(3)H]-citrulline produced from L-[(3)H]-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.