Reversed‐phase high‐performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors |
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Authors: | Cristina Maccallini Mauro Di Matteo Alessandra Ammazzalorso Alessandra D'Angelo Barbara De Filippis Sara Di Silvestre Marialuigia Fantacuzzi Letizia Giampietro Assunta Pandolfi Rosa Amoroso |
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Affiliation: | 1. Dipartimento di Farmacia, Università degli Studi “G. d’Annunzio”, Chieti, Italy;2. Dipartimento di Scienze Sperimentali e Cliniche, Centro Scienze dell’Invecchiamento, Ce.S.I.,‘Fondazione Università G. d’Annunzio’, Università degli Studi “G. d’Annunzio”, Chieti, Italy |
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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. |
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Keywords: | l‐Citrulline Fluorescence detection High‐performance liquid chromatography Inhibitors Nitric oxide synthases |
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