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Background: L-arginine and its metabolites such as L-citrulline play an important role in the functions of biology, many of the arginine metabolites and arginine itself participate the regulation of arginine pathways to cope with pathophysiological conditions.To explore the correlation of their quantity in vivo and biological function, it is necessary to determine the L-arginine and L-citrulline in plasma simultaneously.Therefore, we developed a method for simultaneously determining L-arginine and L-citrulline in plasma using reversed phase high performance liquid chromatography.(RP-HPLC).