Determination of L-citrulline (Cit) by high performance liquid chromatography (HPLC) using an on-column derivatization and a column-switching technique has been studied for estimation of nitric oxide (NO) production by macrophages. The reaction system consisted of a reversed-phase polymer-gel column (Asahipak ODP-50) and the eluant of alkaline borate buffer containing ο-phthalaldehyde (OPA) and N, N-dimethy1-2-mercapto-ethylamine hydrochloride. The OPA-fluorophore formed was subsequently transferred to an analytical column (Asahipak ODP-50) using the column-switching system with the mobile phase of acetonitrile and alkaline borate buffer. Optimization of the HPLC conditions led to a simple and rapid analytical method without any purificatio nprocedure for the simultaneous determination of Cit and L-arginine (Arg), a precursor of Cit, in a macrophage culture supernatant. The detection limit was 5 μM for both Cit and Arg in the culture supernatant. The proposed method was successfully applied to the screening of foods for the estimation of nitric oxide production by macrophages.
