ESTIMATION OF NITRIC-OXIDE PRODUCTION BY MACROPHAGES WITH L-CITRULLINE DETERMINATION USING HPLC

Determination of L-citrulline (Cit) by high performance liquid chromat ography (HPLC) using an on-column derivatization and a column-switchin g technique has been studied for estimation of nitric oxide (NO) produ ction by macrophages. The reaction system consisted of a reversed-phas e polymer-gel column (Asahipak ODP-50) and the eluant of alkaline bera te buffer containing o-phthalaldehyde (OPA) and N,N-dimethyl-2-mercapt o-ethylamine hydrochloride. The OPA-fluorophore formed was subsequentl y transferred to an analytical column (Asahipak ODP-50) using the colu mn-switching system with the mobile phase of acetonitrile and alkaline berate buffer. Optimization of the HPLC conditions led to a simple an d rapid analytical method without any purification