Development of a fully-automated on-line oxidation column-switching HPLC system for the determination of endogenous melatonin in human clinical samples

A fully-automated on-line oxidation column-switching HPLC system has been developed for the determination of endogenous melatonin in human plasma and saliva. This HPLC system consists of four processes. In the first step, melatonin is fractionated using a reversed-phase C4 column (Proteonavi, 75 mm × 1.0 mm i.d.). In the second step, the obtained melatonin fraction is on-line collected, and oxidized to a highly-fluorescent compound, N-[(6-methoxy-4-oxo- 1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), by mixing with hydrogen peroxide under alkaline conditions. In the third step, the produced 6-MOQMA is concentrated, and the oxidation reagents are removed using an alkaline resistive reversed-phase column, Asahipak ODP (35 mm × 1.0 mm i.d.). In the final fourth step, the 6-MOQMA is determined by a microbore-ODS column packed with ultrafine particles (CAPCELL PAK C18 IF, 250 mm × 1.0 mm i.d.). The limit of detection of melatonin using this system is about 200 amol/injection, and the determination of endogenous melatonin in a small volume of human physiological fluids, such as 100 μL of plasma and 300 μL of saliva, was successfully accomplished.