A sensitive internal standard method for the determination of melatonin in mammals using precolumn oxidation reversed-phase high-performance liquid chromatography

A sensitive and accurate internal standard method to determine melatonin in mammalian tissues and physiological fluids has been described. This method includes the oxidation of melatonin to a highly fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), and the determination of 6-MOQMA by a reversed-phase HPLC system. For the accurate and reliable determination, several melatonin analogs were designed and utilized as the internal standards, and ethyl and isopropyl analogs (having the corresponding alkyl group via the amide bond of melatonin instead of the methyl group) were found to be promising. Using these internal standards, highly accurate and sensitive determination could be accomplished using rat pineal gland samples, and the clear circadian rhythms are demonstrated. This method was also successfully applied to the determination of melatonin in a small amount (20 μL) of human saliva.