Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pi neal gland was performed using an RP-HPLC system. Melatonin was separated f ollowing precolumn derivatization and determined with a fluorescence detect or at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melat onin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 mu l). The detection limit of added melaton in was 1 fmol (S / N = 5). Repeatability and day-to-day precision for the m elatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respec tively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, becaus e pineal melatonin of many inbred mouse strains has been reported to be low er than the detection limit. (C) 2000 Academic Press.