Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide . The resultant fluorophore was excited at 247 nm and the emission waveleng th was 384 nm. The Stokes shift was 137 nm, which was longer than that of m elatonin itself (lambda(ex) 280 nm, lambda(em) 330 nm). The melatonin deriv ative was separated by reversed-phase HPLC in about 15 min and the calibrat ion curve was linear from 500 amol to 5 pmol (r>0.999) with the detection l imit of 500 amol (S/N=5). The sensitivity of this method was about ten time s higher than that of previous methods. Both the day-to-day precision and w ithin-day precision were about 5%, and the derivative of melatonin in the a queous solution was stable for more than 10 days. This method was successfu lly applied to the determination of melatonin in rat pineal gland. (C) 1999 Elsevier Science B.V. All rights reserved.