Determination of exogenous melatonin and its 6-hydroxy metabolite in humanplasma by liquid chromatography-mass spectrometry

Melatonin has recently garnered interest as a possible treatment for sleep disorders, and this has created a desire for appropriate pharmacokinetic st udies. No method has yet been published that can measure the concentrations of both melatonin and its main metabolite, 6-hydroxymelatonin, in plasma o r serum. Therefore, a liquid chromatogaphy-mass spectrometry (LC/MS) method including enzymatic hydrolysis and one-step liquid-liquid extraction was d eveloped for this purpose. The mean extraction recovery was 59% for melaton in and 42% for 6-hydroxymelatonin. The mean precision of the method as calc ulated from the interassay coefficient of variation of quality control samp les at high and low concentrations was 18% for 6-hydroxymelatonin and 15% f or melatonin. The inaccuracy was always less than 2%. The limit of detectio n was approximately 2 ng/mL for 6-hydroxymelatonin (signal/noise ratio = 4) and less than 2 ng/mL for melatonin (signal/noise ratio at 2 ng/mL = 10). The method was applied to the analysis of plasma from a healthy volunteer w ho had received a single oral dose of 25 mg melatonin.