Determination of Melatonin in Plasma by Liquid-liquid Extraction and High Performance Liquid Chromatography Coupled with Fluorescence Detection and Its Application for Melatonin Pharmacokinetic Study in Humans

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Nuttawut Jenjirattithigarn
Katcharin Phunikom
Thachanan Kongpan
Sirimas Kanjanawart
Jeffrey Roy Johns
Wichittra Tassaneeyakul


Melatonin is a neurohormone secreted from the pineal gland which regulates the circadian rhythm in human. To date, this compound has been used as a food supplement as well as a chemotherapy adjuvant for several types of cancer. However, data on the pharmacokinetics of melatonin at therapeutic doses is still limited. This study aimed to develop and validate a high performance liquid chromatography method for determination of melatonin in human plasma for purposes of its application to pharmacokinetic study after a therapeutic dose. Plasma melatonin was extracted with dichloromethane under alkaline conditions. A mobile phase consisting of 10 mM phosphate buffer: acetonitrile (80:20), pH 7.2 and a reverse phase, C18 column as well as a fluorescence detector were used. The excitation and emission wavelength were set at 286 and 346 nm and the flow rate of the mobile phase was set at 1 mL/min. Under these chromatographic conditions, the retention times for 5-methoxytryptamine, the internal standard, and melatonin, were 3.5 and 5.5 min, respectively. No interferences from endogenous plasma compounds were recorded at the retention times of the analytes. The lowest limit of quantitation of melatonin was 0.1 ng/mL. The good linearity of the calibration at the range 0.1–35 ng/mL was obtained with the correlation coefficient of 0.99. The inter-day and intra-day accuracy as well as precision were within the acceptance limits. This assay method was successfully applied for pharmacokinetic study of 20 mg oral melatonin dose in male healthy Thai volunteers fasting condition.


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