Simultaneous Determination of Melatonin and its Active Metabolite in Rat Plasma by LC-MS/MS: Application to Pharmacokinetic Study

A selective liquid chromatographic-mass spectrometric method (LC-MS/MS) has been established and validated for simultaneous determination of melatonin and its active metabolite in human plasma. Plasma samples were extracted by one step protein precipitation procedure and separated on a Acquity BEH C18 column (2.1 x 50 mm, 1.7 in particle size), with acetonitrile-0.1% formic acid solution as mobile phase at flow rate of 0.4 ml/min. The linear range was 1-500 ng/mL for melatonin and 0.1-50.0 ng/mL for its active metabolite, with lower limit of quantitation of 1 ng/mL and 0.1 ng/mL, respectively. Intra- and inter day precision and accuracy met the requirements, with the extraction recoveries 90.99-94.49% and 86.01-9432% for the two analytes, respectively. The validated method was successfully applied to a pharmacokinetic study of melatonin and its active metabolite in rat after oral administration and intravenous of melatonin.