A novel LC-MS/MS assay for the simultaneous determination of melatonin and its two major metabolites, 6-hydroxymelatonin and 6-sulfatoxymelatonin in dog plasma: Application to a pharmacokinetic study.

Abstract A convenient and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of melatonin (MEL) and its major metabolites, 6-hydroxymelatonin (6-O-MEL) and 6-sulfatoxymelationin (S-O-MEL) in dog plasma. After plasma samples were deproteinized with acetonitrile, the post-treatment samples were analyzed on a Phenomenex Kinetex C18 column (50 × 2.1 mm, 1.7 μm) interfaced with a triple quadrupole tandem mass spectrometer. Electrospray ionization mode (ESI) and multiple reaction monitoring were used to assay MEL and its metabolites. Acetonitrile and 5 mM ammonium acetate were used as the mobile phase with a gradient elution at a flow rate of 0.2 mL/min. The analytical run time of 6.5 min was divided into two periods according to ionization mode. S-O-MEL was monitored in negative ionization mode (period 1), while MEL and 6-O-MEL were detected in positive ionization mode (period 2). All calibration curves showed good linearity ( r  > 0.991) over the concentration range with a lower limit of quantification (LLOQ) of 0.02 ng/mL for MEL, 0.04 ng/mL for 6-O-MEL and 0.50 ng/mL for S-O-MEL. The intra- and inter-day precision was within 13.5% in terms of relative standard deviation (RSD%) and the accuracy within 13.0% in terms of relative error. This convenient and specific LC–MS/MS method was successfully applied to the pharmacokinetic study of MEL and its metabolites in Beagle dogs after an oral dose of 2.0 mg MEL. After ingestion of MEL, S-O-MEL was the predominant component circulating in blood. 6-O-MEL showed similar pharmacokinetic profile to that of MEL.