Synthesis of internal labeled standards of melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine for their quantification using an on-line liquid chromatography-electrospray tandem mass spectrometry system

Melatonin (N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D-3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D-3) was obtained after photooxidation of melatonin-D-3. The predominant ion [M + H](+) in the full scan mass spectra of melatonin, melatonin-D-3, AFMK and AFMK-D-3 were located, respectively, at m/z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D-3 (loss of the N-acetyl group), and at m/z = 178 for AFMK and AFMK-D-3 (loss of both the N-acetyl and the N-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D-3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D-3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.