Work

Protocol for the quantification of melatonin and AFMK in cerebrospinal fluid by LC-MS/MS

Year: 2025

Type: article

Abstract: Melatonin and its oxidant derivative, N-acetyl-N-formyl-5-methoxykynuramine (AFMK), are indolamines present in higher organisms, which are related to light-darkness circadian rhythms and redox physiology. Here, we present a protocol for the quantification of both in cerebrospinal fluid (CSF). We describe steps for standardization by isotopic dilution or alternatively by internal standard 5-methoxytryptophol (5-MT). We then detail procedures for quantification by using liquid chromatography coupled to tandem mass spectrometry. For complete details on the use and execution of this protocol, please refer to Artime-Naveda et al. 1 • Steps to determine melatonin and AFMK by isotopic dilution and internal standardization • Steps for liquid chromatographic separation of melatonin and derivatives • Details on CSF sample extraction and preparation for mass spectrometry analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Melatonin and its oxidant derivative, N-acetyl-N-formyl-5-methoxykynuramine (AFMK), are indolamines present in higher organisms, which are related to light-darkness circadian rhythms and redox physiology. Here, we present a protocol for the quantification of both in cerebrospinal fluid (CSF). We describe steps for standardization by isotopic dilution or alternatively by internal standard 5-methoxytryptophol (5-MT). We then detail procedures for quantification by using liquid chromatography coupled to tandem mass spectrometry.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104145