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

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.