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

## TL;DR

This paper provides a detailed protocol for measuring melatonin and its derivative AFMK in cerebrospinal fluid using advanced mass spectrometry techniques.

## Contribution

The paper introduces a standardized method for quantifying melatonin and AFMK in CSF using isotopic dilution or internal standardization.

## Key findings

- The protocol includes steps for isotopic dilution and internal standardization using 5-methoxytryptophol.
- It outlines procedures for liquid chromatographic separation and sample preparation for mass spectrometry analysis.

## 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

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.