# Overcoming Challenges in the Determination of Fatty Acid Ethyl Esters in Post-Mortem Plasma Samples with the Use of Targeted Metabolomics and the Quality by Design Approach

## TL;DR

This study develops a reliable method to measure fatty acid ethyl esters in post-mortem blood, which can help determine alcohol consumption before death.

## Contribution

A new, validated analytical method using gas chromatography and mass spectrometry for detecting fatty acid ethyl esters in post-mortem plasma is introduced.

## Key findings

- A method for quantifying six fatty acid ethyl esters was developed and validated according to ICH guidelines.
- The quality by design approach identified critical parameters affecting the method's robustness.
- Plasma concentrations of fatty acid ethyl esters correlate with pre-mortem alcohol consumption and may aid in determining cause of death.

## Abstract

Background: Excessive alcohol consumption constitutes a serious cause of death worldwide. Fatty acid ethyl esters, as metabolites of the non-oxidative elimination pathway of ethanol, have been recognized as mediators of alcohol-induced organ damage. These metabolites serve as potential biomarkers for the assessment of ethanol intake and might be also used in post-mortem studies. Methods: In this study, the development and optimization of a simple, fast, precise, accurate, and cost-effective method with the use of gas chromatography coupled with tandem mass spectrometry for quantitative analysis of six fatty acid ethyl esters, namely ethyl laurate, myristate, palmitate, linoleate, oleate, and stearate, were conducted. Results: The optimized method was fully validated according to ICH guidelines. Additionally, identification of critical method parameters was possible by using the quality by design approach. By carrying out analyses according to the Plackett–Burman plan (design of experiments methodology), the robustness of the analytical method developed was confirmed for four (ethyl palmitate, linoleate, oleate, and stearate) ethyl esters. In the case of ethyl myristate, the variable significantly affecting the results appeared to be the temperature of solvent evaporation after the deproteinization step. Conclusions: Biochemical interpretation of the obtained results with available medical records suggests that plasma concentrations of selected fatty acid ethyl esters are valuable indicators of pre-mortem alcohol consumption and may be one of the key factors helpful in determining the cause and mechanism of death.