# Comprehensive Analysis of Different Subtypes of Oxylipins to Determine a LC–MS/MS Approach in Clinical Research

## TL;DR

This study develops a reliable method to detect different oxylipin subtypes in blood samples, which could help identify biomarkers for heart disease.

## Contribution

A robust aSPE–LC–MS/MS platform was developed for comprehensive oxylipin analysis with high sensitivity and specificity.

## Key findings

- Oxo-oxylipins, resolvin, and eicosanoids show best extraction under SPE protocol.
- 5-HETE, 11-HETE, and 15-HETE are effective biomarkers for coronary heart disease.
- The aSPE–LC–MS/MS method offers wide coverage, high sensitivity, and low matrix effects.

## Abstract

Background/Objectives: Different oxylipin subtypes have unique biological properties, requiring effective analytical protocols. However, establishing a complete pathway detection protocol for comprehensive oxylipin analysis is challenging. This study aimed to evaluate the adaptability and specificity of oxylipin subtypes under different extraction schemes and to develop a robust analytical platform for clinical biomarker investigation. Methods: We revealed the adaptability and specificity of oxylipin subtypes based on different single-step extraction schemes. A high-throughput quantitative automated solid-phase extraction coupled with a liquid chromatography–tandem mass spectrometry (aSPE–LC–MS/MS) analytical platform was established for a broad panel of complex oxylipins. The method was applied to serum samples of patients with coronary heart disease (CHD). Results: Our results verified that oxo-oxylipins, resolvin, and eicosanoids showed the best extraction efficiency under SPE protocol. Most hydroxy-oxylipins, dihydroxy-oxylipins, and HOTrEs are suitable for methanol protocol, HDHA for acetonitrile protocol, and epoxy-oxylipins for the methyl tert-butyl ether protocol, while medium-chain HETE is suitable for ethyl acetate protocol. Importantly, a novel sensitive fast method with wide coverage by the aSPE–LC–MS/MS analytical platform with satisfying sensitivity, accuracy and precision, extraction efficiency, low matrix effect, and linear calibration curves was obtained. Furthermore, we have successfully applied this method and found that 5-HETE, 11-HETE, and 15-HETE can serve as integrated biomarkers for patients with CHD, with high diagnostic performance. Conclusions: The study provides the best protocol for the clinically targeted detection of oxylipins and provides an important means for studying biomarkers of diseases.