# Species-specific optimization of oxylipin ionization in LC–MS: a design of experiments approach to improve sensitivity

## TL;DR

This paper introduces a systematic method using design of experiments to optimize the analysis of oxylipins in mass spectrometry, improving detection sensitivity and consistency.

## Contribution

The study applies a design of experiments approach to optimize oxylipin ionization, revealing species-specific parameter adjustments for improved UHPLC-MS/MS sensitivity.

## Key findings

- Response surface modeling showed distinct ionization behaviors between polar and apolar oxylipins based on interface temperature and CID gas pressure.
- Signal-to-noise ratios improved two- to four-fold for several oxylipin classes, enhancing trace-level detection.
- Species-specific adjustments to entrance/exit potentials and collision energies were necessary for optimal results.

## Abstract

Oxylipins are diverse bioactive signaling molecules, which occur in very low concentrations in complex matrices, posing challenges in achieving consistent and sensitive analysis. UHPLC-MS/MS is the preferred technique to separate and quantify these molecules, often optimized using a time-consuming trial-and-error approach. In this study, we applied the design of experiments (DoE) approach to systematically investigate the ionization properties of multiple oxylipin species. Fractional factorial and central composite designs were employed to detect relevant instrument parameters and optimize signal intensity in ESI–MS/MS analysis. Response surface modeling revealed distinct ionization and fragmentation behaviors between polar and apolar oxylipins, driven by their responses to interface temperature and collision-induced dissociation (CID) gas pressure. Particularly, prostaglandins and lipoxins benefit from higher CID gas pressure and lower temperatures compared to the lipophilic HODEs and HETEs to achieve optimal intensity in multiple reaction monitoring analysis. While global source parameters were optimized, analyte-specific entrance/exit potentials and collision energies required individual adjustments. The final method was applied to analyze seven oxylipin classes including leukotrienes, prostaglandins, lipoxins, resolvins, HETEs, HODES, and HoTrEs. Although improvements in lower limits of quantification were modest (< 1 pg on-column), signal-to-noise ratios increased two-fold for lipoxins and resolvins and three- to four-fold for leukotrienes and HETEs, enhancing detection at trace levels. This DoE-guided strategy provides a powerful tool to improve UHPLC-MS/MS analysis of oxylipins across various instrument vendors, guiding the way towards inter-laboratory comparability.

The online version contains supplementary material available at 10.1007/s00216-025-05759-6.