Oral Presentation AUS-oMicS 2025

Enhancing Eicosanoid Stability for LC-MS Analysis Post-SPE: Investigating the Role of Ethylene Glycol (#106)

Jaya Ajay Singh 1 , Monique Ryan 1 , Manthan Sharma 1 , Robert Clegg 1 , Luke Whiley 1 , Nicola Gray 1 , Jeremy Nicholson 1
  1. Australian National Phenome Centre, Murdoch University, Murdoch, WESTERN AUSTRALIA, Australia

67ab193cd1f10-Screenshot+2025-02-11+at+5.32.35%E2%80%AFPM.pngBackground

Eicosanoids are vital lipid mediators involved in inflammation but are highly volatile

and prone to evaporation during sample preparation steps such as solid-phase

extraction (SPE). Their volatility, driven by low molecular weight, reactive functional

groups, and vacuum-induced evaporation, compromises their accurate quantification

in LC-MS analysis. To address this, ethylene glycol was evaluated as a vacuum

additive to stabilize eicosanoids during evaporation, aiming to enhance recovery and

reliability in LC-MS workflows.

 

Methodology

Solid-phase extraction (SPE) was employed to isolate and concentrate eicosanoids

from plasma samples. Six experimental conditions (Exp A–F) were tested to examine

the effects of mixing, sonication, pH adjustments, and ethylene glycol addition on

eicosanoid stability. These included controls with no additives or stabilization steps

(Exp A–C) and conditions incorporating ethylene glycol as a vacuum additive (Exp D

and Exp F) or pH adjustment (Exp E and Exp F). This systematic design allowed the

evaluation of ethylene glycol’s effectiveness in reducing volatilization and improving

stability during drying steps.

 

Results

Log-transformed LC-MS peak areas across the experimental conditions demonstrated

that Experiment D (with ethylene glycol) consistently produced the highest and most

stable peak areas for all tested eicosanoids, including 16-HETE, 5(6)-EET, and 17-

HDHA. Control experiments (Exp A and Exp B) exhibited significantly lower and more

variable peak areas, indicating substantial analyte loss during sample preparation. The

superior stabilization achieved with ethylene glycol underscores its efficacy in

preventing eicosanoid volatilization, offering a simple and effective solution to enhance

their quantification in LC-MS analysis.

 

Conclusion

Ethylene glycol proves to be a promising vacuum additive, effectively preventing lipid

mediator evaporation and ensuring reproducibility and accuracy in biomarker

quantification. This approach significantly improves the robustness and reliability of

LC-MS workflows for studying eicosanoids and oxidative stress biomarkers, with

critical implications for clinical applications.