Detection of ionisable lipids in equine plasma by supported liquid extraction and liquid chromatography-high-resolution tandem mass spectrometry for doping control of mRNA agents.

Overview

• This research article describes the development of a sensitive laboratory method to detect ionisable lipids in horse blood plasma, which are key components of lipid nanoparticle (LNP)-mRNA drugs, aiming to control doping misuse in equine sports.
• The method uses supported liquid extraction combined with liquid chromatography and high-resolution tandem mass spectrometry to identify these lipids with high sensitivity and robustness.

Introduction and Background

• mRNA therapies have recently gained prominence, especially due to the success of COVID-19 vaccines delivered via lipid nanoparticles (LNPs).
• LNPs contain ionisable lipids which facilitate the delivery of mRNA into cells.
• These advanced therapies pose a doping risk in horse racing and equestrian sports, as they could be misused to enhance performance.
• Detecting such doping agents requires methods sensitive enough to identify trace amounts of ionisable lipids in biological samples such as plasma.

Technical Challenges

• Ionisable lipids bind strongly to plasma proteins, making their extraction and detection difficult.
• Traditional extraction methods did not efficiently separate these lipids from proteins in plasma.

Methods Developed

• Sample Preparation:
  • Plasma was pre-treated by dilution with isopropanol and heptane to disrupt lipid-protein binding.
  • This dilution improved the availability of ionisable lipids for extraction.
• Supported Liquid Extraction (SLE):
  • Aided in rapid and efficient isolation of ionisable lipids from the plasma matrix.
• Analytical Detection:
  • Liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) was employed for precise identification and quantification of nine candidate ionisable lipids and their derivatives.
  • This technique allowed very low detection limits, as low as 5 pg/mL.

Results

• The method provided robust separation and reproducible detection of the targeted ionisable lipids.
• Validated to be sensitive enough for doping control purposes, supporting the detection of extremely low concentrations in plasma.
• Successfully applied to real samples: detected the ionisable lipid ALC-0315 from a horse administered with LNP-mRNA formulation.
• This confirms practical applicability in testing for doping agents in veterinary sports contexts.

Significance and Novelty

• This is the first reported method specifically targeting ionisable lipids in equine plasma.
• It provides a practical detection tool for regulators to screen for unauthorized use of mRNA agents in horses.
• Represents an important advancement in doping control science adapted to new biotechnologies like mRNA-LNP therapeutics.
• Supports integrity and fairness in equine sports by monitoring emerging drug classes.