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.

Messenger ribonucleic acid (mRNA) has emerged as a new class of therapeutic agent with unprecedented potential. In particular, the lipid nanoparticle (LNP)-mRNA formulations enabled safe and effective delivery of mRNA in vivo, leading to the success of mRNA vaccines during the COVID-19 pandemic. To control the potential misuse of LNP-mRNA agents in equine sports, a simple and sensitive method has been developed for the detection of ionisable lipids in equine plasma using supported liquid extraction (SLE) followed by liquid chromatography-high-resolution tandem mass spectrometry. To overcome the significant ionisable lipid-protein binding, plasma was diluted with isopropanol and heptane prior to SLE. This preparation technique yielded rapid and efficient recovery of a selection of nine candidate ionisable lipids and derivatives. Method validation showed adequate sensitivity and robustness, achieving estimated limits of detection as low as 5 pg/mL. The method has been successfully applied for the detection of ALC-0315 in plasma samples from a horse administered with LNP-mRNA. To the best of our knowledge, this is the first report of a detection method for the screening of ionisable lipids in equine biological samples, providing a practical tool for doping control of mRNA agents.