LC-HRMS detection of three ionizable lipids in equine plasma: surrogate markers for long-term LNP-mediated gene-doping surveillance.

Lipid nanoparticles (LNPs) are essential delivery vehicles for messenger ribonucleic acid (mRNA)-based therapies. However, their potential misuse as gene-doping agents in horse racing and equestrian sports necessitates robust analytical surveillance. This study presents a highly sensitive, Orbitrap technology-driven liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous detection of three clinically relevant ionizable lipids: ALC-0315, SM-102, and DLin-MC3-DMA (MC3), which are critical components of approved RNA therapeutics and vaccines. Following a modified Bligh and Dyer extraction from equine plasma, the analytes were detected with high sensitivity. Based on the applicable criteria in the Association of Official Racing Chemists guidelines, the limits of detection were 0.05, 0.01, and 0.05 ng/mL for ALC-0315, SM-102, and MC3, respectively. By comparison, their limits of identification (LOIs) ranged from 0.05 to 0.1 ng/mL. The applicability of the method was demonstrated in an in vivo administration study. In this study, a horse was administered LNP-encapsulated N1-methylpseudouridine-modified mRNA encoding erythropoietin. The result revealed that SM-102 was confirmable for up to 4 days, remaining detectable in plasma for up to 14 days post-administration. To the best of our knowledge, this is the first LC-MS-based strategy for the unequivocal identification of ionizable lipids as surrogate markers for LNP-mediated gene doping. This approach offers the longest known detection windows for LNP components in equine plasma, significantly enhancing the diagnostic capabilities of equine antidoping laboratories.