Comparative Elimination Analysis of Lipid Nanoparticle‐Encapsulated Human Erythropoietin mRNA by Multimodal Approaches Suggests an Effective Doping Control Strategy for mRNA‐Based Agents

Illicit use of messenger ribonucleic acid (mRNA) agents represents an imminent threat to gene doping control in equine sports. The effectiveness and safety of lipid nanoparticle-encapsulated mRNA agents (LNP-mRNA), which have been well-proven during COVID-19 pandemic, have resulted in prominent growth of the interest on LNP-mRNA agents for expressing desired proteins in vivo. The vast number of potential performance-enhancing mRNAs has demanded cost-effective and reliable methods for their detections. This study employed a multimodal analysis approach to detect an LNP-mRNA product ("LNP-epo" hereafter) arising from encapsulation of a black-market product of human erythropoietin (hEPO) mRNA. Lipid nanoparticle (LNP) components were detected by liquid chromatography/high-resolution tandem mass spectrometry after supported liquid extraction; hEPO mRNA was detected by reverse transcription quantitative polymerase chain reaction after RNA extraction; hEPO protein was detected by sandwich enzyme-linked immunosorbent assay. Comparative elimination analysis of LNP-epo in blood samples collected from an administered horse showed the longest detection time achieved by LNP monitoring (at least 34-day postadministration), followed by hEPO mRNA detection (up to 30 days in whole blood; up to 17 days in plasma), and shortest by hEPO protein detection (up to 3 days). Together with its low analysis cost, broad coverage of LNP components, expandable scope and independence from mRNA sequence modifications and the need for specific reagents (e.g., primers, antibodies), the detection of LNP can be a fit-for-purpose screening method for monitoring the misuse of LNP-mRNA in equine sports. The other two approaches could provide insight on the intentional effect elicited by LNP-mRNA.