Direct LC-HRMS/MS characterisation and RT-qPCR detection for potential mRNA-based doping agent

Messenger ribonucleic acid (mRNA) therapeutics offer unprecedented potential for in vivo protein expression but raise emerging risks of misuse for gene doping. We investigated an online-available product purported to be codon-optimised, 5-methoxyuridine-substituted human erythropoietin (EPO) mRNA and developed a corresponding detection assay for equine anti-doping. Product identity was verified using a mass spectrometry (MS)-based bottom-up workflow comprising RNase 4 digestion, liquid chromatography-high-resolution tandem MS analysis, and automated sequence mapping against an mRNA database of potential doping genes. This workflow enabled direct detection of base modifications and achieved mean sequence coverage above 74%, verifying the mRNA sequence and its chemical composition. Method optimisation shows that partial RNase 4 digestion with a 10-min incubation time produced more consistent coverage by generating longer, informative oligoribonucleotides. The enhanced consistency would be advantageous for single-analysis scenarios typical of doping investigations. Building on the verified product identity, we developed a corresponding reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for equine plasma. We explored the simple use of naked mRNA for matrix spiking as a reference material for doping control analysis. The method was validated with a limit of detection at 1250 copies/mL of EPO mRNA in equine plasma. In addition to EPO mRNA, the combined MS and RT-qPCR approach provides a practical framework that can be extended for surveillance of emerging mRNA-based agents in sport.