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Analytical and bioanalytical chemistry2026; doi: 10.1007/s00216-026-06556-5

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

Abstract: 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.
© 2026. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.
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Publication Date: 2026-05-14 PubMed ID: 42135550PubMed Central: 5623340DOI: 10.1007/s00216-026-06556-5Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research developed a highly sensitive liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to detect specific ionizable lipids in horse plasma, which serve as markers for gene doping using lipid nanoparticle (LNP) delivery systems.
  • The method enables surveillance for illicit use of mRNA therapies in horse racing and equestrian sports by detecting key LNP components long after administration.

Introduction and Background

  • Lipid nanoparticles (LNPs) are crucial for delivering mRNA-based therapeutics, including vaccines.
  • Due to their effective delivery properties, LNPs pose a risk of misuse as gene-doping agents in equine sports.
  • Gene doping involves the use of genetic material to enhance performance, which is prohibited in horse racing and equestrian competitions.
  • Detecting LNP components in plasma could provide indirect markers or “surrogate markers” indicating gene doping.

Study Objective

  • The study aimed to develop and validate a method for detecting three clinically important ionizable lipids used in LNP formulations: ALC-0315, SM-102, and DLin-MC3-DMA (MC3).
  • These lipids are integral to approved RNA therapeutics and vaccines, making their detection relevant for identifying illegal gene doping.

Methods

  • The researchers employed liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) using Orbitrap technology, known for its high sensitivity and specificity.
  • Equine plasma samples underwent a modified Bligh and Dyer extraction—a standard lipid extraction technique optimized here for isolating the targeted lipids.
  • Limits of detection (LODs) and limits of identification (LOIs) were established based on guidelines from the Association of Official Racing Chemists (AORC).
  • LOD values achieved were:
    • ALC-0315: 0.05 ng/mL
    • SM-102: 0.01 ng/mL
    • DLin-MC3-DMA (MC3): 0.05 ng/mL
  • LOI values ranged from 0.05 to 0.1 ng/mL for all three lipids.

In Vivo Application and Results

  • The method was tested in a practical gene-doping scenario by administering a horse with LNP-encapsulated N1-methylpseudouridine-modified mRNA encoding erythropoietin (EPO), a well-known performance-enhancing target.
  • Among the three lipids, SM-102 was confirmed to be detectable for up to 4 days post-administration and remained detectable in plasma up to 14 days.
  • This establishes a relatively long detection window for the presence of synthetic LNP components, offering a tool for extended monitoring.

Significance and Novelty

  • This study is the first to describe an LC-MS based strategy to unambiguously identify ionizable lipids as surrogate markers for LNP-mediated gene doping in horses.
  • The extended detection window (up to 14 days) significantly improves the capability of equine anti-doping laboratories to monitor for illicit gene doping.
  • By focusing on LNP components rather than the mRNA itself, detection becomes more reliable given the potential instability or rapid clearance of the nucleic acids.

Conclusion

  • The developed LC-HRMS method provides a valuable diagnostic tool for equine doping control by enabling sensitive, specific, and long-term detection of ionizable lipid components indicative of LNP-based gene doping.
  • The approach supports fair competition and effective regulation in horse racing and equestrian sport by addressing emerging gene-doping threats.

Cite This Article

APA
Shimizu Y, Furukawa R, Kikuchi M, Sugai-Bannai M, Hirano-Kodaira M, Tozaki T, Leung GN. (2026). LC-HRMS detection of three ionizable lipids in equine plasma: surrogate markers for long-term LNP-mediated gene-doping surveillance. Anal Bioanal Chem. https://doi.org/10.1007/s00216-026-06556-5

Publication

Analytical and bioanalytical chemistry
ISSN: 1618-2650
NlmUniqueID: 101134327
Country: Germany
Language: English

Researcher Affiliations

Shimizu, Yoshibumi
  • Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan. y-shimizu@lrc.or.jp.
Furukawa, Risako
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Sugai-Bannai, Michiko
  • Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Hirano-Kodaira, Misato
  • Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Leung, Gary Ngai-Wa
  • Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.

Conflict of Interest Statement

Declarations. Ethical approval: The administration protocols were approved by the Animal Care and Use Committees of the Japan Racing Association (JRA) Equine Research Institute (Review no. 25-9) and the Laboratory of Racing Chemistry (Review no. 20-4). All experiments were performed in accordance with the relevant guidelines and regulations. Conflict of interest: The authors declare no competing interests.

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