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Home / Equine Research Bank / BMC Res Notes / Detection of phosphorothioated (PS) oligonucleotides in hors...
BMC research notes2018; 11(1); 770; doi: 10.1186/s13104-018-3885-5

Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control.

Abstract: Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common. Results: We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1-200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.
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Publication Date: 2018-10-29 PubMed ID: 30373660PubMed Central: PMC6206624DOI: 10.1186/s13104-018-3885-5Google Scholar: Lookup
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  • Journal Article

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.

The research presents a method for detecting illegal use of gene therapy (gene doping) in horse racing through specific detection of phosphorothioated oligonucleotides (PSOs) in horse plasma using liquid chromatography-tandem mass spectrometry (LC/MS/MS).

Introduction and Background

  • Gene therapy has made advances in the field of veterinary medicine, but illegal usage, known as gene doping, is a growing concern in horse racing.
  • The International Federation of Horseracing Authorities classified the use of oligonucleotides and their analogues as a form of gene therapy in 2017.
  • Most oligonucleotide therapeutics developed for humans consist of modified nucleotides for increased stability, with phosphorothioate (PS) modification being quite common.
  • It has become an urgent need to develop therapeutic nucleotide-detection techniques.

Research Methodology and Results

  • The researchers proposed a method for detecting PSOs using LC/MS/MS.
  • PSOs produce a specific product ion (m/z 94.9362) derived from PS. Since PS isn’t derived from substances within the animal’s body, this ion serves as an ideal marker for detecting PSOs.
  • Through this approach, they managed to achieve reproducible analysis for identifying specific substances, with a limit of detection (LOD) of 0.1 ng/mL, and a quantification range of 0.1 – 200 ng/mL in deproteinated plasma.
  • Moreover, the researchers could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix in non-target analyses.
  • The results suggested the viability of PSOs detection in horse blood by targeting the product ion using LC/MS/MS.

Conclusion and Implications

  • This work establishes a potentially viable method for detecting gene doping, an illegal practice in horse racing, through specific detection of PSOs in horse plasma.
  • The technique could be pivotal in maintaining the integrity and fairness of horse racing, and may also pave the way for similar detection techniques in other areas of sport.

Cite This Article

APA
Tozaki T, Karasawa K, Minamijima Y, Ishii H, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. (2018). Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control. BMC Res Notes, 11(1), 770. https://doi.org/10.1186/s13104-018-3885-5

Publication

BMC research notes
ISSN: 1756-0500
NlmUniqueID: 101462768
Country: England
Language: English
Volume: 11
Issue: 1
Pages: 770

Researcher Affiliations

Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan. ttozaki@lrc.or.jp.
Karasawa, Kaoru
  • AB Sciex, 4-7-35 Kitashinagawa, Shinagawa-ku, Tokyo, 140-0001, Japan. Kaoru.Karasawa@sciex.com.
Minamijima, Yohei
  • Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Ishii, Hideaki
  • Drug 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.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Hirota, Kei-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Kusano, Kanichi
  • Racehorse Hospital Ritto Training Center, Japan Racing Association, 1028 Misono, Ritto, Shiga, 520-3085, Japan.
Nagata, Shun-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.

MeSH Terms

  • Animals
  • Blood Chemical Analysis / veterinary
  • Chromatography, Liquid
  • Doping in Sports
  • Genetic Therapy
  • Horses / blood
  • Phosphorothioate Oligonucleotides / blood
  • Plasma / chemistry
  • Tandem Mass Spectrometry

Grant Funding

  • Japan Racing Association (2017-2019) / Japan Racing Association

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Citations

This article has been cited 6 times.
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  3. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control. Genes (Basel) 2020 Apr 23;11(4).
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  4. Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control. Genes (Basel) 2019 Mar 21;10(3).
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