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Rapid communications in mass spectrometry : RCM2024; 38(17); e9856; doi: 10.1002/rcm.9856

Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry.

Abstract: To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. Methods: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. Results: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. Conclusions: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.
© 2024 John Wiley & Sons Ltd.
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Publication Date: 2024-07-01 PubMed ID: 38945695DOI: 10.1002/rcm.9856Google Scholar: Lookup
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  • Journal Article

Summary

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This research details the development of an enhanced screening method for detecting the use of prohibited substances in horses, particularly for horse racing and equestrian sports. The method is capable of detecting over 1000 compounds in both positive and negative ion modes and has proven successful in identifying prohibited substances in both overseas and domestic horse urine samples.

Methodology

  • Research revealed a comprehensive procedure involving a few steps which starts with enzymatic hydrolysis of 3 mL of horse urine samples. This process entails breaking down complex structures in the urine into simpler substances.
  • Post hydrolysis, the samples undergo a solid-phase extraction process using a HF Bond Elut C18 cartridge. This step is crucial for isolating specific substances from the sample.
  • Once the target substances are isolated, they are then separated on a C18 reversed-phase column. This process is carried out to further isolate the substances based on their chemical properties.
  • The final detailed analysis of these separated substances is carried out using a technique called liquid chromatography/high-resolution mass spectrometry (LC/HRMS). Liquid chromatography separates the mixture and the high-resolution mass spectrometry helps identify. It is conducted in both positive and negative modes in two separate injections.
  • The entire screening process, from sample isolation to identification, is conducted in an 11-minute LC run.

Results

  • This developed method has the capacity to detect 1011 compounds in horse urine samples in both positive and negative ion modes. These accurate and threshold sensitive screenings are incredibly important in anti-doping laboratories.
  • Over 95% of the target compounds have detection limits (LODs) of 10 ng/mL or less, while over half have LODs of 0.5 ng/mL or less. The lowest recorded LOD achieved in this study was 0.01 ng/mL, indicating a high detection sensitivity.
  • This method proved its utility by accurately detecting prohibited substances in both overseas and domestic equine urine samples.

Conclusions

  • The researchers successfully developed a regular screening method that can detect more than 1000 compounds at sub-ppb (parts per billion) levels in both positive and negative ion modes.
  • This method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.
  • These advancements in screening methods are crucial to ensure and maintain the integrity of horse racing and equestrian sports by effectively preventing the use of banned substances and doping strategies.

Cite This Article

APA
Uchida T, Kisugi T, Ishii H, Yamada M, Kinoshita K, Leung GN. (2024). Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry. Rapid Commun Mass Spectrom, 38(17), e9856. https://doi.org/10.1002/rcm.9856

Publication

Rapid communications in mass spectrometry : RCM
ISSN: 1097-0231
NlmUniqueID: 8802365
Country: England
Language: English
Volume: 38
Issue: 17
Pages: e9856

Researcher Affiliations

Uchida, Taiga
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.
Kisugi, Takaya
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.
Ishii, Hideaki
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.
Yamada, Masayuki
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.
Kinoshita, Kenji
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.
Leung, Gary Ngai-Wa
  • Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan.

MeSH Terms

  • Animals
  • Horses / urine
  • Doping in Sports / prevention & control
  • Limit of Detection
  • Chromatography, Liquid / methods
  • Substance Abuse Detection / methods
  • Substance Abuse Detection / veterinary
  • Mass Spectrometry / methods
  • Solid Phase Extraction / methods
  • Reproducibility of Results

Grant Funding

  • Japan Racing Association

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Citations

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