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Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control.
Abstract: High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 reversed-phase column using mobile phases of 5 mM ammonium formate and acetonitrile. A 7.5 min LC gradient was employed to elute substances and results indicated that the LC method generated sharp and symmetric chromatographic peaks. An in-house equine doping compound database and a spectral library were built to increase method specificity for substances of interest. Five criteria, i.e. accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions (compound database) and HRMS/MS spectra (spectral library), were employed for targeted screening. We utilized these criteria to validate targeted detection of 451 substances within our in-house equine doping compound database. By using all five criteria in screening, the false screening positive rate is significantly reduced. A screening strategy and a Microsoft Excel macro were developed to facilitate interpretation and reporting of results. As the simultaneous acquisition of the full scan HRMS data provides the opportunity for retrospective non-targeted analysis, our findings highlight the use of this novel methodology as a simple, rapid, and reliably reproducible strategy to meet the challenge of identifying an increasing number of doping substances that could potentially impact the integrity of the horse racing community.
Publication Date: 2021-03-12 PubMed ID: 33710179DOI: 10.1039/d0ay02297gGoogle Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article details the development and validation of a advanced screening method for doping in equine sports using a hybrid high-resolution mass spectrometry (HRMS) technique, potentially enhancing the integrity of horse racing by identifying prohibited substances.
Utilization of High-Resolution Mass Spectrometry
- The research study focused on the application of high-resolution mass spectrometry (HRMS), a potent tool for analyzing substances within a sample. Specifically, a hybrid Orbitrap-based HRMS instrument was used, offering both targeted (specific substances) and non-targeted (non-specific substances) screening analyses.
Method Development and Validation
- A liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) method was developed to detect prohibited substances in horse doping. It involved recovering substances from equine plasma using liquid-liquid extraction (LLE), followed by separation on a C18 reversed-phase column.
- The testing team confirmed the method’s effectiveness, noting that it yielded sharply defined and symmetric chromatographic peaks.
Use of Doping Compound Database and Spectral Library
- An equine doping compound database and a spectral library were developed to enhance specificity in targeting substances of interest.
- Targeted screening involved an evaluation based on five criteria – accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions, and HRMS/MS spectra.
- With the help of the database and the library, a total of 451 substances were validated for targeted detection.
- Application of all five criteria reduced the chance of false positives in screening results significantly.
Development of Screening Strategy and Automation
- A screening strategy, coupled with a Microsoft Excel macro, was created to ease the interpretation and reporting of the results.
Significance of the Study
- The final part of the research emphasizes the technique’s potential for retrospective non-targeted analysis due to the simultaneous acquisition of full scan HRMS data.
- This study suggests that this new method is a quick, straightforward, and highly reproducible strategy to address the increasing threats from various doping substances in horse racing.
Cite This Article
APA
You Y, Proctor RM, Guo K, Li X, Xue E, Guan F, Robinson MA.
(2021).
Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control.
Anal Methods, 13(13), 1565-1575.
https://doi.org/10.1039/d0ay02297g Publication
Researcher Affiliations
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA 19348, USA. ywyou@upenn.edu.
MeSH Terms
- Animals
- Chromatography, Liquid
- Horses
- Liquid-Liquid Extraction
- Mass Screening
- Mass Spectrometry
- Retrospective Studies
Citations
This article has been cited 2 times.- Paula RAO, Gondim CS, Schmidt EM, Diniz MHGM, Lana MAG, Oliveira LS. Critical Evaluation of Two Qualitative Analytical Approaches for Multiclass Determination of Veterinary Drugs in Bovine Muscle Using UHPLC-Q-Orbitrap: The Wind of Change in Brazilian Monitoring. Molecules 2023 May 17;28(10).
- Zakaria N, El-Sayed ASA, Ali MG. Phytochemical fingerprinting of phytotoxins as a cutting-edge approach for unveiling nature's secrets in forensic science. Nat Prod Bioprospect 2025 Jan 3;15(1):1.
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