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Drug testing and analysis2023; doi: 10.1002/dta.3544

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

Abstract: The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.
Drug Testing and Analysis© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.
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Publication Date: 2023-08-07 PubMed ID: 37548131DOI: 10.1002/dta.3544Google 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.

Introduction:

  • The misuse of prohibited substances like peptides and basic small-molecule drugs is a major concern in horseracing. These substances have a high potential to be used as doping agents.
  • They include growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs.
  • Detecting and regulating these compounds can be difficult due to their chemical properties and the possibility of them being quickly eliminated from the body after being administered.

Analytical Techniques:

To detect these substances and their potential metabolites, it’s crucial to use highly sensitive and specific analytical methods. Liquid chromatography-mass spectrometry (LC-MS) is one such technique.

Study Objective:

This research focused on developing and validating a method that can detect over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in horse urine.

Methodology:

  1. Samples were first prepared using solid-phase extraction with a mixed mode weak cation exchange sorbent.
  2. After extraction, the compounds were analyzed using LC-MS.
  3. Chromatographic separation was achieved through a reverse phase gradient.
  4. Detection was done using selective reaction monitoring after introducing the samples to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization.

Validation:

The validation process examined various parameters such as:

  • Limits of detection and quantitation.
  • Accuracy and precision.
  • Linear range.
  • Recovery rate.
  • Stability and matrix effects.

Results:

Most compounds could be detected at sub-ng/mL ranges, with acceptable precision and accuracy suitable for initial testing. Stability studies indicated that most substances remained stable enough for effective screening under conditions typically used in drug testing labs.

Conclusion:

The developed method provides an effective way to screen for a wide range of potential doping agents in horse urine, ensuring the integrity of horseracing and the well-being of the animals.

Cite This Article

APA
Flores L, Hargrave S, Clifford A, Alarcio G, Moeller BC. (2023). Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry. Drug Test Anal. https://doi.org/10.1002/dta.3544

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Flores, Luis
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Hargrave, Sabine
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Clifford, Amel
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Alarcio, Gwendolyne
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Moeller, Benjamin C
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.

Grant Funding

  • California Horse Racing Board

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Citations

This article has been cited 1 times.
  1. Králík F, Kvíčalová A, Salaďáková A, Kuchař M, Setnička V. Growth Hormone-Releasing Peptides: Investigation of Their Secondary Structure, Thermal Stability, and Model Membrane Interactions. Chirality 2026 Feb;38(2):e70083.
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