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Molecules (Basel, Switzerland)2024; 29(21); 4988; doi: 10.3390/molecules29214988

Administration Route Differentiation of Altrenogest via the Metabolomic LC-HRMS Analysis of Equine Urine.

Abstract: Altrenogest, also known as allyltrenbolone, is a synthetic form of progesterone used therapeutically to suppress unwanted symptoms of estrus in female horses. Altrenogest affects the system by decreasing levels of endogenous gonadotrophin and luteinizing and follicle-stimulating hormones, which in turn decreases estrogen and mimics the increase of progesterone production. This results in more manageable mares for training and competition alongside male horses while improving the workplace safety of riders and handlers. However, when altrenogest is administered, prohibited steroid impurities such as trendione, trenbolone, and epitrenbolone can be detected. It has been assumed that greater concentrations of these steroid impurities are present in injectable preparations and, therefore, pose a greater risk of causing anabolic effects when administered. For this reason, and due to the necessity of this therapeutic substance for the safety of thoroughbred racing participants, a metabolomic approach investigating the differentiation of two main administration routes was conducted. Liquid chromatography high-resolution mass spectrometry analysis of equine urine samples found five sulfated compounds, estrone sulfate, testosterone sulfate, 2-methoxyestradiol sulfate, pregnenolone sulfate, and cortisol sulfate, with the potential to differentiate between oral and intramuscularly administered altrenogest using a random forest classification model. The best model results, comparing two horses' administration normalized peak area datasets, gave an AUC score of 0.965 with a confidence level of 95% (between 0.931 and 0.995). Identifications of these compounds were confirmed with assistance from the Shimadzu Insight Explore Assign feature, together with MS/MS spectrum and retention time matching of purchased and synthesized reference standards. This study proposes a new potential application for metabolomic multi-tool workflows and machine learning models in a forensic toxicological context.
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Publication Date: 2024-10-22 PubMed ID: 39519629PubMed Central: PMC11547534DOI: 10.3390/molecules29214988Google 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.

The study investigated different methods of administering Altrenogest, a synthetic form of progesterone used in female horses, and their metabolic effects. Researchers identified five specific compounds that could determine if Altrenogest was given orally or injected, providing a potential new application for metabolomic multi-tool workflows and machine learning models in a forensic toxicological context.

Introduction

  • The research revolves around Altrenogest, a synthetic variant of progesterone utilized to control estrus-induced behavior in mares, making them safer and more manageable in equestrian settings.
  • The study acknowledges the controversy surrounding the use of Altrenogest, due to the presence of prohibited steroid impurities like trendione, trenbolone, and epitrenbolone in its composition, with injectable versions assumed to contain higher concentrations.

Methods

  • The study design required a metabolomic investigation to differentiate between the oral and intramuscular administration of Altrenogest.
  • They used liquid chromatography high-resolution mass spectrometry (LC-HRMS) for the metabolomic analysis of urine samples from horses.
  • The researchers identified the relevant compounds using the Shimadzu Insight Explore Assign feature, combined with the MS/MS spectrum and retention time matching of purchased and synthesized reference standards.

Results

  • The metabolomic LC-HRMS analysis identified five sulfated compounds—estrone sulfate, testosterone sulfate, 2-methoxyestradiol sulfate, pregnenolone sulfate, and cortisol sulfate.
  • These compounds could potentially be used to distinguish between oral and intramuscular administration of Altrenogest using a random forest machine learning model.
  • The performance of the machine learning model was evaluated using the normalized peak area data from two horses.
  • The model achieved a high area under the curve (AUC) score of 0.965 with a confidence level of 95% (ranging from 0.931 to 0.995), indicating its reliability in determining the administration route of Altrenogest.

Conclusion

  • The research introduced a possible new application for metabolomic multi-tool workflows and machine learning models in the field of forensic toxicology. Essentially, this could help in the identification of prohibited substances in horseracing and other applications where substance use needs to be monitored and controlled.

Cite This Article

APA
Elbourne M, Keledjian J, Cawley A, Fu S. (2024). Administration Route Differentiation of Altrenogest via the Metabolomic LC-HRMS Analysis of Equine Urine. Molecules, 29(21), 4988. https://doi.org/10.3390/molecules29214988

Publication

Molecules (Basel, Switzerland)
ISSN: 1420-3049
NlmUniqueID: 100964009
Country: Switzerland
Language: English
Volume: 29
Issue: 21
PII: 4988

Researcher Affiliations

Elbourne, Madysen
  • Centre for Forensic Science, University of Technology Sydney, Sydney, NSW 2007, Australia.
Keledjian, John
  • Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW 2000, Australia.
Cawley, Adam
  • Racing Analytical Services Limited, Flemington, VIC 3031, Australia.
Fu, Shanlin
  • Centre for Forensic Science, University of Technology Sydney, Sydney, NSW 2007, Australia.

MeSH Terms

  • Horses
  • Animals
  • Trenbolone Acetate / analogs & derivatives
  • Trenbolone Acetate / urine
  • Chromatography, Liquid / methods
  • Metabolomics / methods
  • Female
  • Male
  • Administration, Oral
  • Injections, Intramuscular

Conflict of Interest Statement

Author Adam Cawley was employed by the company Racing Analytical Services Limited. Author John Keledjian was employed by Australian Racing Forensic Laboratory, Racing NSW. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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