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Rapid communications in mass spectrometry : RCM2023; 37(18); e9611; doi: 10.1002/rcm.9611

An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry.

Abstract: Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. Methods: To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase extraction, liquid-liquid extraction, and dilute-and-inject, were explored to detect ecdysteroids from equine urine and plasma. Results: The most suitable method of detection was solid-phase extraction using ABS Elut-NEXUS, while liquid-liquid extraction and dilute-and-inject methods encountered difficulties due to the high polarity of ecdysteroids and the presence of significant matrix interferences. Mass spectrometric parameters are optimized on both the Q Exactive high-resolution mass spectrometer and the TSQ Altis triple quadrupole mass spectrometer. However, the study indicated that the triple quadrupole mass spectrometer exhibited improved limit of detection when analyzing samples. To achieve optimal separation of the analytes under investigation from the matrix interferences, various liquid chromatography columns were compared. The Selectra PFPP LC column with a mobile phase consisting of 0.2% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.5 mL/min demonstrated superior performance. Conclusions: The findings of this study will significantly contribute to the accurate identification of ecdysteroids, facilitating the investigation of their illicit use in horse racing.
© 2023 John Wiley & Sons Ltd.
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Publication Date: 2023-08-15 PubMed ID: 37580844DOI: 10.1002/rcm.9611Google 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 research presents an optimized and validated method for detecting and quantifying ecdysteroids in horse urine and plasma. This method, which uses solid-phase extraction and mass spectrometry, was developed in response to reports suggesting these substances could potentially be used to enhance sports performance, making them a potential focus for doping tests.

Methodology

  • The researchers aimed to detect and quantify the presence of several ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C in horse urine and plasma samples.
  • Various extraction techniques were tested including solid-phase extraction, liquid-liquid extraction, and dilute-and-inject.
  • The assessment of ecdysteroids relied on the use of two different mass spectrometers – the Q Exactive high-resolution mass spectrometer and TSQ Altis triple quadrupole mass spectrometer.

Results

  • The research found that the solid-phase extraction using ABS Elut-NEXUS was the most effective method for detecting ecdysteroids. Other methods encountered difficulties due to the high polarity of ecdysteroids and significant matrix interferences.
  • Between the two spectrometers, the TSQ Altis triple quadrupole mass spectrometer showed more improved detection limits when analyzing samples.
  • Various liquid chromatography (LC) columns were compared for optimal separation of the analytes from the matrix interferences. The best performing LC column was the Selectra PFPP LC with a mobile phase consisting of 0.2% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.5 mL/min.

Conclusions

  • The findings facilitate more accurate detection and quantification of ecdysteroids in equine urine and plasma. This improves anti-doping control measures by identifying potential misuse of these substances in horse racing.

By optimizing and validating this solid-phase extraction method, the researchers have made a significant contribution to doping control measures in equine sports. The development of these enhanced detection methods could be critical in maintaining integrity in competitive sports and ensuring a fair and level playing field.

Cite This Article

APA
Karatt TK, Sathiq MA, Laya S, Ajeebsanu MPM, Karakka Kal AK, Subhahar MB, Perwad Z. (2023). An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry. Rapid Commun Mass Spectrom, 37(18), e9611. https://doi.org/10.1002/rcm.9611

Publication

Rapid communications in mass spectrometry : RCM
ISSN: 1097-0231
NlmUniqueID: 8802365
Country: England
Language: English
Volume: 37
Issue: 18
Pages: e9611

Researcher Affiliations

Karatt, Tajudheen K
  • Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
  • Post Graduate and Research Department of Chemistry, Jamal Mohamed College (Affiliated to Bharathidasan University), Tiruchirappalli, India.
Sathiq, M Anwar
  • Post Graduate and Research Department of Chemistry, Jamal Mohamed College (Affiliated to Bharathidasan University), Tiruchirappalli, India.
Laya, Saraswathy
  • Department of Chemistry, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates.
Ajeebsanu, M P Muhammed
  • Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Karakka Kal, Abdul Khader
  • Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Subhahar, Michael Benedict
  • Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Perwad, Zubair
  • Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

MeSH Terms

  • Horses
  • Animals
  • Ecdysteroids
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Liquid
  • Mass Spectrometry
  • Doping in Sports / prevention & control
  • Solid Phase Extraction

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Citations

This article has been cited 2 times.
  1. Sillence MN, Holt K, Li FI, Harris PA, Coyle M, Fitzgerald DM. Sources of Environmental Exposure to the Naturally Occurring Anabolic Steroid Ecdysterone in Horses. Animals (Basel) 2025 Jul 17;15(14).
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  2. Marešová L, Moos M, Opekar S, Kazek M, Eichler C, Šimek P. A validated HPLC-MS/MS method for the simultaneous determination of ecdysteroid hormones in subminimal amounts of biological material. J Lipid Res 2024 Oct;65(10):100640.
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