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Pharmaceuticals (Basel, Switzerland)2021; 14(5); 393; doi: 10.3390/ph14050393

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

Abstract: A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.
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Publication Date: 2021-04-21 PubMed ID: 33919404PubMed Central: PMC8143330DOI: 10.3390/ph14050393Google Scholar: Lookup
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Summary

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A reliable new analytical method has been developed that can simultaneously detect and quantify 17 different steroidal hormones in bovine and equine blood, which could be pivotal for doping control in animal sports.

Method Development

  • The research team developed a fast and simplified analytical method capable of identifying and quantifying 17 endogenous and exogenous steroidal hormones in the blood of cattle (bovine) and horses (equine). This method uses ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
  • For the testing, a total of 500 µL blood sample was deproteinized, meaning, proteins that could interfere with the analysis were removed. This was done using a mixture of methanol (a type of alcohol) and zinc sulfate.
  • The deproteinized samples were then evaporated and reconstituted with a 25% methanol solution, before being injected into the UHPLC-MS/MS system.

Assessment of Method’s Precision

  • The precision and acuity of this method were assessed through the calibration curves of the analysed compounds. The correlation coefficients (a measure of how closely the data fit the regression line on the calibration curve) ranged between 0.9932 and 0.9999, indicating a very high degree of precision.

Sensitivity and Quality Analysis

  • The limits of detection (the smallest amount that can be reliably detected) and quantification (the smallest amount of a compound that can be reliably measured using the method) were determined. These were found to be between 0.023 and 1.833 parts per billion (ppb), and 0.069 and 5.5 ppb, respectively.
  • The study revealed the acclaimable sensitivity and quality aspects of this analytical method, suggesting it was capable of detecting all steroids in equine and bovine blood.

Application of the Method

  • Once validated, the researchers used this method to quantify 17 different steroid hormones in equine and bovine blood samples.
  • The most commonly detected endogenous compounds (those naturally occurring in the body) were corticosterone and cortisol, followed by androstenedione, testosterone, and 11-deoxycortisol.
  • Importantly, this method had a detection limit of 50 parts per trillion (ppt) for testosterone, which is half of the threshold admitted in plasma (100 ppt), making it a potential game-changer for doping control in animal sports.

Cite This Article

APA
Caprioli G, Genangeli M, Mustafa AM, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Laus F, Vittori S, Cortese M. (2021). Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS. Pharmaceuticals (Basel), 14(5), 393. https://doi.org/10.3390/ph14050393

Publication

Pharmaceuticals (Basel, Switzerland)
ISSN: 1424-8247
NlmUniqueID: 101238453
Country: Switzerland
Language: English
Volume: 14
Issue: 5
PII: 393

Researcher Affiliations

Caprioli, Giovanni
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Genangeli, Michele
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Mustafa, Ahmed M
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
  • Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt.
Petrelli, Riccardo
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Ricciutelli, Massimo
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Sagratini, Gianni
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Sartori, Stefano
  • Eureka Lab Division, 60033 Chiaravalle, Italy.
Laus, Fulvio
  • School of Bioscience and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy.
Vittori, Sauro
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.
Cortese, Manuela
  • School of Pharmacy, University of Camerino, 62032 Camerino, Italy.

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

This article has been cited 1 times.
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