FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 2021-04-21 PubMed ID: 33919404PubMed Central: PMC8143330DOI: 10.3390/ph14050393Google 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.
LinkedInCopy
  • Journal Article

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.

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.

References

This article includes 39 references
  1. Genangeli M, Caprioli G, Cortese M, Laus F, Matteucci M, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Vittori S. Development and application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum.. J Mass Spectrom 2017 Jan;52(1):22-29.
    doi: 10.1002/jms.3896pubmed: 27790795google scholar: lookup
  2. Andrews MA, Magee CD, Combest TM, Allard RJ, Douglas KM. Physical Effects of Anabolic-androgenic Steroids in Healthy Exercising Adults: A Systematic Review and Meta-analysis.. Curr Sports Med Rep 2018 Jul;17(7):232-241.
    doi: 10.1249/JSR.0000000000000500pubmed: 29994823google scholar: lookup
  3. Armstrong JM, Avant RA, Charchenko CM, Westerman ME, Ziegelmann MJ, Miest TS, Trost LW. Impact of anabolic androgenic steroids on sexual function.. Transl Androl Urol 2018 Jun;7(3):483-489.
    doi: 10.21037/tau.2018.04.23pmc: PMC6043738pubmed: 30050806google scholar: lookup
  4. Melo Junior AF, Dalpiaz PLM, Sousa GJ, Oliveira PWC, Birocale AM, Andrade TU, Abreu GR, Bissoli NS. Nandrolone alter left ventricular contractility and promotes remodelling involving calcium-handling proteins and renin-angiotensin system in male SHR.. Life Sci 2018 Sep 1;208:239-245.
    doi: 10.1016/j.lfs.2018.07.041pubmed: 30040952google scholar: lookup
  5. Büttler RM, Martens F, Kushnir MM, Ackermans MT, Blankenstein MA, Heijboer AC. Simultaneous measurement of testosterone, androstenedione and dehydroepiandrosterone (DHEA) in serum and plasma using Isotope-Dilution 2-Dimension Ultra High Performance Liquid-Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS).. Clin Chim Acta 2015 Jan 1;438:157-9.
    doi: 10.1016/j.cca.2014.08.023pubmed: 25172041google scholar: lookup
  6. Wong JK, Wan TS. Doping control analyses in horseracing: a clinician's guide.. Vet J 2014 Apr;200(1):8-16.
    doi: 10.1016/j.tvjl.2014.01.006pubmed: 24485918google scholar: lookup
  7. IFHA. [(accessed on 25 March 2021)]; Available online: http://www.ifhaonline.org/default.asp?section=Racing&area=0#a6a.
  8. Pellegrini K.G.M.V. Anabolic Steroids. [(accessed on 25 March 2021)]; Available online: https://www.ncbi.nlm.nih.gov/books/NBK482418/#article-17499.s9.
  9. Caron P, Turcotte V, Guillemette C. A chromatography/tandem mass spectrometry method for the simultaneous profiling of ten endogenous steroids, including progesterone, adrenal precursors, androgens and estrogens, using low serum volume.. Steroids 2015 Dec;104:16-24.
    doi: 10.1016/j.steroids.2015.07.009pubmed: 26254607google scholar: lookup
  10. Görög S. Recent advances in the analysis of steroid hormones and related drugs.. Anal Sci 2004 May;20(5):767-82.
    doi: 10.2116/analsci.20.767pubmed: 15171279google scholar: lookup
  11. Stanczyk FZ, Jurow J, Hsing AW. Limitations of direct immunoassays for measuring circulating estradiol levels in postmenopausal women and men in epidemiologic studies.. Cancer Epidemiol Biomarkers Prev 2010 Apr;19(4):903-6.
    doi: 10.1158/1055-9965.EPI-10-0081pubmed: 20332268google scholar: lookup
  12. Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, Lacroix I, Somma-Delpero C, Boudou P. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children.. Clin Chem 2003 Aug;49(8):1381-95.
    doi: 10.1373/49.8.1381pubmed: 12881456google scholar: lookup
  13. Huang X, Yuan D, Huang B. Determination of steroid sex hormones in urine matrix by stir bar sorptive extraction based on monolithic material and liquid chromatography with diode array detection.. Talanta 2008 Mar 15;75(1):172-7.
    doi: 10.1016/j.talanta.2007.10.052pubmed: 18371864google scholar: lookup
  14. He G, Wu Y, Lu J. Doping control analysis of 13 steroids and structural-like analytes in human urine using Quadrupole-Orbitrap LC-MS/MS with parallel reaction monitoring (PRM) mode.. Steroids 2018 Mar;131:1-6.
    doi: 10.1016/j.steroids.2017.12.011pubmed: 29274404google scholar: lookup
  15. Karatt TK, Nalakath J, Perwad Z, Albert PH, Abdul Khader KK, Syed Ali Padusha M, Laya S. Mass spectrometric method for distinguishing isomers of dexamethasone via fragment mass ratio: An HRMS approach.. J Mass Spectrom 2018 Nov;53(11):1046-1058.
    doi: 10.1002/jms.4279pubmed: 30098588google scholar: lookup
  16. Keevil BG. Novel liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measuring steroids.. Best Pract Res Clin Endocrinol Metab 2013 Oct;27(5):663-74.
    doi: 10.1016/j.beem.2013.05.015pubmed: 24094637google scholar: lookup
  17. You Y, Uboh CE, Soma LR, Guan F, Li X, Liu Y, Rudy JA, Chen J, Tsang D. Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control.. J Chromatogr A 2011 Jul 1;1218(26):3982-93.
    doi: 10.1016/j.chroma.2011.04.087pubmed: 21601862google scholar: lookup
  18. Wong CH, Leung DK, Tang FP, Wong JK, Yu NH, Wan TS. Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation.. J Chromatogr A 2012 Apr 6;1232:257-65.
    doi: 10.1016/j.chroma.2011.12.095pubmed: 22265177google scholar: lookup
  19. Tuzimski T, Petruczynik A. Review of Chromatographic Methods Coupled with Modern Detection Techniques Applied in the Therapeutic Drugs Monitoring (TDM).. Molecules 2020 Sep 3;25(17).
    doi: 10.3390/molecules25174026pmc: PMC7504794pubmed: 32899296google scholar: lookup
  20. Choi TLS, Kwok KY, Kwok WH, Tsoi YYK, Wong JKY, Wan TSM. Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS(2) mode and gas chromatography-tandem mass spectrometry.. J Chromatogr A 2018 Sep 7;1566:51-63.
    doi: 10.1016/j.chroma.2018.06.049pubmed: 29958683google scholar: lookup
  21. Wong JKY, Choi TLS, Kwok KY, Lei ENY, Wan TSM. Doping control analysis of 121 prohibited substances in equine hair by liquid chromatography-tandem mass spectrometry.. J Pharm Biomed Anal 2018 Sep 5;158:189-203.
    doi: 10.1016/j.jpba.2018.05.043pubmed: 29885604google scholar: lookup
  22. Guan F, Uboh CE, Soma LR, Luo Y, Rudy J, Tobin T. Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry.. J Chromatogr B Analyt Technol Biomed Life Sci 2005 Dec 27;829(1-2):56-68.
    doi: 10.1016/j.jchromb.2005.09.045pubmed: 16289956google scholar: lookup
  23. Hintikka L. Development of Mass Spectrometric Methods for Analysis of Anabolic Androgenic Steroids. University of Helsinki; Helsinki, Finland: 2018.
  24. Georgakopoulos CG, Vonaparti A, Stamou M, Kiousi P, Lyris E, Angelis YS, Tsoupras G, Wuest B, Nielen MW, Panderi I, Koupparis M. Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids.. Rapid Commun Mass Spectrom 2007;21(15):2439-46.
    doi: 10.1002/rcm.3103pubmed: 17610244google scholar: lookup
  25. Gray B, Viljanto M, Menzies E, Vanhaecke L. Detection of prohibited substances in equine hair by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry - application to doping control samples.. Drug Test Anal 2018 Feb 12;.
    doi: 10.1002/dta.2367pubmed: 29430877google scholar: lookup
  26. Kaabia Z, Dervilly-Pinel G, Hanganu F, Cesbron N, Bichon E, Popot MA, Bonnaire Y, Le Bizec B. Ultra high performance liquid chromatography/tandem mass spectrometry based identification of steroid esters in serum and plasma: an efficient strategy to detect natural steroids abuse in breeding and racing animals.. J Chromatogr A 2013 Apr 5;1284:126-40.
    doi: 10.1016/j.chroma.2013.02.010pubmed: 23484650google scholar: lookup
  27. Annesley TM. Ion suppression in mass spectrometry.. Clin Chem 2003 Jul;49(7):1041-4.
    doi: 10.1373/49.7.1041pubmed: 12816898google scholar: lookup
  28. Harrison AG. Chemical Ionization Mass Spectrometry. 2nd ed. CRC Press; Boca Raton, FL, USA: 1992. p. 208.
  29. Shao B, Zhao R, Meng J, Xue Y, Wu G, Hu J, Tu X. Simultaneous determination of residual hormonal chemicals in meat, kidney, liver tissues and milk by liquid chromatography–tandem mass spectrometry. Anal. Chim. Acta 2005;548:41–50.
    doi: 10.1016/j.aca.2005.06.003google scholar: lookup
  30. Yamashita K, Okuyama M, Nakagawa R, Honma S, Satoh F, Morimoto R, Ito S, Takahashi M, Numazawa M. Development of sensitive derivatization method for aldosterone in liquid chromatography-electrospray ionization tandem mass spectrometry of corticosteroids.. J Chromatogr A 2008 Jul 25;1200(2):114-21.
    doi: 10.1016/j.chroma.2008.05.034pubmed: 18561939google scholar: lookup
  31. Zuber J, Kroll MM, Rathsack P, Otto M. Gas Chromatography/Atmospheric Pressure Chemical Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Pyrolysis Oil from German Brown Coal.. Int J Anal Chem 2016;2016:5960916.
    doi: 10.1155/2016/5960916pmc: PMC4811094pubmed: 27066076google scholar: lookup
  32. You Y, Uboh CE, Soma LR, Guan F, Li X, Liu Y, Chen J, Tsang D. Simultaneous Determination of Testosterone and Testosterone Enanthate in Equine Plasma by UHPLC-MS-MS. Chromatographia 2010;72:1097–1106.
    doi: 10.1365/s10337-010-1784-6google scholar: lookup
  33. Prokai L, Stevens SM Jr. Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry.. Int J Mol Sci 2016 Jan 16;17(1).
    doi: 10.3390/ijms17010116pmc: PMC4730357pubmed: 26784186google scholar: lookup
  34. Allis O, Dauphard J, Hamilton B, Shuilleabhain AN, Lehane M, James KJ, Furey A. Liquid chromatography-tandem mass spectrometry application, for the determination of extracellular hepatotoxins in Irish lake and drinking waters.. Anal Chem 2007 May 1;79(9):3436-47.
    doi: 10.1021/ac062088qpubmed: 17402708google scholar: lookup
  35. Bonfiglio R, King RC, Olah TV, Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds.. Rapid Commun Mass Spectrom 1999 Jun;13(12):1175-1185.
    doi: 10.1002/(SICI)1097-0231(19990630)13:12<1175::AID-RCM639>3.0.CO;2-0pubmed: 10407294google scholar: lookup
  36. Kaabia Z, Dervilly-Pinel G, Popot MA, Bailly-Chouriberry L, Plou P, Bonnaire Y, Le Bizec B. Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses.. Drug Test Anal 2014 Apr;6(4):376-88.
    doi: 10.1002/dta.1520pubmed: 23949888google scholar: lookup
  37. [(accessed on 25 March 2021)]; Available online: https://www.comsol.nl/multiphysics/the-joule-heating-effect.
  38. Wang YN, Zhao M, Yu YB, Wang M, Zhao CJ. Metabolic profile of Cortex Fraxini in rats using UHPLC combined with Fourier transform ion cyclotron resonance mass spectrometry. RSC Adv 2016;6:39642–39651.
    doi: 10.1039/C6RA01428Cgoogle scholar: lookup
  39. Magnisali P, Dracopoulou M, Mataragas M, Dacou-Voutetakis A, Moutsatsou P. Routine method for the simultaneous quantification of 17alpha-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry.. J Chromatogr A 2008 Oct 10;1206(2):166-77.
    doi: 10.1016/j.chroma.2008.07.057pubmed: 18760785google scholar: lookup

Citations

This article has been cited 2 times.
  1. Keen B, Cawley A, Reedy B, Fu S. Metabolomics in clinical and forensic toxicology, sports anti-doping and veterinary residues. Drug Test Anal 2022 May;14(5):794-807.
    doi: 10.1002/dta.3245pubmed: 35194967google scholar: lookup
  2. Devo P, Cretu V, Radhakrishnan H, Hamilton-Pink D, Boussios S, Ovsepian SV. An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS). J Neural Transm (Vienna) 2025 Dec;132(12):1815-1823.
    doi: 10.1007/s00702-024-02862-3pubmed: 39545973google scholar: lookup
Subscribe to our newsletter
Join 99,780 horse owners like you who receive our email updates on horse health and nutrition.