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Analytical and Bioanalytical Chemistry

, Volume 400, Issue 2, pp 503–516 | Cite as

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

  • Flavia Badoud
  • Elia Grata
  • Julien Boccard
  • Davy Guillarme
  • Jean-Luc Veuthey
  • Serge Rudaz
  • Martial SaugyEmail author
Original Paper

Abstract

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MSE with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MSE analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.

Figure

UHPLC-QTOF-MSE acquisition mode for DHEAG in urine sample

Keywords

Testosterone Steroids Glucuronide and sulfate conjugates Ultra high-pressure liquid chromatography Quadrupole time-of-flight Quantification MSE 

References

  1. 1.
    Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W (2008) J Mass Spectrom 43:877–891CrossRefGoogle Scholar
  2. 2.
    Taylor NF (2006) Methods Mol Biol 324:159–175Google Scholar
  3. 3.
    Sjöqvist F, Garle M, Rane A (2008) Lancet 371:1872–1882CrossRefGoogle Scholar
  4. 4.
    Van Renterghem P, Van Eenoo P, Delbeke FT (2010) Steroids 75:1047–1057CrossRefGoogle Scholar
  5. 5.
    World Anti-doping Agency (WADA) (2010) The world anti-doping code. Endogenous anabolic androgenic steroids, testing and reporting guidance, technical document TD2010EAAS, Montreal. Available at http://www.wada-ama.org. Accessed on Dec 2010
  6. 6.
    Ayotte C (2009) Detecting the administration of endogenous anabolic androgenic steroids. In: Thieme D, Hemmersbach P (ed) Doping in sports: biochemical principles, effects and analysis. Springer, Heidelberg, pp 77–98Google Scholar
  7. 7.
    Van Renterghem P, Van Eenoo P, Geyer H, Schänzer W, Delbeke FT (2011) Steroids 75:154–163CrossRefGoogle Scholar
  8. 8.
    Bowers LD (1997) Clin Chem 43:1299–1304Google Scholar
  9. 9.
    Bowers LD, Sanaullah (1996) J Chromatogr B Biomed Sci Appl 87:61–68CrossRefGoogle Scholar
  10. 10.
    Antignac J-P, Brosseaud A, Gaudin-Hirret I, André F, Bizec BL (2005) Steroids 70:205–216CrossRefGoogle Scholar
  11. 11.
    Strahm E, Kohler I, Rudaz S, Martel S, Carrupt P-A, Veuthey J-L, Saugy M, Saudan C (2008) J Chromatogr A 1196–1197:153–160CrossRefGoogle Scholar
  12. 12.
    Bean KA, Henion JD (1997) J Chromatogr B Biomed Sci Appl 690:65–75CrossRefGoogle Scholar
  13. 13.
    Buiarelli F, Coccioli F, Merolle M, Neri B, Terracciano A (2004) Anal Chim Acta 526:113–120CrossRefGoogle Scholar
  14. 14.
    Pozo OJ, Van Eenoo P, Van Thuyne W, Deventer K, Delbeke FT (2008) J Chromatogr A 1183:108–118CrossRefGoogle Scholar
  15. 15.
    Flores-Valverde AM, Hill EM (2008) Anal Chem 80:8771–8779CrossRefGoogle Scholar
  16. 16.
    Rijk JCW, Lommen A, Essers ML, Groot MJ, Van Hende JM, Doeswijk TG, Nielen MWF (2009) Anal Chem 81:6879–6888CrossRefGoogle Scholar
  17. 17.
    Guillarme D, Ruta J, Rudaz S, Veuthey J-L (2009) Anal Bioanal Chem 397:1069–1082CrossRefGoogle Scholar
  18. 18.
    Hopfgartner G, Bourgogne E (2003) Mass Spectrom Rev 22:195–214CrossRefGoogle Scholar
  19. 19.
    Georgakopoulos CG, Vonaparti A, Stamou M, Kiousi P, Lyris E, Angelis YS, Tsoupras G, Wuest B, Nielen MWF, Panderi I, Koupparis M (2007) Rapid Commun Mass Spectrom 21:2439–2446CrossRefGoogle Scholar
  20. 20.
    Fragkaki AG, Tsantili-Kakoulidou A, Angelis YS, Koupparis M, Georgakopoulos C (2009) J Chromatogr A 1216:8404–8420CrossRefGoogle Scholar
  21. 21.
    Galesio M, Rial-Otero R, Capelo-Martínez JL (2009) Rapid Commun Mass Spectrom 23:1783–1791CrossRefGoogle Scholar
  22. 22.
    Peters RJB, Rijk JCW, Bovee TFH, Nijrolder AWJM, Lommen A, Nielen MWF (2010) Anal Chim Acta 664:77–88CrossRefGoogle Scholar
  23. 23.
    Nielen MWF, Bovee TFH, van Engelen MC, Rutgers P, Hamers ARM, van Rhijn JA, Hoogenboom LAP (2005) Anal Chem 78:424–431CrossRefGoogle Scholar
  24. 24.
    Temesi DG, Martin S, Smith R, Jones C, Middleton B (2010) Rapid Commun Mass Spectrom 24:1730–1736CrossRefGoogle Scholar
  25. 25.
    Plumb RS, Johnson KA, Rainville P, Smith BW, Wilson ID, Castro-Perez JM, Nicholson JK (2006) Rapid Commun Mass Spectrom 20:1989–1994CrossRefGoogle Scholar
  26. 26.
    Purves RW, Gabryelski W, Li L (1998) Rapid Commun Mass Spectrom 12:695–700CrossRefGoogle Scholar
  27. 27.
    Zhang H, Heinig K, Henion J (2000) J Mass Spectrom 35:423–431CrossRefGoogle Scholar
  28. 28.
    Zhang N, Fountain ST, Bi H, Rossi DT (2000) Anal Chem 72:800–806CrossRefGoogle Scholar
  29. 29.
    World Anti-Doping Agency (WAA) (2010) International standard for laboratories V5.0. Available at http://www.wada-ama.org. Accessed on Dec 2010
  30. 30.
    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005) ICH harmonised tripartite guideline, validation of analytical procedures: text and methodology, Q2 (R1). ICH, GenevaGoogle Scholar
  31. 31.
    Food and Drug Administration (2001) Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, FDA, Centre for Drug Evaluation and Research, RockvilleGoogle Scholar
  32. 32.
    Viswanathan C, Bansal S, Booth B, DeStefano A, Rose M, Sailstad J, Shah V, Skelly J, Swann P, Weiner R (2007) AAPS J 9:E30–E42CrossRefGoogle Scholar
  33. 33.
    European Medicines Agency, Committee for Medicinal Products for Human Use (CHMP) (2008) Concept paper/recommendations on the need for a (CHMP) guideline on the validation of bioanalytical methods, London, 18 DecGoogle Scholar
  34. 34.
    Weykamp CW, Penders TJ, Schmidt NA, Borburgh AJ, van de Calseyde JF, Wolthers BJ (1989) Clin Chem 35:2281–2284Google Scholar
  35. 35.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Anal Chem 75:3019–3030CrossRefGoogle Scholar
  36. 36.
    Marchi I, Viette V, Badoud F, Fathi M, Saugy M, Rudaz S, Veuthey J-L (2010) J Chromatogr A 1217:4071–4078CrossRefGoogle Scholar
  37. 37.
    Rozet E, Ceccato A, Hubert C, Ziemons E, Oprean R, Rudaz S, Boulanger B, Hubert P (2007) J Chromatogr A 1158:111–125CrossRefGoogle Scholar
  38. 38.
    Feinberg M, Boulanger B, Dewé W, Hubert P (2004) Anal Bioanal Chem 380:502–514CrossRefGoogle Scholar
  39. 39.
    Baume N, Schumacher Y, Sottas P-E, Bagutti C, Cauderay M, Mangin P, Saugy M (2006) Eur J Appl Physiol 98:329–340CrossRefGoogle Scholar
  40. 40.
    Baume N, Saudan C, Desmarchelier A, Strahm E, Sottas P-E, Bagutti C, Cauderay M, Schumacher YO, Mangin P, Saugy M (2006) Steroids 71:364–370CrossRefGoogle Scholar
  41. 41.
    Strahm E, Sottas PE, Schweizer C, Saugy M, Dvorak J, Saudan C (2009) Br J Sports Med 43:1126–1130CrossRefGoogle Scholar
  42. 42.
    Weinshilboum R, Otterness D (1994) Sulfotransferase enzymes. In: Kauffmann FC (ed) Conjugation–deconjugation reactions in drug metabolism and toxicity. Springer, Berlin, pp 45–78Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Flavia Badoud
    • 1
    • 2
    • 3
  • Elia Grata
    • 1
    • 3
  • Julien Boccard
    • 2
    • 3
  • Davy Guillarme
    • 2
    • 3
  • Jean-Luc Veuthey
    • 2
    • 3
  • Serge Rudaz
    • 2
    • 3
  • Martial Saugy
    • 1
    • 3
    Email author
  1. 1.Swiss Laboratory for Doping AnalysesUniversity Center of Legal Medicine, Geneva and LausanneEpalingesSwitzerland
  2. 2.School of Pharmaceutical SciencesUniversity of Geneva, University of LausanneGeneva 4Switzerland
  3. 3.Swiss Centre for Applied Human Toxicology, CMUGeneva 4Switzerland

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