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


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.


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


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


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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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