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Analytical and Bioanalytical Chemistry

, Volume 407, Issue 16, pp 4709–4720 | Cite as

Determination of Synacthen® in dried blood spots for doping control analysis using liquid chromatography tandem mass spectrometry

  • Laura Tretzel
  • Andreas Thomas
  • Hans Geyer
  • Philippe Delahaut
  • Wilhelm Schänzer
  • Mario ThevisEmail author
Research Paper

Abstract

Dried blood spot (DBS) sampling, a technique used for taking whole blood samples dried on a filter paper, was initially reported in 1963 by Robert Guthrie. While the diagnostic analysis of metabolic disorders in newborns was the focus of investigations at that time, the number of established applications for preclinical drug development, toxicological studies, and therapeutic drug monitoring increased enormously in the last decades. As a consequence of speed, simplicity, and minimal invasiveness, DBS recommends itself as the preferential technique in sports drug testing. The present approach highlights for the first time the development of a screening assay for the analysis of the synthetic human adrenocorticotropic hormone tetracosactide hexaacetate (Synacthen®) in DBS using liquid chromatography tandem mass spectrometry. Highly purified sample extracts were obtained by an advanced sample preparation procedure including the addition of an internal standard (d8-tetracosactide) and immunoaffinity purification. The method’s overall recovery was 27.6 %, and the assay’s imprecision was calculated between 8.1 and 17.9 % for intraday and 12.9 to 20.5 % for interday measurements. Stability of the synthetic peptide in DBS was shown for at least 10 days at room temperature and presents a major benefit, since a rapid degradation in conventionally applied matrices such as urine or plasma is well known. With a limit of detection of 50 pg/mL, a detection window of several hours is expected considering reported steady-state plasma levels of 300 pg/mL after intramuscular application of Synacthen® Depot (1 mg). The analysis of authentic DBS samples within the scope of an administration study with 250 μg Synacthen® (short stimulation test) demonstrated the great potential of the developed assay to simplify the analysis of Synacthen® for doping control purposes.

Keywords

Dried blood spots Doping control analysis Synacthen® ACTH Immunoaffinity purification LC-MS/MS 

Notes

Acknowledgments

The study was carried out with support of Antidoping Switzerland (Bern, Switzerland), the Irish Sports Council (Dublin, Ireland), and the Federal Ministry of the Interior of the Federal Republic of Germany (Berlin, Germany).

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Laura Tretzel
    • 1
  • Andreas Thomas
    • 1
  • Hans Geyer
    • 1
  • Philippe Delahaut
    • 2
  • Wilhelm Schänzer
    • 1
  • Mario Thevis
    • 1
    Email author
  1. 1.Institute of Biochemistry, Center for Preventive Doping ResearchGerman Sport University CologneCologneGermany
  2. 2.Laboratory of HormonologyCentre d’Economie RuraleMarloieBelgium

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