Analytical and Bioanalytical Chemistry

, Volume 408, Issue 18, pp 4883–4895 | Cite as

Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays

  • Johan Juhl Weisser
  • Cecilie Hurup Hansen
  • Rikke Poulsen
  • Lizette Weber Larsen
  • Claus Cornett
  • Bjarne Styrishave
Research Paper


Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS2). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS2 analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS2 method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55–530 pg/mL (LLOQ).


Online cleanup Plasma Testis Gonads H295R cell assay Solid phase extraction 


Compliance with ethical standards

All experimental protocols for collecting plasma and testis samples from male rats were approved by the Danish Animal Experimentation Council (24th of July 2014, license holder: Anne-Marie Heegaard, case no. 2014-15-0201-00031).

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2016_9575_MOESM1_ESM.pdf (536 kb)
ESM 1 (PDF 536 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Johan Juhl Weisser
    • 1
  • Cecilie Hurup Hansen
    • 1
  • Rikke Poulsen
    • 1
  • Lizette Weber Larsen
    • 1
  • Claus Cornett
    • 1
  • Bjarne Styrishave
    • 1
  1. 1.Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical ScienceUniversity of CopenhagenCopenhagenDenmark

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