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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

Abstract

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).

Keywords

Online cleanup Plasma Testis Gonads H295R cell assay Solid phase extraction 

Notes

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)

References

  1. 1.
    Miller WL, Auchus RJ. Endocr Rev. 2011;32:81–151.CrossRefGoogle Scholar
  2. 2.
    Colborn T, Saal FS, Soto AM. Environ Impact Assess Rev. 1994;14:469–89.CrossRefGoogle Scholar
  3. 3.
    Giwercman A, Carlsen E, Keiding N, Skakkebak NE. Environ Health Perspect. 1993;101:65–71.CrossRefGoogle Scholar
  4. 4.
    Grün F, Blumberg B. Endocrinology. 2006;147:50–5.CrossRefGoogle Scholar
  5. 5.
    Grün F, Blumberg B. Mol Cell Endocrinol. 2009;304:19–29.CrossRefGoogle Scholar
  6. 6.
    Joensen UN, Bossi R, Leffers H, Jensen AA, Skakkebaek NE, Jørgensen N. Environ Health Perspect. 2009;117:923–7.CrossRefGoogle Scholar
  7. 7.
    Jørgensen N, Asklund C, Carlsen E, Skakkebaek NE. Int J Androl. 2006;29:54–61.CrossRefGoogle Scholar
  8. 8.
    Priskorn L, Holmboe SA, Jacobsen R, Jensen TK, Lassen TH, Skakkebaek NE. Int J Androl. 2012;35:449–55.CrossRefGoogle Scholar
  9. 9.
    Skakkebæk NE, Meyts ER-D, Main KM. Hum Reprod. 2001;16:972–8.CrossRefGoogle Scholar
  10. 10.
    Nielsen FK, Hansen CH, Fey JA, Hansen M, Jacobsen NW, Halling-Sørensen B, et al. Toxicol in Vitro. 2012;26:343–50.CrossRefGoogle Scholar
  11. 11.
    Hecker M, Newsted JL, Murphy MB, Higley EB, Jones PD, Wu R, et al. Appl Pharmacol. 2006;217:114–24.CrossRefGoogle Scholar
  12. 12.
  13. 13.
    Schloms L, Storbeck KH, Swart P, Gelderblom WCA, Swart AC. J Steroid Biochem Mol Biol. 2012;128:128–38.CrossRefGoogle Scholar
  14. 14.
    Tonoli D, Fürstenberger C, Boccard J, Hochstrasser D, Jeanneret F, Odermatt A, et al. Chem Res Toxicol. 2015;28:955–66.CrossRefGoogle Scholar
  15. 15.
    van der Pas R, Hofland LJ, Hofland J, Taylor AE, Arlt W, Steenbergen J, et al. J Endocrinol. 2012;215:403–12.CrossRefGoogle Scholar
  16. 16.
    Xing Y, Edwards MA, Ahlem C, Kennedy M, Cohen A, Gomez-Sanches CE, et al. J Endocrinol. 2011;209:327–35.CrossRefGoogle Scholar
  17. 17.
    Zhang X, Chang H, Wiseman S, He Y, Higley E, Jones P, et al. Toxicol Sci. 2011;121:320–7.CrossRefGoogle Scholar
  18. 18.
    Iwaoka Y, Hashimoto R, Koizumi H, Yu J, Okabe T. Life Sci. 2010;86:894–8.CrossRefGoogle Scholar
  19. 19.
    Rosenmai AK, Nielsen FK, Pedersen M, Hadrup N, Trier X, Christensen JH, et al. Toxicol Appl Pharmacol. 2013;266:132–42.CrossRefGoogle Scholar
  20. 20.
    Quignot N, Arnaud M, Robidel F, Lecomte A, Tournier M, Cren-Olivé C, et al. Reprod Toxicol. 2012;33:339–52.CrossRefGoogle Scholar
  21. 21.
    Matty AJ. Fish endocrinology, Matty AJ (ED.). 1985; 138-173.Google Scholar
  22. 22.
    Kindler PM, Philipp DP, Gross MR, Bahr JM. Gen Comp Endocrinol. 1989;75:446–53.CrossRefGoogle Scholar
  23. 23.
    Carvalho VM, Nakamura OH, Vieira JGH. J Chromatogr B. 2008;872:154–61.CrossRefGoogle Scholar
  24. 24.
    Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. Clin Chim Acta. 2009;401:14–118.CrossRefGoogle Scholar
  25. 25.
    Koren L, Ng ESM, Soma KK, Wynne-Edwards KE. PLoS ONE. 2012;7:e32496.CrossRefGoogle Scholar
  26. 26.
    Winther CS, Nielsen FK, Hansen M, Styrishave B. Int J Toxicol. 2013;32:219–27.CrossRefGoogle Scholar
  27. 27.
    Jacobsen NW, Hansen CH, Nellemann C, Styrishave B, Halling-Sørensen B. Toxicol in Vitro. 2015;29:1729–35.CrossRefGoogle Scholar
  28. 28.
    Guldvang A, Hansen CH, Weisser JJ, Halling-Sørensen B, Styrishave B. Reprod Toxicol. 2015;58:174–83.CrossRefGoogle Scholar
  29. 29.
    Sørensen AM, Hansen CH, Bonomo S, Olsen L, Jørgensen FS, Weisser JJ, Kretschmann AC, Styrishave B. Toxicol in Vitro. 2016;34:71–80.Google Scholar
  30. 30.
    Abdel-Khalik J, Björklund E, Hansen M. J Chromatogr B. 2013;935:61–9.CrossRefGoogle Scholar
  31. 31.
    Yamashitaa K, Miyashirob Y, Maekubob H, Okuyamab M, Honmab S, Takahashia M, et al. Steroids. 2011;74:920–6.CrossRefGoogle Scholar
  32. 32.
    Fanellia F, Belluomoa I, Lalloa VDD, Cuomoa G, Iasiob RD, Baccinia M, et al. Steroids. 2011;76:244–53.CrossRefGoogle Scholar
  33. 33.
    US National Library of Medicine. ChemIDplus advanced http://chem.sis.nlm.nih.gov/chemidplus/. 2010.
  34. 34.
    Hansen M, Jacobsen NW, Nielsen FK, Björklund E, Styrishave B, Halling-Sørensen B. Anal Bioanal Chem. 2011;400:3409–17.CrossRefGoogle Scholar
  35. 35.
    ICH. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedure: Text and Methodology Q2(R1) 1-17 http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. 2005.
  36. 36.
    U.S. FDA, Guidance for Industry – Bioanalytical Method Validation, May 2001 http://www.fda.gov/downloads/Drugs/.%20.%20./Guidances/ucm070107.pdf.
  37. 37.
    Pouech C, Tournier M, Quignot N, Kiss A, Wiest L, Lafay F, et al. Anal Bioanal Chem. 2012;402:2777–88.CrossRefGoogle Scholar
  38. 38.
    Concas A, Popcu P, Sogliano C, Serra M, Purdy RH, Biggio G. Pharmacol Biochem Behav. 2000;66:39–45.CrossRefGoogle Scholar
  39. 39.
    Serra M, Pisu MG, Muggironi M, Parodo V, Papi G, Sari R, et al. Psychopharmacology (Berlin). 2001;158:48–54.CrossRefGoogle Scholar
  40. 40.
    Zorrilla LM, Gibson EK, Jeffay SC, Crofton KM, Setzer WR, Cooper RL, et al. Toxicol Sci. 2009;107:56–64.CrossRefGoogle Scholar
  41. 41.
    Pollard I. J Endocrinol. 1988;119:275–80.CrossRefGoogle Scholar
  42. 42.
    Abdel-Khalik J, Björklund E, Hansen M. J Chromatogr B. 2013;928:58–77.CrossRefGoogle Scholar

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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