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Journal of Plant Growth Regulation

, Volume 28, Issue 2, pp 125–136 | Cite as

Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS

  • Radim Simerský
  • Ondřej Novák
  • David A. Morris
  • Vladimír Pouzar
  • Miroslav StrnadEmail author
Article

Abstract

We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ4-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10−10 mol L−1 and cross-reactivity with most Δ4-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry [UPLC-ESI(+)-MS/MS]. In multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to 10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species, that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone, were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea.

Keywords

Ultra-performance liquid chromatography (UPLC) Tandem mass spectrometry (MS/MS) Immunoaffinity purification Steroids Plant extracts Digitalis purpurea Nicotiana tabacum Inula helenium 

Notes

Acknowledgments

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (MSM 6198959216) and the Grant Agency of the Academy of Sciences of the Czech Republic (KAN 200380801).

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Radim Simerský
    • 1
  • Ondřej Novák
    • 1
  • David A. Morris
    • 1
  • Vladimír Pouzar
    • 2
  • Miroslav Strnad
    • 1
    Email author
  1. 1.Laboratory of Growth Regulators, Faculty of SciencePalacký University & Institute of Experimental Botany AS CROlomoucCzech Republic
  2. 2.Institute of Organic Chemistry and Biochemistry AS CRPrague 6Czech Republic

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