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


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.


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



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).


  1. Bennett RD, Heftmann E (1965) Biosynthesis of Holarrhena alkaloids from pregnenolone and progesterone. Phytochemistry 4:873–879CrossRefGoogle Scholar
  2. Bennett RD, Ko ST, Heftmann E (1966) Isolation of estrone and cholesterol from the date palm, Phoenix dactylifera L. Phytochemistry 5:231–235CrossRefGoogle Scholar
  3. Bennett RD, Sauer HH, Heftmann E (1968) Progesterone metabolism in Digitalis lanata. Phytochemistry 7:41–50CrossRefGoogle Scholar
  4. Bonner J, Axtman G (1937) The growth of plant embryos in vitro. Preliminary experiments on the role of accessory substances. Proc Natl Acad Sci USA 23:453–457PubMedCrossRefGoogle Scholar
  5. Bosch AMG, Hollande FC, Woods GF (1974) Specificities of antisera against testosterone linked to albumin at different positions (C3, C11, C17). Steroids 23:699–711PubMedGoogle Scholar
  6. Caspi E, Lewis DO (1967) Progesterone: its possible role in the biosynthesis of cardenolides in Digitalis lanata. Science 156:519–520PubMedCrossRefGoogle Scholar
  7. Dohrn M, Faure W, Poll H, Blotevogel W (1926) Tokokinine, Stoff mit sexualhormonartiger Wirkung aus Pflanzenzellen. Med Klin 22:1417–1419Google Scholar
  8. Fantl VE, Wang DY (1983) Simultaneous production of monoclonal antibodies to dehydroepiandrosterone, oestradiol, progesterone and testosterone. J Endocrinol 100:367–376CrossRefGoogle Scholar
  9. Ferguson PL, Iden CR, McElroy AE, Brownawell BJ (2001) Determination of steroid estrogens in wastewater by immunoaffinity extraction coupled with HPLC–Electrospray–MS. Anal Chem 73:3890–3895PubMedCrossRefGoogle Scholar
  10. Gaskell SJ, Brownsey BG (1983) Immunoadsorption to improve gas chromatography/high-resolution mass spectrometry of estradiol 17β in plasma. Clin Chem 29:677–680PubMedGoogle Scholar
  11. Gawienowski AM, Gibbs CC (1968) Identification of cholesterol and progesterone in apple seeds. Steroids 12:545–550PubMedCrossRefGoogle Scholar
  12. Geuns JMC (1978) Steroid hormones and plant growth and development. Phytochemistry 17:1–14CrossRefGoogle Scholar
  13. Graves JMH, Smith WK (1967) Transformation of pregnenolone and progesterone by cultured plant cells. Nature 214:1248PubMedCrossRefGoogle Scholar
  14. Gross H, Bilk L (1968) Zur reaktion N-hydroxysuccinimid mit dicyklohexylcarbodiimid. Tetrahedron 24:6935–6939CrossRefGoogle Scholar
  15. Gruz J, Novak O, Strnad M (2008) Rapid analysis of phenolic acids in beverages by UPLC-MS/MS. Food Chem 111:789–794CrossRefGoogle Scholar
  16. Hauserova E, Swaczynova J, Dolezal K, Lenobel R, Popa I, Hajduch M, Vydra D, Fuksova K, Strnad M (2005) Batch immunoextraction method for efficient purification of aromatic cytokinins. J Chromatogr A 1100:116–125PubMedCrossRefGoogle Scholar
  17. Hernando MD, Ferrer C, Ulaszewska M, Garcia-Reyes JF, Molina-Diaz A, Fernandez-Alba AR (2007) Application of high-performance liquid chromatography–tandem mass spectrometry with a quadrupole/linear ion trap instrument for the analysis of pesticide residues in olive oil. Anal Bioanal Chem 389:1815–1831PubMedCrossRefGoogle Scholar
  18. Hradecka V, Novak O, Havlicek L, Strnad M (2007) Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography–mass spectrometry. J Chromatogr B 847:162–173CrossRefGoogle Scholar
  19. Iino M, Nomura T, Tamaki Y, Yamada Y, Yoneyama K, Takeuchi Y, Mori M, Asami T, Nakano T, Yokota T (2007) Progesterone: its occurence in plants and involvement in plant growth. Phytochemistry 68:1664–1673PubMedCrossRefGoogle Scholar
  20. Janeczko A, Skoczowski A (2005) Mammalian sex hormones in plants. Folia Histochem Cytobiol 43:71–79PubMedGoogle Scholar
  21. Kuronen P, Vaananen T, Pehu E (1999) Reversed-phase liquid chromatographic separation and simultaneous profiling of steroidal glycoalkaloids and their aglycones. J Chromatogr A 25:25–35CrossRefGoogle Scholar
  22. Kushnir MM, Rockwood AL, Roberts WL, Pattison EG, Bunker AM, Fitzgerald RL, Meikle AW (2006a) Performance characteristic of a novel tandem mass spectrometry assay for serum testosterone. Clin Chem 52:120–128PubMedCrossRefGoogle Scholar
  23. Kushnir MM, Rockwood AL, Roberts WL, Pattison EG, Owen WE, Bunker AM, Meikle AW (2006b) Development and performance evaluation of a tandem mass spectrometry assay for 4 adrenal steroids. Clin Chem 52:1559–1567PubMedCrossRefGoogle Scholar
  24. Li J, Biswas MG, Chao A, Russel DW, Chory J (1997) Conservation of function between mammalian and plant steroid 5α-reductases. Proc Natl Acad Sci USA 94:3554–3559PubMedCrossRefGoogle Scholar
  25. Li X, Xiong Z, Ying X, Cui L, Zhu W, Li F (2006) A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric method for the qualitative and quantitative analysis of the constituents of the flower Trollius ledibouri Reichb. Anal Chim Acta 580:170–180PubMedCrossRefGoogle Scholar
  26. Lindemann P, Luckner M (1997) Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata. Phytochemistry 46:507–513CrossRefGoogle Scholar
  27. Ma YC, Kim HY (1997) Determination of steroids by liquid chromatography mass spectrometry. J Am Soc Mass Spectrom 8:1010–1020CrossRefGoogle Scholar
  28. Milanesi L, Monje P, Boland R (2001) Presence of estrogen and estrogen receptor-like proteins in Solanum glaucophyllum. Biochem Biophys Res Commun 289:1175–1179PubMedCrossRefGoogle Scholar
  29. Novak O, Tarkowski P, Tarkowska D, Dolezal K, Lenobel R, Strnad M (2003) Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry. Anal Chim Acta 480:207–218CrossRefGoogle Scholar
  30. Novak O, Hauserova E, Amakorova P, Dolezal K, Strnad M (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochemistry 69:2214–2224PubMedCrossRefGoogle Scholar
  31. Pouzar V, Cerny I (1994) Preparation of 17(E)-3β-hydroxyandrost-5-ene-17-one (O-carboxymethyl)oxime derivatives with short peptide chain. Collect Czech Chem Commun 59:2042–2056CrossRefGoogle Scholar
  32. Rosati F, Danza G, Guarna A, Cini N, Racchi ML, Serio M (2005) New evidence of similarity between human and plant steroid metabolism: 5α-reductase activity in Solanum malacoxylon. Endocrinology 144:220–229CrossRefGoogle Scholar
  33. Saden-Krehula M, Kustrak D, Blazevic N (1991) Δ4-3-ketosteroids in flowers and leaves of Vitex agnus-castus. Acta Pharm Jugoslavica 41:237–241Google Scholar
  34. Sauer HH, Bennett RD, Heftmann E (1967) Pregnenolone metabolism in Digitalis lanata. Phytochemistry 6:1521–1526CrossRefGoogle Scholar
  35. Schlüsener MP, Bester K (2005) Determination of steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants utilising high-performance liquid chromatography/tandem mass spectrometry with electrospray and atmospheric pressure chemical ionisation. Rapid Commun Mass Spectrom 19:3269–3278PubMedCrossRefGoogle Scholar
  36. Schmidt G, Steinhart H (2002) Impact of extraction solvents on steroid contents determined in beef. Food Chem 76:83–88CrossRefGoogle Scholar
  37. Seaman FC (1982) Sesquiterpene lactones as taxonomic characters in the Asteraceae. Bot Rev 48:121–595CrossRefGoogle Scholar
  38. Seidel S, Kreis W, Reinhard E (1990) Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis, in cell cultures and plants of Digitalis lanata Ehrh. Plant Cell Rep 8:621–624CrossRefGoogle Scholar
  39. Shimada K, Mitamura K, Higashi T (2001) Gas chromatography and high performance liquid chromatography of natural steroids. J Chromatogr A 935:141–172PubMedCrossRefGoogle Scholar
  40. Skarzynski B (1933) An oestrogenic substance from plant material. Nature 131:766Google Scholar
  41. Skliar M, Curino A, Milanesi E, Benassati S, Boland R (2000) Nicotiana glauca: another plant species containing vitamin D-3 metabolites. Plant Sci 156:193–199PubMedCrossRefGoogle Scholar
  42. Storbeck KH, Kolar NW, Stander M, Swart AC, Prevoo D, Swart P (2008) The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids. Anal Biochem 372:11–20PubMedCrossRefGoogle Scholar
  43. Strnad M, Hanus J, Vanek T, Kaminek M, Ballantine JA, Fussel B, Hanke DE (1997) Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus x canadensis Moench, cv. Robusta). Phytochemistry 45:213–218CrossRefGoogle Scholar
  44. van Aerden C, Debrauwer L, Tabet JC (1998) Analysis of nucleoside-estrogen adducts by LC-ESI-MS–MS. Analyst 123:2677–2680CrossRefGoogle Scholar
  45. van Rhijn JA, Heskamp HH, Davelaar E, Jordi W, Leloux MS, Brinkman UAT (2001) Quantitative determination of glycosylated and aglycon isoprenoid cytokinins at sub-picomolar levels by microcolumn liquid chromatography combined with electrospray tandem mass spectrometry. J Chromatogr A 929:31–42CrossRefGoogle Scholar
  46. Vulliet E, Baugros JB, Flament-Waton MM, Grenier-Loustalot MF (2007) Analytical methods for the determination of selected steroid sex hormones and corticosteroids in wastewater. Anal Bioanal Chem 387:2143–2151PubMedCrossRefGoogle Scholar
  47. Wendroth S, Seitz UH (1990) Characterization and localization of progesterone 5α-reductase from cell cultures of foxglove (Digitalis lanata Ehrh.). Biochem J 266:41–46PubMedGoogle Scholar
  48. Yang XH, Xu ZH, Xue HW (2005) Arabidopsis Membrane Steroid Binding Protein 1 is involved in inhibition of cell elongation. Plant Cell 17:116–131PubMedCrossRefGoogle Scholar
  49. Yokota T, Baba J, Koba S (1984) Purification and separation of eight steroidal plant-growth regulators from Dolichos lablab seed. Agric Biol Chem 48:2529–2534Google Scholar
  50. Young IJ, Knights BA, Hillman JR (1977) Oestradiol and its biosynthesis in Phaseolus vulgaris L. Nature 267:249CrossRefGoogle Scholar
  51. Zhao XJ, Wang WZ, Wang JS, Yang J, Xu GW (2006) Urinary profiling investigation of metabolites with cis-diol structure from cancer patients based on UPLC–MS and HPLC–MS as well as multivariate statistical analysis. J Separation Sci 29:2444–2451PubMedCrossRefGoogle Scholar

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

Personalised recommendations