Analytical and Bioanalytical Chemistry

, Volume 408, Issue 24, pp 6799–6812 | Cite as

The determination of 22 natural brassinosteroids in a minute sample of plant tissue by UHPLC–ESI–MS/MS

  • Danuše Tarkowská
  • Ondřej Novák
  • Jana Oklestkova
  • Miroslav Strnad
Research Paper

Abstract

The triterpenoid plant hormones brassinosteroids (BRs) are believed to influence almost every aspect of plant growth and development. We have developed a sensitive mass spectrometry-based method for the simultaneous profiling of twenty-two naturally occurring brassinosteroids including biosynthetic precursors and the majority of biologically active metabolites. Using ultra-high performance liquid chromatographic (UHPLC) analysis, the run time was reduced up to three times (to 9 min) in comparison to standard HPLC BRs analyses, the retention time stability was improved to 0.1–0.2 % RSD and the injection accuracy was increased to 1.1–4.9 % RSD. The procedures for extraction and for two-step purification based on solid-phase extraction (SPE) were optimised in combination with subsequent UHPLC analysis coupled to electrospray ionisation tandem mass spectrometry (ESI–MS/MS) using Brassica flowers and Arabidopsis plant tissue extracts. In multiple reaction monitoring (MRM) mode, the average detection limit for BRs analysed was close to 7 pg, and the linear range covered up to 3 orders of magnitude. The low detection limits for this broad range of BR metabolites enabled as little as 50 mg of plant tissue to be used for quantitative analyses. The results of determinations exploiting internal standards showed that this approach provides a high level of practicality, reproducibility and recovery. The method we have established will enable researchers to gain a better understanding of the dynamics of the biosynthesis and metabolism of brassinosteroids and their modes of action in plant growth and development.

Keywords

Brassinosteroids Solid-phase extraction Ultra-high performance liquid chromatography Tandem mass spectrometry Arabidopsis thaliana Brassica napus 

Supplementary material

216_2016_9807_MOESM1_ESM.pdf (1.7 mb)
ESM 1(PDF 1.73 mb)

References

  1. 1.
    Caño-Delgado A, Yin Y, Yu C, Vafeados D, Mora-García S, Cheng J-C, et al. BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development. 2004. doi:10.1242/dev.01403.Google Scholar
  2. 2.
    Grove MD, Spencer FG, Rohwedder WK, Mandava NBN, Worley JF, Warthen JD, et al. Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen. Nature. 1979. doi:10.1038/281216a0.Google Scholar
  3. 3.
    Fujioka S, Sakurai A. Brassinosteroids. Nat Prod Rep. 1997. doi:10.1039/NP9971400001.Google Scholar
  4. 4.
    Bajguz A. Brassinosteroids—occurrence and chemical structures in plants. In: Hayat S, Ahmad A, editors. Brassinosteroids: A Class of Plant Hormone, Springer Science + Business Media B.V. 2011. p. 1–28.CrossRefGoogle Scholar
  5. 5.
    Bajguz A, Tretyn A. The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry. 2003. doi:10.1016/S0031-9422(02)00656-8.Google Scholar
  6. 6.
    Fujioka S, Inoue T, Takatsuto S, Yanagisawa T, Yokota T, Sakurai A. Biological activities of biosynthetically-related congeners of brassinolide. Biosci Biotech Biochem. 1995; doi: http://dx.doi.org/10.1271/bbb.59.1973.
  7. 7.
    Tarkowská D, Novák O, Floková K, Tarkowski P, Turečková V, Grúz J, et al. Quo vadis plant hormone analysis? Planta. 2014. doi:10.1007/s00425-014-2063-9.Google Scholar
  8. 8.
    Yokota T, Nomura T, Nakayama M. Identification of brassinosteroids that appear to be derived from campesterol and cholesterol in tomato shoots. Plant Cell Physiol. 1997. doi:10.1104/pp.126.2.770.Google Scholar
  9. 9.
    Takatsuto S, Ying B, Morisaki M, Ikekawa N. Microanalysis of brassinolide and its analogues by gas chromatography and gas chromatography-mass spectrometry. J Chromatogr. 1982. doi:10.1016/S0021-9673(00)81983-4.Google Scholar
  10. 10.
    Gamoh K, Yamaguchi I, Takatsuto S. Rapid and selective sample preparation for the chromatographic determination of brassinosteroids from plant material using solid-phase extraction method. Anal Sci. 1994. doi:10.2116/analsci.10.913.Google Scholar
  11. 11.
    Takatsuto S. Brassinosteroids: distribution in plants, bioassays and microanalysis by gas chromatography-mass spectrometry. J Chromarogr A. 1994. doi:10.1016/0021-9673(94)85202-2.Google Scholar
  12. 12.
    Swaczynová J, Novák O, Hauserová E, Fuksová K, Šíša M, Kohout L, et al. New techniques for the estimation of naturally occurring brassinosteroids. J Plant Growth Regul. 2007. doi:10.1007/s00344-006-0045-2.Google Scholar
  13. 13.
    Konstantinova OV, Antonchick AP, Oldham NJ, Zhabinskii VN, Khripach VA, Schneider B. Analysis of underivatized brassinosteroids by HPLC/APCI-MS. Occurrence of 3-epibrassinolide in Arabidopsis thaliana. Collect Czech Chem Commun. 2001; doi: 10.1135/cccc20011729.
  14. 14.
    Gamoh K, Abe H, Shimada K, Takatsuto S. Liquid chromatography mass spectrometry with atmospheric pressure chemical ionization of free brassinosteroids. Rapid Commun Mass Spectrom. 1996. doi:10.1002/(SICI)1097-0231(19960610)1.Google Scholar
  15. 15.
    Svatoš A, Antonchick A, Schneider B. Determination of brassinosteroids in the sub-femtomolar range using dansyl-3-aminophenylboronate derivatization and electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2004. doi:10.1002/rcm.1413.Google Scholar
  16. 16.
    Huo F, Wang X, Han Y, Bai Y, Zhang W, Yuan H, et al. A new derivatization approach for the rapid and sensitive analysis of brassinosteroids by using ultra high performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry. Talanta. 2012. doi:10.1016/j.talanta.2012.05.073.Google Scholar
  17. 17.
    Ding J, Mao L-J, Yuan B-F, Feng Y-Q. A selective pretreatment method for determination of endogenous active brassinosteroids in plant tissues: double layered solid phase extraction combined with boronate affinity polymer monolith microextraction. Plant Methods. 2013. doi:10.1186/1746-4811-9-13.Google Scholar
  18. 18.
    Wu Q, Wu D, Shen Z, Duan C, Guan Y. Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2013. doi:10.1016/j.chroma.2013.04.043.Google Scholar
  19. 19.
    Ding J, Wu J-H, Liu J-F, Yuan B-F, Feng Y-Q. Improved methodology for assaying brassinosteroids in plant tissues using magnetic hydrophilic material for both extraction and derivatization. Plant Methods. 2014. doi:10.1186/1746-4811-10-39.Google Scholar
  20. 20.
    Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution with application to the determination of amino acids and fat acids. J Biol Chem. 1940;133:737–44.Google Scholar
  21. 21.
    Lichtenthaler HK, Wellburn AR. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans. 1983. doi:10.1042/bst0110591.Google Scholar
  22. 22.
    Wellburn AR. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol. 1994. doi:10.1016/S0176-1617(11)81192-2.Google Scholar
  23. 23.
    Urbanová T, Tarkowská D, Novák O, Hedden P, Strnad M. Analysis of gibberellins as free acids by ultra performance liquid chromatography–tandem mass spectrometry. Talanta. 2013. doi:10.1016/j.talanta.2013.03.068.Google Scholar
  24. 24.
    Pratt JJ. Isotope dilution analysis using chromatographic separation of isotopic forms of the compound to be measured. Ann Clin Biochem. 1986. doi:10.1177/000456328602300305.Google Scholar
  25. 25.
    Khripach VA, Tarkowská D, Zhabinskii VN, Gulyakevich OV, Ermolovich YV, Drašar P, et al. Synthesis and mass spectral fragmentation patterns of brassinolide early biosynthetic precursors labeled at C-26. Nat Product Commun. 2013;8:771–4.Google Scholar
  26. 26.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003. doi:10.1021/ac020361s.Google Scholar
  27. 27.
    Araujo P. Key aspects of analytical method validation and linearity evaluation. J Chromatogr B. 2009. doi:10.1016/j.jchromb.2008.09.030.Google Scholar
  28. 28.
    Polko JK, Pierik R, van Zanten M, Tarkowská D, Strnad M, Voesenek LACJ, et al. Ethylene promotes hyponastic growth through interaction with ROTUNDIFOLIA3/CYP90C1 in Arabidopsis. J Exp Bot. 2013. doi:10.1093/jxb/ers356.Google Scholar
  29. 29.
    Singh AP, Fridman Y, Friedlander-Shani L, Tarkowska D, Strnad M, Savaldi-Goldstein S. Activity of the brassinosteroid transcription factors BRASSINAZOLE RESISTANT1 and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1/ BRASSINAZOLE RESISTANT2 blocks developmental reprogramming in response to low phosphate availability. Plant Physiol. 2014. doi:10.1104/pp.114.245019.Google Scholar
  30. 30.
    Stirk WA, Bálint P, Tarkowská D, Novák O, Strnad M, Ördög V, et al. Effect of light on growth and endogenous hormones in Chlorella minutissima (Trebouxiophyceae). Plant Physiol Biochem. 2014. doi:10.1016/j.plaphy.2013.05.037.Google Scholar
  31. 31.
    Aremu AO, Stirk WA, Kulkarni MG, Tarkowská D, Turečková V, Grúz J, et al. Evidence of phytohormones and phenolic acids variability in garden-waste-derived vermicompost leachate, a well-known plant growth stimulant. Plant Growth Regul. 2015. doi:10.1007/s10725-014-0011-0.Google Scholar
  32. 32.
    Stirk WA, Tarkowska D, Tureckova V, Strnad M, van Staden J. Abscisic acid, gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima. J Appl Phycol. 2014. doi:10.1007/s10811-013-0062-z.Google Scholar
  33. 33.
    Shahnejat-Bushehri S, Tarkowska D, Sakuraba Y, Balazadeh S. Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signaling. Nature Plants. 2016. doi:10.1038/NPLANTS.2016.13.Google Scholar
  34. 34.
    Winter J, Schneider B, Meyenburg S, Strack D, Adam G. Monitoring brassinosteroid biosynthetic enzymes by fluorescent tagging and HPLC analysis of their substrates and products. Phytochemistry. 1999. doi:10.1016/S0031-9422(98)00760-2.Google Scholar
  35. 35.
    Schmidt J, Altman T, Adam G. Brassinosteroids from seeds of Arabidopsis thaliana. Phytochemistry. 1997. doi:10.1016/S0031-9422(97)00177-5.Google Scholar
  36. 36.
    Fujioka S, Noguchi T, Yokota T, Takatsuto S, Yoshida S. Brassinosteroids in Arabidopsis thaliana. Phytochemistry. 1998. doi:10.1016/S0031-9422(98)00065-X.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Danuše Tarkowská
    • 1
  • Ondřej Novák
    • 1
  • Jana Oklestkova
    • 1
  • Miroslav Strnad
    • 1
  1. 1.Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany Academy of Sciences of the Czech Republic and Palacký UniversityOlomoucCzech Republic

Personalised recommendations