Analytical and Bioanalytical Chemistry

, Volume 411, Issue 8, pp 1623–1632 | Cite as

A new boronic acid reagent for the simultaneous determination of C27-, C28-, and C29-brassinosteroids in plant tissues by chemical labeling-assisted liquid chromatography-mass spectrometry

  • Lei Yu
  • Wen-Jing Cai
  • Tiantian Ye
  • Yu-Qi FengEmail author
Research Paper


Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C27-, C28-, or C29-BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C27-BRs exhibit similar bioactivities to C28- and C29-BRs, the biosynthetic pathways of C27-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C27-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C27-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C27-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C27-, C28-, and C29-BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C27-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C27-BRs (28-norCS and 28-norBL), 3 C28-BRs (CS, BL, and TY), and a single C29-BR (28-homoBL), are found to be 0.10–1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d5 labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C27-BRs.

Graphical abstract


Brassinosteroid 2-methyl-4-phenylaminomethylphenylboronic acid Chemical labeling LC-MS 


Funding information

The work is supported by the National Key R&D Program of China (Grant 2017YFC0906800), the National Natural Science Foundation of China (Grants 21475098, 21635006, and 31670373) and the Postdoctoral Science Foundation of China (Grant 2017M612493).

Compliance with ethical standards

Conflict of interest

The author declares that there are no conflicts of interest.

Supplementary material

216_2019_1612_MOESM1_ESM.pdf (363 kb)
ESM 1 (PDF 362 kb)


  1. 1.
    Yang CJ, Zhang C, Lu YN, Jin JQ, Wang XL. The mechanisms of brassinosteroids’ action: from signal transduction to plant development. Mol Plant. 2011;4(4):588–600.CrossRefPubMedGoogle Scholar
  2. 2.
    Altmann T. Molecular physiology of brassinosteroids revealed by the analysis of mutants. Planta. 1999;208(1):1–11.CrossRefPubMedGoogle Scholar
  3. 3.
    Bajguz A, Hayat S. Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiol Biochem. 2009;47(1):1–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Bajguz A, Tretyn A. The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry. 2003;62(7):1027–46.CrossRefPubMedGoogle Scholar
  5. 5.
    Kim TW, Chang SC, Lee JS, Takatsuto S, Yokota T, Kim SK. Novel biosynthetic pathway of castasterone from cholesterol in tomato. Plant Physiol. 2004;135(3):1231–42.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Joo SH, Jang MS, Kim MK, Lee JE, Kim SK. Biosynthetic relationship between C(2)(8)-brassinosteroids and C(2)(9)-brassinosteroids in rice (Oryza sativa) seedlings. Phytochemistry. 2015;111:84–90.CrossRefPubMedGoogle Scholar
  7. 7.
    Joo SH, Kim TW, Son SH, Lee WS, Yokota T, Kim SK. Biosynthesis of a cholesterol-derived brassinosteroid, 28-norcastasterone, in Arabidopsis thaliana. J Exp Bot. 2012;63(5):1823–33.CrossRefPubMedGoogle Scholar
  8. 8.
    Fujioka S, Noguchi T, Sekimoto M, Takatsuto S, Yoshida S. 28-Norcastasterone is biosynthesized from castasterone. Phytochemistry. 2000;55(2):97–101.CrossRefPubMedGoogle Scholar
  9. 9.
    Deng T, Wu D, Duan C, Guan Y. Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry. J Chromatogr A. 2016;1456:105–12.CrossRefPubMedGoogle Scholar
  10. 10.
    Ding J, Mao LJ, Guo N, Yu L, Feng YQ. Determination of endogenous brassinosteroids using sequential magnetic solid phase extraction followed by in situ derivatization/desorption method coupled with liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2016;1446:103–13.CrossRefPubMedGoogle Scholar
  11. 11.
    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;99(99):420–5.CrossRefPubMedGoogle Scholar
  12. 12.
    Jun Ding J-HW, 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;10:39–50.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Luo XT, Cai BD, Yu L, Ding J, Feng YQ. Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2018;1546:10–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Wu Q, Wu D, Shen Z, Duan C. Y. G. 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;1297(13):56–63.CrossRefPubMedGoogle Scholar
  15. 15.
    Xin P, Yan J, Fan J, Chu J, Yan C. An improved simplified high-sensitivity quantification method for determining brassinosteroids in different tissues of rice and Arabidopsis. Plant Physiol. 2013;162(4):2056–66.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Xin P, Yan J, Fan J, Chu J, Yan C. A dual role of boronate affinity in high-sensitivity detection of vicinal diol brassinosteroids from sub-gram plant tissues via UPLC-MS/MS. Analyst. 2013;138(5):1342–5.CrossRefPubMedGoogle Scholar
  17. 17.
    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;18(7):816–21.CrossRefPubMedGoogle Scholar
  18. 18.
    Wang L, Duan C, Wu D, Guan Y. Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion-tandem solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2014;1359:44–51.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang S, Ye J, Bie Z, Liu Z. Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting. Chem Sci. 2014;5(3):1135–40.CrossRefGoogle Scholar
  20. 20.
    Yu L, Ding J, Wang YL, Liu P, Feng YQ. 4-Phenylaminomethyl-benzeneboric acid modified tip extraction for determination of brassinosteroids in plant tissues by stable isotope labeling-liquid chromatography-mass spectrometry. Anal Chem. 2016;88(2):1286–93.CrossRefPubMedGoogle Scholar
  21. 21.
    Li D, Chen Y, Liu Z. Boronate affinity materials for separation and molecular recognition: structure, properties and applications. Chem Soc Rev. 2015;44(22):8097–123.CrossRefPubMedGoogle Scholar
  22. 22.
    Dicesare N, Adhikari DP, Heynekamp JJ, Heagy MD, Lakowicz JR. Spectroscopic and photophysical characterization of fluorescent chemosensors for monosaccharides based on N-phenylboronic acid derivatives of 1,8-naphthalimide. J Fluoresc. 2002;12(2):147–54.CrossRefGoogle Scholar
  23. 23.
    Guo Z, Shin I, Yoon J. ChemInform abstract: recognition and sensing of various species using boronic acid derivatives. Chem Commun. 2012;48(48):5956–67.CrossRefGoogle Scholar
  24. 24.
    Yu L, Ye T, Bai Y-L, Cai W-J, Ding J, Yuan B-F, et al. Profiling of potential brassinosteroids in different tissues of rape flower by stable isotope labeling - liquid chromatography/mass spectrometry analysis. Anal Chim Acta. 2018;1037:55–62.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of ChemistryWuhan UniversityWuhanChina

Personalised recommendations