Brassinosteroids (BRs) are plant steroidal hormones that play crucial roles in plant growth and development. Accurate quantification of BRs in plant tissues is essential for understanding their biological functions. This study presents a comprehensive overview of the latest methods used for the quantification of BRs in plants. We discuss the principles, advantages and limitations of various analytical techniques, including immunoassays, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry that are used for the detection and quantification of BRs from complex plant matrixes. We also explore the use of isotopically labeled internal standards to improve the accuracy and reliability of BR quantification.

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany Czech Academy of Sciences Šlechtitelů 27 Olomouc CZ 78371 Czech Republic

See more in PubMed

Abe  H., Takatsuto  S., Nakayama  M. and Yokota  T. (1995) 28-Homotyphasterol, a new natural brassinosteroid from rice (Oryza sativa L.) Bran. Biosci. Biotechnol., Biochem.  59: 176–178.

Allevi  P., Anastasia  M., Cerana  R., Ciuffreda  P. and Lado  P. (1988) 24-Epibrassinolide uptake in growing maize root segments evaluated by multiple-selected ion monitoring. Phytochemistry  27: 1309–1313.

An  N., Zhu  Q.F., Yu  L., Chen  Y.T., Chen  S.L. and Feng  Y.Q. (2020) Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids. Talanta  217: 121058. PubMed

Antonchick  A.P., Schneider  B., Zhabinskii  V.N. and Khripach  V.A. (2004) Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester. Steroids  69: 617–628. PubMed

Bajguz  A. (2011) Brassinosteroids—occurrence and chemical structures in plants. In  Brassinosteroids: A Class of Plant Hormone. Edited by Hayat, S. and Ahmad, A. pp. 375–395. Springer, London.

Bajguz  A. and Tretyn  A. (2003) The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry  62: 1027–1046. PubMed

Chen  M., wang  R., Zhu  Y., Liu  M., Zhu  F. and Xiao  J. (2018) 4-Mercaptophenylboronic acid-modified spirally-curved mesoporous silica nanofibers coupled with ultra performance liquid chromatography–mass spectrometry for determination of brassinosteroids in plants. Food Chem.  263: 51–58. PubMed

Deng  T., Wu  D., Duan  C. and Guan  Y. (2016) 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 spektrometry. J. Chromatogr. A  1456: 105–111. PubMed

Deng  Z., Zhang  X., Tang  W., Oses-Prieto  J.A., Suzuki  N., Gendron  J.M., et al. (2007) A proteomics study of brassinosteroid response in Arabidopsis. Mol. Cell. Proteom.  6: 2058–2071. PubMed PMC

Ding  J., Mao  L.J., Guo  N., Feng  Y.Q. and Feng  Y.-Q. (2016) 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  1446: 103–113. PubMed

Ding  J, Wu  J, Liu  J, Yuan  B and Feng  Y. (2014) Improved methodology for assaying brassinosteroids in plant tissues using magnetic hydrophilic material for both extraction and derivatization. Plant Methods. 10: 39 PubMed PMC

Fujioka  S., Noguchi  T., Yokota  T., Takatsuto  S. and Yoshil  S. (1998) Brassinosteroids in Arabidopsis thaliana. Phytochemistry  48: 595–599. PubMed

Grove  M.D., Spencer  G.F., Rohwedder  W.K., Mandava  N., Worley  J.F., Warthen  J.D.  Jr., et al. (1979) Brassinolide, a plant growth-promoting steroid isolated from Brassica Napus pollen. Nature  281: 216–217.

Horgen  P.A., Nakagawa  C.H. and Irvin  R.T. (1984) Production of monoclonal antibodies to a steroid plant growth regulator. Can. J. Biochem. Cell Biol  62: 715–721.

Hou  Y., Qiu  J., Wang  Y., Li  Z., Zhao  J., Tong  X., et al. (2017) A quantitative proteomic analysis of brassinosteroid-induced protein phosphorylation in rice (Oryza sativa L.). Front. Plant Sci.  8: 514. PubMed PMC

Huo  S., Song  X., Li  L., Wang  R., Wang  X. and Ji  W. (2021) Boronic acid-functionalized scholl-coupling mesoporous polymers for online solid-phase extraction of brassinosteroids from plant-derived foodstuffs. J. Agric. Food Chem.  69: 4883–4893. PubMed

Joo  S.H., Jang  M.S., Kim  M.K., Lee  J.E. and Kim  S.K. (2015) Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings. Phytochemistry  111: 84–90. PubMed

Kanwar  M.K., Bajguz  A., Zhou  J. and Bhardway  R. (2017) Analysis of brassonosteroids in plants. J. Plant Growth Regul.  36: 1002–1030.

Khripach  V.A., Khripach  N.B., Zhabinskii  V.N., Zhiburtovich  Y.Y., Schneider  B. and De Groot  A. (2007) Synthesis of [7,7-2H2]epibrassinolide. J. Label. Compd. Radiopharm.  50: 1153–1158.

Khripach  V.A., Litvinovskaya  R.P., Raiman  M.E., Drach  S.V., Zhabinskii  V.N., Sviridov  O.V., et al. (2008a) Synthesis and immunochemical determination of 28-homobrassinosteroids. Vesti NAN Belarusi, ser khim navuk. 1: 47–58.

Khripach  V.A., Zhabinskii  V.N., Antonchick  A.P., Konstantinova  O.V. and Schneider  B. (2002b) Synthesis of hexadeuterated 23-dehydroxybrassinosteroids. Steroids  67: 1101–1108. PubMed

Khripach  V., Zhabinskii  V., Antonchick  A., Litvinovskaya  R., Drach  S., Sviridov  O., et al. (2008b) A new type of modified brassinosteroids for enzyme-linked immunosorbent assay. Nat. Prod. Commun.  3: 735–748.

Khripach  V.A., Zhabinskii  V.N., Ermolovich  Y.V. and Gulyakevich  O.V. (2012b) Synthesis of [26-2H3]-campesterin and [26-2H3]-campestanol, deuterated analogs of biosynthetic precursors of 28C-brassinosteroids. Chem. Nat. Compd.  48: 606–609.

Khripach  V.A., Zhabinskii  V.N., Gulyakevich  O.V. and Ermolovich  Y.V. (2012a) Synthesis of [26-2H3]-6-deoxo-24-epicastasterone. Chem. Nat. Compd.  48: 601–605.

Khripach  V.A., Zhabinskii  V.N., Konstantinova  O.V., Khripach  N.B., Antonchick  A.P. and Schneider  B. (2002a) Synthesis of [26-2H3]brassinosteroids. Steroids  67: 587–595. PubMed

Khripach  V.A., Zhabinskii  V.N., Litvinovskaya  R.P. (2011) Immunoassay of brassinosteroids. In  Brassinosteroids: A Class of Plant Hormone. Edited by Hayat, S. and Ahmad, A. pp. 375–395. Springer, London.

Kim  M.K., Jang  M.S., Youn  J.H., Son  S.H., Lee  J.E., Kim  T.W., et al. (2015) Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum. Physiol. Plant.  153: 58–67. PubMed

Kolbe  A., Marquardt  V. and Adam  G. (1992) Synthesis of tritium labelled 24-epibrassinolide. J. Label. Compd. Radiopharm.  31: 801–805.

Kolbe  A., Schneider  B., Voigt  B. and Adam  G. (1998) Labelling of biogenetic brassinosteroid precursors. J. Label. Compd. Radiopharm.  41: 131.

Li  Y., Deng  T., Duan  C., Ni  L., Wang  N. and Guan  Y. (2019) Dispersive matrix solid-phase extraction method coupled with high performance liquid chromatography-tandem mass spectrometry for ultrasensitive quantification of endogenous brassinosteroids in minute plants and its application for geographical distribution study. J. Agric. Food Chem.  67: 3037–3045. PubMed

Liu  X., Zhong  Y., Li  W., Li  G., Jin  N., Zhao  X., et al. (2022) Development and comprehensive SPE-UHPLC-MS/MS analysis optimization, comparison, and evaluation of 2,4-epibrassinolide in different plant tissues. Molecules  27: 831. PubMed PMC

Li  Q.F., Xiong  M., Xu  P., Huang  L.C.H., Zhang  C.Q., Liu  Q.-Q., et al. (2016) Dissection of brassinosteroid-regulated proteins in rice embryos during germination by quantitative proteomics. Sci. Rep.  6: 34583. PubMed PMC

Luo  X.T., Cai  B.D., Yu  L., Ding  J. and Feng  Y.Q. (2018) Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spektrometry. J. Chromatogr. A  1546: 10–17. PubMed

Manghwar  H., Hussain  A., Ali  Q. and Liu  F. (2022) Brassinosteroids (BRs) role in plant development and coping with different stresses. Int. J. Mol. Sci.  23: 1012. PubMed PMC

Marek  A., Klepetarova  B. and Elbert  T. (2015) A facile method for steroid labeling by heavy isotopes of hydrogen. Tetrahedron  71: 4874–4882.

Marek  A., Patil  M.R., Klepetarova  B., Kohout  L. and Elbert  T. (2012) A stereospecific pathway for the introduction of deuterium on the brassinosteroid skeleton by reductive dechlorination of chlorocarbonates. Tetrahedron Lett.  53: 2048–2050.

Mitchell  J.W., Mandava  N.B., Worley  J.F., Plimmer  J.R. and Smith  M.V. (1970) Brassins: a new family of plant hormones from rape pollen. Nature  225: 1065–1066. PubMed

Oklestkova  J., Tarkowská  D., Eyer  L., Elbert  T., Marek  A., Smržová  Z., et al. (2017) Immunoaffinity chromatography combined with tandem mass spectrometry: a new tool for the selective capture and analysis of brassinosteroid plant hormones. Talanta  170: 432–440. PubMed

Patil  M.R., Elbert  T. and Keri  R.S. (2015) Labelling of brassinosteroids by isotopes of hydrogen and carbon. RSC Adv.  5: 39726–39745.

Pradko  A.G., Litvinovskaya  R.P., Sauchuck  A.L., Drach  S.V., Baranovsky  A.V., Zhabinskii  V.N., et al. (2015) A new ELISA for quantification of brassinosteroids in plants. Steroids  97: 78–86. PubMed

Schmidt  J., Yokota  T., Spengler  B. and Adam  G. (1993) 28-Homoteasterone, a naturally occurring brassinosteroid from seeds of Raphanus sativus. Phytochemistry  34: 391–392.

Swaczynová  J., Novák  O., Hauserová  E., Fuksová  K., Šíša  M., Kohout  L., et al. (2007) New techniques for the estimation of naturally occurring brassinosteroids. J. Plant Growth Regul.  26: 1–14.

Takatsuto  S. (1994) Brassinosteroids: distribution in plants, bioassays, and micro-analysis by gas chromatography-mass spectrometry. J. Chromatogr. A  658: 3–15.

Takatsuto  S. and Ikekawa  N. (1986a) Synthesis of [26, 28-2H6]brassinolide, [26, 28-2H6]castasterone, [26, 28-2H6]typhasterol, and [26, 28-2H6]teasterone. Chem. Pharm. Bull.  34: 1415–1418.

Takatsuto  S. and Ikekawa  N. (1986b) Synthesis of deuterio-labelled brassinosteroids, [26, 28-2H6]brassinolide, [26, 28-2H6]castasterone, [26, 28-2H6]typhasterol, and [26, 28-2H6]teasterone. Chem. Pharm. Bull.  34: 4045–4049.

Takatsuto  S. and Ikekawa  N. (1986c) Synthesis of [26,28-2H6]crinosterol, a synthetic intermediate of [26,28-2H6]brassinolide and [26,28-2H6]castasterone. J. Chem. Soc., Perkin Trans.  1: 591–593.

Takatsuto  S., Kosuga  N., Abe  B., Noguchi  T., Fujioka  S. and Yokota  T. (1999) Occurrence of potential brassinosteroid precursor steroids in seeds of wheat and foxtail millet. J. Plant Res.  112: 27–33.

Takatsuto  S., Yokota  T., Omote  K., Gamoh  K. and Takahashi  N. (1989) Identification of brassinolide, castasterone and n orcastasterone (brassinone) in sunflower (Helianthus annuus L.) pollen. Agric. Biol. Chem.  53: 2177–2180.

Tarkowská  D., Novák  O., Floková  K., Tarkowski  P., Turečková  V., Grúz  J., et al. (2014) Quo vadis plant hormone analysis?  Planta  240: 55–76. PubMed

Tarkowská  D., Novák  O., Oklestkova  J. and Strnad  M. (2016) The determination of 22 natural brassinosteroids in a minute sample of plant tissue by UHPLC–ESI–MS/MS. Anal. Bioanal. Chem.  408: 6799–6812. PubMed

Taylor  P., Spuck  K., Smith  P., Sasse  J., Yokota  T., and Griffiths  P., et al. (1993) Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry  Planta  189: 91–100

Vukašinović  N., Wang  Y., Vanhoutte  I., Fendrych  M., Guo  B., Kvasnica  M., et al. (2021) Local brassinosteroid biosynthesis enables optimal root growth. Nat. Plants  7: 619–632. PubMed

Wang  X.Y., Xiong  C.F., Ye  T.T., Ding  J. and Feng  Y.O. (2020) Online polymer monolith microextraction with in-situ derivatization for sensitive detection of endogenous brassinosteroids by LC-MS. Microchem. J.  158: 105061.

Wierucka  M. and Biziuk  M. (2014) Application of magnetic nanoparticles for magnetic solid-phase extraction in preparing biological, environmental and food samples. TrAC-Trends Anal. Chem.  59: 50–58.

Wiseman  J., Ifa  D., Venter  A. and Cooks  R.G. (2008) Ambient molecular imaging by desorption electrospray ionization mass spectrometry. Nat. Protoc.  3: 517–524. PubMed

Wu  Q., Wu  D., Shen  Z., Duan  C. and Guan  Y. (2013) 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  1297: 56–63. PubMed

Xin  P., Li  B., Yan  J. and Ch  Y. (2018) Pursuing extreme sensitivity for determination of endogenous brassinosteroids through direct fishing from plant matrices and eliminating most interferences with boronate affinity magnetic nanoparticles. Anal. Bioanal. Chem.  410: 1363–1374. PubMed

Xin  P., Yan  J., Fan  J., Chu  J. and Yan  C. (2013a) An improved simplified high-sensitivity quantification method for determining brassinosteroids in different tissues of rice and Arabidopsis. Plant Physiol.  162: 2056–2066. PubMed PMC

Xin  P., Yan  J., Fan  J., Chu  J. and Yan  C. (2013b) A dual role of boronate affinity in high-sensitivity detection of vicinal diol brassinosteroids from sub-gram plant tissues via UPLC-MS/MS. Analyst  138: 1342–1345. PubMed

Xin  P., Yan  J., Li  B., Fang  S., Fan  J., Tian  H., et al. (2016) A comprehensive and effective mass spectrometry-based screening strategy for discovery and identification of new brassinosteroids from rice tissues. Front. Plant Sci.  7: 1786. PubMed PMC

Yan  S., Bhawal  R., Yin  Z., Thannhauset  T.W. and Zhang  S. (2022) Recent advances in proteomics and metabolomics in plants. Mol. hortic.  2: 17. PubMed PMC

Yokota  T., Nakayama  M., Wakisaka  T., Schmidt  J. and Adam  G. (1994) 3-Dehydroteasterone, a 3,6-diketobrassinosteroid as a possible biosynthetic intermediate of brassinolide from wheat grain. Biosci. Biotechnol. Biochem.  58: 1183–1185.

Yokota  T., Watanabe  S., Ogino  Y., Yamaguchi  I. and Takahashi  N. (1990) Radioimmunoasay for brassinosteroids and its use for comparative analysis of brassinosteroids in stems and seeds of Phaseolus vulgaris. J. Plant Growth Regul.  9: 151–159.

Yu  L., Cai  W.J., Ye  T. and Feng  Y.Q. (2019) 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. Anal. Bioanal. Chem.  411: 1623–1632. PubMed

Yu  L., Ye  T., Bai  Y.L., Cai  W.J., Ding  J., Yaun  B.F., et al. (2018) Profiling of potential brassinosteroids in different tissues of rape flower by stable isotope labeling – liquid chromatography/mass spectrometry analysis. Anal. Chim. Acta  1037: 55–62. PubMed

Zhang  C., Žukauskaitė  A., Petřík  I., Pěnčík  A., Hönig  M., Grúz  J., et al. (2021) In situ characterisation of phytohormones from wounded Arabidopsis leaves using desorption electrospray ionisation mass spectrometry imaging. Analyst  146: 2653–2663. PubMed