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Pre-treatment Methods for Plant Hormone Samples

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Plant Hormone

Plant hormones are a class of trace compounds that are synthesized by plants themselves. Their main function is to participate in the regulation of the whole growth process of plants, including seed dormancy, germination, nutrition, growth, reproduction, maturation to senescence and some stress responses to the outside world, etc. They play an important role in every life process of plants. By regulating the metabolism of phytohormones can significantly improve the crop strain and enhance the yield or quality of crops. Accurate detection of the concentration changes and tissue-specific distribution of phytohormones in plants is an important prerequisite for understanding their metabolic pathways and transport processes.

Plant hormones mainly include growth hormone, gibberellin, cytokinin, abscisic acid, jasmonic acid, salicylic acid, oleuropein sterols and peptide hormones. There are three major difficulties in the detection of plant hormones: 1) the content of some important hormones is extremely low, usually in the order of ng/g or even pg/g; 2) plant hormones are generally unstable, easily decomposed, and sensitive to temperature and media environment; 3) the plant sample matrix is very complex, and direct detection will be affected by a large number of matrix interferences. Therefore, there is a high demand for the selectivity and debulking ability of the sample processing method.

Sample pretreatment is extremely important for plant hormone analysis based on chromatography and mass spectrometry. Pre-treatment determines the accuracy and precision of the analysis.

The chemical properties of phytohormones are important factors in determining their pretreatment methods. Phytohormones can be briefly classified into three categories according to their polarity differences. Mesopolar phytohormones, including growth hormone, gibberellin, abscisic acid, cytokinin, jasmonic acid and salicylic acid, are mostly weakly acidic. The others are alkaline and are collectively called acidic or alkaline phytohormones. Next are strongly polar phytohormones, mainly plant polypeptides, which are peptides composed of amino acids and are therefore amphoteric compounds with strong polarity. There are also weakly polar phytohormones, mainly oleuropein sterols, which are neutral compounds and have no ionization ability.

Fragmentation ions of multi-class plant hormones by triple quadrupole mass spectrometerFragmentation ions of multi-class plant hormones by triple quadrupole mass spectrometer (except ET) (Wang et al., 2020).

Acidic or alkaline phytohormones

The functions of these hormones are different, but their chemical properties are similar, as they are small molecules with relative molecular masses between 100 and 400. Therefore most chromatographic methods can achieve simultaneous detection of them. Pre-treatment usually takes advantage of their weak acidity and polarity for separation.

  • Sample extraction

Plant samples are mostly solid and need to be extracted by leaching to transfer the analytes into a liquid solution for subsequent analysis. The extraction process is relatively homogeneous. For weak acidic and weak alkaline phytohormones, leaching is usually performed with a mixture of methanol, isopropanol or acetonitrile and water. In order to prevent decomposition or oxidation of the analytes, the extraction is usually performed at 4°C or even lower, and a small amount of acidic compound is added to the extraction solution. For example, a modified Bieleski solvent with a solution of methanol/formic acid/water (15:1:4, v/v/v) was used for extraction of growth hormone, cytokinin, abscisic acid and jasmonic acid simultaneously.

  • Purification and enrichment

Due to the complexity of plant matrices, it is usually necessary to purify or enrich the extracts before chromatographic analysis. The methods of purification and enrichment usually include solid phase extraction, matrix solid phase dispersion extraction, liquid phase extraction and liquid phase microextraction.

Sample pretreatment methods for the determination of plant hormoneSample pretreatment methods for the determination of plant hormone (Wang et al., 2020).

Solid phase extraction (SPE) is the most widely used method for sample purification and decontamination. Based on the polarity of weak acid phytohormones, the commonly used SPE columns are C18 column, Oasis HLB column, etc. C18 column can remove a large amount of non-polar chlorophyll matrix, and also can remove some strong polar matrix by adjusting the eluent and the wetting solution. However, the removal capacity is limited. When the plant contains a large amount of non-polar substrates, it often generates large substrate interference peaks. Therefore, the C18 column alone can only be used for some samples with less complex matrix. In many cases, single column extraction methods are difficult to handle complex plant tissue extracts, so multiple small column tandem extraction methods are more widely used, such as C18 in combination with OasisMAX, Oa-sis HLB in combination with C1830 1, and Oasis HLB in combination with OasisMAX. Matrix solid phase dispersion extraction (MSPD) combines the solid phase extraction process and the leaching process simultaneously, allowing the extraction and purification steps to be combined into one.

Liquid-phase extraction is usually used in combination with solid-phase extraction in traditional methods to improve the purification effect. However, with the improvement of sensitivity and selectivity of mass spectrometry detection, direct mass spectrometry detection of extracts after liquid-liquid extraction has also been reported. A micro-liquid extraction method combining liquid-phase microextraction and reverse extraction. It uses the pH gradient between the sample phase and the acceptor phase as the mass transfer driving force, and the composition of the intermediate organic phase determines the extraction selectivity, which is well suited for the extraction of organic compounds with ionization ability.

Oleuropein sterols

  • Sample extraction

Unlike the previous acidic phytohormones, oleurosterols are less polar, and are usually extracted using 80% methanol-water solution at 4°C.

  • Purification and enrichment

Oleuropein sterol is in the middle polar region, the interference of phytochrome and fatty acid is more serious, and requires more thorough removal of impurities. The general sample pre-treatment method requires more than two steps of liquid phase extraction, and then tandem solid phase extraction to achieve the purpose of purification. In recent years, this process has been simplified somewhat by the introduction of new solid-phase extraction materials. Currently, solid-phase extraction, solid-phase microextraction, and on-line microsolid-phase extraction are commonly used.

  • Derivatization reaction

There are neither UV-responsive aromatic groups nor co-allelic groups in the structure of oleuropein sterols, nor ionizable groups in response to ESI sources, nor fluorescent response groups, so chemical sensitization derivatization is essential for the detection of oleuropein sterols compounds.


  • Sample extraction

Due to the strong polarity and amphiphilic nature of peptides, their extraction can usually be performed using pure distilled water. For samples with high water content, direct homogenization and filtration is usually sufficient for purification.

  • Purification and enrichment

Plant peptides are strongly polar and amphoteric, and are often purified by diethyl-aminoethyl cellulose (DEAE) columns, and multi-stage extraction using strong cation exchange columns and C18 columns has also been reported. Meanwhile, due to the distilled water extraction, the extracts usually contain a large amount of macromolecular proteins, which often need to be removed by filtration with dextran gel G25 (Sepha-dex G25).

Extraction and quantification method workflow for phytohormones and RNAExtraction and quantification method workflow for phytohormones and RNA (Cao et al., 2020)


  1. Wang, Liyuan, et al. "Recent developments and emerging trends of mass spectrometric methods in plant hormone analysis: a review." Plant Methods 16.1 (2020): 1-17.
  2. Cao, Da, et al. "A rapid method for quantifying RNA and phytohormones from a small amount of plant tissue." Frontiers in Plant Science 11 (2020): 605069.
For Research Use Only. Not for use in diagnostic procedures.


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