I. Introduction.
Plant endogenous hormones are a class of low-molecular-weight organic compounds with important physiological functions produced in plants, which play an important role in the growth, development and adaptation to environmental stress of plants. In recent years, with the development of liquid chromatography-mass spectrometry technology, important progress has been made in the detection and analysis of plant endogenous hormones. This article will introduce the basic principles, methods and applications of liquid chromatography-mass spectrometry of plant endogenous hormones.
Second, the basic principle.
Liquid chromatography-mass spectrometry (LC/MS) is a method that combines liquid chromatography with mass spectrometry to separate samples and then use mass spectrometry to qualitatively and quantitatively analyze the separated components. In the detection of plant endogenous hormones, liquid chromatography-mass spectrometry has the advantages of high sensitivity, high resolution, and high throughput, which can detect multiple hormone components at the same time and provide accurate molecular weight information.
3. Methodology. 1.Sample handling.
For the detection of plant endogenous hormones, plant samples first need to be processed. The methods commonly employed include extraction, purification, concentration, etc., to remove impurities and increase the concentration of hormones. During the extraction process, suitable solvents such as methanol, ethanol, etc., can be selected, depending on the polarity and solubility of the hormone. The clean-up process typically includes methods such as extraction, column chromatography, etc., to further remove impurities. Finally, a concentration step is performed to increase the concentration of the sample to a level where LC/MS can be performed.
2.Liquid chromatography separation.
In liquid chromatography separations, high-performance liquid chromatography (HPLC) is typically used to separate components in a sample by means of columns with different pore sizes. Appropriate columns and mobile phases can be selected based on the polarity, molecular weight, and functional groups of the hormone. During separation, the different components flow out of the column one after the other and pass through the detector for detection.
3.Mass spectrometry.
In mass spectrometry, the components separated by liquid chromatography are introduced into the mass spectrometer. In mass spectrometers, components are subjected to electron bombardment or chemical ionization, resulting in ionic fragmentation. By analyzing the charge-to-mass ratio and abundance of the ion fragments, information such as the molecular weight and functional groups of the components can be determined. At the same time, multi-stage mass spectrometry can be used to perform more in-depth analysis of components and improve the accuracy of identification.
Fourth, the experimental procedure.
1. Sample extraction process.
1) Take out the ultra-low temperature preservation sample, grind it in liquid nitrogen until it is dry powder, and weigh an appropriate amount of fresh plant sample in a glass test tube
2) Add isopropanol-water-hydrochloric acid mixed extract to the glass test tube
3) Add 8 l of 1 g ml of internal standard solution and shake at low temperature for 30 min
4) Add dichloromethane and shake at low temperature for 30 min
5) Centrifuge at 13000 r min for 5 min at low temperature, and remove the organic phase
6) Avoid light, dry the organic phase with nitrogen, and use methanol (01% formic acid) reconstituted;
7) 4 Centrifuge for 10min (13000g) and take the supernatant over 022 m membrane, HPLC-MS MS.
Note: Plant hormones are unstable, and the extraction process should be kept on a 4 ice box throughout the operation.
2.Standard solution preparation.
In methanol (01% formic acid) as a solvent with a gradient of 01ng/ml，0.2ng/ml，0.5 ng ml, 2 ng ml, 5 ng ml, 20 ng ml, 50 ng ml, 200 ng ml of sa, ja, mesa standard solution, and add the final concentration of 20 ng ml of internal standard solution, and the linear outliers were eliminated when the standard curve equation was actually plotted.
3.Chromatography mass spectrometry detection conditions.
The data acquisition system mainly includes high performance liquid chromatography (HPLC) (Agilent 1290, tandem mass spectrometry, MS MS) (Applied Biosystems 6500 Quadrupole Trap, 4.)Qualitative and quantitative analysis.
Mass spectrometry data were processed using the software Analyst. The figure below shows the total ions current (tic) of the sample, that is, the intensities of all ions in the mass spectrum at each time point are added and continuously depicted, the following figure shows the total ion current of plant hormone detection, the abscissa is the retention time (RT) of plant hormone detection, and the ordinate is the ion current intensity (Count per Second, cps) of ion detection
Total ion current plot.
Selective reaction monitoring conditions for protonation or deprotonation of plant hormones ([m+h]+or[m-h]-).
5. Experimental results and analysis.
1.Some of the standard curves are displayed.
MESA calibration curves.
SA calibration curves.
2.Some methodological validation data.
Substance class, name, linear range, equation, correlation coefficient, and weight coefficient.
Fourth, application. 1.Plant growth regulator research.
Plant endogenous hormones have an important impact on the growth and development of plants, so the study of plant growth regulators is of great significance for agricultural production. Through liquid chromatography-mass spectrometry, the hormone components in plant growth regulators can be detected and analyzed, and their mechanism and effect can be understood, which can provide a scientific basis for agricultural production.
2.Research on environmental stress responses.
Plants will produce a series of physiological and biochemical changes in the face of environmental stress, among which the synthesis and metabolism of hormones are one of the important regulatory pathways. Liquid chromatography-mass spectrometry (LC-MS) can detect and analyze the endogenous hormone changes produced by plants under environmental stress, and understand the response mechanism and adaptation strategies of plants to environmental stress.
3.Evaluation of the quality and safety of agricultural products.
The problem of hormone residues in agricultural products has always been one of the focuses of attention. The amount of hormone residues in agricultural products can be detected and analyzed by liquid chromatography-mass spectrometry (LC-MS), which provides a scientific basis for the quality and safety evaluation of agricultural products. At the same time, this method can also be used to detect and analyze pesticide residues to ensure the safety and food health of agricultural products.
V. Conclusions. Plant endogenous hormone liquid chromatography-mass spectrometry is a highly sensitive, high-resolution, and high-throughput assay that can detect multiple hormone components simultaneously and provide accurate molecular weight information. This method has a wide range of application prospects in the fields of plant growth regulator research, environmental stress response research and agricultural product quality and safety evaluation. As the technology continues to evolve and improve, this method will play an even more important role in the future.