A method for detecting plant hormones, a detection kit and its application

Abstract

The present invention provides a method for detecting plant hormones, a detection kit and an application thereof, the method comprising: extracting plant samples with methanol and dimethyl sulfoxide in sequence; combining the extracts and performing detection. The detection method of the present invention can detect many types of plant hormones, has high accuracy, precision and sensitivity, does not require column enrichment or drying and concentration steps, requires a small amount of sample, is simpler to operate, effectively reduces manpower, time and financial costs, and can be used for the simultaneous detection of 34 plant hormones in plant samples or related samples.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry and relates to a detection method, a detection kit and an application of plant hormones. Background Art

[0002] Phytohormones play key roles in plant growth, development, and responses to biotic and abiotic cues. Each class of hormones has characteristic biological effects. To understand the regulatory effects of phytohormones on plant growth and development, accurate and efficient measurements of individual phytohormones at the organ, cellular, and subcellular levels are required. Simultaneous quantitative analysis of multiple classes of hormones provides the basis for defining additive, synergistic, or antagonistic hormone activities and identifying hormone networks that regulate plant functions.

[0003] The existing methods for determining plant hormones mainly include radioimmunoassay and enzyme-linked immunosorbent assay, HPLC assay, GC-MS assay and LC-MS assay. Radioimmunoassay and enzyme-linked immunosorbent assay have the characteristics of high sensitivity, but the cross-reactivity of the antibodies required usually leads to reduced specificity and accuracy of these methods. The HPLC assay is easily affected by the matrix, and its sensitivity and specificity are limited in the measurement of various endogenous plant hormones. The GC-MS assay has been successfully used for high-sensitivity specific identification and quantification of plant hormones, but GC-MS analysis usually requires extraction, purification and chemical derivatization steps before analysis, which is cumbersome and time-consuming. The LC-MS assay has high sensitivity, specificity, accuracy and reproducibility, but the current detection method can only detect several or more than a dozen plant hormones at the same time, and cannot fully reflect the content of more plant hormones at the same time.

[0004] Therefore, in the art, it is desired to develop a method for detecting plant hormones that can extract more plant hormones at one time, has high sensitivity and specificity, and has simple steps and short time consumption. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting plant hormones. The method of the present invention can extract more than 20 plant hormones at one time, can simultaneously detect the presence and specific content of more than 20 plant hormones, and the method has high accuracy, precision and sensitivity, simple steps and short time consumption.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In one aspect, the present invention provides a method for detecting plant hormones, wherein the method uses methanol and dimethyl sulfoxide (DMSO) to extract samples in sequence; the extracts are combined and detected.

[0008] There are many types of plant hormones in samples such as plants and their content is very low, making it difficult to detect them completely. The inventors have conducted a large number of experiments to study the extraction methods of plant hormones, including single extraction using a single solvent or a mixed solvent, including single extraction using methanol, ethanol, DMSO, formic acid aqueous solution, hydrochloric acid aqueous solution or sodium hydroxide aqueous solution, methanol DMSO mixed solution, etc. The extraction effects are not ideal. Further, the inventors conducted a two-step extraction study and unexpectedly found that the most types of plant hormones can be detected by first extracting the sample twice with methanol and then with DMSO, and then combining the two extracts for detection. As shown in Tables 2, 3, and 4, for different plant samples, the most types of plant hormones were detected using a combination of methanol and DMSO, reaching 28 to 33 types.

[0009] Generally, technicians in this field may think that compared with using a single extraction solvent, multiple extractions with different types of solvents can obtain more types of compounds. However, the inventors extracted plant samples twice with methanol solution and first extracted plant samples with aqueous solution and then with methanol, and combined the extracts for testing. The types of plant hormones detected by the two were the same; however, using methanol first and then DMSO significantly increased the types of plant hormones detected. It may be because there is no obvious difference in the solubility of plant hormones in methanol and water, but there is a certain difference in methanol and DMSO. One or more plant hormones are insoluble or difficult to dissolve in methanol, but are easily soluble in DMSO. The specific reason still needs further study.

[0010] In addition, it is worth noting that the inventors found that the samples must be extracted with methanol first and then with DMSO; the effect of extracting with DMSO first and then with methanol is different from the former, and the types of plant hormones detected in the combined extract solution are significantly lower than those under the conditions of first methanol and then DMSO extraction (Table 2 and Table 3). Perhaps it is because methanol has a small molecular weight and good permeability to plant cells, and it is easy to enter the cells to inactivate enzymes in the cells, and these enzymes may hydrolyze plant hormones during the extraction process. For example, alkaline phosphatase in plants can cause the hydrolysis of nucleotide-type cytokinins. Using methanol first reduces the hydrolysis of plant hormones by enzymes, so that more types of plant hormones are obtained by extraction. It may also be that the permeability of plant cells is changed by using methanol first, and when DMSO is used for extraction, DMSO is easier to enter the cells, making the hormones inside the cells easier to be dissolved by DMSO. It may also be the combined effect of the above two, and the specific reasons need further study.

[0011] In some embodiments, the specific steps of sequentially extracting the plant sample with methanol and dimethyl sulfoxide include:

[0012] (1) Take a plant sample, add methanol, shake at a constant temperature, centrifuge or filter to obtain an extract, and take the filter residue for later use;

[0013] (2) Add DMSO to the filter residue of step (1), shake at a constant temperature, and centrifuge or filter to obtain an extract.

[0014] The extracts from step (1) and step (2) are combined to obtain a sample solution.

[0015] Preferably, an internal standard is added before the constant temperature oscillation in step (1), and the internal standard includes IAA-D5 (indoleacetic acid-D5) and / or ABA-D6 (abscisic acid-D6).

[0016] Preferably, the constant temperature oscillation in step (1) is oscillated at 20°C or below (e.g., 20°C, 15°C, 10°C, 5°C, etc.) for no less than 3 hours (e.g., 3 hours, 5 hours, 8 hours, 10 hours, etc.); the constant temperature oscillation in step (2) is oscillated at 20°C or below (e.g., 20°C, 18°C, etc.) for no less than 2 hours (e.g., 3 hours, 5 hours, 8 hours, 10 hours, etc.). The frequency of oscillation in steps (1) and (2) is 1000-1500 rpm (e.g., 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm or 1500 rpm). Plant hormones are unstable, easily decomposed, and relatively sensitive to temperature and other conditions. For example, gibberellin hormones are usually relatively stable under acidic conditions; at the same time, rapid decomposition occurs when the temperature exceeds 40°C. In step (1) and step (2), methanol and DMSO are used for extraction respectively, wherein the temperature below 20°C is generally not lower than the melting point of the extraction solvent to prevent the solvent from solidifying into a solid. The extraction temperature, extraction time and oscillation frequency, etc. will affect the extraction efficiency and the stability of the plant hormone. The constant temperature oscillation conditions of step (1) and step (2) are obtained by the inventor through a large number of experimental optimizations. Under this condition, the extraction efficiency can be improved while minimizing the influence of temperature. In some embodiments, when the sample is a relatively loose and soft plant tissue such as the stem, flower, and leaf of a plant, the oscillation time of step (1) is 3 hours; when the plant sample is a relatively dense plant tissue such as a seed, the time of step (1) can be appropriately extended to 4 hours or more; the extraction time of step (2) can also be appropriately extended according to the different samples.

[0017] Preferably, the sample is a freeze-dried powder of plant tissue; relative to 20-400 mg (e.g., 20 mg, 50 mg, 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg or 400 mg) of the sample, the amount of methanol is 800-1200 μL (e.g., 800 μL, 900 μL, 1000 μL, 1100 μL or 1200 μL); the amount of dimethyl sulfoxide is 200-600 μL (e.g., 200 μL, 300 μL, 400 μL, 500 μL or 600 μL).

[0018] The method of the present invention is particularly suitable for extracting a small amount of plant samples. The amount of plant samples can be as low as 20-400 mg. By first using 800-1200 μL of methanol and then using 200-600 μL of DMSO for extraction, more types and higher concentrations of plant hormones can be obtained, so the extract can be directly taken for detection without going through a concentration process. During the research process, in order to detect more plant hormones, the inventor tried to increase the amount of plant samples and enrich the extremely low concentration of plant hormones through concentration so that these plant hormones can be detected, but plant hormones are unstable, and the concentration process will lead to the loss of plant hormones, and the ideal effect was not achieved.

[0019] Of course, technicians generally know that proportionally increasing the sample size and the volume of the extraction solvent has a negligible effect on the extraction effect. The amounts of the above-mentioned sample, methanol and dimethyl sulfoxide can be proportionally increased. For example, the amounts of the three can be proportionally increased by 2 times, 5 times, 10 times, 20 times, 30 times, etc.

[0020] In some preferred embodiments of the present invention, the dosages of sample, methanol and dimethyl sulfoxide are 50 mg, 1000 μL and 500 μL respectively. For most plant samples or related samples, such as wheat seeds and wheelbarrow flowers, the extraction effect is best under this ratio.

[0021] In some embodiments, the plant hormone comprises one or a combination of at least two of the following: methyl jasmonate (MEJA), 6-benzylaminopurine (6-BA), kinetin (KT), isopentenyl adenine riboside (IPA), brassinolide (BR), isopentenyl adenine (IP), trans-zeatin riboside (TZR), melatonin (MT), cis-zeatin (CZ), trans-zeatin (TZ), dihydrozeatin (DZ), indole-3-acetic acid methyl ester (MeIAA), indolebutyric acid (IBA), indoleacetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC), strigolactone ( GR24), gibberellin 3 (GA3), gibberellin 1 (GA1), gibberellin 4 (GA4), abscisic acid (ABA), gibberellin 7 (GA7), gibberellin 5 (GA5), gibberellin 19 (GA19), gibberellin 24 (GA24), N-jasmonic acid-isoleucine (JA-ILe), glycosylated salicylic acid (SAG), gibberellin 51 (GA51), jasmonic acid (JA), gibberellin 8 (GA8), dihydrojasmonic acid (DHJA), salicylic acid (SA), 3-indolecarboxylic acid (ICA), indole-3-carboxaldehyde (ICAld), and 12-oxophytodienoic acid (OPDA).

[0022] In some embodiments, the plant hormone comprises BR. In some embodiments and comparative examples of the present application, BR can be detected by extracting with methanol first and then with DMSO, while BR cannot be detected by extracting with methanol twice, and BR cannot be detected by extracting with 30% methanol and then with DMSO or other two combined extractions. This indicates that the extraction method of methanol first and then with DMSO is particularly suitable for detecting BR in plant samples. In addition, it is worth noting that in the embodiments of the present application, BR can be detected in different samples, including wheat seeds, peach blossoms, wheelbarrow flowers, honey and Ophiopogon japonicus plants.

[0023] BR is a member of the brassinosteroid class. Brassinosteroids are ubiquitous in plants. They have a unique mechanism of action and a wide range of physiological effects, but their dosage is extremely small, only one thousandth of any of the other five major hormones. In addition, the ionization efficiency of brassinosteroids in mass spectrometry is low, and the detection limit of direct analysis of brassinosteroids by liquid chromatography-mass spectrometry can only reach 1-10 mg / L (Pan Jialiang et al., Research Progress in Analysis of Brassinosteroid Hormones, Chromatography, 2010, No. 2, Vol. 29, 105-110). Enrichment, concentration and / or derivatization are usually required to detect brassinosteroid hormones in plant samples. For example, Chinese Patent 201210486507.3 (Announcement No.: CN102980953B) uses double-layer solid phase extraction columns, liquid-liquid extraction and blow-drying and concentration processes to quantitatively detect brassinosteroids (including BR). The method of the present invention can detect BR in plant samples without the need for enrichment, concentration or derivatization processes.

[0024] In some embodiments, the detection is performed using LC-MS; the LC conditions of LC-MS include: using ACQUITY UPLC CSH C 18 The chromatographic column has an injection volume of 2 μL to 5 μL and a column temperature of 40°C; mobile phase A and mobile phase B are used for gradient elution, wherein mobile phase A is 2 mM ammonium formate 0.05% formic acid water and mobile phase B is 0.05% formic acid methanol; the gradient elution conditions include: 0 to 2 min, 10% mobile phase B; 2 to 4 min, 10 to 30% mobile phase B; 4 to 19 min, 30 to 95% mobile phase B; 19 to 19.10 min, 95 to 10% mobile phase B; 19.10 to 22 min, 10% mobile phase B, and the flow rate is 0.25 mL / min.

[0025] The MS conditions of LC-MS included: using an electrospray ionization source, in positive ion mode, the mass spectrometer voltage was 4500 V, the ion source temperature was 400°C, the curtain gas was 40 psi, the nebulizer gas was 40 psi, and the auxiliary gas was 35 psi; in negative ion mode, the mass spectrometer voltage was -4500 V, the ion source temperature was 400°C, the curtain gas was 40 psi, the nebulizer gas was 40 psi, and the auxiliary gas was 35 psi.

[0026] In some embodiments, the sample is a freeze-dried powder, such as a low-temperature freeze-dried powder at 4°C.

[0027] In some embodiments, the sample is one or a combination of at least two of plant roots, stems, leaves, flowers, fruits, seeds, pollen, galls, branches, etc.

[0028] In some embodiments, the sample is one or a combination of at least two of wheat seeds, peach blossoms, Ophiopogon japonicus, apple fruit, honey, pitaya fruit, Arabidopsis thaliana, and wheelbarrow flower.

[0029] Another aspect of the present invention provides a kit for detecting plant hormones, the kit comprising: an extraction reagent 1, an extraction reagent 2 and a reference substance; the extraction reagent 1 is methanol, the extraction reagent 2 is DMSO; the reference substance comprises a plant hormone compound. The kit comprises the extraction reagent and the reference substance in the above method, and can be used to prepare a sample solution and a series of concentrations of a reference substance solution of plant hormones, so as to facilitate the detection of plant hormones in a sample according to the above method.

[0030] In some embodiments, the kit further comprises a diluent, and the reference substance comprises one or at least two of the following solid compounds: methyl jasmonate, 6-benzylaminopurine, kinetin, isopentenyl adenine nucleoside, brassinolide, isopentenyl adenine, trans-zeatin riboside, melatonin, cis-zeatin, trans-zeatin, dihydrozeatin, indole-3-acetic acid methyl ester, indolebutyric acid, indoleacetic acid, 1-aminocyclopropane-1-carboxylic acid, strigolactone, gibberellin 3, gibberellin 1, gibberellin 4, abscisic acid, gibberellin 7, gibberellin 5, gibberellin 19, gibberellin 24, N-jasmonic acid-isoleucine, glycosylated salicylic acid, gibberellin 51, jasmonic acid, gibberellin 8, dihydrojasmonic acid, salicylic acid, 3-indolecarboxylic acid, indole-3-carboxaldehyde or 12-oxophytodienoic acid; the reference substance can be dissolved in the diluent. Plant hormones have poor stability. Their reference substances can be placed in the test kit in solid form, and corresponding diluents can be prepared. They can be dissolved and diluted when used. This is convenient and can extend the shelf life of the kit.

[0031] In some embodiments, the reference substance is a mixed reference substance solution or a mixed reference substance stock solution with different concentration gradients. The concentration of each reference substance in the mixed reference substance stock solution here is relatively high, and can be 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1000 times, etc. of the injection concentration during detection (e.g., LC-MS detection). The (high concentration) mixed reference substance stock solution is more stable than the mixed reference substance solution of lower concentration, which is conducive to storage and transportation. In addition, a (high concentration) mixed reference substance stock solution can be diluted into a series of mixed reference substances of different concentrations before detection. The mixed reference substance solution and the mixed reference substance stock solution include one or at least two of the following compounds: methyl jasmonate, 6-benzylaminopurine, kinetin, isopentenyl adenine nucleoside, brassinolide, isopentenyl adenine, trans-zeatin nucleoside, melatonin, cis-zeatin, trans-zeatin, dihydrozeatin, indole-3-acetic acid methyl ester, indolebutyric acid, indoleacetic acid, 1-aminocyclopropane-1-carboxylic acid, strigolactone, gibberellin 3, gibberellin 1, gibberellin 4, abscisic acid, gibberellin 7, gibberellin 5, gibberellin 19, gibberellin 24, N-jasmonic acid-isoleucine, glycosylated salicylic acid, gibberellin 51, jasmonic acid, gibberellin 8, dihydrojasmonic acid, salicylic acid, 3-indolecarboxylic acid, indole-3-carboxaldehyde or 12-oxophytodienoic acid.

[0032] In some embodiments, the kit further comprises an internal standard comprising IAA-D5 and ABA-D6.

[0033] The third aspect of the present invention provides the use of the method and the kit in detecting plant hormones.

[0034] In some embodiments, the method or kit can be used to detect plant samples, and the growth state or health state of the plant, or the growth state or health state of a certain part of the plant, can be determined based on the detection results of the plant samples.

[0035] Plants with different growth states or health states have different types, contents, and distributions of plant hormones. The growth state or health state of a plant can be identified by measuring the types, contents, and distributions of plant hormones. For example, the plant hormone content of a plant, or a certain part of a plant, such as a flower or leaf, is measured by the method, and the types, contents, or distributions of plant hormones of healthy plants and plants suffering from one or more diseases are studied. The sample to be tested is then measured, and the types, contents, or distributions of plant hormones of the sample are compared with those of healthy or diseased plants to determine whether the sample is healthy or diseased. Similarly, the method can also be used to distinguish or differentiate whether a diseased plant suffers from a certain disease, or to determine whether a sample is in a certain growth stage or growth age, etc. For example, in one embodiment of the present invention, the method can be used to determine whether the wheel flower is fresh or withered. It is also possible to measure multiple or large quantities of plant samples, and distinguish healthy and diseased plants in the samples through the status of plant hormones, distinguish different types of diseased plants, distinguish plants at different growth stages, or select plants with a certain disease from a whole, etc. For example, all test results can be clustered through clustering methods such as principal component analysis, that is, plants of the same category can be clustered in the same area, and plants of different categories can be clustered in different areas, such as healthy plants clustered in the same area, and diseased plants clustered in another area, thereby distinguishing them.

[0036] In some embodiments, the method or kit is used to detect and determine the freshness of fruits or flowers, which is of great significance for the detection and control of the quality of fruits or flowers in the market.

[0037] In some embodiments, the method or kit is used to detect and identify the germination stage of seeds such as wheat. By identifying and monitoring the germination stage of seeds such as wheat, it will be beneficial to the study of germination conditions and the accurate grasp of sowing period.

[0038] Compared with the prior art, the present invention has the following beneficial effects: the method of the present invention can extract and detect many types of plant hormones, especially BR, and the method has high sensitivity and specificity, good accuracy and precision. At the same time, the method of the present invention is suitable for detecting small samples, does not require concentration, derivatization and other processes, reduces the amount of plant samples, is easy to operate, and effectively reduces manpower, time and financial costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 TIC diagram of reference solution (positive and negative ions);

[0040] Figure 2 EIC diagram of CZ and TZ under the conditions of comparative example 1;

[0041] Figure 3 EIC diagram of CZ and TZ under the conditions of Example 1;

[0042] Among them, TIC is the total ion current and EIC is the extracted ion current. DETAILED DESCRIPTION

[0043] In order to detect plant hormones in samples, mainly plant samples, such as wheat, flowers, pitaya fruits, ophiopogon plants, Arabidopsis plants, and other samples containing plant hormones, such as honey, plant fermentation products, products made from plants as raw materials, etc. The inventors of the present application have studied the extraction conditions, liquid phase and chromatographic conditions of plant hormones, and developed a detection method for simultaneously determining 34 plant hormones in samples. The method has high accuracy, precision and sensitivity, and can simultaneously perform qualitative and quantitative detection of 34 plant hormones.

[0044] The abbreviations, Chinese and English names, CAS numbers of various plant hormones in this application and the source information of the reference substances used in the following examples or comparative examples are shown in Table 1 below.

[0045] Table 1 List of information on 34 plant hormones

[0046]

[0047]

[0048] The following are specific examples and related comparative studies. It should be clear to those skilled in the art that the examples are only used to help understand the present invention and should not be regarded as specific limitations of the present invention. Routine adjustments made by those skilled in the art under the concept of the present invention should be within the scope of protection of the present invention.

[0049] Example 1

[0050] A method for detecting plant hormones

[0051] 1. Solution preparation

[0052] ① Reference solution

[0053] Weigh MEJA, 6-BA, KT, IPA, BR, IP, TZR, MT, CZ, TZ, DZ, Me-IAA, IBA, IAA, GR24, GA3, GA1, GA4, ABA, GA7, GA5, GA19, GA24, JA-ILe, SAG, GA51, JA, GA8, DHJA, SA, ICA, ICAld, OPDA, and ACC reference substances respectively, dissolve and dilute them with methanol to prepare a series of reference substance solutions.

[0054] ②Internal standard mixed solution

[0055] Take IAA-D5 (indoleacetic acid-D5) and ABA-D6 (abscisic acid-D6) reference substances, dissolve them in methanol and dilute them to prepare internal standard mixed standard solutions.

[0056] ③Sample solution

[0057] (1) Take 20-400 mg of sample, add internal standard mixed solution and extraction reagent 1, where extraction reagent 1 is methanol, and the total volume of methanol added is 800-1200 μL. Constant temperature oscillation is performed at 20°C for more than 3 h, and the oscillation frequency is 1000-1500 rpm. Centrifuge to obtain the supernatant or filter to obtain the filtrate to obtain the first extract.

[0058] (2) Add 200-600 μL of extraction reagent 2 to the remaining filter residue, where the extraction reagent 2 is DMSO, and shake at a constant temperature of 20°C for more than 2 hours at a shaking frequency of 1000-1500 rpm. Centrifuge to obtain the supernatant or filter to obtain the filtrate to obtain the second extract.

[0059] (3) Combine the two extraction solutions to obtain the sample solution.

[0060] Note: A. In order to improve the extraction efficiency, the sample in step (1) is generally a powder, such as a powder obtained by low-temperature freeze-drying and grinding.

[0061] B. In step (1), a low concentration internal standard mixed standard solution can be used, and 800-1200 μL of the solution can be directly added, that is, 800-1200 μL of methanol is added. A higher concentration internal standard mixed standard solution can also be used, and methanol is added to make the total volume of methanol added 800-1200 μL, for example, 50 μL of internal standard mixed standard solution is added and then 950 μL of methanol is added, or 200 μL of internal standard mixed standard solution is added and then 800 μL of methanol is added, etc.

[0062] C. In steps (1) and (2), low temperature centrifugation is preferably performed, and the centrifugation rate is not less than 12000 rpm / min. A microporous filter membrane can be used for filtration, for example, a microporous filter membrane with a pore size of 0.45 μm or less.

[0063] D. Alternatively, in step (2), instead of centrifugation or filtration, the first extract obtained in step (1) is added to the solid-liquid mixture after constant temperature shaking in step (2), and then centrifuged or filtered to obtain a sample solution.

[0064] 2. Liquid chromatography conditions

[0065] Exion LC ultra-high performance liquid chromatography system (AB Sciex, USA) using ACQUITY CSH 18 Chromatographic column (2.1×150mm, 1.7μm, Waters, USA), injection volume 2-5μL, column temperature 40℃. Mobile phase A and mobile phase B were used for gradient elution. Mobile phase A-2mM ammonium formate containing 0.05% formic acid in water, B-0.05% formic acid in methanol. Gradient elution conditions were 0-2min, 10% B; 2-4min, 10-30% B; 4-19min, 30-95% B; 19-19.10min, 95-10% B; 19.10-22min, 10% B. Flow rate 0.25mL / min.

[0066] 3. Mass spectrometry conditions

[0067] AB SCIEX 6500+Qtrap mass spectrometer (AB Sciex, USA) was used with multiple reaction monitoring (MRM scanning) and electrospray ionization (ESI) source. In positive ion mode, the mass spectrometer voltage was 4500 V, the ion source temperature was 400 °C, the curtain gas was 40 psi, the nebulizer gas was 40 psi, and the auxiliary gas was 35 psi. The mass spectrometer voltage in negative ion mode was -4500 V, and other ion source temperature, curtain gas, nebulizer gas, and auxiliary gas conditions were the same as in positive ion mode.

[0068] The AB SCIEX 6500+Qtrap mass spectrometer can perform detection in both positive and negative ion modes simultaneously. Therefore, only one injection of sample is needed to obtain the detection results in both positive and negative ion modes. For other mass spectrometers that cannot use positive and negative ion modes simultaneously, one injection of sample can be performed in the positive ion mode first, and then in the negative ion mode, or the negative ion mode can be used first and then in the positive ion mode to obtain the detection results in both modes.

[0069] A series of concentrations of reference solution and sample solution were injected into the HPLC-MS instrument and tested under the above conditions. The TIC diagram of the reference solution in positive and negative ion modes is shown in Figure 2. Figure 1 The concentration of the plant hormone to be tested in the sample solution was calculated using the standard curve method.

[0070] Comparative Example 1

[0071] A solution containing CZ and TZ reference substances was taken and tested according to the following elution gradient, with other conditions being the same as in Example 1.

[0072] The gradient elution conditions were: 0-2 min, 30% B; 2-16 min, 30-95% B; 16-17.1 min, 95-30% B; 17.1-20 min, 30% B.

[0073] Under this elution condition, the 220.1→136.0 ion pair was selected to detect CZ and TZ, such as Figure 2 The two overlap almost completely.

[0074] CZ and TZ are cis-trans isomers with similar structures, which are difficult to separate under reverse phase chromatography conditions. Since they have the same parent ion and daughter ion, they need to be completely separated for qualitative or quantitative detection. The gradient elution conditions of Example 1 were used to achieve complete separation (such as Figure 3 ), which can be used for the simultaneous quantitative detection of CZ and TZ.

[0075] It should be noted that, in addition to the two cis-trans isomers CZ and TZ, the 34 plant hormones also include a variety of gibberellins, such as GA1, GA4, GA7, GA8, GA19, etc. These gibberellins have similar structures, some of which are isomers, such as GA5 and GA7; in addition, the structures of ICA, IAA, IBA, etc. are also similar. It is difficult to achieve simultaneous detection of these compounds with similar structures under the same liquid phase and mass spectrometry conditions, and it is even more difficult to obtain liquid phase and mass spectrometry suitable for simultaneous detection of 34 plant hormones. In addition, the content of plant hormones in plant samples is very low, usually only one ten-thousandth of plant secondary metabolites or even lower, in the range of 0.1 to 50 ng·g-1 fresh weight (Fu Jihong et al., Research Progress in Quantitative Analysis Methods of Plant Hormones, Science Bulletin, Vol. 55, No. 33, 2010: 3163-3176). Therefore, the sensitivity requirements for the instrument are also very demanding. In order to enable 34 plant hormones to be detected by high performance liquid chromatography-mass spectrometry, the inventors optimized a large number of liquid phase and mass spectrometry conditions, including mobile phase composition, gradient, flow rate, column temperature, chromatographic column type, mass spectrometry ion source temperature, voltage, nebulizing gas pressure, etc., and unexpectedly obtained the liquid phase and chromatographic conditions described in Example 1, which can meet the simultaneous detection of 34 trace plant hormones in the sample.

[0076] Comparative Example 1 is only the representative conditions and data of the optimization process that do not meet the analysis requirements. During the research process, some liquid phase conditions do not meet the separation requirements, some mass spectrometry conditions do not meet the sensitivity requirements, and some liquid phase conditions that meet the separation conditions will affect the sensitivity of the mass spectrometry, etc. Other conditions that do not meet the requirements are not listed here one by one.

[0077] The content of phytohormones in plants is extremely low, and the state of phytohormones is unstable. How to extract more phytohormones, avoid the loss of phytohormones during the extraction process, and reduce the interference of other complex matrices in plants are the challenges faced in the extraction process of phytohormones. For different plant samples, the extraction methods of phytohormones and related research processes are as follows.

[0078] Example 2

[0079] The sample is wheat seeds, and the sample solution is prepared as follows:

[0080] (1) Freeze-dry wheat seeds and grind them into powder. Weigh 50 mg and add them to a brown centrifuge tube. Add 950 μL of extraction reagent and 50 μL of internal standard mixed standard solution, where the extraction reagent is methanol and the internal standard mixed standard solution is a methanol solution of IAA-D5 (0.5 μg / ml) and ABA-D6 (1 μg / ml). Oscillate at a constant temperature of 20°C for 4 hours at an oscillation frequency of 1500 rpm, and centrifuge to obtain the supernatant.

[0081] (2) Add 500 μL of DMSO to the remaining residue in step (1), shake the mixture at a constant temperature of 20° C. for 4 h at a shaking frequency of 1500 rpm, and centrifuge to obtain the supernatant.

[0082] (3) Combine the two supernatants to obtain the sample solution.

[0083] The sample solution was tested according to the conditions of Example 1.

[0084] Comparative Example 2

[0085] The extraction reagent in step (2) is methanol, and the rest is the same as in Example 2.

[0086] Comparative Example 3

[0087] The only difference from Example 2 is that in Example 1, methanol in step (1) is replaced by DMSO, and DMSO in step (2) is replaced by methanol.

[0088] The test results are shown in Table 1.

[0089] Table 2 Peak area (EIC) results of plant hormones in wheat seed sample solutions

[0090]

[0091]

[0092] Note: N / A in the table means not detected, which means below the detection limit. The detection limits of various plant hormones are detailed in Table 7 below. N / A in the following tables has the same meaning.

[0093] The results in the table show that the combination of methanol + DMSO (Example 2) has the most types of compounds that exceed the detection limit, that is, the method of Example 2 detects the most types of plant hormones in wheat seeds, 5 more than the combination of methanol + methanol (Comparative Example 2). In addition, the method of Example 2 can detect BR, GA7, GA5 and GA8 in wheat seeds, while Comparative Examples 2-3 did not detect these compounds in wheat seeds. This shows that the combined extraction method of methanol + DMSO in Example 2 is better than the extraction methods of Comparative Example 2 (methanol + methanol) and Comparative Example 3 (DMSO + methanol). It is worth noting that compared with Comparative Example 3, the types of extraction reagents in Example 2 are the same, only the first step extraction reagent and the second step reagent types are replaced, but the results are very different. This shows that the extraction order of methanol first and then DMSO cannot be changed.

[0094] Example 3

[0095] The sample is peach blossom, and the sample solution preparation method is as follows:

[0096] (1) Peach blossom sample (fresh) was freeze-dried and ground into powder, 20 mg was weighed into a brown centrifuge tube, 750 μL of extraction reagent and 50 μL of internal standard mixed standard solution were added, and the extraction reagent and internal standard were added, wherein the extraction reagent was methanol and the internal standard mixed standard solution was a methanol solution of IAA-D5 (0.5 μg / ml) and ABA-D6 (1 μg / ml). The mixture was shaken at a constant temperature of 20°C for 3 h, the shaking frequency was 1200 rpm, and the supernatant was collected by centrifugation.

[0097] (2) Add 200 μL of extraction reagent DMSO to the remaining filter residue in step (1), and shake at a constant temperature of 20° C. for 3 h at a shaking frequency of 1200 rpm.

[0098] (3) The supernatant of step (1) is combined with the liquid after constant temperature shaking in step (2), centrifuged, and the supernatant is collected.

[0099] The sample solution was tested according to the conditions of Example 1.

[0100] Comparative Example 4

[0101] The only difference from Example 3 is that the methanol in step (1) of Example 3 is replaced by a mixed solution of methanol:DMSO in a volume ratio of 60:40; and the DMSO in step (2) is replaced by a mixed solution of methanol:DMSO in a volume ratio of 60:40.

[0102] Comparative Example 5

[0103] The only difference from Example 3 is that the methanol in step (1) of Example 3 is replaced by a mixed solution of 0.1% formic acid water: methanol in a volume ratio of 30:70; and the DMSO in step (2) is replaced by a mixed solution of 0.1% formic acid water: methanol in a volume ratio of 30:70.

[0104] Comparative Example 6

[0105] The only difference from Example 3 is that the methanol in step (1) of Example 3 is replaced by DMSO, and the DMSO in step (2) is replaced by methanol. Other factors (including the volume of the extraction reagent, constant temperature oscillation temperature, frequency, and time) are first extracted according to the conditions of step (2) of Example 3, and then extracted according to the conditions of step (1).

[0106] The results of plant hormone detection in Example 3 and Comparative Examples 4 to 6 are shown in the following table:

[0107] Table 3 Peak area (EIC) of plant hormones in peach blossom sample solution

[0108]

[0109]

[0110] The test results are shown in Table 3, which show that the types of plant hormones exceeding the detection limit in the combination of methanol + DMSO (Example 3) are more than those in other combinations. Example 3 detected BR, GA4, GA24, GA51, and GA8 in the peach flower sample, while Comparative Examples 4-6 did not detect any of them.

[0111] Interestingly, compared with Example 3, in Comparative Example 6, the types of extractant 1 and extractant 2 were replaced, and the extraction order was changed, but the results were very different, indicating that the extraction order of methanol and DMSO cannot be reversed.

[0112] Example 4

[0113] The sample is Ophiopogon japonicus (fresh plant), and the sample solution preparation method is as follows:

[0114] (1) Freeze-dry Ophiopogon japonicus and grind it into powder. Weigh 100 mg and add it to a brown centrifuge tube. Add 1150 μL of methanol and 50 μL of internal standard mixed standard solution, where the extraction reagent is methanol and the internal standard mixed standard solution is a methanol solution of IAA-D5 (0.5 μg / ml) and ABA-D6 (1 μg / ml). Oscillate at a constant temperature of 20°C for 3 hours at an oscillation frequency of 1500 rpm, and centrifuge to obtain the supernatant.

[0115] (2) Add 300 μL of DMSO to the remaining residue in step (1), shake the mixture at a constant temperature of 20° C. for 2 h at a shaking frequency of 1500 rpm, and centrifuge to obtain the supernatant.

[0116] (3) Combine the two supernatants to obtain the sample solution.

[0117] The sample solution was tested according to the conditions of Example 1.

[0118] Comparative Example 7

[0119] The difference from Example 4 is that the methanol in step (1) of Example 4 is replaced by a mixed solution of 0.1% formic acid water: methanol in a volume ratio of 30:70; and the DMSO in step (2) is replaced by a mixed solution of 0.1% formic acid water: methanol in a volume ratio of 30:70.

[0120] Comparative Example 8

[0121] The difference from Example 4 is that the methanol in step (1) of Example 4 is replaced by a mixed solution of 0.1% formic acid water: methanol in a volume ratio of 50:50.

[0122] Comparative Example 9

[0123] The difference from Example 4 is that the methanol in step (1) of Example 4 is replaced by a mixed solution of 0.1% formic acid water: methanol (volume ratio is 40:60).

[0124] Comparative Example 10

[0125] The difference from Example 4 is that the methanol in step (1) in Example 4 is replaced by a mixed solution of 0.1% formic acid water: methanol (volume ratio is 20:80).

[0126] Comparative Example 11

[0127] The difference from Example 4 is that the methanol in step (1) of Example 4 is replaced by 0.1% formic acid methanol, and the DMSO in step (2) of Example 4 is replaced by 0.1% formic acid methanol.

[0128] Comparative Example 12

[0129] The difference from Example 4 is that the methanol in step (1) of Example 4 is replaced by a mixed solution of methanol:DMSO (volume ratio of 60:40).

[0130] Comparative Example 13

[0131] The difference from Example 4 is that the methanol in step (1) in Example 4 is replaced by a mixed solution of methanol:DMSO (volume ratio is 70:30).

[0132] Comparative Example 14

[0133] The difference from Example 4 is that the methanol in step (1) in Example 4 is replaced by a mixed solution of methanol:DMSO (volume ratio is 80:20).

[0134] The experimental results of Example 4 and Comparative Examples 7-14 are shown in the following table:

[0135] Table 4-1 Peak area (EIC) of each plant hormone measured in Comparative Examples 7-11

[0136]

[0137]

[0138] Table 4-2 Peak areas (EIC) of various plant hormones measured in Comparative Examples 12-14 and Example 4

[0139]

[0140]

[0141]

[0142] From the results in Table 4-1 and Table 4-2, it can be seen that the combination of methanol + DMSO (Example 4) is used to extract Ophiopogon japonicus in sequence, and the most types of plant hormones are detected. In particular, under this condition, BR in Ophiopogon japonicus can be detected with the highest peak area, indicating that this condition is particularly suitable for the extraction of BR. Comparative Examples 7-14 add formic acid, and / or add a certain proportion of water or DMSO to the extractant 1 methanol, and / or the extractant DMSO is replaced by methanol, and the types of plant hormones detected are all lower than those in Example 4. In addition, Example 4 detected GA1, GA4 and GA8 in Ophiopogon japonicus, while Comparative Examples 7-14 did not detect any of them.

[0143] Example 5

[0144] The sample is honey, and the sample solution is prepared as follows:

[0145] (1) After freeze-drying the honey, grind it into powder, weigh 400 mg into a brown centrifuge tube, add 1150 μL of methanol and 50 μL of internal standard mixed standard solution, where the extraction reagent is methanol and the internal standard mixed standard solution is a methanol solution of IAA-D5 (0.5 μg / ml) and ABA-D6 (1 μg / ml). Constant temperature oscillation is performed at 20°C for 4 h, the oscillation frequency is 1000 rpm, and the supernatant is obtained by centrifugation.

[0146] (2) Add 600 μL of DMSO as the extraction reagent to the remaining filter residue in step (1), shake at a constant temperature of 20° C. for 2 h at a shaking frequency of 1000 rpm, and centrifuge to obtain the supernatant.

[0147] (3) Combine the two supernatants to obtain the sample solution.

[0148] The sample solution was tested according to the conditions of Example 1.

[0149] The test results of plant hormones in honey are as follows:

[0150] Table 5 Detection results of 34 plant hormones in honey

[0151]

[0152]

[0153] Example 6

[0154] Analytical method validation

[0155] The detection method of Example 3 was verified, and the verification items included: linearity, limit of quantification, limit of detection, precision, and accuracy, as follows:

[0156] 1. Linear

[0157] MEJA, 6-BA, KT, IPA, BR, IP, TZR, MT, CZ, TZ, DZ, Me-IAA, IBA, IAA, GR24, GA3, GA1, GA4, ABA, GA7, GA5, GA19, GA24, JA-ILe, SAG, GA51, JA, GA8, DHJA, SA, ICA, ICAld, OPDA, and ACC reference substances were weighed respectively, dissolved and diluted with methanol to prepare a series of concentrations of reference substance solutions, and detected according to the liquid phase and mass spectrometry conditions of Example 1.

[0158] The linear test results are shown in the following table:

[0159] Table 6 Linearity and range test results

[0160]

[0161]

[0162] 2. Limit of quantification and detection limit

[0163] Take the reference solution prepared by MEJA, 6-BA, KT, IPA, BR, IP, TZR, MT, CZ, TZ, DZ, Me-IAA, IBA, IAA, GR24, GA3, GA1, GA4, ABA, GA7, GA5, GA19, GA24, JA-ILe, SAG, GA51, JA, GA8, DHJA, SA, ICA, ICAld, OPDA, and ACC reference substances, dilute them in sequence and detect them according to the liquid phase and mass spectrometry conditions of Example 1, and take the concentration with S / N (signal-to-noise ratio) not less than 10 as the quantitative limit; further dilute and detect, and take the concentration with S / N not less than 3 as the detection limit. The recovery rate at the quantitative limit concentration should meet the standard requirements.

[0164] The quantitative limit and detection limit results of each plant hormone are as follows:

[0165] Table 7 Limit of quantitation and detection limit

[0166]

[0167]

[0168] 3. Accuracy

[0169] Take peach blossom sample powder, add a certain amount of 34 plant hormone reference substances, extract according to the conditions of Example 3, prepare low concentration (quantitative limit concentration, recorded as LQC), medium concentration (MQC) and high concentration (HQC) spiked solutions, and each concentration is paralleled 6 times. Detect according to the liquid phase and chromatographic conditions of Example 1, and calculate the recovery rate. The recovery rate results are as follows:

[0170] Table 8 Recovery test results

[0171]

[0172]

[0173] 4. Precision

[0174] With the same accuracy, low concentration (limit of quantitation concentration, recorded as LQC), medium concentration (MQC) and high concentration (HQC) spiked solutions were prepared, and 6 replicates of each concentration were injected and tested. The concentrations of 34 plant hormones in the sample solution were calculated by the standard curve method, and the RSD values ​​of the 6 replicates were calculated. The precision test results are shown in the following table:

[0175] Table 9 Precision test results

[0176]

[0177]

[0178] From the above method verification results, it can be seen that the linear coefficients (r) of the 34 plant hormones are all greater than 0.99, and each plant hormone has a good linear relationship within its own linear range. The accuracy recovery rates of low, medium and high concentration points are between 85% and 115%, and the precision RSD is within 15%, indicating that the method has high precision and accuracy. At the same time, the detection limit and quantification limit of the method are in the range of 0.001 to 25 ng / mL, indicating that the method has high sensitivity. Therefore, the method can be used for the precise quantification of 34 plant hormones in peach flowers.

[0179] The method validation results when using samples such as wheat seeds, Ophiopogon japonicus and honey are similar and will not be repeated here.

[0180] Application Examples

[0181] Distinguish between fresh and wilted flowers.

[0182] (1) Take fresh and withered Prunella vulgaris flowers, freeze-dry them and grind them into powder. Weigh 50 mg of each into a brown centrifuge tube, add 950 μL of methanol and 50 μL of internal standard mixed standard solution, where the extraction reagent is methanol and the internal standard mixed standard solution is a methanol solution of IAA-D5 (0.5 μg / ml) and ABA-D6 (1 μg / ml). Oscillate at a constant temperature of 20°C for 4 hours at an oscillation frequency of 1500 rpm, and centrifuge to obtain the supernatant.

[0183] (2) Add 500 μL of extraction reagent DMSO to the remaining filter residue in step (1), and shake at a constant temperature of 20° C. for 2 h at a shaking frequency of 1500 rpm.

[0184] (3) The supernatant of step (1) is combined with the liquid after constant temperature shaking in step (2), centrifuged, and the supernatant is taken to obtain a fresh coleus sample solution and a withered coleus sample solution.

[0185] The sample solution was tested under the conditions of Example 1. The test results of 34 plant hormones in fresh and withered spatholobi are shown in the following table:

[0186] Table 10 Detection results of 34 plant hormones in fresh and withered spatholobi (ng / mL)

[0187]

[0188]

[0189] From the results in the table, it can be seen that the types and contents of plant hormones detected in fresh schisandrae are significantly different from those in withered schisandrae. Further multivariate statistical analysis (including PCA analysis, PLS-DA analysis and OPLS-DA analysis) and differential analysis were performed to find plant hormones that can be used to distinguish fresh schisandrae from withered schisandrae. Finally, it was found that MEJA, ACC, SAG, GA3, etc. were differential metabolites between fresh schisandrae and withered schisandrae.

[0190] An experimenter randomly selected 50 fresh and withered wheel flowers, prepared sample solutions according to the above method, and tested them. Another experimenter then judged whether the sample was fresh or withered wheel flowers based on the above-discovered differential metabolites and test values, with an accuracy rate of 100%. Therefore, the plant hormone detection method can be applied to the discrimination or differentiation of fresh and withered flowers.

[0191] The inventors used the plant hormone detection method to detect fresh apples, the non-rotten parts of rotten apples, and the rotten parts of rotten apples, and conducted comparative analysis, and found that there were obvious differences among the three. They used multivariate statistical analysis and difference analysis to find differential metabolites, and then used the plant hormone method to detect these differential metabolites to achieve differentiation between the three.

[0192] Similarly, the inventors detected plant hormones in ungerminated wheat, germinated but not rooted wheat, and germinated and rooted wheat by the above method and found that there were significant differences among the three. The plant hormone detection method can be used to distinguish between the three growth states of wheat.

[0193] The applicant declares that the present invention illustrates the plant hormone extraction and quantitative detection method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting plant hormones, characterized in that: The method uses methanol and dimethyl sulfoxide to extract samples in sequence; the extracts are combined and tested; The specific steps of sequentially extracting the sample using methanol and dimethyl sulfoxide include: (1) Take a sample, add methanol, extract with constant temperature shaking, centrifuge or filter to obtain an extract, and take the filter residue for later use; (2) adding dimethyl sulfoxide to the filter residue of step (1), shaking at a constant temperature, and centrifuging or filtering to obtain an extract; The constant temperature oscillation in step (1) is oscillation at 20°C or below for not less than 3 hours; the constant temperature oscillation in step (2) is oscillation at 20°C or below for not less than 2 hours; the frequency of oscillation in step (1) and step (2) is 1000rpm~1500rpm.

2. The method according to claim 1, characterized in that An internal standard is added before the constant temperature oscillation in step (1), wherein the internal standard includes indoleacetic acid-D5 and abscisic acid-D6.

3. The method according to claim 1, characterized in that The sample is a freeze-dried powder. Relative to 20-400 mg of the sample, the amount of methanol used is 800-1200 μL; the amount of dimethyl sulfoxide used is 200-600 μL.

4. The method according to claim 1, characterized in that: The plant hormone includes one or a combination of at least two of the following plant hormones: methyl jasmonate, 6-benzylaminopurine, kinetin, isopentenyl adenine riboside, brassinolide, isopentenyl adenine, trans-zeatin riboside, melatonin, cis-zeatin, trans-zeatin, dihydrozeatin, indole-3-acetic acid methyl ester, indolebutyric acid, indoleacetic acid, 1-aminocyclopropane-1-carboxylic acid, strigolactone, gibberellin 3, gibberellin 1, gibberellin 4, abscisic acid, gibberellin 7, gibberellin 5, gibberellin 19, gibberellin 24, N-jasmonic acid-isoleucine, glycosylated salicylic acid, gibberellin 51, jasmonic acid, gibberellin 8, dihydrojasmonic acid, salicylic acid, 3-indolecarboxylic acid, indole-3-carboxaldehyde or 12-oxophytodienoic acid.

5. The method according to claim 1, characterized in that: The plant hormones include brassinolide.

6. The method according to claim 1, characterized in that The detection is performed using LC-MS; The LC conditions for LC-MS included: using ACQUITY UPLC CSH C 18 Chromatographic column, injection volume of 2 μL to 5 μL, column temperature of 40°C; gradient elution with mobile phase A and mobile phase B, mobile phase A is 2 mM ammonium formate 0.05% formic acid water, mobile phase B is 0.05% formic acid methanol; gradient elution conditions include: 0 to 2 min, 10% mobile phase B; 2 to 4 min, 10 to 30% mobile phase B; 4 to 19 min, 30 to 95% mobile phase B; 19 to 19.10 min, 95 to 10% mobile phase B; 19.10 to 22 min, 10% mobile phase B, flow rate of 0.25 mL / min; The MS conditions of LC-MS included: using an electrospray ionization source, in positive ion mode, the mass spectrometer voltage was 4500 V, the ion source temperature was 400°C, the curtain gas was 40 psi, the nebulizer gas was 40 psi, and the auxiliary gas was 35 psi; in negative ion mode, the mass spectrometer voltage was -4500 V, the ion source temperature was 400°C, the curtain gas was 40 psi, the nebulizer gas was 40 psi, and the auxiliary gas was 35 psi.

7. The method according to claim 1, characterized in that The sample is freeze-dried powder of plant tissue; the plant tissue is one or a combination of at least two of roots, stems, leaves, flowers, fruits, seeds, pollen, galls or branches.

8. The method according to claim 1, characterized in that The sample is any one of wheat seeds, peach blossoms, ophiopogon japonicus, apple fruit, honey, pitaya fruit, Arabidopsis thaliana or wheelbarrow flower, or a combination of at least two of them.

9. Use of the method according to any one of claims 1 to 8 in the detection of plant hormones.

10. The use according to claim 9, characterized in that: The method is used to detect plant samples, and to judge the growth state or health state of the plant, or the growth state or health state of a certain part of the plant, through the detection result of the plant sample.

11. The use according to claim 9, characterized in that: The method is used to judge the freshness of fruits or flowers, or to judge the germination stage of wheat.

Images