Method for rapidly detecting plant hormones by using UHPLC-QqQ-MS / MS

Abstract

The invention provides a method for rapidly detecting plant hormones by using UHPLC-QqQ-MS / MS. The method comprises the following steps: S1, extracting plant hormones in a plant sample to obtain a sample to be detected; s2, measuring the sample to be measured by adopting a UHPLC-QqQ-MS / MS method to obtain the type or content of the plant hormones; the UHPLC-QqQ-MS / MS method is a method for establishing a multiple reaction monitoring (MRM) mode for simultaneously monitoring positive ions and negative ions by utilizing an electrospray ionization (ESI) source; the plant hormones comprise striga asiatica lactone GR24, trans-zeatin, gibberellin, abscisic acid, jasmonic acid, methyl jasmonate, salicylic acid, trans-zeatin nucleoside, brassinolide and indole-3-acetic acid.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnological detection, and particularly relates to a method for rapidly detecting phytohormones by using UHPLC-QqQ-MS / MS; the UHPLC-QqQ-MS / MS adopts an electrospray ionization positive and negative ion (ESI+, ESI-) simultaneous scanning detection mode and a multiple reaction monitoring (MRM) mode. Background Art

[0002] Phytohormones play important roles in various growth and development stages of plants, such as plant seed germination, growth, flowering, fruit ripening, and response to environmental stress. Accurately and reliably detecting the content of phytohormones is crucial for deeply understanding the mechanisms of plant growth and development.

[0003] Phytohormones exist in trace amounts in plants, are easily degraded, have many impurity interferences, and are difficult to separate. Therefore, the requirements for hormone extraction methods are relatively strict. The extraction of phytohormones requires fresh plants and involves steps such as leaching, purification, concentration, and re-dissolution. The process is cumbersome and time-consuming, and it is easy to cause losses. Therefore, the requirements for the extraction method, the instrument detection limit and the method detection limit in the test method for determining phytohormones are extremely high.

[0004] Currently, the commonly used hormone test methods include enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). The Elisa method is the simplest, with a short test period and low cost, and is suitable for large-scale screening of samples. However, it has the disadvantages of low data accuracy, high instrument detection limit, and low recognition rate. The instrument sensitivity of HPLC is better than that of ELISA, but it cannot accurately quantify most phytohormones with low contents, and even cannot qualitatively analyze them. The instrument sensitivities of GC-MS and LC-MS are high, but the requirements for sample pretreatment in gas phase are higher than those in liquid phase, and phytohormones are sensitive to temperature. For example, gibberellin GA3 will isomerize above 40°C. In addition, the plant matrix is complex, and there are great differences in the contents of different phytohormones in plants. For example, the contents of IAA, SA, JA, etc. are between 1 and 100 ng / g FW (fresh weight), while GA3 is less than 0.1 ng / g FW. This requires the instrument to have high sensitivity and a wide measurement range to accurately detect. Currently, most methods based on liquid chromatography-mass spectrometry (LC-MS) adopt the full scan mode, so they cannot exclude the interference from compounds with the same molecular mass in the plant matrix, and thus cannot accurately determine the content of hormones in complex plant backgrounds. The sensitivity is not high, and it is difficult to be popularized and applied in the detection of phytohormones. There is an urgent need for a method for high-throughput and high-sensitivity detection of phytohormones based on LC-MS. Summary of the Invention

[0005] To overcome the defects and deficiencies of the above-mentioned prior art, the present invention proposes a method for quantitatively detecting 10 common hormones in plants by using an ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS / MS) system in the multiple reaction monitoring mode. The multiple reaction monitoring mode is a method in which MS1 selects precursor ions with certain or multiple mass-to-charge ratios in the SIM mode, generates fragment ions in the collision cell, and MS2 also monitors one or more specific product ions generated from the precursor ions in the SIM mode. This method can greatly improve the detection sensitivity and quantitative accuracy. The MRM method is used for the quantitative analysis of target compounds and is the mass spectrometry detection method with the highest sensitivity, featuring high efficiency, rapidity, low detection limit, and good reproducibility.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a method for detecting plant hormones by using UHPLC-QqQ-MS / MS, which includes:

[0008] S1: Extract plant hormones from a plant sample to obtain a sample to be tested;

[0009] S2: Use the UHPLC-QqQ-MS / MS method to measure the sample to be tested to obtain the types or contents of plant hormones;

[0010] The UHPLC-QqQ-MS / MS method is a method for establishing a multiple reaction monitoring (MRM) mode for simultaneously monitoring positive and negative ions by using an electrospray ionization source (ESI).

[0011] The plant hormones described above include strigolactone GR24 (SL), trans-zeatin (TZ), gibberellin A3 (GA3), abscisic acid (ABA), jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), trans-zeatin-riboside (TZR), brassinolide (BR), and indole-3-acetic acid (IAA).

[0012] In some embodiments, the method further includes preparing a solvent for the plant hormone standard, formulating a standard stock solution of the plant hormone with an organic solvent, and then diluting it stepwise into a standard solvent to establish a standard curve; preferably, a 1000 μmol / L standard stock solution is prepared with an organic solvent and then diluted stepwise into a 1.0 - 1000 nmol / L standard solvent.

[0013] As a preferred embodiment, the organic solvent is a mixed solution containing methanol, formic acid, and water; as a preferred embodiment, in the organic solvent, the volume percentage of methanol is 50 - 80%, and the volume percentage of formic acid is 0.1% - 5%; more preferably, the extraction solution is a mixed solution formed by mixing methanol, formic acid, and water in a volume ratio of 80:0.1:19.9.

[0014] In some embodiments, step S1 includes extracting the plant hormone from the plant sample using an extraction solution.

[0015] As a preferred embodiment, the extraction solution is a mixed solution containing methanol, formic acid, and water; as a preferred embodiment, in the extraction solution, the volume percentage of methanol is 50 - 80%, and the volume percentage of formic acid is 0.1% - 5%; more preferably, the extraction solution is a mixed solution formed by mixing methanol, formic acid, and water in a volume ratio of 80:0.1:19.9.

[0016] In some embodiments, the plant sample is a fresh plant sample.

[0017] In some embodiments, step S1 includes: grinding the plant sample into powder in liquid nitrogen, adding the extraction solution, performing ultrasonic treatment in an ice-water bath or grinding with steel beads, placing it at -80°C for freezing, centrifuging at 4°C to obtain the supernatant, filtering, drying, re-dissolving, and then transferring it to a sample vial.

[0018] In some embodiments, step S1 is carried out under light-shielded conditions.

[0019] In some embodiments, the time of ultrasonic treatment in an ice-water bath is 20 - 120 min, and the time of freezing at -80°C is 0.5 - 12 h.

[0020] As a preferred embodiment, the time of ultrasonic treatment in an ice-water bath is 20 min, and the time of freezing at -80°C is 1.5 h.

[0021] As a preferred embodiment, filtration is carried out using a 0.22-μm organic filter membrane. More preferably, the 0.22-μm organic filter membrane is a syringe-type filter membrane filter made of Jinteng organic nylon 66 with a pore size of 0.22 μm.

[0022] As a preferred embodiment, in step S1, it is redissolved with a mixed solution containing methanol, formic acid, and water; in the mixed solution, the volume percentage of methanol is 50 - 80%, and the volume percentage of formic acid is 0.1% - 5%; more preferably, in the mixed solution, the volume ratio of methanol, formic acid, and water is 80:0.1:19.9.

[0023] As a preferred embodiment, the conditions for grinding with steel beads are: grinding at 1000 - 2000 rpm for 1 - 3 min, with 3 - 10 cycles; more preferably, the conditions for grinding with steel beads are: grinding at 2000 rpm for 2 min, with 5 cycles.

[0024] In some embodiments, the dosage ratio of the extraction solution to the powder is 1 - 5 μL / mg, preferably 5 μL / mg.

[0025] In some embodiments, relative to 200 mg of the powder, the dosage of the extraction solution is 1.0 mL.

[0026] As a preferred embodiment, step S1 includes: taking a fresh plant sample, grinding it into powder in liquid nitrogen in the dark, weighing 200 mg of the powder, adding 1.0 mL of the extraction solution, carrying out ultrasonic treatment in an ice-water bath for 20 min, freezing at -80°C for 1.5 h, centrifuging at the maximum speed at 4°C for 10 min, taking the supernatant and filtering it with a 0.22-μm organic filter membrane, drying it using a nitrogen blower or a vacuum centrifugal concentrator, and redissolving it with a mixed solution containing methanol / formic acid / water = 80:0.1:19.9 to obtain a sample to be tested.

[0027] In the present invention, the extraction process of plant hormones is carried out in the dark and at low temperature to prevent the photodegradation or enzymatic decomposition of hormones.

[0028] In some embodiments, the sample to be tested is transferred to a brown vial with a liner for sample introduction.

[0029] In some embodiments, the liquid phase conditions of the UHPLC-QqQ-MS / MS method of the present invention are as follows: The mobile phase and flow rate are as follows: Mobile phase A is 0.1% FA-H2O, and mobile phase B is 0.1% FA-CAN; at 0.00 min, mobile phase A is 95% v / v, mobile phase B is 5% v / v, and the flow rate is 0.3 mL / min; as the analysis time increases, the proportion of mobile phase B is gradually increased, and the proportion of mobile phase A is correspondingly decreased. By continuously adjusting the ratio of mobile phase A and mobile phase B, until all target hormone compounds can successfully elute and effective separation is achieved.

[0030] In some embodiments, the liquid phase conditions of the UHPLC-QqQ-MS / MS method are as follows: The column model is ZORBAXRRHD Eclipse Plus C18, 2.1 x 50 mm, 1.8 μm, and the column temperature is set at 25°C; the liquid phase injection tray temperature is set at 4°C, the injection volume is 5.0 μL, the acquisition time is 7.0 min, and the mobile phase and flow rate are as follows:

[0031] Mobile phase A is 0.1% FA-H2O, and mobile phase B is 0.1% FA-CAN; at 0.00 min, mobile phase A is 95% v / v, mobile phase B is 5% v / v, and the flow rate is 0.3 mL / min; at 3.50 min, mobile phase A is 85% v / v, mobile phase B is 15% v / v, and the flow rate is 0.3 mL / min; at 4.00 min, mobile phase A is 40% v / v, mobile phase B is 60% v / v, and the flow rate is 0.3 mL / min.

[0032] In the present invention, the liquid phase injection tray temperature is set at 4°C to better maintain the stability of the hormone and reduce its degradation rate.

[0033] In some embodiments, the mass spectrometry conditions of the UHPLC-QqQ-MS / MS method are as follows: The ion source temperature is 250 - 300°C, the nebulizer pressure is 40 - 60 psi, and the electrospray voltage is 3000 - 3500 V.

[0034] In some embodiments, the mass spectrometry conditions of the UHPLC-QqQ-MS / MS method are as follows: The ion source temperature is 250°C, the nebulizer pressure is 45 psi, and the electrospray voltage is 3500 V.

[0035] In some embodiments, in the electrospray ionization positive and negative ion simultaneous scanning detection mode, the positive ion mode corresponds to compounds IAA, TZ, SL, MeJA, BR, and TZR, and the negative ion mode corresponds to compounds ABA, GA3, JA, and SA.

[0036] In some embodiments, the tuned electron multiplier voltage (EMV) is increased to 200 V in both positive ion or negative ion mode.

[0037] In the present invention, increasing the tuned EMV by 200 V is beneficial to improving the sensitivity and signal-to-noise ratio.

[0038] In some embodiments, UHPLC-QqQ-MS / MS technology is used for quantitative analysis of multiple phytohormones. According to the chemical properties and mass spectrometry response characteristics of the compounds, the target compounds are collected in positive ion mode and negative ion mode. Among them, the positive mode acquisition parameters are set as follows: the fragment voltage of IAA is 90V, and the collision energies are 35V (176.07>103.1), 35V (176.07>128), 35V (176.07>77.2), and 21V (176.07>130); the fragment voltage of TZ is 120V, and the collision energies are 35V (220.12>119), 17V (220.12>136), 13V (220.12>148), and 13V (220.12>202.1); the fragment voltage of TZR is 120V, and the collision energies are 21V (352.2>220.0), 25V (352.2>202.0), 33V (352.2>148.0), and 35V (352.2>136.0); the fragment voltage of MeJA is 90V, and the collision energies are 9V (225.1>150.8), 17V (225.1>147.0), 13V (225.1>133.0), and 29V (225.1>105.1); the fragment voltage of BR is 110V, and the collision energies are 17V (481.36>315.2), 17V (481.36>113.1), 40V (481.36>95.1), and 33V (481.36>71.1); the fragment voltage of SL is 90V, and the collision energies are 17V (321.1>97.1), 25V (321.1>157), 13V (321.1>183.9), and 5V (321.1>185). The negative mode acquisition parameters are set as follows: the fragment voltage of ABA is 120V, and the collision energies are 5V (263.13>153.1), 35V (263.13>203), 21V (263.13>204.1), and 13V (263.13>219.1); the fragment voltage of SA is 90V, and the collision energies are 17V (137.0>93.0) and 33V (137.0>65.1); the fragment voltage of JA is 120V, and the collision energy is 13V (209.1>59.1); the fragment voltage of GA3 is 180V, and the collision energies are 35V (345.13>143.1), 25V (345.13>221.1), 29V (345.13>227.1), and 13V (345.13>239.2).

[0039] In the present invention, different phytohormones correspond to the selection of different ion pairs. For each compound, under its respective positive / negative ion acquisition mode and optimal collision voltage parameters, the respective characteristic ion fragments are selected and combined into the ion pairs of the compound, as shown in Table 5.

[0040] In the present invention, for each phytohormone, under its respective positive / negative ion acquisition mode and optimal collision voltage parameters, different collision energies are set for each pair of ion pairs, and finally the optimal collision energy as shown in Table 5 is selected for the acquisition of quantitative data. As a preferred embodiment, the acquisition conditions of the mass spectrometry of the UHPLC-QqQ-MS / MS method are as shown in Table 5.

[0041] In the present invention, the mass spectrometry ion source adopts the electrospray ionization (ESI) mode. ESI is a soft ionization technique that generally does not cause fragmentation of the analyte. Therefore, in the positive ion mode (ESI+), the main ion of the compound is usually the molecular ion added with a proton, i.e., [M+H]+; while in the negative ion mode (ESI-), the compound mainly forms the ion of [M-H]-. ESI provides a relatively simple method to ionize non-volatile solutions, enabling the mass spectrometer to provide sensitive direct detection.

[0042] In the present invention, data is acquired in the positive and negative polarity switching mode, and the same sensitivity can be obtained as in the single mode acquisition, without obvious loss of sensitivity.

[0043] In the present invention, the plant is an angiosperm.

[0044] In the present invention, the angiosperms include but are not limited to Arabidopsis thaliana, tobacco, wheat, flowering Chinese cabbage, cotton, rice, tomato, corn, poplar, etc.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention uses the UHPLC-QqQ-MS / MS system in the MRM mode to simultaneously test 10 common hormones in plants (indole-3-acetic acid, gibberellin, abscisic acid, trans-zeatin, jasmonic acid, methyl jasmonate, salicylic acid, trans-zeatin riboside, brassinolide, and strigolactone). These 10 hormones can be simultaneously detected in 7 minutes, saving time, with accurate quantification, and having the advantages of high sensitivity, low detection limit, and wide test range, providing a reliable test method for the rapid, efficient, and accurate determination of different phytohormones with large content differences.

[0047] (2) The present invention uses a low-temperature and light-avoiding solvent extraction method to extract the hormones in plants, and uses a nitrogen blower or a vacuum centrifugal concentrator to batch concentrate and redissolve. The pretreatment operation is simple, fast, low-cost, and has a low detection limit;

[0048] (3) In the present invention, the method steps for extracting phytohormones are simple, fast, and the solvents used are inexpensive and low-toxic;

[0049] In the prior art, in the full scan mode of UPLC-MS (i.e., the full scan mode), after obtaining the total ion chromatogram (TIC), quantification is carried out by the abundance of the extracted ion chromatogram (EIC), thereby detecting 34 phytohormones at one time. The Scan method adopted in this scheme refers to scanning a set molecular weight range during mass spectrometry acquisition. Through the full spectrum obtained by scanning, the EIC map of each specific compound ion can be extracted. This mode is mostly used for the qualitative analysis of compounds. In the case of complex matrices (such as plant extracts), it is easy to cause interference, and it is difficult to avoid the interference of other ions to the experiment and improve the sensitivity of a certain ion. Each compound has different characteristics and ionization properties. For example, in the present invention, IAA, TZ, TZR, MeJA, BR, and SL have better responses in the positive ion mode, while ABA, SA, JA, and GA3 have higher responses in the negative ion mode. The MRM mode used in the present invention can alternate between positive and negative modes, more noise and interference are excluded, and the sensitivity signal-to-noise ratio will be higher, especially for complex samples with a high matrix background. The higher the MRM resolution, the stronger the ability to resist background and exclude interference. Therefore, for compound quantification, especially for compound quantification in a plant background, the more complex the background matrix, the more inclined to use the MRM mode, which has a higher signal intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 are the chemical structural formulas of 10 common phytohormones;

[0051] Figure 2A 、 Figure 2B and Figure 2C are the chromatograms and mass spectra of 10 common phytohormones collected by UHPLC-QqQ-MS / MS;

[0052] Figure 3 are the MRM quantitative peak maps of 10 hormones. DETAILED DESCRIPTION OF THE INVENTION

[0053] Before further describing the specific implementation embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation embodiments, rather than for limiting the protection scope of the present invention.

[0054] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any value therebetween can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0055] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not disclosed in the present invention all adopt the conventional techniques in the art.

[0056] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0057] In the present invention, a mixed solution containing methanol / formic acid / water = 80:0.1:19.9 (all ratios in the present invention are volume ratios) is used as the extraction solution, and ultrasonic extraction is carried out at low temperature. After freezing and centrifuging to remove impurities, it is dried by a nitrogen or vacuum centrifugal concentrator, and finally redissolved with 100 μL of a mixed solution containing methanol / formic acid / water = 80:0.1:19.9, and tested by scanning in MRM mode through UHPLC-QqQ-MS / MS. Through UHPLC-QqQ-MS / MS, the contents of 10 hormones, namely strigolactone (SL), trans-zeatin (TZ), gibberellin A3 (GA3), abscisic acid (ABA), jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), brassinolide (BR), trans-zeatin-riboside (TZR), and indole-3-acetic acid (IAA) (the structures are shown in Figure 1 , and the detailed contents are shown in Table 1) are determined and accurately quantified.

[0058] Table 1

[0059]

[0060]

[0061] Example 1: Determination of the Contents of Phytohormones in Arabidopsis Seedlings

[0062] Step S1: Prepare the standard phytohormone solution: Weigh the standard phytohormones respectively. The standard phytohormones include ABA, IAA, SL, SA, JA, MeJA, GA3, TZ, BR, and TZR. Add the mixed solution of methanol / formic acid / water = 80:0.1:19.9 as the solvent respectively, and prepare the mother liquor of phytohormones with a concentration of 1.0 mg / mL for each of the 10 hormone standards. Then take 100 μL from each mother liquor of the standard phytohormones and mix them evenly for dilution. ABA, IAA, SL, SA, JA, MeJA, BR, and GA3 are respectively diluted into a series of concentration control solutions of 1000 nmol / L, 500 nmol / L, 100 nmol / L, 50 nmol / L, 10 nmol / L, and 5 nmol / L. TZ and TZR are diluted into standard solutions with concentrations of 100 nmol / L, 50 nmol / L, 10 nmol / L, 5 nmol / L, 1 nmol / L, and 0.5 nmol / L for subsequent LC-MS / MS analysis to obtain the standard curve.

[0063] Step S2: Optimize the detection conditions of phytohormones and develop a mass spectrometry detection method.

[0064] Taking GA3 as an example, the specific steps are as follows:

[0065] (1) Selection of the scanning mode (Polarity): Select 100 nM of GA3 in the full-scan mode. The peak area in the positive ion mode is 6.7×10 4 , and the peak area in the negative ion mode is 1.5×10 6 . The peak area corresponding to the negative ion mode is much higher than that of the positive ion mode. It shows that the detection effect is poor when choosing the positive ion mode. Therefore, select the negative ion mode for mass spectrometry detection of GA3.

[0066] (2) Selection of the precursor ion: Electrospray ionization can generate ions in two ways in the positive ion mode: one way is to combine with a proton H+ to generate [M+H]+ ions; the other way is to combine with other cations, such as sodium ions to form [M+Na]+. In the negative ion mode, electrospray ionization generates deprotonated ions [M-H]- or combines with other anions. From the results of mass spectrometry data acquisition, GA3 mainly forms [M-H]- ions, and [M-H]- ions have the highest response. Selecting other ions will result in a poorer detection effect. Therefore, select the precursor ion with m / z 345.13 for subsequent data acquisition.

[0067] (3) Selection of collision voltage (Fragmentor): In the negative ion mode, different collision voltages were set, and the parent ion m / z 345.13 was scanned for mass spectrometry data acquisition. For GA3, the set collision voltages and the peak areas collected at each voltage are shown in Table 2 below:

[0068] Table 2

[0069] Collision voltage (Fragmentor) Peak area (Area) 200V <![CDATA[2.0×10 5 > 180V <![CDATA[2.5×10 5 > 150V <![CDATA[1.2×10 5 > 120V <![CDATA[5.5×10 4 > 90V <![CDATA[2.8×10 4 > 60V <![CDATA[2.6×10 4 > 30V <![CDATA[1.4×10 5 >

[0070] Under the condition of 180V, the response effect of the GA3 parent ion m / z 345.13 is the best. Therefore, a collision voltage of 180V was selected for subsequent data acquisition of GA3.

[0071] (4) Selection of precursor ions: In the positive ion mode and under the condition of a collision voltage of 180V, the collision energy was first set to four parameters: 5V, 10V, 20V, and 40V, and the parent ion 345.1 was scanned to observe the obtained characteristic fragment information. GA3 contains characteristic fragments with mass numbers of m / z 239.2, m / z 227.1, m / z 221.1, and m / z 143.1. Subsequently, 4 ion pairs were formed for the detection of the GA3 compound.

[0072] (5) Selection of collision energy: In the positive ion mode and under the condition of a collision voltage of 180V, a gradient collision energy was set, and the 4 ion pairs of 345.1->239.2, 345.1->227.1, 345.1->221.1, and 345.1->143.1 were scanned respectively. The intensities collected for each ion pair at the collision energy are shown in Table 3 below:

[0073] Table 3

[0074]

[0075]

[0076] The above table only shows the information collected by partial parameter settings. According to the results shown in the above table, the collision energy for 345.1->239.2 was selected as 13V, the collision energy for 345.1->227.1 was selected as 19V, the collision energy for 345.1->221.1 was selected as 25V, and the collision energy for 345.1->143.1 was selected as 35V. If other collision energies are selected, the detection effect will be relatively poor and the best detection effect cannot be achieved.

[0077] According to the above steps, the detection conditions of the other 10 hormone compounds were optimized. The steps are similar and will not be elaborated here one by one.

[0078] It should be noted that the response of SL under the positive spectrum condition is higher than that under the negative spectrum condition. Therefore, the positive spectrum is selected for detection. Under the positive spectrum condition, the response of SL to form [M+Na]+ (321.1, response value is 3.0*10 5 ) is much higher than that to produce the [M+H]+ parent ion (299.1, response value is 1.8*10 4 ). This indicates that the detection effect using the [M+H]+ parent ion is much worse than that using the [M+Na]+ parent ion. Therefore, SL is detected using the parent ion m / z 321.1 [M+Na] + .

[0079] The above parameter settings are the optimal parameter values obtained through comparison of different parameters. Changing the settings of some parameters may result in a poor detection effect.

[0080] The liquid phase conditions in (1)-(5) are as follows: the column model is ZORBAX RRHD Eclipse Plus C18, 2.1x50mm, 1.8μm, the column temperature is set at 25°C, the liquid phase injection tray temperature is set at 4°C, the injection volume is 5.0 μL, the acquisition time is 7.0 min, and the mobile phase and flow rate are shown in Table 4.

[0081] Table 4

[0082]

[0083] The mass spectrometry conditions are as follows: the electrospray ionization positive and negative ion simultaneous scanning detection mode and the multiple reaction monitoring mass spectrometry scanning mode are adopted, the ion source temperature: 250°C, the nebulizer pressure: 45 psi, and the electrospray voltage: 3500 V.

[0084] The final formal mass spectrometry conditions are as follows: for the positive ion mode, the corresponding compounds are IAA, TZ, TZR, MeJA, BR, and SL; for the negative ion mode, the corresponding compounds are ABA, JA, SA, and GA3. In addition, the tuned EMV is increased by 200 V in both the positive ion or negative ion mode. The ion pair acquisition conditions for each hormone are shown in Table 5.

[0085] The mass spectrometry MRM acquisition method for the finally developed 10 hormones is shown in Table 5 below:

[0086] Table 5

[0087]

[0088]

[0089] The chromatograms and mass spectra of 10 common plant hormones detected by UHPLC-QqQ-MS / MS are shown in Figure 2 (including Figure 2A 、 Figure 2B andFigure 2C ). MRM quantitative peak diagram is shown in Figure 3 .

[0090] Step S3: Sample analysis, take 200 mg of fresh Arabidopsis seedling powder ground in liquid nitrogen, add 1 mL of a mixture of methanol / formic acid / water = 80:0.1:19.9 by volume, ultrasonicate in an ice-water bath for 20 min, freeze at -80 ° C for 1.5 h, centrifuge at a maximum speed of 15000 rpm at 4 ° C for 10 min, take the supernatant and filter it with a Jinteng organic nylon 66 syringe filter 0.22 μm, blow dry with a nitrogen blower, add 100 μL of a mixture containing methanol / formic acid / water = 80:0.1:19.9 to dissolve, and transfer to a brown liner sample bottle. Perform LC-MS analysis, and convert the analysis results to the corresponding plant hormone concentration using the standard curve constructed in step S1. The content of each plant hormone in the Arabidopsis seedling sample is shown in Table 6 below.

[0091] Table 6 Table of the content of various plant hormones in Arabidopsis seedlings

[0092]

[0093]

[0094] 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 6, which means that the species does not contain the hormone, or the hormone content is very low and not enough to reach the minimum detection limit of the instrument.

[0095] Example 2 Determination of hormones in other plant materials

[0096] Step S1: Preparation of solvent for plant hormone standard. Weigh a certain mass of standard and use a mixture of methanol / formic acid / water = 80:0.1:19.9 as solvent to prepare a 1000 μmol / L standard stock solution, and then dilute it into a 1.0-1000nmol / L solvent to prepare a standard curve.

[0097] Step S2: Select fresh leaf samples of several common plants and grind them into powder in liquid nitrogen in the dark, weigh 100 mg of the powder, add 1.0 mL of an extract containing a mixture of methanol / formic acid / water = 80:0.1:19.9, ultrasonicate in an ice-water bath for 20 min, freeze at -80°C for 1.5 h, centrifuge at 4°C and 15000 rpm for 10 min, take the supernatant and filter it with a 0.22 μm organic filter membrane, blow dry with a nitrogen blower, add 100 μL of a mixture containing methanol / formic acid / water = 80:0.1:19.9 to re-dissolve, and transfer to a brown sample bottle with a liner. All steps are protected from light as much as possible.

[0098] Step S3: The liquid phase conditions are as follows: the column model is ZORBAX RRHD Eclipse Plus C18, 2.1x 50mm, 1.8μm, the column temperature is set at 25°C, the temperature of the liquid phase injection tray is set at 4°C, the injection volume is 5.0 μL, the acquisition time is 7.0 min, and the mobile phase and flow rate are shown in Table 4.

[0099] Step S4: The electrospray ionization positive and negative ion simultaneous scanning detection mode and the multiple reaction monitoring mass spectrometry scanning mode are adopted. Ion source temperature: 250°C, nebulizer pressure: 45 psi, electrospray voltage: 3500 V. For the positive ion mode, the corresponding compounds are IAA, TZ, TZR, MeJA, BR, and SL; for the negative ion mode, the corresponding compounds are ABA, JA, SA, and GA3. In addition, the EMV is increased by 200 V in both the positive ion or negative ion mode, and the acquisition conditions are shown in Table 5. The detection results are shown in Table 7, indicating that this method has universality and can be well applied in different plant materials.

[0100] Table 7 Test Results of Plant Materials

[0101]

[0102]

[0103] Note: N / A in the table represents not detected. Not detected means below the detection limit. The detection limits of each phytohormone are shown in Table 7, indicating that this species does not contain this hormone, or the hormone content is very low and not enough to reach the minimum detection limit of the instrument.

[0104] Example 3 Analysis of the Stability and Accuracy of the Detection Method Provided by the Present Invention

[0105] Using the above liquid phase conditions (Table 4) and mass spectrometry conditions (Table 5), the linearity, detection limit, and accuracy of the detection method are verified as follows:

[0106] (1) Linearity: For the standard solutions of a series of concentrations in Step 1, detect them according to the liquid phase conditions in Table 4 and the mass spectrometry conditions in Table 5. Prepare the standard phytohormone solutions: Weigh the standard phytohormones separately. The standard phytohormones include ABA, IAA, SL, SA, JA, MeJA, GA3, TZ, BR, and TZR. Add the mixed solution of methanol / formic acid / water = 80:0.1:19.9 as the solvent respectively, and prepare the mother solutions of phytohormones with a concentration of 1.0 mg / mL for the 10 hormones respectively. Then take 100 μL from each of the mother solutions of various standard phytohormones, mix them evenly and dilute. ABA, IAA, SL, SA, JA, MeJA, BR, and GA3 are respectively diluted into a series of standard solutions with concentrations of 1000 nmol / L, 500 nmol / L, 100 nmol / L, 50 nmol / L, 10 nmol / L, and 5 nmol / L. TZ and TZR are diluted into standard solutions with concentrations of 100 nmol / L, 50 nmol / L, 10 nmol / L, 5 nmol / L, 1 nmol / L, and 0.5 nmol / L for later LC-MS / MS analysis to obtain the standard curve. The results of the linearity and range tests are shown in Table 6.

[0107] (2) Method detection limit: Analyze the injection results of the samples in Step s1 of Example 1, find the minimum concentration that the instrument can measure, and ensure that the response value is greater than 3 times the signal-to-noise ratio (S / N), which is set as the method detection limit (LOD). Repeat the measurement of the lowest concentration of the standard curve and the blank sample solvent 10 times (the blank sample is the blank solution or the matrix where no detection result is obtained in Step S1), record the response value each time, calculate the signal-to-noise ratio, and further verify the accuracy of the detection limit by diluting or concentrating the low-concentration samples. The detection limits of each hormone are shown in Table 6.

[0108] (3) Accuracy: Add standard substances with concentrations of 1, 2, and 100 times the detection limit to the blank samples (blank solution or the matrix where no detection result is obtained in Step S1) respectively. Prepare 10 parallel injections for each sample for injection, and calculate the recovery rate and relative standard deviation.

[0109] The test method detection limit, standard equation, and correlation coefficient are shown in Table 8. The reproducibility results are shown in Table 9.

[0110] Table 8 Detection Limit Analysis

[0111]

[0112] Table 9 Accuracy Analysis

[0113]

[0114]

[0115] As can be seen from the verification results of the above method, the linear coefficients (r) of the 10 phytohormones are all greater than 0.99. The linear relationships of the phytohormones within their respective linear ranges are good, meeting the quantitative requirements. The precision RSD is within 2%, indicating good reproducibility and high accuracy of the method. At the same time, the detection limit and quantification limit of the method are within the range of 0.5 - 1000 nM, indicating high sensitivity of the method. This method can accurately quantify phytohormones in plant tissues with high throughput and high sensitivity.

[0116] Example 4 Comparison of the detection method provided by the present invention with the prior art

[0117] Through precise calculation and optimization of detection conditions, the present invention realizes the detection of ultra-low detection limits for multiple phytohormones. For example, the detection limit of abscisic acid (ABA) is as low as 0.66 μg / kg, the detection limit of indole-3-acetic acid (IAA) is 0.44 μg / kg, and the detection limits of zeatin (TZ) and zeatin riboside (TZR) are even as low as 0.05 μg / kg and 0.09 μg / kg respectively. This detection sensitivity is significantly better than the prior art. For example, in "Simultaneous determination of five plant hormones in cotton leaves using QuEChERS combined with HPLC-MS / MS" published by the Institute of Cotton Research, Chinese Academy of Agricultural Sciences in the "Journal of Cotton Research", the detection limit of GA3 is 20 μg / kg, and the detection limits of IAA, ZT, TZR, and ABA are all 5 μg / kg. In addition, the QuEChERS pretreatment technique used in this literature not only increases the cost and time, but also reduces the spiked recovery rate, and the recovery rates of the 5 phytohormones purified by different adsorbent combinations are about 80%.

[0118] Further comparison reveals that in the detection method reported in the literature "Simultaneous determination of plant endogenous hormones in green mustard by liquid chromatography-Tandem mass spectrometry", the detection limits of IAA and GA3 are 32.6 ng / g and 5.2 ng / g respectively, which are significantly higher than the detection limits of the present invention. By optimizing the detection conditions and pretreatment techniques, the present invention not only greatly reduces the detection limit, but also improves the detection efficiency and accuracy. The conversion results of the detection limits are shown in Table 10.

[0119] Table 10 Conversion of detection limits

[0120]

[0121] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for detecting plant hormones using UHPLC-QqQ-MS / MS, comprising: S1: extracting plant hormones from plant samples to obtain samples to be tested; S2: Using UHPLC-QqQ-MS / MS method to measure the sample to be tested, and obtain the type or content of plant hormones; The UHPLC-QqQ-MS / MS method is a method of establishing a multiple reaction monitoring (MRM) mode for simultaneously monitoring positive and negative ions using an electrospray ionization source (ESI). The plant hormones include strigolactone GR24, trans-zeatin, gibberellin, abscisic acid, jasmonic acid, methyl jasmonate, salicylic acid, trans-zeatin riboside, brassinolide and indole-3-acetic acid.

2. The method according to claim 1, further comprising preparing a plant hormone standard solvent, preparing the plant hormone standard into a standard mother solution with an organic solvent, and then diluting it into a standard solvent according to a gradient to formulate a standard curve; preferably, the organic solvent is a mixed solution containing methanol, formic acid and water; preferably, in the organic solvent, the volume percentage of methanol is 50-80%, and the volume percentage of formic acid is 0.1%-5%; preferably, the organic solvent is a mixed solution formed by mixing methanol, formic acid and water in a volume ratio of 80:0.1:19.

9.

3. The method according to claim 1, wherein: Step S1 includes extracting plant hormones from plant samples using an extracting solution. Preferably, the extracting solution is a mixture of methanol, formic acid and water; preferably, in the extracting solution, the volume percentage of methanol is 50-80%, and the volume percentage of formic acid is 0.1%-5%; preferably, the extracting solution is a mixture of methanol, formic acid and water in a volume ratio of 80:0.1:19.

9.

4. The method according to claim 3, wherein: Step S1 comprises: grinding the plant sample into powder in liquid nitrogen, adding the extract, ultrasonicating in an ice-water bath or grinding with steel beads, freezing at -80°C, centrifuging at 4°C, taking the supernatant, filtering, drying, re-dissolving, and transferring to a sample loading bottle; Preferably, step S1 is performed under light-proof conditions; Preferably, the ice-water bath ultrasonic time is 20-120 min, and the -80°C freezing time is 0.5-12 h; more preferably, the ice-water bath ultrasonic time is 20 min, and the -80°C freezing time is 1.5 h; Preferably, filter with a 0.22 μm organic filter membrane, Preferably, the mixture is reconstituted with a mixture containing methanol, formic acid and water, wherein the volume percentage of methanol in the mixture is 50-80%, and the volume percentage of formic acid is 0.1%-5%; more preferably, the volume ratio of methanol, formic acid and water in the mixture is 80:0.1:19.9; Preferably, the grinding condition with steel beads is: grinding at 1000-2000 rpm for 1-3 min, and circulating for 3-10 cycles; more preferably, the grinding condition with steel beads is: grinding at 2000 rpm for 2 min, and circulating for 5 cycles.

5. The method according to claim 4, wherein: The usage ratio of the extract to the powder is 1-5 μL / mg, preferably 5 μL / mg.

6. The method according to claim 1, wherein: The liquid phase conditions of the UHPLC-QqQ-MS / MS method are as follows: the column model is ZORBAX RRHD Eclipse Plus C18, 2.1x 50mm, 1.8μm; the column temperature is set to 25°C; the liquid phase injection plate temperature is set to 4°C, the injection volume is 5.0μL, the acquisition time is 7.0min, and the mobile phase and flow rate are as follows: Mobile phase A was 0.1% FA-H2O, and mobile phase B was 0.1% FA-CAN; at 0.00 min, mobile phase A was 95% v / v, mobile phase B was 5% v / v, and the flow rate was 0.3 mL / min; at 3.50 min, mobile phase A was 85% v / v, mobile phase B was 15% v / v, and the flow rate was 0.3 mL / min; at 4.00 min, mobile phase A was 40% v / v, mobile phase B was 60% v / v, and the flow rate was 0.3 mL / min.

7. The method according to claim 1, wherein: The mass spectrometry conditions of the UHPLC-QqQ-MS / MS method are as follows: ion source temperature of 250-300°C, nebulizer pressure of 40-60 psi, and electrospray voltage of 3000-3500 V; Preferably, the mass spectrometry conditions of the UHPLC-QqQ-MS / MS method are: The ion source temperature was 250°C, the nebulizer pressure was 45 psi, and the electrospray voltage was 3500V.

8. The method according to any one of claims 1 to 7, wherein: In the multiple reaction monitoring (MRM) mode of simultaneously monitoring positive and negative ions, the positive ion mode corresponds to compounds IAA, TZ, SL, MeJA, BR and TZR, and the negative ion mode corresponds to compounds ABA, GA3, JA and SA.

9. The method according to claim 8, wherein: The mass spectrum acquisition conditions of the UHPLC-QqQ-MS / MS method are: The positive mode acquisition parameters were set as follows: IAA fragmentation voltage was 90 V, and collision energies were 35 V (176.07>103.1), 35 V (176.07>128), 35 V (176.07>77.2), and 21 V (176.07>130); The TZ fragmentation voltage was 120 V, and the collision energies were 35 V (220.12>119), 17 V (220.12>136), 13 V (220.12>148), and 13 V (220.12>202.1), respectively. The TZR fragmentation voltage was 120 V, and the collision energies were 21 V (352.2>220.0), 25 V (352.2>202.0), 33 V (352.2>148.0), and 35 V (352.2>136.0), respectively. The MeJA fragmentation voltage was 90 V, and the collision energies were 9 V (225.1>150.8), 17 V (2 25.1>147.0), 13V(225.1>133.0) and 29V(225.1>105.1); BR fragmentation voltage was 110V, and the collision energies were 17V(481.36>315.2), 17V(481.36>113.1), 40V(481.36>95.1) and 33V(481.36>71.1); SL ​​fragmentation voltage was 90V, and the collision energies were 17V(321.1>97.1), 25V(321.1>157), 13V(321.1>183.9) and 5V(321.1>185); The negative mode acquisition parameters were set as follows: ABA fragmentation voltage was 120 V, and the collision energies were 5 V (263.13>153.1), 35 V (263.13>203), 21 V (263.13>204.1) and 13 V (263.13>219.1), respectively; SA fragmentation voltage was 90 V, and the collision energies were 17 V (137.0>93.0) and 33 V (137.0>65.1), respectively; JA fragmentation voltage was 120 V, and the collision energy was 13 V (209.1>59.1), respectively; GA3 fragmentation voltage was 180 V, and the collision energies were 35 V (345.13>143.1), 25 V (345.13>221.1), 29 V (345.13>227.1) and 13 V (345.13>239.2), respectively.

10. The method according to claim 9, wherein: Tuning EMV in positive or negative ion mode increases by 200V.

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