A method for simultaneously detecting 11 plant hormones in loquat by UPLC-QQQ-MS / MS and application thereof

Abstract

The application belongs to the technical field of plant hormone detection, and particularly relates to a method for simultaneously detecting 11 kinds of plant hormones in loquat by UPLC-QQQ-MS / MS and application thereof. The method for simultaneously detecting 11 kinds of plant hormones in loquat by UPLC-QQQ-MS / MS comprises the following steps: step one, sample pretreatment; step two, UPLC-QQQ-MS / MS detection and analysis; and step three, quantitative analysis of the contents of the 11 kinds of endogenous hormones. The 11 kinds of endogenous hormones include IAA, ABA, tZR, iPR, DHZ, Zeatin, iP, GA1, GA3, GA4 and GA7. The application establishes an analysis method for simultaneously determining endogenous hormones in various loquat tissues by using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, has the advantages of high sensitivity and high analysis speed, and can accurately distinguish and quantify various plant endogenous hormones.

Description

Technical Field

[0001] This invention belongs to the field of plant hormone detection technology, specifically relating to a method for simultaneously detecting 11 plant hormones in loquat using UPLC-QQQ-MS / MS and its application. Background Technology

[0002] Plant hormones are key signaling molecules that regulate plant growth and development. Among them, auxin (IAA), abscisic acid (ABA), gibberellins (GAs), and cytokinins (CKs) are the core hormone groups that regulate flower bud differentiation, fruit development, and quality formation in fruit trees.

[0003] Loquat (Eriobotrya japonica Lindl.), an important characteristic evergreen fruit tree in southern my country, has a long annual growth cycle. The dynamic balance of hormones in different tissues (such as buds, fruits, seeds, and leaves) at different developmental stages is key to understanding its physiological mechanisms. In the actual detection of endogenous hormone content in loquat, the complex matrix composition of each tissue presents significant challenges. For example, loquat leaves and fruits are rich in polysaccharides, polyphenols, and pigments, while the matrix interference in seeds and inflorescence tissues varies. This complex matrix effect can severely inhibit or interfere with trace hormone signals. Currently, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) are commonly used to determine endogenous plant hormones in loquat. However, these two methods have significant limitations in practical applications: while ELISA is simple to operate, it relies on the specific recognition of antigens and antibodies. Because plant hormones are often composed of multiple homologues with highly similar structures, antibodies are prone to cross-reactions during recognition, leading to misidentification. Furthermore, the complex endogenous matrix of loquat tissue easily interferes with colorimetric reactions, making it difficult to meet the requirements for accurate quantification in terms of sensitivity and repeatability. HPLC mainly relies on the difference in compound polarity for separation and quantification using UV or fluorescence detectors. However, the physicochemical properties of various hormones in loquat tissue (such as GA1, GA3, GA4, and GA7) are extremely similar, easily leading to "co-elution" on conventional chromatographic columns, resulting in overlapping peaks and making accurate differentiation and qualitative analysis impossible. Moreover, for endogenous hormones present in extremely low concentrations, the sensitivity and selectivity of traditional detectors are insufficient, making it difficult to eliminate interference from the complex matrix background and achieve the simultaneous detection of multiple plant hormones.

[0004] Therefore, establishing a highly efficient UPLC-QQQ-MS / MS detection strategy for the complex tissue matrix of loquat, which can simultaneously achieve precise quantification of key endogenous hormones, has important application value for the molecular breeding and efficient cultivation of loquat. Summary of the Invention

[0005] This invention aims to provide a method for the simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS and its applications. This invention establishes an analytical method for the simultaneous determination of multiple endogenous hormones in loquat tissues using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, which has the advantages of high sensitivity and high analysis speed, enabling accurate differentiation and quantification of multiple plant endogenous hormones.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS, comprising the following steps: Step 1: Sample Pretreatment Loquat was pretreated to obtain loquat tissue samples; the loquat tissue samples were weighed and labeled W, and extraction solvent and internal standard solution were added to obtain a first mixture; the first mixture was extracted, and then an extraction solvent was added to obtain a second mixture; the second mixture was extracted, allowed to stand, and allowed to separate into layers. The bottom layer solution was taken and concentrated to obtain a third mixture; a composite solution was added to the third mixture to reconstitute it, and the volume was marked V to obtain a fourth mixture; the fourth mixture was filtered to obtain a reconstituted solution; Step 2: UPLC-QQQ-MS / MS detection and analysis: The reconstituted solution obtained in step one was loaded into a chromatographic injection vial and analyzed by UPLC-QQQ-MS / MS. Chromatographic conditions: HSS T3 column was used, mobile phase A was pure water, mobile phase B was acetonitrile, and elution was performed by gradient elution. Mass spectrometry conditions: Electrospray ionization source, multiple reaction detection mode, ion source temperature 150℃, capillary voltage 0.35 kV, desolvation gas temperature 500℃, nebulizer gas flow rate 1000 L / Hr, cone gas flow rate 50 L / Hr; collisional argon gas 0.17 L / h; Step 3: Quantitative analysis of the levels of 11 endogenous hormones: Standard solutions of 11 endogenous hormones at various concentrations were prepared. Each standard solution was analyzed sequentially under the chromatographic and mass spectrometric conditions described in step two. Standard curves for the 11 endogenous hormones were plotted, and linear regression equations were obtained. During the sample quantification process, the hormone detection results from step two, UPLC-QQQ-MS / MS, were substituted into the linear regression equation to obtain the concentration C of each hormone. The content of endogenous hormones in loquat tissue is calculated using the formula: FW = CV / W, where FW is the content of endogenous hormones in loquat tissue; C is the concentration of endogenous hormones; V is the fixed volume; and W is the mass of the loquat tissue sample. The 11 endogenous hormones include indole-3-acetic acid (IAA), abscisic acid (ABA), trans-zeatin nucleoside (tZR), isopentenyl adenine nucleoside (iPR), dihydrozeatin (DHZ), zeatin, isopentenyl adenine (iP), GA1, GA3, GA4, and GA7.

[0007] Preferably, the pretreatment of loquat involves grinding the loquat tissue into powder in liquid nitrogen.

[0008] Preferably, the extraction solvent is a mixed solution of isopropanol, water and hydrochloric acid pre-cooled to 4°C; the volume ratio of isopropanol, water and hydrochloric acid is (1~3):(0.5~2):(0.001~0.005).

[0009] More preferably, the extraction solvent is a mixed solution of isopropanol, water and hydrochloric acid pre-cooled to 4°C; the volume ratio of isopropanol, water and hydrochloric acid is 2:1:0.002.

[0010] Preferably, after adding the internal standard solution, the final concentration of the internal standard solution is 40~60 ng / mL.

[0011] More preferably, after adding the internal standard solution, the final concentration of the internal standard solution is 50 ng / mL.

[0012] Preferably, the extractant is a dichloromethane solution pre-cooled to 4°C.

[0013] In the detection method of this invention, by using pre-cooled dichloromethane as the extractant, not only can impurities such as alcohols, phenols, and esters in the loquat sample be effectively removed, but more importantly, dichloromethane can have a specific solubility equilibrium effect on the triterpenoid components or thick waxy layer unique to loquat. Dichloromethane can efficiently and selectively transfer the four types of hormones (11 plant hormones) measured in this invention to the organic phase, while retaining the high content of ursolic acid and other interfering substances unique to the loquat matrix in the aqueous phase or at the interface, thereby avoiding the contamination of the mass spectrometry ion source by the high molecular weight acidic substances. Meanwhile, in the detection method of this invention, dichloromethane is selected as the extractant for loquat samples, which balances the various hormones with huge polarity differences contained in loquat. This allows highly polar cytokinins (tZR, iPR, iP, DHZ, Zeatin) and weakly polar abscisic acid (ABA) to simultaneously achieve extremely high recovery rates and reproducibility, thus solving the bottleneck of recovery rate that is difficult to balance in complex matrices.

[0014] Preferably, the extraction conditions for the first mixed solution are: extraction at 4°C and 3000 rpm with shaking and light protection for 1-3 h.

[0015] Preferably, the second mixture is extracted by shaking and extracting under light at 4°C and 3000 rpm for 1-3 hours.

[0016] In the extraction and leaching process of the detection method of this invention, high-frequency oscillation is used to enable the extraction solvent to quickly penetrate into the damaged cell wall, ensuring that the hormones in the deep layers are completely replaced. This further ensures that the detection of 11 plant hormones contained in loquat samples can be achieved simultaneously, with high sensitivity and high analysis speed.

[0017] Preferably, the standing period is: standing at 4°C for 0.5 to 1 hour.

[0018] Preferably, the composite solution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 80:20.

[0019] Preferably, the fourth mixture is filtered by passing it through a 0.22 µm polytetrafluoroethylene membrane.

[0020] Preferably, the HSS T3 chromatographic column has dimensions of 2.1 mm × 100 mm and 2.5 µm.

[0021] Preferably, the gradient elution program is as follows: 0–1.5 min, 10%–60% pure water; 1.5–5.0 min, 60%–90% pure water; 5.0–6.0 min, 90% pure water; 6.0–6.1 min, 90%–10% pure water; 6.1–7.0 min, 10%–10% pure water, with an elution flow rate of 0.4 mL / min. -1 The injection volume was 2 μL, and the column temperature was 40℃.

[0022] Preferably, the preparation of the standard solution includes the following steps: preparing hormone standard solutions of 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL, and adding an equal amount of the corresponding hormone internal standard solution to each concentration of hormone standard solution to make the internal standard concentration 40~60 ng / mL, thus obtaining standard solutions of each concentration.

[0023] More preferably, the preparation of the standard solution includes the following steps: preparing hormone standard solutions of 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL, and adding an equal amount of the corresponding hormone internal standard solution to each concentration of hormone standard solution to make the internal standard concentration 50 ng / mL, thereby obtaining standard solutions of each concentration.

[0024] Preferably, the standard curve is plotted as follows: based on the measured peak area S1 of the hormone standard and the peak area S2 of the internal standard, a standard curve is plotted with the standard quality concentration C1 as the abscissa and S1 / S2 as the ordinate.

[0025] The present invention also claims protection for the application of the method for simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS in loquat growth regulation, breeding optimization, and high-efficiency cultivation.

[0026] By measuring the content of 11 endogenous hormones (GA1, GA3, GA4, GA7, ABA, tZR, iPR, DHZ, Zeatin, iP, and IAA) in different tissues of loquat, including young leaves, old leaves, seeds, buds, pulp, flower buds, stamens, and pistils, the differences in hormone distribution in different organs can be determined. The data on the content of each hormone can provide accurate data support and technical basis for plant growth regulation, breeding optimization, and functional development.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes an analytical method for the simultaneous determination of multiple endogenous hormones in loquat tissues using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry. By selecting and determining the process steps and parameters, such as loquat sample pretreatment, chromatographic testing conditions, and mass spectrometry testing conditions, the method can simultaneously detect 11 endogenous hormones with similar physicochemical properties in loquat. Furthermore, this method possesses the advantages of high sensitivity (the detection limit for isopentenyl adenine can reach 0.011 ng / g) and high analytical speed, making it suitable for accurately distinguishing and quantifying multiple plant endogenous hormones contained in loquat. This method has significant application value for loquat molecular breeding and efficient cultivation. Attached Figure Description

[0028] Figure 1 This is a comparative analysis of the effects of different extraction solvents on the extraction of 11 plant hormones from loquat tissue in this invention. The data in the figure are mean ± standard deviation. "abc" indicates a significant difference at the p < 0.05 level. If two samples contain the same letter, it indicates no significant difference between them.

[0029] Figure 2 This is a comparative analysis chart showing the effects of different extraction methods on 11 plant hormones in this embodiment of the invention. The data in the chart are mean ± standard deviation, and abc indicates a significant difference at the p < 0.05 level. If two samples contain the same letter, it indicates no significant difference between them.

[0030] Figure 3This is a comparative analysis chart showing the effects of different extraction times on 11 plant hormones in this embodiment of the invention. The data in the chart are mean ± standard deviation, and abc indicates a significant difference at the p < 0.05 level. If two samples contain the same letter, it indicates no significant difference between them.

[0031] Figure 4 This is a comparative analysis chart showing the effects of different extraction methods on 11 plant hormones in this embodiment of the invention. The data in the chart are mean ± standard deviation, and abc indicates a significant difference at the p < 0.05 level. If two samples contain the same letter, it indicates no significant difference between them.

[0032] Figure 5 This is a comparative analysis chart showing the effects of different organic mobile phases in embodiments of the present invention. * indicates a significant difference (P < 0.05).

[0033] Figure 6 This is a comparative analysis chart of the effects of different aqueous phases in an embodiment of the present invention. The data in the chart are mean ± standard deviation, and abc indicates a significant difference at the p < 0.05 level. If two samples contain the same letter, it indicates no significant difference between them.

[0034] Figure 7 This is a quantitative ion chromatogram in an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0037] This embodiment describes a method for simultaneously detecting 11 plant hormones in loquat using UPLC-QQQ-MS / MS, which includes the following steps: Step 1: Sample Pretreatment Loquat was pretreated to obtain loquat tissue samples; the loquat tissue samples were weighed and labeled W, and extraction solvent and internal standard solution were added to obtain a first mixture; the first mixture was extracted, and then an extraction solvent was added to obtain a second mixture; the second mixture was extracted, allowed to stand, and allowed to separate into layers. The bottom layer solution was taken and concentrated to obtain a third mixture; a composite solution was added to the third mixture to reconstitute it, and the volume was marked V to obtain a fourth mixture; the fourth mixture was filtered to obtain a reconstituted solution; The pretreatment of loquat involves grinding the loquat tissue into powder in liquid nitrogen. The extraction solvent is a mixed solution of isopropanol, water, and hydrochloric acid pre-cooled to 4°C; the volume ratio of isopropanol, water, and hydrochloric acid is (1~3):(0.5~2):(0.001~0.005). After adding the internal standard solution, the final concentration of the internal standard solution is 40~60 ng / mL.

[0038] The extractant is a dichloromethane solution pre-cooled to 4°C; The extraction conditions for the first mixed solution are: extraction at 4℃ and 3000 rpm with shaking and in the dark for 1~3 h; The second mixture was extracted by shaking and darkening at 4°C and 3000 rpm for 1-3 hours. The settling period is: settling at 4°C for 0.5–1 h; The composite solution is: methanol: distilled water (v / v = 80 / 20). The fourth mixture is filtered by passing it through a 0.22 µm polytetrafluoroethylene membrane. Step 2: UPLC-QQQ-MS / MS detection and analysis: The reconstituted solution obtained in step one was loaded into a chromatographic injection vial and analyzed by UPLC-QQQ-MS / MS. Chromatographic conditions: C 18 The chromatographic column uses pure water as mobile phase A and acetonitrile as mobile phase B, and elution is performed using a gradient elution method. The chromatographic column used was a Waters X Select HSS T3 column, with dimensions of 2.1 mm × 100 mm and 2.5 µm.

[0039] The gradient elution program is as follows: 0–1.5 min, 10%–60% pure water; 1.5–5.0 min, 60%–90% pure water; 5.0–6.0 min, 90% pure water; 6.0–6.1 min, 90%–10% pure water; 6.1–7.0 min, 10%–10% pure water, with an elution flow rate of 0.4 mL / min.-1 The injection volume was 2 μL, and the column temperature was 40℃.

[0040] Mass spectrometry conditions: Electrospray ionization source, multiple reaction detection mode, ion source temperature 150℃, capillary voltage 0.35 kV, desolvation gas temperature 500℃, nebulizer gas flow rate 1000 L / Hr, cone gas flow rate 50 L / Hr; collisional argon gas 0.17 L / h; Step 3: Quantitative analysis of the levels of 11 endogenous hormones: Standard solutions of 11 endogenous hormones at various concentrations were prepared. Each standard solution was analyzed sequentially under the chromatographic and mass spectrometric conditions in step two. Based on the measured peak area S1 of the hormone standard and the peak area S2 of the internal standard, standard curves of the 11 endogenous hormones were plotted with the standard concentration C1 as the abscissa and S1 / S2 as the ordinate, and linear regression equations were obtained. The preparation of the standard solution includes the following steps: preparing hormone standard solutions of 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL, and adding an equal amount of the corresponding hormone internal standard solution to each concentration of hormone standard solution to make the internal standard concentration 40~60 ng / mL, thus obtaining standard solutions of each concentration; During the sample quantification process, the hormone detection results from step two, UPLC-QQQ-MS / MS, were substituted into the linear regression equation to obtain the concentration C of each hormone. The content of endogenous hormones in loquat tissue is calculated using the formula: FW = CV / W, where FW is the content of endogenous hormones in loquat tissue; C is the concentration of endogenous hormones; V is the fixed volume; and W is the mass of the loquat tissue sample. The 11 endogenous hormones include IAA, ABA, tZR, iPR, DHZ, Zeatin, iP, GA1, GA3, GA4, and GA7.

[0041] Example 1: A method for simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS. Step 1, Sample pretreatment, includes: Loquat tissue was ground into powder in liquid nitrogen to obtain various loquat tissue samples; Weigh 300 mg of loquat tissue sample, labeled as W, and add 3 mL of isopropanol / water / hydrochloric acid mixed solvent (2 / 1 / 0.002, v / v / v) pre-cooled to 4°C to the loquat tissue sample. At the same time, add internal standard solution to the loquat tissue sample to make the final concentration of internal standard 50 ng / mL to obtain the first mixture. The first mixture was subjected to extraction at 4°C and 3000 rpm under light for 2 h. Then, 3 mL of pre-cooled dichloromethane extractant was added to the first mixture to obtain the second mixture; The second mixture was subjected to extraction at 4°C and 3000 rpm under light for 2 h with shaking. Let it stand at 4°C for 0.5 to 1 h until the second mixture separates into three layers. Take about 2.5 mL of the bottom layer solution and concentrate it to near dryness by nitrogen blowing to obtain the concentrated third mixture. Add 0.2 mL of 80% mass spectrometry-grade methanol composite solution (methanol to distilled water volume ratio of 80:20) to the third mixture for redissolution, and mark the final volume as V to obtain the fourth mixture; The fourth mixture was filtered through a 0.22 µm polytetrafluoroethylene membrane and then loaded into a chromatographic sample vial.

[0042] Step 2, UPLC-QQQ-MS / MS detection and analysis, which includes: The reconstituted solution was analyzed by UPLC-QQQ-MS / MS. Chromatographic conditions: Waters X Select HSS T3 column (2.1 mm × 100 mm, 2.5 µm), mobile phase A was pure water, mobile phase B was acetonitrile, injection volume was 2 uL, column temperature was 40℃, and elution was performed by gradient elution. The elution program is shown in Table 1.

[0043] Table 1 Gradient elution program Mass spectrometry conditions: Electrospray ionization source, multiple reaction detection mode, ion source temperature 150℃, capillary voltage 0.35 kV, desolvation gas temperature 500℃, nebulizer gas flow rate 1000 L / Hr, cone gas flow rate 50 L / Hr; collision argon gas 0.17 L / h. The relevant mass spectrometry parameters for each hormone are shown in Table 2.

[0044] Table 2. Relevant mass spectrometry parameters of hormones Note: * indicates qualitative ions. Step 3: Quantitative analysis of hormone levels, which includes: Quantitative analysis was performed on each endogenous hormone separately, and the specific steps included: For each hormone, the procedure is as follows: Prepare hormone standard solutions of 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL. Add an equal volume of the corresponding hormone internal standard solution to each concentration of standard solution to make the internal standard concentration 50 ng / mL. Analyze each standard solution sequentially under the chromatographic and mass spectrometric conditions described in step two. Measure the peak area S1 of the hormone standard and the peak area S2 of the internal standard. Plot a standard curve with the standard concentration C1 as the abscissa and S1 / S2 as the ordinate, and obtain the linear regression equation. The corresponding linear curves, linear regression equations, and other data are shown in Table 3-4. During the sample quantification process, the hormone detection results from step two, UPLC-QQQ-MS / MS, were substituted into the linear regression equation to obtain the concentration C of each hormone. The formula for calculating the endogenous hormone content in loquat tissue is: FW = CV / W The content of endogenous hormones in FW loquat tissue; C represents the concentration of endogenous hormones; V is the constant volume; W represents the mass of the loquat tissue sample.

[0045] Table 3. Linear curves, correlation coefficients, limits of detection, limits of quantitation, and precision of 11 plant hormones. Table 4. Spike recoveries and relative standard deviations of 11 plant hormones (n=3) This invention achieves ultra-low detection limits for multiple plant hormones through precise calculation and optimization of detection conditions. Specifically, the detection limits for abscisic acid (ABA) are 0.148 ng / g, for indoleacetic acid (IAA) 0.087 ng / g, for zeatin as low as 0.06 ng / g, for zeatin nucleoside (tZR) as low as 0.041 ng / g, and for isopentenyl adenine (iP) as low as 0.011 ng / g. This detection sensitivity is significantly superior to existing technologies.

[0046] Example 2: Effects of different extraction solvents on the detection results of 11 hormones This example compares the extraction efficiencies of five extraction systems—isopropanol / H2O / HCl (2 / 1 / 0.002, v / v / v), isopropanol / water / acetic acid (80 / 19 / 1, v / v / v), methanol / H2O / formic acid (75 / 20 / 5, v / v / v), acetone / H2O (80 / 20, v / v), and acetonitrile / H2O (80 / 20, v / v)—for 11 hormones from loquat seeds. The experimental methods are the same as in Example 1, and the results are as follows: Figure 1 As shown.

[0047] from Figure 1 The experimental results show that the acetone:water (80 / 20, v / v) system is most effective for active gibberellins, especially GA1 and GA3, which is consistent with previous research results. The isopropanol:water:acetic acid (80 / 19 / 1, v / v / v) system shows excellent performance in the extraction of specific cytokinins such as Zeatin and iP. The isopropanol:water:hydrochloric acid (2 / 1 / 0.002, v / v / v) system achieved significantly the highest response for four hormones: ABA, tZR, GA4, and GA7, and maintained a high response level for the remaining seven hormones. Therefore, to balance the sensitivity and accuracy of four classes of hormones (11 hormones), this embodiment ultimately selected isopropanol:water:hydrochloric acid (2 / 1 / 0.002, v / v / v) as the extraction solvent.

[0048] Example 3: Effects of different extraction methods and extraction times on the detection results of 11 hormones The physical extraction method and processing time have a significant impact on the degree of cell wall disruption and hormone release efficiency. Therefore, after determining the extraction solvent, three methods were further evaluated: vortex extraction (3000 rpm, 4℃, protected from light, 2h), ultrasound-assisted extraction (40kHz, ice bath, protected from light, 2h), and static extraction (static, 4℃, protected from light, 2h). The extraction time was also investigated to obtain the optimal extraction efficiency. The experimental methods are as described in Example 1, and the experimental results are as follows: Figure 2-3 As shown.

[0049] from Figure 2 The experimental results show that vortex extraction exhibits significant superiority in the extraction of four types of hormones, demonstrating the highest response among all 10 hormones except for IAA. Notably, ultrasound-assisted extraction showed extremely strong specific release of IAA, with a response value nearly 10 times higher than that of vortex extraction. In contrast, static extraction had the lowest extraction efficiency for cytokinins such as DHZ and Zeatin, indicating that simple diffusion is insufficient to release endogenous hormones from loquat tissue.

[0050] from Figure 3The results show that different hormones exhibited significant differences in their extraction kinetics. Active gibberellins GA4 and GA7 reached their peak values ​​after 2 hours of extraction, followed by a significant decrease, indicating that prolonged treatment may lead to the degradation of these nonpolar gibberellins. Conversely, the contents of GA3 and iPR increased with time, reaching their highest values ​​at 8 and 12 hours, respectively. ABA, tZR, and IAA showed relative stability within 2 to 12 hours, with no significant differences. In conclusion, vortex extraction for 2 hours and extraction for 2 hours were ultimately determined as the steps for analyzing four classes of endogenous plant hormones (11 plant hormones).

[0051] Example 4: Effects of different extractants on the detection of 11 hormones Because loquat tissue matrix is ​​complex and contains a large number of secondary metabolites, effective purification steps are necessary to reduce matrix inhibition and improve detection sensitivity. This example compares two solid-phase extraction (C2000-C400) methods. 18 The purification effects of SPE solid-phase extraction column, HLB solid-phase extraction column and two liquid-liquid extraction methods (dichloromethane, ethyl acetate) on 11 hormones. The experimental methods are as described in Example 1, and the experimental results are as follows: Figure 4 As shown.

[0052] from Figure 4 The results show that liquid-liquid extraction is significantly more efficient than solid-phase extraction. Dichloromethane achieved the highest response values ​​for IAA, ABA, four active GAs, and two CKs, iPR and iP. Solid-phase extraction, when processing loquat leaf samples, may lead to severe hormone loss due to matrix overload or incomplete elution. Therefore, dichloromethane was ultimately chosen as the extraction agent for four classes of hormones (eleven plant hormones).

[0053] Example 5: Effects of different flow rates on the detection of 11 hormones The mobile phase is a key factor affecting liquid chromatography, significantly influencing not only the separation and retention time of analytes but also directly impacting ion ionization efficiency. To obtain the highest sensitivity and optimal peak shape in the analysis of various plant hormones, this example systematically evaluated the effects of different organic mobile phases (methanol and acetonitrile) and aqueous phases (pure water, 0.05% formic acid solution, 0.1% formic acid solution, 1 mmol / L ammonium formate solution, and 5 mmol / L ammonium formate solution) on the mass spectrometry response. Experimental methods are as described in Example 1, and experimental results are as follows: Figure 5-7 As shown.

[0054] Regarding the selection of organic phase ( Figure 5For other hormones, including GAs, IAA, ABA, and most cytokinins such as iP and tZR, acetonitrile's response was slightly higher than or equal to that of methanol. Although Zeatin and DHZ showed comparable responses in both solvent systems, acetonitrile demonstrated superior overall elution capacity. Therefore, acetonitrile was ultimately chosen as the optimal organic mobile phase (mobile phase B) for subsequent experiments.

[0055] Regarding the choice of aqueous phase, although volatile acids are usually introduced to adjust the pH and enhance ionization, the final results show that the addition of formic acid or ammonium formate actually leads to significant ion suppression for almost all target analytes. Figure 6 Pure water achieved the highest peak area among all 11 plant hormones, significantly outperforming other treatments. Based on the experimental results using both organic and aqueous mobile phases, pure water (mobile phase A) and acetonitrile (mobile phase B) were ultimately selected as the optimal chromatographic system.

[0056] Under the defined UHPLC-QQQ-MS / MS conditions, the obtained chromatograms showed that the peaks of each hormone were smooth and symmetrical, and the retention times were stable. Figure 7 This indicates that the detection method described in Example 1 of the present invention exhibits high sensitivity and stability, making it suitable for the separation of endogenous hormones from loquat tissue.

[0057] Example 6: Practical application of a method for simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS. The detection method described in Example 1 was used to determine the content of 11 endogenous hormones (GA1, GA3, GA4, GA7, ABA, tZR, iPR, DHZ, Zeatin, iP, and IAA) in different tissues of loquat, including young leaves, old leaves, seeds, buds, pulp, flower buds, stamens, and pistils. The test results are shown in Table 5.

[0058] Table 5. Determination of the content of 11 endogenous hormones in different parts of loquat. Table 5 shows that the distribution of hormones in different organs varies significantly. The seeds and pulp contain high levels of gibberellins and cytokinins, while abscisic acid accumulation is evident in the stamens and pistils. The hormone content data are stable and reliable. The results of determining the content of 11 endogenous hormones in different parts of the loquat can provide accurate data support and technical basis for plant growth regulation, breeding optimization, and functional development.

[0059] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for simultaneously detecting 11 plant hormones in loquat by UPLC-QQQ-MS / MS, characterized in that, Includes the following steps: Step 1: Sample Pretreatment Loquat was pretreated to obtain loquat tissue samples; the loquat tissue samples were weighed and labeled W, and extraction solvent and internal standard solution were added to obtain a first mixture; the first mixture was extracted, and then an extraction solvent was added to obtain a second mixture; the second mixture was extracted, allowed to stand, and allowed to separate into layers. The bottom layer solution was taken and concentrated to obtain a third mixture; a composite solution was added to the third mixture to reconstitute it, and the volume was marked V to obtain a fourth mixture; the fourth mixture was filtered to obtain a reconstituted solution; Step 2: UPLC-QQQ-MS / MS detection and analysis: The reconstituted solution obtained in step one was loaded into a chromatographic injection vial and analyzed by UPLC-QQQ-MS / MS. Chromatographic conditions: HSS T3 column was used, mobile phase A was pure water, mobile phase B was acetonitrile, and elution was performed by gradient elution. Mass spectrometry conditions: Electrospray ionization source, multiple reaction detection mode, ion source temperature 150℃, capillary voltage 0.35 kV, desolvation gas temperature 500℃, nebulizer gas flow rate 1000 L / Hr, cone gas flow rate 50 L / Hr; collisional argon gas 0.17 L / h; Step 3: Quantitative analysis of the levels of 11 endogenous hormones: Standard solutions of 11 endogenous hormones at various concentrations were prepared. Each standard solution was analyzed sequentially under the chromatographic and mass spectrometric conditions described in step two. Standard curves for the 11 endogenous hormones were plotted, and linear regression equations were obtained. During the sample quantification process, the hormone detection results from step two, UPLC-QQQ-MS / MS, were substituted into the linear regression equation to obtain the concentration C of each hormone. The content of endogenous hormones in loquat tissue is calculated using the formula: FW = CV / W, where FW is the content of endogenous hormones in loquat tissue; C is the concentration of endogenous hormones; V is the fixed volume; and W is the mass of the loquat tissue sample. The 11 endogenous hormones include IAA, ABA, tZR, iPR, DHZ, Zeatin, iP, GA1, GA3, GA4, and GA7.

2. The method as described in claim 1, characterized in that, The pretreatment of loquat involves grinding the loquat tissue into powder in liquid nitrogen. The extraction solvent is a mixed solution of isopropanol, water, and hydrochloric acid pre-cooled to 4°C; the volume ratio of isopropanol, water, and hydrochloric acid is (1~3):(0.5~2):(0.001~0.005). After adding the internal standard solution, the final concentration of the internal standard solution is 40~60 ng / mL.

3. The method of claim 1, wherein, The extractant is a dichloromethane solution pre-cooled to 4°C.

4. The method as described in claim 1, characterized in that, The extraction conditions for the first mixed solution are: extraction at 4℃ and 3000 rpm with shaking and in the dark for 1~3 h; The second mixture was extracted by shaking and extracting under light at 4°C and 3000 rpm for 1-3 hours.

5. The method as described in claim 1, characterized in that, The settling period is: settling at 4°C for 0.5–1 h; The composite solution is a mixture of methanol and water, with a volume ratio of methanol to water of 80:

20. The fourth mixture is filtered by passing it through a 0.22 µm polytetrafluoroethylene membrane.

6. The method of claim 1, wherein, The HSS T3 column has dimensions of 2.1 mm × 100 mm and 2.5 µm.

7. The method of claim 1, wherein, The gradient elution program is as follows: 0~1.5 min, 10%~60% pure water; 1.5~5.0 min, 60%~90% pure water; 5.0–6.0 min, 90% pure water; 6.0–6.1 min, 90%–10% pure water; 6.1–7.0 min, 10%–10% pure water, elution flow rate 0.4 mL / min. -1 The injection volume was 2 μL, and the column temperature was 40℃.

8. The method of claim 1, wherein, The preparation of the standard solutions includes the following steps: preparing hormone standard solutions of 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL, and adding an equal amount of the corresponding hormone internal standard solution to each concentration of hormone standard solution to make the internal standard concentration 40~60 ng / mL, thus obtaining standard solutions of each concentration.

9. The method of claim 1, wherein, The method for plotting the standard curve is as follows: based on the measured peak area S1 of the hormone standard and the peak area S2 of the internal standard, a standard curve is plotted with the standard quality concentration C1 as the abscissa and S1 / S2 as the ordinate.

10. The application of a method for simultaneous detection of 11 plant hormones in loquat using UPLC-QQQ-MS / MS as described in any one of claims 1 to 9 in loquat growth regulation, breeding optimization, and high-efficiency cultivation.

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