CN111323506B - Method for determining phytohormone in high-fat plant sample - Google Patents
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Abstract
The invention discloses a method for determining phytohormone in a high-fat plant sample, which comprises a pretreatment process and a quantitative analysis process, wherein the pretreatment process comprises solvent extraction, solid-phase microextraction synchronous derivatization reaction and elution collection, and the method also comprises the step of carrying out quantitative determination on the phytohormone by a liquid chromatography-mass spectrometry analysis method; the method is realized by using a novel magnetic nano material Fe 3 O 4 /Ti 3 C 2 The/beta-cyclodextrin has high selectivity on the phytohormone, has simple steps in the analysis and detection process, can realize high-sensitivity and high-accuracy determination on the phytohormone in a high-oil plant sample, and has the advantages of environmental friendliness, simplicity in operation, rapidness, high efficiency, sensitivity and the like.
Description
Technical Field
The invention relates to the field of chemical analysis, in particular to a method for determining phytohormone in a high-fat plant sample.
Background
Plant hormones are highly bioactive small signaling molecules that are involved in regulating plant growth and development and respond to external stimuli. It is known that hormones produced in plants mainly include auxins, gibberellins (GA), cytokinins, abscisic acid (ABA), ethylene, brassinosteroids, etc., and how to directly and accurately measure the variety and concentration change of phytohormones is a key point of phytology research.
Rape, soybean, sunflower, peanut and cottonseed are five major oil crops in the world, and play a significant role in edible vegetable oil supply in China. The plant hormone has obvious influence on seed germination, seedling growth, yield traits and the like of oil crops such as rape, soybean, peanut and the like, but the quantitative determination of the plant hormone is difficult to perform due to the interference of the oil matrix in the high-oil plant sample, and the measurement accuracy is low. At present, the analysis of phytohormones in high-fat plant samples specifically faces three major problems: firstly, the concentration of the phytohormone in the plant body is low, and is only in ng/g or even pg/g grade; secondly, the matrix interference is large, the matrix in the plant body is complex, and the lipid interference of high-oil plant samples such as rape seeds is large; thirdly, the amount of plant samples is small, and with the deep research of the action mechanism of plant hormone molecules, the quantitative detection of tiny plant tissues or organs is needed. Therefore, the method has important research significance for carrying out high-sensitivity detection on the phytohormone in the trace plant sample.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a method for measuring the phytohormone, which can detect the phytohormone in a high-fat plant sample with high sensitivity.
The method provided by the embodiment of the invention comprises a pretreatment process and a quantitative analysis process, wherein the pretreatment process comprises the following steps of:
s1, extracting the treated plant sample by adopting a solvent to obtain a sample solution;
s2, adding a magnetic nano material and a derivatization reagent into the sample solution, carrying out solid-phase microextraction and synchronous derivatization reaction on the phytohormone in the sample solution, and then separating and collecting the magnetic nano material;
s3, eluting the magnetic nano material obtained in the step S2, and collecting supernatant;
wherein the magnetic nano material is Fe 3 O 4 /Ti 3 C 2 Beta-cyclodextrin.
The method provided by the embodiment of the invention has at least the following beneficial effects:the method can realize high sensitivity and high accuracy determination of the phytohormone in the high-oil plant sample through the pretreatment steps of matrix dispersion, magnetic solid-phase microextraction synchronous derivatization and the like, has simple steps in the analysis and detection process, has no transfer in the whole pretreatment process, can reduce loss, and can detect the phytohormone in a trace sample; magnetic nanomaterial Fe used in the invention 3 O 4 /Ti 3 C 2 The first time the/beta-cyclodextrin is based on Ti 2 C 3 The novel nano magnetic extraction adsorbent synthesized by modifying the material has high selectivity to phytohormone.
According to some embodiments of the invention, the magnetic nanomaterial is prepared by:
s01, etching Ti by adopting hydrofluoric acid solution 3 AlC 2 Reacting at 25-35 deg.C for 20-30 h, washing and drying to obtain Ti 3 C 2 Powder;
s02, ti prepared in the step S01 3 C 2 With Fe 3 O 4 Mixing, ultrasonic treating and drying to obtain Fe 3 O 4 /Ti 3 C 2 A complex;
s03, and Fe prepared in the step S02 3 O 4 /Ti 3 C 2 Mixing with beta-cyclodextrin, heating at 55-65 deg.C for 3-5 h, washing and drying to obtain magnetic nano material Fe 3 O 4 /Ti 3 C 2 Beta-cyclodextrin.
The invention discloses a magnetic dispersion solid-phase microextraction technology, which is a rapid and efficient extraction mode generated by combining matrix dispersion and magnetic solid-phase microextraction 3 O 4 /Ti 3 C 2 The/beta-cyclodextrin is used as an adsorbent, high-efficiency extraction of different target phytohormones is realized through a high-selectivity adsorption material, and high-sensitivity analysis of the target is realized.
Preferably, the Ti is 3 C 2 With Fe 3 O 4 The mass ratio of (2-3): 1; said Fe 3 O 4 /Ti 3 C 2 The mass ratio of the beta-cyclodextrin to the beta-cyclodextrin is 1: (15 to 20).
According to some embodiments of the invention, the plant hormone comprises at least one of gibberellins, growth hormones, jasmonates, and abscisic acid plant hormones.
According to some embodiments of the invention, the solvent of the solvent extraction in step S1 is at least one of lower alcohol, acetonitrile, chloroform, dichloromethane, ethyl acetate, acetone and formic acid; preferably, the solvent is methanol. The extraction efficiency using methanol as a solvent is high.
According to some embodiments of the present invention, the extraction process of the plant sample in step S1 further comprises adding a scavenger (florisil) and zirconium beads for shaking and crushing. The purifying agent and the zirconium beads are used for crushing the plant sample, and the matrix dispersion technology is used for reducing the interference of lipid and the like, so that the extraction efficiency of the phytohormone in the plant sample is improved.
Preferably, the extraction process in step S1 includes the following specific steps: adding cold methanol into plant sample, vortex mixing for 30s, leaching at 4 deg.C, 12h, centrifuging at 10000 Xg low temperature for 5min, and collecting supernatant; the temperature of the cold methanol is (-25-0) DEG C. The stability of the phytohormone can be ensured by keeping the operation process at low temperature, and the detection accuracy is improved.
According to some embodiments of the invention, the derivatizing agent is EDC. The invention uses N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (N- (3-dimethyl-laminopropyl) -N' -ethyl carboxyl side, EDC) as a derivatization reagent for derivatization and mass spectrometry of auxin, jasmonic acid and abscisic acid plant hormone for the first time, and performs ammoniation reaction on carboxylic acid of the acid plant hormone in a water phase, thereby improving the mass spectrometry response of the acid plant hormone in a positive mode.
According to some embodiments of the invention, the solid-phase micro-extraction simultaneous derivatization reaction condition in step S2 is (35-45) ° c reaction (70-110) min. By optimizing the derivatization conditions, the method has better derivatization reaction effect under the temperature and time of the optimized conditions, and is favorable for complete derivatization reaction of the phytohormone in the plant sample.
Preferably, the solid-phase microextraction simultaneous derivatization reaction in step S2 specifically includes the following steps: adding 5mg of magnetic nano material, adding 50 mu L of 20mM EDC derivatization agent, carrying out water bath at 40 ℃, and synchronously finishing solid phase micro-extraction and derivatization.
According to some embodiments of the invention, the elution process in step S3 comprises the steps of: using an ultrapure aqueous solution, vortex for 15-25 min. The aqueous solution has the best elution effect.
According to some embodiments of the invention, the quantitative analysis process comprises the steps of: and (4) detecting the supernatant obtained in the step (3) by adopting a high performance liquid chromatography-mass spectrometry combined method, and calculating to obtain the concentration of the phytohormone.
According to some embodiments of the invention, the parameter conditions during the detection by the hplc-ms method are as follows:
liquid chromatography conditions: the mobile phase A is 0.1 percent of formic acid aqueous solution, the mobile phase B is acetonitrile, and the mobile phase B is subjected to gradient elution from 10 percent to 40 percent;
mass spectrum conditions: the temperature of atomizing gas is 300 ℃; flow rate of atomizing gas: 8 mL/min -1 (ii) a Atomizer pressure 30psi; an ion source: 70eV; the scanning mode comprises the following steps: and (4) detecting multiple reactions.
By optimizing the conditions of the liquid mass spectrum, an accurate phytohormone detection result can be obtained.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a test chart of a magnetic nanomaterial in example 1 of the present invention, in which (a) and (b) are each Ti 3 C 2 And Fe 3 O 4 /Ti 3 C 2 Scanning electron microscope picture of/beta-cyclodextrin, wherein (c) and (d) are respectively Ti 3 C 2 And Fe 3 O 4 /Ti 3 C 2 Transmission electron micrographs of/[ beta ] -cyclodextrin, (e) and (f) are Fe respectively 3 O 4 /Ti 3 C 2 A scanning electron microscope energy spectrum analysis chart of the/beta-cyclodextrin;
FIG. 2 is a schematic view of the measurement method in example 2 of the present invention;
FIG. 3 shows a standard solution (100. Mu.g L) of 12 phytohormones in example 2 of the present invention -1 ) Multiple Reaction Monitoring (MRM) chromatograms.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The materials and instruments used in the following examples are commercially available.
1、Ti 3 C 2 preparation of
10g of Ti were weighed 3 AlC 2 Adding 100mL of 40% hydrofluoric acid solution into the powder, magnetically stirring at 30 ℃ for 24h, and etching Ti 3 AlC 2 Al in the Al alloy to prepare two-dimensional layered Ti 3 C 2 Centrifuging, washing the obtained suspension with deionized water repeatedly, washing with ethanol for 3 times until pH reaches neutral, vacuum drying at 80 deg.C to obtain Ti 3 C 2 And (3) powder.
2、Fe 3 O 4 /Ti 3 C 2 Preparation of the Complex
100mg of Ti 3 C 2 And 40mg of Fe 3 O 4 The nanoparticles were dispersed in 80mL and 20mL deionized water with sonication for 30min, respectively. And carrying out ultrasonic treatment for 6h after mixing. Filtering to obtain Fe 3 O 4 /Ti 3 C 2 And drying the prepared compound in a vacuum drying oven at 80 ℃ for 24 hours.
3、Fe 3 O 4 /Ti 3 C 2 Preparation of/beta-cyclodextrin
100mg Fe 3 O 4 /Ti 3 C 2 And 2g of beta-cyclodextrin were dispersed in 60mL of deoxygenated water and stirred for 20min, and then oil-bathed at 60 ℃ for 4h. Finally the synthesized Fe 3 O 4 /Ti 3 C 2 Beta-ringRepeatedly leaching dextrin, and vacuum drying at 50 deg.C for 24 hr.
FIG. 1 is an electron microscope image of the magnetic nanomaterial, wherein Ti is shown in FIG. 1 (a) and Ti is shown in FIG. 1 (b), respectively 3 C 2 And Fe 3 O 4 /Ti 3 C 2 Scanning electron micrographs of/beta-cyclodextrin, ti in FIG. 1 (c) and Ti in FIG. 1 (d), respectively 3 C 2 And Fe 3 O 4 /Ti 3 C 2 (ii) transmission electron micrographs of/beta-cyclodextrin, (e) and (f) are Fe 3 O 4 /Ti 3 C 2 Scanning electron microscopy energy spectrum analysis chart of the/beta-cyclodextrin. As shown in FIGS. 1 (a) and 1 (c), ti 3 C 2 Is a two-dimensional layered structure; (b) The electron micrograph of (d) shows Fe prepared 3 O 4 And beta-cyclodextrin are uniformly loaded on Ti 3 C 2 The above. The energy spectrum analysis charts of FIG. 1 (e) and FIG. 1 (f) confirm that the magnetic nano-material contains Ti and Fe elements.
1. matrix dispersion degreasing: taking a single fresh rape seed (5-8 mg), adding 2mg of purifying agent and a single zirconium bead, shaking and grinding for 4min, adding phytohormone internal standard (see table 2 in detail) into the uniformly ground sample, swirling for 30s, and standing for 10min.
2. And (3) extraction: adding 200 μ L cold methanol, high speed vortex mixing, standing at 4 deg.C overnight for leaching for 12h, centrifuging at 10000rpm for 5min, and collecting the upper layer extractive solution.
3. Magnetic solid phase micro-extraction synchronous derivatization: 5mg of magnetic nanomaterial (Fe) was added 3 O4/Ti 3 C 2 Beta-cyclodextrin), adding 50. Mu.L of 20mM EDC, shaking in a water bath at 40 ℃ for 90min, magnetically separating, discarding the supernatant, adding 50. Mu.L of ultrapure water for elution, vortexing for 20min, and transferring the supernatant to a sample bottle with an internal cannula.
4. And (3) putting the sample feeding bottle into a high performance liquid chromatography-mass spectrometry combined instrument for detection, calculating the concentration of the target object before and after extraction, wherein the chromatographic conditions and mass spectrometry conditions of phytohormone analysis are as follows:
4.1 chromatographic conditions
A chromatographic column: waters ACQUITY UPLC HSST 3 mm × 2.1 μm;
column temperature: 40 ℃;
sample introduction amount: 10 mu L of the solution;
mobile phase: a:0.1% aqueous formic acid; b: acetonitrile, gradient elution according to the following table;
table 1: gradient elution condition of liquid chromatogram mobile phase
4.2 Mass Spectrometry conditions
Liquid chromatogram-mass spectrum coupling interface: electrospray ionization (ESI) positive mode;
temperature of atomizing gas: 300 ℃;
flow rate of atomizing gas: 8 mL/min -1 ；
Atomizer pressure: 30psi;
ion source (EI): 70eV;
the scanning mode comprises the following steps: multiplex reaction detection (MRM);
table 2: mass spectrometric parameters of phytohormone derivatives to be analyzed
The above compound names and abbreviations are respectively: gibberellin (abbreviated GA) stands for gibberellins, indole-3-acetic Acid (abbreviated IAA) for indoleacetic Acid, aabbsciic Acid (abbreviated ABA) for abscisic Acid, and Jasmic Acid (abbreviated JA) for Jasmonic Acid.
5. Measurement results: measurement of phytohormones in fresh rape seeds, 12 hormones in rape seeds were detected, and the results are shown in table 3 (below * RSD relative standard deviation (%):
table 3:12 phytohormones and test data of standard recovery rate thereof
The recovery rate of the method is between 80.4 and 115.1 percent (see table 3), the Relative Standard Deviation (RSD) is 1.1 to 15.1 percent, and the linear correlation coefficient R is 2 >0.9928, the detection limit of the method is 0.002-0.23ng/ml. The method synthesizes a novel magnetic nano material, combines the matrix dispersing and degreasing, the magnetic solid phase micro-extraction and the synchronous derivatization technology, has good detection limit and recovery rate, is sensitive and efficient, can accurately measure various plant hormone substances, and has important application value in the detection of the plant hormone in high-fat plant samples.
In the experimental process, the best experimental effect is obtained by optimizing experimental factors of all links, such as preparation of magnetic nano materials, dosage of adsorbent materials used for magnetic solid-phase microextraction, pH of a system, synchronous derivatization time of solid-phase microextraction, eluent and the like.
In conclusion, the invention has the beneficial effects that:
(1) The invention is based on Ti for the first time 2 C 3 Material, modifying it and synthesizing a new-type magnetic nano material Fe 3 O 4 /Ti 2 C 3 Beta-cyclodextrin to realize high-selectivity extraction of phytohormone;
(2) The invention combines matrix dispersion degreasing, magnetic solid phase micro-extraction and derivatization to realize synchronous extraction and derivatization, and realizes high-sensitivity qualitative and quantitative analysis of the phytohormone in the multi-lipid sample by taking a liquid chromatography-mass spectrometry technology as a basic means;
(3) The method has the advantages of simple analysis and detection steps, no transfer in the whole pretreatment process, loss reduction, capability of detecting 5mg trace plant samples and realization of analysis and test of the phytohormones in the trace plant samples.
The foregoing is illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the invention in any way. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Many possible variations and modifications may be made to the disclosed methods and techniques, or equivalents may be substituted for those skilled in the art without departing from the spirit and scope of the invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (7)
1. A method for measuring phytohormone in a high-fat plant sample is characterized by comprising a pretreatment process and a quantitative analysis process, wherein the pretreatment process comprises the following steps:
s1, extracting a plant sample by using a solvent to obtain a sample solution;
s2, adding a magnetic nano material and a derivatization reagent into the sample solution, carrying out solid-phase microextraction synchronous derivatization reaction on the plant hormone in the sample solution, and then separating and collecting the magnetic nano material;
s3, eluting the magnetic nano material obtained in the step S2, and collecting supernatant;
wherein the magnetic nano material is Fe 3 O 4 /Ti 3 C 2 Beta-cyclodextrin;
the plant hormone comprises at least one of gibberellins, growth hormones, jasmonic acids and abscisic acid plant hormones;
the derivatization reagent is EDC;
the quantitative analysis process comprises the following steps: detecting the supernatant obtained in the step S3 by adopting a high performance liquid chromatography-mass spectrometry combined method, and calculating to obtain the concentration of the phytohormone;
the high performance liquid chromatography-mass spectrometry combined method has the parameter conditions in the process as follows:
liquid chromatography conditions: the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the mobile phase B is subjected to gradient elution from 10% to 40%; the column temperature is 40 ℃, and the sample injection amount is 10 mu L;
the gradient elution conditions included:
time min mobile phase A% mobile phase B% flow rate mL/min
0 90 10 0.3
5 85 15 0.3
6 83 17 0.3
10 78 22 0.3
12 75 25 0.3
16 70 30 0.3
18 63 37 0.3
19 60 40 0.3
21 10 90 0.3
22 90 10 0.3
26 90 10 0.3。
2. The method of claim 1, wherein the magnetic nanomaterial is prepared by:
s01, etching Ti by adopting hydrofluoric acid solution 3 AlC 2 Reacting at the temperature of 25 to 35 ℃ for 20 to 30h, and washing and drying to obtain Ti 3 C 2 Powder;
s02, and the Ti prepared in the step S01 3 C 2 With Fe 3 O 4 Mixing, ultrasonic treating and drying to obtain Fe 3 O 4 /Ti 3 C 2 A complex;
s03, and Fe prepared in the step S02 3 O 4 /Ti 3 C 2 Mixing with beta-cyclodextrin, heating at 55-65 ℃ for 3-5 h, washing and drying to obtain the magnetic nano material Fe 3 O 4 /Ti 3 C 2 Beta-cyclodextrin.
3. The method of claim 1, wherein the solvent in step S1 is at least one of lower alcohol, acetonitrile, chloroform, dichloromethane, ethyl acetate, acetone, and formic acid.
4. The method of claim 1, wherein the step of extracting the plant sample in step S1 further comprises adding a detergent and zirconium beads for shaking and crushing.
5. The method of claim 1, wherein the solid phase microextraction simultaneous derivatization reaction in step S2 is carried out at 35 to 45 ℃ for 70 to 110min.
6. The method of claim 1, wherein the elution process in step S3 comprises the steps of: vortex analysis was performed using ultrapure water for 15 to 25min.
7. The method of claim 1, wherein the parameter conditions during the detection process by the HPLC-MS method are as follows:
mass spectrum conditions: the temperature of atomizing gas is 300 ℃; flow rate of atomizing gas: 8 mL/min -1 (ii) a Atomizer pressure 30psi; an ion source: 70eV; the scanning mode is as follows: and (4) detecting multiple reactions.
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