CN109813824B - Pretreatment method of plant sample - Google Patents
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- CN109813824B CN109813824B CN201711170242.5A CN201711170242A CN109813824B CN 109813824 B CN109813824 B CN 109813824B CN 201711170242 A CN201711170242 A CN 201711170242A CN 109813824 B CN109813824 B CN 109813824B
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- 238000002203 pretreatment Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 21
- 239000011159 matrix material Substances 0.000 claims abstract description 4
- 239000003463 adsorbent Substances 0.000 claims description 87
- 239000007787 solid Substances 0.000 claims description 33
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- 239000006228 supernatant Substances 0.000 claims description 29
- 238000005349 anion exchange Methods 0.000 claims description 26
- 150000001647 brassinosteroids Chemical class 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 19
- 238000005341 cation exchange Methods 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 17
- 238000001212 derivatisation Methods 0.000 claims description 16
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- 238000001514 detection method Methods 0.000 claims description 12
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
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- 238000000227 grinding Methods 0.000 claims description 5
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Abstract
本发明公开了一种植物样品前处理方法，用于提取富集植物样品中的油菜素甾醇，并对其进行衍生。该方法基于微区基质固相分散的方法，并于分散固相萃取方法结合，避免了传统植物样品前处理过程中多步样品转移带来的目标组分损失，将样品前处理时间从数小时缩短至30分钟以内。同时，该方法能处理亚毫克至亚克量级植物样品，萃取效率高，操作简单，重复性好。
The invention discloses a plant sample pretreatment method, which is used for extracting and enriching brassinosteroid in the plant sample and derivatizing it. This method is based on the solid-phase dispersion method of micro-area matrix and combined with the dispersive solid-phase extraction method, which avoids the loss of target components caused by multi-step sample transfer in the traditional plant sample pretreatment process, and reduces the sample pretreatment time from several hours. shortened to less than 30 minutes. At the same time, the method can process sub-milligram to sub-gram plant samples, has high extraction efficiency, simple operation and good repeatability.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a pretreatment method of a plant sample, which is suitable for sample pretreatment when a brassinosteroid compound in the plant sample is analyzed.
Background
The plant hormone is a small molecule metabolite which is synthesized in a plant body and plays an important role in regulating and controlling the growth and development of the plant, and is almost involved in each process of the growth and development of the plant. The method for detecting the types, the contents and the spatial distribution conditions of the hormones in the plant body has important significance for researching the physiological regulation and the signal transmission mechanism of the plant hormones. Among them, the detection of brassinosteroids is particularly difficult. Because the brassinosteroids are lower in plant body content compared with other phytohormones such as gibberellin, cytokinin, jasmonic acid and the like, the brassinosteroids are generally only about 0.01-0.1 ng/g fresh weight in leaf buds and leaves, and most of the gibberellin is 1-100 ng/g fresh weight. Secondly, the basic structure of brassinosterol is 5-cholestane, the substituent is mainly hydroxyl or ketone, the compound with the structure lacks detectable functional groups, the spectroscopic detection method is difficult to adopt, and the mass spectrum signal is weak. At present, a chromatography-mass spectrometry combined method is mainly adopted to detect brassinosteroids in a plant sample, and before entering chromatographic analysis, the brassinosteroids need to be derived so as to enhance the mass spectrometry response. Therefore, the sample pretreatment method is particularly important for detecting brassinosteroids. Especially for milligram-level plant samples, how to eliminate the interference of complex sample matrixes and efficiently extract and enrich trace brassinosteroids is a difficult problem to be solved and is a key factor for restricting the detection sensitivity of the brassinosteroids.
In the prior art, a combination of liquid phase extraction (SPE), Matrix Solid Phase Dispersion (MSPD), and the like is often used to treat plant samples. Most methods require plant samples in the order of several milligrams to several grams fresh weight, and it is difficult to obtain high extraction efficiency and analytical performance for samples below 1mg fresh weight. The loss of the phytohormone in the sample pretreatment process is large due to multiple sample transfers along with the multi-step sample pretreatment operation, and the loss is a main reason for influencing the extraction efficiency.
Disclosure of Invention
The invention aims to provide a pretreatment method of a plant sample, which can extract, purify and enrich brassinosteroids in a sub-milligram-level to sub-gram-level plant sample, improve the selectivity and sensitivity of subsequent chromatography-mass spectrometry analysis and solve the problem of detection of trace brassinosteroids in a small amount of samples.
In order to solve the technical problem, the technical scheme adopted by the invention is as follows:
a plant sample pretreatment method comprises the following steps:
1) putting the plant sample and the solid adsorbent 1 into a centrifugal tube together, mixing, and grinding under liquid nitrogen;
2) adding an extraction solvent into a centrifugal tube, centrifuging at 0-4 ℃, and extracting brassinosteroids in a plant sample to obtain supernatant A;
3) mixing the supernatant A obtained in the step 2) with the solid adsorbent 2, centrifuging again at 0-4 ℃, and removing matrix impurities in the supernatant A to obtain a supernatant B;
4) transferring all the supernatant B into a reaction container, adding a derivatization reagent, and derivatizing the brassinosteroids at the reaction temperature of 50-90 ℃;
5) after the derivatization reaction is finished, blow-drying the product solution, and dissolving the product solution in an organic solvent again for the chromatographic analysis and detection of brassinosteroids; or directly using the reacted solution for chromatographic analysis and detection. The organic solvent used for reconstitution is selected according to the chromatographic separation conditions.
The solid adsorbent 1 is C18 bonded silica gel particles or C8 bonded silica gel particles; the solid adsorbent 2 is a combination of a cation exchange adsorbent and an anion exchange adsorbent, and the particle sizes of the solid adsorbent 1 and the solid adsorbent 2 are 40-60 micrometers.
The extraction solvent is acetonitrile or acetonitrile-water mixed solution, wherein the volume fraction of the acetonitrile is 60-100%. The volume-to-mass ratio (mL/g) of the extraction solvent to the solid adsorbent 1 is 12: 1-50: 1.
After obtaining the supernatant A, the solid adsorbent 2 can be directly added into the centrifugal tube in the step 2), and after the solid adsorbent is mixed and contacted with the supernatant A and centrifuged, the supernatant B is obtained, so that the component loss caused by sample transfer is reduced.
The cation exchange adsorbent is any one of a strong cation exchange adsorbent, a weak cation exchange adsorbent and a mixed cation exchange adsorbent which are sold in the market; the anion exchange adsorbent is any one of a commercially available strong anion exchange adsorbent, a weak anion exchange adsorbent and a mixed anion exchange adsorbent; when the composite adsorbent is used, namely the composite adsorbent is formed by combining a strong cation exchange adsorbent and a strong anion exchange adsorbent, the composite adsorbent is formed by combining a weak cation exchange adsorbent and a weak anion exchange adsorbent, and the composite cation exchange adsorbent is formed by combining a composite anion exchange adsorbent.
The derivatization reagent is one of aminophenylboronic acid and derivatives thereof, and pyridine boronic acid and derivatives thereof, the final concentration of the derivatization reagent in the reaction solution is 0.1-10 mg/mL, wherein the solvent of the aminophenylboronic acid solution contains 1-10% (v/v) pyridine.
The centrifugation time in the step 2) and the step 3) is 2 min-10 min.
The mass ratio of the plant sample to the solid adsorption 1 or 2 is 1: 1-1: 6, and preferably 1: 4.
When the cation exchange adsorbent and the anion exchange adsorbent are combined, the mass ratio of the cation exchange adsorbent to the anion exchange adsorbent is 1: 10-1: 1.
The technical scheme of the invention is mainly different from the prior art in that:
(1) in the prior art, a plant sample is processed by adopting a traditional matrix solid-phase dispersion method, namely, the plant sample and a solid adsorbent are mixed and ground in a mortar, then the mixture is filled into a solid-phase extraction column tube, and a solvent is added for elution. In this method, the sample is inevitably transferred from the mortar to the extraction column tube, and the sample loss caused by the transfer is inevitable. Especially for small samples, such as milligram or even sub-milligram samples, the loss due to this transfer step is very considerable. According to the technical scheme, a sample and the solid adsorbent 1 are ground in a centrifugal tube, and then an extraction solvent is directly added into the tube for extraction. The operation of the step is very simple, the method is suitable for the samples from the sub-milligram level to the sub-gram level, and the loss caused by sample transfer is completely avoided.
(2) In the existing sample pretreatment method aiming at brassinosteroids, a sample is usually purified and decontaminated by two or more solid phase extractions. For example, after matrix solid phase dispersion extraction, the mixed anion exchange adsorbent and the mixed cation exchange adsorbent are still used for carrying out solid phase extraction successively. The invention provides a method for mixing and using two ion exchange adsorbents, adopts a dispersed solid phase extraction mode, directly mixes and centrifuges the mixture with the supernatant of the previous step, does not need to fill a solid phase extraction column, avoids multiple steps of operations such as activation, sample loading, leaching, elution and the like required by the traditional solid phase extraction, and has the advantages of shorter time consumption, less solvent consumption and higher extraction efficiency for a small amount of samples.
The sample pretreatment method can treat plant samples with fresh weight less than 1mg, and compared with the traditional MSPD method, the method grinds a small amount of samples in a centrifugal tube, directly adds a solvent for centrifugal extraction under the condition of not transferring the samples, solves the problem of serious sample loss caused by transfer of a plurality of samples, and simultaneously solves the problem of sample loss caused by adsorption when a small amount of samples are ground in a mortar. According to the technical scheme, the anion exchange adsorbent and the cation exchange adsorbent are combined together to be used as the solid adsorbent 2 for dispersed solid-phase extraction, and compared with the technical scheme of performing dispersed solid-phase extraction step by step, loss caused by sample transfer is avoided. Theoretically, such combined use would reduce the effect of removing impurities, and particularly when solid adsorbent 2 is used in combination with solid adsorbent 1, the extraction efficiency is poor. However, experiments prove that the combination of the anion and cation exchange adsorbents does not cause adverse effect on the extraction of brassinosteroids in a plant sample, but improves the extraction efficiency because the sample loss is eliminated.
The method has the advantages of simple operation, low consumption and high efficiency, and can reduce the pretreatment time of the plant sample from the traditional hours to within 30 min. The absolute recovery rate of brassinosteroids in a sub-mg to sub-g fresh weight plant sample reaches more than 80 percent.
Drawings
FIG. 1 is a chromatogram-mass spectrum detection spectrum obtained after sample treatment according to the pretreatment method described in example 1. Wherein, the components 1 to 5 are respectively: 24-epirassinolide, 24-epicastasterone, teasterone, typhasterol, 6-deoxo-24-epicastasterone.
Detailed Description
The specific implementation mode of the invention comprises the following steps:
(1) mixing the plant sample and C18 bonded silica gel particles or C8 bonded silica gel particles in a centrifugal tube, and grinding under liquid nitrogen; the mass ratio of the plant sample to the silica gel particles is 1: 1-1: 6;
(2) adding an extraction solvent acetonitrile or acetonitrile-water mixed solution into a centrifugal tube, centrifuging for 2min to 10min at the temperature of 0 ℃ to 4 ℃, and extracting brassinosteroids in a plant sample to obtain a supernatant A; wherein the volume-to-mass ratio (mL/g) of the extraction solvent to the silica gel particles is 12: 1-50: 1;
(3) transferring the obtained supernatant A into a centrifugal tube containing a solid adsorbent 2, centrifuging again at 0-4 ℃, and removing matrix impurities in the supernatant A to obtain a supernatant B; the solid adsorbent 2 can also be directly added into a centrifugal tube in the previous step for centrifugation, so that the component loss caused by sample transfer is reduced; the solid adsorbent 2 is a combination of a cation exchange adsorbent and an anion exchange adsorbent, and the mass ratio of the plant sample to the solid adsorbent 2 is 1: 1-1: 6, preferably 1: 4;
(4) transferring the supernatant B into a reaction container, adding a derivatization reagent, wherein the concentration of the derivatization reagent in the reaction solution is 0.1-10 mg/mL, and derivatizing the brassinosteroid molecules at the reaction temperature of 50-100 ℃; wherein the derivative reagent is one of aminophenylboronic acid and derivatives thereof, and pyridine boronic acid and derivatives thereof; when the aminobenzeneboronic acid is adopted, the solvent contains 1-10% (v/v) pyridine.
(5) After the derivatization reaction is finished, blow-drying the product solution, and dissolving the product solution in an organic solvent again for the chromatographic analysis and detection of brassinosteroids; or directly using the reacted solution for chromatographic analysis and detection. The organic solvent used for reconstitution is selected according to the chromatographic separation conditions.
The present invention will now be described with reference to specific embodiments, illustrative examples and descriptions of the present invention are provided to explain the present invention, but not to limit the present invention.
EXAMPLE 1 pretreatment of Arabidopsis leaves
(1) Mixing 2mg of arabidopsis thaliana leaves and 8mg of C8 bonded silica gel particles in a centrifugal tube, adding liquid nitrogen, and grinding with a quartz glass rod;
(2) adding 200 microliters of 80% (v/v) acetonitrile aqueous solution into a centrifugal tube, and centrifuging for 5min at 4 ℃ to obtain supernatant A;
(3) transferring the supernatant A into a centrifugal tube containing a solid adsorbent 2, and centrifuging at 4 ℃ for 3min to obtain a supernatant B; wherein solid adsorbent 2 is a combination of 4mg of a commercially available mixed anion exchange adsorbent and 4mg of a mixed cation exchange adsorbent;
(4) transferring the supernatant B into a reaction vessel, and adding 50 microliters of 2mg/mL aminobenzeneboronic acid solution for derivatization, wherein the solvent of the aminobenzeneboronic acid solution is acetonitrile containing 6.8% (v/v) pyridine;
(5) after the derivatization reaction, the product solution is dried and redissolved in acetonitrile for detection of brassinosteroids by liquid Chromatography-mass spectrometry under the same instrument conditions as those described in the literature (Journal of Chromatography A,2016,1456, 105-112).
FIG. 1 is a chromatogram obtained by the chromatographic-mass spectrometric analysis, and it can be seen that five brassinosteroids can be completely separated.
Example 2 pretreatment of Rice leaves
(1) Mixing 0.1mg of rice leaves and 0.4mg of C18 bonded silica gel particles in a centrifugal tube, adding liquid nitrogen, and grinding with a quartz glass rod;
(2) adding 20 microliters of acetonitrile into a centrifugal tube, and centrifuging for 5min at 4 ℃ to obtain a supernatant A;
(3) continuously adding a solid adsorbent 2 into the centrifugal tube, and centrifuging again for 5min at 4 ℃ to obtain a supernatant B; wherein solid adsorbent 2 is a combination of 0.1mg of a commercially available strong anion exchange adsorbent and 0.1mg of a strong cation exchange adsorbent;
(4) transferring the supernatant B into a reaction container, adding 20 microliters of 2mg/mL aminobenzeneboronic acid, and performing derivatization reaction at 80 ℃ for 10min, wherein the solvent of the aminobenzeneboronic acid solution is acetonitrile containing 2% (v/v) pyridine;
(5) after the derivatization reaction, the product solution is directly injected into a liquid Chromatography-mass spectrometer under the same conditions as those in the literature (Journal of Chromatography A,2016,1456, 105-.
Claims (7)
1. A plant sample pretreatment method is characterized by comprising the following steps:
1) putting the plant sample and the solid adsorbent 1 into a centrifugal tube together, mixing, and grinding under liquid nitrogen;
2) adding an extraction solvent into a centrifugal tube, centrifuging at 0-4 ℃, and extracting brassinosteroids in a plant sample to obtain a supernatant A;
3) mixing the supernatant A obtained in the step 2) with a solid adsorbent 2, centrifuging again at 0-4 ℃, and removing matrix impurities in the supernatant A to obtain a supernatant B;
4) transferring the supernatant B into a reaction container completely, adding a derivatization reagent to perform derivatization on brassinosteroids at normal temperature or 50-100 ℃;
5) after the derivatization reaction is finished, blow-drying the product solution, and dissolving the product solution in an organic solvent again for the chromatographic analysis and detection of brassinosteroids; or directly using the reacted solution for chromatographic analysis and detection;
the solid adsorbent 1 is C18 bonded silica gel particles or C8 bonded silica gel particles; the solid adsorbent 2 is a combination of a cation exchange adsorbent and an anion exchange adsorbent, and the particle sizes of the solid adsorbent 1 and the solid adsorbent 2 are 40-60 micrometers;
the extraction solvent is acetonitrile or an acetonitrile-water mixed solution, wherein the volume fraction of the acetonitrile is 60-100%;
the mass ratio of the plant sample to the solid adsorption 1 or 2 is 1: 1-1: 6;
the sample pretreatment method can treat plant samples with fresh weight less than 1 mg.
2. The method of claim 1, wherein: directly adding the solid adsorbent 2 into the centrifugal tube in the step 2) after obtaining the supernatant A, mixing and contacting the solid adsorbent with the supernatant A, and centrifuging to obtain a supernatant B so as to reduce component loss caused by sample transfer.
3. The method of claim 1, wherein: the cation exchange adsorbent is any one of a strong cation exchange adsorbent, a weak cation exchange adsorbent and a mixed cation exchange adsorbent which are sold in the market; the anion exchange adsorbent is any one of a commercially available strong anion exchange adsorbent, a weak anion exchange adsorbent and a mixed anion exchange adsorbent; when the composite adsorbent is used, the strong cation exchange adsorbent is combined with the strong anion exchange adsorbent, the weak cation exchange adsorbent is combined with the weak anion exchange adsorbent, and the mixed cation exchange adsorbent is combined with the mixed anion exchange adsorbent.
4. The method of claim 1, wherein: the derivatization reagent is one of aminophenylboronic acid or a derivative thereof and pyridine boronic acid or a derivative thereof, and the final concentration of the derivatization reagent in the reaction solution is 0.1-10 mg/mL; wherein the solvent of the aminophenylboronic acid solution contains 1-10% of pyridine, and the unit is v/v.
5. The method of claim 1, wherein: the centrifugation time in the step 2) and the step 3) is 2-10 min.
6. The method of claim 3, wherein: when the cation exchange adsorbent and the anion exchange adsorbent are combined, the mass ratio of the cation exchange adsorbent to the anion exchange adsorbent is 1: 10-1: 1.
7. The method of claim 1, wherein: the volume-to-mass ratio mL/g of the extraction solvent to the solid adsorbent 1 is 12: 1-50: 1.
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