CN103207103A - Sample pretreatment method of endogenous brassinosteroids in plant sample - Google Patents
Sample pretreatment method of endogenous brassinosteroids in plant sample Download PDFInfo
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Abstract
The invention discloses a sample pretreatment method of the endogenous brassinosteroids in a plant sample. The method comprises the following steps that: firstly, the endogenous brassinosteroids are extracted by a solvent; and then the impurities of the solution obtained in the previous step are further removed with boron-affinity material as an extraction medium, so that the sample pretreatment of the endogenous brassinosteroids is realized. The method has the advantages of simplicity, rapidness, solvent conservation and simplicity in operation. Besides, according to the method, high selectivity is shown in the aspect of removing endogenous impurities of plant extracting solution and enrichment and purification of low-content endogenous brassinosteroids can be realized.
Description
Technical field
The present invention relates to the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample, belong to the analytical chemistry field.
Background technology
(brassinosteroids BRs) is the phytosterin compound of polyhydroxylization to the plain sterol of rape, and is extremely low at the plant intensive amount, but regulating and control a series of physiology and the metabolic process of plant.In recent years, about the physiology function of the plain sterol of rape, for example biosynthesizing of BRs, extensive concern is received in the research of degraded and metabolic pathway.But because the plain sterol content of plant extraction liquid mesostroma complexity and rape is extremely low, carrying out of these researchs is subjected to certain limitation.Therefore, setting up efficiently, sample-pretreating method is the key that endogenous BRs detects in conjunction with sensitive detection means also.
The pre-treating method of the plain sterol of rape comprises liquid-liquid extraction, Solid-Phase Extraction, solid-phase microextraction, magnetic Solid-Phase Extraction and liquid chromatography purification process etc. in the plant of having reported at present.These method majorities require tens gram fresh weight plant tissues, and so big sample size is difficult to realize for the precious sample of part, bring problems such as the big and cost height of solvent consumption also for simultaneously the pre-treatment process.In addition, above-mentioned pre-treating method is only realized removal of impurities by the hydrophobic difference of BR and impurity, can cause the co-elute of impurity and BRs inevitably, must bring burden for follow-up liquid phase separation, thereby reduces the mass spectrum Ionization Efficiency of BRs.
Summary of the invention
Technical matters to be solved by this invention is that the plain sterol pre-treating method of existing rape is improved, and a kind of high selectivity and simple and rapid plant sample pre-treating method are provided, and realizes the sample pre-treatments of the plain sterol of endogenous rape.
The present invention addresses the above problem and the technical scheme that adopts is: the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample may further comprise the steps:
Be placed in the mortar behind the accurate weighing plant sample of 1-1, liquid nitrogen frozen be ground to Powdered after, add mark in the isotope [
2H
3] brassinosteroid and [
2H
3] the plain sterone of rape, add solvent extraction then, leave standstill, the centrifuging and taking supernatant obtains the sample solution of not removal of impurities;
Solvent in the sample solution that 2-1 removal previous step obtains, with buffer solution sample dissolution solution, with the extraction of the boron affinitive material after activation sample solution, clean the boron affinitive material, again with the plain sterol of the rape on the stripping liquid desorb boron affinitive material, collect stripping liquid, remove the organic solvent in the stripping liquid, and then realize the sample pre-treatments of the plain sterol of endogenous rape.
Among the described step 2-1, the boron affinitive material is successively activating with organic solvent and the buffering solution that water dissolves each other.
Be inserted with the following step 1-2 between step 1-1 of the present invention and the 2-1:
The sample solution that the step 1-1 of 1-2 obtains dewaters; With the sample solution of the material extraction step 1-2 after the activation after dewatering, desorb, collection also merge goes up sample and flows out liquid and stripping liquid, obtains the sample solution after the preliminary removal of impurities.
Moisture in the sample can have a negative impact to follow-up removal of impurities process, and therefore, the step that dewaters is very important.The removal process of indication of the present invention comprises that chemistry removes water law and physics removes water law.Chemistry is characterized in that except water law, uses the moisture in the chemical reagent consumption system; Physics is characterized in that using physical means that the moisture in the system is volatilized except water law, comprises freeze-drying, and nitrogen such as dries up at method.
The effect that plant tissue extracts solvent is: under the prerequisite that extracts analyte effectively, extract chaff interferences such as impurity as few as possible.The used solvent of extraction is one or more in methyl alcohol, acetonitrile, isopropyl alcohol, chloroform, methylene chloride, ethyl acetate, normal hexane, ether, acetone or the formic acid among the described step 1-1.
Boron affinitive material among the described step 2-1 is a kind of in silica gel particle, magnetic material, amorphous material, gel, packed column or the integral post that contains boric acid base group.
Described preliminary removal of impurities refers to tentatively remove a large amount of pigments, lipid and the hydrophilic impurity that contains in the extract.Therefore, used material is to adopt to be passed through by material with carbon element, water wetted material, hydrophobic material among the step 1-2 of the present invention
Perhaps
Mode makes up the purpose that the silica gel particle, magnetic material, amorphous material, gel, packed column or the integral post that make up with chemistry or physics mode all can reach preliminary removal of impurities.
The boron affinity chromatography is that selectivity is caught the effective means that separation contains the cis hydroxyl groups compound, successfully is used for the concentration and separation of glycoprotein, RNA and carbohydrate.In alkaline environment, boric acid can form stable five yuan or six-membered cyclic borate with the cis glycol, and under acid condition, cis glycols material can be released by open loop again.BRs is at C2, C3 and C22, and two pairs of cis hydroxyl groups on the C23 position make the very suitable boron affinity extraction of the plain sterol of rape.The affine removal of impurities of boron of the present invention refers to, realizes extraction to the plain sterol of rape by silica gel particle, magnetic material, amorphous material, gel, packed column or the integral post of boronic acid containing group.
The instrument that detects the plain sterol of rape refers to liquid chromatography-fluorescence detector, liquid chromatograph-mass spectrometer coupling, Capillary Electrophoresis-fluorescence detector coupling, the perhaps coupling of capillary electrophoresis-mass spectrometry instrument.
The inventive method is simple, quick, province's solvent, easy to operate.In addition, method can realize the enriching and purifying of the plain sterol of endogenous rape of low content showing high selectivity aspect the removal plant extraction liquid endogenous impurity.
Description of drawings
Fig. 1 is the total ion current comparison diagram of the inventive method and bibliographical information method, wherein, A be plant sample with the total ion current figure of literature method gained, B is the total ions chromatogram after handling with method of the present invention, C is the total ions chromatogram after standard model is handled with boron affinity extraction method.
Fig. 2 is many reaction monitorings chromatogram of the plain sterol of the detected endogenous rape of method of the present invention.Wherein A is the abundant safe A leaf sample of paddy rice; B is the abundant safe B leaf sample of paddy rice; C is the rape leave sample; D1～D4 is the petal sample of rape flower; E1～E3 is the sample of rape flower.1 is 28-norbrassinolide, and 2 is 28-norcastasterone, and 3 is brassinolide, and 4 is castasterone.
Embodiment
Below in conjunction with specific embodiment content of the present invention is elaborated.
Embodiment 1
Accurately take by weighing the 1g plant tissue, liquid nitrogen grinding is transferred in the 10mL centrifuge tube to Powdered, mark in the adding isotope [
2H
3] brassinosteroid and [
2H
3] plain each 2ng of sterone of rape, place moments later, add the 5mL acetonitrile and mix.The refrigerator of putting into-18 ℃ extracted 12 hours.The centrifuging and taking supernatant adds 0.5 gram sodium chloride in the supernatant and impels solution layering, aqueous phase discarded.Add 0.5 gram anhydrous magnesium sulfate again and remove residual moisture.Supernatant after centrifuging and taking dewaters discards insolubles.With the Graphon/ethylenediamine-N-propyl group bonded silica gel bilayer Solid-Phase Extraction of supernatant by activation in advance, with methyl alcohol/acetonitrile (1/1, v/v, 2mL) desorb; Sample flows out liquid and stripping liquid and dries up in the collection.With buffer solution (phosphate buffer solution, 20mM, pH9.0,1mL) dissolve the sample residue that dries up, with the boron affinity solid phase micro-extraction integral post of this solution by activating in advance, with buffer solution (phosphate buffer solution, 20mM, pH9.0 1mL) cleans integral post, and is last to contain the stripping liquid (H of 3% hydrogen peroxide
2The O/ acetonitrile, 1/9, v/v, 1mL) desorb is collected stripping liquid and is dried up wherein organic solvent.Finally, sample solution is settled to 100 microlitres, wherein 80 microlitres enter high performance liquid chromatography-electron spray-triple level Four bar tandem mass spectrum analysis.With the total ion current contrast of said method and bibliographical information method, as shown in Figure 1.Compare with literature method, this method can reduce the matrix of sample greatly, and its matrix and standard items are similar, shows that the present invention has good selectivity impurity-eliminating effect.Brassinosteroid (28-norbrassinolide) falls in plain sterol: the 28-of the detected several endogenous rapes of method of the present invention, the plain sterone (28-norcastasterone) of rape falls in 28-, brassinosteroid (brassinolide), many reaction monitorings chromatogram of the plain sterone of rape (castasterone) as shown in Figure 2.The plain sterol testing result of endogenous rape is as shown in table 1 in five kinds of plant samples.
The content of the plain sterol of endogenous rape in five kinds of actual samples of table 1.
Unit: ng/g fresh weight
Embodiment 2
Accurately take by weighing the 1g plant tissue, liquid nitrogen grinding is transferred in the 10mL centrifuge tube to Powdered, mark in the adding isotope [
2H
3] brassinosteroid and [
2H
3] plain each 2ng of sterone of rape, place moments later, add the 5mL acetonitrile and mix.The refrigerator of putting into-18 ℃ extracted 12 hours.The centrifuging and taking supernatant dries up supernatant with nitrogen, again with the acetonitrile dissolved residue.With the carbon nano-tube/pure silicon glue bilayer solid-phase extraction column of the sample after the dissolving by activation in advance, with methyl alcohol (2mL) desorb; Sample flows out liquid and stripping liquid and dries up in the collection.With buffer solution (phosphate buffer solution, 20mM, pH11.0,1mL) dissolve the sample residue that dries up, with the commercialization phenyl boric acid bonded silica gel solid-phase extraction column of this solution by activating in advance, with buffer solution (phosphate buffer solution, 20mM, pH9.0 1mL) cleans extraction column, and is last to contain the stripping liquid (H of 3% hydrogen peroxide
2The O/ acetonitrile, 1/9, v/v, 1mL) desorb is collected stripping liquid and is dried up wherein organic solvent.Finally, sample solution is settled to 100 microlitres, wherein 80 microlitres enter Ultra Performance Liquid Chromatography-atmospheric pressure ionization source-triple level Four bar tandem mass spectrum analysis.
Embodiment 3
Accurately take by weighing the 1g plant tissue, liquid nitrogen grinding is transferred in the 10mL centrifuge tube to Powdered, mark in the adding isotope [
2H
3] brassinosteroid and [
2H
3] plain each 1ng of sterone of rape, place moments later, add the 5mL acetonitrile and mix.The refrigerator of putting into-18 ℃ extracted 12 hours.The centrifuging and taking supernatant adds 0.5 gram sodium chloride in the supernatant and impels solution layering, aqueous phase discarded.Add 0.5 gram anhydrous magnesium sulfate again and remove residual moisture.Supernatant after centrifuging and taking dewaters discards insolubles.The composite material that adds Graphon, C18 bonded silica gel and magnetic particle in the supernatant, vortex mixed 5 minutes, sample flows out liquid in the collection.Be adsorbed on the plain sterol of rape on the material with the desorb of 2mL methyl alcohol again.Sample flows out liquid and stripping liquid in the merging, volatilizes solvent.With 5mL buffer solution (acetonitrile/water of 20mM ammonium formate, 1/4, v/v, pH9.0) sample dissolution residue.The gained sample solution is extracted centrifugal back abandoning supernatant with the phenyl boric acid polymer beads (20mg) after activating.The acetonitrile that contains 0.5% ammoniacal liquor with 2mL cleans phenyl boric acid magnetic Nano material, abandoning supernatant.At last, to contain 3%H
2O
2Water/acetonitrile solution (1/9, v/v) desorb is adsorbed on the plain sterol of rape on the material, dry up solvent after, be settled to 100 microlitres, wherein 80 microlitres enter Ultra Performance Liquid Chromatography-electron spray-triple level Four bar tandem mass spectrum analysis.
Embodiment 4
Accurately take by weighing the 1g plant tissue, liquid nitrogen grinding is transferred in the 10mL centrifuge tube to Powdered, mark in the adding isotope [
2H
3] brassinosteroid and [
2H
3] plain each 1ng of sterone of rape, place moments later, add the 5mL acetonitrile and mix.The refrigerator of putting into-18 ℃ extracted 12 hours.The centrifuging and taking supernatant is removed the solution in the supernatant, dissolves with acetonitrile.In gained solution, add Graphon, C18 bonded silica gel and ethylenediamine-N-propyl group bonded silica gel, vortex mixed 5 minutes, sample flows out liquid in the centrifugal collection.Be adsorbed on the plain sterol of rape on the material with the desorb of 2mL methyl alcohol again.Sample flows out liquid and stripping liquid in the merging, volatilizes solvent.With 5mL buffer solution (acetonitrile/water of 20mM ammonium formate, 1/4, v/v, pH10.0) sample dissolution residue.The gained sample solution is extracted centrifugal back abandoning supernatant with the phenyl boric acid magnetic gel (20mg) after activating.The acetonitrile that contains 0.5% ammoniacal liquor with 2mL cleans phenyl boric acid magnetic Nano material, abandoning supernatant.At last, to contain 3%H
2O
2Water/acetonitrile solution (1/9, v/v) desorb is adsorbed on the plain sterol of rape on the material, dry up solvent after, be settled to 100 microlitres, wherein 80 microlitres enter Ultra Performance Liquid Chromatography-electron spray-triple level Four bar tandem mass spectrum analysis.
Embodiment 5
Accurately take by weighing the 1g plant tissue, liquid nitrogen grinding is transferred in the 10mL centrifuge tube to Powdered, mark in the adding isotope [
2H
3] brassinosteroid and [
2H
3] plain each 1ng of sterone of rape, place moments later, add the 5mL acetonitrile and mix.The refrigerator of putting into-18 ℃ extracted 12 hours.The centrifuging and taking supernatant volatilizes solvent, with 5mL buffer solution (acetonitrile/water of 20mM ammonium formate, 1/4, v/v, pH9.0) sample dissolution residue.Add through the boric acid modified magnetic nano material (100mg) of acetonitrile with the activation of buffering solution in sample solution, vortex carried out the magnetic Solid-Phase Extraction in 10 minutes, then abandoning supernatant under externally-applied magnetic field.Successively with the acetonitrile/water solution that contains 0.5% ammoniacal liquor (1/4, v/v, 2mL), the acetonitrile solution of 0.5% formic acid, acetonitrile (2mL), (1/4, v/v 2mL) cleans the acetonitrile/water solution of 0.5% ammoniacal liquor.At last to contain 6%H
2O
2Water/acetonitrile solution (1/4, v/v, 1mL) desorb is adsorbed on the plain sterol of rape on the material, dry up solvent after, be settled to 100 microlitres, wherein 80 microlitres enter Ultra Performance Liquid Chromatography-electron spray-triple level Four bar tandem mass spectrum analysis.
Above embodiment has disclosed concrete operations content of the present invention, but the present invention is not limited to the above-described embodiment and examples, without departing from the spirit and scope of the present invention, with step 1) and 4) be under the prerequisite of committed step, any those skilled in the art can also make a little modifications and perfect.
Claims (6)
1. the sample-pretreating method of the plain sterol of endogenous rape in the plant sample is characterized in that, may further comprise the steps:
Be placed in the mortar behind the accurate weighing plant sample of 1-1, liquid nitrogen frozen be ground to Powdered after, add mark in the isotope [
2H
3] brassinosteroid and [
2H
3] the plain sterone of rape, add solvent extraction then, leave standstill, the centrifuging and taking supernatant obtains the sample solution of not removal of impurities;
Solvent in the sample solution that 2-1 removal previous step obtains, with buffer solution sample dissolution solution, with the extraction of the boron affinitive material after activation sample solution, clean the boron affinitive material, again with the plain sterol of the rape on the stripping liquid desorb boron affinitive material, collect stripping liquid, remove the organic solvent in the stripping liquid, and then realize the sample pre-treatments of the plain sterol of endogenous rape.
2. the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample according to claim 1 is characterized in that, among the described step 2-1, the boron affinitive material is successively activating with organic solvent and the buffering solution that water dissolves each other.
3. the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample according to claim 1 is characterized in that, is inserted with the following step 1-2 between step 1-1 and the 2-1:
The sample solution that the step 1-1 of 1-2 obtains dewaters; With the sample solution of the material extraction step 1-2 after the activation after dewatering, desorb, collection also merge goes up sample and flows out liquid and stripping liquid, obtains the sample solution after the preliminary removal of impurities.
4. the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample according to claim 1, it is characterized in that the used solvent of extraction is one or more in methyl alcohol, acetonitrile, isopropyl alcohol, chloroform, methylene chloride, ethyl acetate, normal hexane, ether, acetone or the formic acid among the described step 1-1.
5. the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample according to claim 1, it is characterized in that the boron affinitive material among the described step 2-1 is a kind of in silica gel particle, magnetic material, amorphous material, gel, packed column or the integral post that contains boric acid base group.
6. the sample-pretreating method of the plain sterol of endogenous rape in a kind of plant sample according to claim 3 is characterized in that used material is to be passed through by material with carbon element, water wetted material, hydrophobic material among the described step 1-2
Perhaps
Mode makes up a kind of in silica gel particle, magnetic material, amorphous material, gel, packed column or the integral post that the back makes up with chemistry or physical method.
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Cited By (4)
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| CN107543746A (en) * | 2016-11-16 | 2018-01-05 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of the endogenous plant hormone of three class difference chemical property in a kind of plant sample |
| CN107941971A (en) * | 2017-11-22 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | The purification process of plant endogenous brassinosteroid based on the extraction of boron affinity solid phase |
| CN109813824A (en) * | 2017-11-22 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of plant sample pre-treating method |
| CN109975085A (en) * | 2017-12-28 | 2019-07-05 | 武汉绿剑可瑞信科技有限公司 | The sample-pretreating method of endogenous rape element sterol and its pre-treatment solid phase material of use in a kind of plant sample |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107543746A (en) * | 2016-11-16 | 2018-01-05 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of the endogenous plant hormone of three class difference chemical property in a kind of plant sample |
| CN107941971A (en) * | 2017-11-22 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | The purification process of plant endogenous brassinosteroid based on the extraction of boron affinity solid phase |
| CN109813824A (en) * | 2017-11-22 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of plant sample pre-treating method |
| CN109813824B (en) * | 2017-11-22 | 2021-11-26 | 中国科学院大连化学物理研究所 | Pretreatment method of plant sample |
| CN109975085A (en) * | 2017-12-28 | 2019-07-05 | 武汉绿剑可瑞信科技有限公司 | The sample-pretreating method of endogenous rape element sterol and its pre-treatment solid phase material of use in a kind of plant sample |
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Effective date of registration: 20170527 Address after: 430075 No. 666, hi tech Avenue, East Lake Development Zone, Hubei, Wuhan Patentee after: Wuhan Green Credit Suisse Technology Co. Ltd can Address before: 430072 Hubei Province, Wuhan city Wuchang District of Wuhan University Luojiashan Patentee before: Wuhan University |