CN105866299A - GC-MS-based plant non-targeted metabolomics sample pretreatment method - Google Patents
GC-MS-based plant non-targeted metabolomics sample pretreatment method Download PDFInfo
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Abstract
The invention discloses a GC-MS-based plant non-targeted metabolomics sample pretreatment method. The method comprises the steps of 1) mixing up a plant sample, an internal standard substance and a hydrophilic organic solvent, wherein the hydrophilic organic solvent is pre-cooled to be -20 to 4 DEG C; cooling the obtained mixture to be -80 to -10 DEG C, grinding, crushing and conducting the supersonic extraction in the ice-water bath; 2) then respectively adding a lipophilic organic solvent and water, uniformly mixing up, conducting the supersonic extraction in the ice-water bath, conducting the high-speed centrifugation at the temperature of 0 to 16 DEG C, obtaining the aqueous phase and evaporating; 3) adding an oximation reagent, and conducting the oximation reaction at the temperature of 30 to 45 DEG C; 4) finally adding a derivatization reagent and n-hexane, and conducting the derivatization reaction at the temperature of 60 to 80 DEG C. According to the technical scheme of the invention, based on the above pretreatment method, the primary metabolites of a plant sample can be fully extracted, so that the abundant metabolite spectrum data information can be obtained. Meanwhile, both the change of metabolites during the extraction process, and the pollution of liposoluble substances to a gas chromatographic column can be avoided as much as possible. Therefore, the better sample reproducibility is realized.
Description
Technical field
The invention belongs to plant sample preprocess method field, be specifically related to a kind of based on GC-MS plant
Thing non-targeted metabolism group sample pretreating method.
Background technology
At present, Plant Metabolome by one be full of imaginary concept evolution become quickly grow, research valency
It is worth huge sphere of learning.Only comprise hundreds of metabolite from antibacterial and yeast different, it is known that plant time
Raw metabolite has reached about 100,000 kinds.But, Plant Metabolome research is the most still at an early stage,
Wide answering is had in fields such as species identification, transgenic plant discriminating, metabolic pathway and gene functional research
Use prospect.
By to metabolism group research, not only can be appreciated that plant change under difficult environmental conditions, also can grind
Study carefully metabolite composition and the content in same plant different parts or period.Metabolism spectrum can be outstanding as species identification
It is the important evidence that trans genie individual differentiates, is the important means of genotype and phenotype research.
The experiment purpose of Plant Metabolome is to carry out metabolite as much as possible in plant sample simultaneously
Qualitative and quantitative analysis, but due in plant sample metabolites kinds a lot, content difference is very big, generation
The dynamic range thanking to substrate concentration is wider, and complexity is higher, and metabolite is heated, easily reaction causes knot
Structure changes.It addition, containing a lot of fat-soluble secondary metabolites, these material kinds in plant tissue
Class is various, and boiling point is high, and is difficult to reduce boiling point by the method for derivatization, is not suitable for using GC-MS
(gas chromatography-mass spectrography) detects.These materials GC-MS data base (Fiehn data base,
NIST database) in Information in Mass Spectra few, it is difficult to qualitative, be not the main purpose of general sieve, easily
Analysis to primary metabolite produces interference.It addition, the path analysis of secondary metabolites is complicated, at KEGG
Cometabolism path in data base is little, lacks the comparison foundation of cometabolism path, therefore in pre-treatment
In primary metabolite in extraction plant sample as much as possible, grease removal dissolubility secondary metabolites is dry side by side
Disturb.But existing plant based on GC-MS non-targeted metabolism group sample pretreating method repeatability
Bad, versatility is not strong, accomplishes extract at low temperature the most as far as possible, the most strictly control during extraction
Temperature in operating process, the high temperature in operating process is easily caused the structure of metabolite and changes, and
The extraction of metabolite is abundant not, is difficulty with the detection of high flux gamut, differs for character or content
Big material is difficult to be detected simultaneously by, and additionally extracts selectivity the highest, and fat-soluble secondary metabolites fails
Reject, it is impossible to avoid interference.
Summary of the invention
Therefore, the present invention is directed to the existing non-targeted metabolism group specimen preprocessing of plant based on GC-MS
The problems that reason method exists, it is proposed that a kind of highly versatile, extract fully, to extract selectivity high
Favorable reproducibility based on GC-MS plant non-targeted metabolism group sample pretreating method.
The present invention based on GC-MS plant non-targeted metabolism group sample pretreating method, it include step
Rapid:
1) by plant sample, internal standard substance be cooled to the hydrophilic organic solvent of-20～4 DEG C in advance and mix and lower the temperature
To-80～-10 DEG C, then grind, and ice-water bath supersound extraction；
2) lipophylic organic solvents and water it are separately added into again, mixing, and ice-water bath supersound extraction；In 0～16 DEG C
High speed centrifugation, water intaking mutually and volatilizes；
3) add oximate reagent, carry out oximation reaction in 30～45 DEG C；
4) it is eventually adding derivatization reagent and normal hexane, performs the derivatization reaction in 60～80 DEG C, obtain
The non-targeted metabolism group sample of plant based on GC-MS.
Described hydrophilic organic solvent, described lipophylic organic solvents and water are extracting solution, described hydrophilic
Organic solvent and water are used for the polar substances (needing the primary metabolite extracted) extracting in plant sample,
Described lipophylic organic solvents is for separating nonpolar or low pole material (secondary metabolites).
Described hydrophilic organic solvent can be selected for methanol, acetone and/or ethanol miscible with water.Described hydrophilic
Organic solvent volume: described plant sample weight is 0.5～1mL:100mg preferred
0.6～0.75mL:100mg.Described lipophylic organic solvents can be selected for chloroform, ether and/or ethyl acetate.
Described hydrophilic organic solvent: described lipophylic organic solvents: the volume ratio of water is 1:0.4～0.6:0.8～1.2,
Preferably 1:0.5:1.
Described internal standard substance should meet following condition: be not the composition in plant sample and not with in plant sample
Metabolite reaction, described hydrophilic organic solvent can be dissolved in, be derivatized with active Hydrogen Energy.Institute
State internal standard substance and can be selected for chlorophenylalanine, chlorophenylalanine be the chloro-phenylalanine of L-2-, 3,4-dichloro phenylpropyl alcohol
Propylhomoserin, L-4-chlorophenylalanine, D-4-chlorophenylalanine or D-2-chlorophenylalanine, be not excluded for it certainly
The compound being derivatized of his type.Described internal standard substance weight: described plant sample weight is
10～30 μ g:100mg preferably 20 μ g:100mg.Because described internal standard substance consumption is little, it is difficult to directly weigh,
The most in certain embodiments, described internal standard substance is diluted to solubility with described hydrophilic organic solvent in advance and is
The internal standard substance solution of 0.2～0.4mg/mL, the most accurately measure and with described plant sample, described parent
Aqueous organic solvent mixes, and deducts described internal standard substance molten when measuring the described hydrophilic organic solvent of mixing
Liquid amasss, and maintains the total volume of described hydrophilic organic solvent with this.
Described oximate reagent is methoxamine hydrochloride, described methoxamine hydrochloride weight: described plant sample
Weight is 15～30:100 preferred 20:100.Can be excellent by adding 10～20mg/mL in actual process
Select the pyridine solution of methoxamine hydrochloride of 15mg/mL to add described oximate reagent.
Described derivatization reagent is N, O-bis-(trimethyl silane) acetamide, dimethyl dioxy silane, diformazan
Base dioxy silane, hmds, N-methyl-N-(trimethyl silane) trifluoroacetamide, containing 1%
Double (TMS) trifluoroacetamides of trim,ethylchlorosilane or the N-first containing 1% trim,ethylchlorosilane
Base is double (trifluoroacetamide).Described derivatization reagent volume: described plant sample weight is 1～2
UL:1mg, preferably 1.3uL:1mg.
Step 4) in the function of normal hexane be to increase the solvent borne of alkylation material, the consumption of normal hexane
Relevant with the consumption of derivatization reagent, described derivatization reagent volume: normal hexane volume is 2～8:1 preferred
4:1。
In one preferred embodiment of the present invention, step 1) concrete operations are, by described internal standard substance solution,
Described plant sample and be cooled to the hydrophilic organic solvent of-20～4 DEG C preferably-10～0 DEG C in advance and mix and lower the temperature
To-80～-10 DEG C preferably-60～-40 DEG C, put in grinder and grind with 40～80Hz preferred 60Hz frequencies
Pulverize 1～4min preferred 2min, and with the preferred 30min of ice-water bath supersound extraction 20～40min.
Further, step 2) concrete operations are, add after described lipophylic organic solvents in grinder
In mix with the 15～30Hz preferred 2min of preferred 20Hz frequency vortex 1～4min, add water in
Grinder mixes, then with the 15～30Hz preferred 2min of preferred 20Hz frequency vortex 1～4min
With the preferred 30min of ice-water bath supersound extraction 20～40min；Then under the conditions of 0～4 DEG C,
Centrifugal 5～15min preferred 10min under 12000～15000rpm preferred 14000rpm rotating speeds.
Further, step 3) concrete operations are, at humid control at 25～35% preferably 30% time,
Add the pyridine solution of the methoxamine hydrochloride of 10～20mg/mL preferred 15mg/mL, then in concussion
Speed is to carry out oxime in 35～40 DEG C preferably 37 DEG C of 100～300rpm preferred 200rpm concussion incubators
Change reaction 60～120min preferred 90min.
Step 4) concrete operations are, at humid control at 25～35% preferably 30% time, spread out described in addition
Biochemical reagents and normal hexane vortex concussion mix, then in 68～72 DEG C of preferably 70 DEG C derivative reactions
45～75min preferred 60min, are cooled to room temperature, obtain the non-targeted metabolism group of plant based on GC-MS
Imitate product.
Method step can be used for most plants blade, stem and or the pre-place of flesh tissue of root etc.
Reason.For fresh group of sugar content≤15% plant in addition to sugar content is high such as banana pulp, Caulis Sacchari sinensis etc.
Knit, especially sugar content≤5% fresh tissues of plants, can fully extract the primary metabolite in plant tissue
Thing, GC-MS analyzes and can reach higher repeatability.
The key point of the present invention includes following three points:
1, the selection of extracting solution and the selection of consumption proportion thereof.For reducing the interference to subsequent analysis, need
To remove the fat-soluble secondary metabolites that there is no in data base, the lipotropy such as such as flavonoid
Material.Described hydrophilic organic solvent and water are miscible, and described lipophylic organic solvents and certain proportion
The mixed phase of miscible described hydrophilic organic solvent-water is immiscible, described hydrophilic organic solvent-water and
Can be divided into the organic facies two-layer of hydrophilic aqueous phase and lipophilic after the mixing of described lipophylic organic solvents, aqueous phase is
It is dissolved with the described hydrophilic organic solvent-water of polar substances (needing the metabolite extracted), organic facies
For being dissolved with the described lipophylic organic solvents of lipophilic substance.Extracting solution is with methanol, water, chloroform combination
As a example by, volume ratio is that the methanol-water polarity of 1:1 is bigger, it is possible to extract polar substances more, and chloroform
Nonpolar or the lipophilic substance of low pole can be dissolved；Methanol, water, the volume ratio of chloroform are 2:2:1
Time, methanol-water is immiscible with chloroform, and solution is layered, upper strata aqueous phase be dissolved with the methanol of polar substances-
Water, lower floor's organic facies is to be dissolved with the nonpolar or chloroform of low pole material, takes the supernatant and i.e. can be used for
Subsequent treatment carries out GC-MS metabonomic analysis again.First add methanol during concrete operations, can extract absolutely
Most metabolite, then add can not or be not easy propose hydroaropic substance and easily extract lipophilic material
Chloroform, finally add water make aqueous phase and organic facies layering, can reach the effect preferably extracting separation.
2, operating process ensures low temperature.Grinder process of lapping can produce amount of heat, therefore grind
Before plant sample can be placed about 2min in low temperature (-20～-80 DEG C) environment.During water bath sonicator,
Temperature can raise, and ultrasonic cleaning instrument temperature can be set to minimum (0 DEG C), and add ice bag, it is ensured that
Whole ultrasonic procedure temperature is in the temperature of ice-water bath.High speed centrifugation can ensure that the tissue residue of plant has precipitated
Entirely, high speed centrifugation also can produce amount of heat simultaneously, and centrifuging temperature can be set to 0～4 DEG C.Above-mentioned each control
It is too high and cause the structure of metabolite to change that the operation of temperature can be prevented effectively from plant sample temperature.
3, the humidity requirement during derivatization is strict.Derivatization reagent is met water and is decomposed immediately, therefore adds
During oximate reagent, derivatization reagent, normal hexane, humidity level controls about 30%, and
Not can exceed that 35%, derivatization otherwise will be caused insufficient, ultimately result in the metabolite quantity detected relatively
Few.
It addition, use the centrifuge tube added with little steel ball to process container as plant sample, it is put in grinder
Grind, plant tissue can be made to destroy fully, then through supersound extraction, it is possible to fully extract metabolite.
The most progressive effect of the present invention is: the described non-targeted metabolism group of plant based on GC-MS
Sample pretreating method is fully to extract the metabolite of plant sample under cryogenic, it is possible to decrease
The high temperature structure influence to metabolite；And the fat-soluble secondary metabolites effectively removed, can avoid fat
The pollution of gas chromatographic column is disturbed subsequent analysis by the secondary metabolites of dissolubility；Additionally strict control derives
Change process experiment room humidity to ensure abundant derivatization.Use the sample that the preprocess method of the present invention processes
Carry out GC-MS Plant Metabolome analysis and can obtain bigger metabolite peak data, and sample reappears
Property preferable, the extraction of various Plant tissue samples can be generally applicable to, improve large sample amount in metabolism group
Efficiency.Additionally the preprocess method of the present invention is simple and quick, and agents useful for same toxicity is less.
Above-mentioned positive progressive effect is to promoting Plant Metabolome research, setting up unified extracting method standard, add
Data exchange is carried out significant between strong correlation researcher.
Accompanying drawing explanation
Figure 1A～1F is total particle flux chromatogram of the tea leaf of example 1.
Fig. 2 A～2F is total particle flux chromatogram of the Citrullus vulgaris stem of example 2.
Fig. 3 A～3F is total particle flux chromatogram of the Radix Zanthoxyli Bungeani of example 3.
Detailed description of the invention
Below in conjunction with specific embodiment to the present invention based on GC-MS plant non-targeted metabolism group sample
Preprocess method is further described.
The pretreatment of embodiment 1 tea leaf (sugar content 1～5%)
Use following steps fresh tea leaf carries out pretreatment and makees GC-MS detection, be repeated 6 times
(1A～1F) parallel laboratory test.
Pretreatment concretely comprises the following steps:
1) accurately weigh tea leaf 60mg, put in the centrifuge tube of 1.5mL；Be sequentially added into two little
Steel ball, the methanol of 360 μ L 4 DEG C and 40 μ L internal standard substance methanol solutions (the chloro-phenylalanine of L-2-,
0.3mg/mL) ,-80 DEG C of refrigerators are placed 2min；Put into grinding machine for grinding (60Hz, 2min),
Take out from grinder, supersound extraction 30min；
2) add 200 μ L chloroforms, put into grinder mesoscale eddies (20Hz, 2min), add 400 μ L
Water, puts into grinder mesoscale eddies (20Hz, 2min), supersound extraction 30min；
3) low-temperature centrifugation 10min (14000rpm, 4 DEG C), takes 200～700 μ L of supernatant liquid, loads
Glass derives in bottle, volatilizes with quick centrifuge concentrator；
4) at humid control at 30% time, derive to glass and bottle adds 80 μ L methoxamine pyridine hydrochlorides
Solution (15mg/mL), after vortex concussion 2min, 37 DEG C of oximation reaction 90min in concussion incubator；
5) at humid control at 30% time, double (TMS) the trifluoro second of 80 μ L after taking-up, is added
Amide (containing 1% trim,ethylchlorosilane) and 20 μ L normal hexane, after vortex concussion 2min, spread out in 70 DEG C
Taking out after biochemical reaction 60min, room temperature places 30min, carries out GC-MS metabonomic analysis.
Analysis of test results:
In the pre-treatment step of the present embodiment, extracting solution methanol, water, chloroform volume ratio are 2:2:1,
The primary metabolite of polarity is efficiently extracted by first alcohol and water, and by chloroform by fat-soluble secondary generation
Thank to thing to remove, thus avoid the pollution to gas chromatographic column and the interference to subsequent analysis.In operating process
Ensure low temperature, be prevented effectively from high temperature and cause the structure of metabolite to change.And during derivatization
Humidity is strict controlled in about 30%, it is ensured that fully derivatization.
Total particle flux chromatogram (Total of the tea leaf metabolite recorded after 6 parallel pretreatment
Particles Chromatogram, TIC) as shown in Figure 1A～1F；Tea leaf metabolites kinds quantity
Testing result is as shown in table 1.Testing result display internal standard peak and known false positive peak (include noise,
Column bleed and derivative materialization reagent peak) all remove from data matrix, and carry out after de-redundancy and peak merge,
6 parallel laboratory tests all can qualitative go out the metabolite of about 270 kinds, wherein have 206 kinds of metabolite at 6 times
Parallel laboratory test all detects have higher repeatability.
The metabolites kinds quantity testing result of table 1 tea leaf
So that metabolite quantity is more and stable in testing result, sample favorable reproducibility.
The pretreatment (sugar content about 2%) of embodiment two Citrullus vulgaris stem
Use following steps fresh Citrullus vulgaris stem carries out pretreatment and makees GC-MS detection, be repeated 6 times
(2A～2F) parallel laboratory test.
Pretreatment concretely comprises the following steps:
1) accurately weigh Citrullus vulgaris stem 60mg, put in the centrifuge tube of 1.5mL；It is sequentially added into two little steel
Pearl, the ethanol of 360 μ L-20 DEG C and 40 μ L internal standard substance ethanol solution (L-4-chlorophenylalanine,
0.3mg/mL) ,-20 DEG C of refrigerators are placed 2min；Put into grinding machine for grinding (60Hz, 2min),
Take out from grinder, supersound extraction 30min；
2) add 200 μ L ether, put into grinder mesoscale eddies (20Hz, 2min), add 400 μ L
Water, puts into grinder mesoscale eddies (20Hz, 2min), supersound extraction 30min；
3) low-temperature centrifugation 10min (14000rpm, 16 DEG C), takes 200～700 μ L subnatants, dress
Enter glass and derive in bottle, volatilize with quick centrifuge concentrator；
4) at humid control at 30% time, derive to glass and bottle adds 80 μ L methoxamine pyridine hydrochlorides
Solution (15mg/mL), after vortex concussion 2min, 37 DEG C of oximation reaction 90min in concussion incubator；
5) at humid control at 30% time, add 80 μ L N-methyl double (trifluoroacetamide) after taking-up and (contain
1% trim,ethylchlorosilane) and 20 μ L normal hexane, after vortex concussion 2min, in 70 DEG C of derivative reactions
Taking out after 60min, room temperature places 30min, carries out GC-MS metabonomic analysis.
Analysis of test results:
In the pre-treatment step of the present embodiment, extracting solution ethanol, water, ether volume ratio are 2:2:1,
The primary metabolite of polarity is extracted fully by second alcohol and water, and by ether by fat-soluble secondary generation
Thank to thing to remove, thus avoid the pollution to gas chromatographic column and the interference to subsequent analysis.In operating process
Ensure low temperature, be prevented effectively from high temperature and cause the structure of metabolite to change.And during derivatization
Humidity is strict controlled in about 30%, it is ensured that fully derivatization.
Total particle flux chromatogram (Total of the Citrullus vulgaris stem metabolite recorded after 6 parallel pretreatment
Particles Chromatogram, TIC) as shown in Fig. 2 A～2F；The inspection of Citrullus vulgaris stem metabolites kinds quantity
Survey result is as shown in table 1.Testing result display internal standard peak and known false positive peak (include noise,
Column bleed and derivative materialization reagent peak) all remove from data matrix, and carry out after de-redundancy and peak merge,
6 parallel laboratory tests all can qualitative go out the metabolite of about 200 kinds, wherein have 160 kinds of metabolite at 6 times
Parallel laboratory test all detects have higher repeatability.
Table 2 Citrullus vulgaris stem metabolites kinds quantity testing result
The pretreatment (sugar content about 1%) of embodiment three Radix Zanthoxyli Bungeani
Use following steps fresh flowers pepper root carries out pretreatment and makees GC-MS detection, be repeated 6 times flat
Row experiment (3A～3F).
Pretreatment concretely comprises the following steps:
1) accurately weigh Radix Zanthoxyli Bungeani 60mg, put in the centrifuge tube of 1.5mL；It is sequentially added into two little steel
Pearl, the acetone of 360 μ L-10 DEG C and 40 μ L internal standard substance acetone solns (3,4-dichloro-benzenes alanine,
0.3mg/mL) ,-10 DEG C of refrigerators are placed 2min；Put into grinding machine for grinding (60Hz, 2min),
Take out from grinder, supersound extraction 30min；
2) add 200 μ L ethyl acetate, put into grinder mesoscale eddies (20Hz, 2min), add 400 μ L
Water, puts into grinder mesoscale eddies (20Hz, 2min), supersound extraction 30min；
3) low-temperature centrifugation 10min (14000rpm, 10 DEG C), takes 200～700 μ subnatants, loads
Glass derives in bottle, volatilizes with quick centrifuge concentrator；
4) at humid control at 30% time, derive to glass and bottle adds 80 μ L methoxamine pyridine hydrochlorides
Solution (15mg/mL), after vortex concussion 2min, 37 DEG C of oximation reaction 90min in concussion incubator；
5) at humid control at 30% time, 80 μ L N-methyl-N-(trimethyl silane) after taking-up, are added
Trifluoroacetamide and 20 μ L normal hexane, after vortex concussion 2min, in 70 DEG C of derivative reaction 60min
Rear taking-up, room temperature is placed 30min, is carried out GC-MS metabonomic analysis.
Analysis of test results:
In the pre-treatment step of the present embodiment, extracting solution acetone, water, ethyl acetate volume ratio are 2:2:
1, the primary metabolite of polarity is extracted fully by acetone and water, and by ethyl acetate by fat-soluble
Secondary metabolites remove, thus avoid the pollution to gas chromatographic column and the interference to subsequent analysis.Behaviour
Ensure low temperature during work, be prevented effectively from high temperature and cause the structure of metabolite to change.And derivatization
During humidity be strict controlled in about 30%, it is ensured that fully derivatization.
Total particle flux chromatogram (Total of the Radix Zanthoxyli Bungeani metabolite recorded after 6 parallel pretreatment
Particles Chromatogram, TIC) as shown in Fig. 3 A～3F；The inspection of Radix Zanthoxyli Bungeani metabolites kinds quantity
Survey result is as shown in table 1.Testing result display internal standard peak and known false positive peak (include noise,
Column bleed and derivative materialization reagent peak) all remove from data matrix, and carry out after de-redundancy and peak merge,
6 parallel laboratory tests all can qualitative go out the metabolite of about 270 kinds, wherein have 211 kinds of metabolite at 6 times
Parallel laboratory test all detects have higher repeatability.
Table 3 Radix Zanthoxyli Bungeani metabolites kinds quantity testing result
From the testing result of above three embodiment, the preprocess method of the present invention can fully extract newly
Metabolite in fresh plant tissue (including blade, stem and root), can detect that the generation of relatively multiple types
Thank thing, metabolites kinds and relative amount and be respectively provided with higher repeatability, be applicable to except sugar content is high
Sarcocarp (such as pear flesh, watermelon flesh, Fructus Melo sarcocarp etc.) beyond the GC-MS of plant fresh sample plant
The process of thing non-targeted metabolism group sample.
Claims (10)
1. the non-targeted metabolism group sample pretreating method of plant based on GC-MS, its feature exists
In comprising the steps:
1) by plant sample, internal standard substance be cooled to the hydrophilic organic solvent of-20～4 DEG C in advance and mix and lower the temperature
To-80～-10 DEG C, then grind, and ice-water bath supersound extraction；
2) lipophylic organic solvents and water it are separately added into again, mixing, and ice-water bath supersound extraction；In 0～16 DEG C
High speed centrifugation, water intaking mutually and volatilizes；
3) add oximate reagent, carry out oximation reaction in 30～45 DEG C；
4) it is eventually adding derivatization reagent and normal hexane, performs the derivatization reaction in 60～80 DEG C, obtain
The non-targeted metabolism group sample of plant based on GC-MS.
2. the method for claim 1, it is characterised in that step 1) in, described hydrophilic
Organic solvent is methanol, acetone or alcohol, described hydrophilic organic solvent volume: described plant sample weight
Amount is 0.5～1mL:100mg preferably 0.6～0.75mL:100mg；Described internal standard substance is chlorophenylalanine,
Described internal standard substance weight: described plant sample weight is 10～30 μ g:100mg preferably 20 μ g:100mg.
3. method as claimed in claim 2, it is characterised in that described chlorophenylalanine is that L-2-is chloro-
Phenylalanine, 3,4-dichloro-benzenes alanine, L-4-chlorophenylalanine, D-4-chlorophenylalanine or D-2-chlorine
Phenylalanine.
4. method as claimed in claim 2, it is characterised in that step 1) in, described internal standard substance
It is diluted in solubility is 0.2～0.4mg/mL preferred 0.3mg/mL with described hydrophilic organic solvent in advance
Mark thing solution, the most accurately measures and mixes with described plant sample, described hydrophilic organic solvent,
Deduct described internal standard substance solution volume when measuring the described hydrophilic organic solvent of mixing, maintain institute with this
State the volume that hydrophilic organic solvent is total；Step 1) concrete operations are, by described internal standard substance solution, institute
State plant sample and be cooled to the hydrophilic organic solvent of-20～4 DEG C preferably-10～0 DEG C in advance and mix and be cooled to
-80～-10 DEG C preferably-60～-40 DEG C, put in grinder with 40～80Hz preferred 60Hz frequency grounds travel
Broken 1～4min preferred 2min, and with the preferred 30min of ice-water bath supersound extraction 20～40min.
5. the method as described in any one of claim 2～4, it is characterised in that step 2) in, institute
Stating lipophylic organic solvents is chloroform, ether and/or ethyl acetate, described hydrophilic organic solvent: described
Lipophylic organic solvents: the volume ratio of water is 1:0.4～0.6:0.8～1.2, preferably 1:0.5:1.
6. method as claimed in claim 5, it is characterised in that step 2) concrete operations are to add
Enter after described lipophylic organic solvents in grinder with 15～30Hz preferred 20Hz frequency vortexs 1～4min
Preferably 2min mixes, and adds water in grinder with 15～30Hz preferred 20Hz frequency vortexs
1～4min preferred 2min mixes, then with the preferred 30min of ice-water bath supersound extraction 20～40min；
Then, under the conditions of 0～4 DEG C, 5～15min under 12000～15000rpm preferred 14000rpm rotating speeds, it are centrifuged
Preferably 10min.
7. method as claimed in claim 6, it is characterised in that step 3) in, described oximate is tried
Agent is methoxamine hydrochloride, described methoxamine hydrochloride weight: described plant sample weight is 1.5～3:100
Preferably 2:100；Step 3) concrete operations are, at humid control at 25～35% preferably 30% time, add
The pyridine solution of the methoxamine hydrochloride of 10～20mg/mL preferred 15mg/mL, then in concussion speed be
35～40 DEG C of 100～300rpm preferred 200rpm preferably 37 DEG C concussion incubator carries out oximation reaction
60～120min preferred 90min.
8. method as claimed in claim 7, it is characterised in that step 4) in, described derivatization
Reagent be N, O-bis-(trimethyl silane) acetamide, dimethyl dioxy silane, dimethyl dioxy silane, six
Methyl two silicon amine, N-methyl-N-(trimethyl silane) trifluoroacetamide, containing 1% trim,ethylchlorosilane
Double (TMS) trifluoroacetamides or the double (trifluoroacetyl of the N-methyl containing 1% trim,ethylchlorosilane
Amine)；Described derivatization reagent volume: described plant sample weight is 1～2uL:1mg, preferably 1.3
uL:1mg；Described derivatization reagent volume: normal hexane volume is 2～8:1 preferred 4:1；Step 4) concrete
Operation is, at humid control at 25～35% preferably 30% time, adds described derivatization reagent and normal hexane
And vortex concussion mixing, then preferred in 68～72 DEG C of preferably 70 DEG C derivative reactions 45～75min
60min, is cooled to room temperature, obtains the non-targeted metabolism group sample of plant based on GC-MS.
9. the method for claim 1, it is characterised in that described plant sample be plant leaf blade,
Stem or the flesh tissue of root.
10. method as claimed in claim 9, it is characterised in that the sugar content of described flesh tissue≤
15%, preferably≤5%.
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