CN109541075A - Potato metabolic components sample pre-treatments and its analysis method - Google Patents
Potato metabolic components sample pre-treatments and its analysis method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
A kind of potato metabolic components sample pre-treatments and its analysis method, the invention belongs to metabonomic technology fields, it include: that potato samples are extracted, extraction includes: to mix potato samples with extractant and internal standard substance, then carries out ultrasonic extraction and extraction mixed liquor is made；Extractant is water and/or organic solvent, and organic solvent is at least one of methanol, isopropanol and acetonitrile.A kind of analysis method of potato metabolic components comprising: potato metabolic components sample made from above-mentioned potato metabolic components sample pre-treatments is subjected to GC-MS analysis.Solving metabonomic analysis in the prior art there is technical issues that sensitivity is low, resolution ratio is not high and the cover of high abundance analyte is low-abundance.
Description
Technical field
The invention belongs to metabonomic technology fields, in particular to a kind of potato metabolic components sample pre-treatments
And its analysis method.
Background technique
Various spectroscopy techniques in the necessary dependency analysis chemistry of the detection and analysis of metabolin integral level, including magnetic resonant wave
Spectrum, mass spectrum, chromatography, infrared and Raman spectrum, ultraviolet-visible spectrum etc. and its coupling connection instrument method obtain metabolism group data；?
The most common analysis tool is NMR in the research of metabolism group, especially H-NMR, can be realized the non-destructive to sample,
Non-selective analysis, but it also there are two obviously defects: sensitivity is low, resolution ratio is not high, frequently results in high abundance
Analyte covers low-abundance analyte.
Summary of the invention
The purpose of the present invention is to provide a kind of potato metabolic components sample pre-treatments and its analysis methods, it is intended at least
Solve that metabonomic analysis there are sensitivity low, resolution ratio in the prior art is not high and analyte of high abundance covers low abundance
Analyte the technical issues of.
The present invention is implemented as follows:
In a first aspect, the embodiment of the present invention provides a kind of potato metabolic components sample pre-treatments, including to potato sample
Product extract, and extraction includes: to mix potato samples with extractant and internal standard substance, then carry out ultrasonic extraction and are made
Extract mixed liquor；
Extractant is water and/or organic solvent, and organic solvent is at least one of methanol, isopropanol and acetonitrile.
In above-mentioned technical proposal, by single_factor method and orthogonal experimental design method optimizing research extract metabolite because
Element determines influence of the extractant type to extraction results, in order to determine which kind of extractant preferably extraction letter can be obtained using
Breath；The purpose of internal standard substance is added is the influence for offsetting loading volume or even mobile phase, detector to experimental result.
In some specific embodiments, organic solvent is methanol；Optionally, when extractant is water and organic solvent
When, methanol percentage by volume is 50-80%.
In above-mentioned technical proposal, since water has very big advantage in terms of extracting amino acid and sugar, in organic solvent
It is middle that a certain proportion of water is added, richer metabolite information can be obtained, and in organic solvent, methanol extraction effect is most
It is good, richer metabolite information can be obtained in water-methanol mixed system extractant extraction process.
In some specific embodiments, wherein the potato samples of every 100mg, use 300-800 μ L extractant with
And 5-15 μ L internal standard substance is mixed.
In above-mentioned technical proposal, less than enough sample messages, extractant is excessively then easy to make the excessively few then extraction of extractant
It is wasted at extractant, internal standard substance price is relatively expensive, and multipair experimental result improvement was added and is not obvious, therefore suitable use
It measures most important.
In some specific embodiments, the temperature of ultrasonic extraction is 25-65 DEG C, time 3-11min.
In above-mentioned technical proposal, on the one hand, improve extraction time and temperature is conducive to improve the mass transfer rate of metabolin, mention
The recovery rate of hypermetabolism object.On the other hand, extraction time and increasing considerably for temperature result in primary metabolite in sample
It reduces, the interaction between time and temperature is also required to study.
It in some specific embodiments, extracts further include: extraction mixed liquor is subjected to centrifugal treating at 0-10 DEG C
Obtain centrifugal mixture, centrifugation time 5-15min, centrifugal rotational speed 10000-15000rmp, 180- after being then centrifuged
250 μ L supernatants are freeze-dried.
In above-mentioned technical proposal, high speed centrifugation is handled, and solid particle and the liquid in suspension separate, and is taken supernatant, is made
Bulky grain in potato samples is separated with sample to be tested, and freeze-drying avoids influence of the sample to be tested by microbial bacterial.
It further include that the potato samples obtained to extraction perform the derivatization in some specific embodiments, after extracting
Processing, derivatization treatment includes: to carry out oximate processing to potato samples using the first derivatization reagent, by the Ma Ling after oximate
Potato sample and the second derivatization reagent carry out Silanization reaction；
Optionally, every potato samples slice for extracting 100mg, carries out oxime using the first derivatization reagent of 20-40 μ L
Change processing and Silanization reaction is carried out using the second derivatization reagent of 20-40 μ L；And/or first derivatization reagent be first
Oxygen amine hydrochlorate pyridine solution, the second derivatization reagent are N, bis- (trimethyl silicon substrate) trifluoroacetamides of O-.
In above-mentioned technical proposal, the purpose of oximate processing is to stablize carbonyl, inhibits the keto-enol formula interconversion of carbonyl, or suppression
Carbonyl processed is converted to ketal or ethylidene ether structure.The sensitivity of sample detection can be improved in derivatization treatment；Improve sample mixture
Separating degree；It is suitable for further doing Structural Identification.Silylation is introduced into molecule by Silanization reaction, replaces reactive hydrogen.Activity
Hydrogen reduces the polarity of compound after being replaced by silylation, reduce hydrogen bond constraint.Due to the reaction site number containing reactive hydrogen
It reduces, the stability of compound is also enhanced.Silanized compounds polarity weakens, and is tested ability enhancing, and thermal stability improves.
In some specific embodiments, oximate processing temperature be 35-40 DEG C, time 0.8-1.2h；
And/or the temperature of Silanization reaction is 65-75 DEG C, time 0.8-1.2h.
In above-mentioned technical proposal, Silylation object is easier to volatilize, therefore at the temperature and oximate of Silanization reaction
Reason setting is unsuitable excessively high, and Silylation object is avoided to volatilize.
It further include sample pretreatment before extraction in some specific embodiments, sample pretreatment includes: by potato
Sample sections to every piece of diameter is that 50-80mm is subsequently placed in cold in liquid nitrogen with a thickness of 1-3mm, weight 80-120mg
Freeze, is stored under the conditions of -100--70 DEG C.
In above-mentioned technical proposal, due to liquid nitrogen have chemical inertness, can directly and sample sections contact, freeze immediately and
The bioactivity of sample will not be destroyed, therefore can be used to save biopsy tissues, the storage of biological sample.Liquid nitrogen frozen can be most
Metabolic components do not change or are further metabolized in the preservation sample of limits.
Second aspect, the embodiment of the present invention provide a kind of analysis method of potato metabolic components comprising: it will be above-mentioned
Potato metabolic components sample made from potato metabolic components sample pre-treatments carries out GC-MS analysis.
In above-mentioned technical proposal, GC enhances its resolution capability, the structural information with the available metabolic components of combination of MS.
With high sensitivity, high resolution, the advantages of avoiding the analyte of high abundance from covering low-abundance analyte.
In some specific embodiments, HP-5ms chromatographic column, parameter setting are as follows: sample introduction are selected in GC-MS analysis
250-300 DEG C of temperature of mouth, 200-250 DEG C of EI ion source temperature, 120-180 DEG C of quadrupole rod temperature, carrier gas is high-purity helium,
Splitless injecting samples, sample volume are 0.8-1.2 μ L；
Using temperature programming, temperature program are as follows: initial temperature is 60-100 DEG C, keeps 1-3min；With 5-15 DEG C/min
300-350 DEG C is risen to, 4-10min is kept；Balance 4-10min is kept under initial temperature, then reinjects sample next time
Product；Flow rate of carrier gas is 0.8-1.5mL/min；Mass Spectrometer Method is carried out using full scan mode, Mass Spectrometer Method range is 50-550
(m/z)。
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is influence statistical data figure of the extractant type that provides of test example 1 of the present invention to ultrasonic extracting method；
Fig. 2 is influence statistical data figure of the ultrasonic extraction time of test example 2 of the present invention to ultrasonic extracting method；
Fig. 3 is influence statistical data figure of the ultrasonic extraction temperature that provides of test example 2 of the present invention to ultrasonic extracting method；
Fig. 4 is the full ion stream chromatogram of GC-MS for the potato tubers metabolite that test example 4 of the present invention provides；
The accuracy data statistics figure of metabolite in the potato tubers that Fig. 5 provides for test example 5 of the present invention；
The repeated data statistics figure of metabolite in the potato tubers that Fig. 6 provides for test example 5 of the present invention；
The stability data statistical chart of metabolite in the potato tubers that Fig. 7 provides for test example 5 of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The potato metabolic components sample pre-treatments and its analysis method of the embodiment of the present invention are specifically described below.
In a first aspect, the embodiment of the present invention provides a kind of potato metabolic components sample pre-treatments, including to potato sample
Product extract, and extraction includes: to mix potato samples with extractant and internal standard substance, then carry out ultrasonic extraction and are made
Extract mixed liquor；
Extractant is water and/or organic solvent, and organic solvent is at least one of methanol, isopropanol and acetonitrile.It is optional
, in some embodiments, extractant is only water, only organic solvent, or is the mixed liquor of water and organic solvent.In this hair
In bright embodiment, organic solvent selects polar organic solvent.
In some embodiments of the invention, methanol, isopropanol and acetonitrile (chromatographic grade) are public from U.S. Fisher technology
Department, potato samples are potato, and still optionally further, potato samples are from the Exploitation of Agriculture in Heilongjiang academy of sciences and local supermarket
And the market of farm produce, kind includes: black peak (HF), roundpod jute seed (HMZ), excellent into four kinds of 885 (YJ885), gram new 13 (KX13).
In some specific embodiments, organic solvent is methanol；When organic solvent is methanol, extractant is optional
Methanol is used alone, or is mixed using first alcohol and water.Optionally, when extractant is water and organic solvent and this is organic molten
Agent be methanol when, methanol percentage by volume be 50-80% or 55-75 or 60-70, such as, but not limited to, 50%, 55%,
60%, 65%, 70%, 75%, 80%.
Optionally, when extractant is water and organic solvent, methanol percentage by volume is 50-80% or 55-75, or
60-70, such as, but not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%.
In some specific embodiments, wherein the potato samples of every 100mg, using 300-800 μ L extractant,
Or 350-750 μ L or 400-700 μ L or 450-650 μ L or 500-600 μ L, such as, but not limited to, 300 μ L, 400 μ L,
500μL,600μL,700μL,800μL.And 5-15 μ L internal standard substance is mixed or 7-13 μ L or 9-11 μ L, such as
But it is not limited to, 5,7 μ L, 9 μ L, 11 μ L, 13 μ L, 15 μ L.
In some specific embodiments, the temperature of ultrasonic extraction is 25-65 DEG C or 40-50 DEG C or 54-56
DEG C, such as, but not limited to 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, the time of ultrasonic extraction is
3-11min or 8-10min, such as, but not limited to, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min,
11min.In some embodiments of the invention, ultrasonic extraction carries out in supersonic wave cleaning machine, and supersonic wave cleaning machine is selected
KQ2200E type supersonic wave cleaning machine (40kHz, 100W, Kunshan ultrasonic instrument Co., Ltd).
It in some specific embodiments, extracts further include: extraction mixed liquor is subjected to centrifugal treating at 0-10 DEG C
Centrifugal mixture or 2-8 DEG C or 4-6 DEG C are obtained, such as, but not limited to, when 0 DEG C, 2 DEG C, 4 DEG C, 6 DEG C, 8 DEG C, 10 DEG C of centrifugations
Between be 5-15min or 7-13min or 9-11min, such as, but not limited to, 5min, 7min, 9min, 11min, 13min,
15min, centrifugal rotational speed 10000-15000rmp or 11000-14000rmp or 12000-13000rmp, such as but not
It is limited to, 10000,11000rmp, 12000rmp, 13000rmp, 14000rmp, 15000rmp, 180-250 μ after being then centrifuged
L supernatant is freeze-dried or 190-240 μ L or 200-230 μ L or 210-220 μ L, such as, but not limited to 180 μ L,
190μL,200μL,210μL,220μL,230μL,240μL,250μL.In some embodiments of the invention, centrifugal treating from
It is carried out in scheming, supernatant is transferred in GC sample introduction bottle (1.5mL autosampler bottle) after centrifugation, was then freeze-dried
Night.In some embodiments of the invention, Alpha1-2Ldplus freeze drier (German CHRIST public affairs are selected in freeze-drying
Department), TGL-16B supercentrifuge (An Ting Instrument Ltd.) is selected in centrifugal treating operation.
It further include that the potato samples obtained to extraction perform the derivatization in some specific embodiments, after extracting
Processing, derivatization treatment includes: to carry out oximate processing to potato samples using the first derivatization reagent, by the Ma Ling after oximate
Potato sample and the second derivatization reagent carry out Silanization reaction；In order to keep test specimen fresh, in an embodiment of the present invention,
All potato samples are interior for 24 hours after derivatization treatment to be analyzed, and 5 Duplicate Samples are done in all experiments.
Optionally, every potato samples slice for extracting 100mg, carries out oxime using the first derivatization reagent of 20-40 μ L
Change and Silanization reaction or the first derivatization reagent 25-35 μ L are carried out using the second derivatization reagent of 20-40 μ L, or
28-32 μ L, such as, but not limited to, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L or the second derivatization reagent 25-35 μ L, or
28-32 μ L, such as, but not limited to, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L.
And/or first derivatization reagent, it is optionally methoxamine hydrochloride pyridine solution, the second derivatization reagent is N,
Bis- (trimethyl silicon substrate) trifluoroacetamides of O-, in other embodiments of the invention, the second derivatization reagent can also be N-
Methyl-n- (trimethyl silane) trifluoroacetamide.
In some embodiments of the invention, bis- (trimethyl silicon substrate) trifluoroacetamides (BSTFA) of N, O-, methoxy amine salt
Hydrochlorate and pyridine come from Sigma-Aldrich.
In some specific embodiments, the temperature of oximate processing is 35-40 DEG C, such as, but not limited to 35 DEG C, 3637
DEG C, 38 DEG C, 39 DEG C, 40 DEG C, oximate processing time be 0.8-1.2h, such as, but not limited to 0.8h, 0.9h, 1.0h, 1.1h,
1.2h；In some embodiments of the invention, oximate processing carries out in incubator.
And/or the temperature of Silanization reaction be 65-75 DEG C or 67-73 or 69-71, such as, but not limited to 65 DEG C,
67 DEG C, 69 DEG C, 71 DEG C, 73 DEG C, 75 DEG C, time of Silanization reaction are 0.8-1.2h, such as, but not limited to 0.8h, 0.9h,
1.0h,1.1h,1.2h.It should be noted that the "and/or" in the application, such as " option A and/or option b ", each meaning can be with
Individually it is option A, is individually that option b, option A add option b, three kinds of modes.
It further include sample pretreatment before extraction in some specific embodiments, sample pretreatment includes: by potato
Sample sections to every piece of diameter is 50-80mm or 55-75mm, 60-70mm, such as, but not limited to, 50mm, 55mm, 60mm,
65mm, 70mm, 75mm, 80mm, with a thickness of 1-3mm, such as, but not limited to, and 1mm, 1.5mm, 2.0mm, 2.5mm, 3mm, weight
For 80-120mg or 85-115mg, 90-110mg, 95-105mg, such as, but not limited to, 80mg, 85mg, 90mg, 95mg,
100mg, 105mg, 110mg, 115mg, 120mg are subsequently placed in liquid nitrogen and freeze, and store under the conditions of -100--70 DEG C, or -
95--75 DEG C, or -90--80 DEG C, such as, but not limited to, -100 DEG C, -95 DEG C, -90 DEG C, -85 DEG C, -80 DEG C, -
75 DEG C, -70 DEG C.
Second aspect, the embodiment of the present invention provide a kind of analysis method of potato metabolic components comprising: it will be above-mentioned
Potato metabolic components sample made from potato metabolic components sample pre-treatments carries out GC-MS analysis.
In some specific embodiments, HP-5ms chromatographic column, parameter setting are as follows: sample introduction are selected in GC-MS analysis
250-300 DEG C or 260-290 DEG C or 270-280 DEG C of temperature of mouth, such as, but not limited to 250 DEG C, 260 DEG C, 270 DEG C, 280
DEG C, 290 DEG C, 300 DEG C, 200-250 DEG C or 200-250 DEG C or 210-240 DEG C or 220-230 DEG C of EI ion source temperature,
Such as, but not limited to 200 DEG C, 210 DEG C, 220 DEG C, 230 DEG C, 240 DEG C, 250 DEG C, 120-180 DEG C of quadrupole rod temperature or 130-
170 DEG C or 140-160 DEG C, such as, but not limited to, 120 DEG C, 130 DEG C, 140 DEG C, 150 DEG C, 160 DEG C, 170 DEG C, 180 DEG C carry
Gas is high-purity helium, and Splitless injecting samples, sample volume is 0.8-1.2 μ L or 0.9-1.1 μ L, such as, but not limited to 0.8 μ L, 0.9
μL、1.0μL、1.1μL、1.2μL。
In some embodiments of the invention, Milli-q Water warfare of the chromatographic grade water from Millipore Corp, the U.S.
System is used to prepare all aqueous solutions.Every other analytical grade reagent is all from Beijing Chemical Plant (Beijing, China)；And
The EI ion source for selecting GC-MS-QP2010 (Shimadzu Technology Co., Ltd., Japan) to be equipped with, quadrupole mass-synchrometer, AOC-
20i automatic sampler；The standard substance of Structural Identification is from Sigma-Aldrich and national drug and biology system
Product control research institute (Beijing, China).
Using temperature programming, temperature program are as follows: initial temperature be 60-100 DEG C or 70-90 DEG C, such as, but not limited to 60
DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C, keep 1-3min or 1.5-2.5min, such as, but not limited to 1min, 1.5min,
2.0min,2.5min,3min；300-350 DEG C or 7-13 DEG C/min or 9-11 DEG C/min, example are risen to 5-15 DEG C/min
Such as, but not limited to, 5 DEG C/min, 7 DEG C/min, 9 DEG C/min, 11 DEG C/min, 13 DEG C/min, 15 DEG C/min or 310-340 DEG C, or
320-330 DEG C, such as, but not limited to 300 DEG C, 310 DEG C, 320 DEG C, 330 DEG C, 340 DEG C, 350 DEG C keep 4-10min or 5-
9min or 6-8min, such as, but not limited to 4min, 5min, 6min, 7min, 8min, 9min, 10min；It is protected under initial temperature
Maintain an equal level weighing apparatus 4-10min or 5-9min or 6-8min, such as, but not limited to 4min, 5min, 6min, 7min, 8min, 9min,
Then 10min reinjects sample next time；Flow rate of carrier gas is 0.8-1.5mL/min；Mass spectrum inspection is carried out using full scan mode
It surveys, Mass Spectrometer Method range is 50-550 (m/z).
It in some embodiments of the invention, further include performance evaluation, performance evaluation includes: and to the peak region of acquisition
Data are filtered and with 80% rule process, calculate the RSD value of remaining peak region.80% rule refers to removal all
The frequency of occurrences (nonzero value) is both less than 80% ion in (such as model group and normal group) in group.
Parallel five parts of samples are handled, precision is calculated.By to the parallel of same potato tubers sample
Five analyses, calculate reproducibility.In order to obtain stability, makes even row sample solution 5, it is small to place 0,3,6,12,16,20 respectively
When after analyzed.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, 80% methanol and 10 μ of 800 μ L
L internal standard 2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing
Ultrasound 9.0min under the conditions of 35 DEG C, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges,
It is centrifuged 10min with the revolving speed of 12000rmp, 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL automatic sampling after centrifugation
In bottle, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 2
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, the pure methanol of 800 μ L and 10 μ L
Internal standard 2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing 35
Ultrasound 9.0min under the conditions of DEG C, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges, with
The revolving speed of 12000rmp is centrifuged 10min, and 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL autosampler bottle after centrifugation
In, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 3
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, the water of 800 μ L and 10 μ L internal standards
2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing in 35 DEG C of items
Ultrasound 9.0min under part, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges, with
The revolving speed of 12000rmp is centrifuged 10min, and 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL autosampler bottle after centrifugation
In, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 4
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, 80% methanol and 10 μ of 800 μ L
L internal standard 2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing
Ultrasound 11.0min under the conditions of 25 DEG C, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges,
It is centrifuged 10min with the revolving speed of 12000rmp, 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL automatic sampling after centrifugation
In bottle, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 5
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, the pure methanol of 800 μ L and 10 μ L
Internal standard 2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing 65
Ultrasound 3.0min under the conditions of DEG C, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges, with
The revolving speed of 12000rmp is centrifuged 10min, and 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL autosampler bottle after centrifugation
In, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 6
The present embodiment provides a kind of potato metabolic components sample pre-treatments, comprising:
S1, sample pretreatment: potato cylinder is taken out from entire potato using 65 millimeters of diameter of cork borer
Stem tuber removes the thin slice that 2mm thickness is uniformly cut into after the potato skin at both ends, weight 100.0mg, obtained potato tubers
Sample freezes in liquid nitrogen at once, and is stored in be analyzed in -80 DEG C of refrigerators.
S2, sample extraction: the 100.0mg potato tubers sample that will be stored in refrigerator, the water of 800 μ L and 10 μ L internal standards
2- chlorophenylalanine moves into EP pipe the 30s mixing that is quickly vortexed respectively, is put into supersonic wave cleaning machine after homogenizing in 45 DEG C of items
Ultrasound 7.0min under part, each minute acutely shakes once during ultrasonic extraction, and is placed in 4 DEG C of centrifuges, with
The revolving speed of 12000rmp is centrifuged 10min, and 200 μ L supernatants is taken to be transferred to GC sample introduction bottle i.e. 1.5mL autosampler bottle after centrifugation
In, then lyophilized overnight.
S3, derivatization treatment: take 30 μ L methoxamine hydrochloride pyridine solutions in the potato samples to after being freeze-dried, fastly
Speed is mixed to being completely dissolved, and is placed in 37 DEG C of insulating box 1h, the N of 30 μ L, bis- (trimethyl silicon substrate) trifluoroacetyls of O- are added after taking-up
Amine performs the derivatization processing in 70 DEG C of baking oven 1h, and all potato samples are interior for 24 hours after derivatization treatment to be analyzed, and experiment is done
5 Duplicate Samples.
Embodiment 7
The present embodiment provides a kind of analysis methods of potato metabolic components, comprising:
The 1 μ L potato samples solution provided with Autosampler injection embodiment 1, HP-5ms (30m × 0.25mm ×
0.25 μm) chromatographic column, instrument parameter setting are as follows: 280 DEG C of injector temperature, 230 DEG C of EI ion source temperature, quadrupole rod temperature 150
DEG C, high-purity helium (purity is greater than 99.999%) is used as carrier gas, Splitless injecting samples, 1.0 μ L of sample volume.Temperature program are as follows: initial
80 DEG C of temperature, 2min is maintained, the speed of 10 DEG C/min rises to 320 DEG C, and maintains 6min, and equalized temperature is then carried out at 80 DEG C
Then 6min reinjects sample next time.Mass Spectrometer Method is carried out using full scan mode, Mass Spectrometer Method range is 50-550 (m/
Z), and 5 Duplicate Samples are done.And the peak region data of acquisition are filtered and with 80% rule process, calculate residual peak
It is worth the RSD value in region.
Test example 1
With the mixed extractant that water, methanol, isopropanol, these four single extractants of acetonitrile and water and methanol form, press
The potato metabolic components that a kind of potato metabolic components sample pre-treatments and embodiment 7 provided according to embodiment 1 provide
Analysis method is analyzed, using the sum of isolated peak number mesh and peak area as evaluation index.As shown in Figure 1 specify 4 kinds it is molten
The extraction efficiency of agent and equivalent mixture to peak area and peak number.When using 100% organic solvent, acetonitrile, isopropanol are mentioned
Take efficiency lower than methanol.The extraction effect of different organic solvents is not so good as the extraction effect of methanol, slightly better than other two kinds of solvents
Extraction effect.Since water has very big advantage in terms of extracting amino acid and sugar, certain proportion is added in organic solvent
Water, the sum of peak area and detached peaks number are all had a significant impact.From the point of view of detached peaks number, water-methanol mixed solution effect
Fruit is best, followed by water, methanol, isopropanol and acetonitrile.In conjunction with peak number mesh and total peak area with from the point of view of, water-methanol mixed solution
Effect is best, followed by methanol, isopropanol and acetonitrile.The result shows that water-methanol mixed system can obtain richer generation
Thank to resulting information.
Test example 2
The potato that a kind of potato metabolic components sample pre-treatments and embodiment 7 provided according to embodiment 1 provide
The analysis method of metabolic components, has studied that the ultrasonic extraction time includes 3min, 5min, 7min, 9min, 11min and temperature includes
25 DEG C, 35 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, the influence to ultrasonic extracting method.As can be seen that peak area and separation from Fig. 2 and 3
Peak number is gradually increased with the increase of extraction time and temperature, is declined rapidly with the raising of temperature.On the one hand, when improving extraction
Between and temperature be conducive to improve metabolin mass transfer rate, improve the recovery rate of metabolin.On the other hand, extraction time and temperature
Increase considerably the reduction for resulting in primary metabolite in sample, the interaction between time and temperature is also required to study.
According to experimental result, in order to advanced optimize optimum experimental condition, is designed a model, advanced optimized by orthogonal experiment (OED)
The influence of water, the proportion of methanol and volume, extraction time and temperature.
Test example 3
Orthogonal Experiment and Design, according to the experimental result of single argument method, a kind of potato metabolism group provided according to embodiment 1
The analysis method for the potato metabolic components for dividing sample pre-treatments and embodiment 7 to provide, has carried out orthogonal test (L9 (34)),
Extractant (A) (A1, methanol/water (50:50, v/v), A2, methanol/water (65:35, v/v), A3, methanol/water (80:20, v/v)),
The volume (B) (B1,300 μ L, B2,500 μ L, B3,800 μ L) of extractant, the ultrasonic extraction time (C1,7min, C2,9min, C3,
11min), ultrasonic extraction temperature (D1,35 DEG C, D2,45 DEG C, D3,55 DEG C).The sum of peak area and detached peaks number are shown in Table 1.
The orthogonal result table of table 1
In table 1, Kn is average effect of each factor on different level, and R is range.The experimental results showed that influencing suitable
Sequence is successively the type of extractant, the extraction quantity of extractant, extraction temperature, extraction time.According to experimental result, the kind of extractant
Class, the dosage of extractant, Extracting temperature and ultrasonic extraction time are respectively methanol/water (80:20, v/v), 800 μ L, 35 DEG C,
9min is optimum extraction condition.
Test example 4
The analysis method of the potato metabolic components provided according to embodiment 7 carries out GC-MS analysis to sample solution, obtains
It obtains fingerprint chromatogram and sees Fig. 4, as can be seen from the figure isolate and have detected 70 kinds of potato tubers metabolites, and separating effect is good
Alright, baseline stability.The potato tubers figure of different cultivars is similar, there is slight difference, and the analysis result that embodiment 7 provides exists
It is compared and analyzed with NIST standard spectrum library, it is determined that contained by the specific structure of metabolite and every kind of kind potato
Metabolite, experimental result are shown in Table 2.
Metabolite in 2 different cultivars potato tubers of table
In table 2, Unkonwn " indicates that the matching degree of material and database is very low."+" indicates that the compound has been detected
It measures."-" shows that compound is not detected.Five classes, i.e. organic acid, fatty acid, sugar and its derivative are classified as according to result
Object, amino acid and intermediate product.Important member's succinic acid, gluconic acid, malic acid, ribonic acid, ascorbic acid, the wood of organic acid
Saccharic acid and arabic acid etc.；Fatty acid includes pentadecanoic acid, palmitinic acid, octadecadienoic acid, linoleic acid etc.；The sugar detected
And its derivative includes fructose, sucrose, maltose, lactose, ribose, galactolipin, mannose, cellobiose, furanose, sorb
Alcohol, lysol pyranose, lysol furanose, galactoside, mannose, isopropyl sugar etc.；In potato tubers, amino acid includes sweet
Propylhomoserin, leucine, serine, alanine, phenylalanine and aspartic acid etc.；Last one kind product includes inositol, loose sugar alcohol, gallbladder
Sterol, butyraldehyde, glucuronic acid butyraldehyde lactone, uridine, five pyranosides, glucoside, galactitol, gluconolactone, beans
Sterol, tocopherol and hybar X etc..Wherein four kinds of metabolins are not yet parsed into specific structure.
Test example 5
The peak region data of acquisition are filtered and with 80% rule process, calculate the RSD of remaining peak region
Value.
Parallel five parts of samples of the potato samples provided by embodiment 1 are handled, and precision is calculated.Such as Fig. 5
Shown, the RSD value of the accumulation peak area of the peak number mesh and 95.8% metabolin of 82.7% metabolin is below 15%.As a result table
Bright, measuring most of metabolins with current method has good measurement accuracy.
Parallel five analyses of the potato samples provided by embodiment 1 to same potato tubers sample, meter are provided
Calculate reproducibility.Repeated result is shown in Fig. 6, the RSD of the accumulation peak area of the peak number mesh and 91.4% metabolin of 90.4% metabolin
Value is below 30%.The result shows that this method is reliable and stable, favorable reproducibility meets the requirement of biological sample analysis.
The stability of metabolite in potato tubers is evaluated: the potato Duplicate Samples that Example 1 provides
It product solution 5, is analyzed after placing 0,3,6,12,16,20 hour respectively, as a result sees Fig. 7.In 0-24h, metabolism is calculated
The relative standard deviation (RSD) of peak areas.The result shows that there is the peak number mesh of 76.7% metabolin in total metabolism product
The RSD value of the accumulation peak area of 90.5% metabolin is below 30%, stability tolerance interval.
In conclusion the potato metabolic components sample pre-treatments and its analysis method of the embodiment of the present invention, using derivative
The GC-MS technology of change analyzes the metabolite in the different cultivars potato of Northeast Area of China.It is separated with GC-MS
The peak number mesh and total peak area identified are index, extract metabolism using single_factor method and orthogonal experimental design method optimizing research and produce
The factor of object, determines extractant: methanol/water (80:20, v/v)；Solvent quantity: 800 μ L；Extraction temperature: 35 DEG C；When extraction
Between: 9min, 65 kinds of metabolites of separation identification under optimal condition, such as sugar, organic acid, fatty acid, amino acid.It is each in sample
Component separation is good, and baseline stability, this method has preferable precision, reproducibility and stability.This method is to Chinese east
Northern potato metabolin polar component separation identification be have it is far-reaching.This method can be generalized to other plants of analysis
Object sample, to characterize a kind of metabolism status of plant in terms of environment, development or inherent cause.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of potato metabolic components sample pre-treatments, which is characterized in that including extracting to potato samples, extract packet
It includes: the potato samples is mixed with extractant and internal standard substance, then carry out ultrasonic extraction and extraction mixed liquor is made；
The extractant is water and/or organic solvent, and the organic solvent is at least one of methanol, isopropanol and acetonitrile.
2. potato metabolic components sample pre-treatments according to claim 1, which is characterized in that the organic solvent is first
Alcohol；Optionally, when the extractant is water and the organic solvent, methanol percentage by volume is 50-80%.
3. potato metabolic components sample pre-treatments according to claim 1, which is characterized in that the wherein institute of every 100mg
Potato samples are stated, are mixed using internal standard substance described in extractant described in 300-800 μ L and 5-15 μ L.
4. potato metabolic components sample pre-treatments according to claim 1, which is characterized in that the temperature of ultrasonic extraction is
25-65 DEG C, time 3-11min.
5. potato metabolic components sample pre-treatments according to claim 1, which is characterized in that extract further include: by institute
Extraction mixed liquor is stated to carry out centrifugal treating at 0-10 DEG C and obtain centrifugal mixture, centrifugation time 5-15min, centrifugal rotational speed
For 10000-15000rmp, then 180-250 μ L supernatant after the centrifugation is freeze-dried.
6. potato metabolic components sample pre-treatments according to claim 1-5, which is characterized in that extract it
It afterwards further include that the potato samples that extraction obtains are performed the derivatization with processing, derivatization treatment includes: derivative using first
Change reagent to the potato samples carry out oximate processing, by after oximate the potato samples and the second derivatization reagent into
Row Silanization reaction；
Optionally, it is every extract 100mg the potato samples slice, using 20-40 μ L first derivatization reagent into
Row oximate and second derivatization reagent progress Silanization reaction for using 20-40 μ L；And/or first derivatization
Reagent is methoxamine hydrochloride pyridine solution, and second derivatization reagent is N, bis- (trimethyl silicon substrate) trifluoroacetamides of O-.
7. potato metabolic components sample pre-treatments according to claim 6, which is characterized in that oximate processing temperature be
35-40 DEG C, time 0.8-1.2h；
And/or the temperature of Silanization reaction is 65-75 DEG C, time 0.8-1.2h.
8. potato metabolic components sample pre-treatments according to claim 1, which is characterized in that further include sample before extraction
Pretreatment, sample pretreatment include: that the potato samples are sliced to every piece of diameter is 50-80mm, with a thickness of 1-3mm,
Weight is 80-120mg, is subsequently placed in liquid nitrogen and freezes, and is stored under the conditions of -100--70 DEG C.
9. a kind of analysis method of potato metabolic components characterized by comprising claim 1-8 is described in any item
Potato metabolic components sample made from potato metabolic components sample pre-treatments carries out GC-MS analysis.
10. the analysis method of potato metabolic components according to claim 9, which is characterized in that selected in GC-MS analysis
With HP-5ms chromatographic column, parameter setting are as follows: 250-300 DEG C of injector temperature, 200-250 DEG C of EI ion source temperature, quadrupole rod
120-180 DEG C of temperature, carrier gas is high-purity helium, and Splitless injecting samples, sample volume is 0.8-1.2 μ L；
Using temperature programming, temperature program are as follows: initial temperature is 60-100 DEG C, keeps 1-3min；It is risen to 5-15 DEG C/min
300-350 DEG C, keep 4-10min；Balance 4-10min is kept under initial temperature, then reinjects sample next time；It carries
Gas velocity is 0.8-1.5mL/min；Mass Spectrometer Method is carried out using full scan mode, Mass Spectrometer Method range is 50-550 (m/z).
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