CN102565233A - Method for determining volatile and semi-volatile secondary metabolite in fresh tobacco leaves - Google Patents
Method for determining volatile and semi-volatile secondary metabolite in fresh tobacco leaves Download PDFInfo
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Abstract
A method for determining volatile and semi-volatile secondary metabolite in fresh tobacco leaves is characterized by comprising the steps of rapidly collecting the fresh tobacco leaves and quickly freezing the fresh tobacco leaves on site by liquid nitrogen; after removing water through a freeze drying method, smashing the tobacco leaves; ultrasonically extracting the secondary metabolite from tobacco powder by a methanol-chloroform mixed solvent; suctioning supernatant after centrifugation and conducting suspension steaming or centrifugal concentration at low temperature; and directly analyzing the concentrated sample solution by GC-MS (Gas Chromatography-Mass Spectrometer). The method for determining the volatile and semi-volatile secondary metabolite in the fresh tobacco leaves has an obvious advantage that a method applicable for extracting the volatile and semi-volatile secondary metabolite from the fresh tobacco leaves is developed. The method for determining the volatile and semi-volatile secondary metabolite in the fresh tobacco leaves is simple and efficient, has excellent reproducibility and can make up for the defect that the classical derivatization GC-MS can not detect the volatile and semi-volatile secondary metabolite.
Description
Technical field
The present invention relates to a kind of volatilization, volatilize method of secondary metabolites partly of detecting in the new fresh tobacco leaf, this method is simple to operate, efficient, degree of accuracy is high, and what can remedy classical derivatization GC-MS can't detect volatilization, the volatilize deficiency of secondary metabolites partly.
Background technology
Tobacco is as important mode crop and industrial crops, and people have had a lot of understandings to its chemical composition.But great majority research only limits to the understanding to tobacco leaf after modulating, and people comprise that for new fresh tobacco leaf the chemical composition of different growth periods, different parts tobacco leaf is understood still very limited.Secondary metabolites mainly is meant molecular weight at the micromolecule metabolin below 1000, conducts with plant matrix resistance, signal, grows, phenomenon such as pattern of plant, fragrance is relevant.Therefore, the metabolin that understand different times, is in the tobacco leaf of different growth periods forms and content itself is significant, and is the chemical platform of carrying out genomics, proteomics, transcription group research.
Although begun to carry out research work both at home and abroad in recent years about the tobacco leaf secondary metabolites, limited amount, and mostly based on modulation back tobacco leaf.At present; For tobacco leaf and this detection method of giving birth to metabolin of other model plant is main with derivatization GC-MS method still; This is because its sensing range can contain most of secondary metabolites, and the GC-MS method can carry out library searching, is convenient to secondary metabolites is carried out qualitative analysis.Therefore yet derivatization GC-MS need carry out bone dry to extract, can cause volatilization, partly the secondary metabolites that volatilizees loses fully, influences test result.Fail to contain volatilization, the secondary metabolites that volatilizees partly in view of derivatization GC-MS sensing range, set up and a kind ofly measure volatilization in the new fresh tobacco leaf, partly the volatilize GC-MS detection method of secondary metabolites is that ten minutes is necessary.And, do not have relevant report at present according to investigation.
Summary of the invention
The object of the invention provides a kind of volatilization, volatilize method of secondary metabolites partly of detecting simply, efficiently in the new fresh tobacco leaf just to above-mentioned prior art situation.
The objective of the invention is to realize through following technical scheme:
A kind ofly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly; Gather new fresh tobacco leaf fast and carry out quick-frozen with the liquid nitrogen scene; After adopting freeze-drying method to remove moisture tobacco leaf is pulverized, with secondary metabolites in methyl alcohol-chloroform mixed solvent ultrasonic Extraction tobacco powder, centrifugal after; Draw supernatant concentration, the sample solution after concentrating directly adopts GC-MS to analyze.
Concrete steps are following:
(1) collection of tobacco leaf, processing, drying, pulverizing: pluck cigarette strain appointed part tobacco leaf fast, freezing rapidly with the termination life process with liquid nitrogen, freeze drying 24h is to remove moisture content in leaves, and the dry tobacco leaf powder of crossing is broken to the 40-60 order, is put in-20 ℃ of refrigerators and preserves;
(2) extract secondary metabolites: the tobacco powder of weighing 50-250mg; Adding inner mark solution, 6-8ml methyl alcohol-chloroform extraction solvent carry out ultrasonic Extraction; Extract is got the 4-6ml supernatant and is concentrated behind high speed centrifugation, and concentrate is directly analyzed with GC-MS behind 0.45 μ m filtering with microporous membrane;
(3) detect: adopt GC-MS to carry out check and analysis, chromatographic condition is following:
Chromatographic column: DB-35ms (30m * 0.25mm * 0.25 μ m); Carrier gas, He; Flow velocity: 1.0ml/min; Injector temperature: 290 ℃; Sample size: 1 μ L; Split ratio is 5:1; Heating schedule:
60 ℃ keeps 4min; Programming rate with 5 ℃/min is raised to 280 ℃ then; Keep 10 min
MS parameter: adopt the full scan pattern; Acquisition quality is counted scope: 40-350.
Among the present invention; In the processing procedure of step (1), want rapid place to go tobacco leaf master pulse, wrap up blade-section, put into liquid nitrogen behind the mark and stop the tobacco leaf life process with masking foil; And be put in-80 ℃ of freezing preservations of low temperature refrigerator; Be taken out to from refrigerator before freeze drying begins at tobacco leaf, must use the dry ice embedding, shift.
Said extraction solvent is the methyl alcohol-chloroformic solution of 3: 2 or 4: 1, and addition is 6-8ml, and the extraction time is 20-40min.
Inner mark solution is to fit over deuterium in the extraction solvent for nicotine solution, and concentration is 200 μ g/ml, and addition is 100 μ l.
Concentrated mode in the step (2) adopts to revolve steams or traditional vacuum concentrates.
When adopting GC-MS to carry out check and analysis, the blank pipe kapillary that connects the long 0.25mm internal diameter of 1.0m before the used chromatographic column is as pre-column, and pre-column needs periodic replacement weekly.
Outstanding advantage of the present invention is to have developed to be applicable in the new fresh tobacco leaf volatilization, the volatilize method for distilling of secondary metabolites partly.And method is simple, effectively and favorable reproducibility, what can remedy classical derivatization GC-MS can't detect volatilization, the volatilize deficiency of secondary metabolites partly.
Description of drawings
Fig. 1 is a determination step process flow diagram of the present invention.
Fig. 2 is a gained GC-MS full scan chromatogram of the present invention.
Embodiment
The present invention further describes below in conjunction with embodiment, but does not limit the present invention.
Embodiment 1:
Gather the 6 prosperous long-term new fresh tobacco leafs (kind is the big gold dollar of safflower) in laboratory, remove trunk fast and, use liquid nitrogen flash freezer with the masking foil parcel.Take out the tinfoil paper paper bag from liquid nitrogen, clap brokenly gently,, and be milled to the 40-60 order immediately with the freezing 24h taking-up of freeze drier moisture.Take by weighing 200mg tobacco powder in the 10ml centrifuge tube, add 100 μ l deuteriums for the nicotine inner mark solution, methyl alcohol-chloroform of 5.9ml (3:2) solution, ultrasonic Extraction 30min, centrifugal 10min under the 10000r/min.Draw the 4.0ml supernatant in the 5ml centrifuge tube, 35 ℃ are concentrated to about the 1.0ml volume with the traditional vacuum concentrating instrument down.Sample solution after concentrating is directly analyzed with GC-MS behind 0.45 μ m filtering with microporous membrane.
Chromatographic condition is following:
Chromatographic column: DB-35ms (30m * 0.25mm * 0.25 μ m); Carrier gas, He; Flow velocity: 1.0ml/min; Injector temperature: 240 ℃; Sample size: 1 μ L; Split ratio is 5:1; Heating schedule:
60 ℃ keeps 4min; Programming rate with 5 ℃/min is raised to 280 ℃ then, keeps 10 min.
MS parameter: adopt the full scan pattern; Acquisition quality is counted scope: 40-350.
Gained GC-MS full scan chromatogram is seen Fig. 2.
Embodiment 2:
Gathering the zunyi, guizhou zone variety is 3 maturity stage tobacco leaves of NC297, removes trunk fast and with the masking foil parcel, uses liquid nitrogen flash freezer.The tinfoil paper paper bag is taken out from liquid nitrogen, be embedded in the dry ice, air express is put in-80 ℃ of refrigerators to the laboratory.From refrigerator, take out the tinfoil paper paper bag, clap brokenly gently, with the freezing 24h removal of freeze drier moisture, be milled to the 40-60 order immediately.Take by weighing 50mg, 100mg, 150mg, 200mg, 250mg tobacco powder respectively in the 10ml centrifuge tube; Add 100 μ l deuteriums for the nicotine inner mark solution; 5.9 methyl alcohol-chloroform of ml (3:2) solution, ultrasonic Extraction 30min, centrifugal 10min under the 10000r/min; Get the 4.0ml supernatant in other 5ml centrifuge tube, 35
oC is concentrated to about the 1.0ml volume with the traditional vacuum concentrating instrument down.Sample solution after concentrating is directly analyzed with GC-MS behind 0.45 μ m filtering with microporous membrane.
Chromatographic condition such as embodiment 1.
Choosing representative volatilization, the target analytes that volatilizees partly, calculate chromatographic peak area, is the y axle with " peak area/internal standard area ", takes by weighing tobacco powder weight and carries out linear fit for the x axle, and fitting coefficient is listed in the table 1.Fitting coefficient proves that this method is to volatilization, partly the secondary metabolites that volatilizees has good quantitative performance all on 0.999.
Table 1
Claims (7)
1. one kind is used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly; It is characterized in that: gather new fresh tobacco leaf fast and carry out quick-frozen with the liquid nitrogen scene; After adopting freeze-drying method to remove moisture tobacco leaf is pulverized, with secondary metabolites in methyl alcohol-chloroform mixed solvent ultrasonic Extraction tobacco powder, centrifugal after; Draw outstanding down steaming of supernatant low temperature or centrifugal concentrating, the sample solution after concentrating directly adopts GC-MS to analyze.
2. according to claim 1ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly, it is characterized in that: concrete steps are following:
(1) collection of tobacco leaf, processing, drying, pulverizing: pluck cigarette strain appointed part tobacco leaf fast, freezing rapidly with the termination life process with liquid nitrogen, freeze drying 24h is to remove moisture content in leaves, and the dry tobacco leaf powder of crossing is broken to the 40-60 order, is put in-20 ℃ of refrigerators and preserves;
(2) extract secondary metabolites: the tobacco powder of weighing 50-250mg; Adding inner mark solution, 6-8ml methyl alcohol-chloroform extraction solvent carry out ultrasonic Extraction; Extract is got the 4-6ml supernatant and is concentrated behind high speed centrifugation, and concentrate is directly analyzed with GC-MS behind 0.45 μ m filtering with microporous membrane;
(3) detect: adopt GC-MS to carry out check and analysis, chromatographic condition is following:
Chromatographic column: DB-35ms (30m * 0.25mm * 0.25 μ m); Carrier gas, He; Flow velocity: 1.0ml/min; Injector temperature: 290 ℃; Sample size: 1 μ L; Split ratio is 5:1; Heating schedule:
60 ℃ keeps 4min; Programming rate with 5 ℃/min is raised to 280 ℃ then; Keep 10 min
MS parameter: adopt the full scan pattern; Acquisition quality is counted scope: 40-350.
3. according to claim 2ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly; It is characterized in that: in the processing procedure of step (1), want rapid place to go tobacco leaf master pulse, wrap up blade-section, put into liquid nitrogen behind the mark and stop the tobacco leaf life process with masking foil; And be put in-80 ℃ of freezing preservations of low temperature refrigerator; Be taken out to from refrigerator before freeze drying begins at tobacco leaf, must use the dry ice embedding, shift.
4. according to claim 2ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly, it is characterized in that: extraction solvent is the methyl alcohol-chloroformic solution of 3: 2 or 4: 1, and addition is 6-8ml, and the extraction time is 20-40min.
5. according to claim 2ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly, it is characterized in that: inner mark solution is to fit over deuterium in the extraction solvent for nicotine solution, and concentration is 200 μ g/ml, and addition is 100 μ l.
6. according to claim 2ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly, it is characterized in that: the concentrated mode in the step (2) adopts to revolve steams or traditional vacuum concentrates.
7. according to claim 2ly be used for measuring new fresh tobacco leaf volatilization, the volatilize method of secondary metabolites partly, it is characterized in that: when adopting GC-MS to carry out check and analysis, the blank pipe kapillary that connects the long 0.25mm internal diameter of 1.0m before the used chromatographic column is as pre-column.
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| CN104101662A (en) * | 2014-08-01 | 2014-10-15 | 中国烟草总公司郑州烟草研究院 | Metabonomics method for testing terpenoids in fresh tobacco leaves |
| CN104266871A (en) * | 2014-09-19 | 2015-01-07 | 国家烟草质量监督检验中心 | Linear type smoking machine collecting device with inner chamber and analytical test method of collecting device |
| EP2844634A4 (en) * | 2012-05-03 | 2015-10-21 | Dsm Ip Assets Bv | New intermediate compound for preparing vitamin b6 |
| CN105289773A (en) * | 2015-09-25 | 2016-02-03 | 中国农业科学院茶叶研究所 | Sample pretreatment method for plant metabolome analysis |
| CN106018626A (en) * | 2016-07-29 | 2016-10-12 | 云南省烟草农业科学研究院 | Tobacco stigma metabonomic analysis method based on gas chromatography-mass spectrometry |
| CN106018654A (en) * | 2016-07-29 | 2016-10-12 | 云南省烟草农业科学研究院 | Tobacco column metabonomics analysis method based on gas chromatographic mass spectrometry |
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| CN106248836A (en) * | 2016-09-30 | 2016-12-21 | 中国烟草总公司郑州烟草研究院 | The method of discrimination of fresh tobacco leaves sample quality in a kind of Nicotiana tabacum L. metabolism group based on Volatile Metabolites |
| CN106290616A (en) * | 2016-07-29 | 2017-01-04 | 云南省烟草农业科学研究院 | A kind of tobacco petal metabonomic analysis methods based on gaschromatographic mass spectrometry |
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| CN107497412A (en) * | 2017-08-14 | 2017-12-22 | 中国烟草总公司郑州烟草研究院 | A kind of adsorbent and its application for being used to reduce heavy metal ions from tobacco extract content |
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| CN109696503A (en) * | 2019-02-26 | 2019-04-30 | 中国农业科学院作物科学研究所 | The detection method and its application of metabolin in a kind of plant leaf blade |
| CN112362771A (en) * | 2020-10-29 | 2021-02-12 | 上海鹿明生物科技有限公司 | Method for high-throughput analysis of plant secondary metabolites based on LCMS and application |
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