CN106353429B - The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on organic acid - Google Patents
The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on organic acid Download PDFInfo
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- CN106353429B CN106353429B CN201610868645.6A CN201610868645A CN106353429B CN 106353429 B CN106353429 B CN 106353429B CN 201610868645 A CN201610868645 A CN 201610868645A CN 106353429 B CN106353429 B CN 106353429B
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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
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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
- G01N30/04—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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The method of discrimination of fresh tobacco leaves sample quality in the tobacco metabolism group based on organic acid that the present invention provides a kind of, this method is using heptadecanoic acid in fresh tobacco leaves sample and the ratio of butyric acid content as index, selection for metabolism group qualified samples, which provides foundation, to be differentiated to metabolism group fresh tobacco leaves sample quality.The present invention is accurate and reliable, simple possible, can offer reference for the analysis of tobacco metabolic characteristics and related tobacco metabolism group research.
Description
Technical field
The present invention relates to analytical chemistry field, particularly relate to fresh used in a kind of differentiation tobacco metabolism group research
Whether tobacco sample quality meets the method for discrimination of metabolism group research needs.
Background technique
Plant Metabolome is the variation or its change at any time by investigating metabolite after plant is stimulated or disturbance
Change, to study a kind of technology of plant.In the process of metabolism group edge analysis, the acquisition of typical sample be it is primary and
Step of crucial importance obtains qualified samples, is that metabonomic analysis carries out with the metabolism state of the reflection sample of objective
Basis and premise.The acquisition of fresh tobacco leaves sample needs to be quenched at low temperature to stop its physiological activity to guarantee its stopping
Metabolism state in sampling, standing procedure are after plucking a plant sample, and tinfoil package is placed in -196 DEG C of liquid nitrogen frozens, is used for
Dry powder sample is analyzed after fresh tobacco leaves sample analysis or freeze-drying.It picks in sample to this section of process before analysis, needs to guarantee
Fresh tobacco leaves sample is saved in liquid nitrogen and is ground under the conditions of liquid nitrogen, otherwise will cause the enzymatic browning of tobacco leaf.
Kinds of organic acids in tobacco is various, and content difference is big, plays an important role during tobacco growing, and
And there is important influence to the quality of tobacco and cigarette.Wherein volatile organic acids is additive important in tobacco, it can
To increase cigarette perfume, improves and inhale taste, keep pleasant impression pure and mild and comfortable.Such as C4~C6 fatty acid can generate fruity, creamy taste, in flue gas
It plays an important role in fragrant, jealous.And partly volatilize, non-volatile organic acids can be combined into salt with alkaloid, adjust protonation and
Free state nicotine ratio, to greatly influence the strength of flue gas and jealous, some are also important the normal of tobacco essence fragrance
It can be adjusted with ingredient, such as malic acid, citric acid, oxalic acid although they directly act on the fragrance of flue gas without apparent
The pH value for saving tobacco makes to inhale taste improvement, becomes pure and mild, balanced action is played in flue gas.It is such in view of organic acids on tobacco quality
The report of important role, the methods of extraction, detection in recent years in relation to organic acid in tobacco emerges one after another, but someone did not mentioned
Out by fresh tobacco leaves organic acid content or content ratio be used for the differentiation of metabolism group fresh tobacco leaves sample quality.
Summary of the invention
The purpose of the present invention is to provide fresh tobacco leaves sample matter in a kind of tobacco metabolism group based on organic acid
The method of discrimination of amount.This method is using heptadecanoic acid in fresh tobacco leaves sample and the ratio of butyric acid content in metabolism group research
Fresh tobacco leaves sample quality is differentiated that the method is easy to operate quickly, result is accurate and reliable.
The purpose of the present invention is realized by following technique measures：
The method of discrimination of fresh tobacco leaves sample quality is to pass through inspection in tobacco metabolism group based on organic acid of the invention
The content for surveying heptadecanoic acid and butyric acid in fresh tobacco leaves, calculates its ratio, to the fresh tobacco leaves sample quality in metabolism group research
Differentiated；The specific method is as follows：
1）Fresh tobacco leaves after weighing 20 ~ 25mg freeze grinding are placed in screw socket pressure tube, and methylene chloride/acetonitrile is added
Volume ratio is 1:2 mixed solution 1mL is sealed and is placed in supersonic generator ultrasonic extraction 30min at room temperature, is stood
Supernatant is taken, 0.45 μm of filter membrane is crossed, BSTFA 100 μ L, 60 DEG C of derivative 40min are added, is analyzed into GC/MS, detection organic acid contains
Amount；
2）The gas chromatograph parameters of GC/MS analysis method：Chromatographic column：DB-5MS(50 m×0.25 mm i.d.×0.25
μm d.f. )；Temperature programming：280 DEG C are risen to from 50 DEG C with the speed of 4 DEG C/min（20 min）；Carrier gas：Helium；Column flow rate：
1.0 mL/min；Injector temperature：280℃；2 μ L of sample volume, split sampling, split ratio 10:1；
3）The mass spectrometry parameters of GC/MS analysis method：Solvent delay：4 min；Ionization voltage：70 ev；Ion source temperature：
230 ℃；Transmission line temperature：280 ℃；Scanning mode：Choice ion pattern（SIM）, select ion as follows：Formic acid（103）, second
Acid（117）, butyric acid（145）, lactic acid（147）, caproic acid（173）, trans- 2- hexenoic acid（IS）（171）, enanthic acid（187）, octanoic acid
（201）, succinic acid（147）, n-nonanoic acid（215）, malic acid（147）, citric acid（147）, lauric acid/dodecanoic acid（257）, tetradecylic acid（285）,
Pentadecanoic acid（299）, hexadecylic acid（313）, heptadecanoic acid（327）, linoleic acid（337）, linolenic acid（335）, oleic acid（339）, stearic acid
（341）.
Heretofore described fresh tobacco leaves are to save transport through -196 DEG C of liquid nitrogen containers, after liquid nitrogen frozen grinds and is lyophilized, -
80 DEG C of refrigerators save stand-by offal；The trans- 2- hexenoic acid of containing the internal standard in the mixed solution, concentration are 0.303 μ g/mL..
Heretofore described its ratio of calculating is to establish using heptadecanoic acid and the ratio of butyric acid content as index and distinguish metabolism
Group learns the standard of normal specimens and browning sample, i.e., heptadecanoic acid and the ratio of butyric acid content are below for conjunction 9 in sample to be tested
Lattice sample, can be used for tobacco metabonomic analysis, and the ratio of heptadecanoic acid and butyric acid content is rotten unqualified sample 9 or more
Product are not useable for tobacco metabonomic analysis.
The present invention passes through the analysis and research of a large amount of fresh tobacco leaves samples, finds fresh tobacco leaves sample in case of enzymatic browning
Deng reaction, the content of organic acid can be varied widely, and the ratio of heptadecanoic acid and butyric acid content is significantly raised, this sample will not
It can be used for metabonomic analysis, therefore using heptadecanoic acid and the ratio of butyric acid content as a standard in metabolism group research
Fresh tobacco leaves sample quality is differentiated.
Beneficial effects of the present invention are as follows：
The correlation of the ratio and sample quality of heptadecanoic acid and butyric acid content in fresh tobacco leaves sample is utilized for the present invention,
Metabolism group fresh tobacco leaves sample quality is differentiated, so that the invention has the characteristics that：1）It is easy to operate quick；2）Knot
Fruit is accurate and reliable；3）Amount of samples is few, can offer reference for the analysis of tobacco metabolic characteristics and related tobacco metabolism group research.
Specific embodiment
The present invention is further described below with reference to embodiment.
Embodiment 1
Using the organic acid content in GC-MS method measurement fresh tobacco leaves sample, the heptadecanoic acid and fourth of browning sample are found
Acid content ratio is significantly raised.
Sample to be tested includes the fresh cigarette of 6 kinds in the provinces such as Fujian, Guizhou, Hunan, Henan, Yunnan, 19 places of production
111 parts of leaf sample（Each point has 5-6 biology to repeat）.Including 87 parts of qualified samples（Sample in acquisition, transport and pre-
- 80 DEG C are strict controlled in treatment process）, 24 parts of brown stain samples（It is complete due to liquid nitrogen deficiency etc. in storage and transport process
Brown stain）.
Sample analysis step is as described below：
Its specific method is：1）The fresh tobacco leaves sample picked by crop field saves transport in -196 DEG C of liquid nitrogen containers, and liquid nitrogen is cold
Freeze grinding, low temperature freeze-drying, -80 DEG C of refrigerators are saved to subsequent carry out metabonomic analysis；Cigarette after weighing 20 ~ 25mg freeze grinding
End is placed in 4mL screw socket pressure tube, and it is 1 that methylene chloride/acetonitrile volume ratio, which is added,:2 mixed solution 1mL（The trans- 2- of containing the internal standard
The concentration of hexenoic acid is 0.303 μ g/mL）, it seals and is placed in supersonic generator 30 min of ultrasonic extraction at room temperature, it is quiet
It sets and takes supernatant, cross 0.45 μm of filter membrane, BSTFA 100 μ L, 60 DEG C of derivative 40min are added, analyzed into GC/MS, detect organic acid
Content.
2) gas chromatograph parameters of GC/MS analysis method：Chromatographic column：DB-5MS(50 m×0.25 mm i.d.×0.25
μm d.f. )；Temperature programming：280 DEG C are risen to from 50 DEG C with the speed of 4 DEG C/min（20 min）；Carrier gas：Helium；Column flow rate：
1.0 mL/min；Injector temperature：280 ℃；2 μ L of sample volume, split sampling, split ratio 10:1.
3) mass spectrometry parameters of GC/MS analysis method：Solvent delay：4 min；Ionization voltage：70 ev；Ion source temperature：
230 ℃；Transmission line temperature：280 ℃；Scanning mode：Choice ion pattern（SIM）, select ion as follows：Formic acid（103）, second
Acid（117）, butyric acid（145）, lactic acid（147）, caproic acid（173）, trans- 2- hexenoic acid（IS）（171）, enanthic acid（187）, octanoic acid
（201）, succinic acid（147）, n-nonanoic acid（215）, malic acid（147）, citric acid（147）, lauric acid/dodecanoic acid（257）, tetradecylic acid（285）,
Pentadecanoic acid（299）, hexadecylic acid（313）, heptadecanoic acid（327）, linoleic acid（337）, linolenic acid（335）, oleic acid（339）, stearic acid
（341）.
The standard curve of heptadecanoic acid and butyric acid is depicted, with object peak area and internal standard compound peak area（Trans- 2- hexene
Acid）Ratio be ordinate Y, mass concentration X（μg/mL）For abscissa, its standard curve and related coefficient, measurement result are calculated
It is as follows：Y（Heptadecanoic acid）=0.1386x+0.0038, Y（Butyric acid）=1.446x+0.1716, related coefficient are respectively 0.9948
With 0.9999.
The ratio of tobacco sample information and heptadecanoic acid and butyric acid content in 1 embodiment 1 of table
| Sample number into spectrum | The place of production | Kind | Repeat number | Heptadecanoic acid/butyric acid | It is whether qualified |
| C23Y | Guizhou Bijie | Cloud 97 | 6 | 9.3-12.25 | It is unqualified |
| C01K | Kunming, Yunnan | K326 | 5 | 2.8-5.83 | It is qualified |
| C02K | Yunnan Yuxi | K326 | 6 | 4.37-6.98 | It is qualified |
| C03C | Qujing of Yunnan | Cloud 87 | 6 | 4.37-7 | It is qualified |
| C04Y | Honghe, Yunnan | Cloud 97 | 6 | 4.2-8.33 | It is qualified |
| C05Y | Yunnan Pu'er | Cloud 87 | 6 | 4.2-5.83 | It is qualified |
| C06Y | Dali | Cloud 87 | 5 | 5.6-7 | It is qualified |
| C09Y | Southwest Guizhou | Cloud 97 | 6 | 3.5-7 | It is qualified |
| C12C | Henan table mountain | Zhongyan-100 | 6 | 6.13-8.17 | It is qualified |
| C13C | Nanyang, henan | Zhongyan-100 | 5 | 5.8-7 | It is qualified |
| C14C | Henan Zhumadian | Zhongyan-100 | 6 | 5.83-8.17 | It is qualified |
| C15K | Guangdong Shaoguan | K326 | 6 | 4.2-8.16 | It is qualified |
| C17K | Chenzhou, Hunan Province | K326 | 6 | 6.13-7.87 | It is qualified |
| C25N | Tongren district Guizhou Province | Southern river 3 | 6 | 3.18-7 | It is qualified |
| C26Y | Enshi | Cloud 87 | 6 | 6.13-7 | It is qualified |
| GK10 | Zunyi, guizhou | K326 | 6 | 3.5-7 | It is qualified |
| C07C | Fujian Longyan | Cloud 87 | 6 | 10.5-12.25 | It is unqualified |
| C24B | Guizhou the south of Guizhou Province | Your cigarette 2 | 6 | 9.33-14 | It is unqualified |
| C29K | Hunan Zhangjiajie | K326 | 6 | 9.33-10.5 | It is unqualified |
By testing above, inventor has found serious qualitative change（Browning）The ratio of heptadecanoic acid and butyric acid content in sample is aobvious
It writes and increases.The present invention is using heptadecanoic acid and the ratio of butyric acid content as the unqualified sample distinguishing metabolism group normal specimens and going bad
The standard of product.I.e. in sample to be tested the ratio of heptadecanoic acid and butyric acid content 9 it is below be qualified samples, can be used for tobacco metabolism
The ratio of group credit analysis, heptadecanoic acid and butyric acid content is rotten failed test sample 9 or more, is not useable for metabolism group point
Analysis.
Claims (3)
1. the method for discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on organic acid, it is characterised in that：It is logical
The content for crossing heptadecanoic acid and butyric acid in detection fresh tobacco leaves, calculates its ratio, to the fresh tobacco leaves sample in metabolism group research
Quality is differentiated；Described its ratio of calculating is to establish using heptadecanoic acid and the ratio of butyric acid content as index and distinguish metabolism group
The standard of normal specimens and browning sample, i.e., heptadecanoic acid and the ratio of butyric acid content are below for qualified sample 9 in sample to be tested
Product, can be used for tobacco metabonomic analysis, and the ratio of heptadecanoic acid and butyric acid content is rotten failed test sample 9 or more,
It is not useable for tobacco metabonomic analysis；The specific method is as follows：
1）Fresh tobacco leaves after weighing 20 ~ 25mg freeze grinding are placed in screw socket pressure tube, and methylene chloride/acetonitrile volume is added
Than being 1:2 mixed solution 1mL is sealed and is placed in supersonic generator ultrasonic extraction 30min at room temperature, and standing takes
The stillness of night crosses 0.45 μm of filter membrane, and BSTFA 100 μ L, 60 DEG C of derivative 40min are added, and analyzes into GC/MS, detects organic acid content；
2）The gas chromatograph parameters of GC/MS analysis method：Chromatographic column：DB-5MS, specification are：50 m×0.25 mm i.d.×
0.25 μm d.f. ；Temperature programming：280 DEG C are risen to from 50 DEG C with the speed of 4 DEG C/min, keep 20 min；Carrier gas：Helium；Column
Flow velocity：1.0 mL/min；Injector temperature：280℃；2 μ L of sample volume, split sampling, split ratio 10:1；
3）The mass spectrometry parameters of GC/MS analysis method：Solvent delay：4 min；Ionization voltage：70 ev；Ion source temperature：230
℃；Transmission line temperature：280 ℃；Scanning mode：Choice ion pattern（SIM）, select ion as follows：Formic acid（103）, acetic acid
（117）, butyric acid（145）, lactic acid（147）, caproic acid（173）, trans- 2- hexenoic acid（IS）（171）, enanthic acid（187）, octanoic acid（201）,
Succinic acid（147）, n-nonanoic acid（215）, malic acid（147）, citric acid（147）, lauric acid/dodecanoic acid（257）, tetradecylic acid（285）, pentadecanoic acid
（299）, hexadecylic acid（313）, heptadecanoic acid（327）, linoleic acid（337）, linolenic acid（335）, oleic acid（339）, stearic acid（341）.
2. method of discrimination according to claim 1, it is characterised in that：The fresh tobacco leaves are to save fortune through -196 DEG C of liquid nitrogen containers
It is defeated, after liquid nitrogen frozen grinds and is lyophilized, stand-by tobacco slag is saved in -80 DEG C of refrigerators.
3. method of discrimination according to claim 1, it is characterised in that：The trans- 2- hexenoic acid of containing the internal standard, dense in the mixed solution
Degree is 0.303 μ g/mL.
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| CN106932515B (en) * | 2017-04-24 | 2019-11-01 | 山东省食品药品检验研究院 | The analysis method of true and false Rizhao Green Tea is distinguished based on UHPLC series connection high resolution mass spectrum application metabonomic technology |
| CN107102090B (en) * | 2017-06-09 | 2019-08-09 | 云南中烟工业有限责任公司 | A method of differentiating that tobacco aromaticss are rotten |
| CN109212066B (en) * | 2018-09-21 | 2021-06-18 | 中国烟草总公司郑州烟草研究院 | Method for determining non-fat organic acid in tobacco and tobacco products |
| CN109061005B (en) * | 2018-09-21 | 2021-06-18 | 中国烟草总公司郑州烟草研究院 | Method for measuring volatile fatty acid in tobacco leaves |
| CN109406704B (en) * | 2018-11-20 | 2021-03-12 | 中国烟草总公司郑州烟草研究院 | Method for measuring contents of various organic acids in tobacco and tobacco products |
| CN114624354B (en) * | 2022-01-21 | 2023-07-07 | 陕西科技大学 | Method for distinguishing physiological ripening and edible ripening of kiwi fruits through low-temperature storage based on metabonomics |
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