CN104422750A - Method for discriminating quality of fresh tobacco leaf sample based on research of metabonomics - Google Patents
Method for discriminating quality of fresh tobacco leaf sample based on research of metabonomics Download PDFInfo
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Abstract
The invention provides a method for discriminating the quality of a fresh tobacco leaf sample based on the research of metabonomics. According to the method, the quality of fresh tobacco leaves in the research of metabonomics is discriminated by utilizing the ratio of phosphatidyl ethanolamine (18:3/18:3) to triglyceride (16:0/16:0/16:0). The method is simple and quick to operate and accurate and reliable in results, and can be used for providing reference to analysis of tobacco metabolic characteristics and related researches of tobacco metabolic metabonomics.
Description
Technical field
The present invention relates to analytical chemistry field, is a kind ofly sentence method for distinguishing based on the lipid material content in fresh tobacco leaves to the fresh tobacco leaves sample quality in metabolism group research.
Background technology
Plant Metabolome be change by investigating its metabolic product after plant irriate or disturbance or its over time, study a kind of technology of families of plant.By directly can obtain the physiological ecological change of plant to the profile analysis of micromolecular compound metabolism in plant.In Plant Metabolome research, the quality of sample decides the true and reliable property analyzing data; And the preservation condition of sample determines the quality of sample quality.For the plant sample that can not carry out metabonomic analysis after sampling at once, usually advise Excised Embryos; Namely biological sample is preserved under the extremely low temperature environment below-80 DEG C.Under ultra-low temperature surroundings, the metabolic process of plant sample can slow down greatly, keeps the stability of sample with this.
Tobacco is as model organism and important industrial crops, and the metabolism group research in recent years for tobacco gets more and more.But because tobacco sample preservation is improper, quality that is that cause changes and causes the situation of metabolite content distortion to happen occasionally.Be badly in need of a kind of simply, fast analytical approach to Plant Metabolome research in tobacco sample quality differentiate.
Lipid is a class important substance in tobacco, has important biological function.It is the storage form of required fuel in tobacco metabolic process, can be used as the energy; Lipoid (mainly phosphatide) forms biomembranous principal ingredient, the not permeability of film, to the diffusion of various material and transportation function etc., all relevant with it; Lipid, as the surface mass of cell, has substantial connection with cell recognition, histogenic immunity etc.In concerned plant, the report of the method such as extraction, detection of lipoid substance also emerges in an endless stream in recent years, but someone did not propose the differentiation lipid material content in fresh tobacco leaves or content ratio being used for metabolism group fresh tobacco leaves sample quality.
Summary of the invention
The object of the invention there are provided a kind ofly sentences method for distinguishing to the fresh tobacco leaves sample quality in metabolism group research.Utilize the ratio of phosphatidyl-ethanolamine in fresh tobacco leaves (18:3/18:3) and triglyceride (16:0/16:0/16:0) content to differentiate the fresh tobacco leaves sample quality in metabolism group research, this method quick, result simple to operate accurately and reliably.
In order to realize the object of the invention, technical solution of the present invention is:
Based on a method of discrimination for the fresh tobacco leaves sample quality in metabolism group research, by detecting phosphatidyl-ethanolamine and content of triglyceride in fresh tobacco leaves, calculating its ratio, the fresh tobacco leaves sample quality in metabolism group research is differentiated.
To the ratio of phosphatidyl-ethanolamine and content of triglyceride be obtained as the standard distinguishing metabolism group normal specimens, rotten sample and the rotten sample of part, wherein in kind to be measured phosphatidyl-ethanolamine and content of triglyceride ratio 1.8 and above be normal specimens, can be used for metabonomic analysis, the ratio of phosphatidyl-ethanolamine and content of triglyceride is rotten sample below 0.1, and the ratio of phosphatidyl-ethanolamine and content of triglyceride be that part goes bad sample between 0.1-1.8.
Its concrete grammar is:
1) take a certain amount of fresh tobacco leaves sample, add containing interior target methyl alcohol, vortex 0.5-1.5 minute; Add methyl tert-butyl ether again, vortex 0.5-1.5 minute; Finally add water, vortex 0.5-1.5 minute; Centrifugal after leaving standstill, supernatant is diluted 2-4 doubly, move in sample injection bottle and analyze for HPLC-MS.
2) HPLC design parameter: chromatographic column: Waters T3; Column temperature: 55 DEG C; Sample size 4 μ L; Column flow rate: 0.26mL/min; Mobile phase A: 6/4 acetonitrile/water (containing 10mM ammonium acetate); Mobile phase B: 9/1 isopropyl alcohol/acetonitrile (containing 10mM ammonium acetate); Gradient 0-0.01min:0-30%B, 0.01-1.5min:30%-54%B, 1.5-4.0min:54-55%B, 4.0-14.0min:55-80%B, 14-17.0min:80-85%B, 17-17.5min:85-100%B, 17.5-22.5min:100%-30%B, 22.5-27.5min:30%B; MS design parameter: electric spray ion source (ESI), adopts positive ion mode to detect; Use the ionization of high-purity N 2 assistant spray and desolventizing, dry gas temperature 350 DEG C, dry gas flow velocity is 9m L/min, atomization air pressure 45psig; Ion source temperature 350 DEG C, capillary voltage is 4000V, and solvated ion removes a bunch voltage 230V, taper hole voltage 65V, barycenter (Centroid) type collection mass spectrometric data; Mass scan range m/z400 ~ 1000.
Described interior mark and content thereof are: phosphatidyl-ethanolamine 0.02-0.04g/L and triglyceride 0.002-0.004g/L;
Described fresh tobacco leaf, be=10 ~ 30:0.1 ~ 0.3:0.5 ~ 0.7:0.1 ~ 0.15 by mass/volume than addition containing interior target methanol solution, methyl tert-butyl ether and water.
Researchist of the present invention is through the analysis and research of a large amount of fresh tobacco leaves sample, if it is improper that discovery fresh tobacco leaves is preserved, larger change can be there is in the content of its lipid material, phosphatidyl-ethanolamine (18:3/18:3) also can obviously reduce with the ratio of triglyceride (16:0/16:0/16:0) content, and this type of sample can not be used for metabonomic analysis.Therefore can differentiate the fresh tobacco leaves sample quality in metabolism group research using the ratio of phosphatidyl-ethanolamine (18:3/18:3) with triglyceride (16:0/16:0/16:0) content as a standard.
Researchist of the present invention utilizes phosphatidyl-ethanolamine (18:3/18:3) and the ratio of triglyceride (16:0/16:0/16:0) content and the correlativity of sample quality in fresh tobacco leaves sample, carries out quality discrimination to metabolism group fresh tobacco leaves sample.Advantage of the present invention: fast simple to operate, result is accurately and reliably; Amount of samples is few; Be applicable to the fresh tobacco leaves of different growing.This method can be the analysis of tobacco metabolic characteristics and the research of relevant tobacco metabolism group is offered reference.
Embodiment
Embodiment further describes the present invention below, but described embodiment is only for illustration of the present invention instead of restriction the present invention.
The ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content to go bad the standard of sample as distinguishing metabolism group normal specimens, rotten sample and part by the present invention.Namely in kind to be measured phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content ratio 1.8 and above be normal specimens, can be used for metabonomic analysis, the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content is rotten sample below 0.1, and the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content be the sample that partly goes bad between 0.1-1.8.
Phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) are the two kinds of lipid materials extensively existed in plant, and this experiment adopts high performance liquid chromatography MS (HPLC-MS) to carry out relative quantitative assay to the phosphatidyl-ethanolamine (18:3/18:3) in fresh tobacco leaves sample and triglyceride (16:0/16:0/16:0).Analytical procedure is as follows:
1) a certain amount of fresh tobacco leaves sample (10mg-30mg) is taken, add containing interior mark phosphatidyl-ethanolamine (15:0/15:0) (0.02-0.04g/L, and triglyceride (15:0/15:0/15:0) (0.002-0.004g/L avanti), avanti) pure methyl alcohol (100-300 μ L, Merk), vortex 0.5-1.5 minute; Add methyl tert-butyl ether (500-700 μ L, sigma) again, vortex 0.5-1.5 minute; Finally add water (100-150 μ L), vortex 0.5-1.5 minute; Centrifugal after leaving standstill, supernatant is diluted 2-4 doubly, move in sample injection bottle and analyze for HPLC-MS.
2) HPLC(Agilent1200) design parameter: chromatographic column: Waters T3(1.8 μm, 2.1 × 100mm); Column temperature: 55 DEG C; Sample size 4 μ L; Column flow rate: 0.26mL/min; Mobile phase A: 6/4 acetonitrile/water (containing 10mM ammonium acetate, sigma); Mobile phase B: 9/1 isopropyl alcohol/acetonitrile (containing 10mM ammonium acetate, sigma); Gradient 0-0.01min:0-30%B, 0.01-1.5min:30%-54%B, 1.5-4.0min:54-55%B, 4.0-14.0min:55-80%B, 14-17.0min:80-85%B, 17-17.5min:85-100%B, 17.5-22.5min:100%-30%B, 22.5-27.5min:30%B.MS(Agilent QTOF6510) design parameter: electric spray ion source (ESI), adopts positive ion mode to detect; Use the ionization of high-purity N 2 assistant spray and desolventizing, dry gas temperature 350 DEG C, dry gas flow velocity is 9m L/min, atomization air pressure 45psig; Ion source temperature 350 DEG C, capillary voltage is 4000V, and solvated ion removes a bunch voltage 230V, taper hole voltage 65V, barycenter (Centroid) type collection mass spectrometric data; Mass scan range m/z400 ~ 1000.
Under these conditions can 120 kinds of lipid materials in qualitative fresh tobacco leaves, adopt Internal standard correction methods method to carry out relative quantification to lipid material.
Phosphatidyl-ethanolamine (18:3/18:3) is a kind of material, and what represent in bracket is the chain length of fatty acid chain; Phosphatidyl-ethanolamine (15:0/15:0) is interior mark, the error be used in correction analysis process.Both belong to same classification, but fatty acid chain length is different.
Embodiment 1
This batch of sample to be tested comprises 7 provinces, 7 kinds, 15 places of production such as Fujian, Guizhou, Hunan, 36 points totally 206 parts of fresh tobacco leaf samples (each point has 5-7 biology to repeat) of 5 different growing.The wherein Fujian Longyan cloud and mist 87 of middle leaf mature period, the fresh tobacco leaf samples such as No. 1, the Bi Na of the south of Guizhou Province, Guizhou, the K326 of Zhangjiajie, Hunan and Dali Zhongyan-100 in squaring period and the inferior leads maturity stage greatly red, the red large sample of zunyi, guizhou full-bloom stage is because of complete brownization of reason such as liquid nitrogen are not enough, sample portion brownization in Xuchang, Henan, other Sample preservations are intact.
Sample analysis step is as described below:
1) take the fresh tobacco leaves sample of 20mg, add the pure methyl alcohol 200 μ L containing phosphatidyl-ethanolamine (15:0/15:0) (0.03g/L, avanti) and triglyceride (15:0/15:0/15:0) (0.003g/L, avanti), vortex 0.5-1.5 minute; Add 600 μ L methyl tert-butyl ethers (sigma) again, vortex 0.5-1.5 minute; Finally add 120 μ L water, vortex 0.5-1.5 minute; After leaving standstill centrifugal (15000rpm, 10 minutes), doubly (thinning agent: isopropyl alcohol/acetonitrile/water: 65/30/5), moves in sample injection bottle and analyzes for HPLC-MS supernatant to be diluted 2-4.
2) HPLC(Agilent1200) design parameter: chromatographic column Waters T3(1.8 μm, 2.1 × 100mm); Column temperature: 55 DEG C; Sample size 4 μ L; Column flow rate: 0.26mL/min; Mobile phase A: 6/4 acetonitrile/water (containing 10mM ammonium acetate, sigma); Mobile phase B: 9/1 isopropyl alcohol/acetonitrile (containing 10mM ammonium acetate, sigma); Gradient 0-0.01min:0-30%B, 0.01-1.5min:30%-54%B, 1.5-4.0min:54-55%B, 4.0-14.0min:55-80%B, 14-17.0min:80-85%B, 17-17.5min:85-100%B, 17.5-22.5min:100%-30%B, 22.5-27.5min:30%B.MS(Agilent QTOF6510) design parameter: electric spray ion source (ESI), adopts positive ion mode to detect; Use the ionization of high-purity N 2 assistant spray and desolventizing, dry gas temperature 350 DEG C, dry gas flow velocity is 9m L/min, atomization air pressure 45psig; Ion source temperature 350 DEG C, capillary voltage is 4000V, and solvated ion removes a bunch voltage 230V, taper hole voltage 65V, barycenter (Centroid) type collection mass spectrometric data; Mass scan range m/z400 ~ 1000.
The relative content (namely the mass spectrum response of often kind of lipid marks with interior the ratio responded) of 120 kinds of lipid materials in above-mentioned 206 parts of fresh tobacco leaves is obtained under this analysis condition, find that phosphatidyl-ethanolamine (18:3/18:3) is changed significantly, in table 1 with the ratio of triglyceride (16:0/16:0/16:0) content.
The ratio of laboratory sample information used and phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content in table 1 embodiment 1
Through above experiment, inventor finds to adopt the lipid material content in HPLC-MS method mensuration maturity stage fresh tobacco leaves sample, find in brownization sample that most of lipid material content of brownization sample significantly reduces, phosphatidyl-ethanolamine (18:3/18:3) significantly reduces with the ratio of triglyceride (16:0/16:0/16:0) content.The ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content to go bad the standard of sample as distinguishing metabolism group normal specimens, rotten sample and part by the present invention.Namely in kind to be measured phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content ratio 1.8 and above be normal specimens, can be used for metabonomic analysis, the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content is rotten sample below 0.1, and the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content be the sample that partly goes bad between 0.1-1.8.
Embodiment 2
Random choose is from China 3 provinces, and 8 planting sites, 5 different growing, 5 kinds, 20 points are totally 55 parts of fresh tobacco leaf samples (each point has 5-6 biology to repeat), measure lipid material wherein.Sample analysis step is as described below:
1) take the fresh tobacco leaves sample of 20mg, add the pure methyl alcohol 200 μ L containing phosphatidyl-ethanolamine (15:0/15:0) (0.03g/L, avanti) and triglyceride (15:0/15:0/15:0) (0.003g/L, avanti), vortex 0.5-1.5 minute; Add methyl tert-butyl ether 600 μ L(sigma again), vortex 0.5-1.5 minute; Finally add water 120 μ L, vortex 0.5-1.5 minute; After leaving standstill under 15000rpm centrifugal 10 minutes, doubly (thinning agent: isopropyl alcohol/acetonitrile/water: 65/30/5), moved in sample injection bottle and analyzes for HPLC-MS supernatant to be diluted 2-4.
2) HPLC(Agilent1200) design parameter: chromatographic column: Waters T3((1.8 μm, 2.1 × 100mm); Column temperature: 55 DEG C; Sample size 4 μ L; Column flow rate: 0.26mL/min; Mobile phase A: 6/4 acetonitrile/water (containing 10mM ammonium acetate, sigma); Mobile phase B: 9/1 isopropyl alcohol/acetonitrile (containing 10mM ammonium acetate, sigma); Gradient 0-0.01min:0-30%B, 0.01-1.5min:30%-54%B, 1.5-4.0min:54-55%B, 4.0-14.0min:55-80%B, 14-17.0min:80-85%B, 17-17.5min:85-100%B, 17.5-22.5min:100%-30%B, 22.5-27.5min:30%B.MS(Agilent QTOF6510) design parameter: electric spray ion source (ESI), adopts positive ion mode to detect; Use the ionization of high-purity N 2 assistant spray and desolventizing, dry gas temperature 350 DEG C, dry gas flow velocity is 9m L/min, atomization air pressure 45psig; Ion source temperature 350 DEG C, capillary voltage is 4000V, and solvated ion removes a bunch voltage 230V, taper hole voltage 65V, barycenter (Centroid) type collection mass spectrometric data; Mass scan range m/z400 ~ 1000.
The relative content of 120 kinds of lipid materials in above-mentioned 55 parts of fresh tobacco leafs is obtained under this analysis condition.According to standard described in accompanying examples 1, originally quality discrimination is carried out to institute's test sample.In kind to be measured, the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content is normal sample more than 1.8.The ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content to go bad sample for brownization below 0.1, comprising the middle leaf maturity stage sample of Guizhou Bijie cloud and mist 97, the full-bloom stage sample of Dali K326.The ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content is the rotten sample of part between 0.1 and 1.8, comprises the middle leaf maturity stage sample of Xuchang, Henan Zhongyan-100.Liquid nitrogen in this result and Sample preservation process is not enough, and behavior fits like a glove.Table 2 lists the information of experiment sample in the present embodiment and the ratio of phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content.
The ratio of laboratory sample information used and phosphatidyl-ethanolamine (18:3/18:3) and triglyceride (16:0/16:0/16:0) content in table 2 embodiment 2
This result illustrates that the metabolism group fresh tobacco leaves sample quality method of discrimination that the present invention proposes can obtain same differentiation effect in the different tobacco samples of different growing stages, also shows feasibility and the reliability of the inventive method.
Although be described in detail the present invention and confirmed some instantiations, for a person skilled in the art, only otherwise leave the spirit and scope of the present invention, it is obvious for making various changes or revising.
Claims (4)
1. the method for discrimination based on the fresh tobacco leaves sample quality in metabolism group research, it is characterized in that: by detecting phosphatidyl-ethanolamine and content of triglyceride in fresh tobacco leaves, calculate its ratio, the fresh tobacco leaves sample quality in metabolism group research is differentiated.
2. method of discrimination according to claim 1, it is characterized in that: will the ratio of phosphatidyl-ethanolamine and content of triglyceride be obtained as the standard distinguishing metabolism group normal specimens, rotten sample and the rotten sample of part, wherein in kind to be measured phosphatidyl-ethanolamine and content of triglyceride ratio 1.8 and above be normal specimens, can be used for metabonomic analysis, the ratio of phosphatidyl-ethanolamine and content of triglyceride is rotten sample below 0.1, and the ratio of phosphatidyl-ethanolamine and content of triglyceride be that part goes bad sample between 0.1-1.8.
3. method according to claim 1 or 2, is characterized in that: its concrete grammar is:
1) take a certain amount of fresh tobacco leaves sample, add containing interior target methyl alcohol, vortex 0.5-1.5 minute; Add methyl tert-butyl ether again, vortex 0.5-1.5 minute; Finally add water, vortex 0.5-1.5 minute; Centrifugal after leaving standstill, supernatant is diluted 2-4 doubly, move in sample injection bottle and analyze for HPLC-MS.
2) HPLC design parameter: chromatographic column: Waters T3; Column temperature: 55 DEG C; Sample size 4 μ L; Column flow rate: 0.26mL/min; Mobile phase A: 6/4 acetonitrile/water (containing 10mM ammonium acetate); Mobile phase B: 9/1 isopropyl alcohol/acetonitrile (containing 10mM ammonium acetate); Gradient 0-0.01min:0-30%B, 0.01-1.5min:30%-54%B, 1.5-4.0min:54-55%B, 4.0-14.0min:55-80%B, 14-17.0min:80-85%B, 17-17.5min:85-100%B, 17.5-22.5min:100%-30%B, 22.5-27.5min:30%B; MS design parameter: electric spray ion source (ESI), adopts positive ion mode to detect; Use the ionization of high-purity N 2 assistant spray and desolventizing, dry gas temperature 350 DEG C, dry gas flow velocity is 9m L/min, atomization air pressure 45psig; Ion source temperature 350 DEG C, capillary voltage is 4000V, and solvated ion removes a bunch voltage 230V, taper hole voltage 65V, barycenter (Centroid) type collection mass spectrometric data; Mass scan range m/z400 ~ 1000.
4. method according to claim 1 or 2, is characterized in that: its concrete grammar is: described interior mark and content thereof are: phosphatidyl-ethanolamine 0.02-0.04g/L and triglyceride 0.002-0.004g/L;
Described fresh tobacco leaf, be=10 ~ 30:0.1 ~ 0.3:0.5 ~ 0.7:0.1 ~ 0.15 by mass/volume than addition containing interior target methanol solution, methyl tert-butyl ether and water.
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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 |
| CN109001360A (en) * | 2018-10-09 | 2018-12-14 | 南京财经大学 | A kind of method of high-throughput detection phospholipid in lipid content |
| CN110174468A (en) * | 2019-03-26 | 2019-08-27 | 陕西科技大学 | The analysis method of lipid composition in a kind of Fu-brick tea |
| CN112433015A (en) * | 2020-12-10 | 2021-03-02 | 中国烟草总公司郑州烟草研究院 | High performance liquid chromatography-quadrupole time-of-flight mass spectrometry detection method for non-targeted analysis of electronic cigarette smoke liquid |
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