CN104422741A - Amino-acid-based method for determining quality of fresh tobacco leaf samples in tobacco metabonomics - Google Patents
Amino-acid-based method for determining quality of fresh tobacco leaf samples in tobacco metabonomics Download PDFInfo
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Abstract
The invention provides an amino-acid-based method for determining the quality of fresh tobacco leaf samples in tobacco metabonomics. The method is characterized in that by taking a ratio of the content of glutamic acid to the content of glutamine in fresh tobacco leaves as an index, the quality of the fresh tobacco leaf samples in metabonomics is determined, and a basis for selection of qualified samples of the metabonomics is provided. The method is accurate, reliable, simple and practicable, and can be used for providing reference for analyzing metabolic characteristics of tobacco and study of relevant tobacco metabonomics.
Description
Technical field
The present invention relates to analytical chemistry field, is a kind of method of discrimination whether the fresh tobacco leaf sample quality of tobacco metabolism group research institute meets metabolism group research needs that differentiates.
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.In the flow process of metabolism group profile analysis, the collection of typical sample be primary and and important step, obtaining qualified sample, with the metabolism state of objective ground reflected sample, is the metabonomic analysis basis of carrying out and prerequisite.The collection of the fresh leaf sample of plant, metabolism state when needing cancellation at low temperatures to stop its physiological activity to stop at sampling to ensure it, standing procedure is for after sample of plucking a plant, and tinfoil wraps up, be placed in-196 DEG C of liquid nitrogen frozens, for using dry powder sample analysis after fresh tobacco leaf analysis or freeze-drying.Pluck this section of process before analyzing at sample, need ensure that fresh tobacco leaf is preserved and grinds under liquid nitrogen condition in liquid nitrogen, otherwise the enzymatic browning of fresh leaf can be caused.
Amino acid is a compounds important in plant, is the structural units forming protein, the metabolism of the adjustable plant of some proteinase.Most amino acid is hinge that is nascent and secondary metabolism in plant metabolism network, and amino acid metabolism is the key link of plant metabolism.The further metabolic product of amino acid comprises nucleic acid, ester, polyphenol, chlorophyll, the important substance such as alkaloid.Amino acid whose metabolism is as the start-up portion of nitrogen metabolism, and play an important role in coordination C N metabolism, in tobacco plant growth metabolism process, the coordination of C N metabolism determines quality and the quality of tobacco leaf.In addition, in the sequence of phytoprotein nutritional quality, tobacco leaf protein matter occupy the first.With some chief crops as essential amino acid in paddy rice, wheat, corn, soybean protein is compared, essential amino acids content in tobacco protein is higher, essential amino acids content standard in the protein product all formulated higher than international food and agricultural organization (FAO), wherein tyrosine, phenylalanine, threonine and leucine exceed about 1 times of this standard.
The fresh tobacco leaf sample of serious brown stain, its color can become brown from bud green, but the sample that enzymatic browning is reacted slight is difficult to accurately differentiate that whether sample is normal by means of only the observation sample appearance color depth.This patent proposes to adopt the ratio of glutamic acid and glutamine content to differentiate the quality of fresh tobacco leaf first, and the sample quality for metabolism group research differentiates provides foundation.
Summary of the invention
The invention provides and a kind of method for distinguishing is sentenced to the fresh tobacco leaf sample quality in the research of tobacco metabolism group.In order to realize the object of the invention, of the present inventionly a kind ofly sentence method for distinguishing to the fresh tobacco leaf sample quality in metabolism group research, it utilizes the ratio of fresh tobacco leaf Glutamic Acid and glutamine content to differentiate the fresh tobacco leaf quality in metabolism group research.
By detecting fresh tobacco leaf Glutamic Acid and glutamine content, calculating its ratio, the fresh tobacco leaf sample quality in metabolism group research is differentiated.
To add containing interior target Extraction solvent in fresh tobacco leaf, centrifugal filtration after extracting, after adopting 6-aminoquinoline-N-succinimide methyl esters (AQC) derivative, utilize Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) to analyze, calculated the content of glutamine and glutamic acid by typical curve.
Be designated as in described and fall valine, its content is 5mg/L, and described Extraction solvent is the aqueous solution containing ethanol 70%; Described fresh tobacco leaf is=35:1-3 with pressing mass/volume than addition containing interior target Extraction solvent.
The condition of Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) is: chromatographic column Waters UPLC C18column; Mobile phase A: 20mM ammonium formate, 0.5% formic acid, 1% acetonitrile, 98.5% water, Mobile phase B: 1.6% formic acid, 98.4% acetonitrile (v/v); Flow velocity: 0.35mL/min; Column temperature: 55 DEG C; Sampling volume: 1 μ L; Injector temperature: 15 DEG C; Gradient is: 0-1.08min, 0.1%-0.1%B; 1.08-11.48min, 0.1%-9.1%B; 11.48-16.3min, 9.1%-21.2%B; 16.3-16.9min, 21.2%-59.6%B; 16.9-18.1min, 59.6%-59.6%B; 18.1-18.28min, 59.6%-0.1%B, 18.28-20min, 0.1%-0.1%B; Mass spectrometry method: under ESI positive ion mode, segmentation SIR scans, capillary voltage: 3KV, taper hole voltage: 30V, desolventizing temperature degree: 300 DEG C, taper hole temperature: 120 DEG C, desolventizing airshed: 650L/h, taper hole airshed: 50L/h.
Described fresh tobacco leaf preserves transport through-196 DEG C of liquid nitrogen containers, and liquid nitrogen frozen grinds, and freeze-drying ,-80 DEG C of Refrigerator stores are stand-by.
With the ratio of glutamic acid and glutamine content for index, set up the standard distinguishing the normal sample of metabolism group, part brownization and brownization sample, namely sample to be tested Glutamic Acid and glutamine ratio are qualified sample more than 0.67, for tobacco metabonomic analysis; Glutamic acid and the glutamine ratio defective sample for going bad below 0.12, glutamic acid and glutamine ratio are the defective sample of partial qualitative change between 0.12 and 0.67.
Researchist of the present invention is through the analysis and research of a large amount of fresh tobacco leaf sample, if find that the reactions such as enzymatic browning occur fresh tobacco leaf, larger change can be there is in the content of its free amino acid, the ratio of glutamic acid and glutamine content obviously reduces, and this type of sample can not be used for metabonomic analysis.Therefore the fresh tobacco leaf sample quality in metabolism group research is differentiated using the ratio of glutamic acid and glutamine content as a standard.
Advantage of the present invention
Utilize the ratio of glutamic acid and glutamine content to differentiate the fresh tobacco leaf sample quality in metabolism group research, differentiate whether it meets the needs of metabolism group research, the method simple possible, quickness and high efficiency.Specifically, the present invention is using the ratio of glutamic acid and glutamine content as the standard distinguishing the qualified sample of metabolism group, the serious defective sample of qualitative change and the defective sample of partial qualitative change.Namely sample to be tested Glutamic Acid and glutamine ratio are normal sample more than 0.67, can be used for metabonomic analysis, glutamic acid and glutamine ratio are the rotten defective sample of brownization below 0.12, glutamic acid and glutamine ratio are the defective sample of partial qualitative change between 0.12 and 0.67, need to examine further samples sources.
Embodiment
Explain the present invention further below by example, example is only limitted to the present invention is described so that understand, but not limitation of the invention.
1) fresh tobacco leaf plucked by land for growing field crops, preserves transport in-196 DEG C of liquid nitrogen containers, and liquid nitrogen frozen grinds, low-temperature freeze-dry, and-80 DEG C of Refrigerator stores are treated follow-uply to carry out metabonomic analysis.Accurately take 35mg fresh tobacco leaf, add 2mL70%C
2h
5oH (falling valine 5mg/L containing interior mark), shaking table 160rpm, 25 DEG C, 1h extracts, and 3000rpm is centrifugal, 0.22 μm of filtration, before adopting post, 6-aminoquinoline-N-succinimide methyl esters (AQC) (the Waters AccQ-Tag derivation kit derivative reagent box from waters company) derives, Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) is analyzed, and can carry out accurately to fresh tobacco leaf Free Amino Acids, fast quantification by typical curve.
The concrete derivative step adopting 6-aminoquinoline-N-succinimide methyl esters (AQC) derivative is: the borate buffer solution (the Waters AccQ-Tagderivation kit derivative reagent box from waters company) of 70 μ L pH8.8, add in eppendorf pipe (German Eppendorf company), add 10 μ L samples or amino-acid mixed mark solution, vortex 5-10 second, add 20 μ L derivative reagents again, vortex 5-10 second, ambient temperatare puts about 1min, in 55 DEG C of heating 10min.
By typical curve quantification steps: configuration 0.025,0.05,0.1,0.25,0.5,1,2.5,5, the mixed mark solution of 12.5,25,50mg/L, 29 seed amino acids of totally 11 series concentration, each series concentration all adds interior mark, obtains falling 29 seed amino acids of valine 5mg/L and the series standard solution of amine containing interior mark.
The standard solution of variable concentrations is carried out derivative rear sample introduction analysis.Each amino acid whose typical curve by with peak area/face, interior mark peak corresponding to its series concentration for dependent variable, be that independent variable mapping obtains with series concentration, i.e. formula (1):
Ai/AIS=KiCi+Bi……………………………………………….（1）
Wherein: the amino acid whose peak area of Ai-series concentration, target peak area in AIS-; Ci-amino acid series concentration, the slope of Ki-standard working curve; The intercept of Bi-standard working curve.After obtaining typical curve, the peak area in sample/interior mark peak area is substituted into formula, obtains each amino acid whose concentration.
2) free amino acid analysis is carried out to all samples, chromatographic column Waters UPLC C18column (particle diameter 1.7 μm, internal diameter 2.1mm, column length 100mm); Mobile phase A: 20mM ammonium formate, 0.5% formic acid, 1% acetonitrile, 98.5% water, Mobile phase B: 1.6% formic acid, 98.4% acetonitrile (v/v); Flow velocity: 0.35mL/min; Column temperature: 55 DEG C; Sampling volume: 1 μ L; Injector temperature: 15 DEG C; Gradient is: 0-1.08min, 0.1%-0.1%B; 1.08-11.48min, 0.1%-9.1%B; 11.48-16.3min, 9.1%-21.2%B; 16.3-16.9min, 21.2%-59.6%B; 16.9-18.1min, 59.6%-59.6%B; 18.1-18.28min, 59.6%-0.1%B, 18.28-20min, 0.1%-0.1%B.Mass spectrometry method: under ESI positive ion mode, segmentation SIR scans, capillary voltage: 3KV, taper hole voltage: 30V, desolventizing temperature degree: 300 DEG C, taper hole temperature: 120 DEG C, desolventizing airshed: 650L/h, taper hole airshed: 50L/h.
Adopt retention time qualitative in conjunction with the mass number of standard specimen, inner mark method ration.29 kinds of materials such as free amino acid and amine in fresh tobacco leaf can be recorded under these conditions.
Technology path of the present invention is:
Employing sample is Chinese yunnan, Guizhou, the large gold dollar of safflower of Henan San Sheng, K326, Zhongyan-100 three kinds are for a long time prosperous, squaring period, full-bloom stage, inferior leads maturity stage, the fresh tobacco leaf of 5 breeding times of middle leaf maturity stage and the middle leaf maturity stage fresh tobacco leaf material of some other province main breed, comprising the up-to-standard sample meeting the requirement of metabolism group sample, the defective sample of the serious qualitative change of blade and the underproof doubtful sample of partial qualitative change.According to the rule of the qualified sample of Sample Establishing in enormous quantities and the glutamic acid of defective sample and glutamine ratio, whether the quality of judgement sample is qualified.
Researchist of the present invention utilizes the ratio of fresh tobacco leaf sample Glutamic Acid and glutamine content and the correlativity of sample quality, carries out quality discrimination to metabolism group fresh tobacco leaf sample.The present invention has following characteristics: fast simple to operate, and result accurately and reliably; Sample consumption is few; Method is applicable to the differentiation of fresh tobacco leaf of different growing, different location and kind.This method can be the analysis of tobacco metabolic characteristics and the research of relevant tobacco metabolism group is offered reference.
Embodiment 1
Adopt the free aminoacid content in Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) method mensuration maturity stage fresh tobacco leaf sample, find the most of free aminoacid content marked change in underproof brownization sample, the ratio of glutamic acid and glutamine content obviously reduces.
Sample to be tested comprises 7 provinces, 7 kinds, 15 places of production such as Fujian, Guizhou, Hunan, 212 parts of fresh tobacco leaf samples 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, the fresh leaf sample portion qualitative change in Xuchang, Henan, other Sample preservations are intact.
Sample analysis step is as described below:
1, fresh tobacco leaf sample collection:
Fresh tobacco leaf sample, at Different sources field planting, arrives post-sampling certain breeding time ,-196 DEG C of Liquid nitrogen storage, and transport, grinds under liquid nitrogen condition, low-temperature freeze-dry ,-80 DEG C of refrigerator storage.
2, free amino acid assay method:
Fresh tobacco leaf sample takes out from-80 DEG C of refrigerators, and after 4 DEG C of refrigerator overnight are placed, ambient temperatare puts 1 hour.Accurately take 35mg fresh tobacco leaf sample, add 2mL70%C
2h
5oH (falling valine 5mg/L containing interior mark), shaking table 160rpm, 25 DEG C, 1h extracts, 3000rpm is centrifugal, after 0.22 μm of filtration, before adopting post, 6-aminoquinoline-N-succinimide methyl esters (AQC) derives, concrete derivative step is: the borate buffer solution (the Waters AccQ-Tagderivation kit derivative reagent box from waters company) of 70 μ L pH8.8, add in eppendorf pipe (German Eppendorf company), add 10 μ L samples or amino-acid mixed mark solution, vortex 5-10 second, add 20 μ L derivative reagents again, vortex 5-10 second, ambient temperatare puts about 1min, in 55 DEG C of heating 10min.After having derived, utilize Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) to analyze, by typical curve, amino acid in tobacco fresh tobacco leaves is carried out quantitatively.
3) free amino acid analysis is carried out to all samples, chromatographic column Waters UPLC C18column (particle diameter 1.7 μm, internal diameter 2.1mm, column length 100mm); Mobile phase A: 20mM ammonium formate, 0.5% formic acid, 1% acetonitrile, 98.5% water, Mobile phase B: 1.6% formic acid, 98.4% acetonitrile (v/v); Flow velocity: 0.35mL/min; Column temperature: 55 DEG C; Sampling volume: 1 μ L; Injector temperature: 15 DEG C; Gradient is: 0-1.08min, 0.1%-0.1%B; 1.08-11.48min, 0.1%-9.1%B; 11.48-16.3min, 9.1%-21.2%B; 16.3-16.9min, 21.2%-59.6%B; 16.9-18.1min, 59.6%-59.6%B; 18.1-18.28min, 59.6%-0.1%B, 18.28-20min, 0.1%-0.1%B.Mass spectrometry method: under ESI positive ion mode, segmentation SIR scans, capillary voltage: 3KV, taper hole voltage: 30V, desolventizing temperature degree: 300 DEG C, taper hole temperature: 120 DEG C, desolventizing airshed: 650L/h, taper hole airshed: 50L/h.
Obtain the absolute content of 29 kinds of free amino acids and amine in above-mentioned 212 parts of fresh tobacco leafs under this analysis condition, find that the ratio of glutamic acid and glutamine content is changed significantly, in table 1.
The ratio of the experiment material information that table 1 embodiment 1 is used and glutamic acid and glutamine content
Through above experiment, inventor finds that the ratio of glutamic acid in serious qualitative change (brownization) sample and glutamine content significantly reduces.The present invention using the ratio of glutamic acid and glutamine content as the standard distinguishing the rotten defective sample of the normal sample of metabolism group, the defective sample of critical deterioration and part.Namely sample to be tested Glutamic Acid and glutamine ratio are qualified sample more than 0.67, can be used for metabonomic analysis, glutamic acid and glutamine ratio are divided into two parts below 0.67, glutamic acid and glutamine ratio are the defective sample of partial qualitative change between 0.12 and 0.67, less than 0.12 be the defective sample of critical deterioration.
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 free amino acid wherein.
Sample analysis step is as described below:
1, fresh tobacco leaf sample collection:
Fresh tobacco leaf sample, at Different sources field planting, arrives post-sampling certain breeding time ,-196 DEG C of Liquid nitrogen storage, and transport, grinds under liquid nitrogen condition, low-temperature freeze-dry ,-80 DEG C of refrigerator storage.
2, free amino acid assay method:
Fresh tobacco leaf sample takes out from-80 DEG C of refrigerators, and after 4 DEG C of refrigerator overnight are placed, ambient temperatare puts 1 hour.Accurately take 35mg fresh tobacco leaf, add 2mL70%C2H5OH (containing interior mark Nval5mg/L), shaking table 160rpm, 25 DEG C, 1h extracts, and 3000rpm is centrifugal, 0.22 μm of filtration, adopts Waters AccQ-Tag derivation kit to derive post analysis.Before adopting post, 6-aminoquinoline-N-succinimide methyl esters (AQC) derives, and Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) method, measures amino acid content in fresh tobacco leaf.
3) free amino acid analysis is carried out to all samples, chromatographic column Waters UPLC C18column (particle diameter 1.7 μm, internal diameter 2.1mm, column length 100mm); Mobile phase A: 20mM ammonium formate, 0.5% formic acid, 1% acetonitrile, 98.5% water, Mobile phase B: 1.6% formic acid, 98.4% acetonitrile (v/v); Flow velocity: 0.35mL/min; Column temperature: 55 DEG C; Sampling volume: 1 μ L; Injector temperature: 15 DEG C; Gradient is: 0-1.08min, 0.1%-0.1%B; 1.08-11.48min, 0.1%-9.1%B; 11.48-16.3min, 9.1%-21.2%B; 16.3-16.9min, 21.2%-59.6%B; 16.9-18.1min, 59.6%-59.6%B; 18.1-18.28min, 59.6%-0.1%B, 18.28-20min, 0.1%-0.1%B.Mass spectrometry method: under ESI positive ion mode, segmentation SIR scans, capillary voltage: 3KV, taper hole voltage: 30V, desolventizing temperature degree: 300 DEG C, taper hole temperature: 120 DEG C, desolventizing airshed: 650L/h, taper hole airshed: 50L/h.Adopt retention time qualitative in conjunction with the mass number of standard specimen, inner mark method ration.
The absolute content of the material such as 29 seed amino acids and amine 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.The ratio of kind Glutamic Acid to be measured and glutamine content is normal sample more than 0.67.The ratio of glutamic acid and glutamine content is critical deterioration (brownization) sample below 0.12, 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 glutamic acid and glutamine content is the rotten sample of part between 0.12 and 0.67, 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 glutamic acid and glutamine content.
The ratio of the experiment material information that table 2 embodiment 2 is used and glutamic acid and glutamine content
This result illustrates that metabolism group fresh tobacco leaf sample quality method of discrimination that the present invention proposes is in different province, and different cultivars, can obtain same differentiation effect in the different tobacco leaf samples of different growing, also show 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 (6)
1. one kind method for distinguishing is sentenced to the fresh tobacco leaf sample quality in metabolism group research, it is characterized in that: by detecting fresh tobacco leaf Glutamic Acid and glutamine content, calculate its ratio, the fresh tobacco leaf sample quality in metabolism group research is differentiated.
2. method of discrimination according to claim 1, it is characterized in that: its concrete grammar is: will add containing interior target Extraction solvent in fresh tobacco leaf, centrifugal filtration after extracting, after adopting 6-aminoquinoline-N-succinimide methyl esters (AQC) derivative, utilize Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) to analyze, calculated the content of glutamine and glutamic acid by typical curve.
3. method of discrimination according to claim 2, it is characterized in that: be designated as in described and fall valine, its content is 5mg/L, and described Extraction solvent is the aqueous solution containing ethanol 70%; Described fresh tobacco leaf is=35:1-3 with pressing mass/volume than addition containing interior target Extraction solvent.
4. method of discrimination according to claim 1, is characterized in that: the condition of Ultra Performance Liquid Chromatography-mono-level Four bar mass spectrum (UPLC-SQDMS) is: chromatographic column Waters UPLC C18column; Mobile phase A: 20mM ammonium formate, 0.5% formic acid, 1% acetonitrile, 98.5% water, Mobile phase B: 1.6% formic acid, 98.4% acetonitrile (v/v); Flow velocity: 0.35mL/min; Column temperature: 55 DEG C; Sampling volume: 1 μ L; Injector temperature: 15 DEG C; Gradient is: 0-1.08min, 0.1%-0.1%B; 1.08-11.48min, 0.1%-9.1%B; 11.48-16.3min, 9.1%-21.2%B; 16.3-16.9min, 21.2%-59.6%B; 16.9-18.1min, 59.6%-59.6%B; 18.1-18.28min, 59.6%-0.1%B, 18.28-20min, 0.1%-0.1%B; Mass spectrometry method: under ESI positive ion mode, segmentation SIR scans, capillary voltage: 3KV, taper hole voltage: 30V, desolventizing temperature degree: 300 DEG C, taper hole temperature: 120 DEG C, desolventizing airshed: 650L/h, taper hole airshed: 50L/h.
5. method of discrimination according to claim 1, is characterized in that: described fresh tobacco leaf preserves transport through-196 DEG C of liquid nitrogen containers, and liquid nitrogen frozen grinds, and freeze-drying ,-80 DEG C of Refrigerator stores are stand-by.
6. method of discrimination according to claim 1, it is characterized in that: with the ratio of glutamic acid and glutamine content for index, set up the standard distinguishing the normal sample of metabolism group, part brownization and brownization sample, namely sample to be tested Glutamic Acid and glutamine ratio are qualified sample more than 0.67, for tobacco metabonomic analysis; Glutamic acid and the glutamine ratio defective sample for going bad below 0.12, glutamic acid and glutamine ratio are the defective sample of partial qualitative change between 0.12 and 0.67.
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| CN105866300A (en) * | 2016-06-15 | 2016-08-17 | 博莱克科技（武汉）有限公司 | Determination method of amino metabolites |
| CN106053674A (en) * | 2016-08-03 | 2016-10-26 | 贵州省烟草科学研究院 | Chromatographic detection method for simultaneously analyzing ammonium ions, amino acids and biogenic amine in tobacco leaves |
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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 |
| CN106290690A (en) * | 2016-09-30 | 2017-01-04 | 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 alkaloid |
| CN106248836B (en) * | 2016-09-30 | 2018-06-15 | 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 tobacco metabolism group based on Volatile Metabolites |
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| CN110824039B (en) * | 2019-11-04 | 2022-05-17 | 北京市结核病胸部肿瘤研究所 | Method for detecting concentration of cycloserine in bone tuberculosis specimen |
| CN113030338A (en) * | 2021-04-01 | 2021-06-25 | 贵州中烟工业有限责任公司 | Method for measuring amino acids in tobacco shreds |
| CN114280186A (en) * | 2021-12-24 | 2022-04-05 | 陕西科技大学 | Method for judging physiological maturity and edible maturity of kiwi fruits treated by 1-MCP |
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