Method for detecting secondary metabolites in fresh tobacco leaves by using derivatization GC-MS

A secondary metabolite, GC-MS technology, applied in the field of derivatized GC-MS for detecting secondary metabolites in fresh tobacco leaves

Active Publication Date: 2014-06-25
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The derivatized GC-MS method can simultaneously detect most metabolites such as organic acids, amino acids, amines, phenols, alcohols, sugars, etc. in tobacco leaves, and has high-throughput characteristics. related reports

Method used

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  • Method for detecting secondary metabolites in fresh tobacco leaves by using derivatization GC-MS
  • Method for detecting secondary metabolites in fresh tobacco leaves by using derivatization GC-MS
  • Method for detecting secondary metabolites in fresh tobacco leaves by using derivatization GC-MS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Collect 6 fresh tobacco leaves (safflower Dajinyuan variety) from the laboratory in the long-lived tobacco plant, quickly remove the main stem and wrap it with tin foil, and freeze it with liquid nitrogen. Take out the tin foil package from the liquid nitrogen, pat it lightly, and immediately freeze it in a freeze dryer for 24 hours to remove the water, and grind it to 40-60 mesh. Weigh 10mg of tobacco powder into a 1.5ml centrifuge tube, add 200μl deuterated hexadecanoic acid internal standard solution, 1.0ml of 5:2:2 methanol-water-chloroform solution, ultrasonic extraction for 30min, centrifuge at 10000r / min for 10min, Take 200μl of supernatant and place it in another 1.5ml centrifuge tube and blow dry with a nitrogen blower at 40°C. Add 30μl of methoxyamine hydrochloride solution (in pyridine) with a concentration of 20mg / ml, rotate and shake for 1min and keep in a 37℃ water bath for 90min, then add 30μl of TFMSA, rotate and shake for 1min, then place in a 37℃ water ...

Embodiment 2

[0034] Method recovery evaluation: Three mature tobacco leaves of nc297 were collected in Zunyi, Guizhou, the main stems were quickly removed, wrapped in tin foil, and quickly frozen with liquid nitrogen. Take the tin foil package out of liquid nitrogen, bury it in dry ice, air express it to the laboratory, and place it in a -80 degree refrigerator. Take out the tin foil package from the refrigerator, pat it lightly, and immediately freeze it in a freeze dryer for 24 hours to remove the water, and grind it to 40-60 mesh. Weigh 20mg of tobacco powder into a 1.5ml centrifuge tube, add 200μl of deuterated hexadecanoic acid internal standard solution, 1.0ml of 5:2:2 methanol-water-chloroform solution, 100μl of mixed standard solution (addition to each standard Concentrations are 20μg / ml, 100μg / ml, 200μg / ml in three levels, see Table 2), ultrasonic extraction for 30min, centrifugation at 10000r / min for 10min, transfer 0.4ml of supernatant to a 5ml centrifuge tube, at 40℃ Blow dry w...

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Abstract

A method for detecting secondary metabolites in fresh tobacco leaves by using a derivatization GC-MS (Gas Chromatography-Mass Spectrometer) is characterized by comprising the steps: quickly collecting new tobacco leaves and quickly freezing the new tobacco leaves by using liquid nitrogen on the site, crushing the tobacco leaves after removing the moisture in the tobacco leaves by means of freeze drying, extracting the secondary metabolites from tobacco powder by using a solvent, drying the extracted liquid supernatant, then performing oximation reaction and silane derivatization on the dried extractive, and finally performing analysis by the GC-MS. The method has the prominent advantages that firstly, a method suitable for extracting the secondary metabolites in the fresh tobacco leaves is developed. As the area of the tobacco leaves is larger, the secondary metabolites are not uniformly distributed in the whole tobacco leaves, and the tobacco leaves are not applicable to the punching sampling technique. However, according to a tobacco leave collection and extraction method provided by the invention, the collection period and the metabolite composition of the tobacco leaves at the collected part can be truly reflected. Secondly, most of the important metabolites of the tobacco leaves such as saccharides, amino acids, organic acids, terpene alcohols in the fresh tobacco leaves can be simultaneously detected, and the method has the characteristics of high throughput detection.

Description

Technical field [0001] The invention relates to a derivatized GC-MS method for detecting secondary metabolites in fresh tobacco leaves. The method can be used to determine the sugars, amino acids, organic acids, phenols, terpene alcohols and other secondary products in tobacco leaves at different growth stages. Metabolites are a high-throughput determination method. Background technique [0002] Tobacco is an important model crop and economic crop, and people have a lot of understanding of its chemical composition. However, most researches are limited to the understanding of cured tobacco leaves, and people's understanding of the chemical composition of fresh tobacco leaves, including different growth cycles and different parts of tobacco leaves, is still very limited. Secondary metabolites mainly refer to small molecular metabolites with a molecular weight below 1000, which are related to plant matrix resistance, signal transduction, growth and development, plant color, aroma a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 王晓瑜谢复炜秦亚琼张丽刘惠民杨军丁丽王昇贾云祯
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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