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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: 2012-07-18
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

[0030] Collect 6 pieces of fresh tobacco leaves from long-term tobacco plants in the laboratory (Honghua Dajinyuan variety), quickly remove the trunk and wrap them in tin foil, and freeze them with liquid nitrogen. Take out the tinfoil bag from the liquid nitrogen, pat it into pieces, and immediately freeze it in a freeze dryer for 24 hours to remove the water, and grind it to 40-60 mesh. Weigh 10mg smoke powder into a 1.5ml centrifuge tube, add 200μl deuterated hexadecanoic acid internal standard solution, 1.0ml 5:2:2 methanol-water-chloroform solution, ultrasonically extract for 30min, centrifuge at 10000r / min for 10min, Take 200 μl of the supernatant in another 1.5ml centrifuge tube, and dry it with a nitrogen blower at 40°C. Add 30 μl of methoxyamine hydrochloride solution (in pyridine) with a concentration of 20 mg / ml, mediate and shake for 1 min, then keep in a 37°C water bath for 90 min, then add 30 μl of TFMSA, mediate and shake for 1 min, then place in a 37°C water ba...

Embodiment 2

[0040] Method investigation on the recovery rate: 3 mature tobacco leaves of variety nc297 in Zunyi, Guizhou were collected, the stems were quickly removed, wrapped in tinfoil, and quick-frozen in liquid nitrogen. The tinfoil package was taken out of the liquid nitrogen, embedded in dry ice, air-expressed to the laboratory, and placed in a -80°C refrigerator. Take out the tinfoil package from the refrigerator, pat it into pieces, immediately freeze it in a freeze dryer for 24 hours to remove the water, and grind it to 40-60 mesh. Weigh 20mg smoke powder into a 1.5ml centrifuge tube, add 200μl deuterated hexadecanoic acid internal standard solution, 1.0ml 5:2:2 methanol-water-chloroform solution, 100μl mixed standard solution (each standard is added Concentrations are 20μg / ml, 100μg / ml, 200μg / ml, see Table 2), ultrasonic extraction for 30min, centrifugation at 10000r / min for 10min, transfer 0.4ml supernatant to a 5ml centrifuge tube, and store at 40℃ Blow dry with a nitrogen b...

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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

[0001] technical field [0002] The invention relates to a derivatized GC-MS method for detecting secondary metabolites in fresh tobacco leaves, which can be used to measure various secondary metabolites such as sugars, amino acids, organic acids, phenols, and terpene alcohols in tobacco leaves at different growth stages. Metabolites, a high-throughput assay. [0003] Background technique [0004] Tobacco is an important model crop and economic crop, and people have learned a lot about its chemical composition. However, most studies 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 flower color, a...

Claims

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Application Information

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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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