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Method for detecting primary metabolites and secondary metabolites in fresh tobacco leaves with GC-MS (gas chromatography-mass spectrometer)

A technology of secondary metabolites and fresh tobacco leaves, applied in the field of metabolite detection in fresh tobacco leaves of tobacco metabolomics, can solve imperfect data, affect the accuracy of data preprocessing results, batch analysis data repeatability, narrow linear range, etc. question

Inactive Publication Date: 2017-03-08
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this type of method also has some limitations, such as its detector is easily saturated, and its linear range is narrow, which limits the accurate quantification of metabolites whose concentration range spans several orders of magnitude.
In addition, the number of metabolites that can be matched and the accuracy of the peak matching results are affected by the peak matching parameters. Due to the imperfection of the software algorithm and the complexity of the data, some erroneous results will be generated, which will affect the accuracy of the data preprocessing results. Accuracy and repeatability of batch analysis data

Method used

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  • Method for detecting primary metabolites and secondary metabolites in fresh tobacco leaves with GC-MS (gas chromatography-mass spectrometer)
  • Method for detecting primary metabolites and secondary metabolites in fresh tobacco leaves with GC-MS (gas chromatography-mass spectrometer)
  • Method for detecting primary metabolites and secondary metabolites in fresh tobacco leaves with GC-MS (gas chromatography-mass spectrometer)

Examples

Experimental program
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Embodiment 1

[0028] Example 1: Collect 18 fresh tobacco leaves of the 10th leaf of Honghua Dajinyuan planted in Dali, Yunnan, quickly remove the veins and wrap 3 tobacco leaves with tin foil, freeze them quickly in liquid nitrogen, and take out the samples wrapped in tin foil from liquid nitrogen , crushed and vacuum freeze-dried with a lyophilizer for 72 hours to remove water, and ground to 40-60 mesh. Weigh 20~25mg of crushed tobacco leaves into a 2mL centrifuge tube, add 100μL deuterated tridecanoic acid internal standard solution, 1mL extraction solvent, ultrasonic extraction for 30min, after ultrasonic extraction, centrifuge at 4000r / min for 10min, take 200μL supernatant in Conical vials and blow dry with a nitrogen blower. Add 50 μL of methoxyamine hydrochloride pyridine solution with a concentration of 20 mg / mL to the dried extract, vortex and oscillate and react at 37°C for 90 min, then add 50 μL MSTFA to the injection bottle, vortex and oscillate React at 37°C for 60min and detec...

Embodiment 2

[0036]Example 2: Collect 18 pieces of fresh tobacco leaves at the 10th leaf position of China Tobacco 100 planted in Xiangxian County, Henan Province, quickly remove the trunk and wrap 3 pieces of tobacco leaves with tinfoil paper, freeze them quickly in liquid nitrogen, take out the samples wrapped in tinfoil paper from the liquid nitrogen, Smash it into pieces and vacuum freeze-dry it with a freeze dryer for 72 hours to remove moisture, and grind it to 40-60 mesh. Weigh 20~25mg of crushed tobacco leaves into a 2mL centrifuge tube, add 100μL deuterated tridecanoic acid internal standard solution, 1mL extraction solvent, ultrasonic extraction for 30min, after ultrasonic extraction, centrifuge at 4000r / min for 10min, take 200μL supernatant in a tip Bottom sample bottle and blow dry with nitrogen blower. Add 50 μL of methoxyamine hydrochloride pyridine solution with a concentration of 20 mg / mL to the dried extract, vortex and oscillate and react at 37°C for 90 min, then add 50 μ...

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Abstract

The invention provides a method for detecting primary metabolites and secondary metabolites in fresh tobacco leaves with GC-MS (gas chromatography-mass spectrometer). The method comprises steps as follows: the fresh tobacco leaves are rapidly collected and quick-frozen with liquid nitrogen on site, the quick-frozen tobacco leaves are transferred to dry ice and transported to a laboratory at low temperature, moisture is removed by a freeze dryer with a low-temperature freeze drying method, the tobacco leaves are pulverized by a pulverizer and screened by a 40-mesh sieve, the primary metabolites and the secondary metabolites of the pulverized tobacco leaves are extracted ultrasonically with a solvent, a supernatant of an extracting solution is subjected to nitrogen blow-drying, blow-dried extracts are subjected to an oximation reaction and a silane derivatization reaction, and GC-MS analysis is performed finally. The method has the advantages as follows: a tobacco leaf collecting method can really reflect growth and development periods, collecting parts, varieties and production places of the tobacco leaves; a tobacco leaf extracting and detecting method can cover the primary and secondary metabolites such as sugar, amino acid, organic acid, sterol, alkaloid, polyphenol and the like of the fresh tobacco leaves and has high-flux detection characteristics, and a selective ion monitoring method can realize accurately qualitative and quantitative analysis of 199 primary and secondary metabolites in the fresh tobacco leaves, and 135 metabolites are required to be verified in a standard substance.

Description

technical field [0001] The invention relates to the research on the detection of metabolites in fresh tobacco leaves of tobacco metabolomics, specifically a gas chromatography-tandem mass spectrometry method for detecting primary metabolites sugars, amino acids, organic acids and secondary metabolites sterols, alkaloids, and polysaccharides in fresh tobacco leaves. The detection method of phenol etc. Background technique [0002] Tobacco is an important model crop, and the research on its chemical composition is mainly limited to cured tobacco leaves. There is very limited understanding of the chemical composition of fresh tobacco leaves, including different growth and development stages, collection parts, varieties and origins. Although research on tobacco leaf metabolomics has been carried out at home and abroad in recent years, the number of metabolites detected and accurately quantified is limited, and the primary and secondary metabolites in fresh tobacco leaves are rel...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 陈霞周会娜翟妞李锋徐国云王燃郑庆霞陈千思王晨申晓晔
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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