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CN-117405785-B - Method for simultaneously analyzing multiple fat-soluble components in tobacco leaves

CN117405785BCN 117405785 BCN117405785 BCN 117405785BCN-117405785-B

Abstract

The invention belongs to the technical field of chemical components of tobacco, and particularly relates to a method for simultaneously analyzing multiple fat-soluble components in tobacco. A method for simultaneously analyzing multiple fat-soluble components in tobacco leaves includes such steps as drying tobacco leaves to be tested, grinding, adding extractant and internal standard to obtain solution of sample to be tested, analyzing the sample by supercritical fluid chromatography, multi-wavelength detection and internal standard analysis. The method for simultaneously analyzing various fat-soluble components in tobacco provided by the invention is used for detecting the characteristics of high molecular weight, poor volatility, easiness in oxidative decomposition and the like of the fat-soluble components of the tobacco by adopting a supercritical fluid chromatographic analysis technology, can realize simultaneous quantitative analysis of 13 fat-soluble components in the tobacco, has the advantages of less solvent consumption, low cost and high detection efficiency, and can provide an environment-friendly, more efficient and more convenient high-throughput analysis method for quality evaluation of the tobacco.

Inventors

  • WANG YANGZHONG
  • CAI ZHENBO
  • ZHOU MENGQIAN
  • QI DAWEI
  • ZHOU YAN
  • FEI TING
  • WU DA

Assignees

  • 上海烟草集团有限责任公司

Dates

Publication Date
20260505
Application Date
20231016

Claims (8)

  1. 1. A method for simultaneously analyzing multiple fat-soluble components in tobacco leaves, which is characterized by comprising the following steps: 1) Drying and grinding tobacco leaves to be tested, adding an extractant and an internal standard to obtain a sample solution to be tested, wherein the extractant is dichloromethane-ethanol, and adding the extractant amount and the internal standard, and further comprises vortex and vibration; 2) Analyzing a sample by a supercritical fluid chromatography system, detecting by using multiple wavelengths, and analyzing by an internal standard method to determine various fat-soluble components in tobacco leaves, wherein the fat-soluble components are alpha-carotene, beta-carotene, chlorophyll a, chlorophyll B, lutein, zeaxanthin, neoxanthin, vitamin E, alpha-cembratriene glycol, beta-cembratriene glycol, ergosterol, scopoletin and solanesol, the detection conditions of the supercritical fluid chromatography system comprise a chromatographic column stationary phase being a C18 column, a mobile phase A phase being CO 2 , a mobile phase B phase being a methanol/acetonitrile mixed solution (1:1, volume ratio), dynamic back pressure being 2000psi, detection wavelengths being 210nm, 280nm, 330nm and 430nm, gradient elution conditions being 0-3.5 min,0-3% B, 9-12 min,5-10% B, 15-18 min,15-20% B, 19-23 min,20-30% B, 23.5-27 min, and 0-3% B.
  2. 2. The method for simultaneous analysis of multiple fat-soluble ingredients in tobacco leaves according to claim 1, comprising at least one of the following technical features: 11 In the step 1), the tobacco leaves to be tested are cured tobacco leaves or fresh tobacco leaves; 12 In step 1), the internal standard is selected from beta-apo-8' -carotenal; 13 In the step 1), the feed liquid ratio of the tobacco leaves to be detected to the extractant is (1 g: 5 mL) - (1 g: 30 mL).
  3. 3. The method according to claim 2, wherein in the step (1), the extractant is selected from methylene chloride/ethanol (volume ratio V: V is 1:1 to 5:1).
  4. 4. The method for simultaneous analysis of multiple fat-soluble ingredients in tobacco leaf according to claim 2, wherein in feature 12), the concentration of the internal standard is 5-60 μg/mL.
  5. 5. The method for simultaneously analyzing multiple fat-soluble components in tobacco leaves according to claim 2, wherein in the characteristic 13), the ratio of the feed liquid of the tobacco leaves to be tested to the feed liquid of the extractant is 1g to 20mL.
  6. 6. The method for simultaneously analyzing a plurality of fat-soluble components in tobacco leaves according to claim 1, wherein the shaking time is 5-40min.
  7. 7. The method for simultaneously analyzing a plurality of fat-soluble components in tobacco leaves according to claim 1, wherein the pore size of the filter is 0.2-0.4 μm.
  8. 8. The method for simultaneously analyzing a plurality of fat-soluble components in tobacco leaves according to claim 2, wherein in step 2), the detection conditions of the supercritical fluid chromatography system include the following technical features: the chromatographic conditions are as follows: Column temperature is 30-55 ℃; The sample injection amount is 0.5-3 mu L; The flow rate is 0.5-2 mL/min.

Description

Method for simultaneously analyzing multiple fat-soluble components in tobacco leaves Technical Field The invention belongs to the technical field of chemical components of tobacco, and particularly relates to a method for simultaneously analyzing multiple fat-soluble components in tobacco. Background Tobacco contains a plurality of fat-soluble components, such as beta-carotene, fat-soluble vitamins, sterols, cembrane-like, rice-plant, sugar esters, glyceride and the like, which are related to the quality and function of the tobacco, and the accurate measurement of important fat-soluble components in the tobacco has important significance for evaluating the quality of the tobacco. The fat-soluble components in tobacco have the characteristics of large molecular weight, poor volatility, easy oxidative decomposition and the like, and common detection methods include liquid chromatography, liquid chromatography-mass spectrometry, supercritical fluid chromatography and the like. However, the separation of the isomers of the fat-soluble components by liquid chromatography or liquid chromatography-mass spectrometry is difficult, a large amount of organic solvents having relatively large damage such as acetone, methylene chloride and chloroform are required, and the analysis time is long. Supercritical fluid chromatography uses supercritical fluid CO2 as a mobile phase main body, and performs separation and analysis by means of solvation ability of the mobile phase. In theory, supercritical fluid chromatography can not only analyze high boiling, low volatility, thermally unstable samples that are not compatible with gas chromatography, but also provide suitable retention and separation of a variety of substances such as structural analogs, fat-soluble compounds, and thermally unstable compounds. Compared with liquid chromatography, supercritical fluid chromatography can simplify pretreatment process and reduce organic solvent usage when analyzing liposoluble components. At present, supercritical fluid chromatography is widely applied to detection of carotenoid, triglyceride, steroid, fat-soluble vitamin and the like in foods, but only certain compounds are usually detected, the detection efficiency is low, and related analysis reports on cembrane compounds in tobacco are not found. Therefore, the supercritical fluid chromatography technology is utilized to analyze various fat-soluble components in the tobacco at the same time, which is helpful for further defining key substance components of tobacco quality and provides important supporting basis for improving and improving tobacco quality and comprehensively utilizing the tobacco. Disclosure of Invention In view of the defects of the prior art, the invention aims to provide a method for simultaneously analyzing multiple fat-soluble components in tobacco leaves, which is used for solving the defects of high use amount of organic solvents, high analysis cost, complex pretreatment process and low detection efficiency in the prior art, and can realize simultaneous analysis of 13 fat-soluble components in tobacco leaves. To achieve the above and related objects, the present invention provides a method for simultaneously analyzing a plurality of fat-soluble components in tobacco leaves, comprising the steps of: 1) Drying and grinding tobacco leaves to be detected, and then adding an extractant and an internal standard to obtain a sample solution to be detected; 2) The analysis of the sample is carried out by a supercritical fluid chromatographic system, the detection is carried out by a multi-wavelength method, and the analysis is carried out by an internal standard method. Preferably, 11) in step 1), the tobacco leaf to be tested is cured tobacco leaf or fresh tobacco leaf; Preferably, 12) in step 1), the extractant is selected from one or more of chloroform, acetone, dichloromethane, isopropanol, dichloromethane/ethanol (1:1, v: v) and dichloromethane/ethanol (2:1, v: v); Preferably, 13) in step 1), the internal standard is selected from β -apo-8' -carotenal; preferably, 14) in the step 1), the feed liquid ratio of the tobacco leaf to be detected to the extractant is (1 g:5 ml) - (1 g:30 ml); Preferably, in feature 12), the extractant is selected from dichloromethane/ethanol (volume ratio V: V is 1:1 to 5:1), preferably the extractant is selected from dichloromethane/ethanol (2:1, V: V). Preferably, in feature 13), the concentration of the internal standard is 5-60 μg/mL. Preferably, in the feature 14), the feed liquid ratio of the tobacco leaf to be detected to the extractant is 1g to 20mL. Preferably, in step 1), after adding the extractant amount and the internal standard, vortex and shake are also included. Preferably, A1) the time of the shaking is 5-40min, and preferably, the time of the shaking is 10min; Preferably, A2) said shaking further comprises filtering the extracted supernatant with a filter membrane. Preferably, in feature A2), the filter membrane has