CN-121994960-A - Method for detecting polyphenol compounds in tobacco or products thereof based on one-test-multiple-evaluation method and application of method

Abstract

The invention relates to a method for detecting polyphenol compounds in tobacco or products thereof based on a multi-evaluation method and application thereof, wherein the method comprises the steps of mixing and extracting the tobacco or the products thereof with a solvent to obtain a sample solution; mixing the standard substance of the polyphenols with the second solvent to obtain several standard solutions with different concentrations, detecting the sample solution and the standard solution by liquid chromatography, and obtaining the content of the polyphenols in tobacco or its products based on one-test-multiple-evaluation method by taking chlorogenic acid as an internal reference. The method adopts the high performance liquid chromatography diode array detection method, utilizes the relative correction factors between chlorogenic acid and other polyphenol compounds to calculate the content of the polyphenol compounds in the tobacco and tobacco products, has short detection time, low detection limit and quantitative limit, low RSD (reactive species detector) of precision, repeatability and stability and good durability and repeatability, and can be suitable for content analysis, quality evaluation and classification of the tobacco or products thereof.

Inventors

• TANG XIAODONG
• YU FEI
• ZHAO LUCAN
• LIN KEN
• ZHAO PANPAN
• ZHANG CHEN
• SU YAN
• ZHU SHUXIU
• JIANG JIALEI
• CHEN XIAOSHUI
• XIA JUN
• SI WEN
• PAN LI
• LI XIA

Assignees

• 浙江中烟工业有限责任公司

Dates

Publication Date

20260508

Application Date

20260212

Claims (10)

1. 1. A method for detecting polyphenols in tobacco or a product thereof based on a multi-assay method, the method comprising: mixing tobacco or its product with solvent I, extracting to obtain sample solution; Mixing a standard substance of a polyphenol compound with a second solvent to obtain a plurality of standard solutions with different concentrations; Detecting the sample solution and the standard solution by adopting a liquid chromatography method, and obtaining the content of the polyphenol compounds in the tobacco or the products thereof based on a one-test-multiple-evaluation method by taking chlorogenic acid as an internal reference.
2. 2. The method of claim 1, wherein the polyphenolic compound comprises any one or a combination of at least two of chlorogenic acid, neochlorogenic acid, scopoletin, cryptochlorogenic acid, caffeic acid, scopoletin, rutin, or kaempferol-3-O-rutinoside.
3. 3. The method according to claim 1 or 2, wherein the solvent one comprises any one or a combination of at least two of methanol, water, methyl tert-butyl ether, n-hexane or cyclohexane; Preferably, the solvent one is a combination of methanol, water and n-hexane, or the solvent one is a combination of methanol, water and cyclohexane; Preferably, the volume ratio of the methanol to the water to the n-hexane is 1 (0.8-1.2): 1.6-2.4; Preferably, the volume ratio of the methanol to the water to the cyclohexane is 1 (0.8-1.2): 1.6-2.4; preferably, the dosage ratio of the tobacco or the preparation thereof and the solvent I is 1 mg (0.3-0.5) mL; Preferably, the solvent two comprises methanol and/or water.
4. 4. A method according to any one of claims 1-3, wherein the mobile phase in the liquid chromatography comprises mobile phase a comprising a combination of water, methanol and acetic acid and mobile phase B comprising a combination of methanol and acetic acid.
5. 5. The method according to claim 4, wherein the volume ratio of water, methanol and acetic acid in the mobile phase A is 100 (1-3): 1-3; preferably, the volume ratio of methanol to acetic acid in mobile phase B is 100 (1-3).
6. 6. The method according to claim 4 or 5, wherein the elution procedure of the mobile phase in liquid chromatography is gradient elution, in particular: 0-15 min, the volume percentage of the mobile phase A is changed from 88-92% to 68-72% at a constant speed, the volume percentage of the mobile phase B is changed from 8-12% to 28-32% at a constant speed, 15-20 min, the volume percentage of the mobile phase A is changed from 68-72% to 8-12% at a constant speed, the volume percentage of the mobile phase B is changed from 28-32% to 88-92% at a constant speed, then the mobile phase A is kept unchanged to 22 min, 22-22.1 min, the volume percentage of the mobile phase A is changed from 8-12% to 88-92% at a constant speed, the volume percentage of the mobile phase B is changed from 88-92% to 8-12% at a constant speed, and then the mobile phase B is kept unchanged to 30 min.
7. 7. The method according to any one of claims 1 to 6, wherein the packing of the column in the liquid chromatography is C18, has a particle size of 3 to 5 μm, an inner diameter of 3.0 to 4.6mm, and a length of 100 to 250mm; preferably, the column temperature of the chromatographic column in the liquid chromatography is 28-40 ℃.
8. 8. The method according to any one of claims 1-7, wherein the wavelength detection of the liquid chromatography is a segmented detection, in particular: 0 th to 9 th min th, 325nm th, 9 th to 10.8 th min th, 340nm, 10.8 th to 17 th min th, 325nm, 17 th to 20 th min th, 345 nm th, 20 th to 30 th min th, and 350nm.
9. 9. The method according to any one of claims 1 to 8, wherein in the one-test-multiple-evaluation method, chlorogenic acid is used as an internal reference, a relative correction factor between chlorogenic acid and other polyphenols to be tested is calculated, and the content of polyphenols in tobacco or its product is obtained by using the relative correction factor; preferably, the calculation method of the relative correction factor is a slope correction method.
10. 10. Use of a method according to any one of claims 1-9 for detecting polyphenols in tobacco or products thereof based on a multi-test method for quality assessment, classification of tobacco or products thereof.

Description

Method for detecting polyphenol compounds in tobacco or products thereof based on one-test-multiple-evaluation method and application of method Technical Field The invention relates to the technical field of analysis and detection, in particular to a method for detecting polyphenol compounds in tobacco or products thereof based on a multi-evaluation method and application thereof. Background Tobacco and tobacco products are complex in chemical composition, and their inherent quality depends largely on the content of the various chemical components and interactions. The polyphenol compound is used as an important secondary metabolite in the tobacco, not only affects the color and the sensory flavor of the tobacco, but also is one of key indexes for evaluating the quality of the tobacco and the quality of products thereof. For example, the content and proportion of phenolic acids such as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, and flavonoids such as rutin, kaempferol-3-O-rutinoside are closely related to the aroma quality, taste characteristics, and stability of tobacco. Therefore, the establishment of an analysis method for rapidly and accurately measuring the polyphenol compounds in the tobacco has important significance for the quality evaluation of the tobacco and tobacco products. Currently, high Performance Liquid Chromatography (HPLC) and combined technology thereof are mainly used for detecting polyphenol compounds in tobacco, and the detection comprises ultraviolet spectrophotometry, capillary electrophoresis, high Performance Liquid Chromatography (HPLC) and combined technology thereof with mass spectrometry (HPLC-MS/MS). Although the methods can realize simultaneous analysis of various components, the methods still belong to the category of the traditional external standard method, namely, corresponding standard substances are needed to be provided for each polyphenol compound to be tested. However, the standard products of part of polyphenols such as new chlorogenic acid, scopoletin and the like are expensive, not easy to obtain or poor in stability, so that the external standard method is high in detection cost, and the operation flow is complex, so that the efficiency requirements of batch sample screening and conventional quality control are difficult to meet. As an innovative multi-index quality control and detection technology, the multi-evaluation QAMS method can realize synchronous quantitative analysis of various components by only using one or less internal reference substances and establishing relative correction factors between the internal reference substances and other components to be detected. The method has obvious advantages in quality control of complex systems such as traditional Chinese medicines. However, a multi-evaluation technique is not applied in the tobacco field, and meanwhile, the method is applied to analysis of polyphenol components in tobacco and tobacco products, and still faces significant technical bottlenecks and challenges (1) the difficult problem of matrix interference, namely complex chemical components of tobacco samples, and the effective separation and accurate quantification of polyphenols, particularly structural similar isomers (such as neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid) are seriously affected by a large amount of interference substances such as pigments, alkaloids and saccharides. (2) The method has the general problem that the core of the one-test-multiple-evaluation method is the reproducibility and the stability of the relative correction factors. The method can still keep the stability and reliability of the polyphenol compound relative to the correction factor in the complex matrix of the tobacco in the face of different instruments, different chromatographic columns, column flow rates and column Wen Dengse spectral condition variables, and needs to be verified through intensive research. (3) The analysis of batch samples is difficult, the sample amount and the extraction solvent volume used by the method used by the current common industry standard or literature report method are large, and the analysis of batch samples is difficult to realize. (4) The specific application is blank that at present, in the field of tobacco analysis, a multi-evaluation method is not established for eight specific polyphenol combinations of new chlorogenic acid, scopoletin, chlorogenic acid, cryptochlorogenic acid, caffeic acid, scopoletin, rutin and kaempferol-3-O-rutinoside, and is applied to reports of quality evaluation of tobacco and tobacco products. Therefore, there is an urgent need to establish a quality evaluation method for rapidly, accurately and simultaneously measuring one-measurement-multiple-evaluation of the eight key polyphenol compounds at low cost for tobacco and tobacco products, so as to make up for the defects of the prior art and provide data support and theoretical basis for quality