Method for extracting and determining tea polyphenol in tobacco extract

Abstract

The invention provides a method for extracting and determining tea polyphenol in a tobacco extract. The method for extracting and determining the tea polyphenol in the tobacco extract comprises the following steps: preparing a standard solution of a tea polyphenol substance; adding a methanol aqueous solution into the tobacco extract, and fixing the volume to obtain a tea polyphenol diluent; sealing the tea polyphenol purified liquid, and performing ultrasonic extraction to obtain a tea polyphenol extracting solution; preparing a disodium ethylene diamine tetraacetate stabilizing solution, and fixing the volume of the tea polyphenol extracting solution by using the disodium ethylene diamine tetraacetate stabilizing solution to obtain a tea polyphenol purified solution; adding a PSA adsorbent into the tea polyphenol purified liquid, carrying out centrifugal standing to obtain a to-be-detected liquid, and carrying out instrumental analysis on the to-be-detected liquid. Through ultrasonic extraction, the loss of tea polyphenol substances is less, the preparation of a to-be-detected solution is simple, and the analysis time is short; by adding the disodium ethylene diamine tetraacetate and the PSA adsorbent, the interference of other substances on the detection of the tea polyphenol substances is reduced, so that the detection of the tea polyphenol substances in the tobacco extract is more accurate.

Description

Technical Field

[0001] The present invention relates to the detection of phenolic compounds, and in particular to a method for extracting and determining tea polyphenols in tobacco extracts. Background Art

[0002] Tobacco extract contains many phenolic compounds. Tobacco tea polyphenols are mainly divided into tannins (chlorogenic acid), coumarins (scopoletin, scopoletin, esculetin) and flavonoids (rutin, flavonoids, rhamnose), etc. Among them, chlorogenic acid, rutin and scopoletin are the main phenolic substances in tobacco leaves, and chlorogenic acid accounts for 75% to 95% of the total polyphenols. Tobacco phenolic substances and their derivatives are not only metabolites of tobacco plants, but also participate in the metabolic activities of tobacco plant cells and have a regulatory effect on tobacco growth. Tea polyphenols are considered to be one of the main components of tobacco aroma. Under reasonable modulation and aging conditions, polyphenol compounds are degraded through a series of reactions to generate a series of degradation products, which can give tobacco products an elegant aroma and increase the aroma of tobacco products. It plays an important role in improving the quality of tobacco products. Accurate determination of phenolic substances in tobacco is of great significance for the development of tobacco identification and tobacco product quality control.

[0003] At present, the main methods for determining tea polyphenols are spectrophotometry, gas chromatography and liquid chromatography. Spectrophotometry can only determine the total content of polyphenols, but not the type and content of polyphenols. Experimental results show that the color temperature and color development time have a great influence on the absorbance of the chlorogenic acid standard solution; gas chromatography requires complex derivatization and the operation steps are complicated; traditional liquid chromatography requires the use of organic solvents to defatted samples, which is troublesome to operate, consumes more mobile phase, and causes polyphenol loss, making the test results of polyphenol compounds in tobacco extract inaccurate.

[0004] For example, the comparative document CN202110826485.X discloses a method for detecting polyphenol compounds in cigars, which extracts polyphenol compounds in cigars using a methanol solution of appropriate acidity and then uses a high performance liquid chromatograph for detection. This scheme can accurately determine the content of polyphenol compounds in cigars, but the scheme directly detects tobacco leaves and extracts polyphenol compounds in cigars using a methanol solution. The polyphenol compounds are not completely separated from the cigars, and there may be a problem of oxidation loss of tea polyphenols during the extraction of polyphenol compounds.

[0005] Another example is a method for detecting tea polyphenol monomers disclosed in the comparative document CN202410172935.1, which uses a liquid chromatography column to separate tea polyphenol monomers in a sample, and the separated tea polyphenol monomers enter the reactor, and an appropriate amount of dopamine is added to the reactor, and the pH of the reaction solution is adjusted to 12-13, and a post-column derivatization reaction is performed to generate a product with blue-green fluorescence, and a fluorescence detector is provided to generate a liquid chromatogram of the product. This scheme requires complex derivatization and the operation steps are relatively complicated. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for extracting and determining tea polyphenols in tobacco extracts with less loss of tea polyphenols, simple preparation, short analysis time and accurate determination of tea polyphenols.

[0007] The purpose of this disclosure is achieved through the following technical solutions:

[0008] A method for extracting and determining tea polyphenols in tobacco extract comprises the following steps:

[0009] Prepare standard solution of tea polyphenols;

[0010] Obtain tobacco extract, add methanol aqueous solution to the tobacco extract to make up the volume, so as to obtain tea polyphenol dilution solution;

[0011] The tea polyphenols dilution solution is sealed and then subjected to ultrasonic extraction to obtain a tea polyphenols extract solution;

[0012] preparing a disodium ethylenediaminetetraacetic acid stabilizing solution, and using the disodium ethylenediaminetetraacetic acid stabilizing solution to adjust the volume of the tea polyphenols extract to obtain a tea polyphenols purified solution;

[0013] Adding PSA adsorbent to the tea polyphenol purified liquid, centrifuging and standing to obtain a test liquid;

[0014] The test solution and the standard solution of the tea polyphenols are subjected to instrumental analysis.

[0015] In one embodiment, the preparation of the EDTA disodium stabilizer solution comprises the following steps:

[0016] Adding ethylenediaminetetraacetic acid into the acetonitrile solution to dissolve it, so as to obtain an ethylenediaminetetraacetic acid solution;

[0017] The ethylenediaminetetraacetic acid solution is fixed to volume with ultrapure water to obtain a disodium ethylenediaminetetraacetic acid stable solution.

[0018] In one embodiment, after dissolving ethylenediaminetetraacetic acid in acetonitrile solution to obtain ethylenediaminetetraacetic acid solution, the ethylenediaminetetraacetic acid solution is fixed to volume with ultrapure water to obtain ethylenediaminetetraacetic acid disodium stabilized solution, the following steps are also included:

[0019] Prepare ascorbic acid stabilizing solution;

[0020] The ascorbic acid stabilizing solution is added into the ethylenediaminetetraacetic acid dissolving solution.

[0021] In one embodiment, preparing the ascorbic acid stabilizing solution comprises the following steps:

[0022] adding ascorbic acid into the acetonitrile solution to dissolve it, so as to obtain an ascorbic acid solution;

[0023] The ascorbic acid solution was made up to volume with ultrapure water to obtain an ascorbic acid stable solution.

[0024] In one embodiment, preparing a standard solution of tea polyphenols comprises the following steps:

[0025] Weigh tea polyphenols, dissolve them completely with methanol, and then dilute to volume with methanol to obtain a standard stock solution;

[0026] 5-7 portions of the standard stock solution of different volumes were transferred and prepared with a methanol-water solution with a volume fraction of 50:50 to obtain standard working solutions of tea polyphenols of different concentrations.

[0027] In one embodiment, the tea polyphenol dilution is sealed and then subjected to ultrasonic extraction to obtain a tea polyphenol extract, comprising the following steps:

[0028] Sealing the tea polyphenols purified liquid;

[0029] The sealed tea polyphenols purified liquid is placed in an ultrasonic oscillation device for ultrasonic extraction, the ultrasonic frequency is 40kHz-120kHz, and the ultrasonic extraction time is 15min-20min.

[0030] In one embodiment, after adding PSA adsorbent to the tea polyphenols purified solution and centrifuging and standing to obtain a test solution, before subjecting the test solution and the tea polyphenols standard solution to instrumental analysis, the following steps are also included:

[0031] The test solution and the standard solution of tea polyphenols are filtered respectively.

[0032] In one embodiment, the instrumental analysis of the test solution and the standard solution of the tea polyphenols is performed by high performance liquid chromatography-mass spectrometry with an ESI detector.

[0033] In one embodiment, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry instrument are:

[0034] The chromatographic column is a C18 column with a specification of 100 mm × 2.1 mm and a particle size of 1.9 μm or an equivalent column; the column flow rate is 0.30 mL / min, the column oven temperature is 40°C; the injection volume is 5 μL; and / or,

[0035] The mass spectrometry conditions of the HPLC-MS / MS are:

[0036] The ion source was an electrospray ion source (ESI); the ionization mode was a positive ion mode; the electrospray voltage was +3500 V; the ion source temperature was 350° C.; the ion transfer tube temperature was 350° C.; and the scanning mode was multiple reaction monitoring (MRM).

[0037] In one embodiment, the tea polyphenols include at least one of epigallocatechin, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate.

[0038] Compared with the prior art, the present invention has at least the following advantages:

[0039] The above-mentioned method for extracting and determining tea polyphenols in tobacco extract uses ultrasonic extraction to separate tea polyphenols from the tea polyphenol extract, and the tobacco extract does not need to undergo complex derivatization and defatting treatment, so that the loss of tea polyphenols in the detection of the tobacco extract is less, the preparation of the test solution is simple, and the analysis time is short; by adding disodium ethylenediaminetetraacetic acid stabilizing solution, the tea polyphenol purification solution is made more stable, thereby improving the accuracy of the determination of tea polyphenols in the tobacco extract; by using PSA adsorbent to remove organic acids, fatty acids and metal ions in the tea polyphenol purification solution, the interference of other substances on the detection of tea polyphenols is further reduced, thereby making the detection of tea polyphenols in the tobacco extract more accurate.

[0040] Ascorbic acid is added to maintain the pH stability of the tea polyphenol purification solution and inhibit the oxidation of tea polyphenols. Disodium ethylenediaminetetraacetic acid is used to reduce the destructive effect of metals on ascorbic acid, thereby increasing the antioxidant properties of ascorbic acid and prolonging the antioxidant effect of ascorbic acid on tea polyphenols. Ultrasonic extraction of tea polyphenols is used to control the temperature to reduce the decomposition and oxidation of ascorbic acid in the tea polyphenol purification solution, thereby keeping the tea polyphenols stable and reducing the loss of tea polyphenols, thereby improving the accuracy of tea polyphenols measured by tea polyphenol extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 A flowchart of the steps of a method for extracting and determining tea polyphenols in tobacco extract according to an embodiment;

[0043] Figure 2 It is the chromatogram corresponding to tea polyphenols and the calibration curve diagram for quantitative analysis;

[0044] Figure 3 This is the chromatogram of the tea polyphenols in Comparative Example 1;

[0045] Figure 4 is the chromatogram of the tea polyphenols of Example 1;

[0046] Figure 5 This is the chromatogram of the tea polyphenols of Example 2. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of the present disclosure, the present disclosure will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present disclosure are given in the drawings. However, the present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present disclosure more thoroughly and comprehensively understood.

[0048] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0050] In order to better understand the technical solutions and beneficial effects of the present invention, the present invention is further described in detail below in conjunction with specific embodiments:

[0051] The present invention relates to a method for extracting and determining tea polyphenols in tobacco extract. In one embodiment, the method for extracting and determining tea polyphenols in tobacco extract comprises the following steps: preparing a standard solution of tea polyphenols; taking tobacco extract, adding methanol water solution to fix the volume, so as to obtain tea polyphenol dilution solution; sealing the tea polyphenol purified solution and performing ultrasonic extraction to obtain tea polyphenol extract solution; preparing disodium ethylenediaminetetraacetic acid stabilizing solution, and using the disodium ethylenediaminetetraacetic acid stabilizing solution to fix the volume of the tea polyphenol extract solution to obtain tea polyphenol purified solution; adding PSA adsorbent to the tea polyphenol purified solution, and centrifuging and standing to obtain a test solution; instrumentally analyzing the test solution and the standard solution of tea polyphenols.

[0052] See also Figure 1 , which is a method for extracting and determining tea polyphenols in tobacco extract according to an embodiment of the present invention, comprising the following steps:

[0053] S101 prepares a standard solution of tea polyphenols. In this embodiment, the standard solution of tea polyphenols is subjected to spiked detection, and the spiked standard solution can evaluate the accuracy and reliability of the analysis method by adding a certain amount of a standard substance of known concentration to the original sample.

[0054] S103 obtains tobacco extract, and adds methanol aqueous solution to the tobacco extract to fix the volume to obtain tea polyphenol dilution. In this embodiment, pure methanol is well miscible with most organic substances, but pure methanol is volatile, and the volatility of methanol aqueous solution is relatively weakened due to the presence of water, and the components of the tea polyphenol dilution fixed by methanol are relatively stable.

[0055] Furthermore, obtaining tobacco extract includes the following steps: selecting suitable tobacco varieties and parts, such as tobacco leaves; performing preliminary treatment including washing and drying tobacco leaves to remove impurities; extracting effective ingredients through physical methods, such as solvent extraction, and soaking tobacco leaves with organic solvents to dissolve alkaloids and other components into the solvent; and finally filtering and concentrating the extract to obtain tobacco extract. Through the steps of obtaining tobacco extract, the quality of the tobacco extract is controlled and the quality of the tobacco extract is stable.

[0056] S105: sealing the tea polyphenol dilution solution and then performing ultrasonic extraction to obtain a tea polyphenol extract. In this embodiment, the ultrasonic extraction allows the tea polyphenols to separate from the tea polyphenol extract and allows the tea polyphenols to be evenly mixed with the methanol aqueous solution, thereby making it easier to separate the tea polyphenols from the tea polyphenol extract.

[0057] S107 configures disodium ethylenediaminetetraacetic acid stabilizing solution, and uses the disodium ethylenediaminetetraacetic acid stabilizing solution to constant volume the tea polyphenols extract to obtain tea polyphenols purified solution. In the present embodiment, the tea polyphenols purified solution obtained by constant volume is subjected to vortex oscillation to make disodium ethylenediaminetetraacetic acid and tea polyphenols substances evenly mixed. Disodium ethylenediaminetetraacetic acid is a weak acid and weak alkaline ligand, which can form a stable complex with a variety of metal ions, and a corresponding stable complex is formed by the coordination atoms in its molecules and the free electrons or ligands in the metal ions. The formation of the complex can prevent the discoloration, deterioration, turbidity and other phenomena caused by metal ions in the tea polyphenols extract. By constant volume of the tea polyphenols extract with the disodium ethylenediaminetetraacetic acid stabilizing solution, the metal ion concentration in the tea polyphenols extract is reduced, and the tea polyphenols purified solution is improved, thereby improving the accuracy and reliability of the analysis.

[0058] S109: PSA adsorbent is added to the tea polyphenol purified liquid, and centrifuged and allowed to stand to obtain the test liquid. In this embodiment, the molecular structure of the PSA adsorbent is relatively large and has a certain steric hindrance effect. In the solution, this steric hindrance effect can make it easier to adsorb on the surface of the impurity molecules, thereby removing them. It is understandable that organic acids such as citric acid, malic acid and other organic acids in the tobacco extract of the tea polyphenol purified liquid will affect the component analysis and subsequent application of the extract, and the PSA adsorbent can remove them through ion exchange; fatty acids in tobacco, such as palmitic acid, linoleic acid and other fatty acids, may interfere with the separation and identification of effective ingredients in tobacco extracts, and the PSA adsorbent can effectively adsorb fatty acids and improve the purity of the extract; tobacco will absorb metal ions in the soil during growth, such as copper, iron, zinc, etc. These metal ions may catalyze certain chemical reactions and affect the stability and quality of the extract. The PSA adsorbent can also produce chelation with metal ions, thereby removing metal ions and reducing the interference of other substances in the detection of tea polyphenol substances.

[0059] S111 performs instrumental analysis on the test solution and the standard solution of the tea polyphenols. In this embodiment, the test solution obtained by instrumental analysis of the tea polyphenols standard solution and the test solution is stable, so that the structure of the instrumental analysis is accurate. The standard solution and the test solution are spiked and tested, and the analysis results of the samples before and after the addition of the standard substance are compared to determine whether there is a systematic error or deviation in the analysis method, thereby verifying the accuracy of the analysis method.

[0060] The above-mentioned method for extracting and determining tea polyphenols in tobacco extract uses ultrasonic extraction to separate tea polyphenols from the tea polyphenol extract, and the tobacco extract does not need to undergo complex derivatization and defatting treatment, so that the loss of tea polyphenols in the detection of the tobacco extract is less, the preparation of the test solution is simple, and the analysis time is short; by adding disodium ethylenediaminetetraacetic acid stabilizing solution, the tea polyphenol purification solution is made more stable, thereby improving the accuracy of the determination of tea polyphenols in the tobacco extract; by using PSA adsorbent to remove organic acids, fatty acids and metal ions in the tea polyphenol purification solution, the interference of other substances on the detection of tea polyphenols is further reduced, thereby making the detection of tea polyphenols in the tobacco extract more accurate.

[0061] In one embodiment, the preparation of the EDTA disodium stabilizer solution comprises the following steps:

[0062] Adding ethylenediaminetetraacetic acid into the acetonitrile solution to dissolve it, so as to obtain an ethylenediaminetetraacetic acid solution;

[0063] The ethylenediaminetetraacetic acid solution is constant volume with ultrapure water to obtain disodium ethylenediaminetetraacetic acid stable solution. In the present embodiment, disodium ethylenediaminetetraacetic acid has a low solubility in acetonitrile solution, therefore, after ethylenediaminetetraacetic acid is constant volume with ultrapure water, it is more stable to obtain disodium ethylenediaminetetraacetic acid stable solution. When the ethylenediaminetetraacetic acid solution is constant volume with ultrapure water, ethylenediaminetetraacetic acid is added to acetonitrile solution and dissolved in a beaker, the ethylenediaminetetraacetic acid solution is first moved into a volumetric flask, and then the beaker is repeatedly rinsed with ultrapure water, after the rinse solution is transferred to the volumetric flask, the ethylenediaminetetraacetic acid stable solution after constant volume is obtained. Rinse with ultrapure water, the residue of ethylenediaminetetraacetic acid is reduced, thereby improving the stability of the test.

[0064] In one embodiment, after dissolving ethylenediaminetetraacetic acid in acetonitrile solution to obtain ethylenediaminetetraacetic acid solution, the ethylenediaminetetraacetic acid solution is fixed to volume with ultrapure water to obtain ethylenediaminetetraacetic acid disodium stabilized solution, the following steps are also included:

[0065] Prepare ascorbic acid stabilizing solution;

[0066] The ascorbic acid stabilizing solution is added to the ethylenediaminetetraacetic acid dissolving solution. In this embodiment, ascorbic acid can neutralize free radicals such as -OH, R- and O 2-Etc., thus having an antioxidant effect. Metal ions in the solution will have a negative impact on antioxidants such as ascorbic acid, promoting their oxidative decomposition. Disodium EDTA can chelate metal ions in the solution, such as iron, copper, etc., reducing the destructive effect of metals on ascorbic acid, thereby increasing the antioxidant properties of ascorbic acid, making tea polyphenols more difficult to be oxidized. By adding the synergistic effect of disodium EDTA stabilizing solution and ascorbic acid stabilizing solution, the tea polyphenols in the tea polyphenols purified solution are more stable, making the test results of tea polyphenols more accurate.

[0067] In one embodiment, preparing the ascorbic acid stabilizing solution comprises the following steps:

[0068] adding ascorbic acid into the acetonitrile solution to dissolve it, so as to obtain an ascorbic acid solution;

[0069] The ascorbic acid solution is constant volume with ultrapure water to obtain an ascorbic acid stabilizing solution. In the present embodiment, the polarity of acetonitrile is moderate, so that ascorbic acid can be preferably dissolved in the acetonitrile solution, and the solvent of the ascorbic acid stabilizing solution is the same as the solvent of the disodium ethylenediaminetetraacetic acid stabilizing solution, so that the ascorbic acid stabilizing solution is added to the tea polyphenols purified solution without introducing more types of solvents, thereby reducing the interference of the test of the tea polyphenols substance, making the result of the test of the tea polyphenols substance more accurate.

[0070] Furthermore, in one of the embodiments, the pH of the tea polyphenols purification liquid is controlled at 4-8. In this embodiment, since the phenolic hydroxyl groups in tea polyphenols are acidic, their ionization state will be affected by the pH value of the mobile phase. When the pH value is low, the phenolic hydroxyl groups are not easy to ionize, and tea polyphenols exist more in molecular form; when the pH value is high, the degree of ionization of the phenolic hydroxyl groups increases, and the amount of tea polyphenols existing in ionic form increases. Different existence forms will affect their retention behavior and separation effect in the chromatographic column. Therefore, it is necessary to select a pH range that can ensure a good separation effect and maintain the stability of tea polyphenols. Specifically, when the pH is 4-8, the tea polyphenols are very stable within 1.5 hours and have good heat resistance and acid resistance.

[0071] Furthermore, the solution of disodium ethylenediaminetetraacetic acid is a nearly neutral solution, so disodium ethylenediaminetetraacetic acid has little effect on the tea polyphenols purified solution. Ascorbic acid has a certain acidity and can affect the pH value of the solution. The initial concentration of the ascorbic acid stabilizing solution is 0.1 mg / mL-0.4 mg / mL, and the initial addition amount of the ascorbic acid stabilizing solution added to the ethylenediaminetetraacetic acid dissolving solution is 1 mL-10 mL. Therefore, when the ascorbic acid stabilizing solution is added to the tea polyphenols purified solution, the following steps are also included:

[0072] After the ascorbic acid stabilizing solution is prepared, the pH of the ascorbic acid stabilizing solution is tested.

[0073] If the pH of the ascorbic acid stabilizing solution is less than 4, ultrapure water is added to dilute the ascorbic acid stabilizing solution, and the test is performed again. When the pH of the ascorbic acid stabilizing solution is greater than 4, the dilution is stopped.

[0074] The pH of the disodium ethylenediaminetetraacetic acid solution is tested. If the pH is greater than 8, 1 mL-2 mL of ascorbic acid stabilizing solution is added to the disodium ethylenediaminetetraacetic acid solution again; the pH of the disodium ethylenediaminetetraacetic acid solution is tested. If the pH of the disodium ethylenediaminetetraacetic acid solution is still greater than 8, 1 mL-2 mL of the ascorbic acid stabilizing solution is added again. In the present embodiment, by controlling the pH of the ascorbic acid stabilizing solution to be greater than 4, it is ensured that the pH of the ascorbic acid stabilizing solution will not drop below 4 after the disodium ethylenediaminetetraacetic acid solution is added, so that the pH of the disodium ethylenediaminetetraacetic acid solution obtained after the ethylenediaminetetraacetic acid solution is constant to volume is greater than 4, and the pH of the disodium ethylenediaminetetraacetic acid solution is tested, and the ascorbic acid stabilizing solution is appropriately added, so that the pH of the disodium ethylenediaminetetraacetic acid solution drops to below 8, so that the pH of the disodium ethylenediaminetetraacetic acid solution is controlled at 4-8, so that the tea polyphenols remain stable.

[0075] In one embodiment, preparing a standard solution of tea polyphenols comprises the following steps:

[0076] Weigh tea polyphenols, dissolve them completely with methanol, and then dilute to volume with methanol to obtain a standard stock solution;

[0077] 5-7 portions of the standard stock solution of different volumes were taken and prepared with a methanol-water solution with a volume fraction of 50:50 to obtain standard working solutions of tea polyphenols of different concentrations. In this embodiment, the methanol-water solution has low volatility, and the tea polyphenols are diluted by the methanol-water solution, so that the obtained standard working solution of tea polyphenols is relatively stable. By preparing standard working solutions of tea polyphenols of different concentrations, a standard curve of the standard working solution of tea polyphenols is drawn, and tobacco extracts are spiked and tested to evaluate the accuracy of the spiked test.

[0078] In one embodiment, the tea polyphenol dilution is sealed and then subjected to ultrasonic extraction to obtain a tea polyphenol extract, comprising the following steps:

[0079] Sealing the tea polyphenols purified liquid;

[0080] The sealed tea polyphenols purified liquid is placed in an ultrasonic oscillation device for ultrasonic extraction, the ultrasonic frequency is 40kHZ-120kHz, and the ultrasonic extraction time is 15min-20min. In this embodiment, ultrasonic extraction separates tea polyphenols from the tea polyphenols extract, and makes the tea polyphenols extract and methanol water mix evenly; tea polyphenols are easily affected by environmental factors such as temperature and humidity during storage or processing, and undergo oxidation reactions, resulting in reduced stability. The structure of tea polyphenols contains phenolic groups. When the ultrasonic time is greater than 20min, the phenolic groups make tea polyphenols easy to react with oxygen, causing tea polyphenol loss; when the ultrasonic time is less than 15min, the tea polyphenols are not fully separated from the tea polyphenols extract, thereby affecting the accuracy of tea polyphenols detection; the ultrasonic extraction time is within 15min-20min, so that the tea polyphenols are fully separated from the tea polyphenols extract, avoiding the loss of tea polyphenols caused by the reaction of tea polyphenols with oxygen, and thus making the results of tea polyphenols detection more reliable.

[0081] Furthermore, environmental factors such as light, high temperature, and high humidity can also promote the oxidation of tea polyphenols. The tea polyphenols purification liquid is sealed and then ultrasonically extracted. Therefore, when the tea polyphenols purification liquid is sealed and then ultrasonically extracted, the temperature of the ultrasonic extraction is 30°C-60°C. In this embodiment, when the temperature of the ultrasonic extraction is lower than 30°C, the movement speed between molecules is slow, and the tea polyphenols are slowly separated from the tea polyphenols extract; when the temperature of the ultrasonic extraction is greater than 60°C, the boiling point of methanol is 64.7°C, and methanol is easy to volatilize, thereby affecting the components of the tea polyphenols purification liquid, and when the temperature is higher than 60°C, the tea polyphenols are easily oxidized by heat, thereby affecting the components of the tea polyphenols purification liquid. By setting the temperature of the ultrasonic extraction to 30°C-60°C, the boiling point of the aqueous solution of methanol is low, and the methanol in the tea polyphenols purification liquid evaporates slowly, so that the components of the tea polyphenols purification liquid are stable, and the tea polyphenols in the tea polyphenols extract obtained by ultrasonic extraction remain stable in a low temperature environment, so that the accuracy of the detection of tea polyphenols is improved.

[0082] It is understandable that when the pH of the disodium ethylenediaminetetraacetic acid solution is tested, when the pH of the disodium ethylenediaminetetraacetic acid solution is 6-8, the amount of ascorbic acid in the disodium ethylenediaminetetraacetic acid solution is relatively small, and the antioxidant property of the tea polyphenols purified solution is low. When the pH of the disodium ethylenediaminetetraacetic acid solution is controlled at 4-6, the content of ascorbic acid in the disodium ethylenediaminetetraacetic acid stabilizer is higher. When the temperature of the ultrasonic extraction is 40°C-60°C, when the tea polyphenols extract is fixed to volume with the disodium ethylenediaminetetraacetic acid stabilizer, the ascorbic acid in the disodium ethylenediaminetetraacetic acid stabilizer is easily decomposed by heat, which reduces the antioxidant property of the tea polyphenols purified solution, resulting in oxidation loss of tea polyphenols, thereby affecting the accuracy of the test results of tea polyphenols.

[0083] Furthermore, in one embodiment, if the pH of the EDTA solution is 6-8, when preparing the EDTA disodium stabilizing solution, the tea polyphenol dilution solution is sealed and then ultrasonically extracted to obtain the tea polyphenol extract, and the temperature of the ultrasonic extraction is controlled at 30°C-40°C;

[0084] If the temperature of the ultrasonic extraction is between 40°C and 60°C, when preparing the disodium ethylenediaminetetraacetic acid stabilizing solution, the pH of the ethylenediaminetetraacetic acid solution is controlled at 4-6.

[0085] In this embodiment, when the temperature is greater than 60°C, the high temperature destroys the molecular structure and stability of ascorbic acid, which significantly accelerates the decomposition rate of ascorbic acid, while the oxidative decomposition rate of ascorbic acid at a temperature of 20°C-40°C is relatively slow, and the oxidative decomposition rate of ascorbic acid at a temperature of 40°C-60°C is relatively fast. Therefore, the temperature of ultrasonic extraction is controlled at 30°C-40°C, so that the temperature of the obtained tea polyphenol extract is at 30°C-40°C, so that the tea polyphenol extract at 30°C-40°C has less effect on the ascorbic acid decomposition of the disodium ethylenediaminetetraacetic acid stabilized solution, so that the obtained tea polyphenol purified solution is relatively stable. When preparing the disodium ethylenediaminetetraacetic acid stabilizing solution, the pH of the ethylenediaminetetraacetic acid dissolving solution is controlled at 4-6, so that the disodium ethylenediaminetetraacetic acid stabilizing solution obtained after the ethylenediaminetetraacetic acid dissolving solution is fixed to volume has a higher ascorbic acid content, thereby reducing the influence of the temperature increase on the oxidation rate of ascorbic acid, ensuring the ascorbic acid content of the disodium ethylenediaminetetraacetic acid stabilizing solution, so that the ascorbic acid in the tea polyphenol purified solution maintains good antioxidant properties, thereby making the test results of tea polyphenols more accurate.

[0086] It can be understood that the present scheme needs to take into account the pH stability of tea polyphenols, the purification of metal ions in tea polyphenols, the inhibition of oxidation of tea polyphenols to improve the accuracy of detection, and the temperature range of tea polyphenol ultrasonic extraction temperature and ascorbic acid to reduce ascorbic acid decomposition. The metal ions of the tea polyphenol purification solution are purified by disodium ethylenediaminetetraacetic acid, making the tea polyphenol purification solution more stable, thereby improving the accuracy of the determination of tea polyphenols in tobacco extracts; by adding ascorbic acid to maintain the pH stability of the tea polyphenol purification solution and inhibiting the oxidation of tea polyphenols, disodium ethylenediaminetetraacetic acid is used to reduce the destructive effect of metals on ascorbic acid, thereby increasing the antioxidant properties of ascorbic acid, thereby prolonging the antioxidant effect of ascorbic acid on tea polyphenols; by controlling the temperature of tea polyphenol ultrasonic extraction, the decomposition and oxidation of ascorbic acid in the tea polyphenol purification solution are reduced, thereby keeping the tea polyphenols stable and reducing the loss of tea polyphenols, thereby improving the accuracy of tea polyphenols measured by tea polyphenol extracts.

[0087] In one embodiment, after adding PSA adsorbent to the tea polyphenols purified solution and centrifuging and standing to obtain a test solution, before subjecting the test solution and the tea polyphenols standard solution to instrumental analysis, the following steps are also included:

[0088] The liquid to be tested and the standard solution of the tea polyphenols are filtered separately. In this embodiment, by filtering the liquid to be tested and the standard solution of the tea polyphenols separately, impurities in the liquid to be tested and the tea polyphenols are removed, and at the same time, the processing steps of the liquid to be tested and the standard solution of the tea polyphenols are made the same, which improves the consistency of the detection process of the liquid to be tested and the standard solution of the tea polyphenols, so that the detection results of the liquid to be tested and the standard solution of the tea polyphenols are more accurate.

[0089] In one embodiment, the instrumental analysis of the test liquid and the standard solution of the tea polyphenols is detected by a high performance liquid chromatography-mass spectrometer with an ESI detector. In this embodiment, when the test liquid and the standard solution of the tea polyphenols are detected by a high performance liquid chromatography-mass spectrometer with an ESI detector, the test liquid and the standard solution of the tea polyphenols are detected by reversed phase high performance liquid chromatography. The separation principle of reversed phase high performance liquid chromatography is based on the difference in the distribution coefficient of the substance between the stationary phase and the mobile phase. During the separation process, the mobile phase carries the sample components through a chromatographic column equipped with a high-efficiency stationary phase. Different components have different sizes and strengths of interaction with the stationary phase, resulting in different retention times in the stationary phase, so that they flow out of the chromatographic column one after another and enter the detector for detection. Through reversed phase high performance liquid chromatography, the high performance liquid chromatography-mass spectrometer with an ESI detector has a good separation effect and high resolution on the tea polyphenols of the test liquid and the standard solution of the tea polyphenols.

[0090] In one embodiment, the chromatographic conditions of the HPLC-MS / MS are:

[0091] The chromatographic column is a C18 chromatographic column with a specification of 100mm×2.1mm and a particle size of 1.9μm or an equivalent column; the column flow rate is 0.30mL / min, the column oven temperature is 40°C; the injection volume is 5μL; the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry instrument are as follows: the ion source is an electrospray ion source (ESI); the ionization mode is a positive ion mode; the electrospray voltage is +3500V; the ion source temperature is 350°C; the ion transfer tube temperature is 350°C; and the scanning mode is multiple reaction detection (MRM). In this embodiment, the chromatographic conditions and mass spectrometry conditions are set by the high performance liquid chromatography-mass spectrometry instrument to separate, detect and quantitatively analyze the test liquid and the standard solution of the tea polyphenols. The high performance liquid chromatography-mass spectrometry can detect a variety of tea phenols at the same time, thereby saving detection time.

[0092] In one embodiment, the tea polyphenols include at least one of epigallocatechin, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate. In this embodiment, tea polyphenols such as epigallocatechin, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate in tobacco extract can produce tobacco aroma. Tea phenols and their derivatives in tobacco are not only metabolites of tobacco plants, but also participate in the metabolic activities of tobacco plant cells and have a regulatory effect on tobacco growth. Therefore, accurate determination of tea phenols in tobacco is of great significance for the development of tobacco identification and tobacco product quality control.

[0093] Compared with the prior art, the present invention has at least the following advantages:

[0094] The above-mentioned method for extracting and determining tea polyphenols in tobacco extract uses ultrasonic extraction to separate tea polyphenols from the tea polyphenol extract, and the tobacco extract does not need to undergo complex derivatization and defatting treatment, so that the loss of tea polyphenols in the detection of the tobacco extract is less, the preparation of the test solution is simple, and the analysis time is short; by adding disodium ethylenediaminetetraacetic acid stabilizing solution, the tea polyphenol purification solution is made more stable, thereby improving the accuracy of the determination of tea polyphenols in the tobacco extract; by using PSA adsorbent to remove organic acids, fatty acids and metal ions in the tea polyphenol purification solution, the interference of other substances on the detection of tea polyphenols is further reduced, thereby making the detection of tea polyphenols in the tobacco extract more accurate.

[0095] Ascorbic acid is added to maintain the pH stability of the tea polyphenol purification solution and inhibit the oxidation of tea polyphenols. Disodium ethylenediaminetetraacetic acid is used to reduce the destructive effect of metals on ascorbic acid, thereby increasing the antioxidant properties of ascorbic acid and prolonging the antioxidant effect of ascorbic acid on tea polyphenols. Ultrasonic extraction of tea polyphenols is used to control the temperature to reduce the decomposition and oxidation of ascorbic acid in the tea polyphenol purification solution, thereby keeping the tea polyphenols stable and reducing the loss of tea polyphenols, thereby improving the accuracy of tea polyphenols measured by tea polyphenol extracts.

[0096] Some specific examples are listed below, and if % is mentioned, it means percentage by weight. It should be noted that the following examples do not exhaust all possible situations, and the materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0097] Preparation of standard solution: Accurately weigh a certain amount of epigallocatechin, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate standard substances into a brown volumetric flask, dissolve them completely with methanol, and then dilute with methanol to prepare a primary standard stock solution. Take 5 to 7 portions of the primary standard stock solution of different volumes and prepare standard working solutions of different concentrations with 50:50 methanol and water (volume fraction). Perform instrumental analysis.

[0098] Example 1

[0099] Weigh 0.5 g of electronic cigarette liquid to an accuracy of 0.001 g, place it in a 10 mL volumetric flask, add a 50:50 methanol-water (volume fraction) solution to make the volume 10 mL, seal it with a lid and place it in an ultrasonic oscillator, extract it at a frequency of 120 KHZ for 15 min, and obtain a tea polyphenol extract. Take 1 mL of the tea polyphenol extract and place it in a 10 mL volumetric flask;

[0100] Prepare a disodium ethylenediaminetetraacetic acid stabilizing solution, weigh 0.50 g (accurate to 0.0001 g) of disodium ethylenediaminetetraacetic acid into a 500 mL beaker, add 100 mL of acetonitrile solution to dissolve, add 300 mL of ultrapure water to mix thoroughly, transfer to a 1000 mL volumetric flask, rinse the beaker three times with ultrapure water, transfer the rinse solution to a 1000 mL volumetric flask, and finally dilute to the mark with ultrapure water to obtain a disodium ethylenediaminetetraacetic acid stabilizing solution;

[0101] The 1 mL tea polyphenol extract in a 10 mL volumetric flask was fixed to volume with the disodium ethylenediaminetetraacetic acid stabilizing solution, and after vortexing for 2 minutes, a tea polyphenol purified solution was obtained, and 2 mL of the tea polyphenol purified solution was transferred to a 10 mL centrifuge tube, and after adding 0.5 g of PSA adsorbent, centrifuged for 5-10 minutes, and the test solution was obtained after standing. The test solution was filtered through a 0.22 um filter membrane and then analyzed by a high performance liquid chromatography-mass spectrometer; Example 1 A plurality of samples of different electronic cigarette liquids were taken for detection, and a standard solution of tea polyphenols was added to perform a spiked recovery test.

[0102] Example 2

[0103] Weigh 0.5 g of electronic cigarette liquid to an accuracy of 0.001 g, place it in a 10 mL volumetric flask, add a 50:50 methanol-water (volume fraction) solution to make the volume 10 mL, seal it with a lid and place it in an ultrasonic oscillator, extract it at a frequency of 120 KHZ for 15 min, and obtain a tea polyphenol extract. Take 1 mL of the tea polyphenol extract and place it in a 10 mL volumetric flask;

[0104] Prepare disodium ethylenediaminetetraacetic acid solution, weigh 0.50g (accurate to 0.0001g) of disodium ethylenediaminetetraacetic acid into a 500mL beaker, add 100mL of acetonitrile solution to dissolve, add 300mL of ultrapure water to mix thoroughly, transfer to a 1000mL volumetric flask, rinse the beaker three times with ultrapure water, and transfer the rinse solution to a 1000mL volumetric flask of disodium ethylenediaminetetraacetic acid solution;

[0105] Prepare ascorbic acid stabilizing solution, weigh 0.50 g (accurate to 0.0001 g) of ascorbic acid to a 500 mL beaker, add 100 mL of acetonitrile solution to dissolve, add 300 mL of ultrapure water and mix thoroughly, transfer to a 500 mL volumetric flask, rinse three times with ultrapure water, and then adjust the volume with ultrapure water to obtain ascorbic acid stabilizing solution, and gradually add an appropriate amount of ascorbic acid stabilizing solution to a 1000 mL volumetric flask containing disodium ethylenediaminetetraacetic acid stabilizing solution; finally, adjust the volume of disodium ethylenediaminetetraacetic acid solution to 1000 mL with ultrapure water to obtain disodium ethylenediaminetetraacetic acid stabilizing solution;

[0106] The disodium ethylenediaminetetraacetic acid stabilizing solution was used to make up 1 mL of the tea polyphenol extract in a 10 mL volumetric flask, and after vortexing for 2 minutes, a tea polyphenol purified solution was obtained, and 2 mL of the tea polyphenol purified solution was transferred to a 10 mL centrifuge tube, and after adding 0.5 g of PSA adsorbent, centrifuged for 5-10 minutes, and the test solution was obtained after standing. The test solution was filtered through a 0.22 um filter membrane and then analyzed by a high performance liquid chromatography-mass spectrometer; Example 2 A plurality of electronic cigarette liquid samples were taken for detection, and a standard solution of tea polyphenols was added to perform a spiked recovery test.

[0107] Comparative Example 1

[0108] Weigh 0.5 g of e-cigarette liquid to an accuracy of 0.001 g, place it in a 10 mL volumetric flask, prepare an extraction solution with a volume fraction of water: methanol: acetic acid of 10:88:2, mix well, add the extraction solution to the volumetric flask containing the e-cigarette liquid to make the volume up to 10 mL, and obtain the sample extraction solution.

[0109] After vortexing for 2 minutes, 2 mL of the sample extraction solution was transferred to a 10 mL centrifuge tube and allowed to stand to obtain the test solution, which was filtered through a 0.22 um filter membrane and analyzed by a high performance liquid chromatography-mass spectrometer. For the comparative ratio, multiple samples of electronic cigarette liquid were taken for testing.

[0110] Table 1 Test results of tea polyphenols in Example 2

[0111]

[0112] Table 2 Test results of tea polyphenols in comparative example 1

[0113]

[0114] Table 3 Test results of spiked recovery of tea polyphenols in Example 1

[0115]

[0116]

[0117] Table 4: Test results of spiked recovery of tea polyphenols in Example 2

[0118]

[0119]

[0120] Figure 2 In order, they are the chromatograms of tea polyphenols corresponding to epigallocatechin gallate ECG, epicatechin EC, catechin C, epigallocatechin EGC and epicatechin gallate EGCG and the calibration curves of quantitative analysis.

[0121] from Figure 2 , Table 1 and Figure 4 It can be seen that the extraction and determination method of tea polyphenols in tobacco extract detects tobacco extract, obtains a clear chromatogram, and can accurately measure the content of tea polyphenols in tobacco extract, such as epigallocatechin gallate ECG, epicatechin EC, catechin C, epigallocatechin EGC and epicatechin gallate EGCG.

[0122] It can be seen from Tables 3 and 4 that the ECG, EC, C and EGC in tea polyphenols in Example 2 are all greater than 81%, indicating that the standard substances added in Example 2 are well recovered and the test accuracy is high; by detecting tobacco extracts through the extraction and determination method of tea polyphenols in tobacco extracts, the average spiked recoveries of ECG, EC, C and EGC in Example 2 are greater than those in Example 1, and the standard deviation SD and relative standard deviation RSD% of C, EGC and EGCG in Example 2 are smaller, indicating that after the addition of ascorbic acid, ascorbic acid inhibits the oxidation of tea polyphenols and reduces the interference of other impurities, and the results of detecting tea polyphenols through the extraction and determination method of tea polyphenols in tobacco extracts are more stable.

[0123] from Figures 3 to 5 It can be seen that Figure 3 The chromatogram for the comparative example shows that the chromatogram peaks obtained by the traditional liquid chromatography method are messy and the results of the analysis and test are not accurate enough; Figure 4 is the chromatogram of Example 1, Figure 5 is the chromatogram of Example 2, Figure 4 and Figure 5The chromatographic peaks of epigallocatechin gallate ECG, epicatechin EC, catechin C, epigallocatechin gallate EGC and epicatechin gallate EGCG can all be clearly seen, indicating that the test results of tobacco extracts detected by the extraction and determination method of tea polyphenols in tobacco extracts are accurate; Figure 5 The chromatographic peaks are cleaner, indicating that the addition of ascorbic acid inhibits the oxidation of tea polyphenols and reduces the interference of other impurities, making the detection results of tea polyphenols more accurate.

[0124] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the disclosed patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the disclosed patent shall be subject to the attached claims.

Claims

1. A method for extracting and determining tea polyphenols in tobacco extract, characterized in that: The steps include: Prepare standard solution of tea polyphenols; Obtain tobacco extract, add methanol aqueous solution to the tobacco extract to make up the volume, so as to obtain tea polyphenol dilution solution; The tea polyphenols dilution solution is sealed and then subjected to ultrasonic extraction to obtain a tea polyphenols extract solution; preparing a disodium ethylenediaminetetraacetic acid stabilizing solution, and using the disodium ethylenediaminetetraacetic acid stabilizing solution to adjust the volume of the tea polyphenols extract to obtain a tea polyphenols purified solution; Adding PSA adsorbent to the tea polyphenol purified liquid, centrifuging and standing to obtain a test liquid; The test solution and the standard solution of the tea polyphenols are subjected to instrumental analysis.

2. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: The preparation of EDTA disodium stabilizer solution includes the following steps: Adding ethylenediaminetetraacetic acid into the acetonitrile solution to dissolve it, so as to obtain an ethylenediaminetetraacetic acid solution; The ethylenediaminetetraacetic acid solution is fixed to volume with ultrapure water to obtain a disodium ethylenediaminetetraacetic acid stable solution.

3. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: After ethylenediaminetetraacetic acid is added to an acetonitrile solution to dissolve to obtain an ethylenediaminetetraacetic acid solution, the ethylenediaminetetraacetic acid solution is fixed to volume with ultrapure water to obtain a disodium ethylenediaminetetraacetic acid stable solution, and the following steps are also included: Prepare ascorbic acid stabilizing solution; The ascorbic acid stabilizing solution is added into the ethylenediaminetetraacetic acid dissolving solution.

4. The method for extracting and determining tea polyphenols in tobacco extract according to claim 3, characterized in that: The preparation of ascorbic acid stabilizing solution includes the following steps: adding ascorbic acid into the acetonitrile solution to dissolve it, so as to obtain an ascorbic acid solution; The ascorbic acid solution was made up to volume with ultrapure water to obtain an ascorbic acid stable solution.

5. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: The preparation of the standard solution of tea polyphenols includes the following steps: Weigh tea polyphenols, dissolve them completely with methanol, and then dilute to volume with methanol to obtain a standard stock solution; 5-7 portions of the standard stock solution of different volumes were transferred and prepared with a methanol-water solution with a volume fraction of 50:50 to obtain standard working solutions of tea polyphenols of different concentrations.

6. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: The tea polyphenol dilution is sealed and then subjected to ultrasonic extraction to obtain a tea polyphenol extract, comprising the following steps: Sealing the tea polyphenols purified liquid; The sealed tea polyphenols purified liquid is placed in an ultrasonic oscillation device for ultrasonic extraction, the ultrasonic frequency is 40kHz-120kHz, and the ultrasonic extraction time is 15min-20min.

7. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: After adding PSA adsorbent to the tea polyphenols purified liquid and centrifuging and standing to obtain a test liquid, before subjecting the test liquid and the tea polyphenols standard solution to instrumental analysis, the following steps are also included: The test solution and the standard solution of tea polyphenols are filtered respectively.

8. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: The instrumental analysis of the test solution and the standard solution of the tea polyphenols is performed by using a high performance liquid chromatography-mass spectrometer with an ESI detector.

9. The method for extracting and determining tea polyphenols in tobacco extract according to claim 8, characterized in that: The chromatographic conditions of the HPLC-MS / MS are: The chromatographic column is a C18 column with a specification of 100 mm × 2.1 mm and a particle size of 1.9 μm or an equivalent column; the column flow rate is 0.30 mL / min, the column oven temperature is 40°C; the injection volume is 5 μL; and / or, The mass spectrometry conditions of the HPLC-MS / MS instrument are: The ion source was an electrospray ion source (ESI); the ionization mode was a positive ion mode; the electrospray voltage was +3500 V; the ion source temperature was 350° C.; the ion transfer tube temperature was 350° C.; and the scanning mode was multiple reaction monitoring (MRM).

10. The method for extracting and determining tea polyphenols in tobacco extract according to claim 1, characterized in that: The tea polyphenol substance includes at least one of epigallocatechin, catechin, epicatechin, epigallocatechin gallate and epicatechin gallate.

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