Simple and convenient quantitative detection method for excessive coumarin in tobacco essence

Abstract

The invention discloses a simple and convenient quantitative detection method for excessive coumarin in tobacco essence. The simple and convenient quantitative detection method comprises the following steps: extracting coumarin in tobacco essence to be detected; carrying out chromatography on the obtained extract by using a silica gel chromatography plate, and then carrying out ultraviolet color development; and identifying whether the tobacco essence to be detected contains coumarin or not through ultraviolet color development and by referring to a coumarin standard substance. And if the to-be-detected tobacco flavor contains coumarin, further shooting and processing an ultraviolet developing picture after chromatography to obtain a coumarin selected area gray value, and then determining the content of coumarin in the to-be-detected tobacco flavor according to the established relation curve of the coumarin selected area gray value and the coumarin concentration. The method does not need to depend on large analytical instruments and equipment, extraction operation is simple, interference is little, the recognition method is simple and easy to control, and quantitative detection is convenient and intelligent.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco, and in particular to a simple quantitative detection method for excessive coumarin in tobacco flavor. Background Art

[0002] Coumarin, also known as benzopyrone, is a crystalline lactone compound commonly found in plants of the Rutaceae and Umbelliferae families. It has a pleasant aroma similar to "vanilla essence" and has been widely used as a flavoring and fragrance agent for cigarettes, food, detergents, cosmetics, etc. Studies in the 1950s and 1960s found that coumarin can cause permanent liver and kidney damage in animals. Excessive intake can cause severe headaches, vomiting, dizziness and insomnia. Higher doses may also cause gene mutations and cause cancer in the digestive system, urinary system, and blood system. Therefore, coumarin has long been banned from use as a food additive worldwide.

[0003] The detection of coumarin generally requires large instruments such as gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry, which is not conducive to the rapid identification and quantitative detection of excessive coumarin in tobacco or tobacco flavors by ordinary people or tobacco industry personnel without leaving the laboratory. For this reason, it is very necessary to develop a convenient detection method for coumarin that does not rely on large instruments and equipment for ordinary smokers or tobacco practitioners. Given the unique fluorescence properties of coumarin and the popular application of smart phones, it is possible to develop a convenient and intelligent method for detecting excessive coumarin in tobacco flavors and fragrances. Summary of the invention

[0004] Aiming at the problem that the existing quantitative detection of excess coumarin in tobacco flavors widely relies on large-scale equipment and is not simple and fast enough, the present invention provides a simple quantitative detection method for excess coumarin in tobacco flavors.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A simple quantitative detection method for excessive coumarin in tobacco flavoring comprises the following steps:

[0007] Step 1, extracting coumarin from the tobacco flavor to be tested;

[0008] Step 2, using a silica gel chromatography plate to chromatograph the extract obtained in step 1, and then developing the color with ultraviolet light;

[0009] Step 3, identifying whether the tobacco flavor to be tested contains coumarin by ultraviolet color development and reference to coumarin standard;

[0010] Step 4: If the tobacco flavor to be tested contains coumarin, further quantitatively analyze its content according to the following method: take ultraviolet color development photos after chromatography; process the taken photos using Image processing software to obtain the grayscale value of the coumarin selection area, and then determine the coumarin content in the tobacco flavor to be tested based on the established relationship curve between the grayscale value of the coumarin selection area and the coumarin concentration.

[0011] Furthermore, in step 1, the extraction method is: according to the volume ratio of the tobacco flavor to be tested to ethanol being 1 g:5 mL, the tobacco flavor to be tested is extracted with ethanol, the extraction temperature is 25° C., and the extraction time is 30 min.

[0012] Furthermore, in step 2, the chromatography method is: spotting the extract obtained in step 1 on a silica gel chromatography plate, and then chromatography on the silica gel chromatography plate in a developing agent, wherein the developing agent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:1.

[0013] Furthermore, in step 2, the wavelength used for the ultraviolet color development is 250 nm.

[0014] Furthermore, in step 4, the method for establishing the relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration is: using ethanol as a solvent, the coumarin standard is configured into a series of coumarin standard working solutions of concentrations, the configured coumarin standard working solution is chromatographed using a silica gel chromatography plate, and then an ultraviolet color development photograph after chromatography is taken; the photograph is processed using Image image processing software to obtain the grayscale value of the coumarin selected area, thereby establishing a relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration.

[0015] Furthermore, when the extract of the tobacco flavor to be tested is chromatographed, a coumarin standard working solution of a known concentration is chromatographed simultaneously, and then the grayscale value of the coumarin selected area of ​​the tobacco flavor extract to be tested is corrected with the grayscale value of the coumarin selected area of ​​the coumarin standard working solution. The correction method is as follows: first, the grayscale calculated value A of the coumarin standard sample of known concentration is obtained according to the established relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration, and then it is compared with the grayscale measured value B of the coumarin standard sample of known concentration to obtain the magnification or reduction multiple C of the grayscale value of the standard sample; then the grayscale measured value D of the coumarin selected area of ​​the tobacco flavor extract to be tested is divided by C to obtain the grayscale correction value E of the coumarin in the tobacco flavor extract to be tested; finally, the E value is substituted into the relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration to obtain the coumarin concentration of the tobacco flavor extract to be tested.

[0016] Furthermore, based on the grayscale value of the coumarin selected area of ​​the tobacco flavor extract to be tested, the relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration is used to directly obtain the coumarin concentration in the extract, and then the content of coumarin in the tobacco flavor to be tested is further determined according to the extraction rate of the extraction method used in step 1. The method for obtaining the extraction rate is: extracting tobacco flavor with known coumarin content, performing gas chromatography analysis on the obtained extract, and then determining the concentration of coumarin in the extract according to the established standard curve of the coumarin gas chromatography integrated area and its concentration, and determining the extraction rate in combination with the coumarin content in the tobacco flavor. The highest extraction rate of the extraction method screened by the present invention reaches 96.2%.

[0017] Furthermore, the method for establishing the standard curve of the coumarin gas chromatography integrated area and its concentration is: using ethanol as a solvent, the coumarin standard is configured into a series of coumarin standard working solutions of concentrations, and then gas chromatography analysis is performed to establish a standard curve of the coumarin gas chromatography integrated area and its concentration, and the established standard curve can be used all the time.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The method for extracting coumarin from tobacco flavor to be tested provided by the present invention has simple solvents and required operation steps, less interference from impurities and a high extraction rate.

[0020] 2. The standard curve of the coumarin gas chromatography integrated area and its concentration, and the relationship curve between the coumarin selected area gray value and coumarin concentration in the present invention can be used continuously after being established for the first time, further simplifying the steps in subsequent applications.

[0021] 3. The quantitative detection method of excess coumarin in tobacco flavors provided by the present invention can achieve qualitative and quantitative detection of excess coumarin in tobacco flavors through simple chromatography, ultraviolet color development and mobile phone photography, with simple operation and accurate results.

[0022] 4. The extraction and detection method of excess coumarin in tobacco flavors provided by the present invention is continuous, convenient and intelligent, does not rely on large-scale instruments and equipment, and is beneficial for ordinary smokers or tobacco practitioners to simply detect excess coumarin in tobacco flavors and fragrances to reduce the harm to health after smoking.

[0023] 5. When testing the tobacco flavor to be tested, the present invention uses a coumarin solution of known concentration as a reference standard for calibration, thereby avoiding the influence of external factors such as shooting angle, ambient light, and mobile phone performance on the test results, thereby improving the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a gas chromatogram of different coumarin concentrations in Example 1 of the present invention.

[0025] Figure 2 This is the standard curve of the gas chromatographic integrated area and concentration of coumarin in Example 1 of the present invention.

[0026] Figure 3 The gas chromatogram is a graph showing the extraction results of coumarin in tobacco flavoring by different extraction solvents in Example 2 of the present invention.

[0027] Figure 4 It is the extraction rate of coumarin in tobacco flavoring by different extraction solvents in Example 2 of the present invention.

[0028] Figure 5 The extraction rates of coumarin in tobacco flavoring by different volumes of ethanol in Example 2 of the present invention are shown.

[0029] Figure 6 The extraction rates of coumarin in tobacco flavoring at different extraction times in Example 2 of the present invention are shown.

[0030] Figure 7 The chromatographic behavior and relative migration value of coumarin standard on silica gel plate under different developing solvent polarities in Example 3 of the present invention.

[0031] Figure 8 The chromatographic behaviors of coumarin in the extract and coumarin standard on silica gel plates in Example 3 of the present invention are compared.

[0032] Fig. 9 This is a chromatographic photograph of 3 μL of a coumarin-ethanol solution with a gradient concentration of 0.1 g / Kg-0.40 g / Kg on a silica gel plate in Example 4 of the present invention.

[0033] Fig.10 It is the gray value of the chromatographic color photograph of the coumarin-ethanol solution in the gradient concentration range of 0.1 g / Kg-0.40 g / Kg on the silica gel plate in Example 4 of the present invention.

[0034] Fig.11 This is the linear relationship between the coumarin concentration in the gradient concentration range of 0.1 g / Kg-0.40 g / Kg and the grayscale value of the selected area of ​​its color-developed photograph in Example 4 of the present invention.

[0035] Fig.12 This is a simple and convenient example of quantitative detection of the coumarin concentration of a sample with unknown concentration in Example 5 of the present invention.

[0036] Fig.13 The gas chromatography data of the unknown concentration sample in Example 5 of the present invention and its comparison with the standard sample. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the technical solution of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0039] Example 1

[0040] This example establishes a standard curve of coumarin gas chromatographic integrated area and its concentration according to the following steps:

[0041] Coumarin ethanol solutions with concentrations of 0.1 g / Kg, 0.2 g / Kg, 0.3 g / Kg, 0.4 g / Kg and 0.5 g / Kg were prepared respectively, and quantitative solutions were taken for gas chromatography analysis to establish a standard curve between the gas chromatography integrated area of ​​coumarin and its content. The gas chromatogram and the standard curve are shown in Figure 1 and Figure 2 shown.

[0042] Example 2

[0043] This example screens the optimal extraction method of coumarin in tobacco flavors, and the specific steps are as follows:

[0044] Take 1g of tobacco flavor with a coumarin concentration of 0.2g / Kg, and then use 5mL of n-hexane, ethyl acetate, ethanol, methanol and water to extract the coumarin in the tobacco flavor for 30min respectively, and then analyze the extract by gas chromatography ( Figure 3 ), and according to the standard curve established in Example 1, the concentration of coumarin in the extract was calculated. Combined with the known coumarin content in tobacco flavors, the extraction rates when using n-hexane, ethyl acetate, ethanol, methanol and water were determined to be 64.5%, 69.8%, 91.0%, 71.9% and 36.0%, respectively ( Figure 4 ), among which ethanol has the highest extraction rate for coumarin.

[0045] When the extraction time was kept at 30 min and other conditions were unchanged, the extraction rates were approximately 78.1%, 88.5%, 90.8%, 91.0%, 93.1% ( Figure 5). When the extraction solvent is kept at 5 mL of ethanol and other conditions remain unchanged, the extraction time is 5 min, 10 min, 20 min, 30 min, 40 min, and 50 min, and the extraction rates are approximately 60.5%, 72.3%, 82.3%, 91.4%, 94.6%, and 96.2%, respectively ( Figure 6 ). Considering the timeliness and convenience, 5 mL of ethanol and 30 min of extraction time were finally selected as the optimal extraction conditions.

[0046] Example 3

[0047] This example verifies the feasibility of a simple qualitative identification method for excess coumarin in tobacco flavors:

[0048] The silica gel plate chromatography and ultraviolet color development method were used. The specific steps were as follows: three groups of coumarin standard solutions (concentration of 0.5 g / Kg) were taken and thin layer chromatography was performed on silica gel chromatography plates. Petroleum ether / ethyl acetate (PE:EA) was used as the developing solvent. The chromatographic behavior was observed under ultraviolet light. It was found that when PE:EA was 4:1, 3:1, 2:1 and 1:1, the ratio shift values ​​(Rf) were approximately 0.35, 0.4, 0.5 and 0.7 ( Figure 7 ).

[0049] Taking tobacco flavor with a coumarin content of 0.2g / Kg as the analysis object, 5mL of ethanol and n-hexane were used to extract the coumarin therein, respectively. Thin layer chromatography was performed on a silica gel chromatography plate under the condition of PE:EA being 1:2. Under ultraviolet light, it can be observed that the transfer value of coumarin in the extract and the coumarin standard is almost the same ( Figure 8 ); combined with the gas chromatography analysis results of the extract ( Figure 3 ), it can be known that coumarin exists, that is, this silica gel chromatography plate chromatography and ultraviolet color development method can achieve simple qualitative identification of coumarin. The color development point with the smallest shift value of the silica gel chromatography plate, that is, the bottom layer, is the main component of flavors and fragrances. At the same time, the Rf values ​​of other impurities in the extract are quite different from those of coumarin, and none of them interfere with coumarin.

[0050] Example 4

[0051] This embodiment establishes the relationship curve between the gray value of the coumarin selected area and the coumarin concentration according to the following steps:

[0052] Prepare 0.1g / Kg, 0.20g / Kg, 0.30g / Kg, and 0.40g / Kg coumarin-ethanol solutions respectively, and spot 3μL of the liquid on the same silica gel chromatography plate, controlling the sample spotting range within a circle with a diameter of 1.5mm. Then, the silica gel chromatography plate was chromatographed in a developing solvent of PE:EA=2:1, and then a smartphone was used to take photos at the same time ( Fig. 9). The color photos taken were converted into gray photos using the formula of Image J software, where Gray = R × 0.299 + G × 0.587 + B × 0.114 (RGB are the attribute values ​​of the color pictures respectively), and then the brightness was set and the background gray value was removed. The coumarin chromatography point area was selected and its gray value was calculated using the area method ( Fig.10 ), and then according to the relationship between gray value and coumarin concentration, a linear relationship of Y = 51005X + 2311.5 can be established ( Fig.11 ).

[0053] Example 5

[0054] This example verifies the feasibility of the quantitative detection method for excessive coumarin in tobacco flavors:

[0055] In order to eliminate the influence of external factors such as the lighting environment on site and the camera angle of the mobile phone on the quantitative detection, a 0.3g / Kg coumarin standard was selected as a calibration reference, and the sample to be tested with unknown concentration was subjected to sample spotting, chromatography, UV color development, mobile phone photography, and software processing. The grayscale photo of the 0.3g / Kg coumarin standard was used for calibration, and the linear relationship between concentration and grayscale value in Example 4 was substituted to calculate the concentration of the sample to be tested to be approximately 0.231g / Kg ( Fig.12 The unknown concentration sample was analyzed by gas chromatography and combined with the standard curve of integrated area and concentration in Example 1, and its concentration was calculated to be about 0.24 g / Kg ( Fig.13 ). From this conclusion, it can be seen that the detection method provided by the present invention is very close to the result detected directly by large-scale gas chromatography, and the detection method does not need to rely entirely on large-scale instruments, which is convenient for smokers or tobacco practitioners to operate and use.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A simple quantitative detection method for excess coumarin in tobacco flavoring, characterized in that: The steps include: Step 1, extracting coumarin from the tobacco flavor to be tested; Step 2, chromatographing the extract obtained in step 1 using a silica gel chromatography plate, and then developing the color using ultraviolet light; Step 3, identifying whether the tobacco flavor to be tested contains coumarin by ultraviolet color development and reference to coumarin standard; Step 4: If the tobacco flavor to be tested contains coumarin, further quantitatively analyze its content according to the following method: take ultraviolet color development photos after chromatography; process the taken photos using Image processing software to obtain the grayscale value of the coumarin selection area, and then determine the coumarin content in the tobacco flavor to be tested based on the established relationship curve between the grayscale value of the coumarin selection area and the coumarin concentration.

2. The method for quantitatively detecting excessive coumarin in tobacco flavoring according to claim 1, characterized in that: In step 1, the extraction method is: according to the volume ratio of the tobacco flavor to be tested to ethanol being 1 g:5 mL, the tobacco flavor to be tested is extracted with ethanol, the extraction temperature is 25° C., and the extraction time is 30 min.

3. The method for quantitatively detecting excessive coumarin in tobacco flavoring according to claim 1, characterized in that: In step 2, the chromatography method is: spotting the extract obtained in step 1 on a silica gel chromatography plate, and then chromatography on the silica gel chromatography plate in a developing solvent, wherein the developing solvent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:

1.

4. The method for quantitatively detecting excessive coumarin in tobacco flavoring according to claim 1, characterized in that: In step 4, the method for establishing the relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration is as follows: using ethanol as a solvent, preparing a series of coumarin standard working solutions of different concentrations, using a silica gel chromatography plate to chromatograph the prepared coumarin standard working solutions, and then taking ultraviolet color development photos after chromatography; using Image image processing software to process the taken photos to obtain the grayscale value of the coumarin selected area, thereby establishing a relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration.

5. The method for quantitatively detecting excessive coumarin in tobacco flavoring according to claim 1, characterized in that: When the extract of the tobacco flavor to be tested is chromatographed, a coumarin standard working solution of a known concentration is chromatographed simultaneously, and then the coumarin selected area grayscale value of the obtained tobacco flavor extract to be tested is calibrated with the coumarin selected area grayscale value of the coumarin standard working solution.

6. The method for quantitatively detecting excess coumarin in tobacco flavoring according to claim 1, characterized in that: Based on the grayscale value of the coumarin selected area of ​​the tobacco flavor extract to be tested, the coumarin concentration in the extract is directly obtained by using the relationship curve between the grayscale value of the coumarin selected area and the coumarin concentration. Then, according to the extraction rate of the extraction method used in step 1, the coumarin content in the tobacco flavor to be tested is further determined.

7. The method for quantitatively detecting excess coumarin in tobacco flavor according to claim 6, characterized in that: The method for obtaining the extraction rate is as follows: extracting a tobacco flavor with a known coumarin content, performing gas chromatography analysis on the obtained extract, and then obtaining the concentration of coumarin in the extract based on an established standard curve of the gas chromatography integrated area of ​​coumarin and its concentration, and determining the extraction rate in combination with the known coumarin content in the tobacco flavor.

8. The simple quantitative detection method for excessive coumarin in tobacco flavor according to claim 7, characterized in that: The method for establishing the standard curve between the gas chromatography integrated area of ​​coumarin and its concentration is as follows: using ethanol as a solvent, preparing a series of coumarin standard working solutions of different concentrations, and then performing gas chromatography analysis to establish the standard curve between the gas chromatography integrated area of ​​coumarin and its concentration.

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