Method for detecting excessive safrole in tobacco essence through smart phone

Abstract

The invention discloses a method for detecting excessive safrole in tobacco essence by a smart phone. The method comprises the following steps: extracting the safrole in the 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 to-be-detected tobacco essence contains safrole or not through ultraviolet color development and by referring to a safrole standard substance. If the to-be-detected tobacco flavor contains the safrole, further shooting and processing an ultraviolet color developing picture after chromatography to obtain a safrole selected area gray value, and then determining the content of the safrole in the to-be-detected tobacco flavor according to the established relation curve of the safrole selected area gray value and the safrole 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 detection method and application of excessive safrole in tobacco flavors. Background Art

[0002] Safrole is an organic natural compound, a colorless oily liquid, easily soluble in ethanol, miscible with chloroform and ether, insoluble in water and glycerin, and widely present in essential oils of camphor plants such as Cinnamomum camphora and Cinnamomum camphora. This substance has a scent similar to that of a Western "candy shop" and is a natural flavor. It has been widely used as a flavoring and flavor enhancer for food, cigarettes, etc. However, studies in the 1950s and 1960s found that safrole can induce liver tumors in mice and may cause reproductive, gastrointestinal and other functional damage. Literature reports that its oral median lethal dose LD 50 It is about 2.3g / Kg. As a flavor, its addition content exceeding 1% can induce liver cancer in mice. Therefore, safrole has long been banned as a food additive worldwide. However, given its unique aroma, it can improve the aroma quality of cigarettes and enhance smokers' smoking experience, causing some businesses or individuals to add excessive amounts of safrole to various flavors and spices or tobacco, which will cause potential physical harm to smokers. The identification and detection of safrole reported in the literature generally requires large-scale analytical instruments such as gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry in the laboratory, which makes it difficult for ordinary smokers or tobacco industry personnel to easily identify and detect excess safrole in tobacco flavors outside the laboratory. Therefore, it is very necessary to develop a simple, convenient and easy-to-master method for identifying and detecting safrole to reduce the harm of excessive safrole in tobacco flavors to smokers' health. Summary of the invention

[0003] Aiming at the problem that the identification and detection of excess safrole in existing tobacco flavors widely relies on large-scale analytical instruments and equipment such as gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry, which is not conducive to simple mastery and convenient use by ordinary smokers and tobacco industry personnel, the present invention provides a method for detecting excess safrole in tobacco flavors by using a smart phone.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] A method for detecting excessive safrole in tobacco flavors by using a smart phone comprises the following steps:

[0006] Step 1, extracting safrole from the tobacco flavor to be tested;

[0007] Step 2, chromatographing the extract obtained in step 1 using a silica gel chromatography plate, and then developing the color using ultraviolet light;

[0008] Step 3, identifying whether the tobacco flavor to be tested contains safrole by ultraviolet color development and reference to safrole standard product;

[0009] Step 4: If the tobacco flavor to be tested contains safrole, further quantitatively analyze its content according to the following method: take ultraviolet color development photos after chromatography with a smart phone; process the taken photos with Image processing software to obtain the gray value of the safrole selection area, and then determine the content of safrole in the tobacco flavor to be tested based on the established relationship curve between the gray value of the safrole selection area and the safrole concentration.

[0010] Furthermore, in step 1, the extraction method is: according to the volume ratio of the tobacco flavor to be tested to the extraction solvent ethanol of 1g:5mL, the tobacco flavor to be tested is extracted with ethanol, the extraction temperature is 25°C, and the extraction time is 50min.

[0011] 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 solvent, wherein the developing solvent is pure petroleum ether.

[0012] Further, in step 4, the method for establishing the relationship curve between the grayscale value of the safrole selected area and the safrole concentration is: using ethanol as a solvent, preparing a series of concentrations of safrole standard working solutions, using a silica gel chromatography plate to chromatograph the prepared safrole standard working solutions, and then using a smart phone to take ultraviolet color photos after chromatography; using Image image processing software to process the photographed photos to obtain the grayscale value of the safrole selected area, thereby establishing a linear relationship curve between the grayscale value of the safrole selected area and the safrole concentration.

[0013] Furthermore, when the extract of the tobacco flavor to be tested is chromatographed, a safrole standard working solution of a certain known concentration is chromatographed at the same volume, and then the grayscale value of the safrole selected area of ​​the tobacco flavor extract to be tested is corrected by the grayscale value of the safrole selected area of ​​the safrole standard working solution. The correction method is as follows: first, the grayscale calculated value A of the safrole standard sample of known concentration is obtained according to the established relationship curve between the grayscale value of the safrole selected area and the safrole concentration, and then it is compared with the grayscale measured value B of the safrole 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 safrole selected area of ​​the tobacco flavor extract to be tested is divided by C to obtain the grayscale correction value E of the safrole of the tobacco flavor extract to be tested; finally, the E value is substituted into the relationship curve between the grayscale value of the safrole selected area and the safrole concentration to obtain the safrole concentration of the tobacco flavor extract to be tested.

[0014] Further, based on the gray value of the safrole selected area of ​​the tobacco flavor extract to be tested, the safrole concentration in the extract is directly obtained by using the relationship curve between the gray value of the safrole selected area and the safrole concentration, and then the content of safrole 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 a tobacco flavor with a known safrole content, performing gas chromatography analysis on the obtained extract, and then obtaining the concentration of safrole in the extract according to the established standard curve of the safrole gas chromatography integrated area and its concentration, and combining the known content of safrole in the tobacco flavor to determine the extraction rate. The method for establishing the standard curve of the safrole gas chromatography integrated area and its concentration is: using ethanol as a solvent, preparing a series of safrole standard working solutions of concentration, and then performing gas chromatography analysis, thereby establishing a standard curve of the safrole gas chromatography integrated area and its concentration.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. The method for extracting safrole from tobacco flavor to be tested provided by the present invention has simple solvents and required operation steps, less interference from impurities and higher extraction rate.

[0017] 2. The standard curve of safrole gas chromatography integrated area and its concentration, and the relationship curve between safrole selected area gray value and safrole concentration in the present invention can be used continuously after being established for the first time, further simplifying the steps in subsequent applications.

[0018] 3. The quantitative detection method of excess safrole in tobacco flavors provided by the present invention can achieve qualitative and quantitative detection of excess safrole in tobacco flavors through simple chromatography, ultraviolet color development and mobile phone photography, with simple operation and accurate results.

[0019] 4. The extraction and detection method of excess safrole in tobacco flavors provided by the present invention is continuous, convenient, intelligent, low-cost, and does not rely on large-scale instruments and equipment. It is beneficial for ordinary smokers or tobacco practitioners to simply detect excess safrole in tobacco flavors and spices to reduce the harm to health after smoking.

[0020] 5. When the present invention detects safrole in the tobacco flavor to be tested, a safrole solution with a known concentration is used as a reference standard for calibration, which can avoid 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 quantitative test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a gas chromatogram of different safrole concentrations in Example 1 of the present invention.

[0022] Figure 2This is a standard curve of the gas chromatographic integrated area and concentration of safrole in Example 1 of the present invention.

[0023] Figure 3 The gas chromatogram is a graph showing the extraction results of safrole in tobacco flavoring by different extraction solvents in Example 2 of the present invention.

[0024] Figure 4 The extraction rates of safrole in tobacco flavorings by different extraction solvents in Example 2 of the present invention are shown in FIG.

[0025] Figure 5 The extraction rates of safrole in tobacco flavoring at different extraction times in Example 2 of the present invention are shown.

[0026] Figure 6 The chromatographic behaviors and relative migration values ​​of safrole standards on silica gel plates under different developing solvent polarities in Example 3 of the present invention are shown.

[0027] Figure 7 Comparison of the chromatographic behavior of safrole in the safrole standard and ethanol and n-hexane extracts in Example 3 of the present invention (the left is the standard, the middle is the ethanol extract, and the right is the n-hexane extract).

[0028] Figure 8 The gas chromatographic analysis chart of safrole in the safrole standard product and the ethanol extract in Example 3 of the present invention.

[0029] Fig. 9 This is a chromatographic photograph of 5 μL of safrole-ethanol solution with a gradient concentration of 0.16 g / Kg-0.40 g / Kg on a silica gel plate in Example 4 of the present invention.

[0030] Fig.10 It is the gray value of the chromatographic color photograph of safrole-ethanol solution with gradient concentration ranging from 0.16 g / Kg to 0.40 g / Kg on a silica gel plate in Example 4 of the present invention.

[0031] Fig.11 This is the linear relationship between the concentration of safrole in the gradient concentration range of 0.16 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.

[0032] Fig.12 This is a simple and convenient quantitative detection example of the safrole concentration of the unknown concentration sample to be tested in Example 5 of the present invention.

[0033] 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.

[0034] Fig.14 This is a simple and convenient quantitative detection example of the safrole concentration of the unknown concentration sample to be tested in Example 6 of the present invention.

[0035] Fig.15 The gas chromatography data of the sample with unknown concentration in Example 6 of the present invention and its comparison with the standard sample. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments 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.

[0037] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0038] 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 art or according to the product instructions.

[0039] Example 1

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

[0041] Safrole ethanol solutions with concentrations of 0.16 g / Kg, 0.24 g / Kg, 0.32 g / Kg, and 0.4 g / Kg were prepared respectively, and quantitative solutions were taken for gas chromatography analysis to establish a standard curve between the gas chromatography integral area of ​​safrole and its concentration. The gas chromatogram and the standard curve are shown in FIG. Figure 1 and Figure 2 shown.

[0042] Example 2

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

[0044] Take 1g of tobacco flavor with a safrole concentration of 0.2g / Kg, and then use 5mL of n-hexane, ethyl acetate, ethanol, methanol and water to extract the safrole in the tobacco flavor for 50min, and then analyze the extract by gas chromatography ( Figure 3 ), and according to the standard curve established in Example 1, the concentration of safrole in the extract was calculated. Combined with the known content of safrole in tobacco flavors, the extraction rates when using n-hexane, ethyl acetate, ethanol, methanol and water were determined to be 54.2%, 40.7%, 97.4%, 10.9% ( Figure 4), among which ethanol has the highest extraction rate for safrole, while water has almost no extraction effect on it, which is consistent with the immiscible nature of safrole and water.

[0045] When the extraction solvent was 5 mL of ethanol and other conditions were kept unchanged, the extraction time was 20 min, 30 min, 40 min, and 50 min, and the extraction rates were approximately 67.7%, 76.9%, 87.5%, and 97.4%, respectively. Figure 5 Considering timeliness and convenience, 5 mL of ethanol and 50 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 safrole 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 safrole standard solutions (concentration of 0.16 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, 6:1, 8:1 and 1:0 (i.e. pure PE), the ratio (Rf) was approximately 1, 0.95, 0.9 and 0.5 ( Figure 6 ).

[0049] Taking tobacco flavor with a safrole content of 1.0 g / kg as the analysis object, 5 mL of ethanol and n-hexane were used to extract the safrole therein, respectively. Thin layer chromatography analysis was performed on a silica gel chromatography plate again under the condition of pure PE as the developing solvent. Under 250 nm ultraviolet light irradiation, it can be observed that the migration value of safrole in the extract and the safrole standard are almost the same ( Figure 7 ); combined with the gas chromatography analysis results of the extract ( Figure 8 ), it can be known that safrole exists, that is, this silica gel chromatography plate chromatography and ultraviolet color development method can realize the simple qualitative identification of safrole. The color development point with the smallest shift value of the silica gel plate, that is, the bottom layer, is other ingredients in the flavor, and its Rf value is quite different from that of safrole, and will not interfere with the qualitative detection of safrole. In addition, under different polarity conditions and Rf value conditions, other ingredients in the flavor will not interfere with the simple qualitative detection of safrole.

[0050] Example 4

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

[0052] Prepare 0.16g / Kg, 0.24g / Kg, 0.32g / Kg, and 0.40g / Kg safrole-ethanol solutions respectively, and spot 5μL of the solution on the same silica gel chromatography plate, controlling the sample spotting range within a circle with a diameter of 2.0mm. Then, the silica gel chromatography plate was chromatographed in PE developing agent, 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 safrole 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 safrole concentration, a Y = 53722x-145.3 (R 2 =0.9991) Fig.11 ), which laid the foundation for the convenient use of smartphones to qualitatively and quantitatively detect excess safrole in flavors.

[0053] Example 5

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

[0055] In order to eliminate the influence of external factors such as the on-site lighting environment, the shooting angle of the mobile phone, and the brand type of the mobile phone on the quantitative detection, 5 μL of 0.16 g / Kg safrole standard was selected as the calibration reference (left point), and it was subjected to the same sample spotting, chromatography, UV color development, mobile phone photography, and software processing (such as Fig.12 ). After calibration with the grayscale photograph of the 0.16g / Kg safrole standard, and substituting the linear relationship between concentration and grayscale value in Example 4, the concentration of the sample to be tested can be calculated to be about 0.287g / Kg. The unknown concentration sample is analyzed by gas chromatography and combined with the standard curve of integrated area and concentration in Example 1, and the concentration can be calculated to be about 0.3g / 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, with an error of only 4.2%. Moreover, the detection method does not need to rely entirely on large-scale instruments, and is convenient for smokers or tobacco practitioners to operate and use.

[0056] Example 6

[0057] Quantitative detection of excess safrole in tobacco flavors of unknown concentration: In order to eliminate the influence of external factors such as the on-site lighting environment and the shooting angle of the mobile phone on the quantitative detection, 6 μL of 0.4 g / Kg safrole standard was selected as a calibration reference (left point), and the same sample to be tested in Example 5 (right point) was subjected to sample spotting, chromatography, UV color development, mobile phone photography, and software processing (such as Fig.14 ). After calibration with the grayscale photograph of the 0.40g / Kg safrole standard, and substituting the linear relationship between concentration and grayscale value in Example 4, the concentration of the sample to be tested can be calculated to be about 0.298g / Kg. The unknown concentration sample is analyzed by gas chromatography and combined with the standard curve of integrated area and concentration in Example 1, and its concentration can be calculated to be about 0.3g / Kg ( Fig.15 ). 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, with an error of only 1%, 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. Moreover, the concentration and volume of the standard product can be changed, and it can be simply used for the qualitative and quantitative detection of safrole. When the amount of the standard product is large, the experimental error will be smaller.

[0058] 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 method for detecting excessive safrole in tobacco flavors using a smart phone, characterized in that: The steps include: Step 1, extracting safrole 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 safrole by ultraviolet color development and reference to safrole standard product; Step 4: If the tobacco flavor to be tested contains safrole, further quantitatively analyze its content according to the following method: take ultraviolet color development photos after chromatography with a smart phone; process the taken photos with Image processing software to obtain the gray value of the safrole selection area, and then determine the content of safrole in the tobacco flavor to be tested based on the established relationship curve between the gray value of the safrole selection area and the safrole concentration.

2. The method for detecting excessive safrole in tobacco flavors by a smart phone 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 the extraction solvent ethanol of 1g:5mL, the tobacco flavor to be tested is extracted with ethanol, the extraction temperature is 25°C, and the extraction time is 50min.

3. The method for detecting excessive safrole in tobacco flavors by a smart phone 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 pure petroleum ether.

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

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

6. The method for detecting excessive safrole in tobacco flavors by a smart phone according to claim 1, characterized in that: Based on the grayscale value of the safrole selected area of ​​the tobacco flavor extract to be tested, the safrole concentration in the extract is directly obtained by using the relationship curve between the grayscale value of the safrole selected area and the safrole concentration, and then according to the extraction rate of the extraction method used in step 1, the content of safrole in the tobacco flavor to be tested is further determined.

7. The method for detecting excessive safrole in tobacco flavors by using a smart phone according to claim 6, characterized in that: The method for obtaining the extraction rate is as follows: extracting a tobacco flavor with a known safrole content, performing gas chromatography analysis on the obtained extract, and then obtaining the concentration of safrole in the extract according to an established standard curve of the safrole gas chromatography integral area and its concentration, and determining the extraction rate in combination with the known safrole content in the tobacco flavor.

8. The method for detecting excessive safrole in tobacco flavors by using a smart phone according to claim 7, characterized in that: The method for establishing the standard curve between the gas chromatography integrated area of ​​safrole and its concentration is as follows: using ethanol as a solvent, preparing a series of safrole standard working solutions with different concentrations, and then performing gas chromatography analysis to establish the standard curve between the gas chromatography integrated area of ​​safrole and its concentration.

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