Method for determining DDMP content in tobacco leaves based on gas chromatography-mass spectrometry

Abstract

The invention discloses a method for determining the content of DDMP in tobacco leaves based on gas chromatography-mass spectrometry, and relates to the technical field of natural product extraction and detection.The method comprises the following steps that the tobacco leaves and ethyl alcohol are mixed, heated and stirred, subjected to reflux extraction and suction filtration to obtain filtrate I and filter residues I, and after the filtrate I is subjected to microfiltration treatment, the filter residues I are obtained; adding a phenethyl acetate standard solution as an internal standard to prepare a to-be-detected sample I; and detecting the DDMP content in the to-be-detected sample I by adopting a gas chromatography-mass spectrometry (GC-MS) combined method. The method has the beneficial effects of simple and convenient process flow and easy operation; the content of the DDMP in the tobacco leaves can be rapidly detected. And the detection result is rapid and accurate and has high reliability. According to the method, the DDMP is extracted by adopting a solvent extraction technology, the influence of each factor in the extraction process is investigated, the DDMP is quantified by adopting an internal standard method, and the extraction process parameters are optimized, so that a reference is provided for the maximum utilization of the tobacco waste. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and detection, and specifically relates to a method for determining the DDMP content in tobacco leaves based on gas chromatography-mass spectrometry. Background Art

[0002] 2,3-Dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one, abbreviated as DDMP, CAS number: 28564-83-2, is commonly found in cigarette smoke, sugar cracking products and natural extracts. Structurally, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one belongs to the pyran enolone class of compounds. Most of these compounds with cyclic enolones have a caramel-like aroma. Compared with caramel-like aroma substances such as maltol, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one has better water solubility and a more prominent "sweetness" in taste. It is significantly positively correlated with the "sweetness" of cigarette smoke, which can significantly improve the sweetness of cigarette smoke and highlight the "sweet" and "smooth" taste characteristics of cigarettes.

[0003] There have been relevant studies on the extraction and detection of DDMP. For example, [SIDDEEG A, ZENG XN, AMMARA F, et al. Sugar profile, volatile compounds, composition and antioxidant activity of Sukkari date palm fruit [J]. Journal of Food Science and Technology, 2019, 56 (2): 754-762] proposed that the DDMP content in the volatile components of dates was close to 10% by GC-MS analysis. [SAKAMOTO T, LI ZS, NISHIDA A, et al. Identification of majorantioxidant compounds from the edible mushroom Basidiomycetes-X (Echigoshirayukidake) [J]. Frontiers in Bioscience-Elite, 2022, 14 (2): 10] proposed that DDMP was isolated from white snow mushroom, and its content was measured by HPLC to be 3.5 mg / g. [Chen Fan, Liu Yonghong, Liu Yongqiong, et al. Extraction and component analysis of guava fruit aromatic extract [J]. Journal of Wuhan Institute of Technology, 2010, 32(12):13-17; 22] proposed GC-MS to analyze the components of the aromatic extract obtained by guava fruit extraction, and the presence of DDMP was also detected.

[0004] 【RODRIGUEZ JMF, DE SOUZA ARC, RL, et al. Kinetics, composition and antioxidant activity of burdock (Arctium lappa) root extracts obtained with supercritical CO2 and co-solvent [J]. The Journal of Supercritical Fluids, 2018, 135: 25-33] proposed to use supercritical CO2 extraction to extract burdock root, and combined with GC-MS analysis, it was found that the main component of burdock extract was DDMP. However, how to extract DDMP from tobacco leaves and detect and analyze its content has not been reported. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to solve the problem that it is currently difficult to separate DDMP from tobacco leaves and accurately determine its content.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention provides a method for determining the DDMP content in tobacco leaves, comprising the following steps:

[0008] (1) mixing tobacco leaves with ethanol, heating and stirring, reflux extraction, and filtering to obtain a filtrate I and a filter residue I; microfiltration of the filtrate I, adding a phenylethyl acetate standard solution as an internal standard, and preparing a sample I to be tested;

[0009] (2) Detect the DDMP content in the sample I by gas chromatography-mass spectrometry (GC-MS) to obtain the DDMP content; the specific GC-MS conditions are as follows:

[0010] Gas chromatography conditions: chromatographic column: DB-WAX (123-7033, 30m×0.32mm×0.50μm); carrier gas: He, 99.99%; flow rate: 30mL·min -1 ; Injection volume: 1 μL; Injection mode: split injection; Split ratio: 30:1; Injection port temperature: 250°C; Heating program: 50°C for 5 min, 5°C min -1 Raise the temperature to 200℃ and then -1 Heating to 240°C;

[0011] The mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; transfer line temperature: 300°C; analyzer: quadrupole mass analyzer; scan mode: full scan; mass scan range: 50-650 amu; solvent delay: 4.5 min.

[0012] Preferably, in step (1), the material-liquid ratio of tobacco leaf to ethanol is 1 g: (5-15) mL, more preferably 1 g: 10 mL.

[0013] Preferably, in step (1), the ethanol has a concentration of 85-95% (v / v) ethanol, more preferably 90% (v / v) ethanol.

[0014] Preferably, in step (1), the heating temperature is 50-70° C., and the reflux extraction time is 1-5 h, more preferably 60° C. for 3 h.

[0015] Preferably, the method further comprises subjecting the filter residue I to secondary extraction, filtering the obtained filtrate II, and subjecting the filtrate II to microfiltration to prepare a sample II to be tested, the steps being the same as (1); measuring the DDMP content in the sample II to be tested, and then adding the DDMP contents of the obtained sample I and the sample II to be tested to obtain.

[0016] Preferably, the instrument used for gas chromatography-mass spectrometry in step (2) is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

[0017] The beneficial effects of the present invention are:

[0018] The present invention proposes a method for determining the content of DDMP in tobacco leaves based on gas chromatography-mass spectrometry. The process is simple, convenient and easy to operate; the content of DDMP in tobacco leaves can be quickly detected. The detection results are fast, accurate and highly reliable. DDMP is extracted by solvent extraction technology, the influence of various factors in the extraction process is investigated, and the internal standard method is used to quantify DDMP, and the extraction process parameters are optimized, aiming to provide a reference for maximizing the utilization of tobacco waste. It has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the process flow for extracting and quantitatively determining DDMP in tobacco leaves according to Example 1 of the present invention.

[0020] Figure 2 is the chromatogram in Example 1 of the present invention;

[0021] Figure 3 It is the chromatogram in Comparative Example 1 of the present invention;

[0022] Figure 4 This is a response surface diagram of the extraction temperature and ethanol concentration on the extraction amount of DDMP from tobacco leaves in Example 12 of the present invention;

[0023] Figure 5 This is a response surface diagram of the extraction temperature and the solid-liquid ratio on the extraction amount of DDMP from tobacco leaves in Example 12 of the present invention;

[0024] Figure 6 This is a response surface diagram of the extraction temperature and extraction time on the extraction amount of DDMP from tobacco leaves in Example 12 of the present invention;

[0025] Figure 7 This is a response surface diagram of the extraction amount of DDMP from tobacco leaves between the extraction temperature and the number of extractions in Example 12 of the present invention;

[0026] Figure 8 is a response surface diagram of the relationship between ethanol concentration and solid-liquid ratio on the amount of DDMP extracted from tobacco leaves in Example 12 of the present invention;

[0027] Fig. 9 is a response surface diagram of the amount of DDMP extracted from tobacco leaves between ethanol concentration and extraction time in Example 12 of the present invention;

[0028] Fig.10 is a response surface diagram of the relationship between ethanol concentration and extraction times on the amount of DDMP extracted from tobacco leaves in Example 12 of the present invention;

[0029] Fig.11 It is a response surface diagram of the relationship between the solid-liquid ratio and the extraction time on the amount of DDMP extracted from tobacco leaves in Example 12 of the present invention;

[0030] Fig.12 It is a response surface diagram of the relationship between the solid-liquid ratio and the number of extractions on the amount of DDMP extracted from tobacco leaves in Example 12 of the present invention;

[0031] Fig.13 This is a response surface diagram of the extraction amount of tobacco leaf DDMP between the extraction time and the number of extractions in Example 12 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of 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.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0034] 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.

[0035] The tobacco leaves are from southern Anhui, and the specific variety is Yunyan 87.

[0036] Embodiment 1:

[0037] A method for determining the DDMP content in tobacco leaves comprises the following steps:

[0038] (1) Add tobacco leaf powder into a three-necked flask, add 90% ethanol solution as an extractant at a solid-liquid ratio of 1g:10mL, and mechanically stir and reflux for extraction at 60°C for 3h. After the extraction is completed, filter to obtain filtrate I and residue I. After the filtrate I is treated by microfiltration (8μm), phenylethyl acetate standard solution is added as an internal standard to prepare sample I to be tested.

[0039] (2) Add 90% (v / v) ethanol solution as an extractant to the residue I at a ratio of 1 g:10 mL, and repeat the above operation. After the second extraction is completed, continue to filter to obtain filtrate II and residue II. After the filtrate II is treated by microfiltration (8 μm), phenylethyl acetate standard solution is added as an internal standard to prepare the sample II to be tested.

[0040] (3) Based on the principle that the peak area ratio of the compound to the internal standard is positively correlated with the concentration ratio, the content of DDMP in the sample I and the sample II is quantitatively determined by GC-MS, and the contents of the two are added together to obtain;

[0041] Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer was used, and the specific chromatographic mass spectrometric conditions were as follows:

[0042] Gas chromatography conditions: chromatographic column: DB-WAX (123-7033, 30m×0.32mm×0.50μm); carrier gas: He, 99.99%; flow rate: 30mL·min -1 ; Injection volume: 1 μL; Injection mode: split injection; Split ratio: 30:1; Injection port temperature: 250°C; Heating program: 50°C for 5 min, 5°C min -1 Raise the temperature to 200℃ and then -1 Raise the temperature to 240°C;

[0043] The mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; transfer line temperature: 300°C; analyzer: quadrupole mass analyzer; scan mode: full scan; mass scan range: 50-650 amu; solvent delay: 4.5 min.

[0044] Chromatogram Figure 3 As shown (at 18.328 min), the results show that the extraction amount of DDMP in this example is 4.34 mg / g. The detection method is simple and the result is accurate.

[0045] Embodiment 2:

[0046] The difference between this embodiment and embodiment 1 is that 60° C. in step (1) is replaced by “50° C.”, and the rest is the same as embodiment 1.

[0047] The results show that the extraction amount of DDMP is 2.03 mg / g, which is significantly reduced compared to that in Example 1, and the test results are not as ideal as those in Example 1.

[0048] Embodiment 3:

[0049] The difference between this embodiment and embodiment 1 is that 60° C. in step (1) is replaced by 70° C., and the rest is the same as embodiment 1.

[0050] The results show that the extraction amount of DDMP is only 2.32 mg / g, which is much less than that in Example 1. The test results are not as ideal as those in Example 1.

[0051] Embodiment 4:

[0052] The difference between this embodiment and embodiment 1 is that the 90% ethanol solution in step (1) is replaced by "85% ethanol solution", and the rest is the same as embodiment 1.

[0053] The results show that the extraction amount of DDMP is only 2.66 mg / g, which is much less than that in Example 1, and the test results are not as ideal as those in Example 1.

[0054] Embodiment 5:

[0055] The difference between this embodiment and embodiment 1 is that the 90% ethanol solution in step (1) is replaced by "95% ethanol solution", and the rest is the same as embodiment 1.

[0056] The results show that the extraction amount of DDMP is only 2.35 mg / g, which is much less than that in Example 1. The test results are not as ideal as those in Example 1.

[0057] Embodiment 6:

[0058] The difference between this embodiment and embodiment 1 is that the material-liquid ratio of 1:10 in steps (1) and (2) is replaced by a "material-liquid ratio of 1:5", and the rest is the same as embodiment 1.

[0059] The results show that the extraction amount of DDMP is only 2.52 mg / g, which is much less than that in Example 1. The test results are not as ideal as those in Example 1.

[0060] Embodiment 7:

[0061] The difference between this embodiment and embodiment 1 is that the material-liquid ratio of 1:10 in steps (1) and (2) is replaced by a "material-liquid ratio of 1:15", and the rest is the same as embodiment 1.

[0062] The results show that the extraction amount of DDMP is only 2.46 mg / g, which is much less than that in Example 1. The test result is not as ideal as that in Example 1.

[0063] Embodiment 8:

[0064] The difference between this embodiment and embodiment 1 is that the reflux extraction for 3 hours in step (1) is replaced by “reflux extraction for 1 hour”, and the rest is the same as embodiment 1.

[0065] The results show that the extraction amount of DDMP is only 2.51 mg / g, which is much less than that in Example 1. The test result is not as ideal as that in Example 1.

[0066] Embodiment 9:

[0067] The difference between this embodiment and embodiment 1 is that the reflux extraction for 3 hours in step (1) is replaced by “reflux extraction for 5 hours”, and the rest is the same as embodiment 1.

[0068] The results show that the extraction amount of DDMP is only 2.54 mg / g, which is much less than that in Example 1. The test results are not as ideal as those in Example 1.

[0069] Embodiment 10:

[0070] The difference between this embodiment and embodiment 1 is that only one extraction is performed, and the rest is the same as embodiment 1.

[0071] The results show that the extraction amount of DDMP is only 2.76 mg / g, which is much less than that in Example 1. The test results are not as ideal as those in Example 1.

[0072] Embodiment 11:

[0073] The difference between this embodiment and embodiment 1 is that the extraction is performed 3 times, and the rest is the same as embodiment 1.

[0074] The results show that the extraction amount of DDMP is only 4.45 mg / g. Compared with the extraction amount of DDMP in Example 1, the increase is limited. Considering the time cost and production energy consumption, the extraction is performed twice.

[0075] Comparative Example 1:

[0076] The difference between this comparative example and Example 1 is that the model of the chromatographic column is changed to Agilent HP-5 (30m×0.53mm×0.88μm), and the rest is the same as Example 1.

[0077] The results are as follows Figure 4 As shown: no DDMP peak was detected and the DDMP content could not be measured.

[0078] Comparative Example 2:

[0079] The difference between this comparative example and Example 1 is that the heating program is changed to: 60°C for 5 min, 10°C min -1Raise the temperature to 250℃ and then -1 The temperature was raised to 350°C; the rest was the same as in Example 1.

[0080] Result: The chromatographic peaks overlapped and the content of DDMP could not be determined.

[0081] Example 12: (Optimization of extraction process parameters using response surface methodology)

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086]

[0087] Table 3

[0088]

[0089]

[0090] Note: ** indicates extremely significant (P < 0.01); * indicates significant (P < 0.05).

[0091] The response surface diagram of the five factors to the DDMP extraction amount of tobacco leaves is shown in the figure below: Figure 4-13 As shown; combined with Table 1-3, it can be seen that the optimal extraction process parameters are: extraction temperature: 60°C, ethanol concentration: 90%, solid-liquid ratio: 1g:10mL, extraction time: 3h, extraction times: 2 times.

[0092] 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 determining the DDMP content in tobacco leaves, characterized in that: The following steps are involved: (1) mixing tobacco leaves with ethanol, heating and stirring, reflux extraction, and filtering to obtain a filtrate I and a filter residue I; microfiltration of the filtrate I, adding a phenylethyl acetate standard solution as an internal standard, and preparing a sample I to be tested; (2) Detect the DDMP content in the sample I by gas chromatography-mass spectrometry, and obtain the following; the specific chromatography-mass spectrometry conditions are as follows: Gas chromatography conditions: chromatographic column: DB-WAX (123-7033, 30m×0.32mm×0.50μm); carrier gas: He, 99.99%; flow rate: 30mL·min -1 ; Injection volume: 1 μL; Injection mode: split injection; Split ratio: 30:1; Injection port temperature: 250°C; Heating program: 50°C for 5 min, 5°C min -1 Raise the temperature to 200℃ and then -1 Heating to 240°C; The mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; transfer line temperature: 300°C; analyzer: quadrupole mass analyzer; scan mode: full scan; mass scan range: 50-650 amu; solvent delay: 4.5 min.

2. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: In step (1), the material-liquid ratio of tobacco leaf to ethanol is 1 g: (5-15) mL.

3. The method for determining the DDMP content in tobacco leaves according to claim 2, characterized in that: In step (1), the material-liquid ratio of tobacco leaf to ethanol is 1 g:10 mL.

4. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: In step (1), the ethanol has a concentration of 85 to 95% (v / v).

5. The method for determining the DDMP content in tobacco leaves according to claim 4, characterized in that: In step (1), the ethanol is ethanol with a concentration of 90% (v / v).

6. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: In step (1), the heating temperature is 50-70°C.

7. The method for determining the DDMP content in tobacco leaves according to claim 6, characterized in that: In step (1), the heating temperature is 60°C.

8. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: The reflux extraction time is 1 to 5 hours.

9. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: The method further comprises subjecting the filter residue I to secondary extraction, filtering the obtained filtrate II through microfiltration to prepare a sample II to be tested, the steps being the same as (1); measuring the DDMP content in the sample II to be tested, and then adding the DDMP contents of the obtained sample I and the sample II to obtain the product.

10. The method for determining the DDMP content in tobacco leaves according to claim 1, characterized in that: The instrument used for gas chromatography-mass spectrometry in step (2) is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

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