Method for rapidly measuring content of wax compounds in tobacco leaves based on extraction and GC-MS (Gas Chromatography-Mass Spectrometer)

Abstract

The invention discloses a method for rapidly detecting the content of wax compounds in tobacco leaves based on extraction and GC-MS. The method relates to the technical field of tobacco leaf extraction and detection, and comprises the following steps: mixing the tobacco leaves with ethanol, performing ultrasonic treatment and suction filtration, diluting filtrate, and performing stepped extraction to obtain a sample to be detected; the method comprises the following steps: detecting wax compounds in a to-be-detected sample by adopting a gas chromatography-mass spectrometry (GC-MS) technology, selecting a standard substance as a qualitative scale, and comparing retention time to further determine the components of the wax compounds in the tobacco leaves. According to the method, 10 compounds, namely, dehydrobulitol, isoseisserian cycloenol, delta5-oat sterol, cholesterol, dodecane, o-nicotine, 1-tetradecene, 1-hexadecene, hexadecane and 1-heptadecanol, are separated and detected for the first time, the method has great significance, the technological process is simple, convenient and easy to operate, a reference is provided for maximum utilization of the tobacco wax compounds, and the method is suitable for industrial production. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and identification of wax compounds in tobacco leaves, and particularly relates to a method for rapidly measuring the content of wax compounds in tobacco leaves based on extraction and GC-MS. Background Art

[0002] The wax covering the surface of tobacco is a mixture that is insoluble in water but easily soluble in organic solvents. It is a barrier for tobacco to protect itself and plays an important role in adapting to high temperatures, drought, diseases and pests.

[0003] In modern research, gas chromatography-mass spectrometry (GC-MS) is often used to analyze and identify tobacco leaf wax components. For example, [Von Wettstein-Knowles, PM (1993) Waxes, Cutin and Suberin, Lipid Metabolism in Plants. CRC Press, Florida, 127-166] proposed that tobacco epidermal wax is a mixture of alkanes, fatty alcohols, ketones, and free fatty acids. [Kolattukudy, PE (1968) Further Evidence for an Elongation-Dacarboxylation Mechanism in the Biosynthesis of Paraffins in Leaves. Plant Physiology, 43, 375-383] proposed that tobacco leaf wax is composed of straight-chain and branched alkanes containing 25 to 34 carbon atoms. [Xu Jing, Wang Meiling, Li Tingting, Ding Penghui, Yuan Wei. Study on the composition and content of wax on the surface of tobacco leaves [J]. Journal of Northwest Agriculture, 2014, 23(4): 140-145] The wax on the surface of fresh tobacco leaves was eluted with chloroform and identified by GC-MS after derivatization. It was found that the surface wax of fresh tobacco leaves was mainly composed of alkanes, alkanols and triterpenes. [Arrendale, RF, Severson, RF, Chorty, DT and Stephenson, MG (1988) Isolation and Identification of the Wax Esters from the Cuticular Waxes of Green Tobacco Leaf. Beitragezur Tabakforschung International / Contributions to Tobacco Research, 14, 67-84] The wax on the surface of fresh tobacco leaves was eluted with dichloromethane and then separated between 80% methanol aqueous solution and n-hexane. It was found that the n-hexane phase contained wax, hydrocarbons and fatty alcohols. However, these methods isolate and detect a limited number of chemical components from tobacco leaves, most of which are known chemicals, with little exploration of new, potential chemicals. The high number of waxy components in tobacco leaves increases the difficulty of related analysis and identification. How to quickly and efficiently detect the various waxy components in tobacco leaves and discover new chemical components in tobacco leaves remains a challenge.

[0004] Chinese patent application publication number CN110542736A discloses a method for the simultaneous analysis of nine alkaloids and 11 aroma components in tobacco leaves. The method involves immersing the sample in an alkaline solution to release the target alkaloids and aroma compounds from the sample matrix. Chloroform is then added and vortexed to transfer the alkaloids and aroma compounds into a chloroform phase. After centrifugation and separation, the chloroform phase is sampled for instrumental analysis. Compared to existing methods for analyzing alkaloids and aroma components in tobacco leaves, the method is simpler to use and more stable. However, the patent does not address the analysis of tobacco wax compounds and covers a relatively limited range of analysis, leaving room for further improvement. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to propose a separation and detection method for simply, conveniently and accurately identifying multiple waxy compound components in tobacco leaves.

[0006] The present invention solves the above technical problems through the following technical means: The present invention provides a method for rapidly measuring the content of waxy compounds in tobacco leaves based on extraction and GC-MS, comprising the following steps: (1) Mix tobacco leaf powder with ethanol, perform ultrasonic treatment, filter, and retain the filtrate; add deionized water to the obtained filtrate to obtain a diluted tobacco leaf extract; extract with an organic solvent to prepare a sample to be tested; (2) Select internal standard and standard to prepare internal standard solution and standard solution respectively, and use GC-MS technology to test according to the following chromatographic mass spectrometry conditions; use the ratio of the peak area of ​​the target compound to the internal standard as the vertical axis and the ratio of the concentration of the target compound to the internal standard as the horizontal axis to draw the standard working curve, and the linear correlation coefficient R 2 Greater than 0.995; The specific chromatographic mass spectrometry conditions are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: He, 99.99%; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min; heating to 120°C at a rate of 2.5°C / min and hold for 2 min, heating to 200°C at a rate of 1°C / min and hold for 2 min, heating to 250°C at a rate of 1°C / min and hold for 2 min, heating to 300°C at a rate of 2.5°C / min and hold for 2 min; The mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature: 150°C; scan mode: full scan; mass scan range: 50-550 amu; solvent delay: 4.5 min; (3) Adding an internal standard solution to the sample obtained in step (1), and using GC-MS technology according to the chromatographic mass spectrometry conditions in step (2) to detect the content of waxy compounds in the sample.

[0007] Preferably, in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g: (5-15) mL, more preferably 1 g: 10 mL.

[0008] Preferably, in step (1), the concentration of ethanol is 85-95% (v / v), more preferably 90% (v / v).

[0009] Preferably, in step (1), the ultrasonic power is 100-500W, and the ultrasonic treatment time is 0.5-3h, more preferably the power is 350W and the time is 0.5h.

[0010] Preferably, in step (1), deionized water is added to the filtrate until the volume concentration of ethanol drops to 10-50%, more preferably to 20%.

[0011] Preferably, in step (1), the organic solvent comprises one or more of petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA). Further preferably, in step (1), petroleum ether, dichloromethane, and ethyl acetate are sequentially used for stepwise extraction (the tobacco leaf extract and the organic solvent are mixed in a volume ratio of 1:10 and shaken evenly, allowed to stand for stratification, and the corresponding components are collected), and petroleum ether extract, dichloromethane extract, and ethyl acetate extract are sequentially obtained as the sample to be tested.

[0012] Preferably, in step (2) and step (3), the instrument used for GC-MS testing is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

[0013] Preferably, in step (2), the internal standard substances include five kinds, specifically phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosyl alcohol and 1-octacosanol.

[0014] Preferably, in step (2), the standard substances include 46 kinds of alkanes, alkanes, fatty alcohols, fatty acids, sterols and the like, specifically dodecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane, nonadecane, eicosane, heneicosane, docosane, tricosane, tetracosane, hexacosane, heptacosane, octacosane, triacontan, dotriacontane, tricosane, pentatriacontane, hexatriacontane, octatriacontane, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-nonadecane, 1-dodecene, 1-dodecanol, 1-tridecyl alcohol, 1-tetradecyl alcohol, 1-pentadecanol, 1-hexadecanol, 1-heptadecanol, 1-octadecyl alcohol, 1-nonadecanol, 1-docosane, 1-tetracosyl alcohol, 1-hexacosyl alcohol, palmitic acid, stearic acid, linolenic acid, cholesterol, stigmasterol, sitosterol, phytol, and scopoletin. On the one hand, the components of the wax compounds in the tobacco leaves can be further determined by comparing the retention times; on the other hand, the content of the wax compounds in the tobacco leaves can be determined by the standard working curve.

[0015] The beneficial effects of the present invention are: 1. The present invention proposes a method for determining waxy compounds in tobacco leaves based on gas chromatography-mass spectrometry. By pre-treating (extraction) the tobacco leaves and conducting GC-MS, the chromatographic and mass spectrometric conditions are optimized, allowing for more accurate detection and qualitative analysis. A total of 37 waxy compounds were identified from tobacco leaf powder, of which 10 compounds, including dehydrotoluol, isosemol, Δ5-avenasterol, cholesterol, dodecane, o-nicotine, 1-tetradecene, 1-hexadecene, hexadecane, and 1-heptadecanol, were isolated and detected for the first time. This further clarifies the types of chemical components in tobacco leaves and is of great significance.

[0016] 2. The separation and detection method of the present invention features a simple, convenient, and easy-to-use process; it can rapidly detect waxy compounds in tobacco leaves; and its detection results are accurate and highly reliable. By using a stepwise extraction process with organic solvents to separate waxy compounds from tobacco leaves and comparing them to standard samples to further identify the waxy compounds, the method aims to provide a reference for maximizing the utilization of tobacco waxy compounds. The method of the present invention has promising application prospects.

[0017] 3. The method of the present invention is the first to separate and detect chemical components from tobacco leaves that have not been detected before, providing an efficient and rapid analytical method for the qualitative analysis of chemical components in tobacco leaves and their quality control, and providing data support for further elucidation of the chemical basis in tobacco leaves and further resource development and utilization in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a process flow chart for separating and testing waxy compounds in tobacco leaves according to Example 1 of the present invention; Figure 2is a chromatogram of the PE component in Example 1 of the present invention; Figure 3 is a chromatogram of the DCM component in Example 1 of the present invention; Figure 4 is the chromatogram of the EA component in Example 1 of the present invention; Figure 5 is the chromatogram in Example 2 of the present invention; Figure 6 is the chromatogram in Example 3 of the present invention; Figure 7 is the chromatogram in Example 4 of the present invention; Figure 8 is the chromatogram in Example 5 of the present invention; Figure 9 It is the chromatogram in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments 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 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 making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0020] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0021] 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 this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0022] Example 1: A method for rapidly measuring the content of waxy compounds in tobacco leaves based on extraction and GC-MS, comprising the following steps: (1) Tobacco leaf powder from southern Anhui flue-cured tobacco was mixed with 90% (v / v) ethanol at a solid-liquid ratio of 1 g:10 mL. Ultrasonic extraction was performed at 350 W for 30 min under heating conditions at 60 °C. Filtrate I and residue I were obtained by filtration. Sufficient deionized water was added to filtrate I to reduce the ethanol concentration to 20%, thereby obtaining a diluted tobacco leaf extract. The extract was then extracted in a stepwise manner using organic solvents such as petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA). The tobacco leaf extract and the organic solvent were mixed in a volume ratio of 1:10 and shaken evenly. The extract was allowed to stand for stratification and the corresponding components were collected. A petroleum ether extract, a dichloromethane extract, and an ethyl acetate extract were obtained in sequence.

[0023] (2) Select internal standards and standards to prepare a series of standard working solutions of appropriate concentrations. There are five internal standards, namely phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosyl alcohol, and 1-octacosanol, numbered ①, ②, ③, ④, and ⑤, respectively. The standard substances include 46 types of alkanes, alkanes, fatty alcohols, fatty acids, and sterols, including dodecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane, nonadecane, eicosane, heneicosane, docosane, tricosane, tetracosane, hexacosane, heptacosane, octacosane, triacontan, dotriacontane, tricaracontane, pentatriacontane, hexatriacontane, octatriacontane, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-nonadecane, 1-dodecene, 1-dodecyl alcohol, 1-tridecyl alcohol, 1-tetradecyl alcohol, 1-pentadecanol, 1-hexadecanol, 1-heptadecanol, 1-octadecanol, 1-nonadecanol, 1-docosyl alcohol, 1-tetracosyl alcohol, 1-hexacosyl alcohol, palmitic acid, stearic acid, linolenic acid, cholesterol, stigmasterol, sitosterol, phytol, and scopoletin. The corresponding internal standards for different standard substances are shown in Table 2. The GC-MS was used to test under the following chromatographic mass spectrometry conditions. The ratio of the peak area of ​​the target compound to the internal standard was used as the ordinate, and the ratio of the concentration of the target compound to the internal standard was used as the abscissa. The standard working curve was drawn. The linear correlation coefficient R 2 Greater than 0.995.

[0024] The specific chromatographic mass spectrometry conditions are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: He, 99.99%; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection; split ratio: 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min; heating to 120°C at a rate of 2.5°C / min and hold for 2 min, heating to 200°C at a rate of 1°C / min and hold for 2 min, heating to 250°C at a rate of 1°C / min and hold for 2 min, heating to 300°C at a rate of 2.5°C / min and hold for 2 min; Mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature: 150°C; scan mode: full scan; mass scan range: 50–550 amu; solvent delay: 4.5 min. Gas chromatography-mass spectrometry was performed using an Agilent 7820A gas chromatograph coupled to an Agilent 5977B mass spectrometer.

[0025] (3) The petroleum ether extract, dichloromethane extract, and ethyl acetate extract obtained in step (1) are sequentially used as test samples. An appropriate amount of internal standard solution is added to the test sample. The content of the waxy compound in the test sample is detected by GC-MS under the same chromatographic mass spectrometry conditions as in step (2). Standard samples are selected as qualitative scales, and retention times are compared to further determine the composition of the waxy compounds in the tobacco leaves.

[0026] This example uses GC-MS to identify the components of waxy compounds in tobacco leaves. The results are shown in Table 1 and Figures 2-4 . The test results showed that a total of 37 compounds were identified, with a total content of 13628.93 μg / g. Among them, the compounds with higher content include stigmasterol (5298.33 μg / g), neophytadiene (2918.06 μg / g), linolenic acid (1196.98 μg / g), palmitic acid (840.48 μg / g), campesterol (373.08 μg / g), and 3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (352.83 μg / g). The detection method is convenient and the results are accurate and comprehensive. Among them, dehydrotoluol, isosemol, Δ5-avenasterol, cholesterol, dodecane, o-nicotine, 1-tetradecene, 1-hexadecene, hexadecane and 1-heptadecanol are 10 compounds separated and detected for the first time by the present invention.

[0027] Table 1: Summary of the total composition of tobacco wax compounds in this embodiment

[0028]

[0029] Table 2: Summary of qualitative standards used and retention times

[0030]

[0031]

[0032]

[0033] Example 2: The difference between this embodiment and embodiment 1 is that the stepwise extraction with petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (1) is replaced by extraction with petroleum ether (PE) only, and the rest is the same as in embodiment 1.

[0034] The results showed that only 26 waxy compounds were identified. Compared with Example 1, the number of waxy compounds was significantly reduced, and the detection results were not as ideal as those in Example 1.

[0035] Example 3: The difference between this embodiment and embodiment 1 is that the stepwise extraction with petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (1) is replaced by extraction with dichloromethane (DCM) only, and the rest is the same as in embodiment 1.

[0036] The results showed that only 34 waxy compounds were identified. Compared with Example 1, the number of waxy compounds was significantly reduced, and the detection results were not as ideal as those in Example 1.

[0037] Example 4: The difference between this embodiment and embodiment 1 is that the stepwise extraction with petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (1) is replaced by extraction with ethyl acetate (EA) only, and the rest is the same as in embodiment 1.

[0038] The results showed that only 30 waxy compounds were identified, which was significantly reduced compared to the number of waxy compounds in Example 1, and the detection results were not as ideal as those in Example 1.

[0039] Example 5: The difference between this embodiment and embodiment 1 is that the stepwise extraction with petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (1) is replaced by extraction with a mixed solvent of petroleum ether (PE): dichloromethane (DCM): ethyl acetate (EA) = 1:1:1. The rest is the same as in embodiment 1.

[0040] The results showed that only 35 waxy compounds were identified, which was significantly reduced compared to the number of waxy compounds in Example 1, and the detection results were not as ideal as those in Example 1.

[0041] Example 6: The difference between this embodiment and embodiment 1 is that in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g:5 mL, the ethanol concentration is 95% (v / v), the ultrasonic power is 100 W, and the ultrasonic treatment time is 3 h. The rest is the same as in embodiment 1.

[0042] The results of this example are similar to those of Example 1.

[0043] Example 7: The difference between this embodiment and embodiment 1 is that in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g:15 mL, the ethanol concentration is 85% (v / v), the ultrasonic power is 500 W, and the ultrasonic treatment time is 1 h. The rest is the same as in embodiment 1.

[0044] The results of this example are similar to those of Example 1.

[0045] Comparative Example 1: The difference between this comparative example and Example 1 is that the model of the chromatographic column is changed to DB-WAX (123-7033, 30m×0.32mm×0.50μm), and the rest is the same as Example 1.

[0046] The results are as follows Figure 9 As shown: The number of chromatographic peaks is extremely limited, making it impossible to accurately detect waxy compounds in tobacco leaves.

[0047] Comparative Example 2: The difference between this comparative example and Example 1 is that the temperature program of the chromatographic conditions is changed to: 50°C for 1 min; heating to 100°C at a rate of 2.5°C / min and holding for 2 min, heating to 200°C at a rate of 1°C / min and holding for 1 min, heating to 250°C at a rate of 2.5°C / min and holding for 3 min, and heating to 300°C at a rate of 1°C / min and holding for 1 min; the rest is the same as in Example 1.

[0048] Results: The chromatographic peaks were seriously overlapped and there were many impurity peaks, which made it impossible to accurately detect the wax compounds in tobacco leaves.

[0049] Comparative Example 3: The difference between this comparative example and Example 1 is that the stepwise extraction with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by "extraction with acetonitrile only", and the rest is the same as Example 1.

[0050] Results: Only 8 waxy compounds were detected, and the content of each waxy compound was low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0051] Comparative Example 4: The difference between this comparative example and Example 1 is that the stepwise extraction with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by "extraction with methanol only", and the rest is the same as Example 1.

[0052] Results: Only 9 waxy compounds were detected, and the content of each waxy compound was extremely low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0053] 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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various embodiments of the present invention.

Claims

1. A method for rapidly measuring the content of waxy compounds in tobacco leaves based on extraction and GC-MS, characterized in that: The following steps are involved: (1) Mix tobacco leaf powder with ethanol, perform ultrasonic treatment, filter, and retain the filtrate; add deionized water to the obtained filtrate to obtain a diluted tobacco leaf extract; extract with an organic solvent to prepare a sample to be tested; (2) Select internal standard and standard to prepare internal standard solution and standard solution respectively, and use GC-MS technology to test; use the ratio of target compound to internal standard peak area as the vertical axis and the ratio of target compound to internal standard concentration as the horizontal axis to draw the standard working curve, and the linear correlation coefficient R 2 Greater than 0.995; (3) Adding an internal standard solution to the sample obtained in step (1) and detecting the content of waxy compounds in the sample using GC-MS technology.

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

3. The method according to claim 1, characterized in that In step (1), the volume concentration of the ethanol is 85-95%.

4. The method according to claim 1, wherein In step (1), the ultrasonic power is 100 to 500 W, and the ultrasonic treatment time is 0.5 to 3 h.

5. The method according to claim 1, wherein In step (1), deionized water is added to the filtrate until the volume concentration of ethanol is reduced to 10-50%.

6. The method according to claim 1, characterized in that In step (1), the organic solvent includes one or more of petroleum ether, dichloromethane and ethyl acetate.

7. The method according to claim 6, characterized in that In step (1), petroleum ether, dichloromethane, and ethyl acetate are sequentially used for stepwise extraction, and petroleum ether extract, dichloromethane extract, and ethyl acetate extract are sequentially obtained as samples to be tested.

8. The method according to claim 1, characterized in that In step (2) and step (3), the instrument used for GC-MS testing is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

9. The method according to claim 1, characterized in that In step (2), the internal standard substances include phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosyl alcohol and 1-octacosanol.

10. The method according to claim 1, characterized in that The specific chromatographic mass spectrometry conditions in step (2) and step (3) are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: 99.99% pure He; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min, then increase the temperature to 120°C at a rate of 2.5°C / min and hold for 2 min, then increase the temperature to 200°C at a rate of 1°C / min and hold for 2 min, then increase the temperature to 250°C at a rate of 1°C / min and hold for 2 min, then increase the temperature to 300°C at a rate of 2.5°C / min and hold for 2 min. The mass spectrometry conditions were as follows: ion source: electron impact EI; electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature 150°C; scan mode: full scan; mass scan range: 50-550 amu; solvent delay: 4.5 min.

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