Liquid chromatography detection method for six alkaloids in tobacco and tobacco products

Abstract

The present invention discloses a liquid chromatography detection method for six alkaloids in tobacco and tobacco products, which is characterized by comprising the following steps: (1) sample extraction: accurately weigh tobacco and tobacco product powder, wet it with sodium hydroxide solution, add triethylamine / ethyl acetate solution, ultrasonicate for a period of time, take the upper liquid, and obtain filtrate by filtration; (2) detection: use high performance liquid chromatography to detect, that is, obtain the content of the six alkaloids in tobacco and tobacco products. The detection method of the present invention can fully extract the alkaloids in tobacco and tobacco products at one time, the chromatographic peaks of the six alkaloids have a good separation effect, and the chromatographic analysis time is shortened to 10 minutes.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a liquid chromatography detection method for six alkaloids in tobacco and tobacco products. Background Art

[0002] Nicotine and other six alkaloids are important chemical components in tobacco and tobacco products. Alkaloids are organic alkaline substances. Their content and composition will affect the pH value of mainstream smoke and the strength of cigarettes, which will directly affect the quality and taste of tobacco. Common alkaloids in tobacco include nicotine, nornicotine, 2,3'-bipyridine, anabasine, myosmine, cotinine, etc.

[0003] The most commonly used method for the detection of nicotine is gas chromatography-mass spectrometry (GC-MS). In the tobacco industry standard, gas chromatography-mass spectrometry (GC-MS) is used to quantitatively analyze the alkaloid content. It mainly uses sodium hydroxide solution to release the alkaloids in tobacco, and then uses triethylamine chloroform extraction. The method in the industry standard can well separate nornicotine and myosamine, but it is necessary to separate the detection of nicotine and other alkaloids and prepare the working curves of standard substances separately. [1] . Li Yong et al. [2] Disclosed is a method for simultaneously analyzing eight alkaloids in tobacco leaves. The method comprises sample pretreatment and standard curve quantitative analysis steps, wherein the alkaloids are freed from the sample matrix by soaking in a sodium hydroxide solution, the alkaloids are transferred into a chloroform phase by adding chloroform and vortexing, and the chloroform layer is taken for instrumental analysis after centrifugal stratification.

[0004] High performance liquid chromatography has the advantages of simple pre-treatment, high separation efficiency, high detection sensitivity, fast analysis speed, wide application range, good system stability, and multiple detection components with good peak shape. The use of liquid chromatography to detect tobacco alkaloids has attracted the continuous attention of technicians. Since 2006, the use of liquid chromatography to detect nicotine has attracted the attention of technicians. [3] By 2021, nicotine, anatabine, anabasine and nornicotine in tobacco [4] The relatively complex sample pretreatment and special mobile phase have always restricted the use of tobacco alkaloid liquid chromatography detection technology. [5] A liquid chromatography detection method for alkaloids is disclosed, but the sample pretreatment used involves a special sample extraction bottle. [4] In order to quickly and accurately determine the content of alkaloids in tobacco leaves, an HPLC method for determining nicotine, anatabine, anabasine and nornicotine in tobacco was established by optimizing the extraction conditions.

[0005] With the emergence of transformed tobacco strains such as cinnabar tobacco, rapid and accurate detection of alkaloids such as nornicotine and neonicotinoids in tobacco and tobacco products has become an important requirement for the development of the industry.

[0006] References:

[0007] [1] Ding Li, Qin Yaqiong, Yan Jin, Wang Xiaoyu, Cai Junlan, Liu Shaofeng, Gu Liang, Bao Fengwei, Yin Yanqi, Tian Haiying, Fan Zhong, Ji Houwei, Su Mingliang, Jiang Xingyi, Huang Fei, Zang Zhipeng, Wang Chao, Jia Yunzhen, Cui Huapeng, Wang Yipeng, Zhang Xiaojing. Determination of nicotine, nornicotine, anabasine, myosamine, anabasine, cotinine and 2,3'-bipyridine in total particulate matter of cigarettes by gas chromatography-mass spectrometry. In State Tobacco Monopoly Administration, 2018; DOI.

[0008] [2] Li Yong, Pang Tao, Shi Junli, Deng Xiaopeng, Li Yongping, Kong Guanghui. A method for simultaneous analysis of eight alkaloids in tobacco leaves[P]. CN110531006A, 2019-12-03.

[0009] [3] Chen Yanwu, Xiao Kun, Yin Dulin, Lian Shixun. Simultaneous determination of nicotine and polyphenols in tobacco leaves by HPLC[J]. Journal of Shunde Vocational and Technical College, 2006, DOI:(01):27-30.

[0010] [4] Lin Yusheng, Jin Hongshi, Zhang Haonan, Jin Jianghua, Guo Wei, Li Yu'e, Zhao Mingqin, Liu Pengfei. Simultaneous determination of multiple alkaloids in different types of tobacco leaves by high performance liquid chromatography[J]. Chinese Tobacco Science, 2021, v.42(03):83-89.

[0011] [5] Xu Yong, Huang Haitao, Li Jing, Kong Weisong, Wang Jin, Liu Xin, Li Xuemei, Yang Yekun, Xiang Haiying, Zeng Wanli, Zhang Chengming, Xu Li, Zhang Jianduo, Deng Lele, Jiang Jiarui, Yang Guangyu. A method for determining alkaloids in tobacco gene editing materials[P]. CN108519455A, 2018-09-11. Summary of the invention

[0012] The present invention provides a liquid chromatography detection method for six alkaloids in tobacco and tobacco products. The method innovatively optimizes the sample pretreatment method and chromatographic conditions, and uses a high performance liquid chromatograph to achieve rapid detection of the six alkaloids in tobacco and tobacco products.

[0013] The objectives of the present invention are achieved through the following technical solutions.

[0014] A liquid chromatography detection method for six alkaloids in tobacco and tobacco products, characterized in that it comprises the following steps:

[0015] (1) Sample extraction: Accurately weigh tobacco and tobacco product powder, moisten with sodium hydroxide solution, add triethylamine / ethyl acetate solution, sonicate for a period of time, take the upper liquid, and filter to obtain the filtrate;

[0016] (2) Detection: High performance liquid chromatography is used to detect the contents of six alkaloids in tobacco and tobacco products.

[0017] Preferably, the sample extraction in step (1) is specifically as follows: accurately weigh tobacco and tobacco product powder, add 5-10 times by weight of sodium hydroxide solution to wet the sample, then add 5-10 times by weight of triethylamine / ethyl acetate solution to the sodium hydroxide solution, ultrasonicate for 1-30 minutes, take the upper liquid and filter it through a 0.22 μm organic filter membrane to obtain a filtrate for detection in step (2).

[0018] Preferably, the concentration of the sodium hydroxide solution is 3-8 wt %; more preferably, the concentration of the sodium hydroxide solution is 5 wt %.

[0019] Preferably, the concentration of triethylamine / ethyl acetate is 0.005-0.05 wt %; more preferably, the concentration of triethylamine / ethyl acetate is 0.01 wt %.

[0020] Preferably, the liquid chromatography conditions in step (2) are: chromatographic column: C18 column, specifications: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength is 259nm.

[0021] Preferably, the gradient elution conditions are: time 0.00 min, 20wt% mobile phase A + 80wt% mobile phase B; 2 min, 30wt% mobile phase A + 70wt% mobile phase B; 6 min, 40wt% mobile phase A + 60wt% mobile phase B; 10 min, 60wt% mobile phase A + 40wt% mobile phase B.

[0022] Preferably, mobile phase A is methanol and mobile phase B is 0.1 wt% ammonium acetate aqueous solution.

[0023] Beneficial effects of the present invention:

[0024] 1. The method of the present invention can fully extract six alkaloids in tobacco and tobacco products at one time, and complete the content detection in ten minutes, and the time to complete the detection is relatively short.

[0025] 2. The alkaloid extraction solvent ethyl acetate used in the present invention has three advantages: (1) When extracting tobacco alkaloids, the operation is simplified by absorbing the upper solvent; (2) ethyl acetate is less corrosive to liquid chromatographs and is more friendly than dichloromethane and chloroform; (3) ethyl acetate (5-20 μL) entering the liquid chromatograph is more soluble in the mobile phase than dichloromethane and chloroform. [5] Compared with the literature, the sample pretreatment method was simplified and the detection of anabasine was added. [4] Compared with the traditional method, the chromatographic detection time is shortened by 15 minutes, and the detection of myosamine and cotinine is added, realizing the rapid determination of the content of six alkaloids in tobacco and tobacco products. When extracting alkaloids, adding a small amount of triethylamine can alkalize and convert the alkaloid salt into a free state.

[0026] 3. This method is fast, accurate and reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the liquid chromatogram of six alkaloid standards.

[0028] Figure 2 Chromatogram of alkaloids in the sample of Example 1.

[0029] Among them, 1. nornicotine, 2. anabasine, 3. anatabine, 4. cotinine, 5. nicotine, 6. myosamine. DETAILED DESCRIPTION

[0030] The present invention is further described in conjunction with the following examples, but the present invention is not limited thereto:

[0031] Example 1

[0032] The present invention is used to detect alkaloids in cinnabar tobacco leaf powder, and the steps are as follows:

[0033] (1) Sample extraction: Weigh 0.3000 g of cinnabar tobacco leaf powder accurately to 0.001 g and place it in a 25 mL plastic centrifuge tube. Add 2 mL of 5 wt% sodium hydroxide solution to the plastic centrifuge tube to wet the sample. Let it stand for 15 min. Add 20 mL of 0.01 wt% triethylamine / ethyl acetate solution. After sealing with a cap, place it in an ultrasonic generator with a power of 720 W and ultrasonicate it at room temperature for 15 min. After ultrasonication, place it in a centrifuge and centrifuge it for 5 min. After centrifugation, take the upper layer of liquid, filter it through a 0.22 μm organic filter membrane into a sample bottle, and collect about 1 mL of the filtrate.

[0034] (2) Determination: The content of the six alkaloids in tobacco and tobacco products was obtained by high performance liquid chromatography. The liquid chromatography conditions were: chromatographic column: C18 column, specification: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength was 259nm. Mobile phase A was methanol, and mobile phase B was 0.1wt% ammonium acetate aqueous solution. Gradient elution conditions were: time 0.00min, 20wt% mobile phase A+80wt% mobile phase B; 2min, 30wt% mobile phase A+70wt% mobile phase B; 6min, 40wt% mobile phase A+60wt% mobile phase B; 10min, 60wt% mobile phase A+40wt% mobile phase B.

[0035] Using this detection method, the standard curve equations and correlation coefficients of the six alkaloids are shown in the following table. The contents of nicotine, nornicotine, anabasine, anatabine, mysmine and cotinine in red sand tobacco leaves are 1.19, 55.42, 1.18, 4.00, 0.08 and 0.03 mg / g, respectively, and the total content of the six alkaloids is 10.55 mg / g.

[0036] Table 1 Detection of the contents of six alkaloids in red sand tobacco leaves

[0037]

[0038] Example 2

[0039] The present invention is used to detect alkaloids in Yunyan 97 tobacco leaf powder, and the steps are as follows:

[0040] (1) Sample extraction: Weigh 0.3000 g of Yunyan 97 tobacco leaf powder accurately to 0.001 g and place it in a 25 mL plastic centrifuge tube. Add 2 mL of 5 wt% sodium hydroxide solution to the plastic centrifuge tube to wet the sample. Let it stand for 15 min. Add 20 mL of 0.01 wt% triethylamine / ethyl acetate solution. After sealing with a cover, place it in an ultrasonic generator with a power of 720 W. Ultrasonicate at room temperature for 15 min. After ultrasonication, place it in a centrifuge for 5 min. Centrifuge, take the upper layer of liquid, filter it through a 0.22 μm organic filter membrane into a sample bottle, and collect about 1 mL of the filtrate.

[0041] (2) Determination: The content of the six alkaloids in tobacco and tobacco products was obtained by high performance liquid chromatography. The liquid chromatography conditions were: chromatographic column: C18 column, specification: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength was 259nm. Mobile phase A was methanol, and mobile phase B was 0.1wt% ammonium acetate aqueous solution. Gradient elution conditions were: time 0.00min, 20wt% mobile phase A+80wt% mobile phase B; 2min, 30wt% mobile phase A+70wt% mobile phase B; 6min, 40wt% mobile phase A+60wt% mobile phase B; 10min, 60wt% mobile phase A+40wt% mobile phase B.

[0042] Using this detection method, the standard curve equations and correlation coefficients of the six alkaloids are shown in the following table. The contents of nicotine, nornicotine, anabasine, anatabine, myosamine and cotinine in Yunyan 97 tobacco leaves are 15.26, 0.41, 0.15, 0.90, 0.06 and 0.01 mg / g, respectively, and the total content of the six alkaloids is 16.79 mg / g.

[0043] Table 2 Detection of the contents of six alkaloids in Yunyan 97 tobacco leaves

[0044]

[0045] Example 3

[0046] The present invention is used to detect alkaloids in Huize dry tobacco leaves, and the steps are as follows:

[0047] (1) Sample extraction: Weigh 0.3000 g of tobacco leaf powder from Huize dry tobacco accurately to 0.001 g and place it in a 25 mL plastic centrifuge tube. Add 2 mL of 5 wt% sodium hydroxide solution to the plastic centrifuge tube to wet the sample. Let it stand for 15 min. Add 20 mL of 0.01 wt% triethylamine / ethyl acetate solution. After sealing with a cap, place it in an ultrasonic generator with a power of 720 W and ultrasonicate it at room temperature for 15 min. After ultrasonication, place it in a centrifuge and centrifuge it for 5 min. After centrifugation, take the upper layer of liquid, filter it through a 0.22 μm organic filter membrane into a sample bottle, and collect about 1 mL of the filtrate.

[0048] (2) Determination: The content of the six alkaloids in tobacco and tobacco products was obtained by high performance liquid chromatography. The liquid chromatography conditions were: chromatographic column: C18 column, specification: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength was 259nm. Mobile phase A was methanol, and mobile phase B was 0.1wt% ammonium acetate aqueous solution. Gradient elution conditions were: time 0.00min, 20wt% mobile phase A+80wt% mobile phase B; 2min, 30wt% mobile phase A+70wt% mobile phase B; 6min, 40wt% mobile phase A+60wt% mobile phase B; 10min, 60wt% mobile phase A+40wt% mobile phase B.

[0049] Using this detection method, the standard curve equations and correlation coefficients of the six alkaloids are shown in the following table. The contents of nicotine, nornicotine, anabasine, anatabine, myosamine and cotinine in Huize dry tobacco leaves are 22.40, 3.51, 0.13, 4.62, 0.12 and 0.15 mg / g, respectively, and the total content of the six alkaloids is 30.93 mg / g.

[0050] Table 3 Detection of the contents of six alkaloids in tobacco leaves of Huize dry tobacco

[0051]

[0052] Example 4

[0053] The present invention is used to detect alkaloids in tobacco shreds of a certain specification, and the steps are as follows:

[0054] (1) Sample extraction: Weigh 0.3000 g of tobacco powder of a certain specification accurately to 0.001 g and place it in a 25 mL plastic centrifuge tube. Add 2 mL of 5 wt% sodium hydroxide solution to the plastic centrifuge tube to wet the sample. Let it stand for 15 min. Add 20 mL of 0.01 wt% triethylamine / ethyl acetate solution. After sealing with a cover, place it in an ultrasonic generator with a power of 720 W and ultrasonicate it at room temperature for 15 min. After ultrasonication, place it in a centrifuge and centrifuge it for 5 min. Centrifuge and take the upper layer of liquid. Filter it through a 0.22 μm organic filter membrane into a sample bottle and collect about 1 mL of the filtrate.

[0055] (2) Determination: The content of the six alkaloids in tobacco and tobacco products was obtained by high performance liquid chromatography. The liquid chromatography conditions were: chromatographic column: C18 column, specification: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength was 259nm. Mobile phase A was methanol, and mobile phase B was 0.1wt% ammonium acetate aqueous solution. Gradient elution conditions were: time 0.00min, 20wt% mobile phase A+80wt% mobile phase B; 2min, 30wt% mobile phase A+70wt% mobile phase B; 6min, 40wt% mobile phase A+60wt% mobile phase B; 10min, 60wt% mobile phase A+40wt% mobile phase B.

[0056] Using this detection method, the standard curve equations and correlation coefficients of the six alkaloids are shown in the following table. The contents of nicotine, nornicotine, anabasine, anatabine, mysamine and cotinine in a certain specification of tobacco are 19.68, 0.39, 0.10, 4.08, 0.12 and 0.03 mg / g respectively, and the total content of the six alkaloids is 24.28 mg / g.

[0057] Table 4 Detection of the contents of six alkaloids in tobacco of a certain specification

[0058]

[0059] -: Below the detection limit

[0060] Example 5

[0061] The present invention is used to detect alkaloids in Huize dry tobacco slices, and the steps are as follows:

[0062] (1) Sample extraction: Weigh 0.3000 g of Huize tobacco flake powder accurately to 0.001 g and place it in a 25 mL plastic centrifuge tube. Add 2 mL of 5 wt% sodium hydroxide solution to the plastic centrifuge tube to wet the sample. Let it stand for 15 min. Add 20 mL of 0.01 wt% triethylamine / ethyl acetate solution. After sealing with a cover, place it in an ultrasonic generator with a power of 720 W and ultrasonicate at room temperature for 15 min. After ultrasonication, place it in a centrifuge for 5 min. Centrifuge and take the upper layer of liquid. Filter it through a 0.22 μm organic filter membrane into a sample bottle and collect about 1 mL of the filtrate.

[0063] (2) Determination: The content of the six alkaloids in tobacco and tobacco products was obtained by high performance liquid chromatography. The liquid chromatography conditions were: chromatographic column: C18 column, specification: 3μm, 4.6mm×150mm; flow rate: 1.0mL / min; column temperature: 25-35°C; injection volume: 10μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength was 259nm. Mobile phase A was methanol, and mobile phase B was 0.1wt% ammonium acetate aqueous solution. Gradient elution conditions were: time 0.00min, 20wt% mobile phase A+80wt% mobile phase B; 2min, 30wt% mobile phase A+70wt% mobile phase B; 6min, 40wt% mobile phase A+60wt% mobile phase B; 10min, 60wt% mobile phase A+40wt% mobile phase B.

[0064] Using this detection method, the standard curve equations and correlation coefficients of the six alkaloids are shown in the following table. The contents of nicotine, nornicotine, anabasine, anatabine, myosamine and cotinine in Huize dry tobacco slices are 22.40, 3.51, 0.13, 4.62, 0.12 and 0.15 mg / g, respectively, and the total content of the six alkaloids is 24.28 mg / g.

[0065] Table 5 Detection of the contents of six alkaloids in tobacco slices

[0066]

[0067] -: Below the detection limit

[0068] Figure 1 This is the liquid chromatogram of six alkaloid standards; Figure 2 Chromatogram of alkaloids in the sample of Example 1. Therefore, the method of the present invention realizes the rapid determination of the contents of six alkaloids in tobacco and tobacco products.

Claims

1. A liquid chromatography method for detecting six alkaloids in tobacco and tobacco products, wherein the six alkaloids are nicotine, nornicotine, anatabine, anabasine, mysmine and cotinine; characterized in that: The steps include: (1) Sample extraction: Accurately weigh tobacco and tobacco product powder, moisten with sodium hydroxide solution, add triethylamine / ethyl acetate solution, sonicate for a period of time, take the upper liquid, and filter to obtain the filtrate; (2) Detection: High performance liquid chromatography was used to detect the contents of six alkaloids in tobacco and tobacco products; The liquid chromatography conditions were as follows: chromatographic column: C18 column, specification: 3 μm, 4.6 mm×150 mm; flow rate: 1.0 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; detector: diode array detector (DAD), detection wavelength: 259 nm; The gradient elution conditions were as follows: time 0.00 min, 20 wt% mobile phase A + 80 wt% mobile phase B; 2 min, 30 wt% mobile phase A + 70 wt% mobile phase B; 6 min, 40 wt% mobile phase A + 60 wt% mobile phase B; 10 min, 60 wt% mobile phase A + 40 wt% mobile phase B; Mobile phase A was methanol, and mobile phase B was 0.1 wt % ammonium acetate aqueous solution; The sample extraction in step (1) is specifically as follows: accurately weigh tobacco and tobacco product powder, add 5-10 times by weight of sodium hydroxide solution to wet the sample, then add 5-10 times by weight of triethylamine / ethyl acetate solution to the sodium hydroxide solution, ultrasonicate for 1-30 minutes, take the upper liquid and filter it through a 0.22 μm organic filter membrane to obtain a filtrate for detection in step (2); the concentration of the sodium hydroxide solution is 3-8wt%; the concentration of the triethylamine / ethyl acetate is 0.005-0.05wt%.

Images