A method for simultaneous analysis of multiple fat-soluble components in tobacco leaves

Abstract

This invention belongs to the field of tobacco chemical composition technology, specifically relating to a method for the simultaneous analysis of multiple fat-soluble components in tobacco leaves. The method includes the following steps: drying and grinding the tobacco leaves to be tested, adding an extractant and an internal standard to obtain a sample solution; analyzing the sample using a supercritical fluid chromatography system, employing multi-wavelength detection, and using the internal standard method. This invention provides a method for the simultaneous analysis of multiple fat-soluble components in tobacco leaves, addressing the common characteristics of fat-soluble components in tobacco leaves such as large molecular weight, poor volatility, and easy oxidation and decomposition. By using supercritical fluid chromatography, it can achieve simultaneous quantitative analysis of 13 fat-soluble components in tobacco, using less solvent, lower cost, and higher detection efficiency. This provides an environmentally friendly, more efficient, and convenient high-throughput analytical method for tobacco leaf quality evaluation.

Description

Technical Field

[0001] This invention belongs to the field of tobacco chemical composition technology, specifically relating to a method for simultaneous analysis of multiple fat-soluble components in tobacco leaves. Background Technology

[0002] Tobacco contains many fat-soluble components, such as β-carotene, fat-soluble vitamins, sterols, cephalosporins, lysine, glycolipids, and glycerides, which are closely related to the quality and function of tobacco. The accurate determination of important fat-soluble components in tobacco is of great significance for the evaluation of tobacco quality.

[0003] Lipid-soluble components in tobacco generally exhibit characteristics such as large molecular weight, poor volatility, and easy oxidation and decomposition. Commonly used detection methods include liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), and supercritical fluid chromatography (SCLC). However, LC or LC-MS methods face difficulties in separating isomers of lipid-soluble components, requiring the use of large amounts of hazardous organic solvents such as acetone, dichloromethane, and chloroform, and involving long analysis times. Supercritical fluid chromatography (SCLC) uses supercritical CO2 as the main mobile phase, relying on the solvation ability of the mobile phase for separation and analysis. Theoretically, SCLC can analyze not only high-boiling-point, low-volatility, and thermally unstable samples that are unsuitable for gas chromatography, but also provides suitable retention and separation for various substances such as structural analogs, lipid-soluble compounds, and thermally unstable compounds. Compared to LC, SCLC simplifies the pretreatment process and reduces the use of organic solvents when analyzing lipid-soluble components. Currently, supercritical fluid chromatography (SFC) is widely used for the detection of carotenoids, triglycerides, steroids, and fat-soluble vitamins in food. However, it typically only detects one type of compound, resulting in low detection efficiency. Furthermore, there are currently no reports on the analysis of cephalosporin compounds in tobacco. Therefore, using SFC to simultaneously analyze multiple fat-soluble components in tobacco can help to further clarify the key components responsible for tobacco quality, providing important support for improving tobacco quality and its comprehensive utilization. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for simultaneous analysis of multiple fat-soluble components in tobacco leaves, which solves the defects of the prior art, such as large amount of organic solvent used, high analysis cost, cumbersome pretreatment process and low detection efficiency. This invention can realize the simultaneous analysis of 13 fat-soluble components in tobacco leaves.

[0005] To achieve the above and related objectives, the present invention provides a method for simultaneous analysis of multiple fat-soluble components in tobacco leaves, comprising the following steps:

[0006] 1) After drying and grinding the tobacco leaves to be tested, add the extractant and internal standard to obtain the sample solution to be tested;

[0007] 2) The sample was analyzed using a supercritical fluid chromatography system, with multi-wavelength detection and analysis using the internal standard method.

[0008] Preferably, in step 1), the tobacco leaf to be tested is a cured tobacco leaf or a fresh tobacco leaf;

[0009] Preferably, in step 1), the extractant is selected from one or more of chloroform, acetone, dichloromethane, isopropanol, dichloromethane / ethanol (1:1, V:V) and dichloromethane / ethanol (2:1, V:V);

[0010] Preferably, in step 1) of 13), the internal standard is selected from β-apo-8'-carotene;

[0011] Preferably, in step 1), the ratio of the tobacco leaf to the extractant is (1g:5mL)-(1g:30mL);

[0012] Preferably, in feature 12), the extractant is selected from dichloromethane / ethanol (volume ratio V:V is 1:1 to 5:1), and more preferably, the extractant is selected from dichloromethane / ethanol (2:1, V:V).

[0013] Preferably, in feature 13), the concentration of the internal standard is 5-60 μg / mL.

[0014] Preferably, in feature 14), the ratio of the tobacco leaf to the extractant is 1g:20mL.

[0015] Preferably, in step 1), after adding the extraction dose and internal standard, the process further includes vortexing and shaking.

[0016] Preferably, the oscillation time in A1) is 5-40 minutes; preferably, the oscillation time is 10 minutes.

[0017] Preferably, A2) further includes filtering the extracted supernatant using a filter membrane after shaking.

[0018] Preferably, in feature A2), the pore size of the filter membrane is 0.2-0.4 μm.

[0019] Preferably, in step 2), the fat-soluble component is selected from one or more of α-carotene, β-carotene, chlorophyll a, chlorophyll b, lutein, zeaxanthin, neoxanthin, vitamin E, α-cephalotrindiol, β-cephalotrindiol, ergosterol, hyoscyamine, and solanesol.

[0020] Preferably, in step 2), the supercritical fluid chromatography system includes at least one of the following technical features:

[0021] 21) The stationary phase of the chromatographic column is 1-aminoanthracene, high-density glycol, diethylamine, 2-aminomethylpyridine, or C18; preferably, it is C18.

[0022] 22) The flowing phase A is CO2;

[0023] 22) Phase B is one or more of methanol, ethanol, isopropanol, acetonitrile, and methanol / acetonitrile; preferably, it is methanol / acetonitrile.

[0024] 23) The chromatographic conditions are:

[0025] Column temperature: 30-55℃;

[0026] Detection wavelengths: one or more of 180-240, 250-310, 300-360 and 430-470 nm; one or more of 210, 280, 330 and 430 nm; during the measurement of this invention, multiple wavelengths are collected simultaneously in the same time period.

[0027] Injection volume: 0.5-3 μL;

[0028] Flow rate: 0.5-2 mL / min; Dynamic back pressure: 1800-2200 psi;

[0029] Gradient elution conditions: 0-3.5 min, 0-3% B; 9-12 min, 5-10% B; 15-18 min, 15-20% B; 19-23 min, 20-30% B; 23.5-27 min, 0-3% B.

[0030] Preferably, the internal standard method refers to the following steps: First, a series of standard test solutions of different concentrations are prepared by adding at least one of the 13 lipid-soluble components to a solvent and an internal standard solution. Then, a supercritical fluid chromatography system is used to analyze these solutions to obtain the linear relationship between the peak area ratio of at least one of the 13 target compounds (standard / internal standard) and the corresponding concentration ratio. A corresponding standard working curve is then plotted, and the regression equation of the standard working curve is calculated. Next, the sample test solution is analyzed using a supercritical fluid chromatography system to obtain the peak area ratio of at least one of the 13 lipid-soluble components to the internal standard. This ratio is then substituted into the regression equation of the standard working curve to calculate the content of the corresponding target compound in the sample test solution.

[0031] As described above, the method for simultaneous analysis of multiple fat-soluble components in tobacco leaves provided by the present invention has the following beneficial effects:

[0032] This invention provides a method for the simultaneous analysis of multiple fat-soluble components in tobacco leaves. Addressing the common characteristics of fat-soluble components in tobacco leaves, such as large molecular weight, poor volatility, and easy oxidation and decomposition, this method employs supercritical fluid chromatography for detection. It can simultaneously and quantitatively analyze 13 fat-soluble components in tobacco, using less solvent, resulting in low cost and high detection efficiency. This provides an environmentally friendly, more efficient, and convenient high-throughput analytical method for the quality evaluation of tobacco leaves. Attached Figure Description

[0033] Figure 1 The chromatogram of the lipid-soluble component standard at 430 nm is shown.

[0034] Figure 2 The chromatogram of the lipid-soluble component standard at 280 nm is shown.

[0035] Figure 3 The chromatogram of the lipid-soluble component standard at 210 nm is shown.

[0036] Figure 4 This is the chromatogram of the actual sample at 430 nm.

[0037] Figure 5 This is the chromatogram of the actual sample at 280 nm.

[0038] Figure 6 This is the chromatogram of the actual sample at 210 nm.

[0039] Figure 7 This is the chromatogram of the actual sample at 330 nm.

[0040] Figure 8 Chromatograms of actual samples at 210 nm under different dynamic back pressures.

[0041] Figure 9 Chromatograms of actual samples at 210 nm under different B phases. Detailed Implementation

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art.

[0043] The reagents and experimental equipment used in the following examples are all commonly used and commercially available. The specific reagents and instruments used are as follows:

[0044] 1. Reagents

[0045] The standards for α-carotene, β-carotene, chlorophyll a, chlorophyll b, lutein, zeaxanthin, neoxanthin, vitamin E, α-cephalotrindiol, β-cephalotrindiol, ergosterol, hyoscyamine, solanesyl alcohol, and β-apo-8'-carotene were purchased from Bailingwei Company. Dichloromethane, methanol, ethanol, acetonitrile, and isopropanol were of chromatographic grade and purchased from TEDIA Company.

[0046] 2. Instruments

[0047] The Waters ACQUITY UPC2 system is equipped with a PDA detector; an electronic balance (accuracy: 0.0001g, Mettler Toledo, Switzerland); a Milli-Q pure water system (Millpore, USA); and a multi-tube vortex mixer (Anson, USA).

[0048] Example 1

[0049] 1. Preparation of standard solutions

[0050] Standard stock solutions: Accurately weigh 50 mg of each standard substance, dissolve it in dichloromethane-ethanol (2:1, V / V) solution and dilute to 10 mL to prepare 5 mg / mL standard stock solutions. After purging with nitrogen, seal and store at -80°C for later use.

[0051] Mixed stock solution: Take 0.5 mL of each standard stock solution of the target analyte into a 10 mL brown volumetric flask, and dilute to 10 mL with dichloromethane-ethanol (2:1, V / V) solution to prepare a mixed stock solution of 250 μg / mL. After purging with nitrogen, seal and store at -80°C for later use.

[0052] Internal standard solution: Weigh 10 mg of β-apo-8'-carotene, dissolve it in dichloromethane-ethanol (2:1, V / V) solution and dilute to 10 mL to prepare a 1 mg / mL internal standard solution. Seal and set aside for later use.

[0053] Standard working solution: Transfer a certain volume of the mixed stock solution into a 10 mL brown volumetric flask, add 100 μL of internal standard solution, and dilute to 10 mL with dichloromethane-ethanol (2:1, V / V) solution. Prepare the standard working solution according to the target analyte content range of the actual sample, purge with nitrogen, seal and store at -80°C until use.

[0054] 2. Analysis conditions

[0055] Column: Waters Acquity UPC 2 HSS C 18SB column (100 mm × 3.0 mm, 1.8 μm); mobile phase: phase A is CO2, phase B is methanol / acetonitrile mixture (1:1, v / v); gradient elution conditions: 0–3.5 min, 3% B; 9–12 min, 10% B; 15–18 min, 20% B; 19–23 min, 30% B; 23.5–27 min, 3% B; flow rate: 1.0 mL / min; injection volume: 2 μL; column temperature: 45 °C; dynamic back pressure: 2000 psi; detection wavelengths: 210 nm, 280 nm, 330 nm, 430 nm; strong elution solvent: methanol; weak elution solvent: methanol / isopropanol (1:1, v / v).

[0056] 3 Experimental Results

[0057] The prepared standard working solution was analyzed in a supercritical fluid chromatography system, and the results are as follows: Figures 1 to 3 As shown, it can be seen that 13 lipid-soluble components can be well separated and detected at different wavelengths. In particular, this method can effectively separate compounds with similar structures and properties, such as chlorophyll a and chlorophyll b, xanthophyll and zeaxanthin, solanesol and cepertrienidol, which are prone to mutual interference.

[0058] A standard curve was plotted with the concentration ratio of each standard to the internal standard as the abscissa and the peak area ratio (Y) as the ordinate. Linear regression was then performed to obtain the regression equation, correlation coefficient, and linear range. The specific results are shown in Table 1. This method can effectively quantify 13 lipid-soluble components in the sample.

[0059] Table 1. Linear relationships of 13 fat-soluble components

[0060]

[0061] Example 2

[0062] 1 Sample Pretreatment

[0063] Fresh tobacco leaf samples were freeze-dried, ground, and stored in an ultra-low temperature freezer at -80°C for later analysis. Roasted tobacco leaves were directly analyzed by taking tobacco leaf powder. For analysis, 0.5 g of tobacco leaf sample was accurately weighed, and 100 μL of internal standard solution and 9.9 mL of dichloromethane-ethanol (2:1, V / V) extraction solution were added. The mixture was vortexed for 10 min, filtered through a 0.2 μm PTFE membrane, and analyzed under the same conditions as in Example 1.

[0064] 2 Experimental Results

[0065] The prepared tobacco extract was analyzed using a supercritical fluid chromatography system, and the results are as follows: Figures 4 to 6As shown, it can be seen that, except for ergosterol (which belongs to fungal sterols and is found in moldy tobacco leaves), the other 12 fat-soluble components were detected. Among them, hyoscyamine showed a weak response at 280 nm and required analysis at 330 nm for detection. Figure 7 ).

[0066] Example 3

[0067] 1 Sample Pretreatment

[0068] Fresh tobacco leaf samples were freeze-dried, ground, and stored in an ultra-low temperature freezer at -80°C for later analysis. For roasted tobacco leaves, the powder was directly collected for analysis. For analysis, 0.5 g of tobacco leaf sample was accurately weighed, and 100 μL of internal standard solution and 9.9 mL of dichloromethane-ethanol (2:1, V / V) extraction solution were added. The sample was vortexed for 10 min, filtered through a 0.2 μm PTFE membrane, and then directly loaded for analysis.

[0069] 2. Analysis conditions

[0070] Column: Waters Acquity UPC 2 HSS C 18 SB column (100mm × 3.0mm, 1.8μm)

[0071] Mobile phase: Phase A is CO2, and Phase B is a methanol / acetonitrile mixed solution (1:1, volume ratio);

[0072] Gradient elution conditions: 0-3.5 min, 3% B; 9-12 min, 10% B; 15-18 min, 20% B; 19-23 min, 30% B; 23.5-27 min, 3% B;

[0073] Flow rate: 1.0 mL / min;

[0074] Injection volume: 2 μL;

[0075] Column temperature: 45℃;

[0076] Dynamic back pressure: 1800, 1900, 2000, 2100, 2200 psi;

[0077] Detection wavelengths: 210nm, 280nm, 330nm, 430nm;

[0078] Strong cleaning solvent: methanol;

[0079] Mild washing solvent: methanol / isopropanol (1:1, volume ratio).

[0080] 3 Experimental Results

[0081] In supercritical fluid chromatography, dynamic back pressure is a crucial factor affecting the separation process. Under different dynamic back pressures, supercritical carbon dioxide exhibits varying solubility for different analytes. As the back pressure increases, the density of the supercritical fluid increases, enhancing its solubilizing ability, leading to higher column pressure and shorter analyte retention times. Experiments investigated the separation effects of target analytes in fresh tobacco leaf samples under different pressure conditions (1800, 1900, 2000, 2100, and 2200 psi). Figure 8 As shown, at 2000 psi, the separation of solanesol, α-cephalitol and β-cephalitol was good between 10 and 12 min. Under other pressure conditions, solanesol would overlap with the chromatographic peaks of α-cephalitol or β-cephalitol and its subsequent impurities.

[0082] Example 4

[0083] 1 Sample Pretreatment

[0084] Fresh tobacco leaf samples were freeze-dried, ground, and stored in an ultra-low temperature freezer at -80°C for later analysis. For roasted tobacco leaves, the powder was directly collected for analysis. For analysis, 0.5 g of tobacco leaf sample was accurately weighed, and 100 μL of internal standard solution and 9.9 mL of dichloromethane-ethanol (2:1, V / V) extraction solution were added. The sample was vortexed for 10 min, filtered through a 0.2 μm PTFE membrane, and then directly loaded for analysis.

[0085] 2. Analysis conditions

[0086] Column: Waters Acquity UPC 2 HSS C 18 SB column (100 mm × 3.0 mm, 1.8 μm); mobile phase: phase A is CO2, phase B is one of the following: methanol / acetonitrile mixture (1:1, v / v), ethanol / acetonitrile mixture (1:1, v / v), ethanol, acetonitrile, or 0.2% formic acid-methanol / acetonitrile mixture (1:1, v / v); gradient elution conditions: 0–3.5 min, 3% B; 9–12 min, 10% B; 15–18 min 20% B; 19-23 min, 30% B; 23.5-27 min, 3% B; Flow rate: 1.0 mL / min; Injection volume: 2 μL; Column temperature: 45℃; Dynamic back pressure: 1800, 1900, 2000, 2100, 2200 psi; Detection wavelength: 210 nm, 280 nm, 330 nm, 430 nm; Strong wash solvent: methanol; Weak wash solvent: methanol / isopropanol (1:1, volume ratio).

[0087] 3 Experimental Results

[0088] The modifier (phase B) affects the solubility of carbon dioxide in supercritical fluids and also influences the interaction between the analyte and the stationary phase. Therefore, modifying the modifier (phase B) plays a crucial role in the retention and selectivity of the target analyte. The effects of different co-solvents, including ethanol, acetonitrile, ethanol / acetonitrile (1:1), methanol / acetonitrile (1:1), and methanol / acetonitrile (1:1) with the addition of 0.2% formic acid, on the separation of the target analyte in real samples were investigated. Figure 9 As shown, ethanol, acetonitrile, and ethanol / acetonitrile (1:1) showed poor separation effects for solanesol and cepertrienidide, while methanol / acetonitrile (1:1) showed better separation effects.

[0089] This invention establishes a method for the simultaneous analysis of multiple fat-soluble components in tobacco leaves, enabling the simultaneous analysis of 13 fat-soluble components, including α-carotene, β-carotene, chlorophyll a, chlorophyll b, lutein, zeaxanthin, neoxanthin, vitamin E, α-ceftrindiol, β-ceftrindiol, ergosterol, hyoscyamine, and solanesol. The method uses minimal organic solvents, is low-cost, and has high detection efficiency, providing an environmentally friendly, more efficient, and convenient high-throughput analytical method for tobacco leaf quality evaluation.

[0090] Therefore, this invention effectively overcomes the shortcomings of the prior art and has industrial application value.

[0091] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for simultaneous analysis of multiple fat-soluble components in tobacco leaves, characterized in that, Includes the following steps: 1) After drying and grinding the tobacco leaves to be tested, add the extractant and internal standard to obtain the sample solution to be tested; the extractant is dichloromethane-ethanol; after adding the extractant and internal standard, the process further includes vortexing and shaking; after shaking, the supernatant of the extract is filtered through a filter membrane. 2) Supercritical fluid chromatography (SFC) was used to analyze the samples, employing multi-wavelength detection and internal standard analysis to identify various lipid-soluble components in tobacco leaves. These lipid-soluble components included α-carotene, β-carotene, chlorophyll a, chlorophyll b, lutein, zeaxanthin, neoxanthin, vitamin E, α-ceftrindiol, β-ceftrindiol, ergosterol, hyoscyamine, and solanesol. The SFC detection conditions were as follows: stationary phase: C18 column; mobile phase A: CO2; mobile phase B: methanol / acetonitrile mixture (1:1, volume ratio); dynamic back pressure: 2000 psi; detection wavelengths: 210 nm, 280 nm, 330 nm, 430 nm; gradient elution conditions: 0-3.5 min, 0-3% B; 9-12 min, 5-10% B; 15-18 min, 15-20% B; 19-23 min, 10-3% B. min, 20-30% B; 23.5-27 min, 0-3% B.

2. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 1, characterized in that, Includes at least one of the following technical features: 11) In step 1), the tobacco leaves to be tested are either cured tobacco leaves or fresh tobacco leaves; 12) In step 1), the internal standard is selected from β-apo-8'-carotene; 13) In step 1), the ratio of the tobacco leaf to the extractant is (1g : 5mL) - (1g : 30mL).

3. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 2, characterized in that, In step (1), the extractant is selected from dichloromethane / ethanol (volume ratio V:V is 1:1 to 5:1).

4. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 2, characterized in that, In feature 12), the concentration of the internal standard is 5-60 μg / mL.

5. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 2, characterized in that, In feature 13), the ratio of the tobacco leaf to the extractant is 1g: 20mL.

6. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 1, characterized in that, The oscillation lasted for 5-40 minutes.

7. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 1, characterized in that, The filter membrane has a pore size of 0.2 - 0.4 μm.

8. The method for simultaneous analysis of multiple fat-soluble components in tobacco leaves according to claim 2, characterized in that, In step 2), the detection conditions of the supercritical fluid chromatography system include the following technical features: The chromatographic conditions are as follows: Column temperature: 30 - 55℃; Injection volume: 0.5 - 3 μL; Flow rate: 0.5 - 2 mL / min.

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