Method for detecting and analyzing metabolite of caramel-sweet, mellow, sweet and fragrant flue-cured tobacco in Guangdong region

Abstract

The invention belongs to the field of chemical analysis and detection, and particularly provides a method for detecting and analyzing metabolite of caramel-sweet, mellow and sweet flue-cured tobacco, which realizes accurate metabolite identification through pretreatment of flue-cured tobacco, efficient extraction of the metabolite of the flue-cured tobacco and fine sample preparation. The flue-cured tobacco sample obtained through the pretreatment method is subjected to metabolite detection analysis through gas chromatography-mass spectrometry (GC-MS), the detection capacity of the low-abundance flue-cured tobacco metabolite with the detection peak limit being 2000 or above can be remarkably enhanced, interference of the matrix effect is reduced, the reliability and repeatability of data are improved, and the method is suitable for large-scale popularization and application. And the quantity of the detected substances can be maximized. The method for detecting and analyzing the metabolite of the caramel-sweet-sweet-fragrant flue-cured tobacco can be applied to research or solve key scientific research problems of flue-cured tobacco non-targeted metabolome, formation reasons of aroma substances, internal metabolic mechanisms and the like, and is wide in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection. More specifically, it relates to a method for detecting and analyzing metabolites of caramel-sweet and mellow-sweet type flue-cured tobacco in Guangdong region. Background Art

[0002] As the main raw material of tobacco products, the metabolomics research of flue-cured tobacco is of great significance for understanding the quality of flue-cured tobacco, the maturity of tobacco leaves, and the formation mechanism of complex compounds in tobacco leaves. Among the commonly used methods for evaluating the quality of tobacco leaves, the appearance quality evaluation method and the sensory evaluation method are greatly affected by the subjectivity of evaluators, while the analysis method by detecting metabolites is relatively objective and can provide more abundant information on the quality of tobacco leaves. The research on flue-cured tobacco metabolomics can comprehensively reveal the composition, variation law, and biological functions of metabolites in flue-cured tobacco, providing a scientific basis for the improvement of flue-cured tobacco quality, health risk assessment, and the development of tobacco products.

[0003] Tobacco leaf metabolites are the material basis of tobacco leaf quality. Appearance quality, sensory quality, and style characteristics are the manifestations of the inherent properties of tobacco leaf metabolites, and the metabolites of tobacco leaves are closely related to the climate conditions at different growth stages in the field. The cultivation of flue-cured tobacco is affected by various factors such as ecological conditions, cultivation varieties, and cultivation management. Flue-cured tobacco in China has regional characteristics. The flue-cured tobacco production areas in Shaoguan and Qingyuan of Guangdong are the flue-cured tobacco areas in the hilly mountains of southern Hunan, northern Guangdong, and eastern Guangxi, belonging to the Nanling hilly ecological region, and the tobacco leaves are of caramel-sweet and mellow-sweet type; the production area in Meizhou is the flue-cured tobacco area in the hilly mountains of western Fujian, southern Jiangxi, and eastern Guangdong, belonging to the Wuyi hilly ecological region, and the tobacco leaves are of fresh-sweet and mellow-sweet type. At present, the research on metabolites of caramel-sweet and mellow-sweet type flue-cured tobacco mainly focuses on the research of carbohydrate substances. However, it should be noted that low-abundance metabolites also play a key regulatory role in aroma quality. For example, pyrrole and furan substances, although their contents in flue-cured tobacco metabolites are very low, are crucial for the formation of cocoa fragrance (Zhao Mingqin, Wang Ying, Li Yuanshi, etc. Influence of organic substances on the components of neutral aroma substances and smoking quality of flue-cured tobacco [J]. Chinese Tobacco Science, 2008, 29(5).)

[0004] However, the metabolites of caramel-sweet and mellow-sweet type flue-cured tobacco in Guangdong are diverse in types, complex in structure, and large in content differences. There are many low-abundance metabolites, and most of the key flavor substances belong to low-abundance components, which poses challenges to the detection of metabolomics of caramel-sweet and mellow-sweet type flue-cured tobacco in Guangdong. Therefore, establishing a detection and analysis method suitable for metabolites of caramel-sweet and mellow-sweet type flue-cured tobacco in Guangdong has become an important research direction at present. Summary of the Invention

[0005] The present invention aims to better utilize gas chromatography-mass spectrometry (GC-MS) to study the metabolites of caramel-sweet and mellow-sweet type flue-cured tobacco in Guangdong, improve the efficiency and accuracy of GC-MS in detecting the metabolomics of caramel-sweet and mellow-sweet type flue-cured tobacco, provide a method for detecting and analyzing tobacco leaf metabolites, and enhance the detection ability of low-abundance tobacco leaf metabolites.

[0006] The object of the present invention is to provide a method for detecting and analyzing flue-cured tobacco metabolites.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] Previous studies have shown that flue-cured tobacco with a caramel-sweet and mellow fragrance type in Guangdong contains rich low-abundance metabolite components. However, existing detection methods have significant limitations in metabolite coverage and detection sensitivity, making it difficult to systematically analyze key flavor precursor substances and characteristic metabolites, and it is impossible to conduct in-depth research on flue-cured tobacco with a caramel-sweet and mellow fragrance type in Guangdong through metabolites. Therefore, the present invention innovatively optimizes the metabolite extraction process and detection method in a coordinated manner. By improving the flue-cured tobacco metabolite extraction method and simultaneously systematically constructing the gas chromatography-mass spectrometry (GC-MS) analysis conditions adapted to this extraction system, the order of magnitude of detection sensitivity is improved through programmed temperature rise optimization and ion source parameter regulation. This technical system has established a systematic detection scheme covering a broad polar range for the first time, providing reliable technical support for analyzing the metabolomics mechanism of the formation of the fragrance of Guangdong characteristic flue-cured tobacco. Therefore, the present invention claims the following solutions:

[0009] The present invention provides a method for detecting and analyzing metabolites of flue-cured tobacco with a caramel-sweet and mellow fragrance type. The method is as follows: Simultaneous distillation extraction is used to extract flue-cured tobacco metabolites to obtain a flue-cured tobacco metabolite extract, and gas chromatography-mass spectrometry analysis is performed on the flue-cured tobacco metabolite extract. The gas chromatography conditions are as follows: The inlet temperature is 250 - 270 °C, and the split ratio is (18 - 22):1; The carrier gas is helium, with a flow rate of 12 - 15 mL / min, and the injection volume is 0.5 - 1.5 μL; Programmed temperature rise: The initial temperature is maintained at 35 - 45 °C for 2 - 4 min, heated at 5 - 9 °C / min to 210 - 230 °C, maintained for 4 - 6 min, heated at 5 - 9 °C / min to 270 - 290 °C, and the holding time is 13 - 17 min.

[0010] Specifically, the flue-cured tobacco with a caramel-sweet and mellow fragrance type is flue-cured tobacco with a caramel-sweet and mellow fragrance type in Guangdong.

[0011] As an alternative embodiment, the gas chromatography conditions are as follows: The inlet temperature is 260 °C, and the split ratio is 20:1; The carrier gas is helium, with a flow rate of 13.6 mL / min, and the injection volume is 1 μL; Programmed temperature rise: The initial temperature is maintained at 40 °C for 3 min, heated at 7 °C / min to 220 °C, maintained for 5 min, heated at 7 °C / min to 280 °C, and the holding time is 15 min.

[0012] Preferably, the gas chromatography analysis uses a DB-5MS chromatographic column with a specification of 30 m × 0.25 mm × 0.25 μm.

[0013] Specifically, the mass spectrometry conditions in the method are as follows: the transfer line temperature is 240 - 260 °C; the ion source temperature is 190 - 210 °C; the scanning range is 50 - 600 Da; the scanning time is 2.5 - 3.5 min; the ionization mode is the EI source; the ionization energy is 60 - 80 eV.

[0014] As an alternative embodiment, the mass spectrometry conditions are as follows: the transfer line temperature is 250 °C; the ion source temperature is 200 °C; the scanning range is 50 - 600 Da; the scanning time is 3 min; the ionization mode is the EI source; the ionization energy is 70 eV.

[0015] Specifically, the specific method for obtaining the flue-cured tobacco metabolite extract is as follows: using the flue-cured tobacco powder solution as the sample solution, extracting by the simultaneous distillation and extraction method, the distillation and extraction time is 1 - 2 h, after the extract is evaporated to dryness, an organic solvent is added for re-dissolution, and after re-dissolution, filtration is carried out to obtain the flue-cured tobacco metabolite extract.

[0016] As an alternative embodiment, the extraction solvent in the simultaneous distillation and extraction method is dichloromethane.

[0017] As an alternative embodiment, the dosage of the extraction solvent in the simultaneous distillation and extraction method is 30 - 50 mL, preferably 40 mL.

[0018] As an alternative embodiment, the distillation temperature of the extraction solvent in the simultaneous distillation and extraction method is 30 - 50 °C, preferably 40 °C.

[0019] Optionally, the organic solvent is dichloromethane, specifically dichloromethane containing an internal standard (ethyl caprate).

[0020] Preferably, the concentration of the flue-cured tobacco powder solution is 0.02 - 0.04 g / mL.

[0021] More preferably, the concentration of the flue-cured tobacco powder solution is 0.02 g / mL.

[0022] As an alternative embodiment, the solvent of the flue-cured tobacco powder solution is a saturated sodium chloride aqueous solution.

[0023] As an alternative embodiment, the specific method for evaporation to dryness is: adding an excessive amount of anhydrous sodium sulfate for drying to remove water, and then rotary evaporation to dryness.

[0024] As an alternative embodiment, the filtration is carried out using a 0.22 μm filter membrane.

[0025] As a specific alternative embodiment, the method for preparing the flue-cured tobacco metabolite extract is as follows: The flue-cured tobacco is ground with liquid nitrogen and passed through a 40-mesh sieve to obtain flue-cured tobacco powder. Weigh 2-4 g of the flue-cured tobacco powder and place it in a round-bottom flask containing 100 mL of distilled water. Then add 10 g of sodium chloride to the flask and heat it to boiling. On the other side, use 40 mL of dichloromethane as the receiving liquid, and the distillation temperature of dichloromethane is 40 °C. Boil and distill for extraction for 1.5 h. After cooling for 30 min, add an excessive amount of anhydrous sodium sulfate to dry and remove water. Then use a rotary evaporator to evaporate to dryness, add 1 mL of chromatographic-grade dichloromethane containing an internal standard (ethyl caprate), redissolve and filter through a membrane (0.22 μm) to obtain the flue-cured tobacco metabolite extract.

[0026] The application of the above method in studying the flue-cured tobacco metabolome or the aroma of flue-cured tobacco should also be within the protection scope of the present invention.

[0027] The present invention has the following beneficial effects:

[0028] The method for detecting and analyzing the metabolites of the caramel-sweet and mellow-sweet type flue-cured tobacco of the present invention, through the pretreatment of flue-cured tobacco, efficiently extracts the metabolites of flue-cured tobacco, and through fine sample preparation, realizes accurate metabolite identification. The present invention uses gas chromatography-mass spectrometry (GC-MS) to detect and analyze the metabolites of the flue-cured tobacco samples obtained by the pretreatment method of the present invention, which can significantly enhance the detection ability of low-abundance flue-cured tobacco metabolites with a detection peak limit of more than 2000, reduce the interference of matrix effects, improve the reliability and repeatability of the data, and maximize the number of detected substances. The method for detecting and analyzing the metabolites of the caramel-sweet and mellow-sweet type flue-cured tobacco of the present invention can be applied to study or solve key scientific research problems such as the non-target metabolome of flue-cured tobacco, the formation reasons of aroma substances, and the internal metabolic mechanism, and has a broad application prospect. Description of the Drawings

[0029] Figure 1 It is the comparison result of metabolite abundances under different GC / MS conditions.

[0030] Figure 2 It is the gas chromatography detection result of different gas chromatography columns.

[0031] Figure 3 It is the gas chromatography detection result of different extraction methods in the pretreatment method.

[0032] Figure 4 It is the gas chromatography detection result of different dosages of flue-cured tobacco powder in the pretreatment method.

[0033] Figure 5 It is the gas chromatography detection result of different distillation times in the pretreatment method. Detailed Embodiments

[0034] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] In the following examples, the flue-cured tobacco used is the caramel-sweet-scented flue-cured tobacco planted in Shaoguan, Guangdong, and the specific variety is Yueyan 97.

[0037] Example 1 Effects of Different GC / MS Conditions on the Abundance of Metabolites in Flue-Cured Tobacco

[0038] I. Sample Preparation

[0039] Simultaneous distillation extraction method (SDE): Put the caramel-sweet-scented flue-cured tobacco from Guangdong into the oven for half an hour, then use liquid nitrogen to grind it and pass it through a 40-mesh sieve to obtain flue-cured tobacco powder. Immediately weigh 2 g of the flue-cured tobacco powder and place it in a round-bottom flask containing 100 mL of distilled water. Then add 10 g of sodium chloride to the flask to inhibit any other reactions, and heat it to boiling with an electric furnace. On the other side, use 40 ml of dichloromethane as the receiving liquid, and the distillation temperature of dichloromethane is 40 °C. Boil and distill for extraction for 1.5 h. After cooling for 30 min, add an excessive amount of anhydrous sodium sulfate to dry and remove water. Then use a rotary evaporator to evaporate to dryness, add 1 mL of chromatographic-grade dichloromethane containing an internal standard (ethyl decanoate), and after redissolving, filter through a membrane (0.22 μm) to obtain the test solution.

[0040] II. GC / MS Conditions

[0041] Model of gas chromatography-mass spectrometry: SHIMADZU GCMS-TQ8040.

[0042] Model of chromatographic column: DB-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0043] (1) Original GC / MS Conditions

[0044] The inlet temperature is 280 °C, the injection mode is split, and the split ratio of the split injection volume is 10:1; the carrier gas is helium, the flow rate is 1 mL / min, and the injection volume is 1 μL;

[0045] Temperature programming: The initial temperature is 60 °C, the holding time is 2 min, then it is heated to 135 °C at a rate of 4 °C / min; then it is heated to 175 °C at a rate of 2 °C / min, and finally it is heated to 250 °C at a rate of 5 °C / min, and the holding time is 2 min.

[0046] Mass spectrometry conditions: For the TQ series MS, the transfer line temperature: 250 °C; ionization mode: EI source; ionization energy: 70 eV; ion source temperature: 220 °C; scan time: 3 min; scan mass range: 50 - 350 Da.

[0047] (2) Improved GC / MS conditions

[0048] The inlet temperature is 260 °C, the injection mode is split, and the split ratio is 20:1; the carrier gas is helium, the flow rate is 13.6 mL / min, and the injection volume is 1 μL;

[0049] Temperature programming: The initial temperature is 40 °C, the holding time is 3 min, then it is heated to 220 °C at a rate of 7 °C / min and held for 5 minutes; finally, it is heated to 280 °C at a rate of 7 °C / min and held for 15 min.

[0050] Mass spectrometry conditions: For the TQ series MS, the transfer line temperature: 250 °C; ionization mode: EI source; ionization energy: 70 eV; ion source temperature: 200 °C; scan time: 3 min; scan range: 50 - 600 Da.

[0051] III. Experimental operations

[0052] After the system is balanced, using the original GC / MS conditions and the improved GC / MS conditions respectively, inject the test solution into the gas chromatography - mass spectrometry instrument to analyze the test solution of the flue - cured tobacco sample, and record the chromatogram.

[0053] IV. Experimental results

[0054] Analyze the Guangdong flue - cured tobacco samples with caramel - sweet flavor using the original GC / MS conditions and the improved GC / MS conditions respectively. The results are as Figure 1 shown. The results show that when analyzing with the original GC / MS conditions, a total of 91 metabolites with peak limits higher than 2000 were qualitatively identified, and when analyzing with the improved GC / MS conditions, a total of 121 metabolites with peak limits higher than 2000 were qualitatively identified. Compared with the original GC / MS conditions, using the improved GC / MS conditions can significantly improve the separation efficiency, detection sensitivity and qualitative quantity of metabolites, so as to more accurately quantify the metabolite content and achieve effective detection of a wider and more comprehensive variety of metabolites. Therefore, the improved GC / MS conditions are determined to be used.

[0055] Example 2 Determination of the gas chromatography column

[0056] I. Sample preparation

[0057] Simultaneous Distillation and Extraction (SDE) method: Put the Guangdong caramel-sweet and mellow-flavor flue-cured tobacco into the oven for half an hour, then use liquid nitrogen to grind it and pass it through a 40-mesh sieve to obtain flue-cured tobacco powder. Immediately weigh 2 g of the powder and place it in a round-bottom flask containing 100 mL of distilled water. Then add 10 g of sodium chloride to the flask to inhibit any other reactions, and heat it with an electric furnace until it boils. On the other side, use 40 mL of dichloromethane as the receiving liquid, and the distillation temperature of dichloromethane is 40 °C. Boil and distill for extraction for 1.5 h. After cooling for 30 min, add an excessive amount of anhydrous sodium sulfate to dry and remove water. Then use a rotary evaporator to evaporate to dryness, add 1 mL of chromatographic-grade dichloromethane containing an internal standard (ethyl caprate), dissolve it again and filter through a membrane (0.22 μm) to obtain the test solution.

[0058] II. GC / MS Conditions

[0059] Model of gas chromatography-mass spectrometry: SHIMADZU GCMS-TQ8040.

[0060] Model of chromatographic column: DB-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0061] Model of chromatographic column: HP-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0062] (1) Chromatographic Conditions

[0063] Injector temperature: 260 °C, injection mode is split, split ratio of split injection volume is: 20:1; carrier gas is helium, flow rate is 13.6 mL / min, injection volume is 1 μL;

[0064] Temperature programming: Initial temperature is 40 °C, holding time is 3 min, then increase the temperature to 220 °C at a rate of 7 °C / min and hold for 5 minutes; finally, increase the temperature to 280 °C at a rate of 7 °C / min and hold for 15 min.

[0065] (2) Mass Spectrometry Conditions

[0066] TQ series MS transfer line temperature: 250 °C; ionization mode: EI source; ionization energy: 70 eV; ion source temperature: 200 °C; scanning time 3 min; scanning range: 50 - 600 Da.

[0067] III. Experimental Operations

[0068] After the system is balanced, according to the above GC / MS conditions, inject the test solution into the gas chromatography-mass spectrometry, and analyze the test solution of the Guangdong caramel-sweet and mellow-flavor flue-cured tobacco sample using the HP-5MS chromatographic column and the DB-5MS chromatographic column respectively, and record the chromatogram.

[0069] IV. Experimental Results

[0070] The samples of Guangdong flue-cured tobacco with caramel and sweet flavor were analyzed using HP-5MS and DB-5MS chromatographic columns. The results are as follows: Figure 2 As shown, the results indicate that compared with the HP-5MS chromatographic column, using the DB-5MS chromatographic column can significantly improve the separation efficiency and detection sensitivity of metabolites, thus more accurately quantifying the metabolite content. Therefore, the DB-5MS chromatographic column was determined to be used as the analytical column.

[0071] Determination of the pretreatment method in Example 3

[0072] I. Sample preparation

[0073] Simultaneous distillation extraction (SDE) method:

[0074] The Guangdong flue-cured tobacco with caramel and sweet flavor was placed in an oven for half an hour, then ground with liquid nitrogen and passed through a 40-mesh sieve to obtain flue-cured tobacco powder. Immediately, 2 g of the powder was weighed and placed in a round-bottom flask containing 100 mL of distilled water. Then, 10 g of sodium chloride was added to the flask to inhibit any other reactions, and the mixture was heated to boiling on an electric furnace. On the other side, 40 mL of dichloromethane was used as the receiving liquid, and the distillation temperature of dichloromethane was 40 °C. The mixture was distilled and extracted by boiling for 1.5 h. After cooling for 30 min, an excessive amount of anhydrous sodium sulfate was added to dry and remove water. Then, it was rotary evaporated to dryness, 1 mL of chromatographic-grade dichloromethane containing an internal standard (ethyl caprate) was added, and after redissolution, it was filtered through a membrane (0.22 μm) to obtain the test solution 1.

[0075] Traditional ultrasonic extraction method:

[0076] The Guangdong flue-cured tobacco with caramel and sweet flavor was placed in an oven for half an hour, then ground with liquid nitrogen and passed through a 40-mesh sieve. 50 mg was weighed and placed in a 2-mL centrifuge tube. A mixed solution of methanol and dichloromethane (3:2) was added respectively, and the final extraction concentration was 50 mg / mL. It was ultrasonically treated at 50 °C for 10 min, centrifuged at 12,000 r / min for 10 min under the condition of 4 °C, and the supernatant was transferred and filtered through a 0.22-μm membrane into a GC injection vial to obtain the test solution 2.

[0077] II. GC / MS conditions

[0078] Model of gas chromatography-mass spectrometry: SHIMADZU GCMS-TQ8040.

[0079] Model of chromatographic column: DB-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0080] (1) Chromatographic conditions

[0081] The inlet temperature was 260 °C, the injection mode was split, and the split ratio of the split injection volume was 20:1; the carrier gas was helium, the flow rate was 13.6 mL / min, and the injection volume was 1 μL;

[0082] Temperature rising program: The initial temperature is 40 °C, the holding time is 3 min, then it is heated to 220 °C at a rate of 7 °C / min and held for 5 minutes; finally, it is heated to 280 °C at a rate of 7 °C / min and the holding time is 15 min.

[0083] (2) Mass spectrometry conditions

[0084] TQ series MS transfer line temperature: 250 °C; Ionization mode: EI source; Ionization energy: 70 eV; Ion source temperature: 200 °C; Scanning time 3 min; Scanning range: 50 - 600 Da.

[0085] III. Experimental operations

[0086] After the system is balanced, according to the above GC / MS conditions, the test solution 1 and test solution 2 are respectively injected into the gas chromatography - mass spectrometry instrument, and the chromatogram is recorded.

[0087] IV. Experimental results

[0088] The results are as Figure 3 shown. The results show that both the metabolite peak area and the number of ion peaks in the off - machine data of the test solution by the SDE extraction method are much higher than those of the ultrasonic extraction method. A total of 122 metabolites with a detection peak limit higher than 2000 were detected by SDE treatment, and a total of 95 metabolites with a detection peak limit higher than 2000 were detected by ultrasonic extraction. Therefore, SDE is used as the pretreatment method.

[0089] Determination of the concentration of the pretreated flue - cured tobacco powder in Example 4

[0090] I. Sample preparation

[0091] Simultaneous distillation extraction (SDE) method: Put the Guangdong caramel - flavored and sweet - scented flue - cured tobacco into the oven for half an hour, then grind it with liquid nitrogen and pass through a 40 - mesh sieve to obtain flue - cured tobacco powder.

[0092] Set 4 treatments for the dosage of flue - cured tobacco powder;

[0093] Treatment a: The dosage of flue - cured tobacco powder is 2 g;

[0094] Treatment b: The dosage of flue - cured tobacco powder is 4 g;

[0095] Treatment c: The dosage of flue - cured tobacco powder is 6 g;

[0096] Treatment d: The dosage of flue - cured tobacco powder is 10 g.

[0097] For different treatments, according to the amount of flue-cured tobacco powder used, the flue-cured tobacco powder was placed in a round-bottom flask containing 100 mL of distilled water, and then 10 g of sodium chloride was added to the flask to inhibit any other reactions. The mixture was heated to boiling on an electric furnace. On the other side, 40 mL of dichloromethane was used as the receiving liquid, and the distillation temperature of dichloromethane was 40 °C. It was boiled and distilled for extraction for 1.5 h. After cooling for 30 min, an excessive amount of anhydrous sodium sulfate was added for drying to remove water. Then, it was rotary evaporated to dryness using a rotary evaporator. 1 mL of chromatographic grade dichloromethane containing an internal standard (ethyl caprate) was added, and after redissolution, it was filtered through a membrane (0.22 μm) to obtain the test solutions for treatment a, treatment b, treatment c, and treatment d respectively.

[0098] II. GC / MS Conditions

[0099] Model of gas chromatography-mass spectrometry: SHIMADZU GCMS-TQ8040.

[0100] Model of chromatographic column: DB-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0101] (1) Chromatographic Conditions

[0102] The inlet temperature was 260 °C, the injection mode was split, and the split ratio of the split injection volume was 20:1; the carrier gas was helium, the flow rate was 13.6 mL / min, and the injection volume was 1 μL.

[0103] Temperature programming: The initial temperature was 40 °C, the holding time was 3 min, then it was heated to 220 °C at a rate of 7 °C / min and held for 5 minutes; finally, it was heated to 280 °C at a rate of 7 °C / min and held for 15 min.

[0104] (2) Mass Spectrometry Conditions

[0105] TQ series MS transfer line temperature: 250 °C; ionization mode: EI source; ionization energy: 70 eV; ion source temperature: 200 °C; scan time 3 min; scan range: 50 - 600 Da.

[0106] III. Experimental Operations

[0107] After the system was balanced, according to the above GC / MS conditions, the test solutions for treatment a, treatment b, treatment c, and treatment d were respectively injected into the gas chromatography-mass spectrometry, and the chromatograms were recorded.

[0108] IV. Experimental Results

[0109] The detection results are as Figure 4As shown in the figure, the results show that there are significant differences in the number of metabolites detected in the test solution when SDE extraction is carried out using different dosages of Guangdong caramel-sweet-flavored flue-cured tobacco powder. When the dosage of flue-cured tobacco powder is 2 g, metabolites with a peak limit above 2000 can be well separated. Therefore, 2 g of Guangdong caramel-sweet-flavored flue-cured tobacco powder is determined as the final concentration of the sample.

[0110] Determination of Pretreatment Distillation Time in Example 5

[0111] I. Sample Preparation

[0112] Set 3 distillation time treatments;

[0113] Treatment 1: The distillation time is 1 h;

[0114] Treatment 2: The distillation time is 1.5 h;

[0115] Treatment 3: The distillation time is 2 h.

[0116] Simultaneous Distillation Extractor (SDE) Extraction Method: Put the Guangdong caramel-sweet-flavored flue-cured tobacco into the oven for half an hour, then use liquid nitrogen to grind it and pass it through a 40-mesh sieve to obtain flue-cured tobacco powder. Immediately weigh 2 g of the powder and place it in a round-bottom flask containing 100 mL of distilled water. Then add 10 g of sodium chloride to the flask to inhibit any other reactions, and heat it with an electric furnace until it boils. On the other side, use 40 mL of dichloromethane as the receiving liquid, and the distillation temperature of dichloromethane is 40 °C. Carry out boiling distillation extraction according to the distillation time of different treatments. After cooling for 30 min, add an excessive amount of anhydrous sodium sulfate to dry and remove water, then use a rotary evaporator to evaporate to dryness, add 1 mL of chromatographic-grade dichloromethane containing an internal standard (ethyl caprate), redissolve it and filter through a membrane (0.22 μm) to obtain the test solution for Treatment 1, the test solution for Treatment 2, and the test solution for Treatment 3 respectively.

[0117] II. GC / MS Conditions

[0118] Model of gas chromatography-mass spectrometry: SHIMADZU GCMS-TQ8040.

[0119] Model of chromatographic column: DB-5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0120] (1) Chromatographic Conditions

[0121] The inlet temperature is 260 °C, the injection mode is split, and the split ratio of the split injection volume is 20:1; the carrier gas is helium, the flow rate is 13.6 mL / min, and the injection volume is 1 μL;

[0122] Temperature rising program: The initial temperature is 40°C, the holding time is 3 min, then it is heated to 220°C at a rate of 7°C / min and held for 5 minutes; finally, it is heated to 280°C at a rate of 7°C / min and the holding time is 15 min.

[0123] (2) Mass spectrometry conditions

[0124] TQ series MS transfer line temperature: 250°C; Ionization mode: EI source; Ionization energy: 70 eV; Ion source temperature: 200°C; Scanning time 3 min; Scanning range: 50 - 600 Da.

[0125] III. Experimental operations

[0126] After the system is balanced, according to the above GC / MS conditions, the test solutions of Treatment 1, Treatment 2, and Treatment 3 are respectively injected into the gas chromatography - mass spectrometry instrument, and the chromatograms are recorded.

[0127] IV. Experimental results

[0128] The detection results are as Figure 5 shown. The results indicate that different distillation extraction times have no effect on the metabolite precipitation content of Guangdong caramel - sweet - type flue - cured tobacco. Therefore, to ensure the stability of the method, it is determined to uniformly use 1.5 h as the final distillation time.

[0129] Example 6 Detection and analysis method for metabolites of caramel - sweet - type flue - cured tobacco in Guangdong region

[0130] (1) Sample pretreatment

[0131] Put the Guangdong caramel - sweet - type flue - cured tobacco into the oven for half an hour, then grind it with liquid nitrogen and pass through a 40 - mesh sieve to obtain flue - cured tobacco powder. Immediately weigh 2 g of the powder and place it in a round - bottom flask containing 100 mL of distilled water, and then add 10 g of sodium chloride to inhibit any other reactions. Heat it with an electric furnace until boiling. On the other side, use 40 mL of dichloromethane as the receiving liquid, the distillation temperature of dichloromethane is 40°C, boil and distill for extraction for 1.5 h. After cooling for 30 min, add an excessive amount of anhydrous sodium sulfate to dry and remove water, then use a rotary evaporator to evaporate to dryness, add 1 mL of chromatographic - grade dichloromethane containing an internal standard (ethyl caprate), redissolve and filter through a membrane (0.22 μm) to obtain the test solution.

[0132] II. GC / MS conditions

[0133] Model of gas chromatography - mass spectrometry instrument: SHIMADZU GCMS - TQ8040.

[0134] Model of chromatographic column: DB - 5MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm).

[0135] (1) Chromatographic conditions

[0136] The inlet temperature is 260 °C, the injection mode is split, and the split ratio of the split injection volume is 20:1; the carrier gas is helium, the flow rate is 13.6 mL / min, and the injection volume is 1 μL;

[0137] Temperature programming: The initial temperature is 40 °C, the holding time is 3 min, then it is heated to 220 °C at a rate of 7 °C / min and held for 5 minutes; finally, it is heated to 280 °C at a rate of 7 °C / min and held for 15 min.

[0138] (2) Mass spectrometry conditions

[0139] TQ series MS transfer line temperature: 250 °C; ionization mode: EI source; ionization energy: 70 eV; ion source temperature: 200 °C; scan time 3 min; scan range: 50 - 600 Da.

[0140] III. Experimental operations

[0141] After the system is balanced, according to the above GC / MS conditions, inject the test solution into the gas chromatography - mass spectrometry instrument and record the analysis results.

[0142] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for detecting and analyzing metabolites of sweet and mellow flue-cured tobacco, characterized in that: The flue-cured tobacco metabolites are extracted by a simultaneous distillation extraction method to obtain a flue-cured tobacco metabolite extract, and the flue-cured tobacco metabolite extract is analyzed by gas chromatography-mass spectrometry; the gas chromatography conditions are as follows: the injection port temperature is 250-270°C, the split ratio is (18-22):1; the carrier gas is helium, the flow rate is 12-15mL / min, and the injection volume is 0.5-1.5μL; the programmed temperature is as follows: the initial temperature is 35-45°C and maintained for 2-4min, the temperature is increased to 210-230°C at 5-9°C / min, maintained for 4-6min, the temperature is increased to 270-290°C at 5-9°C / min, and the holding time is 13-17min.

2. The method according to claim 1, characterized in that: The gas chromatography conditions are as follows: the injection port temperature is 260°C, the split ratio is 20:1; the carrier gas is helium, the flow rate is 13.6 mL / min, and the injection volume is 1 μL; the programmed temperature is: the initial temperature is 40°C and maintained for 3 min, then the temperature is increased to 220°C at 7°C / min and maintained for 5 min, then the temperature is increased to 280°C at 7°C / min and maintained for 15 min.

3. The method according to claim 1, characterized in that: The mass spectrometry conditions were as follows: the transfer line temperature was 240-260°C; the ion source temperature was 190-210°C; the scanning range was 50-600Da; the scanning time was 2.5-3.5min; the ionization mode was EI source; and the ionization energy was 60-80eV.

4. The method according to claim 3, characterized in that: The mass spectrometry conditions were as follows: the transfer line temperature was 250°C; the ion source temperature was 200°C; the scanning range was 50-600Da; the scanning time was 3min; the ionization mode was EI source; and the ionization energy was 70eV.

5. The method according to claim 1, characterized in that: The specific method for obtaining the flue-cured tobacco metabolite extract is as follows: using flue-cured tobacco powder solution as the sample solution, extracting by simultaneous distillation extraction, the distillation extraction time is 1-2 hours, and then adding an organic solvent to the extract after evaporation and drying, and filtering after re-dissolution to obtain the flue-cured tobacco metabolite extract.

6. The method according to claim 5, characterized in that: The organic solvent is dichloromethane.

7. The method according to claim 5, characterized in that: The amount of the organic solvent used is 30-50 mL.

8. The method according to claim 5, characterized in that: The distillation temperature of the organic solvent is 30-50°C.

9. The method according to claim 5, characterized in that: The concentration of the flue-cured tobacco powder solution is 0.02-0.04 g / mL.

10. The method according to claim 5, characterized in that: The solvent of the flue-cured tobacco powder solution is a saturated sodium chloride aqueous solution.

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