CN113358797A - Chromatographic analysis method for simultaneously analyzing flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves - Google Patents
Chromatographic analysis method for simultaneously analyzing flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/74—Optical detectors
Abstract
The invention discloses a chromatographic analysis method for simultaneously analyzing flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves, which comprises the following steps: A. microwave-assisted extraction of secondary metabolites in fresh tobacco leaves; B. performing ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrometry on the extract; C. and identifying secondary metabolites, and carrying out relative quantitative analysis on the identified 13 flavones, 19 phenylpropanes, 4 coumarins and 6 phenolic amides. The method for simultaneously measuring 13 flavones, 19 phenylpropanes, 4 coumarins and 6 phenolic amides in fresh tobacco leaves by microwave-assisted extraction-ultra-high performance liquid chromatography-ultraviolet detector-series four-stage rod-flight time mass spectrum has the characteristics of multiple and comprehensive varieties of qualitative and quantitative metabolites, and can quickly analyze the secondary metabolites of flavones, phenylpropanes, coumarins and phenolic amides in the fresh tobacco leaves with high sensitivity and good stability and high throughput.
Description
Technical Field
The invention relates to a simple, stable and accurate method for simultaneously analyzing secondary metabolites such as flavone, phenylpropane, coumarin and phenolic amides in fresh tobacco leaves, and belongs to the technical field of determination methods of chemical components of tobacco.
Background
The flavone, the phenylpropane, the coumarin and the phenolic amide in the tobacco are important secondary metabolites, the secondary metabolites mainly refer to small molecular metabolites below 1000, and the small molecular metabolites are closely related to phenomena of plant resistance, signal conduction, growth and development, plant flower color, aroma and the like, so that the research on the composition and the content of the flavone, the phenylpropane, the coumarin and the phenolic amide has great plant physiological significance, and chemical substance support can be provided for functional verification of genomes, transcriptomes and the like.
The tobacco flavonoids compounds are substances with relatively rich contents in tobacco, play an important role in the aspects of growth and development of the tobacco, appearance color and luster of the tobacco leaves, modulation characteristics, flavor and taste, physiological strength of smoke and the like, and are an important factor for measuring the quality of the tobacco. The content and oxidation degree of the tobacco flavone not only affect the appearance quality of the tobacco leaves, but also affect the internal quality of the tobacco leaves. The phenylpropanoids are intermediate products of phenylpropanoid metabolic pathways and lignin synthesis pathway metabolic products, and have close relation with physiological activities such as stress resistance, tobacco characteristic formation and the like. Coumarins are a class of biologically active substances that are widely found in nature. The plant coumarin has multiple structural types, complicated stereochemistry and multiple biological activities, and has important effects in resisting stress and disease of tobacco. The plant phenolic amide is beneficial to the development and the resistance of plants to environmental stressors, has a certain function of resisting plant pathogens, and is also closely related to the flower formation and reproductive differentiation of the plants.
Since flavone, phenylpropane, coumarin and phenolic amides have the characteristics of strong polarity, poor volatility and strong ultraviolet absorption, liquid chromatography is usually utilized for separation and an ultraviolet detector is used for qualitative and quantitative analysis, but only 1 or 2 types of secondary metabolites are usually concerned for analysis, for example, Li and the like invents an analytical method for simultaneously measuring the content of 6 polyphenols in flue-cured tobacco, Liyan and the like invents an HPLC-DAD segmented detection method for simultaneously measuring 14 polyphenols in tobacco, Lianping and the like invents a detection method for polyphenols (10) in tobacco leaves, Kongshiu and the like invent a method for quantitatively analyzing 18 polyphenols in tobacco leaves by using an ultra-high performance liquid chromatography-tandem mass spectrometry, Jinzhanfeng and the like invent a method for detecting the metabolism of various flavonoids (7) in fresh tobacco leaves, Yong and the like invent a detection method for quantitatively analyzing 12 types of flavonoids in tobacco leaves and the like, at present, no comprehensive screening and quantitative analysis for 4 types of secondary metabolites including flavone, phenylpropane, coumarin and phenolic amides in tobacco is reported.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the chromatographic detection method capable of simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic acid amides in the fresh tobacco leaves has the advantages of simplicity, rapidness, high sensitivity, good stability and the like, and can improve the sample detection flux and the data accuracy; in addition, the simultaneous analysis of the 4 types of substances also greatly reduces the workload of analysis and can effectively overcome the defects of the prior art.
The technical scheme of the invention is as follows: a chromatographic analysis method for simultaneously analyzing flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves comprises the following steps:
A. extracting flavone, phenylpropane, coumarin and phenolic amide secondary metabolites from fresh tobacco leaves: weighing freeze-dried fresh tobacco leaf powder in a microwave extraction tank, adding an internal standard, adding an extracting solution for microwave-assisted extraction, centrifuging an extract in a centrifugal tube, and filtering supernatant liquid with an organic filter membrane to obtain a filtrate;
B. chromatographic analysis of flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves: performing chromatographic analysis by adopting ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrometry;
C. qualitative and quantitative analysis of flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves: qualitatively comparing with high-resolution mass spectrogram, and quantitatively comparing with internal standard method.
Preferably, the extract in step A is 0.1% formic acid in methanol (v/v 7: 3).
Preferably, the microwave-assisted extraction conditions in the step A are 800w and extraction is carried out at 90 ℃ for 5 min.
Preferably, the internal standards in the step A are 1.88mg/mL cinnamic acid and 14.796mg/mL adipic acid.
Preferably, the chromatographic analysis condition in the step B is an Acquity UPLC HSS T3 column (2.1mm multiplied by 100mm,1.8 μm,), the column temperature is 35 ℃, and the sample injection amount is 0.5 μ L; mobile phase a was ultrapure water (containing 0.05% HCOOH), B was mass-spec acetonitrile, flow rate was 0.35mL/min, gradient elution was as follows: maintaining the initial 98% A for 1min, 16min for 60% A, 20min for 5% A, maintaining for 24min, 25min for 0% A, maintaining for 27min, 29min for 98% A, and maintaining for 31 min; the mass spectrum condition adopts a primary mass spectrum ESI negative mode, nitrogen is atomizing gas and taper hole gas, the airflow speed of the atomizing gas and the taper hole gas is 800L/h and 50L/h, the atomizing gas temperature is 450 ℃, the source temperature is 120 ℃, and the capillary tube voltage is as follows: 2.0KV, taper hole voltage: 35V, collision capacity of 6eV, scanning time of 0.2s, interval of 0.02s, scanning range of 70-1000 and detector voltage of 1950V; leucine cephalka peptide is used as a real-time calibration solution, the concentration is 200ng/mL, the flow rate is 10 mu L/min, the correction interval is 15s, and the correction result is 3 times of average; MS (Mass Spectrometry)eData acquisition energy of 6-35eV to obtainAnd obtaining a corresponding fragment mass spectrogram.
Preferably, in the step C, high-resolution excimer ions, fragment ions, ultraviolet absorption binding databases Scripps, HMDB, Respect and Massbank are qualitatively used to identify the main metabolite components, and meanwhile, different MSe fragmentation rules of different glycosidic bond positions in the flavone are utilized to determine the accurate structure of the flavone; the quantification is carried out by an internal standard method.
The invention has the beneficial effects that: the invention establishes a method for simultaneously measuring 13 flavones, 19 phenylpropanes, 4 coumarins and 6 phenamides in fresh tobacco leaves by microwave-assisted extraction, ultra-high performance liquid chromatography, ultraviolet detector, serial four-stage rod and flight time mass spectrum, and 42 secondary metabolites in total. In order to obtain the maximum extraction efficiency, the microwave-assisted extraction is optimized by adopting a factorial design and a Box-Behnken design experiment. Compared with the prior art, the invention has the advantages that: 1) the extraction time is reduced from 30-60min to 5min by microwave-assisted extraction; 2) the ultraviolet detector and the tandem quadrupole-time-of-flight mass spectrum are combined, ultraviolet and mass spectrum signals can be obtained simultaneously for qualitative and quantitative determination, and high-resolution mass spectrum information can accurately determine secondary metabolites such as flavone, phenylpropane, coumarin and phenolic amides; 3) 4 kinds of secondary metabolites (13 kinds of flavone, 19 kinds of phenylpropane, 4 kinds of coumarin and 6 kinds of phenolic amide) can be analyzed simultaneously; 4) the method has the characteristics of simplicity, rapidness, high sensitivity and good stability, and can effectively improve the sample detection flux and the data accuracy.
Drawings
FIG. 1 is a one-dimensional chromatogram of ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrum for simultaneously analyzing secondary metabolites of flavones, phenylpropane, coumarin and phenolic amides in fresh tobacco leaves;
FIG. 2 is a two-dimensional chromatogram of ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrum for simultaneously analyzing secondary metabolites of flavones, phenylpropane, coumarin and phenolic amides in fresh tobacco leaves;
FIG. 3MSeHigh resolution quality for determining Kaempferol-rutinoside fragmentation at different substitution positions under the condition of non-ionization energySpectra and fragmentation mechanism (A: MS)e Fragmentation energy 6 eV; b: MS (Mass Spectrometry)eThe fragmentation energy is 10-30 eV; c: MS (Mass Spectrometry)eThe fragmentation energy is 15-40 eV; d: 3 substituted Kaempferol-rutinoside fragmentation mechanism);
FIG. 4 shows the phenylpropanoid metabolic pathways (HLJ, SD, SX, GZ, and CQ) between different ecoregions with middle fragrance.
Detailed Description
Example (b):
the chromatographic analysis method provided by the embodiment of the invention mainly comprises the following steps:
(1) weighing 50mg of freeze-dried fresh tobacco leaf powder into a microwave extraction tank (quality control sample QC is optimized), adding 50 mu L of cinnamic acid internal standard (1.88mg/mL) and 10 mu L of adipic acid internal standard (14.796mg/mL), adding 2.5mL of methanol aqueous solution (v/v 7:3) containing 0.1% formic acid (vortex for 1min before and after microwave extraction to ensure uniform extraction), performing microwave-assisted extraction, optimizing microwave-assisted extraction conditions by using a cause design and a Box-Behnken design experiment, extracting for 5min at 90 ℃ under the optimized conditions of 800w and 5min at 3000rpm, centrifuging the extract in a centrifuge tube for 5min, and passing supernatant through a 0.45 mu m organic filter membrane for analysis.
(2) The ultra performance liquid chromatography separation adopts an Acquity UPLC HSS T3 column (2.1mm multiplied by 100mm,1.8 μm,), the column temperature is 35 ℃, and the sample injection amount is 0.5 μ L. Mobile phase a was ultrapure water (containing 0.05% HCOOH), B was mass-spec acetonitrile, flow rate was 0.35mL/min, gradient elution was as follows: the initial 98% A is maintained for 1min, 16min is changed to 60% A, 20min is changed to 5% A, the maintenance is carried out to 24min, 25min is changed to 0% A, the maintenance is carried out to 27min, 29min is changed to 98% A, and the maintenance is carried out to 31 min. The data acquisition time is 24 min. The PDA assay was a full wavelength scan at 200-400nm while monitoring the wavelength absorption at 340 and 280 nm.
The Waters Xevo Q-TOF mass spectrum conditions are as follows, a first-order mass spectrum ESI negative mode is adopted, nitrogen is atomizing gas and taper hole gas, the flow rates of the atomizing gas and the taper hole gas are 800L/h and 50L/h, the temperature of the atomizing gas is 450 ℃, the source temperature is 120 ℃, and the capillary tube voltage is as follows: 2.0KV, taper hole voltage: 35V, collision power 6eV, scan time 0.2s, interval 0.02s, scan range 70-1000, detector voltage 1950V. Using leucineThe kafirin is a real-time calibration solution, the concentration is 200ng/mL, the flow rate is 10 mu L/min, the correction interval is 15s, and the correction result is 3 times of average. MS (Mass Spectrometry)eThe data acquisition energy is 6-35eV to obtain the corresponding fragment mass spectrum. A one-dimensional chromatogram and a two-dimensional chromatogram of a typical ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrum are shown in fig. 1 and 2.
(3) After a chromatogram spectrogram is collected, the main flavone, phenylpropane, coumarin and phenolic amide secondary metabolites are identified by utilizing accurate excimer ions, fragment ions, ultraviolet absorption and the like in combination with databases of Scripps, HMDB, response and Massbank. 13 flavones, 19 phenylpropanes, 4 coumarins and 6 phenolic amide secondary metabolites are obtained by the method through identification, 42 metabolites are obtained, the error between the measured molecular ion peak and the theoretical value of most substances is less than 10ppm, and the measurement accuracy is good. The accurate molecular ion peak 593.1503 is used for obtaining Kaempferol-rutinoside flavonoid substance by using a Scripps database, collision fragmentation is further carried out by using low energy (10-30eV), only a small amount of fragment peaks (284) are obtained, according to the conclusion of the literature, the judgment is preliminarily 3-rutinoside (3 is not easy to fragment under the condition of low energy, 7 is easy to fragment), fragmentation is further carried out by using high energy (15-40eV), the fragment peaks (284) are remarkably increased, and the fragment peaks (284) can be deduced to be M-rutinoside-2H]-Ion, this cleavage mode is only possible with a 3-substitution mode (7-substitution mode is [ M-rutinoside-H ]]-285), which was further demonstrated to be Kaempferol-3-rutinoside, a 3-substituted species, MSeThe mass spectrum fragmentation pattern at different energies and possible fragmentation equations are shown in figure 3. The major identified metabolites are shown in table 1.
TABLE 1 identification of the major secondary metabolites in tobacco by ultra performance liquid chromatography-UV detector-tandem quadrupole-time-of-flight mass spectrometry
aAnd (3) calculating mass error: absolute value of (theoretical value-measured value)/theoretical value
(4) The 5 QC samples are extracted and injected according to optimized pretreatment conditions and chromatographic conditions, the day precision (5 QC samples and injection samples are repeatedly processed within one day) and the day precision (5 QC samples and injection samples are repeatedly processed within three days) of the method are respectively evaluated, and the results show that the day precision of all secondary metabolites is 0.8-12.5%, and the day precision is 1.4-19.5%, so that the method conforms to the regulation of the American FDA on the repeatability requirement of metabolic research, and the method has good repeatability. The linear range of the method was evaluated using QC samples at different concentrations, since there was no corresponding standard for linear investigation. Weighing 20, 30, 40, 50, 60 and 70mg QC samples for extraction and chromatographic analysis, and performing linear investigation by using peak areas and Pearson correlation coefficients corresponding to the weighing quantities of the QC samples, wherein results show that the Pearson correlation coefficients of 35 secondary metabolites are greater than 0.99, and the others are greater than 0.90, so that the linear relation is good, and the change of the metabolites in the fresh tobacco leaf samples can be accurately reflected.
Application example:
in order to reveal the metabolic difference of related secondary metabolites under the phenylpropane metabolic pathways of different styles of tobacco leaves in different middle-odor type ecological regions, a northeast flue-cured tobacco planting region (HLJ in Nian county of Heilongjiang province), a Luzhong mountain region (SD in Monyin county of Shandong province), a Qinba mountain region (SX in Linan county of Shaanxi province) and a Qiazhong mountain region (GZ in Zunyi county of Guizhou province) are collected, the middle fresh tobacco leaves (12 th leaf positions of tobacco plants from bottom to top) in the physiological maturity period of 5 typical middle cigarette areas in the Wuling mountain area (Wulong CQ in Chongqing city) are immediately put into liquid nitrogen and brought back to a laboratory for vacuum freeze drying, microwave-assisted extraction-ultra-high performance liquid chromatography-ultraviolet detector-serial four-stage rod-flight time mass spectrum is adopted to simultaneously determine 13 flavones, 19 phenylpropane, 4 coumarins and 6 phenolic amides in the fresh tobacco leaves, and the phenylpropane metabolic pathway among different ecological areas with middle fragrance type is shown in figure 4. The tobacco leaves of Heilongjiang and Guizhou show obvious up-regulation on p-coumaroyl branches in the phenylpropane approach, while the tobacco leaves of Shandong show obvious down-regulation. For example, the p-coumaroyl quinic acid and the quercetin glycoside have the highest content in Heilongjiang, the kaempferol glycoside has the highest content in Guizhou, and the p-coumaroyl quinic acid, the quercetin glycoside and the kaempferol glycoside have the lowest content in Shandong tobacco leaves; on the caffeic acid branch, Shanxi shows the highest performance on the caffeoyl polyamine substances, and Heilongjiang tobacco shows the lowest performance; the black Longjiang tobacco leaves have the highest chlorogenic acid substance performance, and the Shandong tobacco leaves have the lowest content; the branches of ferulic acid and sinapic acid show the highest contents of tobacco leaves in Heilongjiang and Guizhou respectively. In addition, phenylalanine appears to be present in the lowest amount, although most phenylpropanoid metabolites are up-regulated in Heilongjiang and Guizhou.
The results show that: the detection method can quickly and accurately obtain the content composition of 13 flavones, 19 phenylpropanes, 4 coumarins and 6 phenamides in fresh tobacco leaves, obtain qualitative and quantitative data of 42 secondary metabolites, and can be applied to the difference analysis of phenylpropane metabolic pathways in different ecological regions.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (6)
1. A chromatographic analysis method for simultaneously analyzing flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves is characterized by comprising the following steps:
A. extracting flavone, phenylpropane, coumarin and phenolic amide secondary metabolites from fresh tobacco leaves: weighing freeze-dried fresh tobacco leaf powder in a microwave extraction tank, adding an internal standard, adding an extracting solution for microwave-assisted extraction, centrifuging an extract in a centrifugal tube, and filtering supernatant liquid with an organic filter membrane to obtain a filtrate;
B. chromatographic analysis of flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves: performing chromatographic analysis by adopting ultra-high performance liquid chromatography-ultraviolet detector-tandem quadrupole-time-of-flight mass spectrometry;
C. qualitative and quantitative analysis of flavone, phenylpropane, coumarin and phenolic amide secondary metabolites in fresh tobacco leaves: qualitatively comparing with high-resolution mass spectrogram, and quantitatively comparing with internal standard method.
2. The chromatographic analysis method for simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic amide secondary metabolites in the fresh tobacco leaves as claimed in claim 1, wherein the chromatographic analysis method comprises the following steps: the extracting solution in the step A is 0.1 percent of methanolic solution of formic acid.
3. The chromatographic analysis method for simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic amide secondary metabolites in the fresh tobacco leaves as claimed in claim 1, wherein the chromatographic analysis method comprises the following steps: and B, extracting for 5min at 90 ℃ under the microwave-assisted extraction condition of 800w in the step A.
4. The chromatographic analysis method for simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic amide secondary metabolites in the fresh tobacco leaves as claimed in claim 1, wherein the chromatographic analysis method comprises the following steps: the internal standards in step A were 1.88mg/mL cinnamic acid and 14.796mg/mL adipic acid.
5. The chromatographic analysis method for simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic amide secondary metabolites in the fresh tobacco leaves as claimed in claim 1, wherein the chromatographic analysis method comprises the following steps: the chromatographic analysis condition in the step B is an Acquity UPLC HSS T3 column (2.1mm multiplied by 100mm,1.8 mu m,), the column temperature is 35 ℃, and the sample injection amount is 0.5 mu L; mobile phase a was ultrapure water (containing 0.05% HCOOH), B was mass-spec acetonitrile, flow rate was 0.35mL/min, gradient elution was as follows: maintaining the initial 98% A for 1min, 16min for 60% A, 20min for 5% A, maintaining for 24min, 25min for 0% A, maintaining for 27min, 29min for 98% A, and maintaining for 31 min; the mass spectrum condition adopts a primary mass spectrum ESI negative mode, nitrogen is atomizing gas and taper hole gas, the flow rates of the atomizing gas and the taper hole gas are 800L/h and 50L/h, the temperature of the atomizing gas is 450 ℃, the source temperature is 120 ℃, and a capillary tube is usedVoltage: 2.0KV, taper hole voltage: 35V, collision capacity of 6eV, scanning time of 0.2s, interval of 0.02s, scanning range of 70-1000 and detector voltage of 1950V; leucine cephalka peptide is used as a real-time calibration solution, the concentration is 200ng/mL, the flow rate is 10 mu L/min, the correction interval is 15s, and the correction result is 3 times of average; MS (Mass Spectrometry)eThe data acquisition energy is 6-35eV to obtain the corresponding fragment mass spectrum.
6. The chromatographic analysis method for simultaneously analyzing the flavone, the phenylpropane, the coumarin and the phenolic amide secondary metabolites in the fresh tobacco leaves as claimed in claim 1, wherein the chromatographic analysis method comprises the following steps: in the step C, high-resolution excimer ions, fragment ions, ultraviolet absorption combined databases Scripps, HMDB, Respect and Massbank are qualitatively adopted to identify main metabolite compositions, and MS with different positions of glycosidic bonds in flavone is utilized to identify main metabolite compositionseFragmentation rules to determine the exact structure of the flavone; the quantification is carried out by an internal standard method.
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