CN115097020B - Method for screening and confirming composite tobacco flavor based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrum - Google Patents

Method for screening and confirming composite tobacco flavor based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrum Download PDF

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CN115097020B
CN115097020B CN202210592242.9A CN202210592242A CN115097020B CN 115097020 B CN115097020 B CN 115097020B CN 202210592242 A CN202210592242 A CN 202210592242A CN 115097020 B CN115097020 B CN 115097020B
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acetate
gamma
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resolution mass
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CN115097020A (en
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罗彦波
王晓如
陈小静
陈伟华
李翔宇
庞永强
何爱民
姜兴益
沈薇
张洪非
朱风鹏
侯宏卫
胡清源
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HEBEI CHINA TOBACCO INDUSTRY CO LTD
National Tobacco Quality Supervision and Inspection Center
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HEBEI CHINA TOBACCO INDUSTRY CO LTD
National Tobacco Quality Supervision and Inspection Center
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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
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Abstract

The invention provides a method for screening and confirming a spice compound for cigarettes based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrum, which comprises the following steps of (1) establishing a high-resolution mass spectrum database; (2) pretreatment of the composite tobacco flavor; (3) sample analysis; and (4) screening and confirming.

Description

Method for screening and confirming composite tobacco flavor based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrum
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for screening and confirming composite tobacco flavor based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry.
Background
In the cigarette production process, composite tobacco flavors such as a flavor base module and the like are required to be applied to tobacco leaves according to the type and style of cigarette products so as to improve the toughness and combustibility of the tobacco, improve the taste and increase the aroma. Therefore, the tobacco flavor compound plays a key role in flavoring the cigarettes, shaping the styles of the cigarettes, improving the fragrance quality of the cigarettes and improving the smoking quality. The key technology of cigarette flavoring is a weak link in scientific research work in the tobacco industry. The cigarette fragrance base module is formed by mixing and blending various natural fragrances and synthetic fragrances, and has complex components. The analysis research on the components of the tobacco flavor-based module is beneficial to analyzing the formula of complex essence and effectively controlling the quality of the essence and the spice, and has important significance in improving the research on the autonomous research and development of the tobacco essence and spice core technology, the self-guarantee capability and the replaceability.
The prior analysis and research on chemical components of the tobacco fragrance-based module mostly adopts a chromatograph-monopole mass spectrometer, but is limited by mass accuracy, sensitivity and dynamic linear range. For example, wei Weiwei uses gas chromatography-mass spectrometry, gas chromatography and chemometric data processing methods to analyze the natural perfume raw materials and part of the tobacco flavor components, and uses NIST02 spectrum library (low resolution mass spectrometry database) to retrieve qualitative properties, and accuracy is to be improved (middle and south university's master paper, 2010). Zhao Jian et al use a method of combining gas chromatography-mass spectrometry with autonomously developed software to analyze the components of essence with unknown components, and use an NIST02 spectrum library and a spectrum retention index in primary characterization; the qualitative results may change with a change in the set threshold in the smart software, resulting in overserved (oversubscription) or underserved (missed substances) (analytical science report, 2012,28, (2): 202-206.). Tandem mass spectrometry (e.g., triple quadrupole mass spectrometry) employs a multiple reaction detection mode (MRM) for targeted detection, which has strong quantitative capability, but has certain limitations in new component and unknown screening and validation. The electrostatic field orbit trap high-resolution mass spectrum (Orbitrap HRMS) has the advantages of high resolution and high quality precision, the acquired mass spectrum information is rich, comprehensive monitoring coverage can be achieved, and the method has remarkable advantages in screening and confirmation.
At present, no report of screening and confirming tobacco flavor, such as flavor module related substances, by adopting an electrostatic field orbit trap high-resolution mass spectrum is found. Therefore, it is necessary to develop a method for rapidly and accurately screening and confirming tobacco flavor (such as a flavor-based module) based on the high-resolution mass spectrum of the electrostatic field orbitrap, thereby creating necessary conditions for analyzing and effectively controlling the quality of the tobacco flavor.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for screening and confirming composite tobacco flavor based on gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry.
In order to solve the technical problems, the application adopts the following technical scheme:
a method for screening and confirming tobacco flavor compounds based on gas chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrum comprises the following steps:
(1) Establishment of a high resolution mass spectrum database (Compounds database, CDB);
The mixed standard solution is measured by adopting gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry, and the retention time of the corresponding compound, the accurate molecular weight of fragment ions and the chemical formula information are obtained in a full scanning mode (FullScan); importing the data into TRACEFINDER to establish the high-resolution mass spectrum database;
(2) Pretreatment of the spice for the composite cigarette;
Accurately weighing the composite tobacco flavor to be measured, dissolving the composite tobacco flavor with ethanol, oscillating for 10-30 min at room temperature, taking 1mL of extract, and filtering the extract with a 0.22 mu m organic phase filter membrane into a chromatographic bottle to obtain a sample solution;
(3) Analyzing a sample;
Taking the sample solution obtained in the step (2), measuring under the same high-resolution mass spectrum conditions of gas chromatography-quadrupole/electrostatic field orbitrap as in the step (1), and collecting necessary data, wherein the data comprise retention time of each chromatographic peak, accurate molecular weight of fragment ions, isotope intensity and isotope distribution;
(4) Screening and validation
Introducing TRACEFINDER the data acquired in the step 3 into software, and automatically screening and matching with the information such as the retention time of the compound, the accurate mass number of fragment ions, the isotope intensity, the isotope distribution and the like in the high-resolution mass spectrum database established in the step 1; when the accurate mass number deviation of the target ion (TargetCompound) is smaller than 5 multiplied by 10 -6, the retention time deviation is smaller than 30s, the accurate mass number deviation of at least one confirmed ion (Confirming AND FRAGMENT) is smaller than 5 multiplied by 10 -6, and the isotope peak distribution is similar (theoretical and actual detection), judging to be detected; and comparing the secondary mass spectrogram of the detected component with a standard substance spectrogram recorded in a database, and confirming the detected component as the same substance.
Preferably, in the step (1), the conditions of the gas chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrum are:
Gas chromatography was tuned and calibrated using perfluorotributylamine;
Chromatographic column: elastic capillary column, specification: 60m×0.25mm×1.4 μm, stationary phase is 6% cyanopropylbenzene, 94% dimethylsiloxane; more preferably an Agilent DB-624 elastic capillary column;
carrier gas: helium with purity > 99.999%;
flow rate: 1.0mL/min;
Sample inlet temperature: 280 ℃;
Sample injection volume: 1 μl;
Sample injection mode: sampling in a split-flow mode;
split ratio: 10:1;
The temperature programming conditions of the column box are as follows: the initial temperature is 80 ℃ and kept for 1min, the temperature is raised to 100 ℃ at the speed of 4 ℃/min, kept for 5min, raised to 180 ℃ at the speed of 4 ℃/min, kept for 2min, raised to 230 ℃ at the speed of 4 ℃/min, and kept for 15min;
Transmission line temperature: 250 ℃;
Ionization mode: electron bombardment ionization (EI);
Ionization energy: 70ev;
Ion source temperature: 280 ℃;
Mass spectrometer scanning mode: full scan, 60000 resolution, scan mass number range 33-495; and using internal locking masses, correction was performed with fragment ions m/z 73.04680, m/z 133.0356, m/z 207.03235, m/z 281.05114, and m/z 355.06993.
Preferably, the mixed standard solution comprises a solvent selected from the group consisting of ethyl acetate, acetic acid, ethyl propionate, propionic acid, isoamyl alcohol, ethyl butyrate, butyric acid, ethyl isovalerate, isoamyl acetate, furfural, 2-methylbutyric acid, furfuryl alcohol, alpha-angelolide, 2, 5-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-acetylfuran, butyl butyrate, furfuryl acetate, butyl acetate, 2,3, 5-trimethylpyrazine, D-limonene, isobutyl acetate, heptyl acetate, isoamyl butyrate, 2-acetylthiazole, 2-acetylpyrazine, 2-acetylpyridine, allyl caproate, methylcyclopentanone, benzyl alcohol, isoamyl isovalerate, 4-hydroxy-2, 5-dimethyl-3 (2H) furanone, linalyl acetate, methyl benzoate, 2-acetylpyrrole, gamma-caprolactone, phenethyl alcohol, maltol, benzyl acetate, ethyl benzoate, 2, 6-trimethyl-2-cyclohexen-1, 4-dione, 3-hydroxy-3, 5-dimethyl furanone, L-menthol, gamma-heptanolide, benzoic acid, ethyl maltol, ethyl phenylacetate, geraniol, ethyl pelargonate, ethyl phenylacetate, L-carvone, p-methoxybenzaldehyde, gamma-octalactone, linalool, nerol, eugenol, beta-damascone, methyl cinnamate, cis-jasmone, anisole acetate, gamma-nonenolactone, beta-ionone, alpha-ionone, myricetin, vanillin, ethyl cinnamate, nerolidol, ethyl vanillin, gamma-decalactone, farnesol, delta-decalactone, dihydroactinolide, raspberry ketone, methyl dihydrojasmonate, and gamma-dodecalactone.
More preferably, the mixed standard solution comprises ethyl acetate, acetic acid, ethyl propionate, propionic acid, isoamyl alcohol, ethyl butyrate, butyric acid, ethyl isovalerate, isoamyl acetate, furfural, 2-methylbutyric acid, furfuryl alcohol, alpha-angelolide, 2, 5-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-acetylfuran, butyl butyrate, furfuryl acetate, butyl acetate, 2,3, 5-trimethylpyrazine, D-limonene, isobutyl acetate, heptyl acetate, isoamyl butyrate, 2-acetylthiazole, 2-acetylpyrazine, 2-acetylpyridine, allyl caproate, methylcyclopentanone, benzyl alcohol, isoamyl isovalerate, 4-hydroxy-2, 5-dimethyl-3 (2H) furanone, linalyl acetate, methyl benzoate, 2-acetylpyrrole, gamma-caprolactone, phenethyl alcohol, maltol, benzyl acetate, ethyl benzoate, 2, 6-trimethyl-2-cyclohexen-1, 4-dione, 3-hydroxy-3, 5-dimethyl furanone, L-menthol, gamma-heptanolide, benzoic acid, ethyl maltol, ethyl phenylacetate, geraniol, ethyl pelargonate, ethyl phenylacetate, L-carvone, p-methoxybenzaldehyde, gamma-octalactone, linalool, nerol eugenol, beta-damascone, methyl cinnamate, cis-jasmone, anisole acetate, gamma-nonolactone beta-ionone, alpha-ionone, myricetin, vanillin, ethyl cinnamate, nerolidol, all of ethyl vanillin, gamma-decalactone, farnesol, delta-decalactone, dihydroactinolide, raspberry ketone, methyl dihydrojasmonate, and gamma-dodecalactone.
Preferably, the TRACEFINDER software version is 4.1 and above.
Preferably, the high resolution mass spectrometry database includes information of compound name, CAS registration number, molecular formula, retention time, exact mass of Target Ion (Target Ion), and exact mass of three confirmed ions (Confirming Fragment).
Preferably, under the condition of the gas chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrum, the high-resolution mass spectrum database is built as follows:
preferably, in the step (2), the sample weighing amount of the composite tobacco flavor is 500+/-25 mg.
Preferably, in the step (2), the mass ratio of the volume of ethanol to the composite tobacco flavor is: 50 ml/1 g.
Preferably, the composite tobacco flavor is a tobacco flavor-based module.
In the specification of the application, the ethanol is ethanol with the volume percentage concentration of 95-100 percent unless specified otherwise.
The invention utilizes the combination of ultra-high resolution, stable mass precision and high sensitivity of gas chromatography-quadrupole/electrostatic field orbit trap mass spectrum and TRACEFINDER software to establish an accurate mass database of the composite tobacco flavor, in particular to tobacco flavor base module related substances. The accurate mass number of the compound is combined with the information such as retention time, isotope intensity, isotope distribution and the like, and a threshold value is not required to be set, so that the sample can be rapidly screened. Compared with the prior art, the invention has the advantages of high detection speed, high detection efficiency, multiple analysis components and accurate screening result, and only needs 70-90min from the preparation of the sample solution to the completion of screening and confirmation of a cigarette fragrance base module, thereby providing a new technical support for high-throughput screening of relevant product additives.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 shows the correlation patterns obtained from different chromatographic columns. Wherein A: is a chromatogram of the mixed standard solution under the DB-5MS chromatographic column; b: a mass spectrum of the mixed standard solution under the DB-5MS chromatographic column; c: is a chromatogram of the mixed standard solution under the DB-624 chromatographic column; d: is a mass spectrum of the mixed standard solution under the DB-624 chromatographic column.
FIG. 2 is a graph showing the separation effect of standard compounds at different temperature elevation procedures.
FIG. 3 shows the results of screening D-limonene in the samples. Wherein, the upper graph shows a list of compound identification results based on fragment ions (. + -.5 ppm range), and the lower left graph shows an extracted ion chromatogram of D-limonene; the lower right panel is a secondary mass spectrum (upper and lower mass spectra show measured and theoretical fragment ions, respectively).
FIG. 4 shows the results of screening for alpha-angelicalactone. Wherein, the upper graph shows a compound identification result list based on fragment ions (±5ppm range), and the lower left graph shows an extraction ion chromatogram of alpha-angelica lactone; the lower right panel is a secondary mass spectrum (upper and lower mass spectra show measured and theoretical fragment ions, respectively).
FIG. 5 shows the results of screening maltol. Wherein, the upper graph shows a list of compound identification results based on fragment ions (±5ppm range), and the lower left graph shows an extracted ion chromatogram of maltol; the lower right panel is a secondary mass spectrum (upper and lower mass spectra show measured and theoretical fragment ions, respectively).
FIG. 6 shows the results of screening D, L-menthol. Wherein, the upper graph shows a list of compound identification results based on fragment ions (. + -.5 ppm range), and the lower left graph shows an extracted ion chromatogram of D, L-menthol; the lower right panel is a secondary mass spectrum (upper and lower mass spectra show measured and theoretical fragment ions, respectively).
FIG. 7 shows the results of screening for alpha-ionone. Wherein, the upper graph shows a list of compound identification results based on fragment ions (±5ppm range), and the lower left graph shows an extracted ion chromatogram of α -ionone; the lower right panel is a secondary mass spectrum (upper and lower mass spectra show measured and theoretical fragment ions, respectively).
FIG. 8 is a screening result of dihydroactinide. Wherein, the upper graph shows a compound identification result list based on fragment ions (within + -5 ppm), and the lower left graph shows an extraction ion chromatogram of dihydroactinolide; the lower right image is a secondary mass spectrogram, and the upper and lower mass spectrograms respectively show measured fragment ions and theoretical fragment ions.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified. Wherein, partial reagent and instrument purchase conditions are as follows:
ThermoScientic TMTRACETM 1310 gas chromatograph, quadrupole/electrostatic field orbitrap high resolution mass spectrum Q-Exactive, thermoScientic TMTriPlusTM RSH autosampler: thermo Fisher company;
TRACEFINDER 4.1.1 software (database with unknown retrieval function and high resolution and accurate quality of common pollutants) is used for data processing;
AlphA-Angelicalactone (purity >98%, TCI); gamma valerolactone standard (purity 98%, alfa); gamma-caprolactone standard (purity 98%, alfa); gamma-heptanolactone standard (purity 98%, aletin); gamma-octalactone standard (purity >99%, adamas); gamma-nonanolactone standard (purity >97%, SIGMA-ALDRICH); gamma-decalactone (purity 98%, alfa); delta-decalactone (purity >97.0%, TCI); gamma-dodecalactone (purity 98%, SIGMA); dihydroactinolide (purity 98%, BLOCK chemistry); ethyl acetate (99.8% pure, SIGMA); butyl acetate (purity 99.7%, aladin); hexyl acetate (purity >99.5%, aladin); isobutyl acetate (purity >99.0%, TCI); isoamyl acetate (purity >98.0%, TCI); heptyl acetate (purity >99%, aletin); anisole acetate (purity 98%, aledine); benzyl acetate (99% pure, aletin); furfuryl acetate (99% pure, aledine); geranyl acetate (purity 98%, alfa); leaf alcohol acetate (purity 99%, alfa); linalyl acetate (purity >98%, adamas); neryl acetate (purity >95%, TCI); phenethyl acetate (purity 98%, aledine); ethyl propionate (99% purity, enoKai); butyl butyrate (purity >99%, aledine); ethyl butyrate (purity 99%, enoKai); isoamyl butyrate (99% pure, aletin); ethyl isovalerate (99% pure, aledine); isoamyl isovalerate (98% purity, aletin); allyl caproate (purity 98%, aletin); ethyl pelargonate (purity >99%, adamas); methyl benzoate (99% purity, enoKai); ethyl benzoate (purity >99%, enokie); methyl dihydrojasmonate (96% purity, aledine); methyl cinnamate (purity 99%, alfa); ethyl cinnamate (99% purity, adamas); d-limonene (purity >99%, TCI); ethanol (chromatographic purity, DIMA):
a tobacco fragrance base module: is provided by the technical center of certain national smoke industry company.
The mixed standard solutions used in the following study and/or examples were prepared by the following methods:
The standard substance is dissolved in ethanol to prepare a single standard substance standard stock solution, and the concentration is generally 0.5-2mg/mL. Then, the standard stock solution of the single standard product with different volumes is removed in a 10mL volumetric flask, and ethanol is used for fixing the volume, so that the mixed standard solution with the concentration of each standard product of 1 mug/mL is obtained.
Study example 1 optimization of chromatographic conditions
1. Optimization selection of gas chromatographic column:
First, a chromatographic column was optimized with a part of ester compounds (ethyl acetate, ethyl propionate, ethyl butyrate, ethyl isovalerate, etc.) as representative compounds.
Gas chromatography conditions:
Chromatographic column: DB-5MS chromatographic column, stationary phase is 5% -phenyl-methyl polysiloxane (30 m x 0.25mm x 1.0 μm) and DB-624 chromatographic column, stationary phase is 6% cyanopropyl benzene-methyl polysiloxane (60 m x 0.25mm x 1.4 μm);
carrier gas: helium with purity > 99.999%;
flow rate: 1.0mL/min;
Sample inlet temperature: 280 ℃;
Sample injection volume: 1 μl;
Sample injection mode: sample injection in a non-split mode;
The temperature programming conditions of the column box are as follows: the initial temperature is 40 ℃, and is kept for 2min, the temperature is increased to 180 ℃ at the speed of 5 ℃/min, the temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 40 ℃/min, and the temperature is kept for 1min;
Transmission line temperature: 280 ℃;
Ionization mode: electron bombardment ionization (EI);
Ionization energy: 70ev;
Ion source temperature: 280 ℃;
Mass spectrum mass analyzer: quadrupole rods;
Mass spectrometer scanning mode: full scan, scan range m/z 30-350.
The related map is shown in figure 1.
As shown in a diagram in fig. 1, when using a DB-5MS column, characteristic ion (m/z 43, 29 and 61) chromatographic peaks of ethyl acetate were not observed at the same time in the chromatogram of the selected ion, characteristic fragment ion (m/z 29) of ethyl acetate was not observed in the B diagram, and the ratio of fragment ion to intensity observed in the mass spectrum was also different from the D diagram in fig. 1 and the standard mass spectrum of ethyl acetate, indicating that ethyl acetate was not detected, ethyl acetate and solvent (ethanol) were eluted together in the mixed standard solution. Therefore, the analysis purpose cannot be achieved by using a DB-5MS chromatographic column. Probably because the boiling point of ethyl acetate is 77℃and is close to that of ethanol (78 ℃), co-elution is caused. When using the DB-624 column, the chromatographic peaks (C plot in FIG. 1) of three characteristic ions (m/z 43, 29 and 61) of ethyl acetate were observed at the same time, and the corresponding mass spectrum (see D plot in FIG. 1) was also consistent with the standard mass spectrum of ethyl acetate. All targets can be well separated within 55 min. Thus, DB-624 chromatographic columns are preferred in the present invention.
2. Optimizing temperature programming conditions
The influence of three temperature programming conditions on the separation effect was examined under the above chromatographic conditions (except the temperature programming conditions) using a DB-624 column:
Program 1: the initial temperature is 80 ℃, and is kept for 1min, the temperature is increased to 180 ℃ at the speed of 4 ℃/min, the temperature is kept for 5min, the temperature is increased to 230 ℃ at the speed of 4 ℃/min, and the temperature is kept for 10min;
Program 2: the initial temperature is 80 ℃ and kept for 1min, the temperature is raised to 120 ℃ at the speed of 4 ℃/min, kept for 5min, raised to 180 ℃ at the speed of 4 ℃/min, kept for 2min, raised to 230 ℃ at the speed of 4 ℃/min, and kept for 15min;
Program 3: the initial temperature was 80℃and maintained for 1min, at a rate of 4℃per minute to 100℃and for 5min, at a rate of 4℃per minute to 180℃and for 2min, at a rate of 4℃per minute to 230℃and for 15min.
The chromatogram is shown in FIG. 2.
As can be seen from the circled portion of fig. 2, none of these standard compounds achieved baseline separation using temperature elevation procedure 1 and 2, and only temperature elevation procedure 3 achieved baseline separation of the compounds. Therefore, the temperature increase program 3 is preferable in the present invention.
Example 1 high resolution database was built based on gas chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry
The chromatographic conditions (chromatographic column and temperature-raising program) selected in research example 1 are adopted, 1 mu L of mixed standard solution is taken for sample injection, and the retention time of the corresponding compound, the accurate molecular weight of fragment ions, the chemical formula and other spectrogram information are obtained in a full scanning mode (FullScan). 4 fragment ions were selected for each compound to obtain ion information (exact mass, chemical formula). The data is imported TRACEFINDER to build a relational database as shown in table 1.
Table 1 high resolution mass spectrum database information for 76 standard compounds
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Example 2 screening and confirmation of tobacco fragrance module related Compounds
1. Pretreatment of cigarette fragrance base module
Accurately weighing 511.6mg of cigarette fragrance base module, placing in a triangular flask, adding 25mL of ethanol solution, oscillating at room temperature for 20min, collecting 1mL of extractive solution, filtering with 0.22 μm organic phase filter membrane, and bottling to obtain sample solution
2. Sample analysis
Under the same chromatographic-mass spectrometry conditions as in example 1, 1 μl of the sample solution is sampled, and the chromatograms are recorded to obtain information such as retention time, accurate fragment ion mass number, isotope intensity and isotope distribution of each compound.
3. Screening and confirmation of Compounds
The data obtained in the previous step are imported into TRACEFINDER software, and are automatically screened and matched with the information such as the retention time of the compounds, the accurate mass number of fragment ions, the isotope intensity, the isotope distribution and the like in the database established in the embodiment 1. When the Target Compound (Target Compound) accurate mass number deviation is smaller than 5×10 -6, the retention time deviation is smaller than 30s, the accurate mass number deviation of at least one confirmed ion (Confirming AND FRAGMENT) is smaller than 5×10 -6, and the isotope peak distribution is similar (theoretical and actual detection) as detected. The secondary mass spectrogram of the detected component is consistent with the spectrogram of the standard substance recorded in the database, and can be confirmed to be the same substance.
The compounds of the present invention such as α -angelic lactone, D-limonene, maltol, D, L-menthol, α -ionone, and dihydroactinolide in table 1 were detected in the samples of the cigarette fragrance base module measured in this example. Taking D-limonene as an example for the screening and confirming process of the method: the corresponding ion extraction ion flow diagram, primary mass spectrum and secondary mass spectrum of the target ions in the sample are shown in figure 3. In the database, the retention time of D-limonene was 21.768min, theoretical target ion m/z 93.06988, corroborating ions m/z 79.05423, 91.05423 and 67.05423; the retention time of suspicious peaks in the target ion extraction ion flow graph is 21.78min, which is consistent with the retention time recorded in the database, and meets the requirement of the retention time. The range of the accurate mass number of the extracted ions in the primary mass spectrogram is 93.06941-93.07035, and compared with a theoretical value, the mass deviation meets the requirement of 5ppm and meets the requirement of the mass number of the target ions. The secondary mass spectrogram has consistent characteristic fragment ions, and the overall distribution trend of the ions is consistent, so that the D-limonene can be confirmed.
Based on the same process, the determination of the compounds of alpha-angelicalactone, maltol, D, L-menthol, alpha-ionone, dihydroactinolide and the like in the sample is detected and confirmed; the results of screening for these compounds are shown in FIGS. 4-8.
While the preferred embodiments and examples of the present invention have been disclosed in connection with the accompanying drawings, the present invention is not limited to the above embodiments and examples, and modifications and improvements may be made by those skilled in the art without departing from the spirit and scope of the invention. The scope of the invention is therefore defined by the appended claims.

Claims (12)

1. A method for screening and confirming tobacco flavor compounds based on gas chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrum comprises the following steps:
(1) Establishing a high-resolution mass spectrum database;
The mixed standard solution is measured by adopting gas chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry, and the retention time of the corresponding compound, the accurate molecular weight of fragment ions and the chemical formula information are obtained in a full scanning mode; importing the data into TRACEFINDER to establish the high-resolution mass spectrum database;
(2) Pretreatment of the spice for the composite cigarette;
Accurately weighing the composite tobacco flavor to be measured, dissolving the composite tobacco flavor with ethanol, oscillating for 10-30 min at room temperature, taking 1mL of extract, and filtering the extract with a 0.22 mu m organic phase filter membrane into a chromatographic bottle to obtain a sample solution;
(3) Analyzing a sample;
Taking the sample solution obtained in the step (2), measuring under the same high-resolution mass spectrum conditions of gas chromatography-quadrupole/electrostatic field orbitrap as in the step (1), and collecting necessary data, wherein the data comprise retention time of each chromatographic peak, accurate molecular weight of fragment ions, isotope intensity and isotope distribution;
(4) Screening and validation
Introducing TRACEFINDER the data acquired in the step 3 into software, and automatically screening and matching with the information such as the retention time of the compound, the accurate mass number of fragment ions, the isotope intensity, the isotope distribution and the like in the high-resolution mass spectrum database established in the step 1; when the accurate mass number deviation of the target ion is smaller than 5 multiplied by 10 -6, the retention time deviation is smaller than 30s, at least one confirmed ion accurate mass number deviation is smaller than 5 multiplied by 10 -6, and the isotope peak distribution is similar, judging to be detected; and comparing the secondary mass spectrogram of the detected component with a standard substance spectrogram recorded in a database, and confirming the detected component as the same substance.
2. The method according to claim 1, wherein in the step (1), the conditions of the gas chromatograph-quadrupole/electrostatic field orbitrap high resolution mass spectrum are:
Gas chromatography was tuned and calibrated using perfluorotributylamine;
chromatographic column: elastic capillary column, specification: 60m×0.25mm×1.4 μm, stationary phase is 6% cyanopropylbenzene, 94% dimethylsiloxane;
carrier gas: helium with purity > 99.999%;
flow rate: 1.0mL/min;
Sample inlet temperature: 280 ℃;
Sample injection volume: 1 μl;
Sample injection mode: sampling in a split-flow mode;
split ratio: 10:1;
The temperature programming conditions of the column box are as follows: the initial temperature is 80 ℃ and kept for 1min, the temperature is raised to 100 ℃ at the speed of 4 ℃/min, kept for 5min, raised to 180 ℃ at the speed of 4 ℃/min, kept for 2min, raised to 230 ℃ at the speed of 4 ℃/min, and kept for 15min;
Transmission line temperature: 250 ℃;
ionization mode: electron bombardment ionization;
Ionization energy: 70ev;
Ion source temperature: 280 ℃;
Mass spectrometer scanning mode: full scan, 60000 resolution, scan mass number range 33-495; and using internal locking masses, correction was performed with fragment ions m/z 73.04680, m/z 133.0356, m/z 207.03235, m/z 281.05114, and m/z 355.06993.
3. The method of claim 2, wherein the chromatographic column is an Agilent DB-624 elastic capillary column.
4. The method of claim 1, wherein the mixed standard solution comprises a solvent selected from the group consisting of ethyl acetate, acetic acid, ethyl propionate, propionic acid, isoamyl alcohol, ethyl butyrate, butyric acid, ethyl isovalerate, isoamyl acetate, furfural, 2-methylbutyric acid, furfuryl alcohol, alpha-angelica lactone, 2, 5-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-acetylfuran, butyl butyrate, furfuryl acetate, butyl acetate, 2,3, 5-trimethylpyrazine, D-limonene, isobutyl acetate, heptyl acetate, isoamyl butyrate, 2-acetylthiazole, 2-acetylpyrazine, 2-acetylpyridine, allyl caproate, methylcycloenolone, benzyl alcohol, isoamyl isovalerate, 4-hydroxy-2, 5-dimethyl-3 (2H) furanone, linalyl acetate, methyl benzoate, 2-acetylpyrrole, gamma-caprolactone, phenethyl alcohol, maltol, benzyl acetate, ethyl benzoate, 2, 6-trimethyl-2-cyclohexen-4-2, 4-dihydroxy-3-2H) furanone, L-menthol, gamma-heptanolide, benzoic acid, ethyl maltol, ethyl phenylacetate, geraniol, ethyl pelargonate, ethyl phenylacetate, L-carvone, p-methoxybenzaldehyde, gamma-octalactone, linalool, nerol, eugenol, beta-damascone, damascenone, methyl cinnamate, cis-jasmone, anisole acetate, gamma-nonalactone, beta-ionone, alpha-ionone, myricetin, vanillin, ethyl cinnamate, nerolidol, ethyl vanillin, gamma-decalactone, farnesol, delta-decalactone, dihydroactinolide, raspberry ketone, methyl dihydrojasmonate, and gamma-dodecalactone.
5. The method of claim 4, wherein the mixed standard solution comprises ethyl acetate, acetic acid, ethyl propionate, propionic acid, isoamyl alcohol, ethyl butyrate, butyric acid, ethyl isovalerate, isoamyl acetate, furfural, 2-methylbutyric acid, furfuryl alcohol, alpha-angelica lactone, 2, 5-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-acetylfuran, butyl butyrate, furfuryl acetate, butyl acetate, 2,3, 5-trimethylpyrazine, D-limonene, isobutyl acetate, heptyl acetate, isoamyl butyrate, 2-acetylthiazole, 2-acetylpyrazine, 2-acetylpyridine, allyl caproate, methylcycloenolone, benzyl alcohol, isoamyl isovalerate, 4-hydroxy-2, 5-dimethyl-3 (2H) furanone, linalyl acetate, methyl benzoate, 2-acetylpyrrole, gamma-caprolactone, phenethyl alcohol, maltol, benzyl acetate, ethyl benzoate, 2, 6-trimethyl-2-cyclohexen-4-2, 4-dihydroxy-3 (2H) furanone, L-menthol, gamma-heptanolide, benzoic acid, ethyl maltol, ethyl phenylacetate, geraniol, ethyl pelargonate, ethyl phenylacetate, L-carvone, p-methoxybenzaldehyde, gamma-octalactone, linalool, nerol, eugenol, beta-damascone, damascenone, methyl cinnamate, cis-jasmone, anisole acetate, gamma-nonalactone, beta-ionone, alpha-ionone, all of myricetin, vanillin, ethyl cinnamate, nerolidol, ethyl vanillin, gamma-decalactone, farnesol, delta-decalactone, dihydroactinolide, raspberry ketone, methyl dihydrojasmonate, and gamma-dodecalactone.
6. The method of claim 1, wherein the TRACEFINDER software release is 4.1 and above.
7. The method of claim 1, wherein the high resolution mass spectrometry database comprises information of compound name, CAS registry number, molecular formula, retention time, target ion exact mass, and three confirmed ion exact masses.
8. A method according to any one of claims 1 to 3, wherein the high resolution mass spectrum database is established under the conditions of the gas chromatograph-quadrupole/electrostatic field orbitrap high resolution mass spectrum as follows:
9. the method of claim 1, wherein in step (2), the composite tobacco flavor is weighed in an amount of 500±25mg.
10. The method according to claim 1 or 9, wherein in the step (2), the mass ratio of the volume of ethanol to the mass of the composite tobacco flavor is: 50 ml/1 g.
11. The method of claim 1, wherein the composite tobacco flavor is a tobacco flavor-based module.
12. The method of claim 10, wherein the composite tobacco flavor is a tobacco flavor-based module.
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