Method for determining characteristic aroma components in clary sage essential oil
Through the combination of double-column qualitative detection technology and GC-MS-O, the accuracy and cost of the characteristic aroma component analysis of perilla essential oil in the prior art was solved, and efficient and low-cost characteristic aroma component screening was achieved.
Patent Information
- Application Number
- CN202510791668.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-09-02
AI Technical Summary
The existing method for analyzing the characteristic aroma components of perilla essential oil relies on gas chromatography-mass spectrometry combined technology (GC-MS), and cannot accurately evaluate sensory contribution, and full-two-dimensional gas chromatography equipment is expensive and complex to operate.
Two-column qualitative detection technology is used, combining polar and non-polar chromatographic columns and optimized gradient heating program, and GC-MS analysis and olfactory smell are combined, and characteristic aroma components are screened using OAV values.
It realizes efficient separation of different polar aroma components in perilla essential oil, and accurately screens out characteristic aroma components, which is low cost and easy to operate, and is more accurate than single column method and full two-dimensional gas chromatography.
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Figure CN120577460A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cigarette flavoring, and in particular to a method for determining characteristic aroma components in clary sage essential oil. Background Art
[0002] Currently, analysis of key aroma components in clary sage essential oil primarily relies on gas chromatography-mass spectrometry (GC-MS), combined with relative content analysis to infer the primary aroma compounds. For example, Wu Jizhong et al. used GC-MS to analyze clary sage essential oil and identified linalyl acetate and linalool as the primary aroma components. However, this method, based solely on compound content, cannot accurately assess their sensory contributions. In recent years, molecular sensory science has been introduced to fragrance analysis, using gas chromatography-mass spectrometry-olfaction (GC-MS-O) coupled with aroma dilution analysis (AEDA) to screen for key aroma-active components. However, this method relies on single-column separation, and for complex aroma systems such as clary sage essential oil, coelution may result in some compounds not being detected. Furthermore, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOF / MS) can improve separation capabilities, but this technique is expensive and requires optimization of two-dimensional column parameters, making it complex to operate. Summary of the Invention
[0003] In order to solve or partially solve the problems existing in the related art, the present invention provides a method for determining characteristic aroma components in Clary Sage essential oil.
[0004] The present invention provides a method for determining characteristic aroma components in clary sage essential oil, comprising: Step a), mixing the clary sage essential oil and the internal standard and then ultrasonically dissolving them in a solvent to obtain a clary sage essential oil internal standard solution; concentrating the clary sage essential oil internal standard solution and filtering the organic phase with a microporous membrane to obtain a sample to be analyzed; Step b), performing GC-MS analysis on the sample to be analyzed on the first chromatographic column and the second chromatographic column, respectively, to obtain GC-MS analysis results of the first chromatographic column and the second chromatographic column, respectively; the first chromatographic column is a polar chromatographic column, and the second chromatographic column is a non-polar chromatographic column; Step c), according to the GC-MS analysis results of the first chromatographic column and the second chromatographic column, calculate the retention index RI value of each compound, according to the corresponding literature retention index RI values, calculating the difference between the two, retaining compounds with a difference of less than 10 as compounds to be analyzed, and constructing data of compounds to be analyzed; the data of compounds to be analyzed includes: compound name, its retention time on the two chromatographic columns, and its retention index on the two chromatographic columns; Step d), connecting the first chromatographic column and the second chromatographic column to an olfactometer, smelling the effluents of the first chromatographic column and the second chromatographic column respectively using the method of step b), and recording the smelled aroma using the time-aroma description method; based on the recorded information and the data of the compound to be analyzed, determining the compound name corresponding to each aroma, and obtaining an olfactory aroma component set; Step e), calculate the compound to be analyzed in the compound data OAV Value, reserved OAV Compounds with values greater than 1 were considered as the active aroma component set; Step f) taking a subset of the olfactory aroma component set and the active aroma component set as characteristic aroma components.
[0005] Furthermore, in the step a), the internal standard is L-menthyl acetate and the solvent is dichloromethane.
[0006] Furthermore, in the step a), the concentration is specifically performed by first performing rotary evaporation and then performing nitrogen blowing.
[0007] Furthermore, in step a), the pore size of the organic phase microporous filter membrane is 0.22 μm.
[0008] Furthermore, in the step b), the conditions for GC-MS analysis of the first chromatographic column are as follows: Chromatographic column: ZB-WAX capillary column, 60 m × 0.25 mm × 0.25 μm; Inlet temperature: 250 °C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 230 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 240 °C; ion source temperature: 250 °C; mass scan range: m / z 35–450; scan mode: full scan.
[0009] Furthermore, in the step b), the conditions for GC-MS analysis of the second chromatographic column are as follows: Chromatographic column: ZB-5 capillary column, 30 m × 0.25 mm × 0.25 μm; Inlet temperature: 250 °C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 300 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 280 °C; ion source temperature: 250 °C; mass scan range: m / z 35–450; scan mode: full scan.
[0010] Furthermore, in step d), the olfactometer operating conditions are as follows: interface temperature 200° C., heating line temperature 250° C., and nitrogen output flow rate 15 mL / min.
[0011] Furthermore, in step e), the odor activity value OAV Calculate according to the following formula: OAV s=c / AT; OAV s is the odor activity value of the compound, c is the concentration of the compound, in mg / L; AT is the aroma threshold of the compound, in mg / L.
[0012] Furthermore, the step f) further comprises: constructing characteristic aroma component data, wherein the characteristic aroma component data comprises: compound name, corresponding aroma description, and corresponding OAV value.
[0013] The present invention also provides a method for preparing a clary sage flavored tobacco flavor, which comprises: Step S1), determining the characteristic aroma components of clary sage essential oil according to any of the above methods; Step S2), compounding the clary sage essential oil with one or more of its characteristic aroma components to obtain a clary sage flavored tobacco flavor.
[0014] The method for determining characteristic aroma components in clary sage essential oil provided by the present invention may have the following beneficial effects: This method uses dual-column qualitative detection technology. It uses polar and non-polar chromatographic columns in combination with an optimized gradient heating program to achieve efficient separation of different polar aroma components in clary sage essential oil, accurately identify them by combining mass spectrometry and olfaction, and combine OAVScreening is performed to ultimately determine the characteristic aroma components. This method has higher detection accuracy than the single-column method and is low-cost and easy to operate compared to comprehensive two-dimensional gas chromatography. In summary, the method for determining the characteristic aroma components in clary sage essential oil provided in this application has the advantages of high accuracy, low cost, and ease of operation.
[0015] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.
[0017] Figure 1 1 is a profile diagram of flavor compounds with different aroma properties in Clary Sage essential oil shown in an embodiment of the present invention. DETAILED DESCRIPTION
[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0019] The terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. The singular forms "a," "the," and "the" used in this invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.
[0020] It should be understood that although the terms "first", "second", "third", etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "plurality" means two or more, unless otherwise clearly and specifically defined.
[0021] The embodiment of the present invention provides a method for determining characteristic aroma components in clary sage essential oil, comprising the following steps: Step a), mixing the clary sage essential oil and the internal standard and then ultrasonically dissolving them in a solvent to obtain a clary sage essential oil internal standard solution; concentrating the clary sage essential oil internal standard solution and filtering the organic phase with a microporous membrane to obtain a sample to be analyzed; Step b), performing GC-MS analysis on the sample to be analyzed on the first chromatographic column and the second chromatographic column, respectively, to obtain GC-MS analysis results of the first chromatographic column and the second chromatographic column, respectively; the first chromatographic column is a polar chromatographic column, and the second chromatographic column is a non-polar chromatographic column; Step c) calculating the retention index of each compound based on the GC-MS analysis results of the first chromatographic column and the second chromatographic column RI Value, according to the corresponding literature retention index RI values, calculating the difference between the two, retaining compounds with a difference of less than 10 as compounds to be analyzed, and constructing data of compounds to be analyzed; the data of compounds to be analyzed includes: compound name, its retention time on the two chromatographic columns, and its retention index on the two chromatographic columns; Step d), connecting the first chromatographic column and the second chromatographic column to an olfactometer, smelling the effluents of the first chromatographic column and the second chromatographic column respectively using the method of step b), and recording the smelled aroma using the time-aroma description method; based on the recorded information and the data of the compound to be analyzed, determining the compound name corresponding to each aroma, and obtaining an olfactory aroma component set; Step e), calculate the compound to be analyzed in the compound data OAV Value, reserved OAV Compounds with values greater than 1 were considered as the active aroma component set; Step f) taking a subset of the olfactory aroma component set and the active aroma component set as characteristic aroma components.
[0022] The above step a) involves sample pretreatment, which serves the following purposes: enrichment; addition of an internal standard to facilitate subsequent calculation of compound content; and preparation of an injection solution suitable for GC-MS injection. The internal standard in this step is preferably L-menthyl acetate, and the solvent is preferably dichloromethane. In this step, ultrasonic dispersion facilitates rapid and uniform dispersion of the clary sage essential oil and the internal standard in the solvent. The ultrasonic frequency is preferably 40-60 Hz, and the ultrasonic time is preferably 25-40 minutes. Most preferably, the ultrasonic frequency is preferably 50 Hz, and the ultrasonic time is 30 minutes.
[0023] After ultrasonic dispersion to obtain the internal standard solution of Clary Sage essential oil, it is concentrated to prepare an injection solution with a concentration suitable for GC-MS injection. Considering that there may be raw material loss during the concentration process, this embodiment first adds the internal standard and then concentrates, so that the relative content of the internal standard and Clary Sage essential oil remains stable. To control the concentration of the internal standard, the concentration in this step is preferably: first perform rotary evaporation and then perform nitrogen blowing. Rotary evaporation is fast, but it is difficult to accurately control the volume of the concentrate. Although nitrogen blowing can accurately control the volume of the concentrate, it is slow. Performing rotary evaporation first and then nitrogen blowing allows for faster concentration in the early stage and slower, controllable concentration to a predetermined volume in the later stage. Specifically, rotary evaporation can be performed to 2-3.5 times the predetermined volume, and then nitrogen blowing can be used to concentrate to the predetermined volume. After concentration, the organic phase is filtered through a microporous membrane to remove impurities such as particulate matter and suspended matter in the concentrated sample, prevent contamination of the injection port or chromatographic column, and reduce baseline noise. Preferably, the pore size of the organic phase microporous membrane is 0.22 μm.
[0024] Step b) is a process of performing preliminary qualitative analysis of the clary sage essential oil by GC-MS analysis. This step can provide a general understanding of the composition of the clary sage essential oil, providing a basis for the subsequent screening and determination of characteristic aroma components.
[0025] Since the analytical results obtained in this step serve as the raw data for the subsequent screening and identification of characteristic aroma components, the integrity of this data is crucial for the identification of these components. Traditional GC-MS utilizes a single chromatographic column for sample injection and separation. The inventors of this application have discovered that due to the complex composition of clary sage essential oil, some compounds with similar polarity or boiling points tend to coelute when using this separation method, resulting in the inability to accurately identify key aroma components. The retention behavior of different aroma compounds varies significantly, making this method difficult to achieve optimal separation of all compounds. This results in a reduced detection rate of active aroma components and compromises the accuracy of screening for key flavor compounds. To enhance separation capabilities, the inventors of this application have also investigated the use of comprehensive two-dimensional gas chromatography (GC×GC). While this method has been found to be helpful in improving separation capabilities, it is expensive, requires optimization of two-dimensional column parameters (e.g., temperature program, carrier gas flow rate), and is complex to operate. The high technical barriers and maintenance costs make it difficult to implement in conventional laboratories, limiting its practical application in the fragrance industry. In order to improve sample separation capabilities while reducing costs and increasing the universality of application, this embodiment adopts a dual-column qualitative technology, that is, two chromatographic columns of different polarities are used for separate sample injection and then MS analysis. This overcomes the co-elution problem of single-column GC-MS-O and achieves a more comprehensive detection of complex aroma components. At the same time, the operation is simple and easy, and there is no need to replace equipment, only the chromatographic column.
[0026] As a preferred embodiment of this step, the conditions for GC-MS analysis of the first chromatographic column are as follows: Chromatographic column: ZB-WAX capillary column, 60 m × 0.25 mm × 0.25 μm; Inlet temperature: 250°C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 230 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 240°C; ion source temperature: 250°C; mass scan range: m / z 35–450; scan mode: full scan.
[0027] The conditions for GC-MS analysis of the second chromatographic column are preferably as follows: Chromatographic column: ZB-5 capillary column, 30 m × 0.25 mm × 0.25 μm; Inlet temperature: 250°C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 300 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 280°C; ion source temperature: 250°C; mass scan range: m / z 35–450; scan mode: full scan.
[0028] Step c) is a step of verifying the above GC-MS analysis results by using the retention index RI value. The retention index RI value is calculated according to formula I: Formula I In Formula 1: n is the number of carbon atoms; t is the retention time of the target compound, min; t n is the retention time of the last normal alkane eluting before the target peak, min; t n+1 The retention time of the first normal alkane eluting after the target peak, min The retention index of each compound in the GC-MS analysis results RI Values and corresponding literature retention index RI Compare the values and calculate the difference Δ between the two RI , the compounds with a difference of less than 10 are retained as the compounds to be analyzed. In this embodiment, two chromatographic columns are used: for the compounds analyzed on both chromatographic columns, the Δ RI If all of them are less than 10, the compound is retained as the compound to be analyzed; for the compound analyzed on only one column, calculate its Δ RI If the index is less than 10, the compound is retained as the compound to be analyzed. RI If the value is Δ RI Less than 10. Through this step, the GC-MS analysis results are verified to obtain the compounds to be analyzed. On this basis, the data of the compounds to be analyzed is constructed to facilitate subsequent further screening. The data of the compounds to be analyzed includes: the name of the compound, its retention time on the two chromatographic columns, and its retention index on the two chromatographic columns. Furthermore, the data of the compounds to be analyzed can also include: the relative content of the compound, and / or the aroma description corresponding to the compound.
[0029] Step d) utilizes gas chromatography-olfactometry (GC-O), a method that combines the separation power of gas chromatography with the sensitive sense of smell of the human nose, facilitating the selection and evaluation of odor-active substances from complex mixtures. In this embodiment, the olfactometer preferably operates under the following conditions: an interface temperature of 200°C, a heating line temperature of 250°C, and a nitrogen output flow rate of 15 mL / min.
[0030] Those skilled in the art will appreciate that the GC conditions for the first and second chromatographic columns should be the same as those for step b). The smelling should be performed by trained evaluators (aged between 25 and 35) familiar with the aroma characteristics of clary sage essential oil and various flavor components. Multiple evaluators should be involved, with each aroma attribute evaluated in parallel by multiple evaluators. The sniffing results, which are consistent with at least half of the evaluations, are recorded. This step yields aroma information data, including a description of the aroma's notes, the time at which the aroma is detected on both chromatographic columns, and the duration of the aroma.
[0031] Based on this recorded information, combined with the aforementioned data on the compounds to be analyzed, the compound names corresponding to each aroma can be determined, thereby obtaining a set of olfactory aroma components. Specifically, this step involves comparing the aroma onset time and duration in the aroma information data with the retention time in the data on the compounds to be analyzed. Simultaneously, the corresponding aroma description in the recorded information is compared with the corresponding compound in the data on the compounds to be analyzed. If the compound matches the aroma description, the compound corresponding to the aroma is determined and used as the olfactory aroma component. If the comparison results are inconsistent, the compound is eliminated and therefore not used as an olfactory aroma component.
[0032] This step also preferably constructs olfactory aroma component data, which includes: compound names and corresponding aroma descriptions. Further, the data also includes: corresponding dilution factors FD Specifically, the AEDA method can be used to calculate the value of each compound in the aroma information data. FD The specific method is as follows: Gradual dilution of the extract sample, the evaluator continues to smell the sample from low dilution to high dilution until no odor can be smelled, and the highest dilution factor of the flavor compound detected by the smell is the FD value.
[0033] Step e) is based on OAV Value, the step of further screening the data of the compound to be analyzed. In this step, retain OAV Compounds with values greater than 1 are considered as active aroma components. OAV Calculate according to the following formula II: OAV s=c / AT formula II OAV s is the odor activity value of the target compound, c is the concentration of the target compound, in mg / L; AT is the aroma threshold of the compound, in mg / L.
[0034] The concentration c of the target compound can be obtained using the internal standard curve method, and the GC conditions can be the same as those of the first chromatographic column. The specific operation can be: The standard solution is serially diluted, and a calibration curve is constructed based on the relationship between the concentration of the added standard solution and the peak area obtained by analysis. Sage essential oil samples are then tested under the same experimental conditions, and the peak areas of the aroma compounds measured are substituting into the calibration curve of the corresponding standard to determine the concentration of the corresponding substance in the sample. All analyses are preferably repeated three times. Gas chromatography (GC) conditions are the same as those used for the first column, and the mass spectrometry (MS) scanning mode is SRM.
[0035] Those skilled in the art will understand that for compounds for which standard products cannot be purchased, i.e., theirOAV For such compounds, they are considered to be retained OAV Compounds with values less than 1 were removed.
[0036] Step f) is a step of determining characteristic aroma components, which is to take a subset of the olfactory aroma component set and the active aroma component set as the characteristic aroma component. Preferably, this step also preferably includes: constructing characteristic aroma component data, the characteristic aroma component data includes: compound name, corresponding aroma description, and corresponding OAV value.
[0037] Another embodiment of the present invention further provides a method for preparing a clary sage flavored tobacco flavoring, comprising: Step S1), determining the characteristic aroma components of Clary Sage essential oil according to the method of the above embodiment; Step S2), compounding the clary sage essential oil with one or more of its characteristic aroma components to obtain clary sage flavored tobacco spices.
[0038] Those skilled in the art can compound clary sage essential oil with its characteristic aroma components according to the requirements of the target fragrance to achieve style enhancement and endogenous fragrance enhancement.
[0039] From the above, it can be seen that the method for determining characteristic aroma components in clary sage essential oil provided by the embodiment of the present invention has the following advantages: This method uses dual-column qualitative detection technology. It uses polar and non-polar chromatographic columns in combination with an optimized gradient heating program to achieve efficient separation of different polar aroma components in clary sage essential oil, accurately identify them by combining mass spectrometry and olfaction, and combine OAV Screening is performed to ultimately determine the characteristic aroma components. This method has higher detection accuracy than the single-column method and is low-cost and easy to operate compared to comprehensive two-dimensional gas chromatography. In summary, the method for determining the characteristic aroma components in clary sage essential oil provided in this application has the advantages of high accuracy, low cost, and ease of operation.
[0040] The technical solution of the present invention will be further described below in conjunction with specific embodiments: Example 1 Determination of characteristic aroma components of a commercially available Clary Sage essential oil 1. Sample Pretreatment Weigh 1.5 g of sample into an Erlenmeyer flask, add 150 mL of dichloromethane, and add 750 µL of 100 mg / L L-menthyl acetate as an internal standard. Ultrasonicate for 30 min at 50 Hz. Concentrate to 3–5 mL by rotary evaporation in a 30°C water bath, 50 mbar vacuum, and 200 rpm. Then, purge with nitrogen to a volume of 1.5 mL. Pass the organic phase through a microporous filter membrane (pore size 0.22 μm) to obtain the sample to be analyzed. Prepare three replicate samples under the same conditions.
[0041] 2. Double-column qualitative analysis The samples to be analyzed were respectively fed into chromatographic columns 1 and 2 for GC-MS analysis.
[0042] GC-MS analysis conditions for column 1: Chromatographic column: ZB-WAX capillary column, 60 m × 0.25 mm × 0.25 μm, Thermo Fisher Scientific (China) Co., Ltd.; injection port temperature: 250 °C; injection volume: 1 μL; split mode split ratio: 101; constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; gas chromatography temperature program: initial temperature 50 °C, increase to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase to 230 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 240 °C; ion source temperature: 250 °C; mass scan range: m / z 35–450; scan mode: full scan.
[0043] GC-MS analysis conditions for column 2: Chromatographic column: ZB-5 capillary column, 30 m × 0.25 mm × 0.25 μm, Thermo Fisher Scientific (China) Co., Ltd. Gas chromatography temperature program: initial temperature 50 °C, increase to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase to 300 °C at a rate of 10 °C / min, hold for 10 min; transfer line temperature: 280 °C; The other conditions were the same as those of column 1.
[0044] The dual-column qualitative analysis results were obtained by searching the NIST 17 spectral library, and the retention index was calculated using the aforementioned method. RI Value, retention index of each compound RI Values and corresponding literature retention index RIThe values are compared to determine the compound to be analyzed. The comparison rules are as follows: A. If the compound is analyzed on both columns, and the retention index of the compound on both columns is RI The values can be found in both columns. RI Values and corresponding literature retention index RI The difference in values Δ RI When all are less than 10, the compound is determined to be the compound to be analyzed; B. If the compound is analyzed on only one column, and the retention index of the compound on the corresponding column is RI The values can be found, then when the compound RI Values and corresponding literature retention index RI The difference in values Δ RI When it is less than 10, the compound is determined to be the compound to be analyzed; C. If the compound is analyzed in one or two columns, and the retention index of the compound in the corresponding column is RI If the value is not published in the literature, it is considered that the compound has RI Values and corresponding literature retention index RI If the difference in values is less than 10, the subsequent judgment is made according to A or B above. According to the above rules, the compounds to be analyzed were screened and the data of the compounds to be analyzed were constructed; the data of the compounds to be analyzed included: the compound name, its retention time on the two chromatographic columns, its retention index on the two chromatographic columns; and its relative content. The results are listed in Table 1.
[0045] Table 1 Data of compounds to be analyzed in Clary Sage essential oil
[0046] 3. GC-O qualitative analysis Both the first and second columns were connected to an olfactometer. The effluent from the first and second columns was sniffed using the GC conditions described in step 2. The olfactometer operating conditions were: interface temperature 200°C, heating line temperature 250°C, and nitrogen output flow rate 15 mL / min. Five trained evaluators (aged 25-35 years) familiar with the aroma characteristics of clary sage essential oil and various flavor components recorded the aroma using the time-to-note description method. This method recorded the aroma note description, the time of onset of the aroma on both columns, and the duration of the aroma. Aroma data were obtained. Each aroma attribute was evaluated in parallel by five evaluators, and the sniffing results were consistent among at least three of the evaluators.
[0047] According to the recorded information, combined with the aforementioned data of compounds to be analyzed, the compound name corresponding to each aroma is determined, and the olfactory aroma component set is obtained. The olfactory aroma component set includes 27 compounds, namely: myrcene, ocimene, linalool oxide, linalool, linalyl butyrate, α -Terpineol, linalyl formate, nerol, linalyl acetate, geranyl formate, neryl acetate, geranyl acetate, 3,7-dimethyl-6-octenyl hexanoate, α -pinene, β - Caryophyllene, myrcenol, caryophyllene oxide, sclareol, linalyl isobutyrate, cubebene, bornyl acetate, terpineol acetate, germacrene D, citral, ethyl palmitate, β - eucalyptol, β -Cyclohomocitral.
[0048] The olfactory aroma information data is established, and the olfactory aroma information data includes: compound name and corresponding fragrance description.
[0049] The AEDA method was used to calculate the aroma information data of each compound. FD The specific method is as follows: Gradual dilution of the extract sample, the evaluator continues to smell the sample from low dilution to high dilution until no odor can be smelled, and the highest dilution factor of the flavor compound detected by the smell is the FD The dilution gradient used in this example is 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187.
[0050] The olfactory aroma information data obtained in this step are listed in Table 3: Table 3 Olfactory aroma information data of Clary Sage essential oil
[0051] 4. OAV value screening For the compounds in Table 1 for which standards are commercially available, their concentrations (mg / L) were calculated using the internal standard calibration curve method and analyzed using GC-MS / MS injection on a ZB-WAX capillary column. The standard was diluted in a gradient, and a calibration curve was plotted based on the relationship between the concentration of the added standard solution and the peak area obtained by instrumental analysis. The clary sage essential oil sample was then tested under the same experimental conditions, and the measured peak areas of the aroma compounds were inserted into the calibration curve of the corresponding standard to obtain the concentration of the corresponding substance in the sample, calculated in mg / L. All analyses were repeated three times. The gas chromatography (GC) conditions were the same as those for the aforementioned chromatographic column 1 in Example 1, and the mass spectrometry (MS) scanning mode was SRM. For specific parameter settings, please refer to Table 4: Table 4 Names, CAS numbers, retention times and SRM parameter settings of the quantitative compounds in Clerodendrum clarytum essential oil
[0052] Note: ① The data in column 1 are the quantitative ion pairs and their collision energies. The data in columns 2, 3, and 4 are the qualitative ion pairs and their collision energies.
[0053] The compound concentration is calculated and the corresponding odor activity value is calculated OAV , odor activity value OAV Calculate according to the following formula: OAV s=c / AT; OAVs is the odor activity value of the compound, c is the concentration of the compound, in mg / L; AT is the aroma threshold of the compound, in mg / L.
[0054] The quantitative results of the above quantitative compounds and the calculated OAV The values are listed in Table 5: Table 5 Quantitative curve, quantitative results and OAV of Clerodendrum sclarea essential oil
[0055] reserve OAV Compounds with values greater than 1 are considered as the active aroma component set; the active aroma component set includes 22 compounds, namely: α -pinene, camphene, β -Pinene, Myrcene, Limonene, Ocimene, Linalool Oxide, Linalool, Linalyl Acetate, Terpinolene, Bornyl Acetate, β - caryophyllene, α - caryophyllene, α -Terpineol, terpineol acetate, geranyl formate, neryl acetate, geranyl acetate, nerol, caryophyllene oxide, sclareol, ethyl palmitate.
[0056] 5. According to the above steps, the following content is obtained: The olfactory aroma component set includes 27 compounds, namely: myrcene, ocimene, linalool oxide, linalool, linalyl butyrate, α -Terpineol, linalyl formate, nerol, linalyl acetate, geranyl formate, neryl acetate, geranyl acetate, 3,7-dimethyl-6-octenyl hexanoate, α -pinene, β - Caryophyllene, myrcenol, caryophyllene oxide, sclareol, linalyl isobutyrate, cubebene, bornyl acetate, terpineol acetate, germacrene D, citral, ethyl palmitate, β - eucalyptol, β -Cyclohomocitral.
[0057] The active aroma component set includes 22 compounds, namely: α-pinene, camphene, β -Pinene, Myrcene, Limonene, Ocimene, Linalool Oxide, Linalool, Linalyl Acetate, Terpinolene, Bornyl Acetate, β - caryophyllene, α - caryophyllene, α -Terpineol, terpineol acetate, geranyl formate, neryl acetate, geranyl acetate, nerol, caryophyllene oxide, sclareol, ethyl palmitate.
[0058] The subsets of the olfactory aroma component set and the active aroma component set were taken as characteristic aroma components. There are 17 characteristic aroma components, including: linalool, ocimene, geranyl acetate, linalool oxide, nerol, neryl acetate, geranyl formate, linalyl acetate, ethyl palmitate, myrcene, α -Terpineol, caryophyllene oxide, terpineol acetate, β - caryophyllene, α -Pinene, sclareol, bornyl acetate.
[0059] Construct characteristic aroma component data, as shown in Table 5, which includes: compound name, corresponding aroma description, and corresponding OAV value.
[0060] Table 5 Characteristic aroma component data of Clary Sage essential oil
[0061] According to Table 5, the aroma characteristics and flavor wheel of clary sage essential oil are formed. Different aroma components have different contributions to the flavor characteristics of clary sage essential oil. The aroma characteristics of flavor substances in clary sage essential oil are divided into four categories, including floral, fruity, herbal and woody. Figure 1 shown.
[0062] Comparative Example 1 Single column GC-MS-O detection method 1. Sample Pretreatment Same as step 1 in Example 1.
[0063] 2. Single column qualitative analysis The sample to be analyzed was fed into a polar chromatography column for GC-MS analysis. The conditions were the same as those for GC-MS analysis of column 1 in Example 1. Retention index obtained by searching NIST 17 spectral library RI The compound to be analyzed is determined by the following steps:
[0064] 3. GC-O qualitative analysis Same as step 3 of Example 1.
[0065] Comparative Example 2 Single column GC-MS-O detection method 1. Sample Pretreatment Same as step 1 in Example 1.
[0066] 2. Single column qualitative analysis The sample to be analyzed was fed into the polar chromatographic column for GC-MS analysis. The conditions were the same as those for GC-MS analysis of chromatographic column 2 in Example 1. Retention index obtained by searching NIST 17 spectral library RI The compound to be analyzed is determined by the following steps:
[0067] 3. GC-O qualitative analysis Same as step 3 of Example 1.
[0068] Comparative Example 3 GC×GC-TOF / MS Detection Method 1. Sample Pretreatment Same as step 1 in Example 1.
[0069] 2. The sample to be analyzed was placed in a GC×GC-TOF / MS instrument (selectable Pegasus 4D 1D / 2D GC×GC-TOF / MS chromatograph, LECO, USA) under the following analysis conditions: The primary column was an RXi-5silMS (60 m × 0.25 mm × 0.25 µm, Restek, USA). The secondary column was a DB-17ht (1.5 m × 0.1 mm × 0.10 µm, Agilent, USA). Carrier gas was helium at a flow rate of 1.0 mL / min and a modulation period of 7.0 s. The first-dimension temperature program was 60°C (hold for 1 min), then increased to 280°C at a rate of 3.0°C / min, and held for 15 min. The second-dimension temperature program was 75°C (hold for 1 min), then increased to 295°C at a rate of 3.0°C / min, and held for 15 min. Inlet temperature was 250°C, injection volume was 1.0 µL, and split ratio was 10:1. Solvent delay was 350 s.
[0070] Electron impact ion source (EI), ionization energy: 70 eV, ion source temperature: 200 °C, transfer line temperature: 250 °C, scan range: m / z 40-450, one-dimensional acquisition rate: 10 spec / s, two-dimensional acquisition rate: 100 spec / s, spectral library search: Wiley7N, Nist08; mass spectrometry detection mode: selected ion; tuning mode: automatic tuning.
[0071] The performance data of the detection methods of Example 1 of the present invention and Comparative Examples 1-3 are summarized and compared, and are listed in Table 6: Table 6 Performance data corresponding to different analysis methods
[0072] It can be seen from Table 6 that the present invention improves the analysis efficiency by 50% and reduces the equipment cost while ensuring that the detection rate is close to that of full two-dimensional gas chromatography.
[0073] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A method for determining characteristic aroma components in clary sage essential oil, characterized in that: include: Step a), mixing the clary sage essential oil and the internal standard and then dissolving them in a solvent by ultrasonication to obtain a clary sage essential oil internal standard solution; The clary sage essential oil internal standard solution is concentrated and then filtered through a microporous filter membrane to obtain a sample to be analyzed; Step b), performing GC-MS analysis on the sample to be analyzed on the first chromatographic column and the second chromatographic column, respectively, to obtain GC-MS analysis results of the first chromatographic column and the second chromatographic column, respectively; the first chromatographic column is a polar chromatographic column, and the second chromatographic column is a non-polar chromatographic column; Step c) calculating the retention index of each compound based on the GC-MS analysis results of the first chromatographic column and the second chromatographic column RI Value, according to the corresponding literature retention index RI The difference between the two values is calculated, and the compounds with a difference less than 10 are retained as the compounds to be analyzed, and the data of the compounds to be analyzed is constructed; The data of the compound to be analyzed include: the name of the compound, its retention time on the two chromatographic columns, and its retention index on the two chromatographic columns; Step d), connecting the first chromatographic column and the second chromatographic column to an olfactometer, smelling the effluents of the first chromatographic column and the second chromatographic column respectively using the method of step b), and recording the smelled aroma using the time-aroma description method; based on the recorded information and the data of the compound to be analyzed, determining the compound name corresponding to each aroma, and obtaining an olfactory aroma component set; Step e), calculate the compound to be analyzed in the compound data OAV Value, reserved OAV Compounds with values greater than 1 were considered as the active aroma component set; Step f) taking a subset of the olfactory aroma component set and the active aroma component set as characteristic aroma components.
2. The determination method according to claim 1, characterized in that In the step a), the internal standard is L-menthyl acetate, and the solvent is dichloromethane.
3. The determination method according to claim 1, characterized in that In the step a), the concentration is specifically performed by first performing rotary evaporation and then performing nitrogen blowing.
4. The determination method according to claim 1, wherein: In the step a), the pore size of the organic phase microporous filter membrane is 0.22 μm.
5. The determination method according to claim 1, characterized in that: In the step b), the conditions for GC-MS analysis of the first chromatographic column are as follows: Chromatographic column: ZB-WAX capillary column, 60 m × 0.25 mm × 0.25 μm; Inlet temperature: 250 °C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 230 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 240 °C; ion source temperature: 250 °C; mass scan range: m / z 35–450; scan mode: full scan.
6. The determination method according to claim 1, characterized in that: In the step b), the conditions for GC-MS analysis of the second chromatographic column are as follows: Chromatographic column: ZB-5 capillary column, 30 m × 0.25 mm × 0.25 μm; Inlet temperature: 250 °C; injection volume: 1 μL; split mode split ratio: 10:1; Constant flow (He) flow rate: 1 mL / min; solvent delay: 9 min; Gas chromatography temperature program: initial temperature 50 °C, increase the temperature to 140 °C at a rate of 10 °C / min, hold for 5 min, then increase the temperature to 210 °C at a rate of 5 °C / min, hold for 5 min, then increase the temperature to 300 °C at a rate of 10 °C / min, hold for 10 min; Ion source: electron impact (EI) source; ionization energy: 70 eV; transfer line temperature: 280 °C; ion source temperature: 250 °C; mass scan range: m / z 35–450; scan mode: full scan.
7. The determination method according to claim 1, wherein: In step d), the olfactometer operating conditions are as follows: interface temperature 200°C, heating line temperature 250°C, and nitrogen output flow rate 15 mL / min.
8. The determination method according to claim 1, characterized in that: In the step e), the odor activity value OAV Calculate according to the following formula: OAV s=c / AT; OAV s is the odor activity value of the compound, c is the concentration of the compound, in mg / L; AT is the aroma threshold of the compound, in mg / L.
9. The determination method according to claim 1, characterized in that: The step f) further comprises: constructing characteristic aroma component data, wherein the characteristic aroma component data comprises: compound name, corresponding aroma description, and corresponding OAV value.
10. A method for preparing a clary sage flavored tobacco spice, characterized in that: include: Step S1), determining the characteristic aroma components of clary sage essential oil according to the method according to any one of claims 1 to 9; Step S2), compounding the clary sage essential oil with one or more of its characteristic aroma components to obtain a clary sage flavored tobacco flavor.