A method of screening natural monomeric flavour raw materials in tobacco flavours

Abstract

The present application belongs to the field of test analysis of tobacco flavor, and particularly relates to a method for screening natural monomer fragrance raw materials in tobacco flavor. The method for screening natural monomer fragrance raw materials in tobacco flavor adopts liquid chromatography-high resolution mass spectrometry to analyze the natural monomer fragrance raw materials, realizes full coverage of volatile, semi-volatile and difficult-to-volatile components in the natural monomer fragrance raw materials, establishes a liquid chromatography-high resolution mass spectrometry database of the natural monomer fragrance raw materials by combining non-target extraction and targeted extraction, and after liquid chromatography-high resolution mass spectrometry analysis and targeted extraction of the tobacco flavor to be analyzed, determines whether the natural monomer fragrance raw materials are added by comparing and analyzing characteristic peaks. The screening method involves ten thousand characteristic peaks, so that the comparison and analysis result has high reliability. In addition, the method can realize batch rapid screening of various natural monomer fragrance raw materials in the tobacco flavor, greatly reduces the workload of the flavoring technicians, and shortens the time.

Description

Technical Field

[0001] This invention belongs to the field of testing and analysis of tobacco flavorings and fragrances, specifically relating to a method for screening natural monomeric flavoring raw materials in tobacco flavorings and fragrances. Background Technology

[0002] The natural single-component flavoring ingredients in the permitted and temporary licensed lists of tobacco additives mainly include extracts, essential oils, and tinctures, such as maple extract, licorice fluid extract, jujube tincture, Paraguayan tea essential oil, apple extract, coffee extract, and fresh grape concentrate. These natural single-component flavoring ingredients constitute various styles of tobacco flavorings and fragrances. Currently, the information characterization of tobacco flavorings and fragrances is still insufficient. The source of many tobacco flavorings and fragrances is unknown, and their quality is uncontrollable, directly affecting the efficiency and effect of cigarette flavoring.

[0003] The composition of the above natural single fragrance raw materials covers volatile, semi-volatile and non-volatile components. In particular, most natural single fragrance raw materials are mainly composed of water-soluble and alcohol-soluble non-volatile components. There are still significant blind spots in the component analysis methods, and many natural single fragrance raw materials cannot be analyzed by traditional analytical and detection methods.

[0004] Liquid chromatography-mass spectrometry (LC-MS) based on liquid chromatography separation can be used to detect non-volatile and semi-volatile components in tobacco flavorings and fragrances. However, tobacco flavorings and fragrances and natural monomeric flavor raw materials have complex compositions, and incomplete separation of multiple components leads to significant component interference and inaccurate qualitative analysis in primary mass spectrometry. Furthermore, this method relies on purchasing standards for qualitative analysis, making it difficult to achieve simultaneous analysis of a large number of components. In addition, the diversity of plant sources and extraction processes results in significant differences in the physical and chemical properties of natural monomeric flavor raw materials, which further poses a great challenge to the screening of natural monomeric flavor raw materials in tobacco flavorings and fragrances. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening natural monomeric flavoring materials in tobacco flavorings and fragrances, so as to solve the problem that existing technologies are unable to achieve efficient and reliable screening of natural monomeric flavoring materials in tobacco flavorings and fragrances.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for screening natural monomeric flavoring ingredients in tobacco flavorings includes the following steps:

[0008] (1) Sample pretreatment was performed on each natural single fragrance raw material, and then liquid chromatography-high resolution mass spectrometry analysis was performed to extract the characteristic peaks of each natural single fragrance raw material in a non-targeted manner.

[0009] (2) Align the feature peaks extracted in step (1) to obtain aligned feature peaks;

[0010] (3) The liquid chromatography-high resolution mass spectrometry analysis results of each natural single fragrance raw material in step (1) are extracted using the alignment characteristic peak features to establish a liquid chromatography-high resolution mass spectrometry database of natural single fragrance raw materials.

[0011] (4) The tobacco flavorings to be analyzed are subjected to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and the alignment characteristic peaks are used for targeted extraction to obtain the characteristic peaks to be compared.

[0012] (5) The characteristic peak to be compared is compared and analyzed with the characteristic peaks of each natural monomeric fragrance raw material in the liquid chromatography-high resolution mass spectrometry database to screen out the natural monomeric fragrance raw materials in tobacco flavorings.

[0013] This invention is pioneering, employing liquid chromatography-high resolution mass spectrometry (LC-HMS) to analyze natural monomeric flavoring raw materials. It achieves full coverage of volatile, semi-volatile, and non-volatile components in these raw materials. A LC-HMS database of natural monomeric flavoring raw materials is established through a combination of non-targeted and targeted extraction. After LC-HMS analysis and targeted extraction of the tobacco flavorings to be analyzed, characteristic peak comparison analysis determines whether natural monomeric flavoring raw materials have been added. This screening method involves tens of thousands of characteristic peaks, ensuring high reliability of the comparison analysis results. Furthermore, this method enables rapid, batch screening of multiple natural monomeric flavoring raw materials in tobacco flavorings, significantly reducing the workload of flavoring technicians and shortening the time required.

[0014] Preferably, in step (3), the natural monomer fragrance raw material liquid chromatography-high resolution mass spectrometry database is a matrix-type liquid chromatography-high resolution mass spectrometry database containing the names of natural monomer fragrance raw materials and the retention times, precise molecular weights and peak intensities of all corresponding characteristic peaks.

[0015] Preferably, in step (5), the comparative analysis includes batch statistically analyzing the number of characteristic peaks of different intensity ranges in the flavorings and fragrances to be analyzed, as well as the number of characteristic peaks of each natural monomer flavoring raw material corresponding to the intensity range, calculating the detection probability of characteristic peaks of different intensity ranges of all natural monomer flavoring raw materials in the flavorings and fragrances to be analyzed, and determining whether the corresponding natural monomer flavoring raw material is added based on the detection probability.

[0016] More preferably, the characteristic peaks of different intensity ranges are divided according to the peak intensity from high to low, and the threshold value of the previous level peak intensity is 2 to 5 times that of the next level peak intensity threshold value.

[0017] More preferably, the characteristic peaks in different intensity ranges include peak intensity > 10,000,000, peak intensity > 5,000,000, peak intensity > 1,000,000, peak intensity > 500,000, peak intensity > 100,000, peak intensity > 50,000, and peak intensity > 10,000.

[0018] Preferably, in step (2), the principle of peak alignment is: retention time difference ≤ 0.2 min, and precise molecular weight difference ≤ 5 ppm.

[0019] Preferably, in steps (3) and (4), the characteristic peaks of the targeted extraction satisfy the following: retention time difference ≤ 0.2 min, and precise molecular weight difference ≤ 5 ppm.

[0020] Preferably, in step (1), the characteristic peak is a peak not present in the blank sample or a peak with an intensity greater than 10 times that of the blank sample.

[0021] Preferably, the chromatographic conditions for the liquid chromatography-high resolution mass spectrometry analysis are as follows: column: Atlantis@T3 column, mobile phase A: 5 mmol / L ammonium acetate solution containing 0.1% formic acid, mobile phase B: acetonitrile; gradient elution;

[0022] Mass spectrometry conditions: ESI source; positive / negative ion mode scanning separately; ion source temperature: 500℃; cluster fragmentation voltage: 40V; collision energy: 10V; spray voltage: +5500V / -4500V; nebulizer gas pressure: 55psi; curtain gas pressure: 35psi; auxiliary gas pressure: 50psi; scan range: m / z 100~1000.

[0023] Preferably, in step (1), the sample pretreatment includes extracting the natural monomeric fragrance raw material using an extraction solvent, wherein the extraction solvent is a 40-60% methanol aqueous solution. Detailed Implementation

[0024] The technical concept of this invention is to identify, rationally extract, and analyze liquid chromatography-high resolution mass spectrometry (LC-HMS) data to screen natural monomeric flavoring raw materials in tobacco flavorings. The LC-HMS data covers volatile, semi-volatile, and low-volatile components, ensuring the comprehensiveness and representativeness of the analytical data. By combining non-targeted and targeted extraction, characteristic peaks are avoided from being missed, and a large number of characteristic peaks are compiled into a LC-HMS database, ensuring the high reliability of subsequent comparative analysis results.

[0025] The aforementioned liquid chromatography-high resolution mass spectrometry database boasts high availability and scalability. The more temporary licensed natural monomeric flavoring raw materials it covers, the more comprehensive and accurate the screening becomes. It can be dynamically expanded as needed to meet the ever-growing or changing requirements for reconstructing tobacco flavoring and fragrance formulations.

[0026] In addition, the powerful qualitative capabilities of the liquid chromatography-high resolution mass spectrometry database enable the screening method for natural monomeric flavoring raw materials in tobacco flavorings and fragrances to be completed without the need for standards, which greatly reduces the economic cost of screening and lowers the barrier to entry.

[0027] Based on the screening results of natural monomeric flavoring raw materials in tobacco flavorings and fragrances in this invention, support can be provided for the analysis of the types of natural monomeric flavoring raw materials added to tobacco flavorings and fragrances, formula reconstruction and style adjustment, thereby helping cigarette manufacturing enterprises to achieve independent control of flavoring and fragrance formulation technology.

[0028] The implementation process of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, "%" refers to volume fraction.

[0029] I. Specific Embodiments of the Method for Screening Natural Monomeric Flavor Raw Materials in Tobacco Flavorings and Fragrances of the Present Invention

[0030] Example 1

[0031] The method for screening natural monomeric flavoring ingredients in tobacco flavorings and fragrances according to this embodiment includes the following steps:

[0032] (1) Sample pretreatment was performed on each natural single fragrance raw material, and then liquid chromatography-high resolution mass spectrometry analysis was performed to extract the characteristic peaks of each natural single fragrance raw material in a non-targeted manner.

[0033] Accurately weigh 20 mg of natural single fragrance raw material into a 50 mL capped centrifuge tube, then accurately add 20 mL of 50% methanol aqueous solution, vortex for 10 min, centrifuge, filter through an organic phase filter membrane (13 mm, 0.22 μm), and take 1.5 mL of filtrate into a 2 mL chromatographic bottle for liquid chromatography-high resolution mass spectrometry analysis.

[0034] The liquid chromatography was performed using an Agilent 1290 high-performance liquid chromatograph, and the analytical conditions were as follows:

[0035] Chromatographic column: Atlantis@T3 column (150 mm length, 2.1 mm inner diameter, 3 μm stationary phase particles); column temperature: 40 °C; flow rate: 0.3 mL / min; injection volume: 10 μL; mobile phase A: ammonium acetate (5 mmol / L) solution containing 0.1% formic acid; mobile phase B: acetonitrile; gradient elution conditions: 0–1 min, 97% A; 1–1.5 min, 97%–85% A; 1.5–18 min, 85%–2% A; 18–25 min, 2% A; 25–25.1 min, 2%–97% A; 25.1–35 min, 97% A.

[0036] High-resolution mass spectrometry was performed using an AB 4600 quadrupole time-of-flight mass spectrometer, under the following conditions:

[0037] ESI source; positive / negative ion mode scanning separately; ion source temperature: 500℃; cluster cleavage voltage: 40V; collision energy: 10V; spray voltage: +5500V / -4500V; nebulizer gas pressure: 55psi; curtain gas pressure: 35psi; auxiliary gas pressure: 50psi; scan range: m / z 100~1000.

[0038] The raw data of natural monomeric fragrance raw materials and blank samples (except for the absence of natural monomeric fragrance raw materials, all other operations were the same) were processed simultaneously using the OS-Q data processing software built into the high performance liquid chromatography-quadrupole time-of-flight mass spectrometer. First, a non-targeted extraction method was established, with key parameters being a moderate peak detection sensitivity (S3), a threshold of 10 for the peak intensity ratio of natural monomeric fragrance raw materials to blank samples, and ensuring that the peak detection time range was consistent with the acquisition time range. By subtracting peaks in the natural monomeric fragrance raw materials whose peak intensities were not more than 10 times greater than the corresponding peak intensities in the blank samples, all characteristic peaks of the natural monomeric fragrance raw materials were obtained. The characteristic peak information included retention time and precise molecular weight.

[0039] (2) Align the feature peaks extracted in step (1) to obtain aligned feature peaks.

[0040] A self-developed Matlab program was used to align the characteristic peaks of all natural monomeric fragrance raw materials. The alignment principle was that the difference in retention time should be ≤0.2 min and the difference in precise molecular weight should be ≤5 ppm. During peak alignment, the characteristic parameters of the first eluting peak were used as the standard. Due to the large number of characteristic peaks, they cannot all be shown. Only four characteristic peaks from three natural monomeric fragrance raw materials are used as examples for illustration. The information of the four characteristic peaks of these three natural monomeric fragrance raw materials is shown in Table 1 below.

[0041] Table 1. Examples of four characteristic peaks in natural single-origin fragrance raw materials.

[0042]

[0043]

[0044] The results of aligning the four characteristic peaks of the above three natural monomeric fragrance raw materials according to the principle of retention time difference ≤ 0.2 min and precise molecular weight difference ≤ 5 ppm are shown in Table 2 below:

[0045] Table 2. Alignment results of four characteristic peaks of natural monomeric fragrance raw materials

[0046]

[0047] (3) The liquid chromatography-high resolution mass spectrometry analysis results of each natural single fragrance raw material in step (1) are extracted using the alignment characteristic peak features to establish a liquid chromatography-high resolution mass spectrometry database of natural single fragrance raw materials.

[0048] Using OS-Q data processing software, a targeted extraction method was established based on the retention time and precise molecular weight of the aligned characteristic peaks. The key parameters were: peak detection based on the preset retention time, retention time difference ≤ 0.2 min, and precise molecular weight difference ≤ 5 ppm. This targeted extraction method was used to extract the analytical results of all natural single fragrance raw materials, obtaining the intensity of all characteristic peaks of all fragrance raw materials, forming a matrix-type liquid chromatography-high resolution mass spectrometry database. This database includes the name of the natural single fragrance raw material and the retention time, precise molecular weight, and peak intensity of all characteristic peaks.

[0049] Due to the large number of columns and rows in a matrix database, it is impossible to display it completely. Instead, the format of a matrix database is illustrated by omitting duplicate content, as shown in Table 3 below.

[0050] Table 3. Targeted extraction results of natural monomeric fragrance raw materials

[0051]

[0052] (In the table, ND means Not Detected)

[0053] (4) The flavorings and fragrances to be analyzed are subjected to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and the alignment characteristic peaks are used for targeted extraction to obtain the characteristic peaks to be compared.

[0054] The sample pretreatment method and liquid chromatography-high resolution mass spectrometry (LC-HMS) analysis conditions for the tobacco flavorings to be analyzed were used in the same way as in step (1). The targeted extraction method in step (3) was used to target the LC-HMS analysis results of the tobacco flavorings to be analyzed (prepared by the flavorist) to obtain relevant information consistent with the matrix-type LC-HMS database format, and then added to the database, as shown in Table 4 below:

[0055] Table 4. Examples of adding tobacco flavorings and fragrances to the database for analysis.

[0056]

[0057] (5) The characteristic peak to be compared is compared and analyzed with the characteristic peaks of each natural monomeric fragrance raw material in the liquid chromatography-high resolution mass spectrometry database to screen out the natural monomeric fragrance raw materials in tobacco flavorings.

[0058] Based on the aforementioned database, the number of characteristic peaks in different intensity ranges in the tobacco flavorings to be analyzed is counted in batches, as well as the number of characteristic peaks in the corresponding intensity ranges of each natural monomer flavoring raw material. The detection probability of characteristic peaks in different intensity ranges of all natural monomer flavoring raw materials in the tobacco flavorings to be analyzed is calculated, and the addition of the corresponding natural monomer flavoring raw material is determined based on the detection probability.

[0059] The comparative analysis results of characteristic peaks of the tobacco flavorings and fragrances to be analyzed and some natural monomeric flavoring raw materials are shown in Table 5 below:

[0060] Table 5. Comparison analysis results of characteristic peaks of tobacco flavorings and some natural single-component flavorings.

[0061]

[0062]

[0063] The detection probability standards for characteristic peaks of different intensities, such as >10,000,000, >5,000,000, >1,000,000, >500,000, >100,000, >50,000, and >10,000, can be set to 100%, 99%, 98%, 95%, 90%, 80%, and 60%, respectively. In other specific cases, the above detection probability standards can be adjusted appropriately. If the characteristic peaks in each intensity range meet the above detection probability standards, it can be determined that the corresponding natural monomeric flavoring raw material is present in the tobacco flavoring and fragrance being analyzed. Using the above detection probability standards and the method of this invention, the present flavoring raw materials can be accurately identified, avoiding false positives.

[0064] The data in the table above shows that the characteristic peaks of maple extract, licorice fluid extract, jujube tincture, apple extract, and fresh grape concentrate have a high detection probability in the analyzed tobacco flavorings and fragrances, and exhibit a trend of higher detection probability with greater peak intensity. Specifically, the detection probability of characteristic peaks >10,000,000 and >5,000,000 is 100%; the detection probability of characteristic peaks >1,000,000 is greater than 98%; the detection probability of characteristic peaks >500,000 is greater than 95%; the detection probability of characteristic peaks >100,000 is greater than 90%; the detection probability of characteristic peaks >50,000 is greater than 80%; and the detection probability of characteristic peaks >10,000 is greater than 60%. This proves that maple extract, licorice fluid extract, jujube tincture, apple extract, and fresh grape concentrate are added to this tobacco flavoring and fragrance.

[0065] In comparison, the characteristic peaks of the natural single-component flavoring raw materials, Brazilian tobacco extract and monk fruit extract, had a low detection probability in the tobacco flavoring and fragrance to be analyzed, indicating that the natural single-component flavoring raw materials, Brazilian tobacco extract and monk fruit extract, were not added to this tobacco flavoring and fragrance.

[0066] Further investigation of other natural monomeric flavoring raw materials in the database revealed that the number of natural monomeric flavoring raw materials that could meet the detection probabilities (100%, 99%, 98%, 95%, 90%, 80%, and 60%) of characteristic peaks corresponding to different intensity ranges (>10,000,000, >5,000,000, >100,000, >50,000, and >10,000) were 82, 51, 0, 16, 25, 39, and 62, respectively. However, no natural monomeric flavoring raw materials were found that could simultaneously meet the detection probabilities of characteristic peaks in all the above different intensity ranges. Therefore, it can be ruled out that these natural monomeric flavoring raw materials were not added to the tobacco flavoring.

[0067] The above screening results were corroborated with those of the personnel who blended the flavorings and fragrances for the tobacco products, confirming the accuracy of the screening results.

[0068] As can be seen from the above embodiments, the screening method of the present invention has the characteristics of high efficiency, high reliability and low cost, and can realize the batch rapid screening of a variety of natural monomeric flavoring raw materials in tobacco flavorings and fragrances, greatly reducing the workload of flavoring technicians.

Claims

1. A method for screening natural monomeric flavoring ingredients in tobacco flavorings, characterized in that, Includes the following steps: (1) Sample pretreatment was performed on each natural single fragrance raw material, and then liquid chromatography-high resolution mass spectrometry analysis was performed to extract the characteristic peaks in each natural single fragrance raw material in a non-targeted manner. (2) Align the feature peaks extracted in step (1) to obtain aligned feature peaks; (3) Using the alignment characteristic peak features, the liquid chromatography-high resolution mass spectrometry analysis results of each natural monomer fragrance raw material in step (1) are targeted and extracted to establish a matrix-type liquid chromatography-high resolution mass spectrometry database of natural monomer fragrance raw materials containing the name of natural monomer fragrance raw materials and the retention time, precise molecular weight and peak intensity of all corresponding characteristic peaks. (4) The flavorings and fragrances to be analyzed are subjected to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and the alignment characteristic peaks are used for targeted extraction to obtain the characteristic peaks to be compared. (5) Compare and analyze the characteristic peaks to be compared with the characteristic peaks of each natural monomeric fragrance raw material in the liquid chromatography-high resolution mass spectrometry database to screen out the natural monomeric fragrance raw materials in tobacco flavorings; The comparative analysis includes batch statistical analysis of the number of characteristic peaks in different intensity ranges in the flavorings and fragrances to be analyzed, as well as the number of characteristic peaks in the corresponding intensity ranges of each natural monomer flavoring raw material, calculating the detection probability of characteristic peaks in different intensity ranges of all natural monomer flavoring raw materials in the flavorings and fragrances to be analyzed, and determining whether the corresponding natural monomer flavoring raw material should be added based on the detection probability. The characteristic peaks of different intensity ranges are divided into those with high to low peak intensity, with the threshold of the previous level peak intensity being 2 to 5 times that of the next level peak intensity threshold. Specifically, these include peak intensity > 10,000,000, peak intensity > 5,000,000, peak intensity > 1,000,000, peak intensity > 500,000, peak intensity > 100,000, peak intensity > 50,000, and peak intensity > 10,000.

2. The method for screening natural monomeric flavoring raw materials in tobacco flavorings as described in claim 1, characterized in that, In step (1), the characteristic peak information includes retention time and precise molecular weight.

3. The method for screening natural monomeric flavoring raw materials in tobacco flavorings as described in claim 1, characterized in that, In step (2), the principle of peak alignment is: retention time difference ≤ 0.2 min, precise molecular weight difference ≤ 5 ppm.

4. The method for screening natural monomeric flavoring raw materials in tobacco flavorings as described in claim 1, characterized in that, In steps (3) and (4), the characteristic peaks extracted by the targeted method satisfy the following conditions: retention time difference ≤ 0.2 min, and precise molecular weight difference ≤ 5 ppm.

5. The method for screening natural monomeric flavoring raw materials in tobacco flavorings and fragrances as described in claim 1, 3, or 4, characterized in that, In step (1), the characteristic peak is a peak not present in the blank sample or a peak with an intensity greater than 10 times that of the blank sample.

6. The method for screening natural monomeric flavoring raw materials in tobacco flavorings as described in claim 1, characterized in that, The chromatographic conditions for the liquid chromatography-high resolution mass spectrometry analysis were as follows: column: Atlantis@T3 column, mobile phase A: 5 mmol / L ammonium acetate solution containing 0.1% formic acid, mobile phase B: acetonitrile; gradient elution; Mass spectrometry conditions: ESI source; positive / negative ion mode scanning separately; ion source temperature: 500 ℃; cluster fragmentation voltage: 40 V; collision energy: 10 V; spray voltage: +5500 V / -4500 V; nebulizer gas pressure: 55 psi; curtain gas pressure: 35 psi; Auxiliary gas pressure: 50 psi; Scan range: m / z 100~1000.

7. The method for screening natural monomeric flavoring raw materials in tobacco flavorings and fragrances as described in claim 1 or 6, characterized in that, In step (1), the sample pretreatment includes extracting natural monomeric fragrance raw materials using an extraction solvent, wherein the extraction solvent is a 40-60% methanol aqueous solution.

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