Method for screening natural monomer aroma raw materials in tobacco essence and perfume

Abstract

The invention belongs to the field of test and analysis of tobacco flavors and fragrances, and particularly relates to a method for screening natural monomer flavor raw materials in tobacco flavors and fragrances. According to the method for screening the natural monomer aroma raw materials in the tobacco essence and perfume, liquid chromatography-high resolution mass spectrometry is adopted to analyze the natural monomer aroma raw materials, so that full coverage of volatile, semi-volatile and non-volatile components in the natural monomer aroma raw materials is realized; the method comprises the following steps: establishing a natural monomer aroma raw material liquid chromatography-high resolution mass spectrometry database in a manner of combining non-targeted extraction and targeted extraction, carrying out liquid chromatography-high resolution mass spectrometry analysis and targeted extraction on to-be-analyzed tobacco flavors and fragrances, and determining whether the natural monomer aroma raw material is added or not through characteristic peak comparative analysis. The screening method relates to tens of thousands of characteristic peaks, so that a comparison analysis result has high reliability; in addition, the method can realize batch rapid screening of various natural monomer perfume raw materials in tobacco flavors and fragrances, so that the workload of flavor blending technicians is greatly reduced, and the time is shortened.

Description

Technical Field

[0001] The invention belongs to the field of testing and analysis of tobacco flavors and fragrances, and particularly relates to a method for screening natural monomer fragrance raw materials in tobacco flavors and fragrances. Background Art

[0002] The natural monomer aroma raw materials in the tobacco additive license and temporary license list mainly include extracts, essential oils, tinctures, etc., such as maple extract, licorice fluid extract, date tincture, Paraguay tea absolute oil, apple extract, coffee extract, fresh grape concentrate, etc. These natural monomer aroma raw materials constitute various styles of tobacco flavors and fragrances. At present, there is still a lack of information characterization of tobacco flavors and fragrances. The source of the ingredients of many tobacco flavors and fragrances is unknown and the quality is uncontrollable, which directly affects the efficiency and effect of cigarette flavoring.

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

[0004] Liquid chromatography-mass spectrometry (LC-MS) based on liquid chromatography separation can be used to detect the non-volatile and semi-volatile components in tobacco flavors and fragrances, but the components of tobacco flavors and fragrances and natural monomer fragrance raw materials are complex. The incomplete separation of multiple components leads to large component interference and inaccurate qualitative analysis in the primary mass spectrometry determination. Moreover, this method relies on the purchase of standard products for qualitative analysis, which makes it difficult to achieve simultaneous analysis of multiple components. In addition, the diversity of plant sources and extraction production processes also leads to large differences in the physical and chemical properties of natural monomer fragrance raw materials, which further leads to great challenges in the screening of natural monomer fragrance raw materials in tobacco flavors and fragrances. Summary of the invention

[0005] The purpose of the present invention is to provide a method for screening natural monomer aroma raw materials in tobacco flavors and fragrances, so as to solve the problem that the prior art is difficult to achieve efficient and reliable screening of natural monomer aroma raw materials in tobacco flavors and fragrances.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for screening natural monomeric aroma raw materials in tobacco flavors and fragrances, comprising the following steps:

[0008] (1) performing sample pretreatment on each natural monomer fragrance raw material, and then performing liquid chromatography-high resolution mass spectrometry analysis to non-target extract characteristic peaks in each natural monomer fragrance raw material;

[0009] (2) performing peak alignment on the characteristic peaks extracted in step (1) to obtain aligned characteristic peaks;

[0010] (3) using the alignment characteristic peak features to perform targeted extraction on the liquid chromatography-high resolution mass spectrometry analysis results of each natural monomer fragrance raw material in step (1), and establish a natural monomer fragrance raw material liquid chromatography-high resolution mass spectrometry database;

[0011] (4) subjecting the tobacco flavors and fragrances to be analyzed to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and using the alignment characteristic peak features to perform targeted extraction to obtain characteristic peaks to be compared;

[0012] (5) comparing and analyzing the characteristic peaks to be compared with the characteristic peaks of each natural monomer aroma raw material in the natural monomer aroma raw material liquid chromatography-high resolution mass spectrometry database to screen out the natural monomer aroma raw materials in tobacco flavors and fragrances.

[0013] The present invention is a pioneering invention. It uses liquid chromatography-high resolution mass spectrometry to analyze natural monomer fragrance raw materials, achieves full coverage of volatile, semi-volatile and non-volatile components in natural monomer fragrance raw materials, and establishes a natural monomer fragrance raw material liquid chromatography-high resolution mass spectrometry database by combining non-targeted extraction and targeted extraction. After liquid chromatography-high resolution mass spectrometry analysis and targeted extraction of the tobacco flavors and fragrances to be analyzed, characteristic peak comparison analysis is performed to determine whether the natural monomer fragrance raw materials are added. The screening method involves tens of thousands of characteristic peaks, so that the comparison analysis results have high reliability; in addition, the method can realize batch rapid screening of a variety of natural monomer fragrance raw materials in tobacco flavors and fragrances, greatly reducing the workload of flavoring technicians and shortening the time.

[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 comprising the names of natural monomer fragrance raw materials and corresponding retention times of all characteristic peaks, precise molecular weights and peak intensities.

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

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

[0017] More preferably, the characteristic peaks in different intensity ranges include peak intensity >10000000, peak intensity >5000000, peak intensity >1000000, peak intensity >500000, peak intensity >100000, peak intensity >50000, peak intensity >100000, peak intensity >50000, and peak intensity >10000.

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

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

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

[0021] Preferably, the chromatographic conditions of the liquid chromatography-high resolution mass spectrometry analysis are: chromatographic 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 respectively; ion source temperature: 500°C; 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; scanning range: m / z 100~1000.

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

[0024] The technical concept of the present invention is to identify, rationalize extraction and analyze liquid chromatography-high resolution mass spectrometry analysis data to complete the screening of natural monomer fragrance raw materials in tobacco flavors and fragrances. Among them, the liquid chromatography-high resolution mass spectrometry analysis data covers volatile, semi-volatile and difficult-to-volatile components, ensuring the comprehensiveness and representativeness of the analysis data. By combining non-targeted extraction and targeted extraction, the omission of characteristic peaks is avoided, and a liquid chromatography-high resolution mass spectrometry database containing a huge number of characteristic peak information is compiled, ensuring the high reliability of subsequent comparison and analysis results.

[0025] The above-mentioned liquid chromatography-high-resolution mass spectrometry database has high availability and easy scalability. The more temporary licensed natural monomer flavor raw materials involved, the more comprehensive and accurate the screening will be. It can be dynamically expanded at any time according to demand, so as to meet the growing or changing needs of tobacco flavor and fragrance formula reconstruction.

[0026] In addition, the powerful qualitative capability of the liquid chromatography-high-resolution mass spectrometry database means that the screening method for natural monomer flavor raw materials in tobacco flavors and fragrances does not require standard samples throughout the process, greatly reducing the economic cost of screening and the threshold for use.

[0027] Based on the screening results of natural monomer aroma raw materials in tobacco flavors and fragrances of the present invention, support can be provided for the analysis of types of natural monomer aroma raw materials added to tobacco flavors and fragrances, formula reconstruction and style adjustment, thereby helping cigarette industry enterprises to achieve independent control of flavor and fragrance formula technology.

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

[0029] 1. Specific embodiments of the method for screening natural monomeric aroma raw materials in tobacco flavors and fragrances of the present invention

[0030] Example 1

[0031] The method of screening natural monomer fragrance raw materials in tobacco flavors and fragrances of this embodiment comprises the following steps:

[0032] (1) Each natural monomer fragrance raw material is subjected to sample pretreatment, and then subjected to liquid chromatography-high resolution mass spectrometry analysis to non-targetedly extract characteristic peaks in each natural monomer fragrance raw material.

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

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

[0035] Chromatographic column: Atlantis@T3 column (column length 150 mm, inner diameter 2.1 mm, stationary phase particles 3 μm); 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, with the following specific conditions:

[0037] ESI source; positive / negative ion mode scanning respectively; 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; scanning range: m / z 100~1000.

[0038] The OS-Q data processing software provided by the high performance liquid chromatography-quadrupole time-of-flight mass spectrometer was used to synchronously process the raw data of natural monomer fragrance raw materials and blank samples (except that no natural monomer fragrance raw materials were added, the rest of the operations were the same). First, a non-targeted extraction method was established, and the key parameters were that the sensitivity of peak detection was medium (S3), the threshold value of the peak intensity ratio of natural monomer fragrance raw materials to blank samples was 10, and the time range of peak detection was consistent with the acquisition time range; by deducting the peaks in the natural monomer fragrance raw materials whose peak intensity was not greater than 10 times the corresponding peak intensity in the blank samples, all the characteristic peaks of the natural monomer fragrance raw materials were obtained, and the characteristic peak information included retention time and precise molecular weight.

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

[0040] The characteristic peaks of all natural monomer fragrance raw materials were aligned using a self-written Matlab program. The alignment principle was that the retention time difference was ≤0.2min and the precise molecular weight difference was ≤5ppm. When aligning the peaks, the characteristic parameters of the first peak were used as the standard. Due to the large number of characteristic peaks, it is impossible to display them one by one. Only the four characteristic peaks of three natural monomer fragrance raw materials are used as examples. The information of the four characteristic peaks of these three natural monomer fragrance raw materials is shown in Table 1 below.

[0041] Table 1 Examples of information on four characteristic peaks of natural monomer fragrance raw materials

[0042]

[0043]

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

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

[0046]

[0047] (3) Utilizing the alignment characteristic peak features, targeted extraction is performed on the liquid chromatography-high resolution mass spectrometry analysis results of each natural monomer fragrance raw material in step (1), and a liquid chromatography-high resolution mass spectrometry database of natural monomer fragrance raw materials is established.

[0048] OS-Q data processing software was used to establish a targeted extraction method based on the retention time and precise molecular weight of the characteristic peaks after the above alignment. The key parameters were that the peak detection was based on the preset retention time, the retention time difference was ≤0.2 min, and the precise molecular weight difference was ≤5 ppm. The targeted extraction method was used to perform targeted extraction on the analysis results of all natural monomer fragrance raw materials to obtain all characteristic peak intensities of all fragrance raw materials, forming a matrix liquid chromatography-high-resolution mass spectrometry database, which included the names of natural monomer fragrance raw materials and all characteristic peak retention times, precise molecular weights and peak intensities.

[0049] Since the matrix database has a large number of columns and rows and cannot be fully displayed, the format of the matrix database is only illustrated by omitting repeated content, as shown in Table 3 below.

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

[0051]

[0052] (In the table, ND means not detected)

[0053] (4) subjecting the tobacco flavors and fragrances to be analyzed to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and using the alignment characteristic peak features to perform targeted extraction to obtain characteristic peaks to be compared.

[0054] The tobacco flavors and fragrances to be analyzed are processed according to the same sample pretreatment method and liquid chromatography-high resolution mass spectrometry analysis conditions as in step (1), and the liquid chromatography-high resolution mass spectrometry analysis results of the tobacco flavors and fragrances to be analyzed (prepared by the flavoring personnel) are subjected to targeted extraction using the targeted extraction method in step (3) to obtain relevant information consistent with the format of the matrix liquid chromatography-high resolution mass spectrometry database, and then added to the database, as shown in Table 4 below:

[0055] Table 4 Examples of adding tobacco flavors and fragrances to the database

[0056]

[0057] (5) comparing and analyzing the characteristic peaks to be compared with the characteristic peaks of each natural monomer aroma raw material in the natural monomer aroma raw material liquid chromatography-high resolution mass spectrometry database to screen out the natural monomer aroma raw materials in tobacco flavors and fragrances.

[0058] According to the above database, the number of characteristic peaks in different intensity ranges in the tobacco flavors and fragrances to be analyzed, as well as the number of characteristic peaks in the corresponding intensity ranges of each natural monomer flavor raw material are counted in batches, and the detection probability of the characteristic peaks in different intensity ranges of all natural monomer flavor raw materials in the tobacco flavors and fragrances to be analyzed is calculated, and it is determined whether the corresponding natural monomer flavor raw materials are added based on the detection probability.

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

[0060] Table 5 Comparison analysis results of characteristic peaks of tobacco flavors and fragrances to be analyzed and some natural monomer flavor raw materials

[0061]

[0062]

[0063] The detection probability standards of characteristic peaks of different intensities such as >10000000, >5000000, >1000000, >500000, >100000, >50000 and >10000 can be set to 100%, 99%, 98%, 95%, 90%, 80% and 60%. In other specific cases, the above detection probability standards can be adjusted appropriately. If the characteristic peaks of each intensity range meet the above detection probability standards, it can be determined that the corresponding natural monomer aroma raw materials exist in the tobacco flavors and fragrances to be analyzed. The above detection probability standards and the method of the present invention can accurately find out the existing aroma raw materials and avoid the occurrence of false positives.

[0064] From the data in the above table, it can be seen that the characteristic peaks of maple extract, licorice fluid extract, jujube tincture, apple extract, and fresh grape concentrate have a high probability of detection in the tobacco flavors and fragrances to be analyzed, and show a trend that the greater the peak intensity, the higher the detection probability. Among them, the detection probability of characteristic peaks >10000000 and >5000000 is 100%; the detection probability of characteristic peaks >1000000 is greater than 98%; the detection probability of characteristic peaks >500000 is greater than 95%; the detection probability of characteristic peaks >100000 is greater than 90%; the detection probability of characteristic peaks >50000 is greater than 80%; the detection probability of characteristic peaks >10000 is greater than 60%. It can be proved that maple extract, licorice fluid extract, jujube tincture, apple extract, and fresh grape concentrate are added to the tobacco flavors and fragrances.

[0065] In comparison, the detection probability of the characteristic peaks of natural monomer aroma raw materials Brazilian tobacco extract and monk fruit extract in the tobacco flavors and fragrances to be analyzed is relatively low, indicating that the natural monomer aroma raw materials Brazilian tobacco extract and monk fruit extract are not added to the tobacco flavors and fragrances.

[0066] Further checking of other natural monomer aroma raw materials in the database, the numbers of natural monomer aroma raw materials that can meet the corresponding characteristic peak detection probabilities (100%, 99%, 98%, 95%, 90%, 80% and 60%) for different intensity ranges (>10000000, >5000000, >1000000, >500000, >100000, >50000 and >10000) are 82, 51, 0, 16, 25, 39 and 62 respectively. However, no natural monomer aroma raw materials that can simultaneously meet the characteristic peak detection probabilities of all the above-mentioned different intensity ranges are found. Therefore, it can be ruled out that these natural monomer aroma raw materials are not added into the tobacco flavors and fragrances.

[0067] The above screening results were verified with the blenders of the tobacco flavors and fragrances, confirming their accuracy.

[0068] It can be seen from the description of the above embodiments that the screening method of the present invention has the characteristics of high efficiency, good reliability and low cost, and can realize the batch rapid screening of various natural monomer fragrance raw materials in tobacco flavors and fragrances, greatly reducing the workload of flavoring technicians.

Claims

1. A method for screening natural monomeric aroma raw materials in tobacco flavors and fragrances, characterized in that: The following steps are involved: (1) performing sample pretreatment on each natural monomer fragrance raw material, and then performing liquid chromatography-high resolution mass spectrometry analysis to non-target extract characteristic peaks in each natural monomer fragrance raw material; (2) performing peak alignment on the characteristic peaks extracted in step (1) to obtain aligned characteristic peaks; (3) using the alignment characteristic peak features to perform targeted extraction on the liquid chromatography-high resolution mass spectrometry analysis results of each natural monomer fragrance raw material in step (1), and establish a natural monomer fragrance raw material liquid chromatography-high resolution mass spectrometry database; (4) subjecting the tobacco flavors and fragrances to be analyzed to the sample pretreatment and liquid chromatography-high resolution mass spectrometry analysis, and using the alignment characteristic peak features to perform targeted extraction to obtain characteristic peaks to be compared; (5) comparing and analyzing the characteristic peaks to be compared with the characteristic peaks of each natural monomer aroma raw material in the natural monomer aroma raw material liquid chromatography-high resolution mass spectrometry database to screen out the natural monomer aroma raw materials in tobacco flavors and fragrances.

2. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to claim 1, characterized in that: 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 corresponding retention times of all characteristic peaks, accurate molecular weights and peak intensities.

3. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to claim 1 or 2, characterized in that: In step (5), the comparative analysis includes batch counting the number of characteristic peaks in different intensity ranges in the tobacco flavors and fragrances to be analyzed, and the number of characteristic peaks in the corresponding intensity ranges of each natural monomer flavor raw material, calculating the detection probability of the characteristic peaks in different intensity ranges of all natural monomer flavor raw materials in the tobacco flavors and fragrances to be analyzed, and determining whether the corresponding natural monomer flavor raw material is added based on the detection probability.

4. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances as claimed in claim 3, characterized in that: The characteristic peaks of different intensity ranges are divided from high to low according to peak intensity, and the peak intensity threshold of the previous level is 2 to 5 times the peak intensity threshold of the next level.

5. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances as claimed in claim 4, characterized in that: 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.

6. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to 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.

7. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to claim 1, characterized in that: In step (3) and step (4), the characteristic peaks of the targeted extraction satisfy the following requirements: retention time difference ≤ 0.2 min, and precise molecular weight difference ≤ 5 ppm.

8. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances as claimed in claim 1, 6 or 7, characterized in that: In step (1), the characteristic peak is a peak that is not contained in the blank sample or a peak whose peak intensity is 10 times greater than the peak intensity of the blank sample.

9. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to claim 1, characterized in that: The chromatographic conditions of the liquid chromatography-high resolution mass spectrometry analysis are as follows: chromatographic 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; ion source temperature: 500°C; cluster fragmentation voltage: 40V; collision energy: 10V; spray voltage: +5500V / -4500V; nebulizer gas pressure: 55psi; curtain gas pressure: 35psi; Auxiliary gas pressure: 50 psi; scanning range: m / z 100~1000.

10. The method for screening natural monomer aroma raw materials in tobacco flavors and fragrances according to claim 1 or 9, characterized in that: In step (1), the sample pretreatment includes extracting the natural monomer fragrance raw material using an extraction solvent, and the extraction solvent is a 40-60% methanol aqueous solution.

Images