Method for detecting polyphenol compounds in tobacco or tobacco products based on quantitative analysis of multi-components by single marker and application of method

Abstract

The invention relates to a method for detecting polyphenol compounds in tobacco or products thereof based on quantitative analysis of multi-components by single marker and application thereof, and the method comprises the following steps: mixing the tobacco or products thereof with a solvent I, and extracting to obtain a test solution; mixing a standard substance of a polyphenol compound with a solvent II to obtain a plurality of standard solutions with different concentrations; and detecting the test solution and the standard solution by adopting a liquid chromatography, and obtaining the content of the polyphenol compounds in the tobacco or the tobacco product based on a quantitative analysis of multi-components by single marker by taking chlorogenic acid as an internal reference substance. According to the method, a high performance liquid chromatography diode array detection method is adopted, the content of the polyphenol compounds in the tobacco and the tobacco products is calculated according to the relative correction factors between chlorogenic acid and other polyphenol compounds, and the method is short in detection time, low in detection limit and quantification limit, low in RSD of precision, repeatability and stability, good in durability and reproducibility, high in accuracy and high in repeatability. The method is suitable for content analysis, quality evaluation and classification of tobacco or products thereof.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multi-evaluation method, and its application. Background Technology

[0002] Tobacco and tobacco products have complex chemical compositions, and their intrinsic quality largely depends on the content and synergistic effects of various chemical components. Polyphenolic compounds, as important secondary metabolites in tobacco, not only affect the color and sensory flavor of tobacco but are also key indicators for evaluating the quality of tobacco and tobacco products. For example, the content and proportion of phenolic acids such as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, as well as flavonoids such as rutin and kaempferol-3-O-rutin, are closely related to the aroma quality, taste characteristics, and stability of tobacco. Therefore, establishing rapid and accurate analytical methods for determining polyphenolic compounds in tobacco is of great significance for the quality evaluation of tobacco and tobacco products.

[0003] Currently, the main methods for detecting polyphenolic compounds in tobacco are high-performance liquid chromatography (HPLC) and its coupled techniques, including ultraviolet spectrophotometry, capillary electrophoresis, and HPLC coupled with mass spectrometry (HPLC-MS / MS). While these methods can achieve simultaneous analysis of multiple components, they still fall under the traditional external standard method, requiring a corresponding standard for each polyphenolic compound to be analyzed. However, some polyphenolic compound standards, such as neochlorogenic acid, scopolamine, and hyoscyamine, are expensive, difficult to obtain, or have poor stability, resulting in high costs and cumbersome procedures for external standard method detection, making it difficult to meet the efficiency requirements of batch sample screening and routine quality control.

[0004] The QAMS method, as an innovative multi-index quality control and detection technology, only requires one or fewer internal reference standards. By establishing a relative correction factor between the reference standard and other analytes, it can achieve simultaneous quantitative analysis of multiple components. This method has shown significant advantages in the quality control of complex systems such as traditional Chinese medicine. However, the QAMS technology has not yet been applied in the tobacco field. At the same time, the application of this method to the analysis of polyphenolic components in tobacco and tobacco products still faces significant technical bottlenecks and challenges: (1) Matrix interference problem: Tobacco samples have complex chemical compositions and contain a large number of interfering substances such as pigments, alkaloids, and sugars, which seriously affect the effective separation and accurate quantification of polyphenols, especially structurally similar isomers (such as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid). (2) Method universality problem: The core of the QAMS method lies in the reproducibility and stability of the relative correction factor. Whether this method can maintain the stability and reliability of the relative correction factor of polyphenolic compounds in the complex matrix of tobacco, under different chromatographic conditions such as different instruments, different chromatographic columns, column flow rates, and column temperatures, needs to be verified through in-depth research. (3) Challenges in batch sample analysis: The methods commonly used in industry standards or literature currently require large sample volumes and extraction solvent volumes, making it difficult to analyze batch samples. (4) Gaps in specific applications: Currently, in the field of tobacco analysis, there are no reports of establishing a multi-evaluation method for the eight specific polyphenol combinations of neochlorogenic acid, scopolamine, chlorogenic acid, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, and kaempferol-3-O-rutin, and applying it to the quality evaluation of tobacco and tobacco products.

[0005] Therefore, there is an urgent need to establish a rapid, accurate, and low-cost quality evaluation method for the simultaneous determination of the above eight key polyphenolic compounds in tobacco and tobacco products, in order to make up for the deficiencies of existing technologies and provide data support and theoretical basis for the quality evaluation and research of tobacco and tobacco products. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for detecting polyphenolic compounds in tobacco or tobacco products based on a single-analysis, multiple-evaluation approach, and its application. This invention employs high-performance liquid chromatography with diode array detection, using chlorogenic acid as an internal reference. The relative correction factor between chlorogenic acid and other polyphenolic compounds is used to calculate the content of polyphenolic compounds in tobacco and tobacco products, establishing a single-analysis, multiple-evaluation approach for tobacco and tobacco products. This method features short detection time, low limits of detection and quantitation, low RSD of precision, repeatability, and stability, and good robustness and reproducibility, making it suitable for content analysis, quality evaluation, and classification of tobacco or tobacco products.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multiple-evaluation method, the method comprising: Tobacco or its products are mixed with a solvent and extracted to obtain a test solution; A standard substance of polyphenolic compound and solvent 2 were mixed to obtain several standard solutions of different concentrations; The test solution and standard solution were detected by liquid chromatography, with chlorogenic acid as an internal reference, and the content of polyphenolic compounds in tobacco or its products was obtained based on the one-test-multiple-evaluation method.

[0008] This invention establishes a method for the first time to measure multiple components in tobacco and tobacco products. It uses the relative correction factor between chlorogenic acid and other polyphenolic compounds to calculate the content of polyphenolic compounds in tobacco and tobacco products. It can simultaneously determine eight polyphenolic compounds. At the same time, the RSD of the method in terms of precision, repeatability and stability is less than 2.28%, indicating that the method can be used to determine the content of eight polyphenolic compounds in tobacco and tobacco products.

[0009] This invention uses chlorogenic acid as an internal reference for a single assay with multiple evaluations to determine the content of polyphenolic compounds in tobacco and tobacco products. Chlorogenic acid, as the most abundant and abundant polyphenolic compound in tobacco, can be stably detected in various tobacco products. This demonstrates that chlorogenic acid in tobacco is stable, readily available, exhibits good chromatographic behavior, and demonstrates excellent separation from other components (no interference from other components in the sample). Furthermore, chlorogenic acid standards are inexpensive, solving the problem of high cost and limited availability of standards such as neochlorogenic acid, cryptochlorogenic acid, and scopolamine, thus providing an experimental basis for the quality evaluation of tobacco and tobacco products.

[0010] Compared to the external standard method, this invention only requires the purchase of one standard, chlorogenic acid, to simultaneously detect eight polyphenolic compounds in tobacco and tobacco products. This simplifies experimental procedures, improves detection efficiency, and is particularly suitable for rapid screening and quality evaluation of large batches of samples.

[0011] Preferably, the polyphenolic compound includes any one or a combination of at least two of the following: chlorogenic acid, neochlorogenic acid, scopolamine, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, or kaempferol-3-O-rutin.

[0012] Preferably, the solvent includes any one or a combination of at least two of methanol, water, methyl tert-butyl ether, n-hexane, or cyclohexane.

[0013] Preferably, the solvent is a combination of methanol, water and n-hexane, or the solvent is a combination of methanol, water and cyclohexane.

[0014] In this invention, a specific methanol-water-n-hexane / or cyclohexane system is selected for extraction and purification. The supernatant is then subjected to HPLC analysis. The operation is simple and rapid, with high throughput and low solvent consumption. It can simultaneously extract and purify eight polyphenolic compounds in tobacco and tobacco products. Compared with the commonly used analytical methods for polyphenolic compounds in tobacco, it greatly reduces the amount of solvent used (by 80%) and facilitates batch sample analysis.

[0015] In addition, tobacco samples have complex chemical compositions and contain a large number of interfering substances such as pigments, alkaloids, and sugars. Compared with traditional purification adsorbents C18 and GCB, using n-hexane or cyclohexane for purification can reduce experimental costs while achieving a very ideal purification effect, providing a clean solution for use, and reducing the maintenance of the liquid chromatography system during use.

[0016] Preferably, the volume ratio of methanol, water and n-hexane is 1:(0.8-1.2):(1.6-2.4).

[0017] Preferably, the volume ratio of methanol, water and cyclohexane is 1:(0.8-1.2):(1.6-2.4).

[0018] The values ​​0.8-1.2 can be independently 0.8, 0.9, 1, 1.1, 1.2, etc., and the values ​​1.6-2.4 can be independently 1.6, 1.8, 2, 2.2, 2.4, etc.

[0019] Preferably, the ratio of the tobacco or its product to solvent one is 1 mg: (0.3-0.5) mL (e.g., 1 mg: 0.3 mL, 1 mg: 0.35 mL, 1 mg: 0.4 mL, 1 mg: 0.45 mL, 1 mg: 0.5 mL, etc.).

[0020] In this invention, the extraction solvent has a small volume, which saves solvent consumption and makes it easy to achieve high-throughput sample analysis.

[0021] Preferably, the second solvent includes methanol and / or water.

[0022] Preferably, the mobile phase in the liquid chromatography method includes mobile phase A and mobile phase B, wherein mobile phase A comprises a combination of water, methanol and acetic acid, and mobile phase B comprises a combination of methanol and acetic acid.

[0023] Preferably, the volume ratio of water, methanol and acetic acid in the mobile phase A is 100:(1-3):(1-3).

[0024] Preferably, the volume ratio of methanol to acetic acid in the mobile phase B is 100:(1-3).

[0025] The values ​​1-3 above can each be 1, 1.5, 2, 2.5, 3, etc., independently.

[0026] Preferably, the elution program of the mobile phase in the liquid chromatography method is gradient elution, specifically as follows: From 0 to 15 min, the volume percentage of mobile phase A changes uniformly from 88-92% (e.g., 88%, 89%, 90%, 91%, 92%, etc.) to 68-72% (e.g., 68%, 69%, 70%, 71%, 72%, etc.), and the volume percentage of mobile phase B changes uniformly from 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.) to 28-32% (e.g., 28%, 29%, 30%, 31%, 32%, etc.); from 15 to 20 min... At min 22, the volume percentage of mobile phase A is uniformly changed from 68-72% (e.g., 68%, 69%, 70%, 71%, 72%, etc.) to 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.), and the volume percentage of mobile phase B is uniformly changed from 28-32% (e.g., 28%, 29%, 30%, 31%, 32%, etc.) to 88-92% (e.g., 88%, 89%, 90%, 91%, 92%, etc.), and then maintained constant until min 22; min 22-22.1 For min, the volume percentage of mobile phase A is uniformly changed from 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.) to 88-92% (e.g., 88%, 89%, 90%, 91%, 92%, etc.), and the volume percentage of mobile phase B is uniformly changed from 88-92% (e.g., 88%, 89%, 90%, 91%, 92%, etc.) to 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.), and then kept constant until min 30.

[0027] In this invention, by specifically selecting the mobile phase and elution procedure, the separation of various polyphenolic compounds can be achieved, thereby improving the detection accuracy.

[0028] Preferably, the chromatographic column in the liquid chromatography method is packed with C18 material, with a particle size of 3-5 μm (e.g., 3 μm, 4 μm, 5 μm, etc.), an inner diameter of 3.0-4.6 mm (e.g., 3.0 μm, 4.0 μm, 4.6 mm, etc.), and a length of 100-250 mm (e.g., 150 mm, 180 mm, 200 mm, 220 mm, 250 mm, etc.).

[0029] In this invention, Waters Symmetry C18, DIKMA Luna 5u C18(2) 100A, and Agilent ZORBAX Eclipse Plus C18 columns are preferred, with 4.6 mm × 250 mm × 5 μm being the most preferred specifications. The separation effect of the above columns is better.

[0030] Preferably, the column temperature of the chromatographic column in the liquid chromatography method is 28-40℃ (e.g., 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, etc.).

[0031] Preferably, the wavelength detection in the liquid chromatography method is segmented detection, specifically: The detection wavelength was 325 nm from 0 to 9 min; 340 nm from 9 to 10.8 min; 325 nm from 10.8 to 17 min; 345 nm from 17 to 20 min; and 350 nm from 20 to 30 min.

[0032] In this invention, if detection is performed using a single wavelength of 340nm, some targets will respond very weakly, resulting in insufficient detection sensitivity. This invention improves the detection sensitivity by employing a segmented wavelength detection method.

[0033] Preferably, in the one-test-multiple-evaluation method, chlorogenic acid is used as an internal reference, the relative correction factor between chlorogenic acid and other polyphenolic compounds to be tested is calculated, and the content of polyphenolic compounds in tobacco or its products is obtained using the relative correction factor.

[0034] Preferably, the relative correction factor is calculated using the slope correction method.

[0035] In this invention, the slope correction method has a smaller average relative error compared to the multi-point correction method or the single-point correction method, making it more suitable for the quantitative analysis of polyphenolic compounds in tobacco and tobacco products. Specifically, the average relative error of the multi-point correction method and the external standard method is less than 5% for all components except scopolamine, which has an average relative error of 6.47%. The average relative error of the slope correction method and the external standard method is less than 3.21%. The average relative error of the single-point correction method and the external standard method is larger; for example, the average relative error of scopolamine reaches 8.11%.

[0036] In this invention, the slope correction method exhibits good robustness and reproducibility. The RSD of the RCF for each analyte at different flow rates is less than 2.18%; the RSD of the RCF for each analyte at different column temperatures is less than 2.41%. The RSD of the RCF under different instruments and columns from different manufacturers is less than 2.96%. Therefore, this method can be widely used in different laboratories with different instruments and under different chromatographic conditions.

[0037] Secondly, the present invention provides an application of the method for detecting polyphenolic compounds in tobacco or its products based on the one-test-multiple-evaluation method described in the first aspect in the quality evaluation and classification of tobacco or its products.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention establishes a high performance liquid chromatography-diode array detection method, which enables the effective separation of eight polyphenolic compounds in a complex tobacco matrix within just 30 minutes (YC / T 202—2006 can only detect three substances, and the analysis time exceeds 40 minutes). At the same time, the method optimizes the use of appropriate wavelengths for different time periods, which greatly improves the detection limit and quantitation limit of the method. It is more suitable for the accurate quantitative analysis of low-content polyphenolic compounds in reconstituted tobacco samples (the mass fraction of polyphenolic compounds in reconstituted tobacco samples is much lower than that in raw tobacco, tobacco flakes and finished cigarette tobacco shreds). It can meet the quantitative analysis of polyphenolic compounds in different types of tobacco and tobacco product samples.

[0039] (2) This invention establishes a method for the first time to measure multiple components in tobacco and tobacco products. It utilizes the relative correction factor between chlorogenic acid and other polyphenolic compounds to calculate the content of each component, enabling the simultaneous determination of eight polyphenolic compounds. Simultaneously, the content of these eight polyphenolic compounds in tobacco and tobacco products is determined using the external standard method. Comparing these two methods, the average relative error is less than 3.21%. The RSD of the method's precision, repeatability, and stability is all less than 2.28%, indicating that the method of this invention can be used to determine the content of eight polyphenolic compounds in tobacco and tobacco products.

[0040] (3) This invention uses chlorogenic acid as an internal reference for the simultaneous determination of polyphenolic compounds in tobacco and tobacco products. Chlorogenic acid in tobacco is stable, readily available, exhibits good chromatographic behavior, and demonstrates excellent separation from other components (no interference from other components in the sample). Furthermore, chlorogenic acid standards are inexpensive, solving the problem of high prices and limited availability of standards such as neochlorogenic acid, cryptochlorogenic acid, and scopolamine, thus providing an experimental basis for the quality evaluation of tobacco and tobacco products. Compared to the external standard method, this invention only requires the purchase of one chlorogenic acid standard to simultaneously detect eight polyphenolic compounds in tobacco and tobacco products, simplifying experimental procedures, improving detection efficiency, and making it particularly suitable for rapid screening and quality evaluation of large batches of samples.

[0041] (4) The method provided by the present invention can be applied to the quality evaluation of tobacco and tobacco products, such as to distinguish between different places of origin, parts and types of tobacco. Attached Figure Description

[0042] Figure 1The chromatogram provided in Example 1 is (1-neochlorogenic acid, 2-scopolamine, 3-chlorogenic acid, 4-cryptochlorogenic acid, 5-caffeic acid, 6-hyoscyamine, 7-rutin, 8-kaempferol-3-O-rutin).

[0043] Figure 2 The chromatogram provided in Example 2 is (1-neochlorogenic acid, 2-scopolamine, 3-chlorogenic acid, 4-cryptochlorogenic acid, 5-caffeic acid, 6-hyoscyamine, 7-rutin, 8-kaempferol-3-O-rutin).

[0044] Figure 3 The chromatogram provided in Example 3 is (1-neochlorogenic acid, 2-scopolamine, 3-chlorogenic acid, 4-cryptochlorogenic acid, 5-caffeic acid, 6-hyoscyamine, 7-rutin, 8-kaempferol-3-O-rutin).

[0045] Figure 4 The chromatogram provided in Example 4 is (1-neochlorogenic acid, 2-scopolamine, 3-chlorogenic acid, 4-cryptochlorogenic acid, 5-caffeic acid, 6-hyoscyamine, 7-rutin, 8-kaempferol-3-O-rutin).

[0046] Figure 5 The chromatogram provided in Example 5 is (1-neochlorogenic acid, 2-scopolamine, 3-chlorogenic acid, 4-cryptochlorogenic acid, 5-caffeic acid, 6-hyoscyamine, 7-rutin, 8-kaempferol-3-O-rutin).

[0047] Figure 6 This is the chromatogram provided in Example 6.

[0048] Figure 7 This is the chromatogram provided in Example 7.

[0049] Figure 8 This is the chromatogram provided in Example 8.

[0050] Figure 9 This is the chromatogram provided in Example 9.

[0051] Figure 10 This is the chromatogram provided in Example 10.

[0052] Figure 11 These are PCA diagrams of different smoke types obtained using the one-test-multiple-evaluation method in Example 1.

[0053] Figure 12 The PCA diagrams for different smoke types were obtained using the external standard method in Comparative Example 1, as shown in Application Example 1.

[0054] Figure 13 This is a PCA diagram of tobacco leaf samples from different origins obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 2.

[0055] Figure 14 The PCA diagrams of different grades of tobacco leaf samples obtained using the one-test-multiple-evaluation method in Example 1 are shown in Application Example 3.

[0056] Figure 15 This is a PLS-DA analysis chart of the polyphenol content of tobacco leaves at different storage times, obtained by using the one-test-multiple-evaluation method in Example 1 in Application Example 4.

[0057] Figure 16 This is a graph showing the trend of chlorogenic acid content in tobacco shreds at different storage times, obtained by using the one-test-multiple-evaluation method in Example 1 in Application Example 4.

[0058] Figure 17 This is a graph showing the trend of rutin content in tobacco shreds at different storage times, obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 4.

[0059] Figure 18 This is a graph showing the trend of cryptochlorogenic acid content in tobacco shreds at different storage times, obtained by using the one-test-multiple-evaluation method in Example 1 in Application Example 4.

[0060] Figure 19 This is a graph showing the trend of chlorogenic acid content in tobacco shreds at different storage times, obtained by using the one-test-multiple-evaluation method in Example 1 in Application Example 4.

[0061] Figure 20 This is a graph showing the trend of kaempferol content in tobacco shreds at different storage times, obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 4.

[0062] Figure 21 This is a graph showing the trend of scopolamine content in tobacco shreds at different storage times, obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 4.

[0063] Figure 22 This is a graph showing the trend of caffeic acid content in tobacco shreds at different storage times, obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 4.

[0064] Figure 23 This is a graph showing the trend of hyoscyamine content in tobacco shreds at different storage times, obtained using the one-test-multiple-evaluation method in Example 1, as shown in Application Example 4. Detailed Implementation

[0065] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0066] The reagents and instruments used in the following examples are as follows: Several samples of tobacco and tobacco products were provided by Zhejiang China Tobacco Industry Co., Ltd.

[0067] Neochlorogenic acid (≥98.0%), chlorogenic acid (≥95.0%), cryptochlorogenic acid (≥98.0%), hyoscyamine (≥99%) (standard, Sigma-Aldrich, USA); scopolamine (97%), caffeic acid (98.0%), rutin (98.0%), kaempferol-3-O-rutin (98.0%) (standard, Trc, Canada); methanol (chromatographic grade, Dima Technology Co., Ltd.); acetic acid (chromatographic grade, Dima Technology Co., Ltd.); Milli-Q ultrapure water (prepared by Millipore ultrapure water system).

[0068] Agilent 1260 high performance liquid chromatograph; Waters Symmetry C18 (4.6mm×250mm×5μm); DIKMA Luna 5u C18(2) 100A (4.6mm×250mm×5μm); Agilent ZORBAX Eclipse Plus C18 (4.6 mm×250mm×5μm); Agilent Microsorb-MV 100-5 C18 (4.6 mm×250mm×5μm); Agilent ZORBAX SB-Aq (4.6 mm×250mm×5μm); XP205 electronic balance (sensitivity 0.0001 g, Mettler Toledo, Switzerland); 4-16K centrifuge (Sigma, Germany).

[0069] Example 1 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multiple-evaluation method, including the following steps: (1) Preparation of standard solutions (1.1) Mixed standard stock solution Accurately weigh 100 mg of chlorogenic acid, 10 mg of caffeic acid, 10 mg of scopolamine and 100 mg of rutin into a 10 mL volumetric flask, accurate to 0.1 mg respectively, and dilute to volume with methanol to prepare mixed standard stock solution 1; store sealed and protected from light at 0-4℃.

[0070] Accurately weigh 10 mg of neochlorogenic acid, scopolamine, cryptochlorogenic acid, and kaempferol-3-O-rutin into a 10 mL volumetric flask, accurate to 0.1 mg, and dilute to volume with methanol to prepare mixed standard stock solution 2; store sealed and protected from light at 0~4℃.

[0071] (1.2) Mixed standard working solutions Accurately transfer 20, 50, 100, 200, 500, and 1000 μL of mixed standard stock solution 1 into different 10 mL volumetric flasks, and then add 12, 30, 60, 120, 300, and 600 μL of mixed standard stock solution 2, respectively. Dilute to the mark with methanol aqueous solution at a volume ratio of 1:1, and these are used as a series of mixed standard solutions 1 to 6.

[0072] (2) Sample processing Accurately weigh 10.0 mg of tobacco or tobacco product sample and place it in a 5 mL centrifuge tube. Accurately add 4 mL of a methanol, water, and n-hexane solution with a volume ratio of 1:1:2 and extract by sonication for 20 min. Collect the supernatant and filter it through a 0.45 μm aqueous filter membrane. Transfer the supernatant to a chromatographic bottle to obtain the test solution.

[0073] (3) High performance liquid chromatography detection High-performance liquid chromatography (HPLC) was used to detect the series of mixed standard solutions 1-6 and the test solution, under the following conditions: Instrument: Agilent 1260 high performance liquid chromatograph; column: Waters Symmetry C18; column temperature: 30℃; injection volume: 10 μL; Mobile phase A: water:methanol:acetic acid = 100:2:2 (volume ratio); Mobile phase B: methanol:acetic acid = 100:2 (volume ratio); Flow rate: 1.0 mL / min; The gradient elution program was as follows (based on a total volume of mobile phases A and B of 100%): 0-15 min, 10-30% B; 15-20 min, 30-90% B; 20-22 min, 90% B; 22-22.1 min, 90-10% B; 22.1-30 min, 10% B. Diode array segmented wavelength detection method: 0-9 min, 325 nm; 9-10.8 min, 340 nm; 10.8-17 min, 325 nm; 17-20 min, 345 nm; 20-30 min, 350 nm; reference wavelength 480 nm.

[0074] (4) Localization of chromatographic peaks of polyphenolic compounds Using chlorogenic acid as an internal reference, the chromatographic peaks were located based on the relative retention times of chlorogenic acid and neochlorogenic acid, scopolamine, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, and kaempferol-3-O-rutin in the high performance liquid chromatogram of the mixed standard solution.

[0075] (5) Calculation of relative correction factor Using the high-performance liquid chromatography (HPLC) chromatograms of a series of mixed standard solutions, with chlorogenic acid as an internal reference, the relative correction factor fs / i = as / ai was rapidly calculated based on the ratio of the slopes of the standard curves of chlorogenic acid to those of neochlorogenic acid, scopolamine, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, and kaempferol-3-O-rutin. In this formula, ai is the slope of the standard curve of the analyte, and as is the slope of the standard curve of the reference.

[0076] (6) Calculation of the content of the analyte Using relative correction factors, the contents of neochlorogenic acid, scopolamine, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, and kaempferol-3-O-rutin in the test solution were calculated. The concentration of the analyte was Ci = Ai / ai = (Ai·fs / i) / (as), where ai is the slope of the standard curve of the analyte, as is the slope of the standard curve of the reference, and Ai is the peak area of ​​the analyte.

[0077] Example 2 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multiple-evaluation method. The only difference between this method and Example 1 is that: In step (2), the extraction solvent is a 3 mL solution of methanol, water and n-hexane in a volume ratio of 1:0.8:1.6.

[0078] In step (3), the mobile phase A: water: methanol: acetic acid = 100:1:1 (volume ratio); the mobile phase B: methanol: acetic acid = 100:3 (volume ratio), and the column temperature is 35℃; The gradient elution program was as follows (based on a total volume of mobile phases A and B of 100%): 0-15 min, 8-28% B; 15-20 min, 28-88% B; 20-22 min, 88% B; 22-22.1 min, 88-8% B; 22.1-30 min, 8% B. Other examples are shown in Example 1.

[0079] Example 3 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multiple-evaluation method. The only difference between this method and Example 1 is that: In step (2), the extraction solvent is 5 mL of a methanol, water, and cyclohexane solution with a volume ratio of 1:1.2:2.4; In step (3), the mobile phase A: water: methanol: acetic acid = 100:3:3 (volume ratio); the mobile phase B: methanol: acetic acid = 100:1 (volume ratio); and the column temperature is 40℃. The gradient elution program was as follows (based on a total volume of mobile phases A and B of 100%): 0-15 min, 12-32% B; 15-20 min, 32-92% B; 20-22 min, 92% B; 22-22.1 min, 92-12% B; 22.1-30 min, 12% B. Other examples are shown in Example 1.

[0080] Example 4 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), a DIKMA Luna 5u C18(2) 100A chromatographic column is used, and the rest is the same as in Example 1.

[0081] Example 5 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), an Agilent ZORBAX Eclipse Plus C18 column is used, and the rest is the same as in Example 1.

[0082] Example 6 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), an Agilent Microsorb-MV 100-5 C18 column is used, and the rest is the same as in Example 1.

[0083] Example 7 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), an Agilent ZORBAX SB-Aq chromatographic column is used, and the rest is the same as in Example 1.

[0084] Example 8 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the mobile phase A is water:methanol = 100:2, the mobile phase B is methanol, and the other steps are the same as in Example 1.

[0085] Example 9 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the gradient elution program is as follows (based on the total volume of mobile phases A and B being 100%): 0-18 min, 10-30% B; 18-20 min, 30-90% B; 20-22 min, 90% B; 22-22.1 min, 90-10% B; 22.1-30 min, 10% B, and the other steps are the same as in Example 1.

[0086] Example 10 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the gradient elution program is as follows (based on the total volume of mobile phases A and B being 100%): 0-18 min, 10-30% B; 18-24 min, 30-90% B; 24-26 min, 90% B; 26-26.1 min, 90-10% B; 26.1-35 min, 10% B, and the other steps are the same as in Example 1.

[0087] Example 11 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a single test and multiple evaluation method. The only difference between this method and Example 1 is that in step (3), a single wavelength of 340 nm is used for detection, while the rest is the same as in Example 1.

[0088] Example 12 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The difference between this method and Example 1 is only that in step (5), the calculation method for the relative correction factor is replaced by a multi-point correction method, specifically: The relative correction factor obtained from multiple mass concentration points ( f s / i The average value is used for quantification, calculated according to the formula. f s / i : f s / i =A s × C i / ( A i × C s In the formula, : A s The peak area of ​​the internal reference material; C i and A i The mass concentration (mg / L) and peak area of ​​the analyte are given.C s The concentration of the internal control (mg / L) is the mass concentration of the internal control; other parameters are as described in Example 1.

[0089] Example 13 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The difference between this method and Example 1 is only that in step (5), the calculation method for the relative correction factor is replaced by a single-point correction method, specifically: The relative correction factor is calculated using the external standard method at concentration points, and then calculated according to the formula. f s / i : f s / i = k s / k i In the formula: k s The slope of a single point of the internal reference. k i The slope is the single-point slope of the component to be tested; other parameters are as described in Example 1.

[0090] Example 14 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the extraction solvent is an equal volume ratio of methanol, water and methyl tert-butyl ether solution of 1:1:2. Other steps are the same as in Example 1.

[0091] Example 15 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the extraction solvent is an equal volume ratio of methanol and aqueous solution of 1:1. Other steps are the same as in Example 1.

[0092] Example 16 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on a one-test-multiple-evaluation method. The only difference between this method and Example 1 is that in step (3), the amount of extraction solvent is adjusted to 2 mL, and the rest is the same as in Example 1.

[0093] Comparative Example 1 This embodiment provides a method for detecting polyphenolic compounds in tobacco or its products based on the external standard method. The only difference between this method and Example 1 is that the one-to-many evaluation method is replaced by the external standard method. Specifically, a series of mixed standard solutions 1 to 6 are taken and analyzed by HPLC. The linear relationship between the peak area and concentration of polyphenolic compounds in each standard working solution is calculated to obtain the regression equation. The concentration of the target compound in the sample is obtained by substituting the peak area obtained from the sample into the regression equation. Other steps are the same as in Example 1.

[0094] Test Example 1 (1) Resolution detection The mixed standard solution 3 was detected using the liquid chromatography method provided in Examples 1-10, and the chromatograms are shown below. Figures 1-10 As shown.

[0095] Figures 1-3 The results show that the chromatographic detection conditions provided in Examples 1-3 have good separation effects on all eight polyphenolic compounds.

[0096] Figure 1 , Figures 4-7 The comparison showed that Waters Symmetry C18 provided in Example 1, DIKMA Luna 5u C18(2) 100A provided in Example 4, and Agilent ZORBAX Eclipse Plus C18 provided in Example 5 could all achieve baseline separation of 8 polyphenol compounds, while Agilent Microsorb-MV 100-5 C18 provided in Example 6 and Agilent ZORBAX SB-Aq provided in Example 7 had poor separation effects.

[0097] Figure 1 , Figure 8 The comparison shows that the separation effect is poor when the methanol-water system of Example 8 is used as the mobile phase because the phenolic hydroxyl groups in polyphenolic compounds are easily oxidized and ionized.

[0098] Figure 1 , Figures 9-10 The comparison shows that the mobile phase elution method provided in Example 1 can quickly complete the analysis of the sample within 30 minutes, improving the detection efficiency; while the mobile phase elution method provided in Example 9 has poor separation effect, and the mobile phase elution method provided in Example 10 has a longer analysis time.

[0099] (2) Response intensity detection The segmented detection time in Example 1 is shown in Table 1. The response results of each target object in Example 1 (segmented detection) and Example 11 (single wavelength detection) are shown in Table 2.

[0100] Table 1 Table 2 Table 2 shows that, by employing a segmented detection method at the maximum absorption wavelength of different target compounds, the response intensity of all polyphenolic compounds except scopolamine and hyoscyamine was significantly enhanced compared to single-wavelength detection. Therefore, the segmented detection method chosen in this invention can improve the detection sensitivity of the method.

[0101] Test Example 2 Methodological Validation (1) Linearity, limit of detection and limit of quantitation The mixed standard working solutions were analyzed using the method provided in Example 1. The linear relationship between the peak area and concentration of polyphenolic compounds in each standard working solution was calculated, and the regression equation and its correlation coefficient were obtained. The mixed standard working solution with the lowest concentration was measured 10 times consecutively. The limit of detection (LOD) was set at three times the standard deviation of the results, and the limit of quantitation (LOQ) was set at ten times the standard deviation of the results. The results are shown in Table 3.

[0102] Table 3 As shown in the table, the eight polyphenolic compounds exhibited good linearity within their respective linear ranges (R0 of the regression equation). 2 All values ​​were greater than 0.999. The limits of detection for the eight polyphenols were between 0.01 and 0.15 mg / L, and the limits of quantitation were between 0.02 and 0.48 mg / L, which met the quantitative analysis requirements of their respective target analytes.

[0103] (2) Precision, repeatability and stability Take 3 mixed standard solutions and perform 6 consecutive injections using the method provided in Example 1 to conduct a precision test; perform 6 parallel determinations of the same sample according to the pretreatment method to conduct a repeatability test; take the same pretreated tobacco sample and place it at room temperature for 0, 4, 8, 12, 16 and 24 h for injection and determination to conduct a stability test. The results are shown in Table 4.

[0104] Table 4 As shown in the table, the precision, repeatability, and stability RSD of the eight polyphenolic compounds are less than 2.28%, indicating that the method has good precision, reproducibility, and stability.

[0105] (3) Spike recovery rate and relative standard deviation In tobacco samples with known contents of 8 polyphenolic compounds, three different concentration levels (high, medium, and low) were added to conduct spiked recovery experiments. At each spiking level, the method of Example 1 was used to determine the recovery rate 6 times in parallel. The recovery rate of each polyphenolic compound in the sample was calculated, and the precision (RSD) of the method was examined. The results are shown in Table 5.

[0106] Table 5 As shown in the table, the average recoveries of the eight polyphenolic compounds at different concentration levels ranged from 92.00% to 102.38%, with relative standard deviations ranging from 1.56% to 4.10%. This indicates that the method has good accuracy and repeatability, and is suitable for the determination of eight polyphenolic compounds in tobacco and tobacco products.

[0107] Test Example 3 Verification of the one-test-multiple-evaluation method (1) Determination of the relative correction factor (RCF) The slope correction method provided in Example 1, the multi-point correction method provided in Example 12, and the single-point correction method provided in Example 13 were used to detect a series of mixed standard solutions. The relative correction factors obtained are shown in Table 6.

[0108] Table 6 The table shows that the RSD of the RCF calculated by the three methods is less than 4.02%.

[0109] (2) Examination of RCF durability To investigate the durability of RCF under different chromatographic conditions, the chromatographic conditions were appropriately varied within a certain range. This invention investigated the effects of changes in flow rate and column temperature on three methods for calculating correction factors.

[0110] The effects of different flow rates and column temperatures on RCF in Example 1 (slope method) are shown in Tables 7 and 8, respectively, and the remaining parameters are the same as in Example 1.

[0111] Table 7 Table 8 The effects of different flow rates and column temperatures on RCF in Example 12 (multi-point correction method) are shown in Tables 9 and 10, respectively, and the other parameters are the same as in Example 12.

[0112] Table 9 Table 10 The effects of different flow rates and column temperatures on RCF in Example 13 (single-point correction method) are shown in Tables 11 and 12, respectively, and the other parameters are the same as in Example 13.

[0113] Table 11 Table 12 The results showed that the slope method had a smaller RSD (less than 2.18%) than the multi-point correction method and the single-point method in terms of the effect of different flow rates on RCF. Similarly, the slope method had a smaller RSD (less than 2.41%) than the multi-point correction method and the single-point method in terms of the effect of different column temperatures on RCF. This indicates that changes in chromatographic conditions have a relatively small impact on the RCF of each analyte and demonstrate good robustness.

[0114] (3) RCF reproducibility test The effects of different instruments and columns from different manufacturers on RCF were investigated. The effects of different instruments and columns from different manufacturers in Example 1 (slope method) are shown in Table 13. The remaining parameters are the same as in Example 1.

[0115] Table 13 The effects of different instruments and chromatographic columns from different manufacturers in Example 12 (multi-point calibration method) are shown in Table 14. The remaining parameters are the same as in Example 12.

[0116] Table 14 The effects of different instruments and columns from different manufacturers in Example 13 (single-point calibration method) are shown in Table 15. The remaining parameters are the same as in Example 13.

[0117] Table 15 The results show that the RSD of RCF calculated by the slope correction method is generally smaller than that calculated by the multi-point correction method and the single-point method, with an RSD of less than 2.96%, indicating that RCF has good reproducibility under different instruments and different chromatographic columns.

[0118] (4) Chromatographic peak localization Using chlorogenic acid as an internal reference, the effects of different instruments and chromatographic columns on the relative retention time (min) of each analyte were investigated. Other parameters were as described in Example 1. The results are shown in Table 16.

[0119] Table 16 The results showed that the RSD of the relative retention times of the seven analytes and the internal reference was less than 5.26%, indicating that the relative retention times of each component fluctuated little when using different chromatographic columns and systems, and could be used for peak localization.

[0120] Test Example 4 Sample content determination (1) Comparison of results between the one-test-multiple-evaluation method and the external standard method Thirty tobacco and tobacco product samples were taken (yp1~yp10 were reconstituted tobacco samples, yp11~yp20 were commercially available cigarette samples, and yp21~yp30 were first-cured tobacco samples). The components other than chlorogenic acid were calculated according to Example 1 (single-test multiple evaluation slope correction method), Example 12 (single-test multiple evaluation multi-point correction method), Example 13 (single-test multiple evaluation single-point correction method), and Comparative Example 1 (external standard method). The results obtained by the single-test multiple evaluation method and the external standard method were compared with the relative deviation as a parameter. The content of each component (mg / g) is shown in Tables 17-19, and the relative deviation (%) of each component is shown in Tables 20-21.

[0121] Table 17 Table 18 Table 19 Table 20 Table 21 The results showed that, except for scopolamine (6.47%), the average relative error of the multi-point correction method and the external standard method was less than 5% for all other components. The average relative error of the slope correction method and the external standard method was less than 3.21%. The average relative error of the single-point correction method and the external standard method was larger; for example, the average relative error of scopolamine reached 8.11%. These results indicate that both the established multi-evaluation method and the external standard method can be used to determine the content of phenolic compounds in tobacco and tobacco products. Further comparison of the three correction factor calculation methods showed that, except for cryptochlorogenic acid, the average relative error of the slope correction method for other components was significantly lower than that of the other two correction methods. This indicates that the slope correction method and the external standard method yielded more consistent results in the determination of polyphenolic compound content in tobacco and tobacco products.

[0122] (2) Comparison of detection limit and quantitation limit results for segmented and unsegmented detection. The detection limit and quantitation limit results were obtained by using the segmented wavelength detection method of Example 1 and the non-segmented wavelength detection method of Example 11, respectively, as shown in Table 22.

[0123] Table 22 The results show that the segmented determination method established in this invention has lower limits of detection and quantitation. This indicates that the method has higher sensitivity.

[0124] The reconstituted tobacco samples yp1~yp10 were tested using the method provided in Example 1, and the results are shown in Table 23.

[0125] Table 23 The polyphenol content in reconstituted tobacco leaves is significantly lower than that in virgin flue-cured tobacco leaves and commercially available cigarette tobacco. As shown in the table, the caffeic acid content in some samples (YP3, YP8, and YP9) is below the limit of quantitation for the non-segmented method. This indicates that the traditional non-segmented method is not suitable for the determination of some reconstituted tobacco leaf samples, while the segmented method can meet the requirements for these samples. Therefore, the segmented method is more suitable for the detection of polyphenols in reconstituted tobacco leaf samples, especially for the determination of low-content polyphenols. (4) Comparison of extraction effects The same tobacco sample was pretreated using the extraction systems of Examples 1-3 and Examples 14-16, respectively. The results (mg / g) of the determination of eight polyphenolic compounds are shown in Table 24.

[0126] Table 24 The results showed that the methanol-water-n-hexane and methanol-water-cyclohexane systems had better extraction and purification effects. The methanol-water-methyl-tert-butyl ether system resulted in the loss of polyphenols such as caffeic acid, rutin, hyoscyamine, and kaempferol-3-O-rutin. The methanol-water system could not achieve effective extraction of the target substances within the same volume, resulting in lower concentrations. The extraction efficiency decreased when the extraction system size was too small.

[0127] Application Example 1 The contents of eight polyphenolic compounds in different types (reconstituted tobacco leaves, finished cigarettes, and raw tobacco) were determined by the one-test-multiple-evaluation method (Example 1) and the external standard method (Comparative Example 1), respectively. The different types of samples were classified based on the PCA statistical method.

[0128] Example 1: Results of the one-test-multiple-evaluation method are as follows Figure 11 As shown, the results of the external standard method in Comparative Example 1 are as follows: Figure 12 As shown in the figure, the results indicate that both methods can classify different types of samples, meaning that the one-test-multiple-evaluation technique has promising applications in the quality control and evaluation of batch samples.

[0129] Application Example 2 The content of eight polyphenolic compounds in tobacco leaf samples from different origins was determined using the method described in Example 1. The samples from different origins were then classified using PCA statistical methods. Sample information is shown in Table 25, and the results are as follows: Figure 13 The results show that the eight polyphenolic compounds identified using the one-test-multiple-evaluation technique can be used for tobacco origin identification.

[0130] Table 25 Application Example 3 The content of eight polyphenolic compounds in tobacco leaf samples of different grades from Yunnan was determined using the method described in Example 1. The samples were then classified according to different grades using PCA statistical methods. Sample information is shown in Table 26, and the results are as follows: Figure 14 The results show that the eight polyphenolic compounds determined using the one-test-multiple-evaluation technique can be used for tobacco leaf grading.

[0131] Table 26 Application Example 4 The method described in Example 1 was used to evaluate tobacco shreds stored for different times and tobacco leaves stored for different times. The PLS-DA analysis results of polyphenol content in tobacco shreds stored for different times are as follows: Figure 15 As shown in the figure, the trend of polyphenol content in tobacco shreds at different storage times is as follows: Figures 16-23 As shown (A, B, and C represent different brands of cigarette samples), the results show that the method provided by this invention can be used to evaluate tobacco shreds with different storage times and tobacco leaves with different storage times.

[0132] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting polyphenolic compounds in tobacco or its products based on a single-test, multiple-evaluation approach, characterized in that, The method includes: Tobacco or its products are mixed with a solvent and extracted to obtain a test solution; A standard substance of polyphenolic compound and solvent 2 were mixed to obtain several standard solutions of different concentrations; The test solution and standard solution were detected by liquid chromatography, with chlorogenic acid as an internal reference, and the content of polyphenolic compounds in tobacco or its products was obtained based on the one-test-multiple-evaluation method.

2. The method according to claim 1, characterized in that, The polyphenolic compounds include any one or a combination of at least two of the following: chlorogenic acid, neochlorogenic acid, scopolamine, cryptochlorogenic acid, caffeic acid, hyoscyamine, rutin, or kaempferol-3-O-rutin.

3. The method according to claim 1 or 2, characterized in that, The solvent includes any one or a combination of at least two of methanol, water, methyl tert-butyl ether, n-hexane or cyclohexane; Preferably, the first solvent is a combination of methanol, water and n-hexane, or the first solvent is a combination of methanol, water and cyclohexane; Preferably, the volume ratio of methanol, water, and n-hexane is 1:(0.8-1.2):(1.6-2.4). Preferably, the volume ratio of methanol, water, and cyclohexane is 1:(0.8-1.2):(1.6-2.4). Preferably, the ratio of the tobacco or its product to solvent one is 1 mg: (0.3-0.5) mL; Preferably, the second solvent includes methanol and / or water.

4. The method according to any one of claims 1-3, characterized in that, The mobile phase in the liquid chromatography method includes mobile phase A and mobile phase B. Mobile phase A includes a combination of water, methanol and acetic acid, and mobile phase B includes a combination of methanol and acetic acid.

5. The method according to claim 4, characterized in that, The volume ratio of water, methanol, and acetic acid in the mobile phase A is 100:(1-3):(1-3). Preferably, the volume ratio of methanol to acetic acid in the mobile phase B is 100:(1-3).

6. The method according to claim 4 or 5, characterized in that, The elution program of the mobile phase in the liquid chromatography method is gradient elution, specifically as follows: From 0 to 15 min, the volume percentage of mobile phase A changed uniformly from 88-92% to 68-72%, and the volume percentage of mobile phase B changed uniformly from 8-12% to 28-32%. From 15 to 20 min, the volume percentage of mobile phase A changed uniformly from 68-72% to 8-12%, and the volume percentage of mobile phase B changed uniformly from 28-32% to 88-92%, then remained constant until 22 min. From 22 to 22.1 min, the volume percentage of mobile phase A changed uniformly from 8-12% to 88-92%, and the volume percentage of mobile phase B changed uniformly from 88-92% to 8-12%, then remained constant until 30 min.

7. The method according to any one of claims 1-6, characterized in that, The chromatographic column used in the liquid chromatography method is packed with C18 material, with a particle size of 3-5 μm, an inner diameter of 3.0-4.6 mm, and a length of 100-250 mm. Preferably, the column temperature of the chromatographic column in the liquid chromatography method is 28-40℃.

8. The method according to any one of claims 1-7, characterized in that, The wavelength detection in the liquid chromatography method is segmented detection, specifically: The detection wavelength was 325 nm from 0 to 9 min; 340 nm from 9 to 10.8 min; 325 nm from 10.8 to 17 min; 345 nm from 17 to 20 min; and 350 nm from 20 to 30 min.

9. The method according to any one of claims 1-8, characterized in that, In the aforementioned one-test-multiple-evaluation method, chlorogenic acid is used as an internal reference, and the relative correction factor between chlorogenic acid and other polyphenolic compounds to be tested is calculated. The content of polyphenolic compounds in tobacco or its products is obtained using the relative correction factor. Preferably, the relative correction factor is calculated using the slope correction method.

10. The application of the method for detecting polyphenolic compounds in tobacco or its products based on a single-test-multiple-evaluation method according to any one of claims 1-9 in the quality evaluation and classification of tobacco or its products.

Images