Quantitative detection method for tobacco components

Abstract

The invention discloses a quantitative detection method for tobacco components. The quantitative detection method comprises the following steps: extracting a tobacco sample to obtain extract liquor; performing component analysis on the extracting solution by adopting high-resolution liquid chromatography-mass spectrometry to obtain a spectrogram of the tobacco sample; the high-resolution liquid chromatography mass spectrometry adopts two ion sources of an ESI source and an APCI source for detection, and the two ion sources both adopt reversed-phase chromatographic columns for detection; in detection, under the ESI source condition, positive and negative ion modes are adopted for scanning respectively, and under the APCI source condition, a positive ion mode is adopted for scanning. According to the quantitative detection method, two different ion sources are adopted, and high coverage and detection precision are both considered; corresponding chromatographic columns are adopted for liquid phase separation, separation of tobacco metabolism components with different properties in a complex natural tobacco matrix is facilitated, and the separation efficiency and the coverage degree are improved; by combining the ultrahigh resolution and high precision of the high-resolution liquid chromatography-mass spectrometry, the to-be-detected substance and the interfering substance are effectively distinguished, and the detection accuracy of various samples is improved.

Description

Technical Field

[0001] This application relates to the field of tobacco component detection technology, and in particular to a quantitative detection method for tobacco components. Background Technology

[0002] The chemical composition of tobacco is closely related to its sensory quality, originating directly or indirectly from metabolic substances accumulated in tobacco, such as plastid pigments, polyphenols, and free amino acids. These components are closely linked to the appearance, smoking quality, and aroma component content of tobacco. Establishing a correlation chain between tobacco chemical composition analysis and tobacco quality, and further understanding the impact of chemical characteristic components on tobacco sensory quality, can provide technical means for tobacco sensory quality control and a technical basis for tobacco sensory quality management. Therefore, it is necessary to screen tobacco components related to tobacco sensory quality and perform accurate, rapid, and high-throughput analysis and detection.

[0003] Research conducted by the tobacco industry in the field of tobacco component analysis and testing shows that the components that can comprehensively reflect the style characteristics, quality properties, and aging process of flue-cured tobacco are mainly concentrated in sugars, alkaloids, total nitrogen, polyphenolic compounds, polybasic acids and higher fatty acids, Amadori compounds, solanesol, neophytadiene, aroma components, pigments, and volatile acids. Among these, the analysis of water-soluble and alcohol-soluble non-volatile components reflects the correlation between metabolomics and sensory quality, and has received increasing attention in recent years.

[0004] The aforementioned water-soluble and alcohol-soluble non-volatile components mainly include polyphenolic compounds, polybasic acids and higher fatty acids, Amadori compounds, solanesol, and pigments, totaling 56 substances. Of these, 26 substances currently lack standard detection methods, while the remaining 30 substances involve 5 standard detection methods and 3 different instruments. In summary, current industry standard methods for different tobacco components typically only target the detection of one type of substance. However, characterizing tobacco quality usually requires analyzing and detecting multiple types of substances, conducting multiple rounds of experiments, resulting in a significant waste of human, time, and economic resources. Summary of the Invention

[0005] This invention provides a quantitative detection method for tobacco components, thereby solving the technical problem that existing technologies cannot simultaneously detect multiple substances in tobacco components.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows: This invention provides a method for quantitative detection of tobacco components, comprising the following steps: S1. Extract the tobacco sample to obtain the extract; S2. The extract obtained in step S1 is subjected to high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) for component analysis to obtain the spectrum of the tobacco sample. The HPLC-MS / MS uses two ion sources, ESI and APCI, and both ion sources are detected using a reversed-phase column. During the detection, positive and negative ion modes are used under the ESI source condition, and positive ion mode is used under the APCI source condition.

[0007] Furthermore, the reversed-phase chromatographic column is a C18 chromatographic column.

[0008] Further, in step S2, the mobile phase for positive ion mode scanning under ESI source conditions includes mobile phase A1 and mobile phase B1 for detecting Amadori compounds and amino acids. Mobile phase A1 includes an aqueous solution of formic acid containing phthalic acid, wherein the concentration of phthalic acid is 0.1 mM to 0.2 mM and the volume content of formic acid is 0.02% to 0.2%. Mobile phase B1 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of the mobile phase B1 is 10%; From 1.0 to 19.0 min, the volume of the mobile phase B1 increased from 10% to 90%. During the 19.0–19.1 min interval, the volume of the mobile phase B1 decreased from 90% to 10%. The volume of the mobile phase B1 is 10% during the 19.1–21.0 min period.

[0009] Further, in step S2, the mobile phase for negative ion mode scanning under ESI source conditions includes mobile phase A2 and mobile phase B2 for detecting polyphenols and polyacids. Mobile phase A2 includes an aqueous solution of acetic acid, with a volume content of 0.01% to 0.1%. Mobile phase B2 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of the mobile phase B2 is 10%; From 1.0 to 19.0 min, the volume of the mobile phase B2 increased from 10% to 90%. During the 19.0–19.1 min interval, the volume of the mobile phase B2 decreased from 90% to 10%. The volume of the mobile phase B2 is 10% during the 19.1–21.0 min period.

[0010] Further, in step S2, the mobile phase for positive ion mode scanning under APCI source conditions includes mobile phase A2 and mobile phase B2 for detecting solanesol, lutein, and β-carotene. Mobile phase A2 includes an aqueous solution of acetic acid with a volume content of 0.05% to 0.1%. Mobile phase B2 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 0.5 min, the volume of the mobile phase B2 is 5%; Over a period of 0.5–18.0 min, the volume of the mobile phase B2 increased from 10% to 95%. During the 18.0–18.1 min interval, the volume of the mobile phase B2 decreased from 90% to 5%. The volume of the mobile phase B2 is 5% during the 18.1–21.0 min period.

[0011] Furthermore, the high-resolution liquid chromatography-mass spectrometry (HPLC-MS) using an ESI source for component analysis is as follows: scanning in positive and negative ion modes respectively; sheath gas flow rate: 50 Arb; auxiliary gas flow rate: 10 Arb; purge gas flow rate: 1 Arb; ion transfer tube temperature: 350℃; evaporation chamber temperature: 320℃; scanning mode: PRM mode; primary scan parameters: scan range: 70–800 m / z; orbitrap resolution: 60,000; RF lens: 70%; spray voltage: 2800 V (-) and 5000 V (+); intensity threshold: 1.0 e 5 Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

[0012] Furthermore, the mass spectrometry conditions for component analysis using the APCI source in the high-resolution liquid chromatography-mass spectrometry are as follows: positive ion mode scanning; sheath gas flow rate: 45 Arb; auxiliary gas flow rate: 5 Arb; purge gas flow rate: 1 Arb; ion transfer tube temperature: 300℃; evaporation chamber temperature: 350℃; scanning mode: PRM mode; primary scan parameters: scan range: 70–800 m / z; orbitrap resolution: 60,000; RF lens: 70%; positive ion exchange current: 4 μA; intensity threshold: 1.0 e 5 Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

[0013] Furthermore, the solvent used in the extraction step S1 is an aqueous solution containing 10% to 50% methanol, used to detect Amadori compounds and amino acids.

[0014] Furthermore, the solvent used in the extraction step S1 is an aqueous solution containing 30% to 80% methanol, which is used to detect polyphenols and polybasic acids.

[0015] Further, in step S1, the extraction is selected from one of the following: ultrasonic extraction for 10-20 min, oscillation extraction for 20-25 min, oscillation extraction for 5-10 min followed by ultrasonic extraction for 5-10 min, and ultrasonic extraction for 10-15 min followed by oscillation extraction for 10-15 min.

[0016] Further, the tobacco components obtained by positive ion mode scanning under the ESI source conditions in step S2 include: 1-deoxy-1-L-aminobutyric acid-D-fructose, 1-deoxy-1-L-histidine-D-fructose, 1-deoxy-1-L-proline-D-fructose, 1-deoxy-1-L-valine-D-fructose, 1-deoxy-1-L-threonine-D-fructose, 1-deoxy-1-L-alanine-D-fructose, 1-deoxy-1-L-asparagine-D-fructose, 1-deoxy-1-L-aspartic acid-D-fructose, and 1-deoxy-1-L-glutamine-D-fructose. 1-Deoxy-1-L-glutamic acid-D-fructose, 1-Deoxy-1-L-isoleucine-D-fructose, 1-Deoxy-1-L-leucine-D-fructose, 1-Deoxy-1-L-tyrosine-D-fructose, 1-Deoxy-1-L-phenylalanine-D-fructose, 1-Deoxy-1-L-tryptophan-D-fructose, proline, aspartic acid, threonine, serine, asparagine, glutamic acid, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, 4-aminobutyric acid, lysine, histidine, tryptophan, arginine, glucosamine.

[0017] Furthermore, the tobacco components obtained by negative ion mode scanning under the ESI source conditions in step S2 include: chlorogenic acid, rutin, scopolamine, neochlorogenic acid, cryptochlorogenic acid, malic acid, citric acid, vanillic acid, tetradecanoic acid, hexadecanoic acid, linoleic acid, oleic acid, linolenic acid, stearic acid, eicosanoic acid, lactic acid, and fructose acid.

[0018] Furthermore, the tobacco components obtained by positive ion mode scanning under the APCI source conditions in step S2 include: solanesol, lutein, and β-carotene.

[0019] The quantitative detection method for tobacco components provided by this invention employs two different ion sources, balancing high coverage and detection accuracy, making it suitable for the general and accurate analysis of various types of samples. The use of corresponding chromatographic columns for liquid phase separation facilitates the separation of tobacco metabolic components with different properties from complex natural tobacco matrices, improving separation efficiency and coverage, and making it suitable for the general analysis of various types of samples. Combined with the ultra-high resolution and high precision of high-resolution liquid chromatography-mass spectrometry, the separated components are precisely analyzed, effectively distinguishing analytes from interfering substances and improving the detection accuracy for various types of samples. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figures 1 to 8 This is a chromatogram of extracted ions of 53 components obtained by scanning the positive and negative ion modes of an electrospray ionization source (ESI) in an embodiment of the present invention. Figure 9 This is a chromatogram of extracted ions of three components obtained by positive ion mode scanning using an atmospheric pressure chemical ionization source (APCI) in an embodiment of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0023] It should be noted that the structures, proportions, sizes, etc., shown in the accompanying drawings of this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed in the specification, and are not intended to limit the conditions under which this application can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size should still fall within the scope of the technical content disclosed in this application, provided that they do not affect the effects and purposes that this application can produce.

[0024] Metabolic component analysis methods based on liquid chromatography support the simultaneous detection of multiple substances and are suitable for the detection of non-volatile and semi-volatile components in tobacco. However, as a natural product, tobacco has a complex composition, and incomplete separation of multiple components leads to problems such as significant component interference, difficulty in quantification, and the need to improve detection stability in mass spectrometry quantitative detection, making it difficult to achieve simultaneous quantitative analysis of multiple components. In addition, the complex and diverse metabolic components of natural products and the diversity of processing techniques also result in significant differences in the physical and chemical properties of tobacco, which further poses great challenges to tobacco sample pretreatment and analysis.

[0025] This application provides a method for quantitative detection of tobacco components, comprising the following steps: S1. Extract the tobacco sample to obtain the extract; S2. The extract obtained in step S1 was subjected to high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) for component analysis to obtain the spectrum of the tobacco sample. The HPLC-MS / MS was performed using two ion sources: ESI and APCI. Both ion sources were detected using a reversed-phase column. During the detection, positive and negative ion modes were used for scanning under ESI source conditions, and positive ion mode was used for scanning under APCI source conditions.

[0026] The quantitative detection method for tobacco components provided in this application employs two different ion sources, balancing high coverage and detection accuracy, making it suitable for the general and accurate analysis of various types of samples. The use of corresponding chromatographic columns for liquid phase separation facilitates the separation of tobacco metabolic components with different properties within the complex natural tobacco matrix, improving separation efficiency and coverage, and making it suitable for the general analysis of various types of samples. Combined with the ultra-high resolution and high precision of high-resolution liquid chromatography-mass spectrometry, the separated components are precisely analyzed, effectively distinguishing analytes from interfering substances and improving the detection accuracy for various types of samples. Furthermore, the reversed-phase chromatographic column is a C18 column. The column temperature is 20℃~60℃; the flow rate is 0.2~0.6mL / min; and the injection volume is 1μL~10μL.

[0027] In this embodiment, the spectrum of the tobacco sample obtained in step S2 is compared with the standard spectrum of each component in the tobacco to quantify each component in the tobacco sample. The aforementioned standard spectrum consists of the spectra of water-soluble and alcohol-soluble non-volatile components in tobacco, mainly including polyphenolic compounds, polybasic acids and higher fatty acids, Amadori compounds, solanesol, and pigments, totaling 56 substances. Standard spectra are measured for each of these substances using solutions of different concentrations.

[0028] In some embodiments, the mobile phase used for positive ion mode scanning under ESI source conditions in step S2 includes mobile phase A1 and mobile phase B1 for detecting Amadori compounds and amino acids. Mobile phase A1 includes an aqueous solution of formic acid containing phthalic acid, with a phthalic acid concentration of 0.1 mM to 0.2 mM and a formic acid volume content of 0.05% to 0.2%. Mobile phase B1 includes acetonitrile. The gradient elution method is as follows: based on the total volume of the mobile phase as 100%, the volume of mobile phase B1 is 10% from 0 to 1.0 min; from 1.0 to 19.0 min, the volume of mobile phase B1 increases from 10% to 90%; from 19.0 to 19.1 min, the volume of mobile phase B1 decreases from 90% to 10%; and from 19.1 to 21.0 min, the volume of mobile phase B1 is 10%.

[0029] This application compares the chromatographic separation effects of adding phthalic acid to an aqueous mobile phase containing formic acid and not adding phthalic acid. Experimental results show that phthalic acid can significantly improve the retention behavior and peak shape of amino acids and Amadori compounds in a reverse-phase chromatographic column (referring to the C18 column used in this study). Without phthalic acid, most amino acids and Amadori compounds were not retained in the C18 column, and the few retained amino acids and Amadori compounds exhibited poor peak shape and severe tailing. After adding trace amounts of phthalic acid, the retention time of amino acids and Amadori compounds in the reverse-phase chromatographic column was significantly prolonged, the peak shape was significantly improved, the half-maximum width decreased, the column efficiency increased, and the detection sensitivity improved.

[0030] The reason for this is likely that phthalic acid may act as an ion-pairing reagent and shielding agent, improving the retention of amino acids and Amadori compounds on the C18 column. (1) Positively charged amino acid ions and Amadori compounds may form ion pairs with negatively charged phthalate anions. The hydrophobicity of the ion pairs formed is stronger than that of amino acid cations, making them easier to be retained by the C18 stationary phase, resulting in a significant delay in retention time. (2) Positively charged amino acids and Amadori compounds may react with free silanol groups on the surface of the C18 packing material to generate electrostatic attraction, resulting in peak tailing. Phthalic acid may preferentially adsorb onto the active sites on the surface of the stationary phase, shielding the non-specific interaction of silanol groups with amino acids, thereby improving the peak shape of amino acids.

[0031] The effect of phthalic acid on the retention enhancement of Amadori compounds in reverse chromatographic columns is a first discovery in this study.

[0032] In the embodiments of this application, the gradient elution method was used to investigate the difference in separation performance of the C18 reversed-phase column under multi-segment gradient elution and single-segment gradient elution conditions.

[0033] Multi-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B1 was 5% from 0 to 0.5 min; from 0.5 to 15.0 min, the volume of mobile phase B1 increased from 10% to 35%; from 15 to 17 min, the volume of mobile phase B1 increased from 35% to 95%; from 17 to 19 min, the volume of mobile phase B1 remained at 95%; from 19.0 to 19.1 min, the volume of mobile phase B1 decreased to 10%; and at 21 min, the volume of mobile phase B1 was 10%.

[0034] Single-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B1 is 10% from 0 to 1.0 min; from 1.0 to 19.0 min, the volume of mobile phase B1 increases from 10% to 90%; from 19.0 to 19.1 min, the volume of mobile phase B1 decreases from 90% to 10%; and from 19.1 to 21.0 min, the volume of mobile phase B1 is 10%.

[0035] Experimental results show that for the C18 reversed-phase chromatography system, single-segment gradient elution significantly improves the separation efficiency of amino acids and Amadori compounds compared to multi-segment gradient elution. This may be because multi-segment gradients can cause baseline fluctuations at slope change points and increase the risk of peak broadening due to repeated changes in elution intensity. Single-segment gradients provide a more stable separation environment, which helps to obtain sharp peak shapes; moreover, the parameters that need to be optimized for single-segment gradients (initial / final ratio, time) are far fewer than those for multi-segment gradients, making the method simpler, more robust, and more reproducible. Therefore, for the complex and diverse metabolic components containing various structures and polarities in this study, the single and continuous gradient change of single-segment gradient elution avoids the "step" perturbations of multi-segment gradients, thus achieving more efficient and stable separation.

[0036] In other embodiments, the mobile phase for negative ion mode scanning under ESI source conditions in step S2 includes mobile phase A2 and mobile phase B2 for detecting polyphenols and polybasic acids. Mobile phase A2 includes an aqueous solution of acetic acid, with a volume content of 0.05% to 0.1%; mobile phase B2 includes acetonitrile; the gradient elution method is as follows: based on the total volume of the mobile phase as 100%, the volume of mobile phase B2 is 10% from 0 to 1.0 min; from 1.0 to 19.0 min, the volume of mobile phase B2 increases from 10% to 90%; from 19.0 to 19.1 min, the volume of mobile phase B2 decreases from 90% to 10%; from 19.1 to 21.0 min, the volume of mobile phase B2 is 10%.

[0037] This application compares the chromatographic separation effects of adding different volume ratios of formic acid / acetic acid as the mobile phase. Experimental results with different acid addition volumes (0%, 0.05%, 0.1%, 0.5%, 1%, and 2% formic acid / acetic acid) show that as the acid addition increases from 0% to 0.1%, the retention effect of polyphenols and polybasic acids continuously improves, and the peak shape becomes sharper; from 0.1% to 0.5%, the retention time and peak shape remain essentially unchanged; from 0.5% to 2%, the retention effect decreases. Furthermore, the addition of acetic acid results in better retention of polyphenols and polybasic acids. For example, neochlorogenic acid exhibits a symmetrical and sharp peak shape when using 0.1% acetic acid aqueous solution as the mobile phase. At other volume ratios and with the addition of formic acid, the peak shape of neochlorogenic acid broadens and tails significantly, leading to insufficient separation of chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid isomers. Vanillic acid, myristic acid, and linolenic acid show the best retention effect when using 0.1% acetic acid aqueous solution as the mobile phase, but are not retained when using formic acid aqueous solution as the mobile phase. Therefore, a 0.1% aqueous acetic acid solution is the optimal mobile phase.

[0038] The reasons for this are as follows: When the acid concentration increases from 0% to 0.1%, the ionization of acidic functional groups in polyphenols and polybasic acids is suppressed, and the hydrophobic interaction between the compounds and the C18 stationary phase is enhanced, resulting in improved retention. When the acid concentration increases from 0.1% to 0.5%, the ionization of polyphenols and polybasic acids is completely suppressed, and the retention time and peak shape plateau. When the concentration increases from 0.5% to 2%, the high concentration of acid significantly enhances the ionic strength of the mobile phase. The ions in the mobile phase surround the analyte and stationary phase, compressing the electric double layer and weakening the hydrophobic interaction between the analyte and stationary phase, leading to a shorter retention time. Compared to formic acid, acetic acid has a lower ionic strength, which is more conducive to the interaction between the analyte and stationary phase in this study. Therefore, a 0.1% aqueous acetic acid solution was chosen as the optimal mobile phase.

[0039] In the embodiments of this application, the gradient elution method was used to investigate the difference in separation performance of the C18 reversed-phase column under multi-segment gradient elution and single-segment gradient elution conditions.

[0040] Multi-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B1 was 5% from 0 to 0.5 min; from 0.5 to 15.0 min, the volume of mobile phase B1 increased from 10% to 35%; from 15 to 17 min, the volume of mobile phase B1 increased from 35% to 95%; from 17 to 19 min, the volume of mobile phase B1 remained at 95%; from 19.0 to 19.1 min, the volume of mobile phase B1 decreased to 10%; and at 21 min, the volume of mobile phase B1 was 10%.

[0041] Single-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B1 is 10% from 0 to 1.0 min; from 1.0 to 19.0 min, the volume of mobile phase B1 increases from 10% to 90%; from 19.0 to 19.1 min, the volume of mobile phase B1 decreases from 90% to 10%; and from 19.1 to 21.0 min, the volume of mobile phase B1 is 10%.

[0042] Experimental results show that for the C18 reversed-phase chromatography system, single-segment gradient elution significantly improves the separation efficiency of polyphenols and polybasic acids compared to multi-segment gradient elution. This may be because multi-segment gradients can cause baseline fluctuations at slope change points and increase the risk of peak broadening due to repeated changes in elution intensity. Single-segment gradients provide a more stable separation environment, which helps to obtain sharp peak shapes; moreover, the parameters that need to be optimized for single-segment gradients (initial / final ratio, time) are far fewer than those for multi-segment gradients, making the method simpler, more robust, and more reproducible. Therefore, for the complex and diverse metabolic components containing multiple structures and polarities in this study, the single and continuous gradient change of single-segment gradient elution avoids the "step" perturbations of multi-segment gradients, thus achieving more efficient and stable separation.

[0043] In other embodiments, the mobile phase used for positive ion mode scanning under APCI source conditions in step S2 includes mobile phase A2 and mobile phase B2 for detecting solanesol, lutein, and β-carotene. Mobile phase A2 includes an aqueous solution of acetic acid, with a volume content of 0.05% to 0.1%; mobile phase B2 includes acetonitrile; the gradient elution method is as follows: based on the total volume of the mobile phase as 100%, the volume of mobile phase B2 is 5% from 0 to 0.5 min; from 0.5 to 18.0 min, the volume of mobile phase B2 increases from 10% to 95%; from 18.0 to 18.1 min, the volume of mobile phase B2 decreases from 90% to 5%; and from 18.1 to 21.0 min, the volume of mobile phase B2 is 5%.

[0044] This application compares the differences between using an electrospray ionization (ESI) source and an atmospheric pressure chemical ionization (APCI) source for the detection of solanesol, lutein, and β-carotene. Experiments showed that using ESI, solanesol, lutein, and β-carotene showed no mass spectrometry response signal; while using APCI, these compounds showed significant responses. The reason for this is that solanesol, lutein, and β-carotene are typical weakly polar and hydrophobic compounds with extremely low protonation efficiency, making it difficult to form stable charged ions, resulting in almost no detectable signal with ESI. APCI does not require the compounds to be charged in solution, therefore it is suitable for the detection of solanesol, lutein, and β-carotene.

[0045] The experiment compared the chromatographic separation effects of adding formic acid / acetic acid to the mobile phase in aqueous solution. The results showed that the addition of acetic acid resulted in better retention of solanesol, lutein, and β-carotene, with sharper peak shapes. Therefore, aqueous acetic acid was chosen as the mobile phase.

[0046] The reason for this is that, compared to formic acid, acetic acid has a lower ionic strength, which is more conducive to the interaction between the analyte and the stationary phase in this study. Therefore, choosing an aqueous solution of acetic acid as the mobile phase is the optimal choice.

[0047] In the embodiments of this application, the gradient elution method was used to investigate the difference in separation performance of the C18 reversed-phase column under multi-segment gradient elution and single-segment gradient elution conditions.

[0048] Multi-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B1 was 5% from 0 to 0.5 min; from 0.5 to 15.0 min, the volume of mobile phase B1 increased from 10% to 35%; from 15 to 17 min, the volume of mobile phase B1 increased from 35% to 95%; from 17 to 18 min, the volume of mobile phase B1 remained at 95%; from 18.0 to 18.1 min, the volume of mobile phase B1 decreased to 10%; and at 21 min, the volume of mobile phase B1 was 5%.

[0049] Single-stage gradient elution: With the total volume of the mobile phase as 100%, the volume of mobile phase B2 is 5% from 0 to 0.5 min; from 0.5 to 18.0 min, the volume of mobile phase B2 increases from 10% to 95%; from 18.0 to 18.1 min, the volume of mobile phase B2 decreases from 90% to 5%; and from 18.1 to 21.0 min, the volume of mobile phase B2 is 5%.

[0050] Experimental results show that for the C18 reversed-phase chromatography system, single-segment gradient elution significantly improves the separation efficiency of solanesol, lutein, and β-carotene compared to multi-segment gradient elution. This may be because multi-segment gradients can cause baseline fluctuations at slope change points and increase the risk of peak broadening due to repeated changes in elution intensity. Single-segment gradients provide a more stable separation environment, which helps to obtain sharp peak shapes; moreover, the parameters that need to be optimized for single-segment gradients (initial / final ratio, time) are far fewer than those for multi-segment gradients, making the method simpler, more robust, and more reproducible. Therefore, for solanesol, lutein, and β-carotene in this study, the single, continuous gradient change of single-segment gradient elution avoids the "step" perturbations of multi-segment gradients, thus achieving more efficient and stable separation.

[0051] In some embodiments, the mass spectrometry conditions for high-resolution liquid chromatography-mass spectrometry (HPLC-MS) using an ESI source for component analysis are as follows: scanning in positive and negative ion modes respectively; sheath gas flow rate: 50 Arb; auxiliary gas flow rate: 10 Arb; purge gas flow rate: 1 Arb; ion transfer tube temperature: 350°C; evaporation chamber temperature: 320°C; scanning mode: PRM mode; primary scan parameters: scan range: 70–800 m / z; orbitrap resolution: 60,000; RF lens: 70%; spray voltage: 2800 V (-) and 5000 V (+); intensity threshold: 1.0 e 5 Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z;

[0052] This application compares the mass spectrometry detection effects of polyphenols, polybasic acids, amino acids, and Amadori compounds with and without using the PRM mode. The results show that without the PRM mode and using normalized collision energy for detection, the mass spectrometry response signals for malic acid, malic acid, citric acid, tyrosine, and arginine are low. Using the PRM mode, which optimizes the collision energy for each component and selects the optimal collision energy during mass spectrometry acquisition, significantly improves the mass spectrometry response.

[0053] The experiment compared the mass spectrometry detection effects of dynamic exclusion on polyphenols, polybasic acids, amino acids, and Amadori compounds. The results showed that without dynamic exclusion, the number of acquired secondary mass spectra was significantly reduced, and most components showed no signal response. This was because the mass spectrometer repeatedly acquired secondary data from components with the highest response intensities, resulting in a large number of lower-ranking components failing to acquire secondary data. By incorporating dynamic exclusion into the mass spectrometry method, we avoided repeated acquisition of the same component, ensuring that secondary mass spectra were acquired for all components.

[0054] The experiment compared the mass spectrometry detection effects of different spray voltages on polyphenols, polybasic acids, amino acids, and Amadori compounds. Experiments in positive ion mode at different spray voltages (2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000) showed that: at spray voltages of 2000–4000, the mass spectrometry response intensities of asparagine, glycine, cysteine, and methionine were extremely low, possibly because the ionization degree of asparagine, glycine, cysteine, methionine, 1-deoxy-1-L-leucine-d-fructose, and 1-deoxy-1-L-isoleucine-d-fructose was low at this voltage; at positive ion spray voltages of 4500–5000, the mass spectrometry response values ​​of asparagine, glycine, cysteine, and methionine continuously increased, reaching a plateau at 5000V; when the voltage increased to 5500V, the ion fragmentation degree of the components was too high, affecting the detection specificity. Therefore, 5000V was selected as the optimal voltage for detecting amino acids and Amadori compounds. Experimental results under negative ion mode at different spray voltages (2000, 2500, 2800, 3000, 3500V) showed that the response intensity of polyphenols and polyacids continuously increased when the spray voltage was between 2000 and 2800V, reaching a plateau at 2800V. When the voltage increased to 3000V, the degree of ion fragmentation was too high, affecting the detection specificity. Therefore, 2800V was selected as the optimal voltage for detecting polyphenols and polyacids.

[0055] In other embodiments, the mass spectrometry conditions for high-resolution liquid chromatography-mass spectrometry (HPLC-MS) using an APCI source for component analysis are as follows: scanning in positive ion mode; sheath gas flow rate: 45 Arb; auxiliary gas flow rate: 5 Arb; purge gas flow rate: 1 Arb; ion transfer tube temperature: 300°C; evaporation chamber temperature: 350°C; scanning mode: PRM mode; primary scan parameters: scan range: 70–800 m / z; orbitrap resolution: 60,000; RF lens: 70%; positive ion exchange current: 4 μA; intensity threshold: 1.0 e 5 Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

[0056] This application compares the mass spectrometry detection effects of different exchange currents on solanesol, β-carotene, and lutein. Experimental results in positive ion mode at different exchange currents (1, 2, 3, 4, 5, 6 μA) show that the mass spectrometry response values ​​of solanesol, β-carotene, and lutein continuously increase when the spray voltage is 1–5 μA, reaching a plateau at 5 μA. When the voltage increases to 6 μA, the ion fragmentation of the components becomes too high, affecting the detection specificity. Therefore, 5 μA was selected as the optimal exchange current for the detection of solanesol, β-carotene, and lutein.

[0057] In this application, during the extraction step, different methanol contents were used in the extractant to detect different tobacco components. Different components have different solubilities, hence the different methanol contents in the extractant. This application investigated the solubility and extraction capacity of different concentration ratios (0, 10%, 30%, 50%, 70%, 90%) of methanol / acetonitrile / acetone aqueous solutions for polyphenols, polybasic acids, amino acids, Amadori compounds, solanesol, and pigments in a tobacco matrix. The results showed that polyphenols and polybasic acids were readily soluble and exhibited excellent extraction capacity in a 70% methanol aqueous solution; amino acids and Amadori compounds were readily soluble and exhibited excellent extraction capacity in a 30% methanol aqueous solution; and solanesol and pigments were readily soluble and exhibited excellent extraction capacity in a 70% acetone aqueous solution. In some embodiments, the solvent used in step S1 of the extraction step was an aqueous solution containing 10%–50% methanol for detecting Amadori compounds and amino acids. In other embodiments, the solvent used in step S1 of the extraction step was an aqueous solution containing 30%–80% methanol for detecting polyphenols and polybasic acids. In other embodiments, the solvent used in the extraction step S1 is an aqueous solution containing 30% to 70% acetone, used to detect solanesol and pigments.

[0058] Further, in step S1, the extraction is selected from one of the following: ultrasonic extraction for 10-20 min, oscillation extraction for 20-25 min, oscillation extraction for 5-10 min followed by ultrasonic extraction for 5-10 min, or ultrasonic extraction for 10-15 min followed by oscillation extraction for 10-15 min.

[0059] In this embodiment of the application, the tobacco components obtained by positive ion mode scanning under ESI source conditions in step S2 include: 1-deoxy-1-L-aminobutyric acid-D-fructose, 1-deoxy-1-L-histidine-D-fructose, 1-deoxy-1-L-proline-D-fructose, 1-deoxy-1-L-valine-D-fructose, 1-deoxy-1-L-threonine-D-fructose, 1-deoxy-1-L-alanine-D-fructose, 1-deoxy-1-L-asparagine-D-fructose, 1-deoxy-1-L-aspartic acid-D-fructose, and 1-deoxy-1-L-glutamine-D-fructose. 1-Deoxy-1-L-glutamic acid-D-fructose, 1-Deoxy-1-L-isoleucine-D-fructose, 1-Deoxy-1-L-leucine-D-fructose, 1-Deoxy-1-L-tyrosine-D-fructose, 1-Deoxy-1-L-phenylalanine-D-fructose, 1-Deoxy-1-L-tryptophan-D-fructose, proline, aspartic acid, threonine, serine, asparagine, glutamic acid, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, 4-aminobutyric acid, lysine, histidine, tryptophan, arginine, glucosamine.

[0060] Under the above ESI source conditions, positive ion mode scanning detected a total of 36 Amadori compounds and amino acid components.

[0061] In this embodiment of the application, the tobacco components obtained by negative ion mode scanning under ESI source conditions in step S2 include: chlorogenic acid, rutin, scopolamine, neochlorogenic acid, cryptochlorogenic acid, malic acid, citric acid, vanillic acid, tetradecanoic acid, hexadecanoic acid, linoleic acid, oleic acid, linolenic acid, octadecanoic acid, eicosanoic acid, lactic acid, and fructose acid.

[0062] A total of 17 polyphenol and polyacid components were detected in the negative ion mode scanning under the above ESI source conditions.

[0063] In this embodiment, the tobacco components obtained by positive ion mode scanning under APCI source conditions in step S2 include: solanesol, lutein, and β-carotene. The positive ion mode under the aforementioned APCI source conditions is a beneficial supplement to the ESI source, enabling the detection of components that the ESI source cannot detect.

[0064] The above quantitative detection method will be explained in more detail below through specific examples.

[0065] Example A method for quantitatively detecting the components of tobacco shreds in a finished cigarette product includes the following steps: 1. The finished cigarette tobacco was extracted using a solvent. When the detected components were Amadori compounds and amino acids, the extraction solvent was a 30% methanol aqueous solution; when the detected components were polyphenols and polybasic acids, the extraction solvent was a 70% methanol aqueous solution. When the detected components were solanesol and pigments, the extraction solvent was a 70% acetone aqueous solution. Ultrasonic extraction was performed for 20 minutes. After extraction, the extract was obtained by centrifugation and filtration.

[0066] 2. The above extract was analyzed by high-resolution liquid chromatography-mass spectrometry.

[0067] (1) Under ESI source conditions, positive and negative ion modes were used for scanning, and the chromatographic column was a C18 column.

[0068] The mass spectrometry conditions for high-resolution liquid chromatography-mass spectrometry are as follows: Sheath gas flow rate: 50 Arb; Auxiliary gas flow rate: 10 Arb; Purge gas flow rate: 1 Arb; Ion transfer tube temperature: 350℃; Evaporation chamber temperature: 320℃; Scan mode: PRM mode; Primary scan parameters: Scan range: 70~800 m / z; Orbitrap resolution: 60000; RF lens: 70%; Spray voltage: 2800V(-) and 5000V(+); Intensity threshold: 1.0e 5Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

[0069] The mobile phase for positive ion mode scanning under ESI source conditions includes mobile phase A1 and mobile phase B1 for the detection of Amadori compounds and amino acids. Mobile phase A1 consists of an aqueous solution of formic acid containing phthalic acid at a concentration of 0.12 mM and a volume content of 0.2%. Mobile phase B1 consists of acetonitrile.

[0070] The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of mobile phase B1 is 10%; From 1.0 to 19.0 min, the volume of mobile phase B1 increased from 10% to 90%; From 19.0 to 19.1 min, the volume of mobile phase B1 decreased from 90% to 10%; 19.1–21.0 min, the volume of mobile phase B1 is 10%.

[0071] The components detected by the reversed-phase column in positive ion mode under ESI source conditions are 1-deoxy-1-L-aminobutyric acid-D-fructose, 1-deoxy-1-L-histidine-D-fructose, 1-deoxy-1-L-proline-D-fructose, 1-deoxy-1-L-valine-D-fructose, 1-deoxy-1-L-threonine-D-fructose, 1-deoxy-1-L-alanine-D-fructose, 1-deoxy-1-L-asparagine-D-fructose, 1-deoxy-1-L-aspartic acid-D-fructose, 1-deoxy-1-L-glutamine-D-fructose, 1-deoxy- 1-L-Glutamic acid-D-fructose, 1-deoxy-1-L-isoleucine-D-fructose, 1-deoxy-1-L-leucine-D-fructose, 1-deoxy-1-L-tyrosine-D-fructose, 1-deoxy-1-L-phenylalanine-D-fructose, 1-deoxy-1-L-tryptophan-D-fructose, proline, aspartic acid, threonine, serine, asparagine, glutamic acid, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, 4-aminobutyric acid, lysine, histidine, tryptophan, arginine, glucosamine. The collision energies and retention times of the above components are shown in Table 1.

[0072] The mobile phases for negative ion mode scanning under ESI source conditions include mobile phase A2 and mobile phase B2 for detecting polyphenols and polybasic acids. Mobile phase A2 consists of an aqueous solution of acetic acid with a volume content of 0.1%; mobile phase B2 consists of acetonitrile.

[0073] The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of the mobile phase B2 is 10%; From 1.0 to 19.0 min, the volume of the mobile phase B2 increased from 10% to 90%. During the 19.0–19.1 min interval, the volume of the mobile phase B2 decreased from 90% to 10%. The volume of the mobile phase B2 is 10% during the 19.1–21.0 min period.

[0074] Tobacco components obtained by reverse-phase column scanning in negative ion mode under ESI source conditions include: chlorogenic acid, rutin, hyoscyamine, neochlorogenic acid, cryptochlorogenic acid, malic acid, citric acid, vanillic acid, myristic acid, hexadecanoic acid, linoleic acid, oleic acid, linolenic acid, stearic acid, eicosanoic acid, lactic acid, and fructose. The collision energies and retention times of each of these components are shown in Table 1.

[0075] See the extracted ion chromatograms of tobacco components obtained by reverse-phase column scanning in positive and negative ion modes under ESI source conditions. Figures 1 to 8 (Contains polyphenols, polyacids, amino acids, and Amadori compounds. Because some peaks have such strong mass spectrometry responses that other peaks are easily overlooked in the same image, the 53 components are divided into 8 images based on peak response intensity. Peaks marked with RT are the target components. Components with consistent retention times are distinguished by high-resolution mass spectrometry using different mass-to-charge ratios, without affecting the specificity of multi-component detection.)

[0076] in, Figure 1 Including citric acid, malic acid, lactic acid, vanillic acid, and fructose; Figure 2 Including linolenic acid, linoleic acid, oleic acid, tetradecanoic acid, stearic acid, and eicosanoic acid; Figure 3 Including cryptochlorogenic acid, rutin, and hexadecanoic acid; Figure 4 Including chlorogenic acid, hyoscyamine, and neochlorogenic acid; Figure 5 Including lysine, proline, histidine, threonine, arginine, valine, methionine, 1-deoxy-1-L-tyrosine-D-fructose, tyrosine, 1-deoxy-1-L-phenylalanine-D-fructose, phenylalanine, and tryptophan; Figure 6 Including 1-deoxy-1-L-glutamine-D-fructose, glucosamine, glycine, serine, 1-deoxy-1-L-alanine-D-fructose, glutamic acid, 1-deoxy-1-L-glutamic acid-D-fructose, and 1-deoxy-1-L-valine-D-fructose; Figure 7 Including 1-deoxy-1-L-aminobutyric acid-D-fructose, 4-aminobutyric acid, alanine, 1-deoxy-1-L-histidine-D-fructose, and 1-deoxy-1-L-tryptophan-D-fructose; Figure 8 Including 1-deoxy-1-L-threonine-D-fructose, asparagine, 1-deoxy-1-L-asparagine-D-fructose, aspartic acid, 1-deoxy-1-L-proline-D-fructose, 1-deoxy-1-L-aspartic acid-D-fructose, 1-deoxy-1-L-leucine-D-fructose, 1-deoxy-1-L-isoleucine-D-fructose, cystine, leucine, and isoleucine.

[0077] (2) Under APCI source conditions, positive ion mode scanning was used, and the chromatographic column was a C18 column.

[0078] The mass spectrometry conditions for high-resolution liquid chromatography-mass spectrometry are as follows: Sheath flow velocity: 45 Arb; Auxiliary flow velocity: 5 Arb; Purging flow velocity: 1 Arb; Ion transfer tube temperature: 300℃; Evaporation chamber temperature: 350℃; Scanning mode: PRM mode; Primary scanning parameters: Scan range: 70–800 m / z; Orbitrap resolution: 60,000; RF lens: 70%; Positive ion exchange current: 4 μA; Intensity threshold: 1.0 e 5 Dynamic exclusion time: 5s~6s; Dynamic exclusion quality deviation: 5ppm; Background noise exclusion; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

[0079] The mobile phases for positive ion mode scanning under APCI source conditions include mobile phase A2 and mobile phase B2. Mobile phase A2 consists of an aqueous solution of acetic acid with a volume content of 0.1%; mobile phase B2 consists of acetonitrile.

[0080] The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 0.5 min, the volume of mobile phase B2 is 5%; Over a period of 0.5–18.0 min, the volume of mobile phase B2 increased from 10% to 95%. Between 18.0 and 18.1 min, the volume of mobile phase B2 decreased from 90% to 5%. The time was 18.1–21.0 min, and the volume of mobile phase B2 was 5%.

[0081] Tobacco components obtained by positive ion mode scanning under APCI source conditions include: solanesol, lutein, and β-carotene. The extracted ion chromatograms of each component are shown below. Figure 9 The collision energy and retention time are shown in Table 1.

[0082] Table 1. Collision energy and retention time of various components in a finished cigarette tobacco product of an example.

[0083] 3. Prepare standard working solutions for 56 components respectively, and quantify them using the external standard method based on the concentration of the series of working standard solutions and the response peak area of ​​each target component.

[0084] Preparation of standard working solutions: Accurately transfer different volumes of the component-mixed standard solutions. Polyphenols and polybasic acids were prepared using 70% methanol aqueous solution, while amino acids and Amadori compounds were diluted to volume using 30% methanol aqueous solution. High-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to determine the peak areas of each component at known concentrations. Calibration curves for the 56 components were established using peak area as the ordinate and concentration as the abscissa. Linear regression (R²) was performed on the calibration data. 2 >0.99). Then, the tobacco extract samples of this embodiment were measured, and the concentrations (mg / mL) of the 56 components in each tobacco extract sample were calculated from the peak areas of each component in the sample. The concentrations were calculated using the following formula:

[0085] In the formula: —Component content per gram of sample, in milligrams per gram (mg / g); c — the concentration of the analyte in the extracted sample, in milligrams per milliliter (mg / mL). V — Volume of the extraction solution, in milliliters (mL); m — the mass of the sample, in grams (g).

[0086] —Percentage of moisture content in the sample; The average of the two measurements was taken as the result, accurate to 0.001 mg / g.

[0087] The results of the experimental studies on the limit of quantitation, recovery rate and precision of this method are shown in Table 2 below, and the content measurement results of each component in the tobacco sample are shown in Table 3.

[0088] Table 2. Test data of polyphenols, polyacids, amino acids, Amadori compounds, solanesol, and pigment compounds in tobacco samples.

[0089] Table 3. Measurement results of polyphenols, polyacids, amino acids, Amadori compounds, solanesol, and pigment compounds in tobacco samples.

[0090] The quantitative detection method for tobacco components in this application, through a simple and rapid sample pretreatment and analysis method, can target and detect 56 components in tobacco, and achieve accurate quantitative detection of the above components. It realizes the simultaneous detection of multiple components, saves measurement time and cost, and greatly improves detection efficiency. It provides good technical support for comprehensively reflecting the style characteristics, quality characteristics and aging process of flue-cured tobacco, as well as the digital characterization of tobacco.

[0091] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for quantitative detection of tobacco components, characterized in that, Includes the following steps: S1. Extract the tobacco sample to obtain the extract; S2. The extract obtained in step S1 is subjected to high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) for component analysis to obtain the spectrum of the tobacco sample. The HPLC-MS / MS uses two ion sources, ESI and APCI, and both ion sources are detected using a reversed-phase column. During the detection, positive and negative ion modes are used under the ESI source condition, and positive ion mode is used under the APCI source condition.

2. The method for quantitative detection of tobacco components according to claim 1, characterized in that, The reversed-phase chromatographic column is a C18 column.

3. The method for quantitative detection of tobacco components according to claim 1, characterized in that, In step S2, the mobile phase for positive ion mode scanning under ESI source conditions includes mobile phase A1 and mobile phase B1 for detecting Amadori compounds and amino acids. Mobile phase A1 includes an aqueous solution of formic acid containing phthalic acid, wherein the concentration of phthalic acid is 0.1 mM to 0.2 mM and the volume content of formic acid is 0.02% to 0.2%. Mobile phase B1 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of the mobile phase B1 is 10%; From 1.0 to 19.0 min, the volume of the mobile phase B1 increased from 10% to 90%. During the 19.0–19.1 min interval, the volume of the mobile phase B1 decreased from 90% to 10%. The volume of the mobile phase B1 is 10% during the 19.1–21.0 min period.

4. The method for quantitative detection of tobacco components according to claim 1, characterized in that, In step S2, the mobile phase for negative ion mode scanning under ESI source conditions includes mobile phase A2 and mobile phase B2 for detecting polyphenols and polybasic acids. Mobile phase A2 includes an aqueous solution of acetic acid with a volume content of 0.01% to 0.1%. Mobile phase B2 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 1.0 min, the volume of the mobile phase B2 is 10%; From 1.0 to 19.0 min, the volume of the mobile phase B2 increased from 10% to 90%. During the 19.0–19.1 min interval, the volume of the mobile phase B2 decreased from 90% to 10%. The volume of the mobile phase B2 is 10% during the 19.1–21.0 min period.

5. The method for quantitative detection of tobacco components according to claim 1, characterized in that, In step S2, the mobile phase for positive ion mode scanning under APCI source conditions includes mobile phase A2 and mobile phase B2 for detecting solanesol, lutein, and β-carotene. Mobile phase A2 includes an aqueous solution of acetic acid with a volume content of 0.05% to 0.1%. Mobile phase B2 includes acetonitrile. The gradient elution method is as follows: with the total volume of the mobile phase being 100%, From 0 to 0.5 min, the volume of the mobile phase B2 is 5%; Over a period of 0.5–18.0 min, the volume of the mobile phase B2 increased from 10% to 95%. During the 18.0–18.1 min interval, the volume of the mobile phase B2 decreased from 90% to 5%. The volume of the mobile phase B2 is 5% during the 18.1–21.0 min period.

6. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The mass spectrometry conditions for component analysis using the high-resolution liquid chromatography-mass spectrometry with an ESI source are as follows: Scanning was performed using both positive and negative ion modes. Sheath flow rate: 50 Arb; Auxiliary flow rate: 10 Arb; Purging flow rate: 1 Arb; Ion transfer tube temperature: 350℃; Evaporation chamber temperature: 320℃; Scanning mode: PRM mode; Primary scanning parameters: Scan range: 70–800 m / z; Orbitrap resolution: 60,000; RF lens: 70%; Spray voltage: 2800V (-) and 5000V (+); Intensity threshold: 1.0 e 5 ; Dynamic removal time: 5s~6s; Dynamic removal quality deviation: 5ppm; Background noise elimination; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

7. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The mass spectrometry conditions for component analysis using the APCI source in the high-resolution liquid chromatography-mass spectrometry are as follows: Positive ion mode scanning was used; Sheath flow velocity: 45 Arb; Auxiliary flow velocity: 5 Arb; Purging flow velocity: 1 Arb; Ion transfer tube temperature: 300℃; Evaporation chamber temperature: 350℃; Scanning mode: PRM mode; Primary scanning parameters: Scan range: 70–800 m / z; Orbitrap resolution: 60,000; RF lens: 70%; Positive ion exchange current: 4 μA; Intensity threshold: 1.0 e 5 ; Dynamic removal time: 5s~6s; Dynamic removal quality deviation: 5ppm; Background noise elimination; Secondary scan parameters: Orbitrap resolution: 15000, separation window: 2m / z.

8. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The solvent used in the extraction step S1 is an aqueous solution containing 10% to 50% methanol, used to detect Amadori compounds and amino acids.

9. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The solvent used in the extraction step S1 is an aqueous solution containing 30% to 80% methanol, which is used to detect polyphenols and polybasic acids.

10. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, In step S1, the extraction is selected from one of the following: ultrasonic extraction for 10-20 min, oscillation extraction for 20-25 min, oscillation extraction for 5-10 min followed by ultrasonic extraction for 5-10 min, or ultrasonic extraction for 10-15 min followed by oscillation extraction for 10-15 min.

11. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The tobacco components obtained by positive ion mode scanning under the ESI source conditions in step S2 include: 1-deoxy-1-L-aminobutyric acid-D-fructose, 1-deoxy-1-L-histidine-D-fructose, 1-deoxy-1-L-proline-D-fructose, 1-deoxy-1-L-valine-D-fructose, 1-deoxy-1-L-threonine-D-fructose, 1-deoxy-1-L-alanine-D-fructose, 1-deoxy-1-L-asparagine-D-fructose, 1-deoxy-1-L-aspartic acid-D-fructose, 1-deoxy-1-L-glutamine-D-fructose, 1 -Deoxy-1-L-glutamic acid-D-fructose, 1-deoxy-1-L-isoleucine-D-fructose, 1-deoxy-1-L-leucine-D-fructose, 1-deoxy-1-L-tyrosine-D-fructose, 1-deoxy-1-L-phenylalanine-D-fructose, 1-deoxy-1-L-tryptophan-D-fructose, proline, aspartic acid, threonine, serine, asparagine, glutamic acid, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, 4-aminobutyric acid, lysine, histidine, tryptophan, arginine, glucosamine.

12. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The tobacco components obtained by negative ion mode scanning under the ESI source conditions in step S2 include: chlorogenic acid, rutin, scopolamine, neochlorogenic acid, cryptochlorogenic acid, malic acid, citric acid, vanillic acid, tetradecanoic acid, hexadecanoic acid, linoleic acid, oleic acid, linolenic acid, octadecanoic acid, eicosanoic acid, lactic acid, and fructose acid.

13. The method for quantitative detection of tobacco components according to any one of claims 1 to 5, characterized in that, The tobacco components obtained by positive ion mode scanning under the APCI source conditions in step S2 include: solanesol, lutein, and β-carotene.

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