Multi-stage mass spectrum identification method for chemical structures of sucrose esters in tobacco and tobacco products

Abstract

The invention relates to the technical field of tobacco chemical analysis, in particular to a multistage mass spectrometry identification method for chemical structures of sucrose esters in tobacco and tobacco products, which comprises the following steps: crushing and extracting a tobacco product sample; carrying out solid-liquid separation and filtration to obtain a liquid to be detected; separating the liquid to be detected by adopting hydrophilic interaction chromatography; performing triple quadrupole-ion trap tandem mass spectrometry analysis on the components separated in the step S2 by adopting an electrospray ion source in a positive ion mode to sequentially obtain mass spectrometry information of parent ions, daughter ions and grandchild ions; and determining the chemical structure of the sucrose ester based on the mass spectrum information of the parent ion, the daughter ion and the grandchild ion measured in the step S3. According to the method, complex pretreatment steps are not needed, sucrose esters with different configurations can be identified more efficiently, interference can be effectively eliminated, and substituent group structure information of a low-content target object can be scientifically confirmed.

Description

Technical Field

[0001] This invention relates to the technical field of tobacco chemical analysis, and in particular to a multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products. Background Technology

[0002] Sucrose esters (SEs) are mixtures formed by the esterification reaction of sucrose and fatty acids. They are the main components of the secretions from the glandular trichomes on the surface of plants such as tobacco, tomato, and datura. They not only possess antibacterial and plant growth-regulating activities but are also important aroma precursors in tobacco, significantly influencing its sensory quality. Their structure is as follows: Figure 1 As shown.

[0003] The composition of sucrose esters (SEs) in tobacco is extremely complex. The differences among all reported SEs lie primarily in the length and number of fatty acid chains, as well as the number and position of acetyl groups. For example, acetyl groups can be located at the C-6 position of the glucose unit or the C-3 position of the fructose unit. These subtle structural differences lead to the coexistence of numerous isomers and components with different chemical formulas but similar molecular weights. Furthermore, the complex matrix of tobacco itself and the presence of many interfering substances make the identification of sucrose esters in tobacco, especially their precise structures, a significant challenge.

[0004] Existing technologies often employ multidimensional preparative chromatography combined with mass spectrometry (MS / MS) for the determination of sucrose esters. For example, invention patent application number CN201911051738.X discloses a method for analyzing sucrose esters in tobacco using multidimensional liquid chromatography-mass spectrometry (LC-MS / MS). This method uses a two-dimensional LC-MS / MS approach, requiring a complex two-dimensional C-type chromatography system. 18 Separation is performed using a reversed-phase liquid chromatography system, involving column switching and multiple gradient elutions. The pretreatment and analysis procedures are time-consuming and require a high level of operational skill. Subsequently, mass spectrometry is used to qualitatively identify the sucrose ester components and internal standard in the sample solution, and the internal standard sample in the sample solution is quantified using a standard curve method. Finally, the relative content of sucrose ester components in the sample solution with structures similar to the quantified internal standard sample is determined. This method focuses on the construction of chromatographic components and semi-quantitative analysis of sucrose esters, and can determine the relative content of six sucrose esters, but it cannot accurately identify the configurations of various sucrose esters.

[0005] Therefore, there is an urgent need to develop a more efficient and accurate analytical method that can systematically identify sucrose esters with different configurations and scientifically characterize the specific chemical structure information of trace target substances without the need for complex multidimensional chromatographic separation and instrument setup. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products. This method eliminates the need for complex pretreatment steps, systematically identifies sucrose esters with different configurations, and scientifically characterizes the specific chemical structure information of trace target substances.

[0007] The technical solution to the problem solved by the present invention is as follows: The present invention provides a multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products, comprising the following steps:

[0008] S1. The tobacco product sample is crushed and extracted, and after solid-liquid separation and filtration, the test solution is obtained;

[0009] S2. The test solution is separated using hydrophilic interaction chromatography (HILIC);

[0010] S3. Using an electrospray ionization source in positive ion mode, perform multi-stage mass spectrometry analysis on the components separated in step S2 to obtain the mass spectrometry information of the parent ion, daughter ion, and granddaughter ion in sequence;

[0011] S4. Based on the mass spectrometry information of the parent ion, daughter ion, and granddaughter ion obtained in step S3, determine the chemical structure of sucrose ester.

[0012] Preferably, step S3 includes:

[0013] S3.1. Perform a first-order mass spectrometry scan to obtain the mass spectrometry information of the parent ion with a mass-to-charge ratio in the range of m / z 400~1000;

[0014] S3.2. Fragment the parent ion into daughter ions, perform secondary mass spectrometry scanning, and determine the mass spectrometry information of the daughter ions;

[0015] S3.3. Based on preset triggering conditions, the daughter ions that meet the triggering conditions are captured and fragmented again, and a three-stage mass spectrometry scan is performed to obtain the mass spectrometry information of the grandchild ions.

[0016] Preferably, the preset triggering condition is that the signal intensity of the daughter ion exceeds a preset threshold.

[0017] Preferably, step S4 includes:

[0018] The molecular formula of sucrose esters is determined based on the mass spectrometry information of the parent ion; the structure of the fructose moiety is confirmed based on the characteristic ion generated by the loss of a fructose group from the parent ion in the mass spectrometry information of the daughter ion; and the composition and structure of the fatty acid chains attached to the glucose moiety are determined based on the fragment ions corresponding to different fatty acid chains in the mass spectrometry information of the grandchild ion.

[0019] Preferably, in step S1, the solvent used for extraction is an aqueous solution of acetonitrile with a volume concentration of 50-100%.

[0020] Preferably, in step S2, the chromatographic mobile phase of the hydrophilic interaction chromatography includes an aqueous phase and an organic phase.

[0021] Preferably, the organic phase is acetonitrile or an acetonitrile solution containing a volatile basic modifier.

[0022] Preferably, the volatile alkaline modifier includes ammonium hydroxide at a concentration of 0.01 to 0.1%.

[0023] Preferably, the gradient elution conditions for hydrophilic interaction chromatography include: 0-10 min, 100% organic phase; 20-21 min, 70% organic phase; 21-28 min, 50% organic phase; 28.1-35 min, 100% organic phase.

[0024] Preferably, the scanning interval for the second-stage mass spectrometry scan is 10 to 15 m / z, and the scanning interval for the third-stage mass spectrometry scan is 1 to 2 m / z.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. In this application, by adopting a simplified sample pretreatment process (only extraction-centrifugation-filtration is required) and without the need to add internal standard substances, and combined with the efficient HILIC separation technology, the operation process is significantly simplified and the analysis efficiency is greatly improved. Moreover, the time from sampling to obtaining the structural identification results is ≤35 min, which significantly reduces the detection cost and time cost.

[0027] 2. In this application, the HILIC chromatographic mode is utilized to effectively separate sucrose esters, thus avoiding the separation of sucrose esters in traditional C1 chromatography. 18 The problem of low resolution on reversed-phase chromatography columns was addressed by achieving a certain degree of separation of sucrose esters with different structures and molecular weights, laying a solid foundation for accurate identification by subsequent mass spectrometry.

[0028] 3. In this application, by systematically adopting a data-dependent three-stage mass spectrometry scanning strategy, the interference of complex tobacco matrix can be effectively eliminated, and rich fragment ion information can be obtained in stages. This enables precise analysis of sucrose ester precursor ions, acyl substitution positions and fatty acid chain composition, systematic identification of sucrose esters with different configurations, and scientific characterization of the specific chemical structure information of trace target substances. Attached Figure Description

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. In these drawings, similar reference numerals are used to denote similar elements. The drawings described below are some embodiments of the invention, but not all embodiments. Other drawings will be readily available to those skilled in the art based on these drawings without any inventive effort.

[0030] Figure 1 This is a schematic diagram of the chemical structure of sucrose esters (R1, R2, R3 = C2-C9 acids).

[0031] Figure 2 The fragmentation paths of sucrose esters under primary, secondary, and tertiary mass spectrometry.

[0032] Figure 3 The mass spectrum of the daughter ion fragment is shown (parent ion is 659.3, daughter ion is 497.2).

[0033] Figure 4 The mass spectrum of the daughter ion fragment is shown (parent ion is 589.3, daughter ion is 427.1).

[0034] Figure 5 This is the mass spectrum of the daughter ion fragment (parent ion is 701.3, daughter ion is 539.2). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example

[0037] (1) Sample processing

[0038] Tobacco leaf samples were ground into powder, and cigarette and new tobacco products were separated into tobacco shreds and ground into powder. 0.2 g (accurate to 0.1 mg) of tobacco and tobacco product powder samples were accurately weighed and placed in a 50 mL plastic centrifuge tube. The supernatant was filtered through a 0.22 μm PTFE filter membrane and then analyzed.

[0039] (2) Chromatographic conditions

[0040] Mobile phase: A was water, and B was an acetonitrile solution containing 0.03% ammonium hydroxide. An Agilent Poroshell 120 Hilic column (2.1 × 100 mm, 2.7 µm) was used, with a column temperature of 25 °C, an injection plate temperature of 15 °C, an injection volume of 1 μL, and a flow rate of 0.2 mL / min. Gradient elution conditions: 0–10 min, 100% B; 20–21 min, 70% B; 21–28 min, 50% B; 28.1–35 min, 100% B.

[0041] (3) Mass spectrometry conditions

[0042] Mass spectrometry features daughter ion scanning and multi-stage ion scanning modes, specifically including:

[0043] Electrospray ionization source (ESI); positive ion mode; electrospray voltage, 3000 V; capillary temperature, 350 °C; sheath gas flow rate, 30 arb; auxiliary heating gas flow rate, 10 arb; auxiliary heating gas temperature, 350 °C; running time, 35 min.

[0044] Mass spectrometry scanning employs daughter ion scanning and multi-stage ion scanning modes: in MS 1 In precursor ion scanning mode, the scanning range is m / z 400 ~ 1000; in MS... 2 In daughter ion scanning mode, according to MS 1 The precursor ion setting MS 2 Fragmentation was performed with a scanning range of m / z 400 ~ 1000 and a scanning interval of m / z 12; MS was used. 3 An exhaustive scanning method was used. In daughter ion scanning mode, the scanning range was 400~1000 m / z, and the scanning interval was 1 m / z. The ionization time, fragmentation Q point, fragmentation time, and fragmentation energy were optimized to obtain the parent ion, daughter ion, and granddaughter ion sequentially.

[0045] (4) Structural identification

[0046] Based on the reported chemical formulas of sucrose esters, the precise molecular weights of the parent ions of various types of sucrose esters were calculated. Furthermore, according to the reported fragmentation pathways of sucrose esters, the precise molecular weights of the daughter and granddaughter ions were calculated, thus deriving their configurations. Since glucose (GLU) in sucrose esters has a high affinity for Na, MS... 1 The middle ions are all sodium salt parent ions [M+Na]. + According to research reports on sucrose esters, MS 2 The fragmentation mainly occurs between the O atom and the fructose group (FRU), therefore, MS 2 Mostly MS 1-The fragmentation form of FRU, with a difference in m / z of MS 1 -162. MS 3 The main process involves the breakage of the GLU group to generate fatty acids of different molecular weights.

[0047] After extraction from actual tobacco samples, analysis was performed using the methods described above:

[0048] In the positive ion mode of the ESI ion source, sucrose esters form [M+Na]. + The parent ion, theoretically, has the following fragmentation paths for its parent ion, daughter ion, and granddaughter ion: Figure 2 As shown.

[0049] The molecular formula of sucrose ester is C 29 H 48 O 15 The parent ion m / z was 659.3, and the corresponding daughter ion m / z was 497.2 in the second-stage mass spectrometry. Analysis was performed, and the fragment information spectrum of the grandchild ion in the third-stage mass spectrometry is shown below. Figure 3 The R1, R2, and R3 fragment information is inferred and shown in Table 1. The molecular formula obtained is C1. 29 H 48 O 15 The sucrose esters R1, R2, and R3 mainly have the following five compositions: 1. -CH3COOH, -C3H7COOH, -C8H 17 COOH; 2, -C2H5COOH, -C2H5COOH, -C8H 17 COOH; 3, -C2H5COOH, -C5H 11 COOH, -C5H 11 COOH; 4. -C3H7COOH, -C4H9COOH, -C5H 11 COOH; 5. -C4H9COOH, -C4H9COOH, -C4H9COOH.

[0050] Table 1 Sucrose Ester C 29 H 48 O 15 Information on the mass-to-charge ratios of the parent ion, daughter ion, and granddaughter ion, as well as possible substituents.

[0051]

[0052] Based on the possible fragmentation paths of sucrose esters using primary, secondary, and tertiary mass spectrometry, the molecular formula of sucrose esters, C2... 25 H 42 O 14 The parent ion m / z was 589.3, and the corresponding daughter ion m / z was 427.1 in the second-stage mass spectrometry. Analysis of the tertiary mass spectrometry fragment information spectrum of the grandchild ion is shown below. Figure 4The information of its R1, R2, and R3 fragments is inferred and shown in Table 2.

[0053] Based on the information of sine fragments from the third-order mass spectrometry, the molecular formula C was obtained. 25 H 42 O 14 The sucrose esters R1, R2, and R3 are mainly composed of the following four types: 1. -C2H5COOH, -CH3COOH, -CH3COOH; 2. -CH3COOH, -C2H5COOH, -C3H7COOH; 3. -C3H7COOH, -C3H7COOH, -CH3COOH; 4. -C2H5COOH, -C2H5COOH-C3H7COOH.

[0054] Table 2 Sucrose Ester C 25 H 42 O 14 Information on the mass-to-charge ratios of the parent ion, daughter ion, and granddaughter ion, as well as possible substituents.

[0055]

[0056] Based on the possible fragmentation paths of sucrose esters using primary, secondary, and tertiary mass spectrometry, the molecular formula of sucrose esters, C2... 32 H 54 O 15 The parent ion m / z was 701.3, and the corresponding daughter ion m / z was 539.2 in the second-stage mass spectrometry. Analysis was performed, and the fragment information spectrum of the grandchild ion in the third-stage mass spectrometry is shown below. Figure 5 The information of its R1, R2, and R3 fragments is inferred and shown in Table 3.

[0057] Based on the information of sine fragments from the third-order mass spectrometry, the molecular formula C was obtained. 32 H 54 O 15 The sucrose esters R1, R2, and R3 are mainly composed of the following two types: 1. -C5H 11 COOH, -C5H 11 COOH, -C5H 11 COOH; 2, -C4H9COOH, -C5H 11 COOH, -C6H 13 COOH.

[0058] Table 3 Sucrose Ester C 32 H 54 O 15 Information on the mass-to-charge ratios of the parent ion, daughter ion, and granddaughter ion, as well as possible substituents.

[0059]

[0060] The above-described contents can be implemented individually or in various combinations, and these variations are all within the protection scope of this invention.

[0061] In the description of this invention, it should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0062] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific embodiments of the present invention are limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered within the scope of protection of the present invention.

Claims

1. A multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products, characterized in that, Includes the following steps: S1. The tobacco product sample is crushed and extracted, and after solid-liquid separation and filtration, the test solution is obtained; S2. The test solution is separated using hydrophilic interaction chromatography; S3. Using an electrospray ionization source in positive ion mode, perform multi-stage mass spectrometry analysis on the components separated in step S2 to obtain the mass spectrometry information of the parent ion, daughter ion, and granddaughter ion in sequence; S4. Based on the mass spectrometry information of the parent ion, daughter ion, and granddaughter ion obtained in step S3, determine the chemical structure of sucrose ester.

2. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 1, characterized in that, Step S3 includes: S3.

1. Perform a first-order mass spectrometry scan to obtain the mass spectrometry information of the parent ion with a mass-to-charge ratio in the range of m / z 400~1000; S3.

2. Fragment the parent ion into daughter ions, perform secondary mass spectrometry scanning, and determine the mass spectrometry information of the daughter ions; S3.

3. Based on preset triggering conditions, the daughter ions that meet the triggering conditions are captured and fragmented again, and a three-stage mass spectrometry scan is performed to obtain the mass spectrometry information of the grandchild ions.

3. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 2, characterized in that, The preset trigger condition is that the signal intensity of the daughter ion exceeds a preset threshold.

4. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 1, characterized in that, Step S4 includes: The molecular formula of sucrose esters is determined based on the mass spectrometry information of the parent ion; the structure of the fructose moiety is confirmed based on the characteristic ion generated by the loss of a fructose group from the parent ion in the mass spectrometry information of the daughter ion; and the composition and structure of the fatty acid chains attached to the glucose moiety are determined based on the fragment ions corresponding to different fatty acid chains in the mass spectrometry information of the grandchild ion.

5. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 1, characterized in that, In step S1, the solvent used for extraction is an aqueous solution of acetonitrile with a volume concentration of 50-100%.

6. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 1, characterized in that, In step S2, the chromatographic mobile phase of hydrophilic interaction chromatography includes an aqueous phase and an organic phase.

7. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 6, characterized in that, The organic phase is acetonitrile or an acetonitrile solution containing a volatile basic modifier.

8. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 7, characterized in that, Volatile alkaline modifiers include ammonium hydroxide at a concentration of 0.01 to 0.1%.

9. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 6, characterized in that, The gradient elution conditions for hydrophilic interaction chromatography include: 0-10 min, 100% organic phase; 20-21 min, 70% organic phase; 21-28 min, 50% organic phase; 28.1-35 min, 100% organic phase.

10. The multi-stage mass spectrometry method for identifying the chemical structure of sucrose esters in tobacco and tobacco products according to claim 2, characterized in that, The scanning interval for secondary mass spectrometry is 10 to 15 m / z, and the scanning interval for tertiary mass spectrometry is 1 to 2 m / z.

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