Tea polyphenol and organic acid imaging analysis method based on combination of desorption electrospray ionization and Xevo G2-XS mass spectrometry

Abstract

The invention discloses an imaging analysis method for 15 polyphenols and organic acids in tea based on combination of desorption electrospray ionization and Xevo G2-XS mass spectrometry, which comprises the following steps: (1) sample pretreatment: cutting the tea into slices, and fixing the dried tea after moisture regaining and transfer printing treatment; (2) DESI-Xevo G2-XS combined detection: acquiring data through a DESI ion source and an Xevo G2-XS mass spectrometer; (3) imaging and qualitative analysis: extracting a characteristic ion signal, and generating a spatial distribution thermodynamic diagram; according to the method, qualitative identification and spatial distribution visual imaging of tea polyphenol are realized, 11 polyphenols and 4 organic acids can be imaged and analyzed at the same time, and technical support is provided for tea quality evaluation and processing technology optimization.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to an imaging analysis method for tea polyphenols and organic acids based on desorption electrospray ionization coupled with XevoG2-XS mass spectrometry. Background Technology

[0002] Polyphenols in tea are the core components determining its flavor, color, and health benefits. Their types, content, and spatial distribution are significantly influenced by the climate, soil characteristics, and cultivation techniques of the producing region. For example, the content of polyphenols such as catechins and epicatechins can vary by more than 30% in teas from different origins, and the distribution patterns of polyphenols in the tea peel, leaf tissue, and veins directly affect the release efficiency of active ingredients after brewing. Furthermore, organic acids in tea are key components regulating the acidity, freshness, and brewing endurance of tea. Their composition, proportion, and form are significantly affected by tea variety, harvesting season, and processing techniques. For example, the content of organic acids such as gallic acid and caffeoylquinic acid in tea can vary by more than 40%, and the distribution characteristics of organic acids in tea tissue directly affect the pH value and flavor harmony of the tea infusion. Therefore, achieving in-situ spatial imaging analysis of tea polyphenols and organic acids is crucial for tea quality analysis and industry promotion.

[0003] Currently, methods for detecting polyphenols and organic acids in tea have significant technical limitations: High-performance liquid chromatography (HPLC) requires complex pretreatment processes such as grinding, ultrasonic extraction, and solid-phase extraction, which is time-consuming and damages the tea's tissue structure, easily leading to the degradation of heat-sensitive polyphenols and making it impossible to obtain spatial distribution information; Conventional mass spectrometry, while possessing qualitative capabilities, lacks spatial resolution, making it difficult to distinguish the distribution differences of polyphenols and organic acids in different tissues of tea; The waxy layer and complex matrix on the surface of tea interfere with ionization efficiency, resulting in weak signals and high detection limits in traditional mass spectrometry imaging techniques, which cannot meet the needs of trace polyphenol and organic acid analysis.

[0004] Although recent studies have attempted to improve the process of in-situ ionization technology, reliable analytical methods have yet to be developed for optimizing the parameters of desorption electrospray ionization (DESI) coupled with Xevo G2-XS mass spectrometry, as well as for tea sample pretreatment and spatial imaging data processing. This limits the application and promotion of this technology. To address these issues, this invention achieves simultaneous spatial imaging distribution analysis of tea polyphenols and organic acids by innovating sample pretreatment, optimizing instrument parameters, and constructing a multi-dimensional analytical method. Summary of the Invention

[0005] The purpose of this invention is to provide an imaging analysis method for tea polyphenols and organic acids based on desorption electrospray ionization coupled with Xevo G2-XS mass spectrometry, solving the problems of complex pretreatment, lack of spatial information, and weak resistance to matrix interference in existing technologies. This method, through in-situ ionization-high-resolution mass spectrometry combined with optimized sample pretreatment and data processing workflows, achieves non-destructive in-situ detection, highly sensitive analysis, and spatial visualization of tea polyphenols and organic acids.

[0006] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows:

[0007] This invention provides a method for imaging and analyzing tea polyphenols based on desorption electrospray ionization coupled with Xevo G2-XS mass spectrometry, comprising the following steps:

[0008] (1) Sample pretreatment: Cut the tea sample into thin slices. For dry samples, perform atomization and rehydration transfer treatment first.

[0009] (2) DESI-Xevo G2-XS coupled detection: DESI ion source was used to ionize the fixed tea sample in situ, combined with Xevo G2-XS mass spectrometer to collect ion signals, and spatial distribution data of polyphenols and organic acids were obtained by two-dimensional scanning.

[0010] (3) Imaging and qualitative analysis: The characteristic ion signals of polyphenols and organic acids are extracted using mass spectrometry workstation software, and spatial distribution imaging maps are generated. The qualitative identification of polyphenols and organic acids is completed based on the characteristic mass-to-charge ratio.

[0011] Further, in step (1), the tea leaf slices are cut to a size of 1cm × 1cm; the atomization rehydration treatment uses deionized water atomization at a rate of 0.5-1mL / min and a treatment time of 30-60s; then the tea leaf slices are placed between two porous Teflon sheets, and pressure is applied to adsorb the metabolites in the tea leaf slices onto the porous Teflon sheets. After adsorption is complete, the porous Teflon sheets carrying the metabolites are directly placed on the DESI sample stage for analysis.

[0012] Further, the feature is that in step (2), the parameters of the DESI ion source are set as follows: the spray solvent is methanol-water with a volume ratio of 7:3 and a flow rate of 2-3 μL / min; the spray voltage is 3.5-4.5 kV; the ion source temperature is 35-45℃; the sampling cone voltage is 30-40 V; the angle between the nozzle and the sample surface is 30-45° and the distance is 2-3 mm.

[0013] Further, in step (2), the detection mode of the XevoG2-XS mass spectrometer is positive ion mode; the mass scan range is m / z 100-1000; the scan rate is 0.5-1s / time; the resolution is ≥10000FWHM; and the ion pairs of the target polyphenol are monitored using multiple reaction monitoring mode.

[0014] Further, in step (3), the qualitative analysis is based on the precise mass number of polyphenols, with a mass deviation ≤ 5 ppm; the extraction threshold for characteristic ion signals is set to 3 times the signal-to-noise ratio; the polyphenols include 11 characteristic components of tea, such as gallocatechin, catechin, epicatechin 3-O-gallate, epigallocatechin gallate, phensorinol-(4α→8)-catechin-3-O-gallate, 3-galloylgallocatechin, theaflavin C, proanthocyanidins B1, dipyranosyl trihydroxyflavanone, safflower disodium isomer, and kaempferol 3-O-galactosylrutin. The organic acids include four types: coumaroyl quinic acid, gallic acid, galloyl quinic acid, and caffeoyl quinic acid.

[0015] Furthermore, in step (3), the step size of the two-dimensional scan is 50-100μm, and mass spectrometry data is collected 1-2 times at each scanning point; the scanning area covers the entire thin slice of the tea sample.

[0016] Furthermore, in step (1), the atomization rehumidification treatment is carried out in an environment with a humidity of 60%-70% and a temperature of 25±2℃, and the rehumidification time is 2-3h.

[0017] Compared with the prior art, the present invention achieves the following beneficial technical effects:

[0018] This invention utilizes innovative in-situ mass spectrometry imaging technology to achieve non-destructive, high-sensitivity detection and spatial distribution visualization of tea polyphenols and organic acids. Without damaging the sample structure, it simultaneously completes qualitative and spatial imaging analysis of multiple polyphenol and organic acid components, effectively overcoming the technical bottlenecks of complex preprocessing and lack of spatial information in traditional methods. This provides comprehensive and reliable technical support for tea quality evaluation, origin traceability, and processing technology optimization. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Distribution imaging of coumaroyl quinic acid (m / z 337.0925). Figure 2 Gallacatechin (m / z 305.0666)

[0021] Figure 3 Gallic acid (m / z 169.0138)

[0022] Figure 4 Catechins (m / z 289.0723)

[0023] Figure 5 Galloylquinic acid (m / z 343.0667)

[0024] Figure 6 Caffeoylquinic acid (m / z 353.0883)

[0025] Figure 7 Epicatechin 3-O-gallate (m / z 441.0824)

[0026] Figure 8 : Table gallic acid catechin gallate (m / z 457.0771)

[0027] Figure 9 Proanthocyanidin B1 (m / z 577.1361)

[0028] Figure 10 : Diglucosyl trihydroxyflavone (m / z 595.1650)

[0029] Figure 11 Theaflavins C (m / z 609.1283)

[0030] Figure 12 : Safflower diglucoside isomer (m / z 611.1612)

[0031] Figure 13 : Feselonol-(4α→8)-catechin-3-O-gallate (m / z 713.1506)

[0032] Figure 14 Kaempferol 3-O-galactosylrutin (m / z 755.2045)

[0033] Figure 15 3-Galloylgallocatechin (m / z 915.1633) Detailed Implementation

[0034] 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.

[0035] This embodiment describes in detail a method for imaging analysis of tea polyphenols and organic acids based on desorption electrospray ionization coupled with Xevo G2-XS mass spectrometry, including the following steps:

[0036] Step 1: Sample Pretreatment

[0037] Three tea samples from different origins were selected (e.g., Yunnan Pu'er, Zhejiang Longjing, and Fujian Tieguanyin). For dried tea leaves, they were first rehydrated in a constant temperature and humidity chamber (65% humidity, 25℃) for 2.5 hours. Then, the tea leaves were cut into 1cm × 1cm slices along the veins. Fresh tea leaves were directly cut into the same size. The tea slices were placed between two porous Teflon sheets, and pressure was applied to adsorb metabolites from the tea slices onto the porous Teflon sheets. After adsorption, the porous Teflon sheets carrying the metabolites were directly placed on the DESI sample stage for analysis.

[0038] Step 2: DESI-Xevo G2-XS coupled detection

[0039] A system was set up to connect the DESI ion source and the Xevo G2-XS mass spectrometer, and the MassLynx workstation software was used for control and data acquisition. Key instrument parameter settings are as follows:

[0040] DESI ion source: spray solvent is methanol-water (99:1, v / v), flow rate is 2.5 μL / min; spray voltage is 4.0 kV; ion source temperature is 40℃; sampling cone voltage is 35 V; the nozzle and sample surface are at an angle of 40° and a distance of 2.5 mm; gas pressure is 0.45 MPa.

[0041] Xevo G2-XS mass spectrometer: scanning mode is positive ion mode; mass scan range m / z 100-1000; scan rate 0.8 s / s; resolution 12000 FWHM; adopts multiple reaction monitoring mode, and the collision energy is optimized for different polyphenols and organic acids (such as catechin 25 eV, epicatechin 28 eV and gallic acid 22 eV, etc.); dynamic exclusion time is set to 10 seconds.

[0042] First, a series of mixed working standard solutions of varying concentrations were tested, with each concentration repeated three times, to establish a standard curve. Then, a two-dimensional scan was performed on the fixed tea sample slices, with a scanning area of ​​0.8 cm × 0.8 cm and a step size of 80 μm. Data was collected twice at each scanning point, and each sample was tested three times.

[0043] Step 3: Imaging and Qualitative Analysis

[0044] The raw mass spectrometry data acquired in step two were imported into MassLynx software for preprocessing, including: removing baseline drift using linear baseline correction; setting a noise threshold of 3 times the signal-to-noise ratio to filter background interference; and using an automatic peak detection algorithm to identify target peaks with a minimum peak area of ​​1000. Based on the precise mass number of the standard (mass deviation ≤ 5 ppm), the characteristic mass-to-charge ratios of 15 target polyphenols and organic acids were determined, and the corresponding ion signals in the tea samples were extracted accordingly. Using the HighDefinition Imaging module, a two-dimensional spatial distribution heatmap of polyphenols and organic acids was generated using the "HotMetal" color scale, where red represents high-content areas and blue represents low-content areas. This step allows for a direct observation of the distribution differences of different polyphenols and organic acids in various tissues of tea (such as leaf veins, leaf mesophyll, and epidermis). The ion addition methods and characteristic ions corresponding to the 15 polyphenols and organic acids are shown in Table 1, and the distribution imaging maps of the 15 polyphenols and organic acids are shown in Table 2. Figures 1-15 .

[0045] Table 1. Mass spectrometry parameters and standard curve parameters of 15 tea polyphenols and organic acids

[0046]

[0047]

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for imaging analysis of 11 polyphenols and 4 organic acids in tea based on desorption electrospray ionization coupled with Xevo G2-XS mass spectrometry, characterized in that, Includes the following steps: (1) Sample pretreatment: The tea sample is cut into thin slices and then subjected to atomization transfer treatment. (2) DESI-Xevo G2-XS coupled detection: DESI ion source was used to ionize the fixed tea sample in situ, combined with Xevo G2-XS mass spectrometer to collect ion signals, and spatial distribution data of polyphenols and organic acids were obtained by two-dimensional scanning. (3) Imaging and qualitative analysis: The characteristic ion signals of polyphenols and organic acids are extracted using mass spectrometry workstation software, and spatial distribution imaging is generated. The qualitative identification of polyphenols is completed based on the characteristic mass-to-charge ratio.

2. The method as described in claim 1, characterized in that: In step (1), the tea slices are cut to a size of 1cm × 1cm; the atomization transfer process uses deionized water atomization at a rate of 0.5-1mL / min and a processing time of 30-60s; then the tea slices are placed between two porous Teflon sheets, and pressure is applied to adsorb the metabolites in the tea slices onto the porous Teflon sheets. After adsorption is complete, the porous Teflon sheets carrying the metabolites are directly placed on the DESI sample stage for analysis.

3. The method as described in claim 1, characterized in that: In step (2), the parameters of the DESI ion source are set as follows: the spray solvent is methanol-water with a volume ratio of 99:1 and a flow rate of 2-3 μL / min; the spray voltage is 3.5-4.5 kV; the ion source temperature is 35-45℃; the sampling cone voltage is 30-40 V; the angle between the nozzle and the sample surface is 30-45° and the distance is 2-3 mm.

4. The method as described in claim 1, characterized in that: In step (2), the Xevo G2-XS mass spectrometer is used in positive ion mode; the mass scan range is 100-1000 m / z; the scan rate is 0.5-1 s / s; the resolution is ≥10000 FWHM; and the multiple reaction monitoring mode is used to monitor the ion pairs of the target polyphenols and organic acids.

5. The method as described in claim 1, characterized in that: In step (3), the qualitative analysis is based on the precise mass numbers of polyphenols and organic acids, with a mass deviation ≤ 5 ppm; the extraction threshold for characteristic ion signals is set to 3 times the signal-to-noise ratio; the polyphenols include 11 characteristic components of tea, such as gallocatechin, catechin, epicatechin 3-O-gallate, epigallocatechin gallate, phensorinol-(4α→8)-catechin-3-O-gallate, 3-galloylgallocatechin, theaflavin C, proanthocyanidins B1, dipyranosyl trihydroxyflavanone, safflower disodium isomer, and kaempferol 3-O-galactosylrutin. The organic acids include 4 types: 3-coumaroylquinic acid, gallic acid, galloylquinic acid, and caffeoylquinic acid.

6. The method as described in claim 1, characterized in that: In step (3), the step size of the two-dimensional scan is 50-100μm, and mass spectrometry data is collected 1-2 times at each scanning point; the scanning area covers the entire thin slice of the tea sample.

7. The method as described in claim 1, characterized in that: In step (1), the atomization rehumidification treatment is carried out in an environment with a humidity of 60%-70% and a temperature of 25±2℃, and the rehumidification time is 2-3h.

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