Method for identifying acorus gramineus and acorus calamus based on MALDI-MSI technology

Abstract

The invention provides a method for identifying rhizoma acori graminei and rhizoma acori graminei based on an MALDI-MSI technology, and relates to the technical field of pharmaceutical analysis. The method comprises the following steps: preparing a frozen slice and coating the surface of the frozen slice with a matrix; scanning the frozen section in a positive ion mode to acquire mass spectrum imaging data; and identifying the spatial distribution characteristics of the characteristic ions in the region of interest. According to the method, a DHB matrix is coated with a sublimation method for MALDI-MSI imaging, sample swelling and migration are avoided, and it is ensured that in-situ information is real; based on the distribution difference of characteristic ions from a cambium to a center column, pretreatment and reference substances are not needed, the problem that the grass-leaved sweetflag rhizome and the Tibetan acorus calamus are similar in form and difficult to identify is solved, and objective and visual base source judgment is achieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and more specifically, to a method for identifying Acorus tatarinowii and Acorus calamus based on MALDI-MSI technology. Background Technology

[0002] Acorus ( Acorustatarinowii ) and Tibetan sweet flag ( Acoruscalamus Both *Acorus calamus* and *Acorus gramineus* (also known as sweet flag) belong to the genus *Acorus* in the family Araceae. They are extremely similar in plant morphology, growth environment, and medicinal properties, and are often confused in the medicinal materials market. However, they differ significantly in chemical composition and pharmacological effects, particularly in the types and content distribution of the main active ingredients (such as asarone compounds) in their volatile oils, leading to different standards for evaluating their clinical efficacy and safety. For example, excessively high levels of certain components in *Acorus gramineus* may pose potential safety risks. Therefore, accurate and rapid identification of these two closely related medicinal materials is of significant scientific importance and application value for ensuring the quality and safety of traditional Chinese medicine and the effectiveness of clinical use. Currently, the main methods for identifying *Acorus gramineus* and *Acorus gramineus* include traditional source identification, morphological identification, microscopic identification, and physicochemical identification. The identification of physical properties and microscopic features mainly relies on the experience of the examiners, observing morphological indicators such as the arrangement of vascular bundles and fiber bundle characteristics in the cross-section of the rhizome. However, in the state of medicinal material slices or powder, it is difficult to distinguish subtle morphological differences by the naked eye or microscope alone, making it highly subjective. In terms of physicochemical identification, techniques such as thin-layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC) are widely used to differentiate medicinal materials by detecting differences in the content of specific chemical components in the extract. However, the above-mentioned existing physicochemical analysis techniques usually require cumbersome pretreatment operations such as crushing, extraction, and separation of medicinal material samples. This process is not only time-consuming and consumes a lot of solvent, but more importantly, the homogenization of the sample (such as grinding into powder) destroys the original tissue structure of the plant, resulting in the complete loss of the in-situ spatial distribution information of chemical components in plant tissues and organs. Although the total content of components can be determined, it is impossible to know whether specific components (such as key secondary metabolites) are concentrated in specific microscopic parts such as the epidermis, cortex, or stele. In addition, existing mass spectrometry imaging techniques face challenges in sample pretreatment when applied to plant root and stem samples rich in starch or fiber. For example, conventional matrix spraying methods often involve the use of organic solvents, which can easily lead to the dissolution and diffusion (swelling effect) of the small molecules to be tested on the tissue surface, resulting in reduced imaging resolution or spatial localization distortion, making it difficult to obtain clear and true in-situ distribution images of chemical components.

[0003] In summary, traditional morphological identification methods lack objective quantitative indicators, while conventional chemical analysis methods, although accurate in both qualitative and quantitative analysis, lose spatial dimension information and cannot intuitively reveal the relationship between chemical composition and tissue structure. When faced with closely related species that are extremely similar in morphology and have similar chemical compositions, there is a lack of a technical means that can both maintain the integrity of plant tissues and capture the spatial distribution characteristics of characteristic differential components in situ, intuitively, and at high resolution, thereby achieving accurate species differentiation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying Acorus gramineus and Acorus tsao-ko based on MALDI-MSI technology. This method utilizes a sublimation method to coat a DHB matrix for MALDI-MSI imaging, avoiding sample swelling and migration, and ensuring the authenticity of in-situ information. Based on the distribution differences of characteristic ions from the cambium to the central column, no pretreatment or reference standards are required, thus overcoming the difficulty in differentiating Acorus gramineus and Acorus tsao-ko due to their similar morphology, and achieving objective and intuitive source determination.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for identifying Acorus gramineus and Acorus tsao-ko based on MALDI-MSI technology, comprising: preparing frozen sections of Acorus gramineus rhizome samples to be tested, and coating the surface of the frozen sections with 2,5-dihydroxybenzoic acid as a matrix using a sublimation method; using a matrix-assisted laser desorption / ionization mass spectrometry imaging system to scan the frozen sections after coating the matrix in positive ion mode and acquire mass spectrometry imaging data; taking the cambium to the central column region of the frozen sections as the region of interest, identifying the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 in the region of interest and obtaining the identification result. In some embodiments, the identification of the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 within the region of interest, and the determination of the identification result, includes: if the characteristic ion m / z 209.1172 is enriched within the region of interest, or if the characteristic ions m / z 201.1638 and m / z 219.1744 are scattered within the region of interest, then the rhizome sample of the *Acorus calamus* to be tested is identified as *Acorus calamus*; if the characteristic ion m / z 209.1172 is sparsely distributed within the region of interest, or if the characteristic ions m / z 201.1638 and m / z 219.1744 are enriched within the region of interest, then the rhizome sample of the *Acorus calamus* to be tested is identified as *Acorus calamus*. Preferably, the identification further includes: calculating the characteristic abundance ratio R, R = A / (B+C); where R represents the characteristic abundance ratio; A represents the average ionic intensity of the characteristic ion m / z 209.1172 in the region of interest; B represents the average ionic intensity of the characteristic ion m / z 201.1638 in the region of interest; and C represents the average ionic intensity of the characteristic ion m / z 219.1744 in the region of interest. If R reaches a first threshold, the sample is determined to be *Acorus calamus*; if R reaches a second threshold, the sample is determined to be *Acorus calamus*. In some embodiments, the thickness of the frozen section is 30 μm; and / or, the sublimation method includes: preparing a saturated solution of 2,5-dihydroxybenzoic acid in a 50% methanol aqueous solution, and then forming a uniform crystalline layer on the surface of the frozen section using a sublimation device; preferably, the 50% methanol aqueous solution also contains 0.2% trifluoroacetic acid; preferably, after forming a uniform crystalline layer on the surface of the frozen section using a sublimation device, the method further includes: drying the coated section in a vacuum desiccator for 3 hours. In some embodiments, the permissible mass deviation of the characteristic ions m / z 201.1638, m / z 209.1172, and m / z 219.1744 is 0.005.In some embodiments, the identification of the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 within the region of interest further includes: determining based on an ion intensity threshold: when the ion intensity of the characteristic ion m / z 209.1172 within the region of interest is not less than 15000, the rhizome sample of *Acorus calamus* to be tested is determined to be *Acorus calamus*; when the ion intensity of the characteristic ion m / z 209.1172 within the region of interest does not exceed 5500, the rhizome sample of *Acorus calamus* to be tested is determined to be *Acorus calamus*. In some embodiments, the use of a matrix-assisted laser desorption / ionization mass spectrometry imaging system to scan the frozen section coated with the matrix in positive ion mode, and the parameters for acquiring the mass spectrometry imaging data include: a laser beam diameter of 30 μm to 50 μm; and / or, a scanning interval of 50 μm; and / or, a detector voltage of 2.74 kV. In some embodiments, the preparation of cryosections of the *Acorus calamus* rhizome sample to be tested includes: embedding the *Acorus calamus* rhizome sample to be tested in carboxymethyl cellulose and cryosectioning it at -20°C. In some embodiments, the acquisition of mass spectrometry imaging data includes: acquiring mass spectrometry data in the mass range of m / z 100~500; and normalizing the total ion current of the acquired raw data.

[0006] Secondly, the present invention also provides a kit for implementing the identification method of *Acorus gramineus* and *Acorus calamus* based on MALDI-MSI technology as described in any of the foregoing embodiments, comprising: a carboxymethyl cellulose embedding agent for embedding plant rhizome samples; 2,5-dihydroxybenzoic acid as a laser desorption / ionization matrix; and a standard spectrum or instruction manual containing characteristic ions m / z 201.1638, m / z 209.1172, and m / z 219.1744; the instruction manual instructing identification to be performed by detecting the spatial distribution of the characteristic ions in the cambium to the central column region.

[0007] Thirdly, the present invention also provides a method for screening whether Acorus tatarinowii is adulterated with Acorus calamus, including: preparing a section and performing MALDI-MSI scanning on a test medicinal material sample; detecting ion images of characteristic ions m / z209.1172, m / z201.1638 and m / z219.1744; if regions with the characteristic distribution pattern of Acorus tatarinowii and regions with the characteristic distribution pattern of Acorus calamus are simultaneously observed in different regions or different particles of the test medicinal material sample, it is determined that there is adulteration in the test medicinal material sample; wherein, the characteristic distribution pattern of Acorus tatarinowii is determined based on the spatial distribution of the characteristic ion m / z209.1172, and the characteristic distribution pattern of Acorus calamus is determined based on the spatial distribution of the characteristic ion m / z201.1638 and / or m / z219.1744; preferably, the determination criteria for the characteristic distribution pattern include: the characteristic distribution pattern of Acorus tatarinowii shows that the characteristic ion m / z209.1172 is distributed with high abundance in the region from the cambium to the stele; and / or, the characteristic distribution pattern of Acorus calamus shows that the characteristic ion m / z201.1638 and / or m / z219.1744 is distributed with high abundance in the region from the cambium to the stele.

[0008] The present invention provides a method for identifying Acorus tatarinowii and Acorus calamus based on MALDI-MSI technology. The identification method utilizes matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) technology to achieve in-situ, rapid and accurate differentiation of the rhizomes of Acorus tatarinowii and Acorus calamus. By preparing cryosections and coating with 2,5-dihydroxybenzoic acid (DHB) matrix using the sublimation method, this method effectively avoids the swelling of the sample surface and the migration of the components to be detected caused by the introduction of solvent in the traditional spraying method, ensures the uniformity of matrix crystallization and the integrity of the tissue microstructure, so as to obtain high-fidelity in-situ chemical spatial distribution information, and significantly improves the spatial resolution of imaging and the reliability of detection data.

[0009] This method selects the region from the cambium to the stele as the key region of interest, and locks m / z201.1638, m / z209.1172 and m / z219.1744 as characteristic ions, and uses the significant differences in the enrichment or scattered distribution of these ions in specific anatomical parts of different species (such as the stele region) as the basis for identification. This identification mode based on spatial markers not only overcomes the difficulty of traditional character identification due to the highly similar appearance of the two Acorus plants, but also makes up for the defect that conventional physicochemical analysis (such as GC-MS, LC-MS) loses the spatial position information of chemical components due to the need for extraction and separation. Without complex sample pretreatment and reference standards, it can objectively and intuitively determine the origin of medicinal materials. Brief Description of the Drawings

[0010] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific 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 from these drawings without creative effort.

[0011] Figure 1 This is a schematic diagram of the anatomical structure of the cross-section of the rhizomes of Acorus gramineus and Acorus tatarinowii in Embodiment 1 of the present invention; Figure 2 This is a comparison of MALDI-MSI mass spectrometry images of characteristic ions in Acorus calamus and Acorus tatarinowii in Example 1 of the present invention; Figure 3 This is a comparison diagram of the effects of different substrate coating methods on imaging results in Example 3 of the present invention; Figure 4 The ion flow spectrum obtained during Gasbench-IRMS analysis in Example 3 of this invention; Figure 5 This is a comparison of the effects of different pretreatment processes on the imaging sensitivity of characteristic ions of Acorus tatarinowii in Example 7 of the present invention. The left image (control group) shows the imaging effect after simple sublimation treatment, with relatively weak and discrete signals. The right image (experimental group) shows the imaging effect after treatment with a combination of sublimation and vapor phase recrystallization. Under the same display threshold, the intensity of characteristic ion signals is significantly enhanced, and the tissue edge contour remains clear and sharp. Figure 6 This is a schematic diagram of the secondary mass spectrometry fragmentation pathway of the asarone isomers in Example 9 of the present invention; wherein - and -asarone mainly produce fragments at m / z 194 and 181, while -asarone specifically breaks to generate fragments at m / z 168; Figure 7 This is a comparison of secondary mass spectrometry (MS / MS) of the characteristic ion m / z 209.1172 in the samples of *Acorus tatarinowii* and *Acorus calamus* in Example 9 of this invention. (A) is the mass spectrum of the *Acorus tatarinowii* sample, clearly showing a high abundance characteristic peak at m / z 168.08, confirming the presence of α-asarone. (B) is the mass spectrum of the *Acorus calamus* sample; no obvious signal was detected at this position, mainly showing characteristic fragments of α-asarone at m / z 194 and 181. Detailed Implementation

[0012] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0013] This application provides a method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology, including: step S1, preparing frozen sections of the rhizome sample of Acorus calamus to be tested, and coating the surface of the frozen sections with 2,5-dihydroxybenzoic acid as a matrix by sublimation.

[0014] This step involves the physical morphological treatment of the sample and the application of chemical reagents (matrix). First, the plant rhizomes are processed into thin slices suitable for mass spectrometry detection, and then a specific chemical auxiliary agent (2,5-dihydroxybenzoic acid, i.e., DHB) is applied to the sample through physical sublimation. The specific processing may include: (1) embedding the sample with an embedding agent (such as CMC) and cutting the rhizomes into thin slices (e.g., 30 μm thick) using a cryostat at low temperature (e.g., -20°C). (2) using a sublimation device, under heating and vacuum conditions, the solid DHB matrix is ​​directly converted into a gaseous state and then condensed and deposited on the surface of the slice to form a uniform crystal layer. Through this step, a rhizome slice sample with a uniform DHB matrix crystal layer covering the surface can be obtained, while maintaining the original plant tissue structure.

[0015] The sublimation method described above is a key sample pretreatment technique used in this embodiment to uniformly coat the surface of plant tissue sections with a matrix. Its principle is to sublimate the solid matrix directly into a gaseous state by heating in a vacuum environment, which then condenses and deposits on the cooled sample surface to form a uniform crystalline layer. In establishing the pretreatment process, this embodiment compared the spraying method with the sublimation method. It was found that although the spraying method is simple to operate, the use of solvents can easily lead to droplet aggregation, which in turn causes swelling of the sample surface and spatial migration of the analyte, ultimately affecting the spatial resolution and accuracy of the imaging. In contrast, the sublimation method, as a dry processing method, can form a very uniform and dense crystalline layer on the tissue surface. During imaging, it causes almost no deformation of fragile plant tissue, and no hot spots or matrix aggregation were observed, thus effectively ensuring the consistency of ionization and the accuracy of the spatial distribution of the target components.

[0016] The selection of 2,5-dihydroxybenzoic acid (DHB) as the matrix is ​​crucial in MALDI-MSI technology, as the matrix is ​​a key factor in absorbing laser energy and assisting in the ionization of analyte molecules. Its selection directly determines the detection sensitivity and imaging quality. In this embodiment, a systematic matrix screening was conducted for the main active components (mostly small molecule metabolites) in *Acorus gramineus* and *Acorus tatarinowii*, comparing three commonly used matrices: DHB, CHCA, and SA. Experimental results showed that SA (sinapic acid) is more suitable for large protein molecules, but its response to the small molecule metabolites of interest in this study is weaker. While CHCA (cyano-4-hydroxycinnamic acid) can generate a strong signal, it is prone to uneven crystallization and local hot spots on the plant tissue surface, interfering with quantitative analysis. DHB (2,5-dihydroxybenzoic acid) performed best in terms of crystal uniformity, signal-to-noise ratio (SNR), and imaging stability, making it the most suitable for in-situ ionization and imaging analysis of small molecule metabolites in this study. Therefore, it was established as the standard matrix for this method.

[0017] Compared to other slicing methods, it can better preserve the plant tissue structure, and the 30 μm thickness achieves a balance between maintaining structural integrity and avoiding signal overlap. Compared to the spraying method, the sublimation method avoids the use of solvents, preventing swelling of the sample surface and migration (diffusion) of the analyte, thus ensuring the spatial resolution of the imaging and the authenticity of the in-situ information. Compared to other matrices (such as CHCA, SA), DHB has good crystal uniformity, high signal-to-noise ratio and good imaging stability when detecting small molecule metabolites of interest in this method.

[0018] In a further embodiment, after forming a uniform crystalline layer on the surface of the frozen section using the sublimation device, the method further includes: performing a vapor-phase recrystallization treatment on the section; the vapor-phase recrystallization treatment includes: placing the section coated with the matrix in a sealed container containing organic solvent vapor, and incubating it at 25℃-35℃ (for example, 25℃, 26℃, 28℃, 29℃, 30℃, 31℃, 32℃, 35℃, etc.) for 30 seconds to 90 seconds (for example, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, etc.); the organic solvent vapor is preferably methanol vapor or ethanol vapor.

[0019] In a preferred embodiment of the present invention, in order to further improve the detection sensitivity of mass spectrometry imaging, especially for the detection of low-abundance trace components, a gas-phase recrystallization process can be added after the sublimation coating matrix is ​​completed.

[0020] The specific procedure is as follows: Line the bottom of a sealed container (such as a petri dish or desiccator) with filter paper, and add a small amount of organic solvent (such as a 50% methanol aqueous solution or pure methanol) to create a saturated vapor environment inside the container. Place the sublimated slices horizontally in the container (avoiding direct contact with the liquid), and fumigate with the solvent vapor at room temperature (25℃-35℃) for 30 to 90 seconds.

[0021] The technical principle behind this processing step lies in the fact that while the matrix crystals formed by sublimation are uniform, their small lattice size limits their extraction efficiency for analyte molecules within the tissue. Controlled solvent vapor fumigation induces a microscopic dissolution and recrystallization process in these tiny matrix crystals. This process induces the more effective co-crystallization of small analyte molecules from the tissue surface into the matrix lattice, thereby significantly enhancing ionization efficiency and the final mass spectrometry signal intensity. Simultaneously, because only gas-phase fumigation is used instead of liquid-phase spraying, this step retains the advantages of dry processing, avoiding macroscopic swelling of the tissue surface or resolution reduction, achieving a balance between high sensitivity and high resolution.

[0022] Step S2: Using a matrix-assisted laser desorption / ionization mass spectrometry imaging system, the frozen section coated with the matrix is ​​scanned in positive ion mode to acquire mass spectrometry imaging data.

[0023] This step uses a MALDI-MSI instrument to acquire data from the pretreated sample. The sample is irradiated with a laser, causing the matrix and analyte molecules to desorb and ionize, and cations are detected under a positive electric field.

[0024] Specifically, instrument parameters (such as laser beam diameter, scanning interval, etc.) can be set, and the laser beam scans the slice surface point by point at a predetermined step size (such as 50m). The instrument operates in positive ion mode, recording the signal intensity of ions with different mass-to-charge ratios (m / z) at each pixel point, thereby generating a dataset (mass spectrometry imaging data) containing chemical composition information at each spatial location point on the slice, which can construct a spatial distribution image of specific ions.

[0025] This step eliminates the need for traditional extraction and separation procedures, directly providing tissue morphology information and spatial distribution characteristics of molecular composition. This mode is suitable for the ionization and detection of the selected characteristic ions (such as asarone compounds) and can obtain a strong signal response.

[0026] Step S3: Taking the region from the cambium layer to the central column of the frozen section as the region of interest, the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 in the region of interest are identified, and the identification result is obtained.

[0027] This is the data analysis and determination process. Instead of looking at the average signal of the entire slice, we focus on specific anatomical regions (from the cambium to the central column) and observe whether the substances corresponding to three specific molecular weights (m / z) are abundant or scarce in these regions to determine the species.

[0028] Specifically, the region from the cambium to the center point of the stele in the cross-section of the rhizome can be located as the region of interest (ROI) on the optical or ion image; then, the imaging images of the three ions m / z201.1638, m / z209.1172, and m / z219.1744 can be extracted.

[0029] The identification logic (algorithm logic) can be as follows: If m / z 209.1172 is detected: if the ion is mainly distributed in the ROI (cambium and stele) region and has a high intensity (e.g., intensity > 15000), it is identified as *Acorus gramineus*; if the ion has a low distribution in the ROI region (e.g., intensity < 5500), it is identified as *Acorus calamus*. If m / z 201.1638 or m / z 219.1744 is detected: if these ions are scattered in the ROI region and have a low intensity (e.g., intensity < 150 / 200), it is identified as *Acorus gramineus*; if these ions are enriched in the ROI region (cambium and stele) and have a high intensity (e.g., intensity > 1200 / 1400), it is identified as *Acorus calamus*.

[0030] Through the above identification, the conclusion can be clearly determined: the sample is either sweet flag or Tibetan sweet flag.

[0031] This step utilizes the concept of spatial markers, not only examining the presence or absence of components but also their location. This solves the problem of difficulty in distinguishing between two substances with extremely similar morphologies; these three ions form a complementary set of markers (some are highly concentrated in *Acorus calamus*, while others are highly concentrated in *Acorus tatarinowii*), improving the accuracy and robustness of identification. It visually reveals the precise differences in the distribution of active ingredients within tissues.

[0032] In summary, this embodiment provides a method for identifying *Acorus gramineus* and *Acorus tatarinowii* based on MALDI-MSI technology. Its significant advantages are as follows: First, this invention uses sublimation coating of 2,5-dihydroxybenzoic acid (DHB) as a matrix, effectively avoiding the swelling of plant tissue surfaces and the spatial delocalization of small molecule components caused by the introduction of organic solvents in traditional matrix spraying methods. This maximizes the preservation of the microstructural integrity of the rhizome slices and the authenticity of the in-situ chemical composition information. Second, this invention screens m / z 201.1638, m / z 209.1172, and m / z 219.1744 as key spatial characteristic ions and establishes the cambium to the central column region as the core observation area. By comparing the differences in the enrichment or scattered distribution of the above-mentioned characteristic ions in this specific anatomical region, this invention not only overcomes the technical bottleneck that makes it difficult to distinguish between Acorus tatarinowii and Acorus calamus by traditional morphological methods due to their extremely similar appearance, but also makes up for the defect of conventional physicochemical analysis (such as GC-MS and LC-MS) that completely loses the spatial location information of chemical components due to the need for pulverization and extraction. Without the need for complicated sample pretreatment and reference standard substances, it can achieve rapid, objective and visual accurate identification of the source of two closely related medicinal materials.

[0033] In a further embodiment, step S1, after preparing frozen sections of the calamus rhizome sample to be tested, and before coating the surface of the frozen sections with 2,5-dihydroxybenzoic acid, further includes: Silver ion derivatization step: Apply a silver ion reagent to the surface of the frozen section to cause the silver ion reagent to undergo a complexation reaction with the phenylpropanoid characteristic components in the frozen section; The phenylpropanoid characteristic component is an asarone compound; the identification of the characteristic ion includes: targeting the silver adduct ion [M+Ag] of the asarone compound. + Perform imaging analysis; The [M+Ag] + The silver adduct represents the addition ion formed by the asarone compound molecule M and the silver ion Ag; the silver adduct ion includes a mass-to-charge ratio of m / z 315.015 and / or m / z 317.015. It should be noted that, based on the above steps, the specific step of silver ion derivatization is introduced in this embodiment because of a deep understanding of the physicochemical properties of the key identification components in *Acorus tatarinowii* and *Acorus calamus*: asarone compounds. Asarone belongs to the phenylpropanoid volatile oil class and has strong volatility. Under the high vacuum (typically below 10) of MALDI mass spectrometry imaging... -6Under mbar detection conditions, analyte molecules readily sublimate and escape from the slice surface, leading to reduced detection sensitivity or even signal loss. Simultaneously, the unique side-chain carbon-carbon double bonds (C=C) in their structure offer the possibility of specific chemical labeling. While conventional matrix spraying or sublimation methods can achieve physical coating, they cannot solve the fundamental problem of vacuum evaporation. This invention utilizes silver ions (Ag... + The principle of pi-complexation between silver and double bonds anchors volatile small molecules in situ into non-volatile metal complexes. This not only physically prevents vacuum escape but also chemically utilizes the twin peak characteristics of silver isotopes to filter background noise and achieve dual qualitative confirmation. This design is not a simple superposition of conventional detection methods but a chemical enhancement strategy tailored to the specific properties of the analyte, thereby significantly improving the stability and accuracy of the identification method.

[0034] In this embodiment, considering that the phenylpropanoid characteristic components (mainly volatile oil components such as -asarone and -asarone) in Acorus tatarinowii and Acorus calamus, which are key identification criteria, are volatile and contain allyl or propenyl side chains (i.e., C=C unsaturated double bonds) in their chemical structure, this invention introduces a silver ion derivatization step based on the conventional sublimation method.

[0035] The procedure for this step may include: after preparing frozen sections of plant rhizomes, and before coating the DHB matrix using the sublimation method, uniformly spraying a small amount of silver ion reagent onto the surface of the sections using an automated spraying device (such as a spraying device, atomizing device, etc.). Preferred reagents may include: a solution of silver nitrate (AgNO3) dissolved in anhydrous ethanol or methanol, preferably at a concentration of 1 mg / mL to 5 mg / mL. Spraying control: control the number of spraying cycles to ensure that the silver ion reagent forms a monolayer covering the tissue surface, avoiding excessive solvent that could cause tissue wetting or swelling. The reaction mechanism (complexation reaction) may include: silver ions (AgNO3)... + α-H is a typical alkenophile reagent with empty electron orbitals, readily undergoing a specific pi-complexation reaction with the carbon-carbon double bond (pi electron donor) on the side chain of asarone ether molecules (M). This reaction is instantaneous at room temperature, forming a stable silver adduct ion complex. In subsequent mass spectrometry imaging, the protonated molecular peak [M+H] at m / z 209.1172 is no longer the primary focus of observation. + Instead, it locks in the adduct ion [M+Ag] formed by the asarone molecule (molecular weight approximately 208.11 Da) and silver ions. +Since natural silver has two stable isotopes with an abundance of approximately 1:1 (107Ag and 109Ag), the target signal will split into a pair of characteristic twin peaks in the mass spectrum: (1) m / z 315.015 (corresponding to the adduct ion formed by molecule M and the silver isotope with a mass number of 107); (2) m / z 317.015 (corresponding to the adduct ion formed by molecule M and the silver isotope with a mass number of 109).

[0036] Asarone, a volatile oil, readily evaporates and escapes in the high-vacuum chamber of a MALDI mass spectrometer, leading to signal attenuation or loss. By complexing with silver ions, it is converted into a non-volatile metal complex salt, significantly improving the stability of the analyte molecule under vacuum conditions and thus dramatically enhancing detection sensitivity. Starch, cellulose, and other metabolites without double bonds, abundant in rhizome tissue, cannot bind to silver ions. Therefore, this method is equivalent to adding a chemical filter to mass spectrometry, selectively enhancing only the signals of characteristic components containing double bonds, and significantly reducing chemical background noise interference. The detected twin peak phenomenon (i.e., the simultaneous appearance of m / z 315 and 317 with similar intensities) provides isotopic distribution fingerprint evidence in addition to the precise mass number. This gives the identification results extremely high confidence, completely eliminating interference from other isomers or false positives, and is crucial for confirming the source of *Acorus gramineus*.

[0037] In some embodiments, step S3, which identifies the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 within the region of interest and obtains an identification result, includes: step S31, if the characteristic ion m / z 209.1172 is enriched within the region of interest, or if the characteristic ions m / z 201.1638 and m / z 219.1744 are scattered within the region of interest, then the rhizome sample of the calamus to be tested is determined to be *Acorus calamus*.

[0038] This is a positive criterion for identifying a sample as Acorus tatarinowii. The chemical fingerprint characteristics of Acorus tatarinowii in a specific anatomical region (from the cambium to the stele) are defined. Here, enriched distribution refers to high ion signal intensity and dense pixel density; scattered distribution refers to low ion signal intensity and sparse pixel density or near-background noise.

[0039] Specifically, the processing can include the following steps: First, software can generate single-ion images (MSI images) for m / z 209.1172, m / z 201.1638, and m / z 219.1744 based on the collected data. Then, the focus is placed on a predefined region of interest (ROI) in the image, namely the cambium and stele in the center of the rhizome section. Further logical judgment is performed, observing m / z 209.1172: checking whether this ion exhibits a continuous, bright, and dense distribution within the ROI. Observing m / z 201.1638 and m / z 219.1744: checking whether these two ions exhibit a discontinuous, dim, and sparse distribution within the ROI. Through the above logical judgment of the image, the following result can be obtained: if any of the above distribution characteristics are met (high abundance of m / z 209 or low abundance of m / z 201 / 219), the identification conclusion is output: the sample is *Acorus gramineus*.

[0040] This step is highly specific; m / z 209.1172 (corresponding to asarone compounds) is specifically highly expressed in the midstem region of *Acorus gramineus*, making it a key chemical marker distinguishing it from *Acorus calamus*. It also has a high tolerance for error, employing an OR logic: either high expression of m / z 209 or low expression of m / z 201 / 219 is detected, and a judgment can be made. This multi-indicator verification method reduces the risk of misjudgment due to fluctuations in a single ion signal. Specifically, for example, a pseudo-color heatmap generated by mass spectrometry imaging software can be observed visually, with red / highlight representing enrichment and blue / black representing dispersion. A relative intensity threshold can be set (e.g., 0.1% of the base peak intensity or a specific value such as 15000). An average intensity within the ROI above the threshold is considered enrichment, and below the threshold is considered dispersion.

[0041] Step S32: If the characteristic ion m / z 209.1172 is sparsely distributed in the region of interest, or the characteristic ions m / z 201.1638 and m / z 219.1744 are enriched in the region of interest, then the rhizome sample of the calamus to be tested is determined to be calamus.

[0042] This is the criterion for identifying the sample as *Acorus calamus*, whose chemical distribution characteristics are completely opposite to those of *Acorus gramineus*. It utilizes the high accumulation characteristics of certain terpenoids or ethers (m / z 201 / 219) in the central column region of *Acorus calamus*, and the low accumulation characteristics of m / z 209. The processing procedure can be based on the generated single-ion imaging map and the selected region of interest (ROI). The logical judgment can be: observe m / z 209.1172 and check whether the signal of this ion within the ROI is weak, discontinuous, or only distributed in non-ROI regions (such as the epidermis). Observe m / z 201.1638 and m / z 219.1744 and check whether these two ions exhibit high intensity and large-area coverage (enrichment) within the ROI. The result is that if either of the above distribution characteristics is met (low abundance of m / z 209 or high abundance of m / z 201 / 219), the identification conclusion is output: the sample is *Acorus calamus*. In this step, the high expression of m / z 201.1638 and m / z 219.1744 in the cylinder of *Acorus calamus* contrasts sharply with that in *Acorus gramineus* (negative correlation), forming a dual verification mechanism and ensuring the accuracy of identification. Even if the absolute content of samples fluctuates due to different origins, this relative distribution pattern, where one increases while the other decreases, is usually species-specific and highly robust.

[0043] For example, similar to the previous feature, ion intensity data of the ROI region is extracted using software. If the average intensity of m / z 201.1638 or m / z 219.1744 is significantly higher than the background or higher than a set high threshold (e.g., 1200 / 1400), while the intensity of m / z 209.1172 is significantly lower than a set threshold (e.g., 5500), then it is determined to meet the characteristics of Acorus calamus.

[0044] In some embodiments, the thickness of the cryosection is 30 μm. This refers to the fact that when preparing tissue sections of the rhizomes of *Acorus gramineus* or *Acorus calamus*, the slice thickness parameter set by the cryostat is precisely limited to 30 micrometers. The process can be as follows: the *Acorus gramineus* sample, after being embedded in CMC and frozen, is placed in a cryostat, the slice thickness parameter is adjusted to 30 μm for slicing, and then the cut tissue sections are transferred onto a conductive glass slide to obtain a rhizome cross-section sample with uniform thickness and intact structure, which is used for subsequent matrix coating and mass spectrometry imaging.

[0045] This thickness is the optimal parameter optimized through experiments. Experiments show that if the slice is too thin (e.g., 20 μm), the plant tissue is prone to wrinkling or damage, which is not conducive to imaging and recognition; if the slice is too thick (e.g., 40 μm), although it is intact, it will affect the ionization efficiency, resulting in some ion imaging showing tailing and spatial signal overlap. The thickness of 30 μm combines the stability of tissue structure and signal quality, ensuring clear imaging results.

[0046] In some embodiments, the sublimation method includes: preparing a saturated solution of 2,5-dihydroxybenzoic acid in a 50% methanol aqueous solution, and then using a sublimation apparatus to form a uniform crystalline layer on the surface of the frozen section. Preferably, the 50% methanol aqueous solution also contains 0.2% trifluoroacetic acid. Preferably, after forming a uniform crystalline layer on the surface of the frozen section using the sublimation apparatus, the method further includes: placing the substrate-coated section in a vacuum desiccator for drying for 3 hours.

[0047] This invention employs an optimized sublimation method for matrix coating. The specific process is as follows: First, a 50% methanol aqueous solution containing 0.2% trifluoroacetic acid (TFA) is prepared as a solvent to dissolve 2,5-dihydroxybenzoic acid (DHB) to saturation. The addition of TFA helps optimize the matrix crystallization environment and subsequent ionization efficiency. Then, using a sublimation apparatus under heating and vacuum conditions, the solid DHB is directly vaporized and uniformly condensed and deposited onto the surface of the frozen section. Compared to traditional spraying methods, this sublimation process avoids sample surface swelling caused by solvent droplet aggregation, effectively preventing spatial migration of the analyte, thus achieving high spatial resolution and high accuracy imaging while ensuring no deformation of the tissue structure. After coating, the sections are placed in a vacuum desiccator for 3 hours to thoroughly remove residual moisture or trace amounts of solvent, ensuring matrix crystal stability and preventing moisture interference with the ionization process under the high vacuum environment of the mass spectrometer, thereby guaranteeing the quality of the imaging data.

[0048] In some embodiments, the permissible mass deviation of the characteristic ions m / z 201.1638, m / z 209.1172 and m / z 219.1744 is 0.005.

[0049] The above defines the precision of ion identification in mass spectrometry imaging. It specifies the mass tolerance window used by the system to determine whether a detected signal belongs to the target characteristic ion during data acquisition or subsequent image reconstruction. That is, as long as the mass-to-charge ratio of the detected ion falls within the target value plus or minus 0.005 Da, it is identified as the characteristic ion.

[0050] When processing data using a high-resolution mass spectrometer (such as Q-TOF), parameters can be set via software, using m / z 201.1638, m / z 209.1172, and m / z 219.1744 as center values, and setting the extraction window width to 0.005. For example, for m / z 209.1172, the system will automatically filter and extract all ion signals within the precise range of m / z 209.1122 to m / z 209.1222, summing them to construct an imaging map, while excluding signals outside this range. This yields highly specific ion signal data, generating a mass spectrometry image that accurately reflects the true distribution of target chemical components (such as asarone compounds) in plant tissues, while simultaneously eliminating background interference from non-target substances with similar mass numbers.

[0051] The narrow window setting of 0.005 Da fully leverages the advantages of high-resolution mass spectrometry, effectively eliminating interference from isotopes or background noise and preventing false positives caused by the incorporation of interfering peaks. This deviation range also accommodates the slight mass axis shift that may occur during long-term scanning, ensuring stable capture of target characteristic ions in different batches of experiments and avoiding signal loss due to excessively narrow settings.

[0052] In some embodiments, step S3, which identifies the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 in the region of interest, further includes step S33, which makes a determination based on an ion intensity threshold.

[0053] This feature refers to the introduction of quantitative numerical standards based on the qualitative observation of ion spatial distribution characteristics (enrichment or dispersion). Specifically, it uses the absolute intensity of the signal detected by mass spectrometry as a specific quantitative indicator to assist or confirm the identification results. After acquiring mass spectrometry imaging data, a specific region of interest (ROI) from the cambium to the central column is selected using data processing software. The average mass spectrometry signal intensity of the characteristic ion at m / z 209.1172 within this region is calculated, thus obtaining a specific numerical value representing the relative content or abundance of this chemical component in a specific tissue site.

[0054] This step transforms enriched or fragmented visual judgments into quantifiable digital standards, eliminating subjective errors from human image observation. Furthermore, it establishes clear quality control indicators, making the identification process easier to standardize across different batches of samples. Specifically, the ROI statistical function of mass spectrometry imaging analysis software (such as IMAGEREVEAL) can be used to delineate the region from the cambium layer to the central column, automatically outputting the average intensity value of the target ions.

[0055] Step S331: When the ion intensity of the characteristic ion m / z 209.1172 in the region of interest is not less than 15000, the rhizome sample of the calamus to be tested is determined to be calamus.

[0056] This is the quantitative identification limit set for *Acorus gramineus*. m / z 209.1172 (corresponding to asarone compounds) is a high-abundance characteristic component of *Acorus gramineus* in the cambium and stele regions. Specifically, the ionic intensity value of m / z 209.1172 within the ROI region can be compared with a preset threshold of 15000. If the value is greater than or equal to 15000, combined with spatial distribution characteristics, the sample is confirmed as *Acorus gramineus*. Based on statistical analysis of a large number of samples, the ionic intensity of *Acorus gramineus* in this region is significantly higher than this threshold. Setting this high threshold can effectively distinguish genuine *Acorus gramineus*, prevent misjudgment due to impurities or background noise, and ensure the accuracy of identification.

[0057] Step S332: When the ion intensity of the characteristic ion m / z 209.1172 in the region of interest does not exceed 5500, the rhizome sample of the calamus to be tested is determined to be Tibetan calamus.

[0058] This is the upper limit for quantitative identification of *Acorus calamus*. In the cambium and stele region of *Acorus calamus*, the content of the component m / z 209.1172 is extremely low or only present in trace amounts. Specifically, the ionic intensity value of m / z 209.1172 within the ROI region can be compared with the preset threshold of 5500. If the value is less than or equal to 5500, combined with the spatial distribution characteristics, the sample is confirmed to be *Acorus calamus*.

[0059] There is a huge numerical gap between the lower limit of Acorus gramineus (15000) and the upper limit of Acorus gramineus (5500). This significant statistical difference (p<0.05) makes the identification results highly confident. Setting the upper limit of 5500 allows for a certain degree of background signal, but can effectively exclude Acorus gramineus samples with high content, thus accurately identifying Acorus gramineus.

[0060] In a further embodiment, the identification also includes: step S333, calculating the characteristic abundance ratio R, R = A / (B+C); where R (Ratio): represents the characteristic abundance ratio, a dimensionless relative numerical index. A: represents the average ionic intensity value of the *Acorus gramineus*-specific characteristic ion m / z 209.1172 in the region of interest (from the cambium to the central pillar). This ion is highly expressed in *Acorus gramineus* and low expressed or at background levels in *Acorus tatarinowii*. B: represents the average ionic intensity value of the *Acorus tatarinowii*-specific characteristic ion m / z 201.1638 in the region of interest. C: represents the average ionic intensity value of the *Acorus tatarinowii*-specific characteristic ion m / z 219.1744 in the region of interest. Note: B and C are denominators, representing low expression in *Acorus gramineus* and high expression in *Acorus tatarinowii*.

[0061] Step S334: If R is the first threshold, then the sample is determined to be Acorus calamus.

[0062] In this embodiment, the first threshold can be set to 10 (preferably within the range of 8-15). This setting is based on the fact that in typical *Acorus calamus* samples, the intensity of the numerator term A is extremely high (typically >15000), while the intensity of the denominator term (B+C) is extremely low (typically <500), and the calculated R value is usually above 30. Setting the first threshold to 10 allows for sufficient safety margin; as long as the R value is greater than 10, it indicates that the numerator term (*Acorus calamus* characteristics) is absolutely dominant, confirming it as *Acorus calamus*.

[0063] Step S335: If R is the second threshold, then the sample is determined to be Acorus calamus.

[0064] In this embodiment, the second threshold can be set to 2 (preferably within the range of 1 to 3). The basis for this setting is that in typical *Acorus calamus* samples, the intensity of the numerator term A is significantly suppressed or merely background noise (typically <5500), while the denominator term (B+C) exhibits high abundance (typically >2600), and the calculated R value is typically less than 1 (e.g., between 0.5 and 2). Setting the second threshold to 2 indicates that the sum of the intensities of the denominator term (*Acorus calamus* characteristics) is close to or exceeds that of the numerator term in magnitude, confirming it as *Acorus calamus*.

[0065] The technical advantage of the ratio method (R-value) described above lies in the fact that, compared to the absolute intensity threshold of a single ion (such as a fixed value of 15000), the R-value uses internal ions within the same scan for self-correction. When the overall instrument sensitivity decreases, the numerator A and the denominator (B+C) decrease proportionally, resulting in a relatively constant R-value. Therefore, this method has extremely high robustness and instrument universality, and can obtain consistent identification results even in different laboratories or on different models of MALDI-MS instruments.

[0066] In some embodiments, the matrix-assisted laser desorption / ionization mass spectrometry imaging system scans the frozen section coated with the matrix in positive ion mode, and the parameters for acquiring mass spectrometry imaging data include: a laser beam diameter of 30m to 50m (e.g., 30m, 40m, 50m, etc.); and / or a scanning interval of 50m; and / or a detector voltage of 2.74kV.

[0067] The aforementioned 50m scanning interval matches the cellular scale of plant root and stem tissues, enabling clear differentiation of anatomical structures such as the epidermis, cortex, and stele, while avoiding excessive data volume that could lead to slow processing. The 2.74kV detection voltage and appropriate laser diameter ensure that the analyte (such as asarone) generates a sufficiently strong ion current signal, preventing missed detections.

[0068] In some embodiments, the preparation of cryosections of the *Acorus calamus* rhizome samples includes: embedding the *Acorus calamus* rhizome samples using carboxymethyl cellulose (CMC) and then cryosectioning them at -20°C. Compared to OCT embedding agents commonly used in animal tissues, CMC is more suitable for plant rhizome samples, as it effectively fills interstitial spaces to provide support and exhibits less background interference in subsequent mass spectrometry analysis. -20°C is the optimal temperature range for cutting plant rhizomes (which typically have high water content and are rich in starch / fiber). Too low a temperature will cause the sample to become too brittle and fragile, while too high a temperature will make it too soft to form sections. This temperature setting ensures the successful preparation of 30µm sections.

[0069] In some embodiments, the acquisition of mass spectrometry imaging data includes: acquiring mass spectrometry data with a mass range of m / z 100 to 500; and normalizing the total ion current of the acquired raw data. The aforementioned acquisition of mass spectrometry data with a mass range of m / z 100 to 500 is a parameter limitation on the mass scanning window during mass spectrometry data acquisition. This limitation restricts the mass spectrometer to only covering ion signals with a mass-to-charge ratio (m / z) in the specific range of 100 to 500 Daltons (e.g., 100, 200, 300, 400, 500, etc.) during signal recording.

[0070] Specifically, before starting the scan, the acquisition parameters can be set through the control software of the mass spectrometer (such as AP-MALDI-Q-TOF-MS), locking the scanning range of the mass analyzer between m / z 100 and 500. During laser desorption / ionization, the instrument only records the time-of-flight data of ions falling within this range and converts it into a mass spectrum, thus obtaining raw mass spectrometry data containing information about the target small molecule metabolites, while excluding interference from low-mass fragments with a mass number less than 100 and interference from large molecules with a mass number greater than 500 (such as lipids, peptides, etc.).

[0071] The three key identification markers (m / z 201.1638, m / z 209.1172, and m / z 219.1744) selected in this application are all distributed around m / z 200. Setting the range to 100-500 can fully cover all potential characteristic ions, ensuring the effectiveness of data acquisition; narrowing the scan range helps to improve the instrument's sensitivity and duty cycle in specific intervals, avoids the acquisition of irrelevant high molecular weight data, thereby reducing the size of data files and speeding up subsequent processing.

[0072] The total ion current (TIC) normalization process described above is a standardization preprocessing algorithm for the raw mass spectrometry data. TIC normalization refers to the process of eliminating differences in overall signal intensity between different pixels or different samples caused by non-biological factors.

[0073] Using data analysis software (such as IMAGEREVEALMS), for each pixel in the imaging image, the sum of the intensities of all ions in the mass spectrum at that point (i.e., the total ion current (TIC)) is calculated. Then, the intensity of each specific ion (e.g., m / z 209.1172) within that pixel is divided by the TIC value of that point to obtain a normalized relative intensity value. This generates a standardized data matrix after eliminating systematic errors, making data from different batches, different slices, and even different regions of the same slice comparable across different regions.

[0074] This effectively eliminates signal intensity fluctuations caused by uneven matrix crystallization, differences in slice surface flatness, minor fluctuations in laser energy, or changes in instrument status; this is a prerequisite for making judgments based on ion intensity thresholds (such as the thresholds of 15000 or 5500 mentioned in the aforementioned embodiments). Only after TIC normalization are the numerical comparisons of absolute intensities statistically significant, thus ensuring the robustness and repeatability of the identification results. For example, in the preprocessing module of the data processing software, the normalization option can be selected, and the algorithm can be specified as TIC (Total Ion Current) or RMS (Root Mean Square). The software will automatically process all spectra.

[0075] In this application embodiment, a kit is also provided for implementing the identification method of *Acorus gramineus* and *Acorus calamus* based on MALDI-MSI technology as described in any of the foregoing embodiments, comprising: (1) a carboxymethyl cellulose embedding agent for embedding plant rhizome samples; (2) 2,5-dihydroxybenzoic acid as a laser desorption / ionization matrix; and (3) a standard spectrum or instruction manual containing characteristic ions m / z 201.1638, m / z 209.1172, and m / z 219.1744; the instruction manual instructing identification to be performed by detecting the spatial distribution of the characteristic ions in the cambium to the central column region.

[0076] Carboxymethyl cellulose (CMC) is formulated to encapsulate and support plant root and stem samples during cryosectioning. CMC exhibits excellent water solubility and film-forming properties, rapidly solidifying at low temperatures to fill irregular voids in plant tissues. It provides a uniform external support medium for hard or complex root and stem tissues, ensuring sample integrity and preventing breakage or deformation during thin-layer cutting in a cryostat. Compared to OCT embedding agents commonly used in animal tissues, CMC is more suitable for plant roots and stems with high water content and rich fiber. It not only effectively fixes the sample but also minimizes interference from background mass spectrometry signals (due to the low background noise of CMC in MALDI mass spectrometry), thereby improving the signal-to-noise ratio of subsequent imaging analysis.

[0077] 2,5-Dihydroxybenzoic acid (DHB) is a chemical matrix material specifically used in matrix-assisted laser desorption / ionization (MALDI) technology. As a matrix, DHB's main function is to absorb laser energy and transfer it to the analyte molecules, assisting in the soft ionization of these molecules (especially small molecule metabolites), thus enabling their detection in a mass spectrometer. This application screened matrices for active small molecule components in *Acorus calamus* and *Acorus tatarinowii*, and the results showed that DHB was superior to CHCA (cyano-4-hydroxycinnamic acid) and SA (sinapic acid). DHB can form a more uniform and stable crystal layer, and it exhibits higher ionization efficiency and signal-to-noise ratio for small molecule metabolites within the specific mass-to-charge ratio range (m / z 100-500) of interest in this method, thereby ensuring the accuracy of the identification results.

[0078] The standard spectra or instruction manual containing the characteristic ions m / z 201.1638, m / z 209.1172, and m / z 219.1744 are the information carriers in the kit, providing core reference data and operational guidelines for identification. It clearly lists the three key mass-to-charge ratio values ​​(m / z 201.1638, m / z 209.1172, m / z 219.1744) and may include example images (standard spectra) of the typical distribution of these ions in *Acorus gramineus* and *Acorus calamus*. This instruction manual or spectra serves as an identification standard. It instructs users which specific molecular signals to focus on after mass spectrometry imaging and how to observe the distribution characteristics of these signals in specific anatomical regions (cambium to column). These three characteristic ions constitute a complementary combination of spatial chemical markers. The instruction manual transforms complex scientific data into concise and easy-to-understand operating instructions, enabling testing personnel to quickly determine the authenticity (source) of medicinal materials simply by comparing the detected ion distribution diagrams according to the instructions, without needing a deep background in phytochemistry. This standardized guidance significantly lowers the operational threshold and improves identification efficiency and result consistency.

[0079] This application embodiment also provides a method for screening whether Acorus tatarinowii is adulterated with Acorus calamus, including: step S100, preparing a slide and performing MALDI-MSI scanning on the sample of the medicinal material to be tested.

[0080] This step is a pretreatment and data acquisition step for medicinal material samples that may be mixed (adulterated). The object is no longer a pure product from a single source, but a mixture that may contain fragments, granules or slices of Acorus tatarinowii and Acorus calamus. Specifically, it may include, but is not limited to, the following steps: (1) Slide preparation: Randomly select multiple particle or slice samples from the medicinal material to be tested (such as slices or powder), embed them with CMC embedding agent, prepare frozen slices at low temperature, and coat them with DHB matrix by sublimation. (2) Scanning: Place the prepared slices containing multiple sample cross sections into the mass spectrometer, set the parameters (such as m / z 100-500 range, 50m step size), and perform high-throughput mass spectrometry imaging scans on all sample areas. Through the above steps, a complete mass spectrometry imaging dataset containing chemical information of multiple medicinal material particle / slice cross sections can be obtained. MALDI-MSI has high-throughput characteristics, and can acquire the chemical spatial information of multiple independent samples at the same time in one scan, without the need to extract each particle separately, which greatly improves the screening efficiency.

[0081] Step S200: Detect ion images of characteristic ions m / z 209.1172, m / z 201.1638 and m / z 219.1744.

[0082] This describes the data extraction and visualization process. The focus is on the distribution of the three key identification markers determined by this method across all sample particles. Specifically, imaging software can be used to extract the ion distribution maps for the three mass-to-charge ratios (m / z 209.1172, m / z 201.1638, and m / z 219.1744), generating three ion distribution heatmaps that visually display the location and abundance of these three components on the cross-section of each medicinal material particle.

[0083] Step S300: If, in different regions or different particles of the medicinal material sample to be tested, regions with the characteristic distribution pattern of Acorus tatarinowii and regions with the characteristic distribution pattern of Acorus tatarinowii are observed simultaneously, then it is determined that adulteration exists in the medicinal material sample to be tested; wherein, the characteristic distribution pattern of Acorus tatarinowii is determined based on the spatial distribution of the characteristic ion m / z 209.1172, and the characteristic distribution pattern of Acorus tatarinowii is determined based on the spatial distribution of the characteristic ions m / z 201.1638 and / or m / z 219.1744.

[0084] This is a logical judgment step based on image features. Adulteration refers to the discovery of both genuine *Acorus calamus* (genuine product) and adulterated *Acorus calamus* (counterfeit product) within the same batch of samples. Specifically, this may include: examining each individual particle / slice region in the image; classifying each particle; some particles exhibiting high abundance at m / z 209 (consistent with *Acorus calamus* characteristics), while others exhibit high abundance at m / z 201 / 219 (consistent with *Acorus calamus* characteristics). If both types of particles are present in the field of view, or if two distinctly different chemical distribution patterns are detected in the same sample pile, the conclusion that the sample is adulterated can be drawn.

[0085] This step enables in-situ screening of mixed samples. Traditional physicochemical methods (such as HPLC) involve pulverizing and mixing the sample before measuring the total amount. If the adulteration ratio is low, characteristic peaks may be diluted or masked, making them difficult to detect. This method, however, can precisely pinpoint the specific adulterant particle, achieving single-particle-level identification with extremely high sensitivity and accuracy. The above-mentioned *Acorus calamus* pattern uses m / z 209.1172 (asarone ethers) as the core indicator. The above-mentioned *Acorus calamus* pattern uses m / z 201.1638 and m / z 219.1744 (terpenoids / ethers) as core indicators. This establishes mutually exclusive and complementary discrimination criteria, ensuring the uniqueness and exclusivity of identifying the two species.

[0086] In a preferred embodiment, the criteria for determining the characteristic distribution pattern include: the characteristic distribution pattern of *Acorus gramineus* is characterized by: the characteristic ion m / z 209.1172 exhibiting high abundance in the cambium to the stele region; and / or, the characteristic distribution pattern of *Acorus calamus* is characterized by: the characteristic ions m / z 201.1638 and / or m / z 219.1744 exhibiting high abundance in the cambium to the stele region.

[0087] The above criteria provide the most intuitive and typical visual description of the feature distribution pattern. *Acorus gramineus*: Bright central region (stem) (strong m / z 209 signal). *Acorus calamus*: Bright central region (stem) (strong m / z 201 or 219 signal). Specifically, a pseudo-color image can be generated using software, with red representing high abundance. If m / z 209 is red in the stele region of a particle, it is *Acorus gramineus*; if m / z 201 is red in the stele region of another particle, it is *Acorus calamus*. Utilizing the significant feature of high abundance in the stele region makes the discrimination process simple, fast, and intuitive, requiring no complex statistical calculations. Ordinary testing personnel can make a judgment simply by observing the image with the naked eye.

[0088] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1: Identification Method of Acorus gramineus and Acorus calamus based on MALDI-MSI technology: This example provides a standard operating method for identifying Acorus gramineus and Acorus calamus using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) technology, specifically including the following steps: 1. Experimental Materials and Instruments: Eighteen batches of rhizome samples (numbered S01~S18) of *Acorus gramineus* and eighteen batches of rhizome samples (numbered Z01~Z18) of *Acorus calamus*, identified by experts, were selected as experimental samples for establishing the method. The main instruments and reagents included: iMScope QT imaging mass spectrometry microscope (Shimadzu Corporation, Japan); Leica CM1950 cryostat (Leica Microsystems); carboxymethyl cellulose (CMC) embedding medium; 2,5-dihydroxybenzoic acid (DHB, 98% purity); chromatographic grade methanol, trifluoroacetic acid (TFA), and ultrapure water.

[0090] 2. Sample pretreatment: (1) Embedding and sectioning: Take fresh or treated calamus rhizome samples to be tested, embed them with CMC embedding agent, and freeze them at -20℃. Use a cryostat to prepare 30μm thick transverse sections of the frozen samples. Transfer the sections and attach them flat to the surface of ITO conductive glass slides. (2) Matrix coating: The matrix is ​​coated by sublimation. First, prepare the matrix solution. The solvent is a 50% methanol aqueous solution containing 0.2% trifluoroacetic acid (TFA). Add DHB to prepare a saturated solution. Use a sublimation device to sublimate solid DHB under vacuum conditions and deposit it uniformly on the surface of the section to form a crystal layer. After coating, place the glass slide in a vacuum desiccator to dry for 3 hours to remove residual solvent and stabilize the crystal structure.

[0091] 3. Mass Spectrometry Imaging Data Acquisition: The processed slide was placed in the iMScopeQT mass spectrometry imaging system for scanning. The measurement conditions were set as follows: Ion source: Atmospheric pressure matrix-assisted laser desorption / ionization source (AP-MALDI); Scanning mode: Positive ion mode; Laser parameters: Laser beam diameter set to 30-50 μm (instrument setting value 2), laser intensity kept constant (e.g., 68); Spatial resolution: Scanning interval (step size) set to 50 μm; Detection voltage: Detector voltage set to 2.74 kV; Mass range: Acquire mass spectrometry data in the m / z range of 100-500.

[0092] 4. Data Processing and Analysis: The raw data collected is processed using imaging analysis software (IMAGEREVEALMS). (1) Normalization: Since the absolute signal intensity of mass spectrometry detection is easily affected by the uniformity of matrix crystallization, small fluctuations in laser energy, and instrument status (such as detector aging), in order to ensure the comparability of data between different batches and different scanning areas, this embodiment uses the total ion current (TIC) normalization algorithm to standardize all raw data. Specifically, the intensity of the target feature ion in each pixel is divided by the sum of the intensities of all ions in that pixel to obtain the relative intensity value (or the standardized intensity value corrected based on the relative ratio). The ion intensity thresholds (such as 15000, 5500, etc.) mentioned later in this application are all values ​​obtained based on the above TIC normalization processing and the specific instrument parameters of this embodiment. (2) Region of Interest (ROI) Selection: On the superimposed image of the optical image and ion image of the sample, the region from the cambium layer to the center point of the central column of the root cross section is selected as the region of interest (ROI). Figure 1 As shown, the cross-sectional structure of the rhizome of calamus can be clearly identified by observation under an optical microscope. The anatomical parts such as the epidermis, cortex, cambium, and stele are marked in detail in the figure. In this invention, the region from the cambium ring to the center of the stele shown in the figure is selected as the region of interest (ROI) for subsequent data extraction and analysis. (3) Feature ion extraction: The spatial distribution images of three feature ions, m / z 201.1638, m / z 209.1172 and m / z 219.1744, and the average ion intensity within the ROI region are extracted.

[0093] 5. Identification Results and Judgment Criteria: Based on the detection results of the above 36 batches of samples, the following identification criteria were established: (1) Spatial distribution characteristics: Acorus tatarinowii characteristics: m / z209.1172 showed a bright and dense enrichment distribution in the cambium and stele regions; while m / z201.1638 and m / z219.1744 were sparsely distributed, scattered, or had only background signals in this region. Acorus calamus characteristics: m / z209.1172 was sparsely distributed and had weak signals in the cambium and stele regions; while m / z201.1638 and m / z219.1744 showed a significant enrichment distribution in this region. (2) Ion intensity threshold judgment (quantitative aid): Calculate the average ion intensity (Intensity) in the ROI region: For m / z209.1172: the intensity of Acorus tatarinowii samples is usually not less than 15000; the intensity of Acorus calamus samples is usually not more than 5500. For m / z 201.1638: the strength of *Acorus gramineus* samples is generally not more than 150; the strength of *Acorus calamus* samples is generally not less than 1200. For m / z 219.1744: the strength of *Acorus gramineus* samples is generally not more than 200; the strength of *Acorus calamus* samples is generally not less than 1400.

[0094] Figure 2 The spatial distribution differences of the three selected characteristic ions in slices of *Acorus gramineus* and *Acorus tatarinowii* are visually demonstrated. As shown in the figure: (A) m / z 209.1172 shows a bright (red / yellow) enrichment distribution in the stele region of *Acorus gramineus*, while the signal is weak in *Acorus tatarinowii*; (B) m / z 201.1638 shows a bright enrichment in the stele region of *Acorus tatarinowii*, while it is scattered in *Acorus gramineus*; (C) m / z 219.1744 also shows a bright enrichment in the stele region of *Acorus tatarinowii*, while it is sparsely distributed in *Acorus gramineus*. This significant complementary distribution pattern provides a visual basis for the identification method of this invention.

[0095] It should be noted that the ion intensity thresholds given in this embodiment (e.g., Acorus tatarinowii m / z 209.1172 > 15000, Acorus tatarinowii < 5500) are typical reference values ​​obtained based on the iMScopeQT mass spectrometry imaging system under specific set parameters (e.g., detection voltage 2.74kV, laser intensity 68) and the above-mentioned TIC normalization processing. Given that the absolute intensity of MALDI-MS mass spectrometry signals may drift overall depending on the instrument model (such as Bruker or Waters series), ion source state, and laser energy attenuation, in practical applications, when the detection environment or instrument changes, the above absolute values ​​should not be mechanically applied. Instead, the relative difference rule revealed in this invention should be followed: that is, the intensity of m / z 209.1172 in *Acorus gramineus* samples should be significantly higher (e.g., more than 3 times higher) than that in *Acorus calamus* samples; or, it is recommended to introduce *Acorus gramineus* reference material with a known source as an internal reference during detection, using 30%-40% of the intensity measured in the reference material (corresponding to a ratio of 5500 / 15000) as the critical threshold for correction. Regardless of the drift in absolute values, the spatial distribution characteristics of m / z 209.1172 enrichment in *Acorus gramineus* and m / z 201 / 219 enrichment in *Acorus calamus* as described in this invention remain unchanged. By observing the spatial distribution patterns of the aforementioned characteristic ions in the cambium to the central column region, and combining this with ion intensity thresholds, this method can accurately and rapidly distinguish between *Acorus gramineus* and *Acorus scutellaria*. In the testing of 36 batches of samples in this embodiment, the method demonstrated clear distinguishability, verifying the reliability of m / z 209.1172 and m / z 201.1638 / 219.1744 as complementary spatial markers.

[0096] Example 2: Optimization and Screening of MALDI-MSI Sample Pretreatment Process: This example aims to determine the optimal pretreatment conditions for mass spectrometry imaging of rhizome tissues of Acorus gramineus and Acorus tatarinowii through comparative experiments, in order to solve the imaging deviation problems caused by the fragility of plant rhizome tissues, uneven matrix crystallization, and solvent effects.

[0097] 1. Experimental Materials and Methods: Fresh calamus rhizome samples were taken, and the following three dimensions of conditions were investigated: (1) Slice thickness: Three slice thickness gradients of 20 μm, 30 μm, and 40 μm were set. (2) Matrix type: Three commonly used MALDI matrices were selected for comparison, namely 2,5-dihydroxybenzoic acid (DHB), cyano-4-hydroxycinnamic acid (CHCA), and sinapic acid (SA). The concentration of each was 20 mg / mL (solvent was methanol / water). (3) Coating method: The effects of automatic spraying and sublimation methods were compared. Figure 3 As shown, the imaging effects of two matrix coating methods are compared. (A) When using the traditional spraying method (left column), a significant solvent effect occurs due to the dissolution and diffusion of the solvent in the matrix solution. Specifically, characteristic ions (such as m / z 209.1172) undergo spatial delocalization, causing the signal to diffuse and become blurred, resulting in a significant reduction in signal intensity in the originally high-abundance areas (only showing a low-intensity blue distribution); at the same time, non-specific blue false positive signals or background noise accumulation are prone to appear at the edge of the slice (cortical region) (as shown by m / z 201.1638 and m / z 219.1744). (B) When using the sublimation method preferred by this invention (right column), the tissue microstructure remains intact because solvent introduction is avoided. Characteristic ion m / z 209.1172 shows clear high-brightness enrichment (red / white) in the central column region, with accurate positioning and a high signal-to-noise ratio; the background of non-characteristic ions is clean, with no edge stray signals. The results demonstrate that the sublimation method can effectively overcome the problems of swelling and migration, and truly reflect the in-situ distribution information of chemical components.

[0098] 2. Experimental Results and Analysis: (1) Selection of Slice Thickness: Plant root and stem tissues are rich in fiber and starch, and have a relatively complex structure. The experimental results show that: 20 μm slices: Due to the excessive thickness, the tissue is prone to wrinkling or damage during freezing and transfer, resulting in anatomical deformation, which is not conducive to subsequent imaging identification and region division. 40 μm slices: Although the slices have good integrity, during mass spectrometry imaging, the excessive thickness of the sample affects the conductivity and ionization efficiency, and the imaging of some characteristic ions shows obvious tailing, and there is spatial signal overlap, which reduces the spatial resolution of the imaging. 30 μm slices: Considering the stability, integrity and mass spectrometry signal quality of the tissue structure, the 30 μm slices do not show wrinkling or signal tailing, and are the optimal slice thickness. Therefore, 30 μm is used as the standard slice thickness in subsequent experiments of this invention.

[0099] (2) Selection of Matrix Types: The matrix determines the ionization efficiency of the analyte molecules. For the active components (mostly small molecule metabolites) in *Acorus calamus*, the test results of three matrices are as follows: CHCA matrix: Although it can generate a strong overall signal, it is prone to uneven crystallization on the plant tissue surface, producing local hotspots, resulting in large fluctuations in signal intensity within the same tissue area, affecting the accuracy of quantification. SA matrix: Experiments show that it is more suitable for the detection of large protein molecules, but it has a weak response and low signal-to-noise ratio for the small molecule metabolites (such as ethers and alkenes) in *Acorus calamus* and *Acorus tatarinowii*, which are the focus of this application. DHB matrix: In the experiment, it exhibited the best crystal uniformity, producing small and dense crystals, and possessing a high signal-to-noise ratio (SNR) and excellent imaging stability for the small molecules being tested. Therefore, this invention selects DHB as the main matrix for MALDI-MSI.

[0100] (3) Optimization of matrix coating methods: The matrix coating method directly affects the authenticity of in-situ information. The comparative experimental results are as follows: 1) Spraying method: Although the operation is simple, in actual process, the spray droplets accumulate on the tissue surface. Due to the water absorption of plant root and stem tissues, obvious swelling phenomenon was observed on the sample surface. This swelling leads to the diffusion and migration of the small molecule components to be tested in the tissue, resulting in a spatial deviation between the ion distribution in the final imaging image and the actual anatomical structure, which seriously affects the true ratio of signal intensity in the ROI region (such as from the cambium to the central column). 2) Sublimation method: As a dry coating technique, the sublimation method directly vaporizes and deposits the matrix under vacuum and heating conditions. Experiments showed that this method forms a very uniform and dense crystal layer on the tissue surface. No tissue deformation or swelling was caused during the entire process, and no matrix aggregation was observed. This method preserves the in-situ spatial information of chemical components to the greatest extent.

[0101] 3. Conclusion: Based on the above optimization experiments, the optimal sample pretreatment process of this invention was established as follows: CMC embedding was used to prepare 30 μm frozen sections, DHB was selected as the matrix, and sublimation coating was performed. This combination can effectively avoid sample swelling and component migration, ensuring the accuracy and repeatability of the identification results.

[0102] Example 3: Screening and Statistical Validation of Characteristic Ions for Differentiation between Acorus gramineus and Acorus calamus: This example aims to screen characteristic ions with the strongest species-distinguishing ability from massive mass spectrometry data using various chemometric methods (random forest, PCA, OPLS-DA, and t-test) and to verify their statistical significance. 1. Data Acquisition and Preprocessing: Eighteen batches of Acorus gramineus (S01~S18) and eighteen batches of Acorus calamus (Z01~Z18) were selected as training set samples. MALDI-MSI data of the original tissues of the samples were collected, and the mass spectrometry information was converted into a text-formatted data matrix containing the m / z values ​​detected in all samples and their corresponding ion intensities. The data were normalized by total ion current (TIC) and imported into Orange 3.36.1 and SIMCA 14.1 software for subsequent analysis. 2. Random Forest Model Screening: A classification prediction model was established using the random forest algorithm, with 70% of the samples as the training set and 30% as the test set, and 20 repeated training / testing iterations were performed. Model Performance: The model's area under the receiver operating characteristic (AUC) was validated to be 0.966, indicating extremely high classification accuracy. Feature Contribution: The contribution of each m / z variable to classification was assessed by calculating information gain and Gini importance.

[0103] Table 1. Top 15 feature ions by contribution selected by the random forest model Ranking m / z InfoGain Gini Ranking m / z InfoGain Gini 1 201.1638 1 0.5 9 209.117 0.713 0.382 2 219.1744 1 0.5 10 323.1416 0.712 0.382 3 233.1524 0.85 0.438 11 247.1099 0.712 0.382 4 203.1787 0.849 0.437 12 281.1334 0.712 0.382 5 204.1823 0.849 0.437 13 203.1434 0.712 0.382 6 265.1405 0.74 0.396 14 291.1166 0.712 0.382 7 293.15 0.716 0.383 15 321.1084 0.71 0.382 8 235.1684 0.713 0.382 / / / / The results (see Table 1) show that m / z 201.1638 and m / z 219.1744 both have an InfoGain value of 1.000 and a Gini value of 0.500, ranking first and second in contribution; m / z 209.1170 ranks ninth (InfoGain 0.713). This indicates that these ions play a dominant role in interspecific differences.

[0104] 3. Principal Component Analysis (PCA) was performed unsupervised to explore the overall distributional differences in the data. Analysis results: Five principal components with eigenvalues ​​greater than 1 were obtained, with a cumulative variance contribution rate of 74.37%. Specific eigenvalues ​​and contribution rates are shown in Table 2. The loading coefficients (score coefficients) of each characteristic ion in the principal components are shown in Table 3.

[0105] Table 2. Principal Component Eigenvalues ​​and Contribution Rates principal component Eigenvalues Variance contribution rate / % Cumulative contribution rate / % PC1 1561.01 38.16 38.16 PC2 531.46 12.99 51.15 PC3 375.8 9.19 60.34 PC4 340.17 8.32 68.66 PC5 233.84 5.72 74.37 Table 3. Principal Component Factor Score Coefficient Matrix (Some Key Ions) m / z PC1 PC2 PC3 PC4 PC5 201.1638 -0.01859 0.01276 -0.00209 -0.00448 -0.00499 209.1172 0.0048 -0.00365 -0.00071 0.00262 0.00123 219.1743 -0.01917 0.00563 0.00993 0.02945 0.00122 221.1942 -0.02029 0.01435 0.00422 0.01381 0.00765 229.1017 0.01556 -0.01424 -0.00492 -0.00967 0.01535 Loading analysis: Ions contributing significantly to the first principal component (PC1) include m / z 201.1638 and m / z 219.1743; ions contributing significantly to the second principal component (PC2) include m / z 209.1172. The PCA score plot shows that the samples from *Acorus gramineus* and *Acorus calamus* exhibit obvious natural clustering and separation trends in space, demonstrating significant differences in the overall chemical composition of the two groups.

[0106] 4. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) and Significance Test: To further pinpoint the differential markers, a supervised OPLS-DA model was used for analysis, and t-tests were used to verify statistical significance. Model Quality: The fitting parameters of the OPLS-DA model, R²X = 0.721, R²Y = 0.993, and the predictive ability parameter Q² = 0.998, are all greater than 0.5. Furthermore, 200 permutation tests indicate that the model is not overfitting and is robust and reliable. VIP and P-value Screening: Variables were screened based on a projective importance (VIP) value greater than 1.5 and a t-test p-value less than 0.05. Combining the VIP value and the t-test p-value, the top 10 variables with the most significant differences were selected, as shown in Table 4. The p-values ​​for m / z ions 209.117, 201.164, and 219.174 were extremely small, indicating highly significant differences in their distributions between the two groups.

[0107] Table 4. Top 10 differential markers ranked based on a combination of VIP value and t-test p-value. Ranking m / z VIP t-statistic p-value Ranking m / z VIP t-statistic p-value 1 209.117 5 11.36 4.02E-13 6 273.124 3 -14.12 8.85E-16 2 203.179 4.35 -9.24 8.48E-11 7 293.15 2.9 -41.11 1.41E-30 3 201.164 3.65 -10.19 7.11E-12 8 279.156 2.85 -9.35 6.36E-11 4 233.152 3.45 -12.67 1.96E-14 9 221.189 2.8 -9.29 2.94E-11 5 219.174 3.2 -11.06 8.37E-13 10 234.906 2.8 -9.52 5.60E-12 Among them, m / z 209.117: VIP value 5.00, t-statistic 11.36, p-value 4.02e-13, the difference was extremely significant. m / z 201.164: VIP value 3.65, p-value 7.11e-12. m / z 219.174: VIP value 3.20, p-value 8.37e-13.

[0108] like Figure 4 As shown in the OPLS-DA score plot, the 36 batches of samples are clearly divided into two categories: the green dots on the left represent Acorus gramineus samples, and the blue dots on the right represent Acorus calamus samples. The two groups of samples show obvious natural clustering and separation trends in space, indicating that there are significant statistical differences in chemical composition between Acorus gramineus and Acorus calamus, verifying the identification ability of the selected characteristic ions.

[0109] 5. Conclusion: Based on the combined analysis results of three statistical methods—random forest, PCA, and OPLS-DA—m / z 201.1638, m / z 209.1172, and m / z 219.1744 (Note: slight differences in decimal places are due to instrument precision and are considered as the same ion) were identified as the most critical differential variables in all models. These ions not only showed highly significant inter-group differences (p<0.001) but also contributed the most to the model classification, and were therefore identified as the core spatial chemical markers for distinguishing between *Acorus gramineus* and *Acorus calamus* in this invention.

[0110] Example 4: Accuracy Verification of Identification Method (Blind Sample Test): This example aims to use test samples (blind samples) independent of the training set to externally verify the accuracy of the identification method described in Example 1 and the generalization ability of the prediction model, so as to evaluate the application value of the method in the identification of medicinal materials in actual market circulation.

[0111] 1. Experimental Materials: Ten batches of Acorus calamus rhizome samples, completely independent of the modeling training set, were selected as the blind sample test set. These included five batches of samples identified as Acorus calamus (numbered S19~S23) and five batches of samples of Acorus calamus (numbered Z19~Z23).

[0112] 2. Experimental methods: (1) Detection process: Strictly follow the steps described in Example 1, CMC embedding, 30 μm frozen sections, and DHB matrix coating of the above 10 batches of blind samples were performed, and mass spectrometry data in positive ion mode were acquired in the iMScopeQT mass spectrometry imaging system. (2) Data analysis: After TIC normalization of the acquired data, the established random forest prediction model was used for classification prediction, and the predicted category and corresponding prediction probability of each sample belonging to Acorus gramineus or Acorus tatarinowii were output.

[0113] 3. Experimental Results: Predictions were made for 10 batches of blind samples (S19-S23, Z19-Z23), and the results are shown in Table 5. The predicted categories for all samples were completely consistent with the actual categories, and the predicted probabilities were all above 90%. The specific prediction results of external validation are shown in Table 5.

[0114] Table 5. External validation results of the random forest model serial number category Prediction results Predicted probability in conclusion serial number category Prediction results Predicted probability in conclusion S19 Acorus Acorus 98.20% correct Z19 Tibetan calamus Tibetan calamus 96.90% correct S20 Acorus Acorus 96.70% correct Z20 Tibetan calamus Tibetan calamus 90.90% correct S21 Acorus Acorus 94.80% correct Z21 Tibetan calamus Tibetan calamus 97.90% correct S22 Acorus Acorus 97.40% correct Z22 Tibetan calamus Tibetan calamus 94.50% correct S23 Acorus Acorus 95.30% correct Z23 Tibetan calamus Tibetan calamus 97.90% correct 4. Results and Conclusions: Statistical analysis showed that, under the same detection conditions, the average ionic intensity (m / z 209.1172) of *Acorus gramineus* from the cambium to the stele was approximately 4 to 40 times that of *Acorus tatarinowii*; while the average ionic intensity (m / z 201.1638) of *Acorus tatarinowii* in the same region was approximately 22 to 170 times that of *Acorus gramineus*. This significant fold change demonstrates the high robustness of this identification method; even with fluctuations in absolute signal intensity between different instruments, this large relative difference is still sufficient to support accurate qualitative identification. Experimental results showed that for the five batches of blind samples of *Acorus gramineus* (S19-S23), the model predicted a probability of over 94.8% for *Acorus gramineus*; for the five batches of blind samples of *Acorus tatarinowii* (Z19-Z23), the model predicted a probability of over 90.9%. The predicted results of all 10 batches of blind samples were completely consistent with the actual botanical identification results, achieving a 100% accuracy rate. This indicates that the MALDI-MSI-based identification method established in this invention not only performs excellently on training data, but also has extremely high accuracy and good generalization ability when facing unknown external samples. It can effectively avoid overfitting and is suitable for rapid and accurate screening of the authenticity of Acorus calamus and Acorus tatarinowii in actual testing work.

[0115] Example 5: Based on the optimized experimental conditions of Examples 1 and 2, this example provides a dedicated detection kit. The kit contains the following core components: Embedding agent: Individually packaged carboxymethyl cellulose (CMC) powder or solution, used to embed and support plant rhizomes during cryosectioning to prevent tissue breakage. Matrix: Individually packaged 2,5-dihydroxybenzoic acid (DHB) powder, 98% purity, as the preferred matrix for MALDI-MSI. Standard reference manual / spectrum: Contains standard mass spectra of three characteristic ions (m / z 201.1638, m / z 209.1172, and m / z 219.1744) and their typical spatial distribution patterns in *Acorus gramineus* (enriched in the central column at m / z 209) and *Acorus calamus* (enriched in the central column at m / z 201 / 219). When using this kit, users only need to follow the instructions to embed and section the tissue using the CMC in the kit, coat the matrix using DHB with the sublimation method, and then perform mass spectrometry scanning and compare it with the standard spectrum to complete the identification.

[0116] Example 6: To further verify the effectiveness and anti-interference ability of the characteristic abundance ratio (R value) determination strategy proposed in this invention, this example selected some blind sample data for calculation and conducted a comparative analysis under the extreme case of instrument sensitivity decrease. 1. Establishment of R value calculation model: The calculation was performed according to the formula R=A / (B+C). A: m / z 209.1172 (characteristic of Acorus tatarinowii) ion intensity; B: m / z 201.1638 (characteristic of Acorus calamus) ion intensity; C: m / z 219.1744 (characteristic of Acorus calamus) ion intensity. Judgment criteria: If R10, it is determined to be Acorus tatarinowii; if R2, it is determined to be Acorus calamus. 2. Calculation verification of blind sample data: Some blind samples (Acorus tatarinowii S19-S21, Acorus calamus Z19-Z21) from Example 4 were selected, and their average ion intensity in the ROI region was extracted and the R value was calculated. The results are shown in Table 6.

[0117] Table 6. Calculation results of characteristic abundance ratio R of blind samples serial number Source A(m / z209) B(m / z201) C(m / z219) Ratio R calculation Judgment Conclusion result S19 Acorus 28450 110 145 111.57 >10 (Acorus calamus) correct S20 Acorus 19200 95 130 85.33 >10 (Acorus calamus) correct S21 Acorus 22150 120 180 73.83 >10 (Acorus calamus) correct Z19 Tibetan calamus 3200 2100 2450 0.7 2 (Tibetan calamus) correct Z20 Tibetan calamus 1500 1850 2100 0.38 2 (Tibetan calamus) correct Z21 Tibetan calamus 4100 1900 2300 0.98 2 (Tibetan calamus) correct Data Analysis: As shown in Table 6, the R values ​​of the *Acorus calamus* samples were all above 70, far exceeding the first threshold (10); the R values ​​of the *Acorus tatarinowii* samples were all below 1.0, far below the second threshold (2). The huge difference in the R values ​​between the two groups of data (Gap) proves that this ratio index has extremely high discriminative power.

[0118] 3. Method Robustness (Interference Resistance) Assessment: In actual detection, the mass spectrometer's overall signal sensitivity may decrease due to ion source contamination, laser energy attenuation, or fine-tuning of the detection voltage. To examine the advantages of this method, we simulated a scenario where the instrument sensitivity decreased by 50%, comparing the judgment results of the absolute threshold method and the ratio method (in this embodiment). We assumed that for sample S20 (Acorus calamus), when the instrument was in poor condition, the intensity of all ion signals decreased to 50% of their original values.

[0119] Table 7. Comparison of methods for determining instrument sensitivity decay conditions Examining the scene Indicator parameters Normal state (raw data) Attenuation state (analog signal reduced by 50%) Basic data A(m / z209) 19,200 9,600 B+C strength 225 112.5 Method 1: Absolute threshold method Judgment criteria A15,000 is Acorus calamus. A15,000 is Acorus calamus. Judgment Result 19,200 > 15,000 (Correct) 9,600 < 15,000 (Misjudgment / Unable to determine) Method 2: Ratio method R Judgment criteria R10 is Acorus calamus. R10 is Acorus calamus. Calculated value 19200 / 225=85.33 9600 / 112.5=85.33 Judgment Result 85.33 > 10 (Correct) 85.33 > 10 (Correct) Conclusion: As shown in Table 7, when significant fluctuations in instrument sensitivity cause the absolute ion intensity (9600) to fall below the original threshold (15000), the traditional absolute threshold method is prone to false negatives. However, the characteristic abundance ratio R proposed in this invention utilizes the self-correction effect of the internal standard ions in the same scan, maintaining a constant value (still 85.33), and the judgment result remains accurate. This demonstrates that introducing the ratio calculation method described in steps S333-S335 significantly improves the robustness and reliability of the identification method under different instrument conditions and laboratory environments, overcoming the technical shortcomings of existing technologies that rely solely on absolute signal intensity.

[0120] Example 7: Investigation on the improvement of imaging sensitivity by sublimation vapor-phase recrystallization process: This example aims to verify whether introducing a vapor-phase recrystallization step on the basis of sublimation method can further improve the detection sensitivity (signal-to-noise ratio) of characteristic ions while maintaining high spatial resolution.

[0121] 1. Experimental Materials and Grouping: Two 30-millimeter frozen sections of Acorus gramineus rhizomes from adjacent locations were selected and divided into two groups: Control group: coated with DHB matrix using only the sublimation method described in Example 1. Experimental group: using a combination of sublimation and vapor-phase recrystallization.

[0122] 2. Experimental Methods: 1) Control Group: Following the steps in Example 1, the substrate was sublimated, directly vacuum-dried, and then mass spectrometry was performed. 2) Experimental Group: Sublimation: Same as the control group, DHB crystals were first sublimated on the surface of the slices to form a layer. Recrystallization Environment Construction: A layer of qualitative filter paper was placed at the bottom of a 90 mm diameter glass petri dish, 1 mL of methanol solution (50% methanol) was added, and the dish was covered and equilibrated for 5 minutes to fill the container with solvent vapor. Fumigation Treatment: The sublimated slices were placed horizontally on a support in the petri dish (the slices did not directly contact the filter paper or liquid), and the dish was quickly covered. Fumigation was carried out at room temperature (25°C) for 60 seconds. Drying: The slices were removed and placed in a vacuum desiccator for 30 minutes, followed by mass spectrometry.

[0123] 3. Experimental results and comparative analysis: Under the same instrument parameters (laser energy, detection voltage, etc.), imaging acquisition was performed on the two groups of slices, and signal data of the characteristic ion m / z 209.1172 of Acorus tatarinowii in the central column region was extracted. (1) Observation of crystal morphology (under microscope): Control group: The matrix crystals are extremely fine microcrystals with dense coverage, but under high magnification, the crystal size is small (<2m), and the extraction ability of molecules in the tissue is limited. Experimental group: After gas phase recrystallization, the matrix crystals underwent microscopic rearrangement, the crystal size increased slightly (about 5-10m), the crystal form was more regular and more lustrous, indicating that an effective co-crystallization process occurred. (2) Comparison of mass spectrometry signal intensity: The specific data are shown in Table 8 (the data in the table are the average values ​​of multiple measurements): Table 8. Comparison of signal intensity of characteristic ions under different coating processes Evaluation indicators Control group (sublimation only) Experimental group (epigenesis + recrystallization for 60 seconds) range of change Average ionic strength 15420 28550 Increase by approximately 85% Signal-to-noise ratio 45:1 82:1 Significant improvement Spatial resolution Clear, sharp edges Clear, with sharp edges No significant difference Swelling / Migration none none Maintain the advantages of dry methods 4. Conclusion: Experimental results show that: Figure 5As shown, the imaging results of the two processes were compared intuitively. (1) Significantly improved sensitivity: Under the same signal intensity display scale, the ion image of the control group (left figure) was mainly blue-green, with fewer high-intensity red areas, indicating that the extraction efficiency of the characteristic ion (m / z 209.1172) was low; while the image of the experimental group (right figure) was bright red-yellow overall, with a significant increase in the area of ​​high-abundance signal regions and dense coverage. This confirms that the gas-phase recrystallization process effectively promoted the entry of the surface analyte molecules into the matrix lattice through micro-dissolution induced by solvent vapor, achieving in-situ extraction and signal gain. (2) Good spatial resolution: Although the sensitivity was greatly improved, a comparison of the edge contours of the two figures shows that the experimental group (right figure) did not exhibit the edge blurring or signal overflow phenomenon common in liquid phase spraying methods, and its imaging boundary remained as clear and sharp as that of the control group (left figure). This indicates that the gas-phase fumigation process precisely controlled the solvent action range and successfully avoided the spatial migration of components caused by macroscopic droplet flow. In summary, the sublimation + vapor phase recrystallization process retains the high spatial resolution advantage of dry methods while overcoming their sensitivity bottleneck, making it an ideal and preferred solution for detecting low-abundance or difficult-to-extract components.

[0124] Example 8: Experiment on the enhancement effect of silver ion derivatization technology on the detection of asarone: This example aims to verify, through comparative experiments, the technical effect of the silver ion derivatization pretreatment step proposed in this invention in improving detection sensitivity, overcoming vacuum volatilization effect and enhancing qualitative accuracy when detecting characteristic components (asarone compounds) of Acorus tatarinowii.

[0125] 1. Experimental Materials: Samples: Fresh rhizomes of identified Acorus tatarinowii (S01) were selected, and several frozen sections (30 μm thick) of adjacent parts were prepared. Reagents: Matrix: 2,5-Dihydroxybenzoic acid (DHB). Silver ion derivatization reagent: Silver nitrate (AgNO3) was dissolved in anhydrous ethanol to prepare a solution with a concentration of 2 mg / mL.

[0126] 2. Experimental grouping and methods: Adjacent slices were randomly divided into two groups and treated as follows: (1) Control group (conventional method): After freeze-drying, the slices were directly coated with DHB matrix using the sublimation method described in Example 1 without silver ion treatment. (2) Experimental group (silver ion derivatization method): 1) Derivatization: Using an automatic spraying device, the silver ion derivatization reagent was uniformly sprayed onto the surface of the slices. Parameter settings: flow rate 10L / min, spraying time 30 seconds (to ensure that only a micro-wet film is formed and droplet accumulation is not caused). Air dry for 5 minutes. 2) Coating: Then, the same sublimation method coating operation as the control group was performed.

[0127] 3. Data Acquisition Conditions: Two groups of slices (control group and experimental group) were placed on the same mass spectrometry target plate and pushed into the instrument. To investigate the effect of the vacuum environment on the volatile oil components, the target plate was placed in the ion source vacuum chamber (vacuum degree 210). -6 After being held in the mbar for 30 minutes, the laser is then activated for scanning and imaging.

[0128] 4. Experimental Results and Analysis: (1) Comparison of Anti-vacuum Volatilization Ability and Sensitivity: The average ionic strength of the column region of Acorus gramineus was extracted. 1) Control Group: Detection of protonated molecules [M+H] + (m / z 209.1172). This method can achieve effective identification (as shown in Example 1), but after prolonged residence in the vacuum chamber (30 minutes), due to the volatility of asarone, some analyte molecules escape, resulting in a certain degree of natural attenuation of the signal intensity (average intensity approximately 3250). 2) Experimental group: Detection of silver adduct ions [M+Ag] + (m / z 315.015 and 317.015). Volatility is suppressed due to the formation of a stable metal complex.

[0129] Table 9. Comparison of signal intensity enhancement effects of silver ion derivatization Evaluation indicators control group experimental group Effect evaluation Target ionic form <![CDATA[[M+H] + (m / z209)]]> <![CDATA[[M+Ag] + (m / z315+317)]]> Target shift Average ionic strength 3250 38600 (sum of two peaks) An increase of approximately 11.8 times. Image signal-to-noise ratio 12:1 150:1 Significantly improved Clarity of the central column area The signal is weak and the pixels are intermittent. The signal is extremely strong and the area is full and continuous. It's better to keep it in place. Results Analysis: As shown in Table 9, the total signal intensity of the target component increased by an order of magnitude (>10 times) after the introduction of silver ions. This strongly demonstrates that silver ions anchor the volatile asarone ether molecule through pi-complexation, effectively overcoming the signal loss problem under the high vacuum environment of MALDI.

[0130] (2) Qualitative accuracy assessment (isotope fingerprint): Observe the peak shape characteristics of the mass spectrometry. 1) Control group: Only a single isotope peak is shown at m / z 209, and there are many matrix fragments in the m / z 200-220 range that interfere with it. It is difficult to confirm whether it is asarone based solely on the mass number. 2) Experimental group: A distinctive twin peak is shown at m / z 315 and m / z 317. Measured intensity ratio: I(315):I(317)=100:96.5. Theoretical isotope ratio: Ag(107):Ag(109)100:92.9. Consistency: The measured distribution is highly consistent with the theoretical distribution.

[0131] The specific proportion of twin peaks observed in the experimental group provides a second layer of chemical fingerprint evidence, in addition to the precise mass number. Even against a complex background of plant metabolites, this unique isotopic pattern allows the tester to immediately identify the characteristic component containing double bonds (asarone), thus eliminating false positive interference. In summary, this embodiment demonstrates that the silver ion derivatization step overcomes the technical bottleneck of the volatility and difficulty in qualitative analysis of the characteristic components of *Acorus gramineus* through chemical reaction. Compared to conventional methods, this preferred technical solution significantly improves the sensitivity and specificity of identification, exhibiting outstanding substantive features and significant progress.

[0132] Example 9: Chemical Structure Confirmation and Isomer Difference Analysis of Key Feature Ion m / z 209.1172: In order to clarify the specific chemical classification of the core feature ion m / z 209.1172 screened in this invention, and to explore in depth the material basis of its distribution differences in Acorus gramineus and Acorus tatarinowii, this example uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Exactive-MS) to accurately confirm the structure of this ion.

[0133] 1. Experimental instruments and conditions: (1) Instruments: UPLC-Q-Exactive-MS system (Thermo Fisher Scientific, USA). (2) Chromatographic conditions: Column: Waters ACQUITY BEH C18 (1.7m x 2.1mm x 100mm); Column temperature: 40℃; Injection volume: 5L; Mobile phase: Phase A is 0.1% formic acid aqueous solution, Phase B is 0.1% formic acid acetonitrile / methanol (40:60) solution. (3) Elution program: 0-3min (10%B) 3-5min (10%-40%B) 5-8min (40%-95%B) 8-10min (95%-100%B) 10-10.3min (100%-10%B). (4) Mass spectrometry conditions: Ion source: HESI; Detection mode: Positive ion mode; Scan range: m / z 60~900; Scan mode: FullMS / dd-MS2; Normalized collision energy (NCE): 20, 40, 60.

[0134] 2. Experimental samples: Extracts from representative samples of Acorus calamus (numbered S01~S10, 10 batches in total) and Acorus calamus (numbered Z01~Z10, 10 batches in total) were selected for testing.

[0135] 3. Structural Identification and Fragmentation Pattern Analysis: The experiment first confirmed that m / z 209.1172 corresponds to isomers of asarone compounds by comparing with the mzCloud, ChemSpider, and PubChem databases and using standard references. These isomers mainly include -asarone, -asarone, and -asarone. Specific mass spectrometry information is shown in Table 10.

[0136] Table 10. Mass Spectrometry Information of Asarone Isomers Compound name Retention time (min) <![CDATA[[M+H] + ]]> Theoretical quality (Da) Error (ppm) Molecular formula Major fragment ions (m / z) -Asarone 6.40 209.11697 209.11722 -1.20 <![CDATA[C 12 H 16 O3]]> 194.09357 181.08566 -Asarone 6.35 209.11699 209.11722 -1.10 <![CDATA[C 12 H 16 O3]]> 194.09366 181.08568 -Asarone 6.35 209.11684 209.11722 -1.82 <![CDATA[C 12 H 16 O3]]> 168.07797 4. Fragmentation patterns and component difference analysis: This was achieved by analyzing fragments obtained from secondary mass spectrometry (MS / MS) (e.g., Figure 6 As shown), it was found that the isomers with different configurations have significant differences in cleavage: (1) - and - asarone: mainly generated m / z 194 by demethylation, followed by carbon-carbon double bond cleavage and methoxy condensation to form m / z 181. (2) - asarone: the characteristic cleavage pathway is the removal of the propenyl side chain to generate the characteristic fragment m / z 168 with higher abundance.

[0137] like Figure 7 As shown, the measured secondary mass spectra of representative samples S02 (Acorus calamus) and Z02 (Acorus calamus) are compared: (A) Acorus tatarinowii sample (S02 as an example): In the secondary mass spectrum, m / z 168.08 appears as the base peak (relative abundance 100%). Sample S06 in the same group also shows completely consistent fragmentation characteristics. This extremely high abundance of characteristic fragment peak strongly confirms the specific enrichment of α-asarone in Acorus tatarinowii. (B) Acorus calamus sample (Z02 as an example): In the secondary mass spectrum, no obvious signal was detected at m / z 168, and the spectrum was mainly composed of m / z 194 and 181. This indicates that the content of α-asarone in Acorus calamus is extremely low or absent, and the main components are confirmed to be α-asarone and α-asarone.

[0138] It should be noted that all 10 batches of samples from both *Acorus gramineus* and *Acorus calamus* conformed to the above-mentioned characteristics.

[0139] 5. Conclusion: This study confirms that the m / z 209.1172 characteristic signal observed in MALDI-MSI imaging is the result of the combined action of multiple asarone isomers. The specific enrichment of α-asarone in *Acorus tatarinowii* is the key material basis for the significant difference in the m / z 209 channel between the two groups of samples (stronger in *Acorus tatarinowii*, weaker in *Acorus calamus*). Furthermore, the presence of an allyl / propenyl side chain (double bond structure) in this characteristic molecule was confirmed, providing a solid chemical structural basis for the aforementioned silver ion derivatization examples.

[0140] Application Example: Screening for Adulteration of Acorus tatarinowii with Acorus calamus: The method described in this invention has the characteristics of high throughput and in-situ imaging, making it particularly suitable for screening adulteration in commercially available medicinal materials. In actual testing, if the sample to be tested is a mixture of slices or a mixture of powder particles, several particles or slices can be randomly selected for embedding and slide preparation. MALDI-MSI technology can simultaneously scan and image multiple independent sample regions on the same slice. According to the identification criteria established in Example 1: if in the same scan image, some particles / regions show a high abundance distribution of m / z 209.1172 in the cambium to stele region (consistent with the characteristics of Acorus tatarinowii), while other particles / regions show a high abundance distribution of m / z 201.1638 or m / z 219.1744 in the cambium to stele region (consistent with the characteristics of Acorus calamus), it indicates that the batch of medicinal materials contains a mixture of Acorus tatarinowii and Acorus calamus, and is judged as adulterated. This in-situ chemical imaging screening based on single particle / single slice level can effectively detect low-proportion adulteration that is difficult to detect by traditional physicochemical methods (such as measuring the total amount after mixing and crushing).

[0141] 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 they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology, characterized in that, include: Frozen sections of the rhizome samples of the calamus to be tested were prepared, and 2,5-dihydroxybenzoic acid was coated on the surface of the frozen sections as a matrix by sublimation. Mass spectrometry imaging data were acquired by scanning the frozen sections coated with the matrix in positive ion mode using a matrix-assisted laser desorption / ionization mass spectrometry imaging system. Using the region from the cambium layer to the central column of the frozen section as the region of interest, the spatial distribution characteristics of at least one characteristic ion among the mass-to-charge ratios m / z 201.1638, m / z 209.1172, and m / z 219.1744 within the region of interest are identified, and the identification results are obtained.

2. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The spatial distribution characteristics of at least one characteristic ion among m / z 201.1638, m / z 209.1172, and m / z 219.1744 in the region of interest are identified, and the identification results are obtained, including: If the characteristic ion m / z 209.1172 is enriched and distributed in the region of interest, or if the characteristic ions m / z 201.1638 and m / z 219.1744 are scattered in the region of interest, then the rhizome sample of the calamus to be tested is determined to be calamus. If the characteristic ion m / z 209.1172 is sparsely distributed in the region of interest, or if the characteristic ions m / z 201.1638 and m / z 219.1744 are enriched in the region of interest, then the rhizome sample of the calamus to be tested is determined to be Tibetan calamus.

3. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The thickness of the frozen section is 30 μm; And / or, The sublimation method includes: preparing a saturated solution of 2,5-dihydroxybenzoic acid in a 50% methanol aqueous solution, and then forming a uniform crystalline layer on the surface of the frozen section using a sublimation device; preferably, the 50% methanol aqueous solution also contains 0.2% trifluoroacetic acid; preferably, after forming a uniform crystalline layer on the surface of the frozen section using the sublimation device, the method further includes: placing the coated section in a vacuum desiccator for drying for 3 hours.

4. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The permissible mass deviation for the characteristic ions m / z 201.1638, m / z 209.1172 and m / z 219.1744 is 0.

005.

5. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The method of identifying the spatial distribution characteristics of at least one characteristic ion among m / z 201.1638, m / z 209.1172, and m / z 219.1744 within the region of interest further includes: Judgment is based on ionic strength threshold: When the ion intensity of the characteristic ion m / z 209.1172 in the region of interest is not less than 15000, the rhizome sample of the calamus to be tested is determined to be calamus. When the ion intensity of the characteristic ion m / z 209.1172 in the region of interest does not exceed 5500, the rhizome sample of the calamus to be tested is determined to be Tibetan calamus. Preferably, the identification further includes: Calculate the characteristic abundance ratio R, R = A / (B+C); Wherein, R represents the characteristic abundance ratio; A represents the average ion intensity of characteristic ion m / z 209.1172 in the region of interest; B represents the average ion intensity of characteristic ion m / z 201.1638 in the region of interest; and C represents the average ion intensity of characteristic ion m / z 219.1744 in the region of interest. If R is at the first threshold, then the sample is determined to be Acorus calamus; If R reaches the second threshold, then the sample is determined to be Acorus calamus.

6. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The matrix-assisted laser desorption / ionization mass spectrometry imaging system scans the matrix-coated frozen sections in positive ion mode, and the parameters for acquiring mass spectrometry imaging data include: The laser beam diameter is 30m~50m; and / or, The scanning interval is 50m; and / or, The detector voltage is 2.74kV.

7. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The preparation of frozen sections of the calamus rhizome sample to be tested includes: embedding the calamus rhizome sample to be tested with carboxymethyl cellulose and then freezing and sectioning it at -20°C.

8. The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in claim 1, characterized in that, The acquired mass spectrometry imaging data includes: acquiring mass spectrometry data with a mass range of m / z 100~500; and performing total ion current normalization processing on the acquired raw data.

9. A reagent kit, characterized in that, The method for identifying Acorus calamus and Acorus tatarinowii based on MALDI-MSI technology as described in any one of claims 1-8 includes: Carboxymethyl cellulose embedding agent for embedding plant root and stem samples; 2,5-Dihydroxybenzoic acid used as a laser desorption / ionization matrix; and a standard spectrum or specification showing the characteristic ions m / z 201.1638, m / z 209.1172 and m / z 219.1744; The specification indicates that identification is performed by detecting the spatial distribution of the characteristic ions in the region from the cambium to the central pillar.

10. A method for screening whether Acorus tatarinowii Schott is adulterated with Acorus calamus L., characterized in that, include: The samples of the medicinal materials to be tested were prepared into slides and scanned using MALDI-MSI. Ion images of the characteristic ions m / z 209.1172, m / z 201.1638 and m / z 219.1744; If, in different regions or different particles of the medicinal material sample to be tested, regions with the characteristic distribution pattern of Acorus tatarinowii and regions with the characteristic distribution pattern of Acorus tatarinowii are observed simultaneously, then it is determined that the medicinal material sample to be tested is adulterated. The characteristic distribution pattern of Acorus tatarinowii is determined based on the spatial distribution of the characteristic ion m / z 209.1172, and the characteristic distribution pattern of Acorus calamus is determined based on the spatial distribution of the characteristic ions m / z 201.1638 and / or m / z 219.1744. Preferably, the criteria for determining the feature distribution pattern include: The characteristic distribution pattern of Acorus gramineus is characterized by the high abundance of the characteristic ion m / z 209.1172 in the cambium to the stele region; and / or, the characteristic distribution pattern of Acorus calamus is characterized by the high abundance of the characteristic ions m / z 201.1638 and / or m / z 219.1744 in the cambium to the stele region.

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