Mass Spectrometry Imaging Empowers Traditional Chinese Medicine Research: Spatial Molecular Evidence from Atractylodes macrocephala’s Cinnabar Stains to the Evaluation of Authenticity

Research into traditional Chinese medicine is introducing a new analytical tool: mass spectrometry imaging. Unlike traditional methods of component analysis, mass spectrometry imaging not only detects the chemical constituents present in traditional Chinese medicinal materials but also reveals the spatial distribution of these constituents within the tissue of the medicinal materials, thereby providing spatial-level molecular evidence for the evaluation of medicinal material quality, the authentication of authentic origins, and research into the mechanisms of medicinal processing. Recent research findings indicate that the application value of mass spectrometry imaging in the field of traditional Chinese medicine is gaining recognition within the academic community.

Limitations of Traditional Analysis of Chinese Medicinal Materials

The chemical analysis of Chinese medicinal materials primarily relies on techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS). Whilst these techniques can accurately identify and quantify the chemical constituents in Chinese medicinal materials, they share a common limitation: the materials must be ground and extracted, and the analytical results represent an ‘average’ of all constituents, with information on the spatial distribution of these constituents being entirely lost.

Significant tissue differentiation exists within the medicinal parts of Chinese herbal materials (roots, stems, leaves, flowers and fruits) – the chemical constituents accumulated in different tissue regions, such as the cork layer, cortex, phloem, xylem and pith, vary considerably. Traditional extraction and analysis methods smooth out all these differences, failing to answer the crucial question of ‘in which tissue region is the target constituent actually concentrated?’.

The evaluation of the authenticity of traditional Chinese medicinal materials faces a similar dilemma. Whilst there may be differences in the external morphology, tissue structure and spatial distribution of chemical constituents between authentic and non-authentic medicinal materials, traditional analytical methods can only compare differences in the types and concentrations of constituents; they are unable to compare the spatial distribution patterns of constituents within the tissue.

How a mass spectrometry imaging system works

A mass spectrometry imaging system scans the surface of a herbal medicine section point by point, simultaneously acquiring mass spectrometry data at each coordinate point. It then reconstructs the signals with the same mass-to-charge ratio onto a spatial coordinate system to obtain a two-dimensional distribution map of that molecule within the tissue.

The advantages of this method lie in the fact that it requires no extraction or grinding, allowing analysis to be carried out directly on the section; it enables the simultaneous detection of hundreds of molecules through non-targeted screening; each molecule has an independent spatial distribution map, allowing it to be localised to specific tissue regions; and the section remains intact after imaging, permitting further traditional microscopic identification.

The detection capability of a mass spectrometry imager depends on the ionisation performance of its imaging ion source. Conventional DESI imaging sources can only effectively ionise highly polar molecules, resulting in detection blind spots for non-polar compounds (many active constituents in traditional Chinese medicine are weakly polar or non-polar compounds such as aglycones, terpenes and steroids). This limitation is particularly pronounced in traditional Chinese medicine research—as the polarity of active constituents in traditional Chinese medicine varies widely, detection within a single polarity range would lead to a significant number of active constituents being missed.

Study on cinnabar spots on Atractylodes macrocephala: Spatial evidence unravels the mystery of their origin

Atractylodes macrocephala is a commonly used Chinese medicinal herb; some specimens exhibit red ‘cinnabar spots’ on their surface, the cause of which has long remained unclear. Is it external staining? Is it discolouration resulting from processing at the place of origin? Or is it the spatial enrichment of the herb’s own chemical substances? This issue is directly related to the quality assessment of Atractylodes macrocephala.

Hefei Xinceyuan Instrument Technology Co., Ltd., in collaboration with the China Academy of Chinese Medical Sciences, utilised a mass spectrometry imaging (MSI) system equipped with an MSI DPI imaging source to perform spatial molecular imaging analysis on Atractylodes macrocephala sections.

The MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source employs a dual-ionisation architecture combining DESI and photochemical secondary ionisation, thereby eliminating the polarity discrimination inherent in traditional DESI and enabling the simultaneous detection of both polar and non-polar molecules. During the imaging of Atractylodes macrocephala sections, the DESI/PI mode detected non-polar components that could not be covered by traditional DESI.

The imaging results showed that signals at m/z 183.0807 and m/z 301.2164 were highly correlated with the cinnabar-coloured spots. Combined with an analysis of DESI/PI-MSI results for other polyacetylene and sesquiterpene compounds, the study confirmed that the substantial accumulation of Atractylodes macrocephala and specific polyacetylene compounds is the cause of the cinnabar-coloured spots—not external staining, nor colour changes due to processing, but rather the spatial localisation of Atractylodes macrocephala’s own chemical substances in specific tissue regions, which leads to the colour differences.

This finding, published in *Molecular Horticulture* (2025, 5:11), represents a flagship application of mass spectrometry imaging in the field of Traditional Chinese Medicine research. It demonstrates how spatial molecular imaging can provide conclusive evidence to address questions that traditional analytical methods cannot resolve.

Broader Application Prospects

The study of cinnabar spots in Atractylodes macrocephala is merely the starting point for the application of mass spectrometry imaging in the field of traditional Chinese medicine. Several areas of application are currently being explored.

Authenticity assessment. There may be differences in the spatial distribution of chemical constituents within the tissue between authentic and non-authentic medicinal materials. Mass spectrometry imaging can compare the spatial distribution patterns of molecules in medicinal materials from different origins, providing a spatial dimension for authenticity assessment. The full-polarity detection capability of the MSI DPI mass spectrometry imaging source ensures that active constituents of varying polarity—such as aglycones, terpenoids and steroids—can be detected and spatially localised.

Research into the mechanisms of medicinal processing. During the processing of traditional Chinese medicinal materials, the chemical constituents within the materials undergo transformation and migration. Mass spectrometry imaging instruments can perform imaging analysis on the same medicinal material before and after processing to observe spatial changes in constituents within the tissue, thereby revealing the impact of processing on the spatial distribution of these constituents. The signal intensity of the MSI DPI’s DESI/PI mode is 1–3 orders of magnitude higher than that of conventional DESI, making it more sensitive to changes in trace constituents during the processing.

Localisation of active constituents. In which tissue regions are the active constituents of Chinese medicinal materials concentrated? Is it the cortex, phloem or xylem? Mass spectrometry imaging can directly answer this question, providing a basis for selecting harvesting sites and optimising processing techniques. The spatial resolution of the MSI DPI, adjustable from 20 to 200 μm as required, enables the localisation of specific tissue structures.

Formulation studies. How do the constituents of individual herbs in traditional Chinese medicine formulations interact during decoction? Mass spectrometry imaging (MSI) can perform imaging analysis on compound preparations to observe the spatial distribution and co-existence relationships of constituents from different sources. The reduced ion suppression effect of the MSI DPI allows complex samples to be analysed directly without the need for cumbersome pre-treatment.

Technical Support: Advantages of the Post-Photoionisation Mass Spectrometry Imaging Source

The effectiveness of mass spectrometry imaging in Traditional Chinese Medicine research is closely related to the detection capabilities of the imaging ion source. The polarity discrimination of traditional DESI imaging sources is particularly detrimental to TCM research—as active ingredients in TCM encompass polar, weakly polar and non-polar compounds, whilst DESI can only detect polar components, resulting in a significant number of active ingredients being missed.

Hefei Xinceyuan’s MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source overcomes this limitation through post-photoionisation technology. The ionisation energy of VUV photons (Kr lamp, 10.0/10.6 eV) covers the ionisation energy range of the vast majority of organic compounds (7–10 eV) and does not exhibit selectivity based on molecular polarity; consequently, weakly polar and non-polar active constituents such as aglycones, terpenoids and steroids are afforded the same opportunities for ionisation and detection as polar constituents.

In validation studies involving imaging of various plant samples, the DESI/PI mode of the MSI DPI detected 100 additional secondary metabolites compared to the DESI mode. In tea leaves, the signal for theanine increased 14.5-fold and that for caffeine 61-fold; whilst in sage, monoterpenes, diterpenes and triterpenes were newly detected; in ginkgo, flavonoids, ginkgolic acid and cashewol were newly detected—these compound types show a high degree of overlap with the chemical classes of active ingredients in traditional Chinese medicine, thereby validating the detection coverage capabilities of MSI DPI in the analysis of plant-derived natural products.

For research scenarios requiring higher spatial resolution, Hefei Xinceyuan’s MSI LDPI laser/photochemical ionisation mass spectrometry imaging source offers a resolution of 2–3 μm, capable of resolving the cellular-level structure of medicinal plant tissues. This product requires no matrix spraying and is free from matrix background interference; it can detect compounds with a minimum charge-to-mass ratio as low as m/z 70, covering a molecular weight range of 70–1,000, making it suitable for the precise spatial localisation of small-molecule active ingredients.

Advancement through Industry-Academia-Research Collaboration

The advancement of mass spectrometry imaging applications in the field of traditional Chinese medicine has been facilitated by in-depth collaboration between industry, academia and research institutions.

The collaboration between Hefei Xinceyuan and the China Academy of Chinese Medical Sciences on the study of cinnabar spots in Atractylodes macrocephala is a prime example of synergy between a mass spectrometry imaging ion source manufacturer and a traditional Chinese medicine research institution. The company provides the mass spectrometry imaging ion source equipment and technical support, whilst the research institution supplies traditional Chinese medicinal material samples and research questions; together, they produce high-calibre academic outcomes.

This collaborative model is expanding to cover a wider range of Chinese herbal medicine varieties and research areas. The multi-brand compatible design of the MSI DPI mass spectrometry imaging source (compatible with Agilent, AB SCIEX and Thermo) enables more Chinese medicine research institutions that already own mass spectrometers to introduce mass spectrometry imaging capabilities with minimal barriers to entry, thereby accelerating the widespread adoption of mass spectrometry imaging technology in the field of traditional Chinese medicine.

Industry Outlook

The application of mass spectrometry imaging technology in the field of traditional Chinese medicine is still in its early stages, but it has already demonstrated unique value. From the aetiology of cinnabar spots on Atractylodes macrocephala to the evaluation of authenticity, and from the mechanisms of medicinal processing to the identification of active constituents, mass spectrometry imaging instruments provide a spatial analytical dimension for traditional Chinese medicine research that cannot be replaced by traditional analytical techniques.

With the continuous improvement in the detection capabilities of domestically produced mass spectrometry imaging ion sources and the gradual increase in the prevalence of such equipment, mass spectrometry imaging is expected to become one of the standard analytical tools for quality assessment and mechanistic research in TCM. This holds significant promise for advancing the modernisation and standardisation of TCM research.

Hefei Xinceyuan has stated that the company will continue to advance the validation of mass spectrometry imaging sources in the field of TCM, conduct collaborative research with TCM research institutions, and provide technical support for spatial molecular analysis to facilitate the modernisation of TCM.