Jinying Zhang ^1,2 Zhiguo Mao ^1,2 Ding Zhang ^1,2 Lin Guo ^1,2 Hui Zhao ^1,2 Mingsan Miao ^1,2*

• ¹ School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, Henan Province, China
• ² Henan Collaborative Innovation Center for Research and Development on the Whole Industry Chain of Yu-Yao,, Zhengzhou, China

Herbal medicines (HMs) have long played a pivotal role in preventing and treating various human diseases and have been studied widely. However, the complexities present in HM metabolites and their unclear mechanisms of action have posed significant challenges in the modernization of traditional Chinese medicine (TCM). Over the past two decades, mass spectrometry imaging (MSI) has garnered increasing attention as a robust analytical technique that enables the simultaneous execution of qualitative, quantitative, and localization analyses without complex sample pretreatment. With advances in technical solutions, MSI has been extensively applied in the field of HMs. MSI, a label-free ion imaging technique can comprehensively map the spatial distribution of HM metabolites in plant native tissues, thereby facilitating the effective quality control of HMs. Furthermore, the spatial dimension information of small molecule endogenous metabolites within animal tissues provided by MSI can also serve as a supplement to uncover pharmacological and toxicological mechanisms of HMs. In the review, we provide an overview of the three most common MSI techniques. In addition, representative applications in HM are highlighted. Finally, we discuss the current challenges and propose several potential solutions. We hope that the summary of recent findings will contribute to the application of MSI in exploring metabolites and mechanisms of action of HMs.