New Methods For Matrix-assisted Laser Desorption Ionization Mass Spectrometry Analysis Of Small-molecule Metabolites In Traditional Chinese Medicine Based On The Novel Nano-matrix

Purpose:In response to the demand for a more comprehensive and in-depth study of the metabolomics of traditional Chinese medicine（TCM）and the current shortcomings of the traditional matrix-assisted laser desorption ionization mass spectrometry（MALDI-MS）,this study systematically carried out the metabolomics of TCM based on MALDI technology,and established a series of efficient and practical MALDI-MS and mass spectrometry imaging（MSI）analysis methods for small-molecule metabolites of TCM by combining the performance advantages of nanomaterials.These methods break through the problems of poor detection of small-molecule analytes by routine MALDI-MS,complicated sample pre-treatment and high sample requirements in MSI.Meanwhile,the qualitative and quantitative detection and spatial metabolomics research of small-molecule metabolites of TCM were carried out based on these development methods,which will expand the application of MALDI mass spectrometry in the field of TCM.This study provides new methods for the in-depth analysis of small molecules in TCM and also provides technical support for future research in the field of spatial metabolomics of TCM and the detection of small-molecule metabolites.Methods:1.The Pt@MXene nanocomposite was constructed by a self-reduction reaction using single/few-layer MXene nanosheets and H₂PtCl₆·6H₂O as raw materials,and also used as a MALDI-MS matrix for qualitative and quantitative analysis of small-molecular natural products of TCM.2.The Ag NWs@MXene composite nanomaterial was prepared using a direct doping method using silver nanowires（Ag NWs）and single/few-layer MXene nanosheets as raw materials.It was used as a novel substrate for MALDI-MSI by self-assembled film formation strategy for MSI analysis of Taraxaci Herba root tissue sections.3.The Ag NWs@MXene free-standing film was prepared by vacuum-assisted filtration using Ag NWs@MXene as raw materials.It was used as a novel substrate in combination with the molecular blotting method to achieve in-situ MALDI-MSI analysis of endogenous metabolites of Scutellaria baicalensis Georgi root,Zingiberis Rhizoma Recens and Lonicerae Japonicae Flos.Results:1.Pt nanoparticles were successfully introduced to the MXene surface by a one-step reduction to form a noble metal/MXene nanocomposites（Pt@MXene）.Compared with the commonly used organic matrix CHCA,precursor MXene and the popular inorganic nanosubstrate GO,it has the following advantages:（i）with a quit low background peaks,suitable for the detection of small-molecules in TCM;（ii）significantly enhanced ionization efficiency of analytes and improved detection sensitivity;（iii）universally applicable to various small molecules in TCM,including amino acids,phytohormones,carbohydrates,organic acids,alkaloids,flavonoids,iridoid,and fatty acids;（iv）excellent signal reproducibility（coefficient of variation<8%）,salt(5 mg·m L^-1)and protein（0.5 M）tolerance.In addition,a novel MALDI-MS method was established for the analysis of natural products of TCM,which can quantify gentiopicroside（GP）in Gentiana scabra Bge and tanshinone IIA（TS）in Salvia miltiorrhiza Bge.The above results indicate that the matrix makes up for the shortcomings of traditional organic matrices and exhibits higher performance than some popular inorganic nano-matrices for the high-throughput detection and quantitative analysis of small-molecule natural products of TCM.2.Ag NWs@MXene composite nanomatrix was successfully prepared by direct doping method.The matrix improved the UV absorption capacity and detection sensitivity,and the signal response of the analytes was 2～4 times higher than that of MXene.Application of this matrix to MALDI-MSI analysis provides lower background interference and higher molecular detection capability than commonly used organic matrices（DHB,CHCA,SA）.A novel method of MALDI-MSI analysis based on Ag NWs@MXene matrix was established for MSI of tissue sections from different parts of Taraxaci Herba root to achieve in-situ visual characterization of 16 known endogenous small-molecule metabolites and 4 unknown endogenous metabolites from Taraxaci Herba root.Among them,carbohydrates were mainly distributed in the cortex and phloem and concentrated in the middle and upper ends closer to the above-ground parts;amino acids were unevenly distributed throughout the root tissues;organic acids were slightly higher in the cortex and phloem,with similar distribution patterns as carbohydrates in the root;and choline was more enriched in the middle end of the root.3.The Ag NWs@MXene free-standing film was successfully prepared by vacuum-assisted filtration method.The surface modification of MXene by adding Ag NWs improved the surface hydrophobicity of the free-standing film and reduced the detection error.The Ag NWs@MXene film was used as a novel substrate for MSI with excellent signal distribution uniformity and signal stability（coefficient of variation<10%）.A novel method for MSI analysis of TCM was established using Ag NWs@MXene free-standing film as a substrate combined with in-situ molecular imprinting technique.Compared with conventional MALDI-MSI based on frozen sections and matrix spraying,this method is particularly suitable for MALDI-MSI analysis of some TCMs with brittle mass,highly fibrotic and no obvious histological structure,which are difficult to prepare frozen sections,and has the advantages of being simpler,faster and less costly.This method successfully obtained high-quality MSI images of endogenous small-molecule metabolites in Scutellaria baicalensis Georgi root and Zingiberis Rhizoma Recens,and characterized for the first time the spatial distribution information of 15 endogenous metabolites,including flavonoids,fatty acids,phenolic acids,organic acids,carbohydrates and amino acids,in Lonicerae Japonicae Flos.