Analysis Of Saponins In Panax Species Using MALDI-TOF Mass Spectrometry Imaging

In Panax genus,Panax ginsengC.A.Mey.,PanaxnotoginsengF.H.Chen.and PanaxquinquefoliusL.are three most widely used traditional Chinese medicines with saponins as their main bioactive compounds.Different ginsenosides have variedpharmacological effects,and the contents of saponins vary with the ages and species of Panax genus.Recently,somemethodsdepend on the chromatography,mass spectrometry and spectroscopy were developedto accomplish the quality control,bioactive compounds selection and pharmacological analysis of the saponins.However,the application of these methods was limited by the complex sample preparation procedure.Matrix assisted laser desorption/ionization mass spectrometry imaging(MALDI MSI)is a rapid,in-situ and high-throughput technique better than traditional methods in the analysis of clinical or animal samples.Howeveronly few analysis on plants,especiallytraditional Chinese medicinesusing MALDI MSI were reported.In this work,we first established a series of methods of MALDI MSI,including sample preparation,data collection and data analysis,for the analysis of Panaxspecies and differentiated Panax ginsengwith three ages.Then all three kinds of Panax plants were also analyzed and differentiated.The methodsshowed high sensitivity and reproducibility in all the three herbs and thus have great potentials in the analysis of plant metabolites.In theanalysis of Panax ginsengwith three ages,sample preparation method,which greatly affects the micro-extraction,co-crystallization and then the ion yields,was first optimized.2,5-dihydroxybenzoic acid(DHB)was selected as the best matrix from 9-aminoacridine(9-AA)and?-cyano-4-hydroxy cinnamic acid(CHCA),and then coated to the sample slices of 30 ?m thickduring an optimized coating procedure.By using the optimized detection parameters,such as laser energy,spot size and spatial resolution,data of Panax ginsengwith three ages were collected.18 peaks corresponding to 31 saponins,including 3 rarely reported saponins,with distinct localization were detected and identified,showing the high sensitivity of the analysis.Additionally,according to the ion images,the data collected from the cork,which accumulated most of the saponins,of the sample were more suitable for the principle component analysis(PCA)than the whole data set to differentiate the ages of Panax ginseng.The established MALDI MSI methods were also used to analyze Panax ginseng,Panaxnotoginseng and Panaxquinquefolius.51 saponins were identified and located in the three Panax species,indicatingthe good universality of the methods.Saponins were also found located and accumulated differently in the three species.According to the ion image,data from the area where contains the most saponins were subjected to PCA analysis to differentiate the three species.Then 16 peaks of saponins were found with the highest variation between the groups,and could be used as chemical markers for the identification of the three Panax species.In addition,it was also found that the relative concentration of the saponin in different samples could be directly compared according to the lightness in the ion images,which relies on the high reproducibility of the methods.