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Introduction Traditional Chinese medicine(TCM)represents a holistic healthcare system that is based on human experience over the past 3,000 years and has become increasingly popular in Western countries during the last decade.In Chinese herbal medicine(CHM),one aspect of TCM,the pharmaceutical efficacy is regarded to depend on the synergistic effects of multiple components of the herbs1-3.The analysis and authentication of CHM is a challenging task because of the complexity of the herbs used and the natural variability of the plants4.Generally,chromatographic fingerprinting is regarded as an effective method for authentication of CHM1-4.For a complete characterization of CHM,one-dimensional liquid chromatography(1D-LC)does not provide enough resolving power.Therefore,comprehensive two-dimensional liquid chromatography(2D-LC)is the method of choice for their analysis.Methods A comprehensive 2D-LC method for the analysis of decoctions of herbs used in CHM was developed based on reversedphase LC in the first and second dimension.The accessible two-dimensional separation space could be enlarged by designing a complex gradient for the second-dimension separation.Furthermore,Si-Wu-Tang,a preparation of four herbs,as well as the individual herbs contained in Si-Wu-Tang were analyzed to investigate the possibility of authentication of CHM.Results An important aspect of the analysis and authentication of CHM is to ensure that the correct herbs are contained in the preparation.To enable authentication of Si-Wu-Tang,characteristic components of each herb that were tentatively identified using detection by Q-TOF mass spectrometry as well as further high abundant peaks were selected to construct a template.The templates were then matched to the peaks detected in Si-Wu-Tang in terms of first and second dimension retention times,as well as agreement of the base peak in the respective mass spectra.Generally 75%or more of the template peaks could be matched to peaks detected in Si-Wu-Tang,which shows the possibility to detect characteristic components of the individual herbs in a preparation.To investigate the possibility to detect an adulteration of Si-Wu-Tang,changes in template matching following omission and replacement of one herb from Si-Wu-Tang were examined.The matching of a considerably smaller number of template peaks disclosed the adulteration.Further,it is illustrated that one or a few marker compounds are not sufficient for authentication of CHM when those compounds are not uniquely contained in one herb.Novel Aspect Comprehensive 2D-LC offers increased resolving power for a complete characterization and authentication of Chinese Herbal Medicine.