Rapid characterization and identification of the chemical constituents and the metabolites of Du-zhi pill using UHPLC coupled with quadrupole time-of-flight mass spectrometry

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A reliable method using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was established to conduct a comprehensive analysis of the chemical constituents of Du-zhi pill (DZP) as well as their metabolites in rat plasma, urine and feces after gastric perfusion. The efficient on-line mass data acquisition modes combined the various off-line mass data mining strategy was applied. A full mass scan was performed, and then accurate MS/MS datasets were obtained through the use of a multiple mass defect filter (MMDF) and dynamic background subtraction (DBS)-dependent data acquisition method. Furthermore, post-acquisition data processing was conducted using various data-mining tools, including extracted ion chromatography (XIC), mass defect filtering (MDF), product ion filtering (PIF), and neutral loss filtering (NLF) (MetabolitePilot™). Finaly, a total of 176 compounds were identified or tentatively characterized in DZP. Moreover, a total of 233 components in vivo, which includes 92 prototype components and 141 metabolites, were unambiguously or tentatively identified in rat plasma, urine and feces. The metabolic pathways, including phase I reactions (hydroxylation, dehydroxylation and hydrogenation) and phase II reactions (acetylation, sulfation, glucuronidation and methylation), for the absorbed constituents, were explored and summarized. This is the first systematic study on the components of DZP and their metabolites in vivo. This study provide a valid analytical strategy for the characterization of chemical compounds and metabolites of TCM formulas. Moreover, an integrative strategy was proposed for the characterization and identification of chemical constituents and metabolites for additional TCM prescriptions.