Characterization and determination of metabolites to monitor metabolic pathways play a paramount role in evaluating the efficacy and safety of medicines. However, the separation and quantification of metabolites are rather difficult due to their limited contents in vivo, especially in the case of Chinese medicine, due to its complexity. In this study, an effective and convenient method was developed to simultaneously quantify bufalin and its nine metabolites (semi-quantitation) in rat plasma

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... er an oral administration of 10 mg/kg to rats. The prototype and metabolites that were identified were subsequently quantified using positive electrospray ionization in multiple reaction monitoring (MRM) mode with transitions of m/z 387.4→369.6 and 387.4→351.3 for bufalin, m/z 513.7→145.3 for IS, and 387.4→369.6, 419.2→365.2, and 403.2→349.2 for the main metabolites (3-epi-bufalin, dihydroxylated bufalin, and hydroxylated bufalin, respectively). The method was validated over the calibration curve range of 1.00–100 ng/mL with a limit of quantitation (LOQ) of 1 ng/mL for bufalin. No obvious matrix effect was observed, and the intra- and inter-day precisions, as well as accuracy, were all within the acceptable criteria in this method. Then, this method was successfully applied in metabolic profiling and a pharmacokinetic study of bufalin after an oral administration of 10 mg/kg to rats. The method of simultaneous determination of bufalin and its nine metabolites in rat plasma could be useful for pharmacokinetic–pharmacodynamic relationship research of bufalin, providing experimental evidence for explaining the occurrence of some adverse effects of Venenum Bufonis and its related preparations.