Objective To detect and analyze the in vitro metabolites of amfepramone using a rat liver microsomal model by liquid chromatography coupled with high-resolution tandem mass spectrometry(LC-HR-MS/MS),and compare them with those derived from authentic urine samples collected from amfepramone users,so as to evaluate the consistency of the in vitro rat liver microsomal model in predicting the metabolites in vivo.Methods Wistar rat liver microsomes were exposed to 5 μg amfepramone and subsequently incubated for 1 h to simulate the metabolic process in the human body.The authentic urine samples collected from amfepramone users were precipitated with acetonitrile.Results A total of of ten phase I metabolites of amfepramone were produced in the in vitro rat liver microsome model.Eight out of the ten in vitro phase I metabolites were also found in authentic urine samples collected from amfepramone users.The primary metabolic pathways were deduced to be carbonyl reduction,N-dealkylation,and hydroxylation.This in vitro metabolic model-based approach proved to be a simple and rapid method for predicting amfepramone metabolites in vivo.However,it was observed that certain metabolites of amfepramone,including M2(ethcathinone)and M4(cathinone),possess non-exclusive origins,limiting their suitability as standalone biomarkers for detecting amfepramone use in urine specimens.Conclusion This study revealed the involvement of the hydroxylation pathway in amfepramone metabolism.Additionally,four new metabolites were identified in real urine samples from amfepramone users.These preliminary results contribute valuable insights into the pharmacokinetics and forensic analysis of amfepramone,providing a theoretical foundation for further research in these domains.