Abstract: A method of microdialysis coupled with ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) was developed to simultaneously determine the nicotine(Nic) and its metabolites cotinine(Cot),nornicotine(NNic),nicotine-N'-oxide(NNO),norcotinine(NCot) and trans-3'-hydroxycotinine(3-HC)in rat brain.After the addition of internal standards,Nic-d₃,Cot-d₃,NNic-d₄ and 3-HC-d₃,the microdialysed sample of rat brain tissue was directly separated by an HILIC column,and determined by mass spectrometry under multi-reaction monitoring(MRM) mode.The results showed that the inter-and intra-day precision and accuracy of Nic,Cot,NNic,NNO,NCot and 3-HC were all less than 11%.The limits of detection ranged from 0.012 to 0.029 ng/mL,and the recoveries of microdialysates ranged from 4.7% to 9.3%. This method is simple,rapid,sensitive,stable,and can be used to quantify nicotine and its metabolites in rat brain in vivo.