Abstract: In order to accurately determine the N-nitrosonornicotine(NNN) and its metabolites in mouse blood,a UPLC-Q Exactive orbit trap high-resolution mass spectrometry method was established by optimizing chromatographic and mass spectrometric conditions and using NNN-d₄ and HPB-d₄ as internal standards.Wherein,after protein precipitating with acetonitrile,the sample was separated directly by an HILIC column in UPLC spectrometer.NNN and its metabolites in mouse plasma were analyzed by mass spectrometry under full scan/data-dependent MS2(FS/ddMS²) mode.The results showed that:1) The limits of detection and the recoveries of NNN and its metabolites ranged from 0.019 to 0.067 ng/mL and from 78.0% to 117.5%,respectively,with the intra-day precision of 0.47%-8.38%.2) The developed method was suitable for the simultaneous analysis of NNN and its seven metabolites in mouse plasma.