Abstract： Nitrosamine compounds,due to the N-N=O bonds in their structure,can easily cause isomerization.When an HPLC method was used to analyze 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK),double peaks were observed.NNK and its isomers were effectively separated by PFP chromatographic column.Then a LC/MS method was used to investigate the secondary fragment ions of the two separated sample peaks.NMR investigation further confirmed that the impurity peaks in NNK belonged to its isomer,and the isomerization ratio range of NNK was determined by NMR quantitative method through variable temperature experiment.And the NMR results were confirmed through the temperature change test of HPLC.This study verified structure of the isomer through a variety of methods,which was conducive to the accurate analysis of NNK content and provided a theoretical basis for the detection of genotoxic impurities.