As a dual-use substance，nitrogen mustard is not only on a list of the Convention on the Prohibition of Chemical Weapons，but its derivatives are traditional clinical chemotherapy drugs. In this paper，the adducts formed by incubation of three typical nitrogen mustards（HN1，HN2 and HN3） with 4 bases，4 deoxyribonucleosides，4 deoxyribonucleotides and salmon DNA respectively，were analyzed and identified by high-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry. The HN-DNA adducts in all 39 incubation systems were chromatographically separated and the data were collected by Full MS/dd-MS

2

mode to obtain their mass spectrometry characteristics. The results showed that the

mono-adducts formed by three kinds of nitrogen mustard with guanine，adenine and cytosine could be identified in each incubation system and the di-adducts formed by nitrogen mustard with guanine or adenine could be identified. Although the mono-adducts formed by three kinds of nitrogen mustard with thymine were detected in the base incubation system，they were not detected in the salmon DNA incubation system，indicating that adducts formed by HNs with thymine base were not primary adducts for DNA. By exploring the mass spectrometry characteristic fragments of each adduct，the specific ion pairs of series of DNA adducts for the multi-reaction monitoring mode of mass spectrometry have been summarized，which lays a foundation for the establishment of a method for structural analysis，accurate traceability and rapid quantification of nitrogen mustard-DNA adducts in complex biological samples.