IDENTIFICATION OF PHOSPHORAMIDE MUSTARD DNA ADDUCTS USING TANDEM MASS-SPECTROMETRY

The reaction pathway of alkylating agents is often exploited in the design of bifunctional anti‐cancer drugs. These drugs form mono‐DNA adducts as well as inter‐ and intra‐strand cross‐linked adducts, notably by reaction at DNA bases, including the N‐7‐position of guanine (G). A positive‐ion fast‐atom bombardment (FAB) mass spectrum of an in vitro preparation of DNA alkylated with phosphoramide mustard (the active metabolite of the anti‐cancer drug cyclophosphamide) indicated the presence of the two mono‐DNA adducts N‐(2‐chloroethyl)‐N‐[2‐(7‐guaninyl)ethyl] amine, designated NORG, and N‐(2‐hydroxyethyl)‐N‐[2‐(7‐guaninyl)ethyl] amine, designated NORGOH, (MH+ 257/259 and 239, respectively) but not the presence of the cross‐linked adduct N,N‐bis‐[2‐(7‐guaninyl)ethyl] amine, designated GNORG (MH+ 372). Using synthetic standards, daughterion spectra of NORG, NORGOH and GNORG were obtained (matrix 0.2 M p‐toluene sulphonic acid in glycerol) by positive‐ion FAB tandem mass spectrometry (FAB‐MS/MS). The daughter‐ion spectra of both mono‐DNA adducts NORG and NORGOH contained a fragment ion at m/z 152 [G + H]+, whereas the cross‐linked adduct, GNORG, showed an ion at m/z221, [MH − G]+. Evidence for the presence of NORG, NORGOH and GNORG in the in vitro preparation was obtained by performing a double parent‐ion scan on m/z 152 and 221. The presence of GNORG was further supported by performing a single parent‐ion scan on m/z 221. The use of this MS/MS technique should eliminate the need for intricate sample purification in the identification of GNORG in biological extracts. Copyright © 1990 John Wiley & Sons Limited