Identification of phosphoramide mustard/DNA adducts using tandem mass spectrometry

Abstract

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, daughter-ion 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/z 221, [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.(ABSTRACT TRUNCATED AT 250 WORDS)