Characterization of nitrogen mustard formamidopyrimidine adduct formation of bis(2-chloroethyl)ethylamine with calf thymus DNA and a human mammary cancer cell line.

A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS3) method was established to characterize and measure five guanine adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM–G) adduct and its cross-link (G–NM–G), the ring-opened formamidopyrimidine (FapyG) monoadduct (NM–FapyG) and cross-links in which one (FapyG–NM–G) or both (FapyG–NM–FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limi...