Equilenin-2 '-deoxynucleoside adducts: analysis with nano-liquid chromatography coupled to nano-electrospray tandem mass spectrometry

The interaction of Q-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupli ng of miniaturized liquid chromatography (LC) to electrospray tandem mass s pectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reac tion of 8-hydroxyequilenin with the main 2 ' -deoxynucleosides were separat ed on a Hypersyl C-18 BDS nano-HPLC column (15 cm X 75 mum i.d.) at a now-r ate of 300 nl min(-1) using gradient elution with CH3OH-0.2% CH3COOH in H2O . The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution lo w-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts wer e found. The nano-HPLC column-switching ES-MS system was tested for its sen sitivity on a triple-quadrupole instrument, and detection limits down to 19 7 fg in the single reaction monitoring mode were obtained for semi-preparat ively isolated equilenin-2 ' -deoxyguanosine adduct. Copyright (C) 2001 Joh n Wiley & Sons, Ltd.