Simultaneous analysis of four stereoisomers of anti-benzo[ a]pyrene diol epoxide–deoxyguanosine adducts in short oligodeoxynucleotides using reversed-phase high-performance liquid chromatography

Abstract

anti-Benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide ( anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[ a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon–DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereosiomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE–deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligoncleotide adducts.