Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

An ultra-performance liquid chromatography tandem mass spectrometry with multiple reaction monitoring method (UPLC-MRM/MS) is developed for fast and sensitive analysis of four genotoxic stereoisomers of anti-benzo[a]pyrene diol epoxide (BPDE)–N2dG adducts (trans-(+), trans-(−), cis-(+) and cis-(−)), which result from environmental exposure to ubiquitous pollutant benzo[a]pyrene (B[a]P). The developed method displays a low limit of detection of <0.7 fmol (S/N = 3) for the four stereoisomers of anti-BPDE–N2dG, a dynamic range of 2 orders of magnitude (2.3–630 fmol, R2 ≥ 0.997), and one separation of 2–4 min. The developed method enables us to use the stereoisomers of anti-BPDE–N2dG as a biomarker and to study the stereoselectivity of metabolic activation of B[a]P in human lung A549 cells. The UPLC-MRM/MS analysis of cellular DNA exposed to B[a]P show that activation of B[a]P in A549 cells predominantly induces trans-(+)-anti-BPDE–N2dG with cis-(+)-anti-BPDE–N2dG and one syn-BPDE–N2dG as two minorities, while trans-(−)-anti-BPDE–N2dG and cis-(−)-anti-BPDE–N2dG are absent. The observed preferential formation of trans-(+)-anti-BPDE–N2dG in B[a]P treated A549 cells may result from combined stereoselectivity of the metabolic activation of B[a]P and the reaction of anti-BPDE with dsDNA. The results also suggest that a number of key optical intermediates are formed during activation of B[a]P in A549 cells, including trans-(+)-B[a]P-7,8-dihydrodiol and trans-(−)-B[a]P-7,8-dihydrodiol and their corresponding downstream metabolites (+)-anti-BPDE and (+)-syn-BPDE.