Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (B[a]PDE) with 20-deoxynucleoside 30-monophosphates. The B[a]PDE plus 20-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M2H]2 consistent with B[a]PDE adducted (either on the base or phosphate group) 20-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)2H]2, resulting from cleavage of the glycosidic bond between the 20-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 20-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 20- deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)2H]2 and [(20-deoxyribose+phosphate+B[a]Ptriol)2H]2, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 20-deoxynucleotides.