Detection and Identification of Benzo[a]pyrene Diol Epoxide Adducts to DNA Utilizing Capillary Electrophoresis-Electrospray Mass Spectrometry

Abstract

Capillary zone electrophoresis (CZE) coupled with negative ion electrospray mass spectrometry (ES-MS) is used for the detection and identification of adducts formed from the reaction of DNA with (+/-)-anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE),an active metabolite of benzo[a]pyrene (BaP). Results presented in this paper demonstrate low nanogram detection limits ( < 10 ng or < 15 pmol) for normal scan spectra and collision-induced dissociation spectra of the main nucleotide adduct formed from this reaction. (BPDE reacts predominantly with the exocyclic amino group of guanine.) Exploitation of selective reaction monitoring (SRM) produces detection limits in the low picogram range ( < 85 pg or < 130 fmol). The application of sample stacking significantly increases the concentration detection limit (to approximately 10(-8) M). Nucleotide adducts are negatively charged at most pHs and are therefore ideally suited to the stacking process used in this research. These techniques have been applied to the analysis of the adducts formed from the in vitro reaction of BPDE with DNA. In addition it is shown that CZE-ES-MS, combined with solid-phase sample cleanup, can detect adducts at levels of four adducts in 10(7) unmodified bases or less.

Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection

“…The authors used an online sample stacking technique for the pre-concentration of the negatively charged adducts and removal of sample buffer (196). A similar approach was used by Barry et al to analyse enzymatically hydrolysed calf thymus DNA treated with benzo[a]pyrene diol epoxide for which the authors noted the presence of an adducted thymidine-guanine dinucleotide in addition to a 2 0 -deoxyguanosine 5 0 -monophosphate adduct of benzo[a]pyrene diol epoxide (197). Again the sample stacking approach in combination with CZE-ESI-MS was used by Deforce et al to detect the formation of adducts following the reaction of phenyl glycidyl ether with calf thymus DNA (145).…”

Recent developments in the determination of urinary cancer biomarkers by capillary electrophoresis

“…Since CE-ESI-MS was first applied for analysis of DNA adducts in 1994 by Janning et al [75], numerous studies have been conducted to detect modified oligonucleotides and modified nucleotides. The researches that have performed on determination of modified oligonucleotides included styrene oxide-modified oligonucleotides [75,82], alkylated oligonucleotides and oligonucleotides containing halogenated nucleobases [79], N-(acetylamino) fluorine-modified oligonucleotides [83], and unmodified oligonucleotides [77]. The modified nucleotides that have been quantitatively studied included N 7 , O-6, N 2 -modified guanosine monophosphate [82],…”

“…-modified guanosine monophosphate and adenosine monophosphate [72], anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol-9,10-epoxidemodified nucleotides [79], phenyl glycidyl ether-modified nucleotides, and phenyl glycidyl ether 5' phosphate-modified nucleotides [78], NAD 1 and FAD 1 dinucleotides [74], modified nucleosides including benzo[g]chrysene and 5,6-dimethylchrysene-modified deoxypurines [84], and styrene oxide-modified adenosine adducts [85]. By using sample stacking technique, CE-MS reached the detection limit of 6.3610…”

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

“…A number of methods have been developed for detection of BPDE-DNA adducts, including capillary electrophoresis (CE)-laserinduced fluorescence (LIF) [13][14][15][16], accelerator mass spectrometry [17,18], liquid chromatography or capillary electrophoresis-mass spectrometry [19][20][21][22][23][24][25][26], and 32 P-postlabelling [27]. However, only few methods demonstrate the capability of resolving the stereoisomers of BPDE-DNA adducts.…”