Novel LC-ESI/MS/MSⁿ method for the characterization and quantification of 2′-deoxyguanosine adducts of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D linear quadrupole ion trap mass spectrometry

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MSⁿ) technique has been developed for the characterization and quantification of 2′-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP-DNA adducts were analyzed by MS/MS and MSⁿ scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 10⁸ DNA bases, and the limit of quantification (LOQ) was 3 adducts per 10 ⁸ DNA bases in both MS/MS and MS³ scan modes, using 27 μg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6- phenylimidazo[4,5-b]pyridine (dG-C8-[²H₃C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H - 116]⁺). The consecutive reaction monitoring (CRM) scan modes in MS³ and MS⁴ were used to measure and further characterize product ions of the aglycone ion (BH₂⁺) (Guanyl-PhIP). The MS³ scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MSⁿ scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LC-ESI/MS/MSⁿ method is the first reported application on the use of the MS³ scan mode for the analysis of DNA adducts in vivo.