Applicability of HILIC-SPE pretreatment for the quantitation of N²-Ethyl-2'-deoxyguanosine in LC-ESI/MS/MS

DNA adducts analysis is crucial for elucidating the mechanism of mutagenesis and carcinogenesis induced by various chemical compounds. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) is commonly employed to determine DNA adduct. Pretreatment is essential in DNA adduct analysis due to a significant concentration disparity between normal 2'-deoxynucleosides (dNs) and DNA adducts. The level of DNA adducts is typically assessed by the ratio of DNA adducts to normal 2'-deoxyguanosine (dG) when the DNA adducts originate from dG. Consequently, the determination of dG and DNA adducts is necessary for accurate evaluation. Reversed-phase (RP)-solid-phase extraction (SPE) is the first choice for pretreatment of DNA adducts analysis. This pretreatment using RP-SPE effectively enriches DNA adducts while removing a substantial amount of dNs, particularly thymidine (T) and 2'-deoxyadenosine (dA), which may suppress the ionization of DNA adducts in the electrospray ionization (ESI) process. However, two separate measurements are required to determine the concentrations of dG and DNA adducts, as dG is removed during the SPE process. Therefore, an SPE method that simultaneously extracts dG and DNA adducts is desirable. In this study, we investigated the applicability of hydrophilic interaction chromatography (HILIC)-type SPE sorbents developed by our team, for the pretreatment of DNA adduct analysis. N²-ethyl-2'-deoxyguanosine (EtdG), an acetaldehyde-derived DNA adduct, was selected as a model DNA adduct. By optimizing the washing and eluting solutions, dG and EtdG were simultaneously extracted from the sample solution while eliminating T and dA. This pretreatment method enables the simultaneous measurements of dG and DNA adducts concentration, thereby enhancing the accuracy and rapidity of the DNA adducts analysis.