Quantitative detection of N⁷‐(2‐hydroxyethyl)guanine adducts in DNA using high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

High-performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to develop a sensitive and selective method for the quantitative measurement of N(7)-(2-hydroxyethyl)guanine (N(7)-HEG) adducts in DNA obtained from ethylene oxide-exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N(7)-HEG (m/z 196) was monitored. The detection limits for N(7)-HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R(2) = 0.999) was observed for signal intensities obtained by injecting 16 fmol--33 pmol of N(7)-HEG into the HPLC/ESI-MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose-response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N(7)-HEG formation in other biological systems.

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

“…For example Ravanat et al showed that for the analysis of 8-oxo-2 0 -deoxyguanosine standards using SIM the limit of detection was much higher at 5 pmol compared with 20 fmol when using SRM (98). A similar finding was observed by Liao et al who showed that a lower limit of detection was obtained for the analysis of N-7(2-OHEt)G standards with calf thymus DNA as the matrix using SRM (16 fmol) when compared with SIM (1 pmol) (99).…”

“…These labile base adducts can be released from the DNA by thermal hydrolysis and then separated from the depurinated DNA backbone by filtration using molecular weight cut-off filters. DNA adducts formed by ethylene oxide, ethylating agents and acrylamide have been studied using this approach (99,110,111). Doerge et al detected the N-3 adenine and N-7 guanine adducts derived from glycidamide, the reactive epoxide metabolite of acrylamide, in mice and rats following neutral thermal hydrolysis.…”

“…This transition of the [M þ H] þ precursor ion to guanine (m/z 152) can be used as a generic method for monitoring N-7 alkylguanines. This approach has been used for the detection of N-7(2-OHEt)G, N-7 (1-or 2-hydroxybutyl)guanine, N-7 (trihydroxybutyl)guanine, N-7EtG and N-7 (2-carbamoyl-2-hydroxyethyl)guanine adducts by SRM (99,(109)(110)(111)(112).…”

The application of mass spectrometry in molecular dosimetry: Ethylene oxide as an example

“…By following the sample preparation procedures of the LC/ MS method (Leclercq, Laurent, & De, 1997), an LC/MS/MS method was developed to quantify N7-HEG levels in Hep G2 cells incubated with EO (Liao et al, 2001). DNA samples were hydrolyzed by neutral thermal hydrolysis, and filtered with Microcon YM-3 microconcentrator with a cutoff mass at 3,000 Da.…”

Structural Characterization of the DNA Adducts Induced by 1,3-Bis-(2-Chloroethyl)-1-Nitrosourea Using Electrospray Ionization Tandem Mass Spectrometry

“…Especially, it is widely held that the cytotoxic activity may be related to the formation of DNA interstrand cross-link [9]. N 7 -(2-hydroxyethyl)guanine (N 7 -HEG) adducts in DNA has been quantitative measured with high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) [10]. Rajinder et al [11,12] applied HPLC-MS/MS method with multiple reaction monitoring (MRM) in the detection of N 7 -ethyldG.…”