Simultaneous Quantification of Three Lipid Peroxidation-Derived Etheno Adducts in Human DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N-6-etheno-2'-deoxyadenosine (epsilon dAdo), 3,N-4-etheno-2'-deoxycytidine (epsilon dCyt), and 1,N-2-etheno-2'-deoxyguanosine (1,N-2-epsilon dGuo) in tissue DNA. Typically, [C-13(1),N-15(2)]epsilon dAdo, [N-15(3])epsilon dCyd, and [C-13(1),N-15(2)]1,N-2-epsilon dGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilon dAdo, epsilon dCyd, and 1,N-2-epsilon dGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilon dAdo, epsilon dCyd, and 1,N-2-epsilon dGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilon dAdo, epsilon dCyd, and 1,N-2-epsilon dGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mu g of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mu g of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.