Simultaneous quantitative analysis of N-ethyladenine and N-ethylguanine in human leukocyte deoxyribonucleic acid by stable isotope dilution capillary liquid chromatography–nanospray ionization tandem mass spectrometry

Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N³-ethyladenine (3-EtAde), N⁷-ethylguanine (7-EtGua), and regioisomers of ethylthymine. Among them, 3-EtAde and 7-EtGua are present in human urine and their levels are higher in smokers than in nonsmokers. The amount of ethylated DNA adducts in tissue DNA represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of 3-EtAde and 7-EtGua by stable isotope dilution capillary liquid chromatography–nanospray ionization tandem mass spectrometry (capLC–NSI/MS/MS). Under the highly selective reaction monitoring (H-SRM) mode, the detection limit of 3-EtAde and 7-EtGua injected on-column was 5.0 fg (31 amol) and 10 fg (56 amol), respectively. The quantification limit for the entire assay was 50 and 100 fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 10⁹ normal nucleotides, respectively, starting with 20 μg of DNA isolated from <1 mL of blood and injecting an equivalent of 4 μg of DNA on-column. The mean (±SD) levels of 3-EtAde and 7-EtGua in leukocyte DNA from 20 smokers were 16.0 ± 7.8 and 9.7 ± 8.3 in 10⁸ normal nucleotides, respectively, which were statistically significantly higher than those of 5.4 ± 2.6 3-EtAde and 0.3 ± 0.8 7-EtGua in 10⁸ normal nucleotides from 20 nonsmokers (p < 0.0001). The levels of 3-EtAde and 7-EtGua in these 40 leukocyte DNA samples are positively correlated (γ = 0.6970, p < 0.0001). Furthermore, there are statistically significant associations between the number of cigarettes smoked per day, as well as the smoking index, and the levels of 3-EtAde and 7-EtGua. Levels of 3-EtAde and 7-EtGua are compared to those of ethylthymidine adducts. To our knowledge, this is the first assay for simultaneous quantification of 3-EtAde and 7-EtGua in the same DNA sample and is the first report of 3-EtAde in human DNA. This highly sensitive and specific stable isotope dilution capLC–NSI/MS/MS assay should be useful in measuring 3-EtAde and 7-EtGua in human leukocyte DNA as potential biomarkers for smoking-related cancers.     

Strongly nonlinear beats in the dynamics of an elastic system with a strong local stiffness nonlinearity: Analysis and identification

We consider a linear cantilever beam attached to ground through a strongly nonlinear stiffness at its free boundary, and study its dynamics computationally by the assumed-modes method. The nonlinear stiffness of this system has no linear component, so it is essentially nonlinear and nonlinearizable. We find that the strong nonlinearity mostly affects the lower-frequency bending modes and gives rise to strongly nonlinear beat phenomena. Analysis of these beats proves that they are caused by internal resonance interactions of nonlinear normal modes (NNMs) of the system. These internal resonances are not of the classical type since they occur between bending modes whose linearized natural frequencies are not necessarily related by rational ratios; rather, they are due to the strong energy-dependence of the frequency of oscillation of the corresponding NNMs of the beam (arising from the strong local stiffness nonlinearity) and occur at energy ranges where the frequencies of these NNMs are rationally related. Nonlinear effects start at a different energy level for each mode. Lower modes are influenced at lower energies due to larger modal displacements than higher modes and thus, at certain energy levels, the NNMs become rationally related, which results in internal resonance. The internal resonances of NNMs are studied using a reduced order model of the beam system. Then, a nonlinear system identification method is developed, capable of identifying this type of strongly nonlinear modal interactions. It is based on an adaptive step-by-step application of empirical mode decomposition (EMD) to the measured time series, which makes it valid for multi-frequency beating signals. Our work extends an earlier nonlinear system identification approach developed for nearly mono-frequency (monochromatic) signals. The extended system identification method is applied to the identification of the strongly nonlinear dynamics of the considered cantilever beam with the local strong nonlinear stiffness at its free end.