2.15. DDA-CNL/MS3 Gas-Phase Fractionation Method

Purified DNA extracted from rat liver was available from previous studies and was used as the matrix for our method development. DNA was enzymatically hydrolyzed and purified as reported above. A standard mixture of six isotopically labelled DNA adducts ([¹⁵N₅]N²-ethyl-dG, [¹⁵N₅]N⁶-methyl–dA, [D₄]O⁶-POB-dG, [D₄]O⁶-POB-dT, [D₄]O⁶-PHB-dG, [¹⁵N₅]8-OH-PdG (structures in SI)) was prepared and spiked in the matrix previously reconstituted in 20 µL of LC-MS H₂O prior to LC-MS analysis.

The MS analysis was performed with Orbitrap detection (resolution of 60,000) in: (i) gas-phase fractionation mode with the mass range of interest split into four scan segments (m/z 197–310, m/z 305–380, m/z 375–450, and m/z 445–750) or (ii) in standard mode with a single scan segment (m/z 197–750). In each partial or full scan, quadrupole filtering was used with a maximum injection time of 200 ms and an automatic gain control (AGC) setting of 5.0 × 10⁴.

For each scan segment, the top five ions were selected for MS² fragmentation with quadrupole isolation of 1.5 m/z, using collision induced dissociation (CID) with a normalized collision energy of 30%, maximum injection time of 200 ms, and Orbitrap detection at a resolution of 30,000. An exclusion list of 95 ions (SI) with a mass tolerance of 5 ppm was used, as was dynamic exclusion of 30 s and an intensity threshold of 2.0 × 10³. MS³ fragmentation was triggered upon observation of the accurate-mass neutral loss of 2′-deoxyribose (-dR: 116.0474 ± 0.0006 m/z, 5 ppm) upon MS² fragmentation. MS³ fragmentation was performed with high-energy collisional dissociation (HCD) with a normalized collision energy of 50%, maximum injection time of 250 ms, and Orbitrap detection at a resolution of 15,000. This gas-phase fractionation MS method was used for DNA-adduct profiling.