Gas-Phase Radical Ion Formation from Protonated 1-Methyl-4-nitrosopiperazine: An ESI-MS/MS, Broken-Symmetry DFT, and...

The formation of odd-electron radical ions (open-shell) from even-electron precursor ions (closed-shell) in electrospray ionization (ESI) tandem mass spectrometry (MS) represents a violation of the "even-electron rule" and has been observed in several classes of compounds. Such processes often involve homolytic bond cleavage and may exhibit multireference character, which is not always reliably described by conventional single-reference methods, such as density functional theory (DFT). In this paper, protonated 1-methyl-4-nitrosopiperazine (MeNP) was employed as a model system to investigate the formation mechanism of an odd-electron fragment ion under ESI-MS/MS conditions. The study integrated high-resolution ESI-MS/MS experiments with conventional DFT, broken-symmetry DFT (BS-DFT), and multireference methods such as second-order perturbation theory (CASPT2) based on the complete active space self-consistent field (CASSCF) approach. Experimentally, the radical ion at m/z 100 was identified as the base peak at low collision energies, with an intensity much higher than that of the even-electron ion at m/z 99. Conventional closed-shell singlet and open-shell triplet DFT calculations predicted the formation of the radical ion with prohibitively high energies, inconsistent with the experimental observations. In contrast, the multireference character of the homolytic N-N bond cleavage was indicated by T1 diagnostic and diradical character y values. BS-DFT and CASPT2//CASSCF calculations support a plausible fragmentation pathway leading to the radical ion at m/z 100, which was kinetically favorable and consistent with experimental results. These findings highlight the importance of multireference treatments for rationalizing radical ion formation in protonated MeNP under ESI-MS/MS conditions.