Rationale. The mechanism underlying dopant-assisted APPI's formation of ions is unclear and still under debate for many chemical classes. In this study, we reexamined the gas-phase reaction mechanisms responsible for the generation of [M-H] + precursor ions, resulting from the loss of a single hydrogen atom, in a series of N-alkyl-substituted thieno[3,4-c]-pyrrole-4,6-dione (TPD) derivatives. Methods. Atmospheric pressure photoionization along with higher-order MS/MS n using high-resolution mass spectrometry (APPI-HR-CID-MS n ) and electronic structures calculations using density functional theory were used to determine the chemical structure of observed [M-H] + ions. As a result, the higher-order MS n (n=3) experiments revealed a reversed-Diels-Alder fragmentation cleavage mechanism leading to a common fragment ion at m/z 322 from the studied [M1-5-H]+ ion species. In addition, the calculation for two chemical structure models (N-alkyl-TPD1 and N-alkyl-TPD5) revealed that the fragment structure by removing the H atom connected to the 3rd carbon atom of the N-Alkyl side chain has a more stable cyclic form compared to the linear one. Conclusions. The proposed chemical structure of the N-alkyl TPD ion species following the loss of a single hydrogen atom was revealed during APPI-HRMS-CID-MS n (n=3) experiments of the [M-H] + ion species. A hydrogen radical (H • ) abstraction from the alkyl side chain (e.g. hexyl, heptyl, octyl, 2-ethylhexyl, and nonyl) activated a rearrangement of the N-alkyl-TPD derivatives radical cation structure initiating a cyclization and forming a six-membered ring that links the oxygen atom to the 3rd carbon atom in the alkyl chain. In addition, the theoretical calculations supported the APPI-HR-CID-MS n (n=3) experiments by revealing that the proposed chemical structure resulted from the intramolecular cyclization of the N-alkyl-TPD ion species was stable in the presence of chlorobenzene. These research results will facilitate structural determination and elucidation of molecules with similar basic structures.