Reference-Free Structure Elucidation of N–O Heterocyclic Ring Systems with Infrared Ion Spectroscopy: A Test Case of the Isoxazoline Insecticide Isocycloseram and Its MS/MS Fragment

Five-membered heterocyclic molecules with an N-O bond are emerging as active ingredients in the agrochemical and pharmaceutical industry. Characterization of these molecules in biological matrices is commonly done using liquid chromatography and mass spectrometry, which is not always diagnostic for the exact molecular structure. We demonstrate how infrared ion spectroscopy (IRIS) can be used to structurally characterize the ions formed in mass spectrometry on a set of 12 isoxazolidinone analogues. Combining IRIS with quantum-chemical methods, we assign the gas-phase structure and show that most of the isoxazolidinone analogues protonate on the carbonyl oxygen. This approach is then extended to the isoxazoline insecticide isocycloseram, which is much larger in size and possesses an IR spectrum that is more congested. In addition to "standard" harmonic frequency calculations at the Density Functional Theory (DFT) level, we compare the experimental spectrum to computations performed using Born-Oppenheimer molecular dynamics (BOMD), which implicitly accounts for anharmonicity and conformational dynamics at the finite temperatures of the experiment. Collision-induced dissociation of protonated isocycloseram yields a low-intensity fragment ion with the charge retained at the isoxazolinone; we assign its structure by comparing the IR spectrum of the fragment ion to the IR spectra from the set of small-molecule analogues.