Differentiating Isomeric Urea Derivatives by Cyclic Ion Mobility–Mass Spectrometry, Host–Guest Chemistry, and Tandem Mass Spectrometry

Alkyl pyridyl ureas are crucial precursors in medicinal and agricultural chemistry. Their isomeric forms, which are distinguished by the position of pyridyl nitrogen relative to the urea functionality, pose challenges in mass spectrometric identification due to their isobaric molecular ions. This study presents orthogonal methods to differentiate 1-phenyl-3-(2-pyridyl) urea (ortho), 1-phenyl-3-(3-pyridyl) urea (meta), and 1-phenyl-3-(4-pyridyl) urea (para). Cyclic ion mobility separation of protonated molecules and their complexes with β-cyclodextrin was employed. While the protonated ortho and para isomers were baseline resolved after six passes, the meta and para isomers were not separated after 40 passes. After complexation with β-cyclodextrin, we observed two peaks for each isomer. The peak that arrived first (peak A) is a doubly protonated dimer of the host-guest complex, while the later arriving peak (peak B) corresponds to the singly protonated host-guest complex. Peak B for the β-cyclodextrin complex of the meta and para compounds was resolved after three passes. Thus, the differentiation of meta and para isomers was achieved after complexation with β-cyclodextrin. Overall, the two methods (ion mobility of protonated molecules and ion mobility of host-guest complexes) led to orthogonal results. Collision-induced dissociation of protonated molecules revealed identical fragment ions at <i>m</i>/<i>z</i> 95 and 121, but their relative intensities varied among isomers, enabling their clear differentiation. Additionally, collision-induced dissociation of host-guest complexes at 20 eV shows that the meta isomer forms the most stable complex with β-cyclodextrin and the para isomer forms the least stable complex, which provides additional orthogonal information to differentiate the three isomers. These findings provide a basis for cyclic traveling wave ion mobility and tandem mass spectrometry-based identification and characterization of other isomeric phenyl pyridyl ureas.