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 m/z 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.

烷基吡啶基脲是医药与农业化学领域的关键前驱体。该类化合物存在同分异构体，其差异源于吡啶基氮原子相对于脲官能团的位置不同，由于分子离子等压，给质谱鉴定带来了挑战。本研究提出了可区分1-苯基-3-(2-吡啶基)脲（邻位）、1-苯基-3-(3-吡啶基)脲（间位）与1-苯基-3-(4-吡啶基)脲（对位）的正交分析方法。研究采用质子化分子及其与β-环糊精复合物的循环离子迁移分离技术。经六次循环后，质子化的邻位与对位异构体实现基线分离，但间位与对位异构体在四十次循环后仍未实现分离。当与β-环糊精形成复合物后，每种异构体均产生两个峰：最先到达的峰（峰A）为主客体复合物的双质子化二聚体，而后到达的峰（峰B）对应单质子化主客体复合物。间位与对位化合物的β-环糊精复合物的峰B在三次循环后即可实现基线分离。因此，通过与β-环糊精复合即可实现间位与对位异构体的区分。总体而言，质子化分子的离子迁移技术与主客体复合物的离子迁移技术这两种方法得到了正交的分析结果。对质子化分子的碰撞诱导解离分析显示，三种异构体均生成了质荷比为95和121的碎片离子，但各碎片离子的相对强度存在差异，这可实现对三种异构体的明确区分。此外，在20 eV能量下对主客体复合物进行碰撞诱导解离发现，间位异构体与β-环糊精形成的复合物稳定性最高，对位异构体形成的复合物稳定性最低，该结果为区分三种异构体提供了额外的正交信息。本研究成果为基于循环行波离子迁移与串联质谱技术鉴定、表征其他同分异构体苯基吡啶基脲提供了理论依据与技术基础。