Elucidation of Fragmentation Pathways and GC-HRMS Nontargeted Screening of V-Series Nerve Agent-Related Compounds: Alkylthiophosphonates

Alkylthiophosphonates, listed in Schedule 2.B.04 of the Chemical Weapons Convention, are key environmental markers and stable degradation products of V-series nerve agents. However, their reliable identification is hampered by some fundamental challenges: over one million theoretical structures are possible, whereas standard reference databases contain only a limited number of entries. This disparity highlights the need for high-throughput, nontargeted screening strategies capable of detecting compounds beyond library coverage. Previous research has indicated that liquid chromatography-mass spectrometry shows limited sensitivity for sulfur-rich analogues, exhibiting an exponential signal attenuation as the number of sulfur atoms increases. To address these gaps, a set of over 100 typical compounds was synthesized and their fragmentation behaviors were systematically investigated using gas chromatography-high resolution mass spectrometry. The analysis revealed that McLafferty (+1) rearrangement and α-cleavage are the dominant mechanisms generating their characteristic fragment ions. Building on these mechanistic insights and leveraging the complementary strengths of electron ionization and chemical ionization, 33 groups of alerting ions and 171 theoretical accurate masses were derived. These were used to construct a nontargeted screening strategy for millions of alkylthiophosphonates. The strategy was successfully applied to identify unknown chemicals in authentic samples, enabling effective discrimination between phosphonothiolate/phosphonothionate and phosphonodithiolate/phosphonothiolothionate isomeric pairs. The method demonstrated high sensitivity, with limits of detection as low as 1-10 ppb, along with good repeatability. These results confirm the practical utility of the developed strategy for the verification and monitoring of alkylthiophosphonates in environmental and forensic contexts.