UHPLC–MS Used to Track Hydrolysis of Nerve Agent A-234 in Multiple Decontaminants

University of Helsinki researchers, led by Susanne Wiedmer (professor of the Department of Chemistry), developed a new approach for determining the rate of hydrolysis of Novichok A-234, a type of chemical warfare agent. Their findings were published in the Journal of Chromatography Open (1).

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Novichok nerve agents are a class of organophosphorus (OP) chemical warfare agents that were developed in Russia during the 1970s and gained international attention in 2018. Following an incident where an English civilian and two Russian nationals were poisoned in Salisbury, England, the Organization for the Prohibition of Chemical Weapons (OPCW) confirmed that a Novichok nerve agent identified as A-234, also known as O-ethyl (1-diethylamino)ethylidene) phosphoramidofluoridate, was involved in the incident (2). Increasing concerns regarding these agents led to great interest into Novichok’s agents, including the recent developments of novel sensing technologies.

Decontamination studies in the past, according to the scientists, have exclusively focused on hydrolysis inducing reactions to degrade Novichoks into their hydrolysate form. Previous techniques used to measure either hydrolysate formation or agent degradation using liquid chromatography–tandem mass spectrometry (LC–MS/MS), high-resolution MS (HRMS), nuclear magnetic resonance (NMR) spectroscopy, gas chromatography–MS (GC–MS), or GC-flame photometric detection (GC-FPD).

In this study, the researchers tested the neutralization of Novichok A-234. Three decontaminants were used, each with different compositions. Namely, the decontaminants were alkaline potassium hydroxide in ethanol, GDS-2000, and Biodecon-83, a deep eutectic solvent. After 24 and 120 hours of exposure to each decontaminant, the degradation products were characterized by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and UHPLC-tandem mass spectrometry (UHPLC–MS/MS). The rate of hydrolysis of Novichok A-234 in each decontaminant was measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy.

Each contaminant showed large variations in the hydrolysis rates of Novichok A-234. The total required decontamination time until Novichok A-234 was no longer observed by ³¹P NMR, ranged from 5 to 44 h. In silico calculations were performed to predict the main decontamination products and their toxicity levels.

While every tested decontaminant proved effective at degrading A-234, each decontaminant produced different degradation products of lower toxicity than A-234, as predicted by the in silico calculations. Decontamination with the quaternary salts of Biodecon-83 in the presence of an oxidizer not only induced hydrolysis of the fluoride substituent but also of the amidine moiety. This helped fully oxidizing the agent to a low toxicity compound. GDS-2000 degraded the majority of A-234 at the fastest rate, though it was slow to degrade the remaining amounts of the compound; this may stem from a stereospecific lack of efficacy. That said, the degradation products formed after GDS-2000 exposure were persistent and of low toxicity, possibly perpetuating some of the initial Novichok agent’s hazardous nature.

Using nucleophiles to degrade A-234 instead of hydrolysis could be a promising strategy, the scientists wrote. A-234 being highly reactive to the decontaminant, which contained ethanol. Further, the transesterification product was observed in large amounts in addition to the hydrolysate. However, in neutral conditions, hydrolysis may not be the most effective mechanism for degrading A-234, and alternative neutralization pathways are to be evaluated. The overall research demonstrates the importance of considering various methods for the decontamination of chemical warfare agents and the characterization of the formed decontamination products.

References

(1) Youngren, C.; Kiljunen, H.; Heikkinen, H. A.; et al. Characterization of Hydrolysis Products and Determination of the Rate of Hydrolysis of Novichok A-234 in Different Decontaminants. J. Chromatogr. Open 2025, 7, 100210. DOI: 10.1016/j.jcoa.2025.100210

(2) Note by the Technical Secretariat: Summary of the Report on Activities Carried Out in Support of a Request for Technical Assistance by the United Kingdom of Great Britain and Northern Ireland (Technical Assistance Visit TAV/02/18). OPCW 2018. https://www.opcw.org/sites/default/files/documents/S_series/2018/en/s-1612-2018_e___1_.pdf (accessed 2025-4-21)