Using GC to Investigate Nerve Agent Presence on Indoor Surfaces

A key technique in the investigation of nerve agents, gas chromatography (GC) is a well-established application for analysts both on site and conducting forensic assays in the laboratory. Furthermore, GC offers analysts the ability to identify non-volatile decomposition products of chemical warfare agents (CWAs) following their derivatization to improve volatility or enhance their analytical properties. Such an analyst is Tomáš Rozsypal of the Nuclear, Biological and Chemical Defence Institute of the University of Defence (Vyskov, Czech Republic). LCGC International recently spoke to Rozsypal about his work utilizing GC in researching the persistence of A-234 nerve agent on indoor surfaces, and the paper that resulted from it.

The aim of your paper (1) was to determine the persistence of A-234 (nerve agent) on selected indoor surfaces. What inspired this research?

This research was inspired by a high-profile event that occurred in 2018 in Salisbury, United Kingdom. Former Russian agent Sergei Skripal and his daughter Yulia were poisoned by an unknown substance, later identified as A-234 (ethyl N-[2-(diethylamino)ethyl]-N-(2-methylpropyl)phosphoramidofluoridate), a nerve agent developed in Soviet Russia during the Cold War. The investigation led authorities to Sergei's home, where traces of the poison were found—on the front door handle and inside the house. The aim of this study was to test the persistence of A-234 on various common indoor materials, with implications for assessing risks to first responders, crime scene investigators, and decontamination efforts.

Are you aware of other researchers performing similar research, either regarding A-234 or on other nerve agents?

Yes, I closely follow global research on these compounds, not only A-234 but also other toxic derivatives developed under the same research program in Russia. The challenge is that, until recently, the chemical structures of these agents were not publicly confirmed. Now, A-234 is listed under the control schedules of the Organization for the Prohibition of Chemical Weapons (OPCW), meaning its production is strictly regulated worldwide, and handling it requires formal declarations. As a result, very few research organizations work with these chemicals today, and even fewer publish their findings in scientific journals. Most current studies on A-series nerve agents rely on in silico computational methods, with significantly fewer experimental studies being conducted.

Briefly state your overall findings in your research. Which material indoor surfaces presented the most persistent half-life for the nerve agent?

When classifying chemical warfare agents as persistent or nonpersistent, A-234 has been assessed as super persistent, especially when compared to CWAs typically considered long-lasting, such as VX or sulfur mustard. Surfaces contaminated with A-234 remain toxic for extended periods, which, while hazardous, also aids in preserving evidence for crime scene investigations. The tested surfaces included a computer keyboard, acrylic paint, laminated chipboard, PVC flooring, ceramics, a PET bottle, and an aluminum can, listed in order from the least persistent to the longest-lasting contamination. Monitoring was conducted over three months, and the agent was still detectable on most surfaces at the end of this period. Although A-234 is almost nonvolatile, its extreme toxicity, which is largely unknown in many areas, necessitates strict safety protocols.

Do your findings correlate with what you had hypothesized or any preconceptions you might have had prior to your work?

There was no preliminary data available, nor were the basic physical properties of this agent accessible. I had to rely solely on fragmented statements from Vil Mirzayanov, the Russian defector who worked in the research organization that developed A-234, and on our previous study, where we examined its hydrolysis, which was found to be very slow. I initially anticipated a persistence level like VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate), or possibly higher. In reality, A-234's persistence far exceeded that of VX, which came as a significant surprise.

Was there anything particularly unexpected that stands out from your perspective?

In addition to its remarkable persistence, I was also surprised by A-234’s very low rate of evaporation. After three months, only trace concentrations of A-234 were detected in vapors, and only above compact, nonporous surfaces. However, this does not automatically mean the agent is not a respiratory hazard, as nerve agents are harmful even at very low concentrations. The challenge arises when using on-site detectors based on ion mobility spectrometry (IMS), detector tubes, flame spectrometry, and so forth, where the detection limits may be higher than the actual vapor concentrations, potentially leading to false negatives.

What were the major challenges you encountered in your work?

One of the main challenges was working with an extremely toxic substance, whose toxicological and chemical properties remain largely unknown. In the past, I have worked with agents like sarin, soman, and sulfur mustard, which are highly dangerous but well-researched, so their behavior is more predictable. With A-234, I worked with high concentrations to simulate real-world scenarios, where an attacker might apply the agent to a surface with the intent to kill. This required strict adherence to safety protocols and the use of personal protective equipment. Additionally, all materials that encountered the substance had to be thoroughly decontaminated.

What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?

This type of analysis was based on the quantification of a known substance. Given that A-234 is sufficiently volatile for analysis, gas chromatography with flame ionization detection (FID) is suitable. I also equipped the exhaust of the gas chromatograph with a charcoal cartridge for capturing harmful substances. If the analyte is unknown and needs to be identified first, mass spectrometry (MS) is essential. For nerve agents and their degradation products, a nonpolar DB-5 column has proven effective, with an oven program utilizing a relatively slow temperature gradient from low temperatures up to at least 280 °C. Other parameters need to be optimized depending on the specific analyte. For headspace analysis, it was necessary to heat the vial to a higher temperature due to the agent's low volatility.

In terms of data analysis, standardization is key. I typically use an internal standard, with tributyl phosphate (C12H29O4P) being the choice for nerve agents. One challenge can be the limited availability of mass spectra for A-series agents in user libraries.

Can you please summarize the feedback that you have received from others regarding this work? I would imagine that most the feedback came from a very focused, specialized group.

Yes, that's correct. I have received inquiries from several foreign state laboratories interested in this article and wanting to learn more. I also work for a state organization, but we publish the results of our research in public journals, which is not the norm.

Do you believe that the method can easily be adapted to perhaps other nerve agents, or other surfaces?

Absolutely, there are no obstacles to this. The wipe sampling method has been optimized for A-234, but most nerve agents can be effectively extracted using the mentioned solvent. It would only be necessary to verify the recovery. The subsequent chromatographic method and column I use are very versatile and have proven successful for other chemical warfare agents. Additionally, there are no restrictions on testing other surfaces; however, it is essential to always verify the matrix effect and potential solubility.

What are the next steps in this research?

Currently, I am focused on expanding analytical methods for this and related compounds. I am also collaborating with colleagues to enhance our understanding of their behavior in different types of matrices. In addition, we are exploring various decontamination options.

Tomáš Rozsypal holds a Master’s degree in Military Chemistry (2015) and a PhD in Weapons of Mass Destruction (2018) from the University of Defense in Brno, Czech Republic. He previously served as Chief of the Deployable Chemical Laboratory in the 311th CBRN Defense Battalion of the Army of the Czech Republic. During his deployment, he commanded a unit of chemical corps laboratory specialists within the 1st Czech Army Task Force in Iraq. He is currently an Assistant Professor at the Nuclear, Biological, and Chemical Defence Institute, University of Defence, Czech Republic, where he also holds the military rank of Captain. His lectures focus on the chemistry of chemical warfare agents and the theory of chemical weapons. Additionally, he serves as a guest lecturer at the University of Bundeswehr in Hamburg, Germany, where he lectures on the chemistry of explosives. His research primarily focuses on environmental and forensic chemistry related to chemical warfare agents and their degradation products. He contributes to the development of field analytical methods and decontamination techniques, collaborating with the Czech chemical corps' analytical laboratories and sampling teams. Photo courtesy of Tomáš Rozsypal.

References

1. Rozsypal, T. Persistence of A-234 Nerve Agent on Indoor Surfaces. Chemosphere 2024, 357, 141968. DOI: 10.1016/j.chemosphere.2024.141968