Keeping your nerve: VX in drinking water

Nerve gas attacks

Terrorist attacks are not limited to bombs and explosives. In the modern era, we have to be aware of threats from a variety of agents like bacteria, viruses, radioactive materials and chemical warfare agents. One of the recognised targets for lethal contamination is the water supply, to which it would be relatively easily to add quantities of chemicals without being detected.

In the USA, the authorities are acutely aware of this type of threat and have been taking steps to increases their preparedness for attack and their subsequent response. This includes having the ability to detect the agents in water and to monitor their levels during remediation. For one nerve agent, VX, the Centers for Disease Control and Prevention and the National Homeland Security Research Center of the EPA have combined to develop a sensitive method for its analysis.

VX is a synthetic organo-phosphorus compound. It is related to sarin, which was released into the Tokyo subway system in 1994. After this attack, traces of sarin were also found in a nearby pond, illustrating a secondary route by which nerve agents can find their way into water.

The method, described by senior author Rudolph Johnson from the Emergency response Branch at the CDC in Atlanta, GA, exploits an immunomagnetic collection method for VX that was developed earlier by the CDC, EPA and the US Army Edgewood Chemical Biological Center. It gave parts-per-trillion detection limits with LC-tandem-MS.

In the new study, the researchers wanted to address the stability of VX in collected water samples. Large numbers of samples might be taken after a contamination event leading to a delay before analysis, so stability becomes a critical factor in the detection and measurement of the nerve agent.

Immunotrapping VX

In the initial stages, the method was tested on solutions of VX in HPLC-grade water that had been stabilised with two additives. Sodium omadine is an antimicrobial preservative and sodium thiosulphate is a dechlorinating agent which is intended to remove oxidants like chlorine that have been added for drinking water disinfection.

VX was extracted with the antibody-coated magnetic beads conjugated with butyrylcholinesterase, an enzyme that forms covalent bonds with organo-phosphorus nerve agents. The VX adduct was then released by treating with pepsin, which digests the enzyme to give a peptide-VX adduct that was extracted for analysis. The peptide fragment was FGESAGAAS, corresponding to the active site of the enzyme.

The analysis was carried out by LC/MS/MS using a stable-isotope-labelled peptide analogue as internal standard. Two transitions were monitored for the peptide-VX adduct and one for the internal standard.

The procedure was then tested on five samples of tap water of various qualities collected from across the US. They were stored for up to 91 days in the dark at 4°C then prepared for analysis, waiting either 15 minutes or 5 hours after preparation before proceeding.

Preservation during storage

The peptide-VX adduct eluted from the HPLC column within 2.2 minutes, helping towards a rapid, high-throughput procedure. The method detection limit was 5.6 ng/L and an upper limit was also defined at 4.0 µg/L. At VX concentrations higher than this, the nerve agent saturates the binding sites on the magnetic beads, so more concentrated samples must be diluted before analysis.

Two of the three possible reactions that degrade VX in water are oxidation by residual oxidants and microbial degradation, which the researchers attempted to prevent by the addition of sodium thiosulphate and sodium omadine, respectively. The third main reaction is hydrolysis, which is a complex pH-dependent process that makes VX unstable in water. However, the compound has a relatively long half-life of about 90 days when stored carefully to minimise hydrolysis.

The stability studies revealed that the VX recoveries were 81-92% if the samples were analysed immediately after their preparation. However, storage for 90 days reduced this figure to 30-45%. While this marks a significant drop in the amount of VX, the main point is that it is not totally degraded.

The team calculated that the method detection limit remained well below the target level determined using risk-based criteria, even allowing for this VX loss during storage. In reality, the 90-day scenario is a worst case because most methods for the analysis of drinking water recommend a storage time of no more than 28 days, so there would be less degradation.

So, the preservation steps combined with immunomagnetic separation and the sensitive LC/MS/MS measurement make this a good candidate method for tracking VX concentrations in the water supply following a poisoning incident and the subsequent remediation.