Improved skin decontamination efficacy for the nerve agent VX.

Early initiated decontamination is demonstrated to be crucial to avoid systemic effects of highly toxic and low volatile agents exposed on the skin. Skin decontamination can be performed by simple procedures, such as washing with soap and water, or by using advanced decontamination products containing absorption and agent degradation properties. Reactive Skin Decontamination Lotion (RSDL) has demonstrated high efficacy to remove nerve agents from the skin. However, contrary to the current operational recommendations, experimental studies have shown that prolonged skin contact time of RSDL is important for efficient decontamination of VX. In the present study, several RSDL-protocols were evaluated for the efficacy to remove neat VX from human skin in vitro. The decontamination efficacies of the RSDL-procedures were compared with the efficacy of the simple procedure of washing off the skin with soapy water. The RSDL-protocols containing repeated swabbing with the sponge and a 10 min skin contact time of RSDL-lotion demonstrated the greatest decontamination efficacy of all procedures evaluated. Repeating the protocol 2 h after the initial decontamination step resulted in a transient increased skin penetration of remaining intact agent on skin and was followed by rapidly declined agent penetration rate. Decontamination performed with soapy water significantly increased agent amounts penetrating skin, most likely caused by skin hydration and agent dilution. In conclusion, a slightly extended procedure for RSDL-decontamination showed improved efficacy and is therefore recommended for removal of nerve agents from the skin. In addition, it is of highest importance that skin decontamination of nerve agents should consist of procedures using low water content.

[Analysis of Countermeasures against Chemical Terrorism].

As a countermeasure against terrorism involving highly toxic chemical warfare agents, the rapid identification of the causative toxic substances is extremely important. This symposium review describes analytical methods the author's group has developed for detecting nerve gases after either high level or low level exposure. As a method for assessing human exposure to high levels of nerve gases, a technology that detects nerve gas hydrolysis products, i.e., strong anion exchange extraction-tert-butyldimethylsilyl derivatization-selectable one-dimensional or two-dimensional GC-MS, is explained. As a method for assessing human exposure to low levels of nerve gases, two technologies that detect adducts of nerve gas with blood cholinesterase, i.e., adduct purification-enzymatic digestion-LC/MS and fluoride-mediated regeneration-solid phase extraction-large volume introduction GC-MS, are explained.

In-situ derivatization and headspace solid-phase microextraction for gas chromatography-mass spectrometry analysis of alkyl methylphosphonic acids following solid-phase extraction using thin film.

A headspace solid-phase microextraction (HS-SPME) method, involving solid-phase extraction and in-situ derivatization using polymeric thin film, was developed for the gas chromatography-mass spectrometry (GC-MS) analysis of the degradation products of nerve agents. The solid-phase extraction (SPE) was performed using poly([2-(Methacryloyloxy)ethyl]trimethylammonium chloride) film on a gold plate prepared via surface-initiated polymerization. The extracted analytes were directly derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) on the plate. Various parameters like fiber type, headspace time, temperature, and amount of BSTFA were optimized. Under the optimized conditions, the relative standard deviations (RSDs) were in the range 7.0-13.1% and the limits of detection (LODs) were measured to be between 10 and 20 pg mL-1. The application of the developed method was tested using the 35th Organization for Prohibition of Chemical Weapons (OPCW) proficiency test sample.

Rapid quantification of two chemical nerve agent metabolites in serum.

Organophosphorus compounds (OPs) continue to represent a significant chemical threat to humans due to exposures from their use as weapons, their potential storage hazards, and from their continued use agriculturally. Existing methods for detection include ELISA and mass spectrometry. The new approach presented here provides an innovative first step toward a portable OP quantification method that surmounts conventional limitations involving sensitivity, selectivity, complexity, and portability. DNA affinity probes, or aptamers, represent an emerging technology that, when combined with a mix-and-read, free-solution assay (FSA) and a compensated interferometer (CI) can provide a novel alternative to existing OP nerve agent (OPNA) quantification methods. Here it is shown that FSA can be used to rapidly screen prospective aptamers in the biological matrix of interest, allowing the identification of a 'best-in-class' probe. It is also shown that combining aptamers with FSA-CI enables quantification of the OPNA metabolites, Sarin (NATO designation "G-series, B", or GB) and Venomous Agent X (VX) acids, rapidly with high selectivity at detection limits of sub-10 pg/mL in 25% serum (by volume in PBS). These results suggest there is potential to directly impact diagnostic specificity and sensitivity of emergency response testing methods by both simplifying sample preparation procedures and making a benchtop reader available for OPNA metabolite quantification.

Simultaneous Time-concentration Analysis of Soman and VX Adducts to Butyrylcholinesterase and Albumin by LC-MS-MS.

A sensitive method for the purification and determination of two protein adducts, organophosphorus (OP)-BChE and OP-albumin adducts, in a single sample using a simultaneous sample preparation method was developed and validated using liquid chromatography-tandem mass spectrometry. First, we isolated O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) and O-pinacolyl methylphosphonofluoridate (soman, GD)-BChE adducts using an immunomagnetic separation (IMS) method and the HiTrap™ Blue affinity column was subsequently used to isolate and purify VX and GD-albumin adducts from the plasma of rhesus monkeys exposed to nerve agents. Additionally, we examined the time-concentration profiles of two biomarkers, VX and GD-nonapeptides and VX and GD-tyrosines, derived from OP-BChE and OP-albumin adducts up to 8 weeks after exposure. Based on the results, we determined that VX and GD-tyrosine is more suitable than VX and GD-nonapeptide as a biomarker owing to its longevity. This integrated approach is expected to be applicable for the quantification of other OP-BChE and OP-albumin adducts in human plasma, thus serving as a potential generic assay for exposure to nerve agents.

Polymeric Sorbent with Controlled Surface Polarity: An Alternate for Solid-Phase Extraction of Nerve Agents and Their Markers from Organic Matrix.

Extraction and identification of lethal nerve agents and their markers in complex organic background have a prime importance from the forensic and verification viewpoint of the Chemical Weapons Convention (CWC). Liquid-liquid extraction with acetonitrile and commercially available solid phase silica cartridges are extensively used for this purpose. Silica cartridges exhibit limited applicability for relatively polar analytes, and acetonitrile extraction shows limited efficacy toward relatively nonpolar analytes. The present study describes the synthesis of polymeric sorbents with tunable surface polarity, their application as a solid-phase extraction (SPE) material against nerve agents and their polar as well as nonpolar markers from nonpolar organic matrices. In comparison with the acetonitrile extraction and commercial silica cartridges, the new sorbent showed better extraction efficiency toward analytes of varying polarity. The extraction parameters were optimized for the proposed method, which included ethyl acetate as an extraction solvent and n-hexane as a washing solvent. Under optimized conditions, method linearity ranged from 0.10 to 10 µg mL-1 ( r2 = 0.9327-0.9988) for organophosphorus esters and 0.05-20 µg mL-1 ( r2 = 0.9976-0.9991) for nerve agents. Limits of detection (S:N = 3:1) in the SIM mode were found in the range of 0.03-0.075 µg mL-1 for organophosphorus esters and 0.015-0.025 µg mL-1 for nerve agents. Limits of quantification (S:N = 10:1) were found in the range of 0.100-0.25 µg mL-1 for organophosphorus esters and 0.05-0.100 µg mL-1 for nerve agents in the SIM mode. The recoveries of the nerve agents and their markers ranged from 90.0 to 98.0% and 75.0 to 95.0% respectively. The repeatability and reproducibility (with relative standard deviations (RSDs) %) for organophosphorus esters were found in the range of 1.35-8.61% and 2.30-9.25% respectively. For nerve agents, the repeatability range from 1.00 to 7.75% and reproducibility were found in the range of 2.17-6.90%.

Comparison of Efficiency of Purification (from Human Plasma) of a Nerve Agent Adduct of Butyrylcholinesterase Between the Affinity Gel Method and Immunomagnetic Separation.

O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) is a highly toxic chemical warfare agent because it inhibits cholinesterase (ChE) activity in the nervous system. Inhibition of butyrylcholinesterase (BChE) activity by VX is due to formation of a phosphorylated BChE adduct; this adduct in human plasma can serve as a biomarker of exposure to nerve agents. We compared purification efficiency between the procainamide affinity gel method and immunomagnetic separation (IMS) for the nerve agent adduct of BChE in plasma and then optimized the sample preparation by purifying BChE to measure biomarkers of human exposure to organophosphorus nerve agents. The purification efficiency of IMS was 5-fold greater than that of the procainamide affinity gel method because the antibody conjugate with protein G magnetic beads ensured highly selective capture and high recovery of VX-inhibited BChE from plasma. Protein isolation and extraction of the adduct of VX-inhibited BChE from plasma were made more specific by IMS. A 50 µL of the IMS solution was enough to bind VX-inhibited BChE in up to 0.5 mL of plasma. Nonetheless, the IMS method has a limitation in terms of reutilization of the complexes antibody-magnetic beads. We expect that this approach can be used to quantify other types of organophosphorus adducts in human plasma, thus serving as a possible general assay for biomarkers of exposure to nerve agents.

Zirconium Hydroxide-coated Nanofiber Mats for Nerve Agent Decontamination.

Diverse innovative fabrics with specific functionalities have been developed for requirements such as self-decontamination of chemical/biological pollutants and toxic nerve agents. In this work, Zr(OH)4 -coated nylon-6,6 nanofiber mats were fabricated for the decontamination of nerve agents. Nylon-6,6 fabric was prepared via the electrospinning process, followed by coating with Zr(OH)4 , which was obtained by the hydrolysis of Zr(OBu)4 by a sol-gel reaction on nanofiber surfaces. The reaction conditions were optimized by varying the amounts of Zr(OBu)4 ,the reaction time, and the temperature of the sol-gel reaction. The composite nanofibers show high decontamination efficiency against diisopropylfluorophosphate, which is a nerve agent analogue, due to its high nucleophilicity that aids in the catalysis of the hydrolysis of the phosphonate ester bonds. Composite nanofiber mats have a large potential and can be applied in specific fields such as military and medical markets.

Fully integrated ready-to-use paper-based electrochemical biosensor to detect nerve agents.

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems.

Mesoporous Non-stacked Graphene-receptor Sensor for Detecting Nerve Agents.

A novel gas sensor consisting of porous, non-stacked reduced graphene oxide (NSrGO)-heaxfluorohydoroxypropanyl benzene (HFHPB) nanosheets was successfully fabricated, allowing the detection of dimethyl methyl phosphonate (DMMP), similar to sarin toxic gas. The HFHPB group was chemically grafted to the NSrGO via a diazotization reaction to produce NSrGO-HFHPB. The NSrGO-HFHPB 3D film has a mesoporous structure with a large pore volume and high surface area that can sensitively detect DMMP and concurrently selectively signal the DMMP through the chemically-attached HFHPB. The DMMP uptake of the mesoporous NSrGO-HFHPB was 240.03 Hz, 12 times greater than that of rGO-HFHPB (20.14 Hz). In addition, the response rate of NSrGO-HFHPB was faster than that of rGO-HFHPB, an approximately 3 times more rapid recovery due to the mesoporous structure of the NSrGO-HFHPB. The NSrGO-HFHPB sensor exhibited long-term stability due to the use of robust carbon and resulting high resistance to humidity.