Highly specific and sensitive chromo-fluorogenic detection of sarin, tabun, and mustard gas stimulants: a multianalyte recognition approach.

Nerve agents are the most notorious substances, which can be fatal to an individual because they block the activity of acetylcholinesterase. Fighting against unpredictable terrorist assaults and wars requires the simple and quick detection of chemical warfare agent vapor. In the present contribution, we have introduced a rhodamine-based chemosensor, BDHA, for the detection of nerve gas-mimicking agents diethylchlorophosphate (DCP) and diethylcyanophosphonate (DCNP) and mustard gas-mimicking agent 2-chloroethyl ethyl sulfide (CEES), both in the liquid and vapor phase. Probe BDHA provides the ability for detection by the naked eye in terms of colorimetric and fluorometric changes. It has been revealed that the interaction between nerve agents mimics and probe BDHA facilitates spirolactam ring opening due to the phosphorylation process. Thus, the highly fluorescent and colored species developed while probe BDHA is colorless and non-fluorescent due to the intramolecular spirolactam ring. Moreover, probe BDHA can effectively recognize DCP, DCNP, and CEES in the µM range despite many toxic analytes and could be identified based on the response times and quantum yield values. Inexpensive, easily carried paper strips-based test kits were developed for the quick, on-location solid and vapor phase detection of these mustard gas imitating agents (CEES) and nerve gas mimicking agents (DCP and DCNP) without needing expensive equipment or skilled personnel. More remarkably, the test strips' color and fluorescence can be rapidly restored, exposing them to triethyl amine (TEA) for cyclic use, suggesting a potential application in the real-time identification of chemical warfare agents. To accomplish the on-location application of BDHA, we have experimented with soil samples to find traces of DCP. Therefore, the chromo-fluorogenic probe BDHA is a promising, instantaneous, and on-the-spot monitoring tool for the selective detection of DCP, DCNP, and CEES in the presence of others.

Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo.

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Visual and Rapid Detection of Nerve Agent Mimics in Gas and Solution Phase by a Simple Fluorescent Probe.

Chemical nerve agents are highly toxic organophosphorus compounds that are easy to obtain and can be utilized by terrorists to threaten homeland security and human safety. Those organophosphorus nerve agents contain nucleophilic ability that can react with acetylcholinesterase leading to muscular paralysis and human death. Therefore, there is great importance to explore a reliable and simple method to detect chemical nerve agents. Herein, the o-phenylenediamine-linked dansyl chloride as a colorimetric and fluorescent probe has been prepared to detect specific chemical nerve agent stimulants in the solution and vapor phase. The o-phenylenediamine unit serves as a detection site that can react with diethyl chlorophosphate (DCP) in a rapid response within 2 min. A satisfied relationship line was obtained between fluorescent intensity and the concentration of DCP in the range of 0-90 µM. In the optimized conditions, we conducted the fluorescent titration to measure the limits of detection (0.082 µM) with the fluorescent enhancement up to 18-fold. Fluorescence titration and NMR studies were also conducted to explore the detection mechanism, indicating that the formation of phosphate ester causes the intensity of fluorescent change during the PET process. Finally, probe 1 coated with the paper test is utilized to detect DCP vapor and solution by the naked eye. We expect that this probe may give some admiration to design the small molecule organic probe and applied in the selectivity detection of chemical nerve agents.

Organic-Molecule-Based Fluorescent Chemosensor for Nerve Agents and Organophosphorus Pesticides.

Organophosphorus (OP) compounds are typically a broad class of compounds that possess various uses such as insecticides, pesticides, etc. One of the most evil utilizations of these compounds is as chemical warfare agents, which pose a greater threat than biological weapons because of their ease of access. OP compounds are highly toxic compounds that cause irreversible inhibition of enzyme acetylcholinesterase, which is essential for hydrolysis of neurotransmitter acetylcholine, leading to series of neurological disorders and even death. Due to the extensive use of these organophosphorus compounds in agriculture, there is an increase in the environmental burden of these toxic chemicals, with severe environmental consequences. Hence, the rapid and sensitive, selective, real-time detection of OP compounds is very much required in terms of environmental protection, health, and survival. Several techniques have been developed over a few decades to easily detect them, but still, numerous challenges and problems remain to be solved. Major advancement has been observed in the development of sensors using the spectroscopic technique over recent years because of the advantages offered over other techniques, which we focus on in the presented review.

Analysis of Organophosphorus-Based Nerve Agent Degradation Products by Gas Chromatography-Mass Spectrometry (GC-MS): Current Derivatization Reactions in the Analytical Chemist's Toolbox.

The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to stress the importance of developing methods for their rapid and unambiguous detection. Although the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must rely on the detection of the products arising from their degradation. To this end, derivatization reactions mainly in the form of silylations and alkylations employing a vast array of reagents have played a pivotal role in the efficient detection of these products that can be used retrospectively to identify the original OPNA.

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro.

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

Fluorescence umpolung enables light-up sensing of N-acetyltransferases and nerve agents.

Intramolecular charge transfer (ICT) is a fundamental mechanism that enables the development of numerous fluorophores and probes for bioimaging and sensing. However, the electron-withdrawing targets (EWTs)-induced fluorescence quenching is a long-standing and unsolved issue in ICT fluorophores, and significantly limits the widespread applicability. Here we report a simple and generalizable structural-modification for completely overturning the intramolecular rotation driving energy, and thus fully reversing the ICT fluorophores' quenching mode into light-up mode. Specifically, the insertion of an indazole unit into ICT scaffold can fully amplify the intramolecular rotation in donor-indazole-π-acceptor fluorophores (fluorescence OFF), whereas efficiently suppressing the rotation in their EWT-substituted system (fluorescence ON). This molecular strategy is generalizable, yielding a palette of chromophores with fluorescence umpolung that spans visible and near-infrared range. This strategy expands the bio-analytical toolboxes and allows exploiting ICT fluorophores for light-up sensing of EWTs including N-acetyltransferases and nerve agents.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

MIL-101(Cr) with incorporated polypyridine zinc complexes for efficient degradation of a nerve agent simulant: spatial isolation of active sites promoting catalysis.

Development of an efficient catalyst for degradation of organophosphorus toxicants is highly desirable. Herein, an MIL-101(Cr)LZn catalyst was fabricated by incorporating polypyridine zinc complexes into a MOF to achieve the spatial isolation of active sites. Compared with a terpyridine zinc complex without an MIL-101 support, this catalyst was highly active for detoxification of diethyl-4-nitrophenylphosphate.

Cloisite Microrobots as Self-Propelling Cleaners for Fast and Efficient Removal of Improvised Organophosphate Nerve Agents.

Naturally available microclays are well-known materials with great adsorption capabilities that are available in nature in megatons quantities. On the contrary, artificial nanostructures are often available at high cost via precision manufacturing. Such precision nanomanufacturing is also typically used for fabrication of self-propelled micromotors and nanomachines. Herein, we utilized naturally available Cloisite microclays to fabricate autonomous self-propelled microrobots and demonstrated their excellent performances in pesticide removal due to their excellent adsorption capability. Six different modified Cloisite microrobots were investigated by sputtering their microclays with platinum (Pt) for the fabrication of platinum-Cloisite (Pt-C) microrobots. The obtained microrobots displayed fast velocities (v > 110 µm/s) with fast and efficient enhanced removal of the pesticide fenitrothion, which is also considered as improvised nerve agent. The fabricated Pt-C microrobots exhibited low cytotoxicity even at high concentrations when incubated with human lung carcinoma epithelial cells, which make them safe for human handling.

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.

Gas chromatography-mass spectrometry with spiral large-volume injection for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum.

A sensitive method for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum was developed using gas chromatography-mass spectrometry (GCMS) with large-volume injection. The GC injection was administered using stomach-type spiral injector (LVI, AiSTI SCIENCE) enabling introduction of only target compounds from 50 µL ethyl acetate extract after purging the solvent. For GCMS analysis of sarin (GB), 670 times higher sensitivity, based on limit of detection (LOD, S/N = 3, on extracted ion chromatogram (EIC) at m/z 99), was achieved using this injection (50 µL) compared to that achieved using 1 µL split injection (ratio 20:1). Ethyl (EtGB), isopropyl (GB), n-propyl (nPrGB), isobutyl (iBuGB), pinacolyl (GD), cyclohexyl (GF) methylphosphonofluoridates, and O-ethyl N, N-dimethylphosphoramidofluoridate (GAF) were detected with low LOD (15-75 pg/mL) and sharp peak shapes (high practical plate number (defined as 5.54 x (tR/Wh)2, where tR is the retention time and Wh is the bandwidth at half-height): 1100000-2400000) in GCMS using a polar separation column, electron ionization, and quadruple mass analyzer. During the analysis of fluoridated phosphonate-spiked ethyl acetate extract of solid phase extraction (SPE, Bond Elut NEXUS) from fluoride-mediated regeneration of blank human plasma, LOD (on EIC at m/z 99 except for GAF (m/z 126)) were 25-140 pg/mL with sharp peak shapes. The reaction recoveries in fluoride-mediated regeneration of plasma, which was inhibited by GB, GD, GA, GF, VX, and Russian VX (10 ng/mL), were 49-114% except for GD (10%). The concentration levels of 0.3-1 ng/mL of nerve agents in plasma could be determined.

An unusual double radical homolysis mechanism for the unexpected activation of the aldoxime nerve-agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions.

We have recently shown that the pyridinium aldoximes, best-known as therapeutic antidotes for chemical warfare nerve-agents, could markedly detoxify the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damages even when 2-PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two-consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin-trapping, HPLC/MS and nitrogen-15 isotope-labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2-PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2-PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2-CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double-steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2-PAM against TCBQ-induced biological damages, and also the potential side-toxic effects induced by 2-PAM and other pyridinium aldoxime nerve-agent antidotes.

Temperature dependent time resolved mid-IR photoacoustic spectroscopy of a nerve gas simulant DMMP.

The paper reports the temperature dependent pulsed photoacoustic spectroscopy of Dimethyl methylphosphonate (DMMP) a nerve gas simulant between 50 and 180 °C temperature range. The time domain PA spectra are recorded using two mid-IR wavelengths i.e. 3374 nm, 3495 nm of pulse duration 1.5 ns at 1 kHz repetition rate obtained from optical parametric oscillator. Two anti-symmetric stretching vibrational modes of (CH3P) and (CH3O) groups of DMMP molecules have very strong vibrational peaks at 2861.2 cm-1 (3495 nm) and 2963.8 cm-1 (3374 nm), respectively. In addition, we have also recorded the PA spectra of acetone at the vibrational frequency 3115.2 cm-1 (3210 nm), which is the strong vibrational mode of CH band. The comparison of two PA spectra of DMMP and acetone recorded using similar PA cavity help us to understand the effect of other functional groups with respect to different excitation wavelengths. The presence of additional acoustic modes in the PA spectra of DMMP (3374 nm) above the boiling point confirms the slow process of thermal decomposition. Finally, the low level detection limit of DMMP in air is of the of the order of 0.91 ppbV.

Mechanisms of acetylcholinesterase protection against sarin and soman by adenosine A1 receptor agonist N6-cyclopentyladenosine.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A1 receptor agonist N6-cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development.

Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin.

Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by µLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 µM nerve agent for tyrosine-adducts, 1-3 µM nerve agent for CysPro-adducts, and 6 µM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent. Graphical abstract Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS).

Identification of V-type nerve agents in vapor samples using a field-portable capillary gas chromatography/membrane-interfaced electron ionization quadrupole mass spectrometry instrument with Tri-Bed concentrator and fluoridating conversion tube.

A field-portable gas chromatography-mass spectrometry (GC-MS) system (Hapsite ER) was evaluated for the detection of nonvolatile V-type nerve agents (VX and Russian VX (RVX)) in the vapor phase. The Hapsite ER system consists of a Tri-Bed concentrator gas sampler, a nonpolar low thermal-mass capillary GC column and a hydrophobic membrane-interfaced electron ionization quadrupole mass spectrometer evacuated by a non-evaporative getter pump. The GC-MS system was attached to a VX-G fluoridating conversion tube containing silver nitrate and potassium fluoride. Sample vapors of VX and RVX were converted into O-ethyl methylphosphonofluoridate (EtGB) and O-isobutyl methylphosphonofluoridate (iBuGB), respectively. These fluoridated derivatives were detected within 10 min. No compounds were detected when the VX and RVX samples were analyzed without the conversion tube. A vapor sample of tabun (GA) was analyzed, in which GA and O-ethyl N,N-dimethylphosphoramidofluoridate were detected. The molar recovery percentages of EtGB and iBuGB from VX and RVX vapors varied from 0.3 to 17%, which was attributed to variations in the vaporization efficiency of the glass vapor container. The conversion efficiencies of the VX-G conversion tube for VX and RVX to their phosphonate derivatives were estimated to be 40%. VX and RVX vapors were detected at concentrations as low as 0.3 mg m-3 . Gasoline vapor was found to interfere with the analyses of VX and RVX. In the presence of 160 mg m-3 gasoline, the detection limits of VX and RVX vapor were increased to 20 mg m-3 . Copyright © 2017 John Wiley & Sons, Ltd.

Target analysis of tert-butyldimethylsilyl derivatives of nerve agent hydrolysis products by selectable one-dimensional or two-dimensional gas chromatography-mass spectrometry.

A target analysis method for the sensitive and discriminative determination of the nerve agent hydrolysis products alkyl methylphosphonic acids as their tert-butyldimethylsilyl (TBDMS) derivatives was developed using a combination of selectable one- and two-dimensional (1D/2D) GC-MS, and applied to the analysis of samples with significant interfering matrices. After sample drying, the alkylmethylphosphonic acids and methylphosphonic acid (MPA) were converted to TBDMS derivatives by addition of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide with heating, and subjected to 1D/2D GC-MS. The apparatus consisted of an initial low thermal mass DB-5 column and a second DB-17 column together with an electron ionization quadrupole mass spectrometer, offering simple and flexible switching between one- and two-dimensional GC-MS analysis in a single GC-MS system. Using 1D/2D GC-MS, analytes that do not co-elute with matrix components can be separated using 1D GC mode alone. Only those parts of the chromatogram that are negatively affected by the co-elution of matrix components need to be transferred and separated with 2D GC. Quantitation can be performed by a combination of both separations and mass spectrometric detection. The TBDMS derivatives of ethyl-, isopropyl-, isobutyl-, pinacolyl-, and cyclohexyl-MPA (cHMPA) and MPA itself were well separated within 3min and determined in 1D GC-MS mode with detection limits of around 10ng/ml of reaction mixture (except for the cHMPA derivative, whose mass spectrum contained noisy background peaks). In 2D-GC-MS mode, where each 0.04min elution window from the 1D GC was subjected to heart-cut (H/C) and transferred to the second column after back-flushing the first column, the peak for the cHMPA TBDMS derivative was isolated and afforded a clean mass spectrum within 6min. The recoveries of all the derivatives on 2D GC from 1D GC were estimated to be over 66%, and the detection limits were around 10ng/ml of reaction mixture. In the presence of urine extract, the target compounds were not detected as separated peaks in 1D GC-MS mode (except for isobutyl-MPA), and quantification based on extracted ion monitoring could not be achieved. However, 2D GC-MS of the H/C fractions of the target derivatives gave single peaks with well-defined mass spectra, and the recoveries of the derivatives were over 70% except for cHMPA (31% at 1.25µg/ml). Phosphonic acids could be detected at less than 60ng/ml. Sulfuric acid and phosphoric acid also negatively affected the determination of alkyl methylphosphonic acid TBDMS derivatives in 1D GC-MS, and the MPA-TBDMS-derivative peak was completely obscured by the large sulfuric-acid-derivative peak. However, under 1D/2D GC-MS conditions, baseline separation of the MPA derivative and sulfuric acid derivative was achieved, enabling highly sensitive MPA detection at 20ng/ml.

Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

SERS-based ultrasensitive detection of organophosphorus nerve agents via substrate's surface modification.

Highly efficient detection of the organicphosphor nerve agents such as sarin and soman, based on surface enhanced Raman scattering (SERS) effect, has been in challenge due to their weak adsorption property on coin metals. In this paper, a new strategy is presented to achieve the SERS-based ultrasensitive detection of sarin-simulated agent methanephosphonic acid (MPA) via the surface modification of SERS-substrates. The Au-coated Si nanocone array is surface-modified with 2-aminoethanethiol and used as SERS-substrate for detection of MPA. It has been shown that the modified substrate could preferentially capture MPA molecules in the solution with coupling agent and induce amidation reaction. The reaction products are still bound or anchor on the substrate's surface. The MPA molecules can thus be detected by Raman spectral measurement of the solution-soaked SERS-substrate. The minimum detection level is down to ∼1ppb. The Raman peak intensity versus the MPA concentration is subject to a linear double logarithmic relation from ∼1ppb to ∼1000ppm, which is attributed to Freundlich adsorption of MPA on the surface-modified SERS substrate. This study provides a new way for the highly efficient SERS-based detection of the organophosphorus nerve agents and some other target molecules weakly interacted with metal substrates.