Methyl Phosphonothioate Research Articles

Determination of o-isobutyl-s-[(2-diethylamino)ethyl]-methyl phosphonothioate and the hydrolysis toxic product traces by liquid chromatography-tandem mass-spectrometry

To date, chemical weapons in the Russian Federation have been completely destroyed. Former chemical weapons destruction facilities (CWDFs) are subject to re-profiling. The most important stage of this activity is to assess the safety of engineering elements of the facilities to find out whether they can be involved in national economy. The most toxic and persistent substance processed at the CWDFs was a V-series agent. The certified gas chromatographic methods previously used for control of residual amounts of this substance on the surfaces and in deep layers of materials were based on its conversion to acid fluoride on silver fluoride pads. The latter are no longer produced, and these methods are impossible to use. The use of liquid tandem chromatography-mass spectrometry (HPLC-MS/MS) provided a direct determination of VR in the different matrices at the level of the toxicity threshold. At the same time HPLC-MS/MS method allows to determine the toxic acid formed by the hydrolysis of the P–O bond in the VR molecule. Previously no methods for the determination of this acid were available. A unified procedure for the sensitive target determination of VR (O-isobutyl S- (2-diethylamino)ethyl methylphosphonothioate) and its most toxic hydrolysis product S-[(2-diethylamino)ethyl] methylphosphonothioic acid in surface wipes and in deep layers of various materials is proposed. The method involves the addition of an internal standard (paraoxon) to the sample, extraction with methanol, concentration of the extract, and analysis by HPLC-MS/MS in the multiple reaction monitoring (MRM) mode. The detection limits of the analytes are close to their toxic threshold levels. Special emphasis in the procedure is paid to the reliability of identification of the analytes, which is of key importance in international investigations under the Chemical Weapons Convention.

In situ microfluidic SERS assay for monitoring enzymatic breakdown of organophosphates.

In this paper, we report on a method to probe the breakdown of the organophosphate (OP) simulants o,s-diethyl methyl phosphonothioate (OSDMP) and demeton S by the enzyme organophosphorous hydrolase (OPH) in a microfluidic device by surface enhanced Raman spectroscopy (SERS). SERS hotspots were formed on-demand inside the microfluidic device by laser-induced aggregation of injected Ag NPs suspensions. The Ag NP clusters, covering micron-sized areas, were formed within minutes using a conventional confocal Raman laser microscope. These Ag NP clusters were used to enhance the Raman spectra of the thiol products of OP breakdown in the microfluidic device: ethanethiol (EtSH) and (ethylsulfanyl) ethane-1-thiol (2-EET). When the OPH enzyme and its substrates OSDMP and demeton S were introduced, the thiolated breakdown products were generated, resulting in changes in the SERS spectra. With the ability to analyze reaction volumes as low as 20 nL, our approach demonstrates great potential for miniaturization of SERS analytical protocols.

Effects of Repeated Low-Dose Exposure of the Nerve Agent VX on Monoamine Levels in Different Brain Structures in Mice

In a previous report, alterations of the serotonin metabolism were previously reported in mice intoxicated with repeated low doses of soman. In order to better understand the effects induced by repeated low-dose exposure to organophosphorus compounds on physiological and behavioural functions, the levels of endogenous monoamines (serotonin and dopamine) in different brain areas in mice intoxicated with sublethal dose of (O-ethyl-S-[2(di-isopropylamino) ethyl] methyl phosphonothioate) (VX) were analysed by HPLC method with electrochemical detection. Animals were injected once a day for three consecutive days with 0.10 LD50 of VX (5 μg/kg, i.p). Neither severe signs of cholinergic toxicity nor pathological changes in brain tissue of exposed animals were observed. Cholinesterase (ChE) activity was only inhibited in plasma (a maximum of 30% inhibition 24 h after the last injection of VX), but remained unchanged in the brain. Serotonin and dopamine (DA) metabolism appeared significantly modified. During the entire period of investigation, at least one of the three parameters investigated (i.e. DA and DOPAC levels and DOPAC/DA ratio) was modified. During the toxic challenge, an increase of the serotonin metabolism was noted in hippocampus (HPC), hypothalamus/thalamus, pons medulla and cerebellum (CER). This increase was maintained 4 weeks after exposure in HPC, pons medulla and CER whereas a decrease in cortex 3 weeks after the toxic challenge was observed. The lack of correlation between brain ChE activity and neurochemical outcomes points out to independent mechanisms. The involvement in possibly long-lasting behavioural disorders is discussed.

Extraction of chemical impurities for forensic investigations: A case study for indoor releases of a sarin surrogate

A solvent extraction approach was developed and examined for extraction of targeted organophosphorus compounds as well as nerve agent simulants from painted wallboard (PWB). Painted wallboard was chosen as a substrate due to its presence as large surface area media in an indoor environment that is applicable to a chemical agent release scenario. Three different solvent systems were examined with a 1:1 methylene chloride: acetone mixture having the most robust and consistent extraction for four target organophosphorus compounds [dimethyl methyl phosphonate (DMMP), diethyl methyl phosphonate (DEMP), diethyl methyl phosphonothioate (DEMPT), and diisopropyl methyl phosphonate (DIMP)]. An average extraction efficiency of approximately 60% was obtained for these four compounds. The extraction approach was further demonstrated by extracting and detecting the chemical impurities present in neat DMMP that was vapor deposited onto painted wallboard tickets as a simulant to an agent release. Five chemical impurities that were present in DMMP – dimethyl phosphate, trimethyl ester phosphoric acid, ethyl methyl methylphosphonate, O,O,S-trimethyl ester phosphorothioic acid, and biphenyl were detected on the PWB and were utilized to determine the source/supplier of the DMMP.

Percutaneous penetration and distribution of VX using in vitro pig or human excised skin : Validation of demeton- S-methyl as adequate simulant for VX skin permeation investigations

The organophosphorus (OP) chemical warfare V agent O-ethyl- S-[2(di-isopropylamino)ethyl] methyl phosphonothioate (VX), is a highly toxic compound which mainly penetrates the body via percutaneous pathways. Hence, the following prerequisite: to ascertain compound absorption and percutaneous profile distribution with a view to further assessing the efficacy of topical skin protectants. We first selected the most appropriate receptor fluid to carry out in vitro VX absorption experiments, namely: Hanks's Balanced Salt Solution (HBSS). After a 24-h topical exposure time lapse, we measured altogether the percentage of applied dose unabsorbed and absorbed, penetration rate, lag time, permeability coefficient ( K p), and dose of VXeq present in skin. To such an end, we used full-thickness and split-thickness pig-ear or human abdominal skin membranes. Further, we scrutinised the potential use of two specific molecules as suitable surrogates for VX percutaneous penetration analyses: thus, we compared the present VX toxicokinetic parameters to earlier findings from our research unit, with respect to OP insecticides demethon- S-methyl (DSM) and paraoxon (POX). Within the framework of our study, we wish to highlight the following evidence: (a) pig-ear skin proves a relevant model to predict in vitro human abdominal skin, taking into account a 2-fold higher skin permeability to VXeq; (b) both full or split-thickness skin membranes may be used indiscriminately to gauge penetration rate and absorbed dose; (c) DSM applied on full-thickness pig-ear skin is the most relevant model to mimic the in vitro VX absorption through full-thickness skin model.

Anticonvulsants for Nerve Agent-Induced Seizures: The Influence of the Therapeutic Dose of Atropine

Two guinea pig models were used to study the anticonvulsant potency of diazepam, midazolam, and scopolamine against seizures induced by the nerve agents tabun, sarin, soman, cyclosarin, O-ethyl S-(2-(diisopropylamino)ethyl)methylphosphonothioate (VX), and O-isobutyl S-(2-diethylamino)ethyl)-methyl phosphonothioate (VR). Animals instrumented for electroencephalogram recording were pretreated with pyridostigmine bromide (0.026 mg/kg i.m.) 30 min before challenge with 2 x LD50 (s.c.) of a nerve agent. In model A, atropine sulfate (2.0 mg/kg i.m.) and pyridine-2-aldoxime methylchloride (2-PAM; 25.0 mg/kg i.m.) were given 1 min after nerve agent challenge, and the tested anticonvulsant was given (i.m.) 5 min after seizure onset. In model B, a lower dose of atropine sulfate (0.1 mg/kg i.m.) was given along with 2-PAM 1 min after nerve agent challenge, and the anticonvulsant was given at seizure onset. With the lower dose of atropine, seizure occurrence increased to virtually 100% for all agents; the time to seizure onset decreased for sarin, cyclosarin, and VX; the signs of nerve agent intoxication were more severe; and coma resulted frequently with cyclosarin. The anticonvulsant ED50 doses for scopolamine or diazepam were, in general, not different between the two models, whereas the anticonvulsant ED50 values of midazolam increased 3- to 17-fold with the lower atropine dose. Seizure termination times were not systematically effected by the different doses of atropine. The order of anticonvulsant effectiveness within each model was scopolamine > or = midazolam > diazepam. The findings indicate that the dose of atropine given as antidotal therapy can significantly influence measures of nerve agent toxicity and responsiveness to anticonvulsant therapy.

Absorption of the nerve agent VX ( O-ethyl- S-[2(di-isopropylamino)ethyl] methyl phosphonothioate) through pig, human and guinea pig skin in vitro

The physico-chemical properties of VX make the skin the most likely route of absorption into the human body. The development of effective medical countermeasures against such percutaneous threat agents relies on the use of appropriate animal models, as the inherent toxicity of nerve agents precludes the use of human volunteers. Previous studies have characterised the mechanism of nerve agent toxicity in rodent models, however, it is generally accepted that one of the most appropriate animal models for human skin absorption is the domestic pig. The purpose of the present study was to measure and compare the skin absorption kinetics of VX in vitro using pig, human and guinea pig skin to highlight any potential species differences in skin permeability. When undiluted VX was applied directly to the skin, the permeability of guinea pig skin was approximately 7-fold greater than human skin. There was no significant difference in the permeability of pig and human skin. When VX diluted with isopropyl alcohol was applied to the skin, the permeability of guinea pig skin was approximately 4-fold greater than human skin. There was no significant difference in the permeability of pig and human skin. From this data it may be inferred that dermatomed, abdominal pig skin is an appropriate model for the human skin absorption of VX.

Gas Chromatographic Determination of Traces of O-Isobutyl S-2-(N,N-Diethylamino)ethyl Methyl Phosphonothioate (VX-Like Compound) in Working Area and Community Air

A gas-chromatographic procedure is proposed for the determination of O-isobutyl S-2-(N,N-diethylamino)ethyl methyl phosphonothioate (mixed ester) at the maximum permissible level for the working area air (5 × 10–6 mg/m3) and at the safe reference level of impact for the community air (5 × 10–8 mg/m3). The procedure is based on trapping the mixed ester from air by a chemisorbent. Next, the analyte is extracted by hexane; transformed to O-methyl-O-isobutyl methyl phosphonate by silver nitrate in methanol; and, in the case of the working area air, the methanol solution of the derivative is chromatographed. In the determination of the mixed ester in the community air, a solution of O-methyl-O-isobutyl methyl phosphonate (dialkyl methyl phosphonate) in methanol is passed through a column with an adsorbent and transferred to the chromatographic system with a thermal desorption system. The relative error of the determination of the mixed ester in the working area and community air is no more than 28 and 40 rel. %, respectively.

Existence of an inactive pool of acetylcholinesterase in chicken brain.

We analyzed acetylcholinesterase (AcChoEase; EC 3.1.1.7) activity and AcChoEase immunoreactive protein in chicken brain by using five monoclonal antibodies raised against chicken AcChoEase. Four of them specifically recognized AcChoEase catalytic subunits in Western blots and one, C-131, recognized only enzymatically active AcChoEase. We observed considerable differences in the ratio of immunoreactive protein to catalytic activity in various fractions, indicating the existence of inactive AcChoEase protein. This inactive AcChoEase component was more abundant in a low-salt-soluble extract than in a subsequent detergent-soluble extract. On the basis of the ratio between activity and immunoreactivity, we calculated that the inactive component represents about 30% of the total AcChoEase subunits in chicken brain. The immunoreactive AcChoEase protein sedimented in sucrose gradients like the active molecular forms; the G1 and G2 peaks contained inactive molecules, whereas the G4 peak appeared to contain only active AcChoEase. The bulk of inactive AcChoEase reacted with the organophosphate cholinesterase inhibitor O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (MTP) but was found to bind the active site affinity ligand N-methylacridinium poorly and was not recognized by the active-form-specific monoclonal antibody, C-131. In addition, most of this fraction is sensitive to endoglycosidase H and binds the lectin wheat germ agglutinin poorly, suggesting that it was not processed in the Golgi apparatus. From these observations, we propose that the active and inactive AcChoEase components are differently folded.

Interaction of an organophosphate with a peripheral site on acetylcholinesterase

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.

Stereospecific coupling reaction for internucleotide methyl phosphonothioate linkage

The (Sp)- and (Rp)- diastereomers of 5′-dimethoxytritylthymidyl-(3′–5′)methylphosphono-5′-thio-3′-acetylthymidine were prepared by the stereospecific reaction of 5′-deoxy-5′-iodo-3′-acetylthymidine with the (Sp)- or (Rp)- diastereomers of 5′-dimethoxytritylthymidyl-3′methylphosphonothioate (Li + salt) in DMF. At 50°C, the coupling reaction was complete within 6 hours.

Enzymatic method for the estimation of residual organophosphorus compounds in water after treatment with chlorine-containing oxidant

A sensitive method for the estimation of residual organophosphorus compounds in water after treatment with chlorine-containing oxidants has been developed. The method is based on the inhibiting action of organophosphorus compounds towards the enzyme-catalyzed hydrolysis of esters. After the oxidants have been quantitatively destroyed by a mixture of NaHCO3 and NaNO2 small amounts of O-ethyl S-(N, N-diisopropylamino)ethyl methylphosphonothioate (MESP), dichlorvos (DDVP) and dimethoate are determined by using horse-serum cholinesterase with 2,6-dichloroindophenyl acetate as the substrate. A linear response range of 2 of 3 orders of magnitude and recovery within 100 ± 5% has been generally obtained.

Reactivation of VX-inhibited cholinesterase by 2-PAM and HS-6 in rats.

Atropinized rats intoxicated with ethyl-S-2-diisopropyl aminoethyl methyl phosphonothioate (VX), 15 mg/kg iv, were divided into three groups and were treated with normal saline, iv, 30 mg/kg of 2-PAM C1, iv, and 30 mg/kg of HS-6, iv. One hr after administration of therapy they were decapitated and cholinesterase (ChE) activity was determined on blood, brain and diaphragm tissue. Both 2-PAM C1 and HS-6 markedly reactivated VX-inhibited blood and diaphragm ChE. Brain ChE activity was not significantly reactivated by either oxime. The effectiveness of these oximes in restoration of VX-inactivated ChE in vivo offers an explanation as to why conventional atropine/oxime therapy is so effective against VX intoxication.