Hydrolysis Products Of Nerve Agents Research Articles

A Modified QuEChERS-DART-MS/MS Technique for High-Throughput Detection of Organophosphate Nerve Agent Hydrolysis Products in Environmental Samples

A Modified QuEChERS-DART-MS/MS Technique for High-Throughput Detection of Organophosphate Nerve Agent Hydrolysis Products in Environmental Samples

Analysis of degradation products of nerve agents in biological fluids by ion chromatography-tandem mass spectrometry.

The detection of hydrolysis products of nerve agents (alkyl methylphosphonic acids; RMPAs) in biological samples from victims is important to confirm exposure to nerve agents. However, analysis of RMPAs is difficult due to their high hydrophilicity. The aim of this study was to develop ion chromatography-tandem mass spectrometry (IC-MS/MS) methods using commercially available equipment and columns to analyze RMPAs in human urine and serum with high sensitivity and without using complicate techniques. A Dionex IonPac AS11-HC anion-exchange column was used to analyze six RMPAs (MPA, EMPA, IMPA, iBuMPA, CHMPA, and PMPA). For pretreatments of biological fluids, we developed two pretreatment methods (Method 1: dilution and ultrafiltration; Method 2: removal of chloride ions with Ag cartridges). Six RMPAs including highly hydrophilic methylphosphonic acid and ethyl methylphosphonic acid could be analyzed with sufficient retention times and peak shape. The detection limits of RMPAs were improved using Dionex OnGuard II Ba/Ag/H cartridges and MetaSEP IC-Ag cartridges (urine: 0.5-5ng/mL; serum: 1-5ng/mL). These methods were also applied to the test samples for the Organisation for the Prohibition of Chemical Weapons Biomedical Proficiency Tests. RMPAs could be sufficiently analyzed by IC-MS/MS. In addition, the limits of detection were superior to those obtained in our previous study involving LC-MS/MS or derivatization-LC-MS/MS method. For analysis of biological samples, an appropriate pretreatment method can be chosen according to the amount of sample available for analysis and expected RMPA concentrations.

A review on analysis methods for nerve agent hydrolysis products

This review article provides an overview of the background of nerve agents (NAs) and their hydrolysis products. The analytical techniques that are used for the analysis of hydrolysis products are also discussed. The analysis methods include the chromatographic, mass spectrometric, electrophoretic, and sensing technologies that are currently available for the detection of exposure to a number of hydrolysis products of NAs. All reviewed information was gathered through a detailed search of Scopus, PubMed, and the World Wide Web using relevant keywords, e.g., NA, hydrolysis products, and methylphosphonic acid. Most analyses of NA hydrolysis products still rely on chromatographic techniques such as gas and liquid chromatography. Analyses based on mass spectrometry are now fairly routine and can be further improved by providing analyses with precise and accurate masses and faster scan speeds that provide further gains in sensitivity. Although these techniques have high selectivity and sensitivity, most of them are rarely applied to real samples or suffer from destructive real sample analysis. In addition, there is still a lack of studies on the development of portable sensors for the detection of NA hydrolysis products. Although the analysis of NA hydrolysis products has experienced a transition from qualitative analysis toward rapid and quantitative identification, portable, and fast sensing technologies have not been well established or investigated extensively. In the future, more analysis methods are expected to be developed and may involve the development of sensors and lab-on-chip devices that should address specificity, sensitivity, stability, response time, and repeatability/reproducibility.

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.

Analysis of nerve agent metabolites from nail clippings by liquid chromatography tandem mass spectrometry.

While several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure, these methods are generally limited in the amount of time after an exposure that markers of exposure can be detected (due to rapid metabolism from biological matrices). In this study, a method for the analysis of nerve agent hydrolysis products from nail clippings was developed to allow evaluation of nails as a long-term repository of these markers. Pinacolyl methylphosphonic acid (PMPA) and isopropyl methylphosphonic acid (IMPA) were extracted from nail samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.3μg/kg and 7.5μg/kg and linear ranges were 0.75-300μg/kg and 30-1500μg/kg, respectively. Precision was within 10% and 8% for PMPA and IMPA, respectively, and accuracy was 100±12% for both analytes. The approach presented here is complementary to current methods for nerve agent exposure verification, and should allow for long-term determination of nerve agent poisoning.

Effective methylation of phosphonic acids related to chemical warfare agents mediated by trimethyloxonium tetrafluoroborate for their qualitative detection and identification by gas chromatography-mass spectrometry

The effective methylation of phosphonic acids related to chemical warfare agents (CWAs) employing trimethyloxonium tetrafluoroborate (TMO·BF4) for their qualitative detection and identification by gas chromatography-mass spectrometry (GC-MS) is presented. The methylation occurs in rapid fashion (1 h) and can be conveniently carried out at ambient temperature, thus providing a safer alternative to the universally employed diazomethane-based methylation protocols. Optimization of the methylation parameters led us to conclude that methylene chloride was the ideal solvent to carry out the derivatization, and that even though methylated products can be observed surfacing after only 1 h, additional time was not found to be detrimental but beneficial to the process particularly when dealing with analytes at low concentrations (∼10 μg mL−1). Due to its insolubility in methylene chloride, TMO·BF4 conveniently settles to the bottom during the reaction and does not produce additional interfering by-products that may further complicate the GC-MS analysis. The method was demonstrated to successfully methylate a variety of Schedule 2 phosphonic acids, including their half esters, resulting in derivatives that were readily detected and identified using the instrument's spectral library. Most importantly, the method was shown to simultaneously methylate a mixture of the organophosphorus-based nerve agent hydrolysis products: pinacolyl methylphosphonate (PMPA), cyclohexyl methylphosphonate (CyMPA) and ethyl methylphosphonate (EMPA) (at a 10 μg mL−1 concentration each) in a fatty acid ester-rich organic matrix (OPCW-PT-O3) featured in the 38th Organisation for the Prohibition of Chemical Weapons (OPCW) Proficiency Test. In addition, the protocol was found to effectively methylate N,N-diethylamino ethanesulfonic acid and N,N-diisopropylamino ethanesulfonic acid that are products arising from the oxidative degradation of the V-series agents VR and VX respectively. The work described herein represents the first report on the use of TMO·BF4 as a viable, stable and safe agent for the methylation of phosphonic acids and their half esters and within the context of an OPCW Proficiency Test sample analysis.

Determination of the hydrolysis products of nerve agents in natural waters by liquid chromatography–mass spectrometry

An approach to the determination of markers of the application of four nerve agents, S-(2-diethylaminoethyl)methylphosphonothioate, S-(2-diisopropylaminoethyl)methylphosphonothioate, bis(2N,N-diethylaminoethyl)disulfide, and bis(2-N,N-diisopropylaminoethyl)disulfide, in aqueous environmental samples by high-performance liquid chromatography with tandem mass spectrometric detection (HPLC–MS2) is developed. The conditions of the mass spectrometric detection (selection of optimal pairs of ionic reactions) are optimized, and the program for the gradient elution in HPLC separation is adjusted. The proposed approach was tested in the analysis of real water bodies using the standard addition method. Good accuracy, reproducibility, and selectivity of determination were shown. It was demonstrated that the matrix components in the water samples have no interfering effect. Using the proposed approach, the detection limits of S-(2-diethylaminoethyl)methylphosphonothioate, S-(2-diisopropylaminoethyl)methylphosphonothioate, bis(2-N,N-diethylaminoethyl)disulfide, and bis(2-N,N-diisopropylaminoethyl)disulfide in water bodies, determined by HPLC–MS2, were found to be 0.0003, 0.003, 0.3, and 0.05 µg mL–1, respectively.

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

On-matrix derivatization for dynamic headspace sampling of nonvolatile surface residues

The goal of this study is to extend sampling by the field and laboratory emission cell (FLEC) dynamic headspace technique to applications that target nonvolatile residues. On-matrix derivatization of residues to render analytes stable and more volatile is explored to achieve this goal. Results show that on-matrix derivatizations of nerve agent hydrolysis products (monoalkyl methylphosphonic acids and methylphosphonic acid [MPA]) with diazomethane were successful on glass and painted wallboard (at the 10-μg level). It also was successful on the more difficult concrete (at the 500-μg level) and carpet (at the 20-μg level), substrates that cannot be successfully sampled using swipe techniques. Analysis of additional chemical warfare (CW)-associated residues can be approached by on-matrix derivatization with trifluoroacetic anhydride (TFAA). For example, amines (used as stabilizers or present as decomposition products of the nerve agent VX) or thiodiglycol (hydrolysis product of sulfur mustard) could be sampled as their TFAA derivatives from glass, painted wallboard, and concrete (at the 40-μg level), as well as carpet (at the 80-μg level) surfaces. Although the amine and thiodiglycol are semi-volatile and could be sampled directly, derivatization improves the recovery and chromatographic behavior of these analytes.

Determination of alkyl methylphosphonates and methylphosphonic acid by capillary electrophoresis with a diode-array spectrophotometric detector

Method of capillary electrophoresis with a diode-array detector was used to study the conditions of the separation of three alkyl methylphosphonates and methylphosphonic acid, hydrolysis products of nerve agents. The optimal conditions providing their separation and determination are reported (background electrolyte based on 100 mM boric acid with the addition of 10 mM of phenylphosphonic acid (pH 6.0); voltage +30 kV, detection wavelength 210 nm, electrokinetic introduction of the sample (10 kV, 10 sec), separation time below 10 min). Calibration curve for all the compounds was linear in the concentration range 0.05–1.0 mg/mL, the correlation coefficient was 0.999 in all cases. The procedure was tested for river and ground waters containing a mixture of isopropyl, isobutyl, pinacolyl methylphosphonates and methylphosphonic acid. Relative standard deviations of the migration times under the conditions of the repeatability of the results lie in the range 1.9–2.7% (n = 5), the relative standard deviation of the reduced peak areas vary from 1.2 to 7.0%.

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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 3 min and determined in 1D GC–MS mode with detection limits of around 10 ng/ml of reaction mixture (except for the cHMPA derivative, whose mass spectrum contained noisy background peaks). In 2D-GC–MS mode, where each 0.04 min 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 6 min. The recoveries of all the derivatives on 2D GC from 1D GC were estimated to be over 66%, and the detection limits were around 10 ng/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 1.25 μg/ml). Phosphonic acids could be detected at less than 60 ng/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 20 ng/ml.

Detection of nerve agent hydrolytes in an engineered nanopore

We report a stochastic nanopore sensing method for the detection of organophosphorus nerve agent hydrolysis products. By employing an engineered α-hemolysin single pore embedded in a planar lipid bilayer as the stochastic sensing element and β-cyclodextrin as a host molecule, trace amounts of soman and cyclosarin hydrolytes could be detected, with detection limits of 53 nM and 102 nM, respectively. Importantly, sarin, tabun, and VX hydrolysis products, as well as other common pesticides, do not interfere with detection of the analytes. The method offers the potential as a rapid and sensitive sensing technique for use in on-site analysis of nerve agents in environmental monitoring applications at the single-molecule level.

Detection Of Nerve Agent Hydrolytes In An Engineered Nanopore

We report a stochastic nanopore sensing method for the detection of organophosphorus nerve agent hydrolysis products. By employing an engineered α-hemolysin single pore embedded in a planar lipid bilayer as the stochastic sensing element and β-cyclodextrin as a host molecule, trace amounts of soman and cyclosarin hydrolytes could be detected, with detection limits of 53 nM and 102 nM, respectively. Importantly, sarin, tabun, and VX hydrolysis products, as well as other common pesticides, do not interfere with detection of the analytes. The method offers the potential as a rapid and sensitive sensing technique for use in on-site analysis of nerve agents in environmental monitoring applications at the single-molecule level.

Analysis of VX nerve agent hydrolysis products in wastewater effluents by ion chromatography with amperometric and conductivity detection

An analytical method, based on the use of ion chromatography, was developed to monitor the levels of three regulated VX hydrolysis products in the effluent from a biological wastewater treatment process—ethylmethylphosphonic acid, methylphosphonic acid and 2-(diisopropyl)aminoethanethiol. Previous methods have not been applied to wastewater matrices or 2-(diisopropyl)aminoethanethiol. Despite the specificity and sensitivity constraints of this method, it was possible to measure the compounds in bioreactor effluents down to a level substantially below the US Army discharge limit of 0.1% (w/v). Analytical data was confirmed by liquid chromatography–mass spectrometry (LC–MS) at an independent laboratory.

Determination of alkylphosphonic acids by microcolumn liquid chromatography with gradient elution coupled on-line with flame photometric detection

Microcolumn liquid chromatography with gradient elution and on-line flame photometric detection has been used for the selective and rapid determination of non-volatile alkylphosphonic acids, which are hydrolysis products of nerve agents, in aqueous samples. A make-up of water was used to maintain stable introduction of the eluent during a gradient run. The detection limits are strongly dependent on quenching, which is determined by the mass flow of methanol in the eluent. Large-volume injections of 100 μl of aqueous solutions of short- and long-chain alkylphosphonic acids resulted in detection limits in the range 6–800 ng/ml. The repeatability of the retention times and analyte response – peak area or peak height – were 0.7–0.9% and 4–11%, respectively. The method was successfully applied to a local tap water sample and an aqueous soil extract.

Effect of cation-exchange pretreatment of aqueous soil extracts on the gas chromatographic–mass spectrometric determination of nerve agent hydrolysis products after tert.-butyldimethylsilylation

The efficency of pretreatment of aqueous soil extracts using a cation-exchange resin has been investigated by gas chromatographic–mass spectrometric (GC–MS) determination of nerve agent hydrolysis products after tert.-butyldimethylsilyl (TBDMS) derivatization. An aqueous solution containing methylphosphonic acid (MPA) and its monoalkyl esters, ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methylphosphonic acid, was dried, and these phosphonic acids were derivatized with N-methyl- N-( tert.-butyldimethylsilyl)trifluoro-acetamide and analyzed by GC–MS. The yields of TBDMS derivatives were significantly decreased by the addition of calcium and magnesium ions to an aqueous solution (≈0.5 m M) before derivatization. The extent of lowered yields was related to the hydrophilicity of phosphonic acids. MPA and its monoalkyl esters were spiked into soil samples (sand, alluvial soil and volcanic ash soil), extracted with distilled water, dried, silylated and applied to GC–MS. The yields of TBDMS derivatives of monoalkyl esters from soil samples were low (3–42%) and MPA derivative was scarcely detected (yield: <0.7%). By desalting the aqueous soil extract by passage through a strong cation-exchange resin, the yields of TBDMS derivatives of monoalkyl esters were significantly improved (12–69%) and MPA derivative was detected (yield: 2–36%). The extent of improved yields was related to the concentrations of divalent metal cations in aqueous soil extracts. In combination with desalting by the cation-exchange resin, GC–MS after TBDMS derivatization enables detection of nerve agent hydrolysis products in soils at sub-ppm (0.2 μg/g) concentrations.

Analysis of degradation products of organophosphorus chemical warfare agents and related compounds by liquid chromatography–mass spectrometry using electrospray and atmospheric pressure chemical ionisation

A qualitative screening procedure was developed for the detection and identification of phosphonic acid hydrolysis products of organophosphorus chemical warfare agents, using liquid chromatography–mass spectrometry with electrospray ionisation (LC–ESP-MS). A mixed C8/C18 reversed-phase column with gradient elution gave good chromatography for a series of phosphonic acids. Detection limits for aqueous solutions of standards were <50 ng/ml (<0.25 ng injected), providing an increase in sensitivity up to an order of magnitude compared with LC–MS using atmospheric pressure chemical ionisation (LC–APCI-MS). The methodology provides a rapid screening procedure for the hydrolysis products of nerve agents in aqueous samples and extracts. The application of LC–ESP/APCI-MS and LC–ESP/APCI-MS–MS to the identification of dialkyl esters of phosphonic acids is also described. Examples are given of the application of LC–MS to spiked water samples analysed during the course of proficiency testing.

Capillary electrophoresis coupled on-line with flame photometric detection: Determination of alkylphosphonic acids

The separation performance and sensitivity of an earlier developed system which uses capillary electrophoresis coupled on-line with a flame photometric detector (originally developed for gas chromatography) are considerably improved. The liquid junction used in order to decouple the electrical field shows a negligible influence on the performance of the system as compared with on-line UV detection. The system is used to determine alkylphosphonic acids, which are the hydrolysis products of nerve agents. By using on-column sample concentration techniques, the detection limits for the alkylphosphonic acids in water are in the range of 0.1–0.5 μg/mL. The first results obtained during interlaboratory tests using capillary electrophoresis coupled with flame photometric detection as fast screening method to detect the presence of alkylphosphonic acids in aqueous extracts or water samples are promising. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 253–259, 1997

Identification of degradation products of some chemical warfare agents by capillary electrophoresis—ionspray mass spectrometry

Capillary zone electrophoresis—ionspray mass spectrometry (CZE—IS-MS) in the negative-ion mode was applied in the identification of five organophosphonic acids, which are the primary hydrolysis products of nerve agents. The spectra exhibit a very abundant (M  H) − ion with minimal fragmentation. Fragment ions were produced by raising the nozzle — skimmer voltage difference in the first vacuum stage between the atmospheric pressure ion source and the mass analyzer. CZE—IS-MS provides extremely good separation efficiency and very high sensitivity for the phosphoric acids. Sensitivity in the range 10–30 pg was achieved with standard solutions using selected ion monitoring. Linear regression analysis data showed correlation coefficients to be between 0.994 and 0.999, indicating good quantitative linearity of the method.

Determination of organophosphorus acids by thermospray liquid chromatography-mass spectrometry

The determination of thirteen organophosphorus acids, hydrolysis products of nerve agents and pesticides, by a combination of ion-pair liquid chromatography on a reversed-phase C 18 column and thermospray mass spectrometry was investigated. Ammonium acetate and three tetraalkylammonium salts with different alkyl groups (methyl, ethyl and n-butyl) were applied as ion-pair reagents. All the organophosphorus acids could be eluted using water or water-methanol mixtures. Capacity factors ( k′) were measured for some selected acids using water as eluent. The recorded thermospray mass spectra using ammonium acetate as electrolyte gave the [M + NH 4] + ion as a predominant peak, whereas with the tetraalkylammonium salts cluster ions were found. This difference in ionization mechanism was also reflected in the sensitivity. An amount of 100 pg of dimethylthiophosphoric acid could be detected by selected ion monitoring using ammonium acetate, whereas with tetramethylammonium hydroxide the amount was 5 ng. To obtain lower detection levels preconcentration could be achieved with a Sep-Pak C 18 cartridge pretreated with a tetra- n-butylammonium salt.