2-chlorovinylarsonous-acid and lewisite

The purpose of this study was to develop a sensitive and simple method, based on dispersive derivatization liquid-liquid microextraction-gas chromatography-mass spectrometry (DDLLME-GC-MS) in scanning and selected-ion-monitoring (SIM) modes, for detection of 2-chlorovinylarsonous acid (CVAA) as a hydrolysis product and urinary metabolite of lewisite in urine samples. Chloroform (65 μL), methanol (500 μL), and ethanedithiol (10 μL) were used as extraction solvent, dispersive solvent, and derivatizing reagent, respectively. Critical conditions of the proposed method were optimized. The nucleophilic reactions of dithiol and monothiol compounds with CVAA were also studied using a competitive method. In view of the high affinity of trivalent arsenic for sulfhydryl groups, the interaction between CVAA and bis(2-chlorovinyl)arsonous acid (BCVAA) and free cysteine (Cys) was also investigated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The interference of Cys, present in human urine, with the detection of CVAA was evaluated using dithiol and monothiol chemicals as derivatization agents. The developed method provided a preconcentration factor of 250, and limits of detection of 0.015 and 0.30 μg L(-1) in SIM and scanning modes, respectively. The calibration curves were linear over the concentration range of 1-400 μg L(-1) in full-scan mode. The relative standard deviation (RSD) values were calculated to be 5.5 and 3.2% at concentrations of 20 and 100 μg L(-1), respectively. Collision-induced dissociation studies of the major electron-impact (EI) ions were performed to confirm the proposed fragment structure of CVAA-dithiols derivatives. Results indicated that the developed method for analysis of CVAA is suitable not only for verification of human exposure to lewisite, but also for quantification of CVAA in urine samples.

Topics: Arsenicals; Biotransformation; Chemical Warfare Agents; Chloroform; Cysteine; Gas Chromatography-Mass Spectrometry; Humans; In Vitro Techniques; Limit of Detection; Liquid-Liquid Extraction; Mercaptoethanol; Methanol

A sensitive and simple method for the quantification and for the detection of 2-chlorovinylarsonous (CVAA) and 2-chlorovinylarsonic (CVAOA) acids was developed. CVAA and CVOA are important biological markers in human and rat urine specific to lewisite (chlorovinylarsonous chloride compounds) exposure. The developed assay was based on the use of solid-phase extraction (SPE) followed by liquid-chromatography coupled to electrospray ionization (negative ion-mode) low-energy collision dissociation-tandem mass spectrometry (ESI-CID-MS/MS). The method demonstrated linearity over at least three orders of magnitude and had a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. The relative standard deviations for the quality control samples ranged from 6 to 11%. Application of this procedure was demonstrated in the lewisite animals exposure model. Rats were exposed intravenously by no lethal doses of lewisite and markers levels in urine samples were analyzed for 21 days post-exposure.

Topics: Animals; Arsenicals; Chromatography, Liquid; Humans; Limit of Detection; Male; Rats; Regression Analysis; Reproducibility of Results; Solid Phase Extraction; Tandem Mass Spectrometry

A high-throughput method has been developed for determining Lewisite [dichloro(2-chlorovinyl)arsine] exposure by measuring the urine metabolite 2-chlorovinylarsonous acid (CVAA) and the oxidized metabolite 2-chlorovinylarsonic acid (CVAOA). The rapid sample preparation included a simple dilution of 400 microL of urine with 40 microL of water and 1 mL of buffer containing an internal standard and brief centrifugation prior to analysis by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (ICP-MS). CVAOA and CVAA were eluted isocratically with retention factors of approximately 3.0 and 4.2, respectively, from a reversed-phase polar embedded column with a cycle time of 5 min per sample. The dynamic reaction cell, typically used to remove polyatomic isobaric interferences, was not required for ICP-MS analysis because of the resolution of chloride from arsenical peaks of interest. This method was used to detect CVAA and CVAOA in the urine of a rat administered Lewisite up to 24 h after exposure. The method demonstrated linearity over at least three orders of magnitude and had a method detection limit of 1.3 microg/L as CVAA (1.4 microg/L CVAOA). The relative standard deviations for quality control samples ranged from 3 to 6%. The method was sensitive and selective with no false positives in 100 different urine samples collected from individuals with no known exposure to Lewisite. Ninety-six samples could be analyzed in an 8-h day.

Topics: Animals; Arsenicals; Calibration; Chromatography, High Pressure Liquid; Environmental Exposure; Humans; Mass Spectrometry; Oxidation-Reduction; Rats; Reproducibility of Results

2-Chlorovinylarsonic acid (CVAOA) is a stable abiotic metabolite of lewisite 1 that has been identified in lewisite dumps. There have been no reports of microbial degradation of CVAOA, so we isolated and examined CVAOA-degrading microorganisms. CVAOA contains arsine, which is toxic to microbial growth. We therefore used the simple organic chemical, ethylene, as a sole carbon source in initial screening for suitable microbes. We isolated several microorganisms from sewage sludge and soil. Two strains, NK0505 and NK0506, could be grown on CVAOA as the sole carbon source and were identified by 16S rRNA sequencing as Nocardia carnea NK0505 and Rhodococcus opacus NK0506. Because N. carnea NK0505 was slightly more active in degrading CVAOA, we used it for further degradation studies. Strain NK0505 utilized about 90% of CVAOA (50 ppm) within 5 days; at higher concentrations of CVAOA no degradation occurred over a 10-day period. We identified 1-chloro-1,2-dihydroxyethane, ethylene glycol, glycolic acid, and arsenic acid as degradation products of CVAOA. Epoxy formation on alkylarsine was not confirmed. CVAOA is probably further metabolized via these compounds in the tricarboxylic acid cycle. Strain NK0505 could also degrade but-3-enylarsonic acid, trichloroethylene, isoprene, and 1,3-butadiene, but utilization of tetrachloroethylene and acetylene did not occur.

Topics: Arsenicals; Bacteria; Biodegradation, Environmental; Ethylenes; Industrial Waste; Nocardia; Organic Chemicals; Rhodococcus

Lewisite [dichloro(2-chlorovinyl)arsine] is a highly toxic chemical warfare agent with vesicant properties. The accidental exposure to lewisite or its intentional use as a chemical terrorism weapon are a public health threat and warrant investigations for the development of analytical methods to detect biomarkers of exposure to lewisite. Under aqueous conditions, lewisite rapidly hydrolyzes to the non-volatile 2-chlorovinylarsonous acid (CVAA). We have developed a sensitive, simple, and automated method for measuring CVAA in human urine. The assay is based on the use of solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) after derivatization of the CVAA with 1,3-propanedithiol (PDT). The volatile CVAA-PDT is adsorbed onto a SPME fiber and analyzed by GC-MS. The assay was validated on human urine samples spiked with CVAA to determine the accuracy, precision, and limit of detection (LOD). The LOD was 7.4 pg in 1 ml of urine.

Topics: Arsenicals; Automation; Calibration; Gas Chromatography-Mass Spectrometry; Humans; Reproducibility of Results; Sensitivity and Specificity

The development of a procedure for retrospective detection and quantitation of exposure to the arsenical dichloro(2-chlorovinyl)arsine (lewisite; L1) has been initiated. Upon incubation of human blood with [14C]L1 (20 nM-0.2 mM) in vitro, more than 90% of the total radioactivity was found in the erythrocytes and 25-50% of the radioactivity becomes associated with globin. Evidence was obtained for the presence of several binding sites. One type of binding was identified as L1-induced crosslinking of cysteine residues 93 and 112 of the beta-globin chain. A method was developed for extraction of bound and unbound 2-chlorovinylarsonous acid (CVAA), a major metabolite of L1, from whole blood after treatment with 2,3-dimercapto-1-propanol (BAL). Subsequent to derivatization with heptafluorobutyryl imidazole, the CVAA-BAL derivative could be analysed at a 40-fmol level by means of gas chromatography-mass spectroscopy (GC-MS) under electron impact conditions. With this procedure, in vitro exposure of human blood to 1 nM L1 could be determined. The same procedure was applied to the analysis of human urine samples spiked with CVAA. In vivo exposure of guinea pigs could be established at least 240 h after subcutaneous administration of the agent (0.25 mg/kg) by the determination of bound and unbound CVAA in the blood. In the urine of these animals, CVAA could be detected for 12 h after exposure.

Topics: Animals; Arsenicals; Binding Sites; Carbon Radioisotopes; Chelating Agents; Dimercaprol; Environmental Exposure; Erythrocytes; Gas Chromatography-Mass Spectrometry; Globins; Guinea Pigs; Hemoglobins; Humans; Imidazoles; Male; Protein Binding; Spectrometry, Mass, Electrospray Ionization