The development of an analytical method for urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS).

The analysis of biomedical samples such as urine and blood can provide evidence of exposure to chemicals for a range of applications including occupational exposure monitoring, detection of drugs of abuse, performance enhancement in sport and investigations of poisoning and incapacitation. This paper reports the development of an analytical method for two suspected urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS): 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine. 2-Chlorohippuric acid was identified in all 2h post-exposure samples from a set of urine samples taken from army recruits exposed to low levels of thermally dispersed CS during training. 2-Chlorobenzyl-N-acetylcysteine, a metabolite known to be formed in the rat, was not identified in any of the samples. The lower limit of detection (LLOD) for 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine was 1ng/ml and 0.5ng/ml in pooled urine from the pre-exposed subjects. 2-Chlorohippuric acid was rapidly excreted but was detectable in the urine of 17 of the 19 subjects tested 20h after exposure.

Biological markers of exposure to organophosphorus nerve agents.

Organophosphorus nerve agents are the most toxic chemical warfare agents that are known to have been produced, stockpiled and weaponised. Their development, production, stockpiling and use are prohibited under the terms of the Chemical Weapons Convention and, together with their precursors, are subject to strict controls and verification procedures. The detection and identification of biological markers of exposure to nerve agents are required for three main purposes: confirmation of exposure for forensic purposes in cases of alleged use; diagnosis to guide appropriate medical countermeasures in the event of an exposure; and occupational health monitoring of workers in defence laboratories and demilitarisation facilities. Biomarkers of nerve agents fall into two main groups, free metabolites and adducts to proteins. These are reviewed together with analytical methods for their identification. Examples are provided of applications in cases of human exposure.

Analysis of clothing and urine from Moscow theatre siege casualties reveals carfentanil and remifentanil use.

On October 26, 2002, Russian Special Forces deployed a chemical aerosol against Chechen terrorists to rescue hostages in the Dubrovka theatre. Its use confirmed Russian military interest in chemicals with effects on personnel and caused 125 deaths through a combination of the aerosol and inadequate medical care. This study provides evidence from liquid chromatography-tandem mass spectrometry analysis of extracts of clothing from two British survivors, and urine from a third survivor, that the aerosol comprised a mixture of two anaesthetics--carfentanil and remifentanil--whose relative proportions this study was unable to identify. Carfentanil and remifentanil were found on a shirt sample and a metabolite called norcarfentanil was found in a urine sample. This metabolite probably originated from carfentanil.

Biomarkers of organophosphorus nerve agent exposure: comparison of phosphylated butyrylcholinesterase and phosphylated albumin after oxime therapy.

Organophosphorus nerve agents inhibit the activity of cholinesterases by phosphylation of the active site serine. In addition, sarin, cyclosarin, soman and tabun have been shown to phosphylate a tyrosine residue in albumin. Therapies against nerve agent poisoning include the use of oximes to reactivate inhibited cholinesterases by displacement of the phosphyl moiety and hence detectable levels of adducts with cholinesterases may be reduced. Adducts with tyrosine have been shown to be persistent in the guinea pig in the presence of oxime therapy. Plasma samples obtained from an animal study aimed at improving therapy against nerve agent poisoning were used to compare the suitability of tyrosine and butyrylcholinesterase (BuChE) adducts as biomarkers of nerve agent exposure after treatment with therapeutic oximes. Under the terms of the project licence, these samples could be collected only on death of the animal, which occurred within hours of exposure or when culled at 23 or 24 days. Tyrosine adducts were detected in all samples collected following intra-muscular administration of twice the LD50 dose of the respective nerve agent. Aged BuChE adducts were detected in samples collected within a few hours after administration of soman and tabun, but not after 23 or 24 days. No BuChE adducts were detected in animals exposed to sarin and cyclosarin where samples were collected only after 23 or 24 days.

History and perspectives of bioanalytical methods for chemical warfare agent detection.

This paper provides a short historical overview of the development of bioanalytical methods for chemical warfare (CW) agents and their biological markers of exposure, with a more detailed overview of methods for organophosphorus nerve agents. Bioanalytical methods for unchanged CW agents are used primarily for toxicokinetic/toxicodynamic studies. An important aspect of nerve agent toxicokinetics is the different biological activity and detoxification pathways for enantiomers. CW agents have a relatively short lifetime in the human body, and are hydrolysed, metabolised, or adducted to nucleophilic sites on macromolecules such as proteins and DNA. These provide biological markers of exposure. In the past two decades, metabolites, protein adducts of nerve agents, vesicants and phosgene, and DNA adducts of sulfur and nitrogen mustards, have been identified and characterized. Sensitive analytical methods have been developed for their detection, based mainly on mass spectrometry combined with gas or liquid chromatography. Biological markers for sarin, VX and sulfur mustard have been validated in cases of accidental and deliberate human exposures. The concern for terrorist use of CW agents has stimulated the development of higher throughput analytical methods in support of homeland security.

An overview of biological markers of exposure to chemical warfare agents.

An overview is given of biological markers of exposure to chemical warfare agents. Metabolites, protein, and/or DNA adducts have been identified for most nerve agents and vesicants and validated in experimental animals or in a small number of human exposures. For several agents, metabolites derived from hydrolysis are unsatisfactory biomarkers of exposure because of background levels in the human population. These are assumed to result from environmental exposure to commercial products that contain these hydrolysis products or chemicals that are metabolized to them. In these cases, metabolites derived from glutathione pathways, or covalent adducts with proteins or DNA, provide more definitive biomarkers. Biomarkers for cyanide and phosgene are unsatisfactory as indicators of chemical warfare exposure because of other sources of these chemicals or their metabolites.

Phosphylated tyrosine in albumin as a biomarker of exposure to organophosphorus nerve agents.

The organophosphorus nerve agents sarin, soman, cyclosarin and tabun phosphylate a tyrosine residue on albumin in human blood. These adducts may offer relatively long-lived biological markers of nerve agent exposure that do not 'age' rapidly, and which are not degraded by therapy with oximes. Sensitive methods for the detection of these adducts have been developed using liquid chromatography-tandem mass spectrometry. Adducts of all four nerve agents were detected in the blood of exposed guinea pigs being used in studies to improve medical countermeasures. The tyrosine adducts with soman and tabun were detected in guinea pigs receiving therapy 7 days following subcutaneous administration of five times the LD(50) dose of the respective nerve agent. VX also forms a tyrosine adduct in human blood in vitro but only at high concentrations.

Analysis of the sulphur mustard metabolites thiodiglycol and thiodiglycol sulphoxide in urine using isotope-dilution gas chromatography-ion trap tandem mass spectrometry.

A sensitive method has been developed for the trace analysis of the sulphur mustard metabolite thiodiglycol (TDG) in urine, and its oxidation product thiodiglycol sulphoxide (TDGO) after reduction to thiodiglycol. Thiodiglycol was extracted from urine by solid phase extraction onto a polymeric cartridge and, after isolation, converted to its bis-heptafluorobutyryl derivative with heptafluorobutyryl imidazole. An ion trap mass spectrometer in selected reaction monitoring mode detected spiked concentrations down to 0.2 ng/ml with a signal to noise ratio>3:1. Urine, from human volunteers with no known exposure to sulphur mustard, contained detectable but very low concentrations (<0.2 ng/ml) of thiodiglycol, consistent with previous observations using different methodologies. Combined concentrations of thiodiglycol and thiodiglycol sulphoxide were determined after reduction of the latter with titanium trichloride. In this case higher background levels (up to 3 ng/ml) were observed, consistent with the sulphoxide being the major excretion product of the two metabolites. The method was applied to urine samples, stored frozen for 13 years, from two casualties of accidental mustard poisoning. Levels of thiodiglycol were 1 and 3 ng/ml, which increased to 78 and 104 ng/ml after treatment of the urine with titanium trichloride.

The trace analysis of alkyl alkylphosphonic acids in urine using gas chromatography-ion trap negative ion tandem mass spectrometry.

A sensitive method has been developed for the trace analysis of alkyl alkylphosphonic acids, metabolites of nerve agents, in urine using a benchtop ion trap mass spectrometer. The acids were isolated from urine by simple solid phase extraction and converted to their pentafluorobenzyl esters. An ion trap mass spectrometer in selected reaction monitoring mode provided limits of detection of 0.1 ng/ml for isopropyl, isobutyl, pinacolyl and cyclohexyl methylphosphonic acids and for ethyl ethylphosphonic acid. The detection limit for ethyl methylphosphonic acid was higher (0.5 ng/ml) due to a lower recovery.

Analysis of beta-lyase metabolites of sulfur mustard in urine by electrospray liquid chromatography-tandem mass spectrometry.

A method is described for the analysis of b-lyase metabolites of sulfur mustard, 1-methylsulfinyl-2- [2-(methylthio)ethylsulfonyl]ethane and 1,1'-sulfonylbis [2-(methylsulfinyl)ethane], in human urine. The analytes were concentrated from urine on an ENV+ solid-phase extraction cartridge and analyzed by liquid chromatography-positive ion electrospray-tandem mass spectrometry in the selected reaction monitoring mode. Quantitation was performed against deuterated internal standards. Limits of detection were 0.1-0.5 ng/mL. The metabolites were detected in samples of urine from human casualties of sulfur mustard poisoning. The method provides a simpler alternative to gas chromatography-tandem mass spectrometry analysis, avoiding the need for reduction to less polar analytes.

Analysis of the sulfur mustard metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] in urine by negative ion electrospray liquid chromatography- tandem mass spectrometry.

A method is described for the analysis of the sulfur mustard metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] in human urine. The analyte was concentrated from urine on a polymeric SPE cartridge and analyzed by liquid chromatography-negative ion electrospray tandem mass spectrometry in the selected reaction monitoring mode. The limit of detection was 0.5-1 ng/mL. The metabolite was detected at concentrations close to the detection limit in samples of urine from two casualties accidentally exposed to sulfur mustard from a First World War munition.

Identification of iso- and n-propylphosphonates using liquid chromatography-tandem mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy.

Organophosphorus nerve agents and their precursors, specifically listed in the schedules of chemicals in the Annex to the Chemical Weapons Convention (CWC), include analogues with C1-C3 alkyl groups on phosphorus. The Organisation for the Prohibition of Chemical Weapons (OPCW) requires designated laboratories to unequivocally identify isomeric propyl groups bonded to phosphorus in analytes that may be present in samples submitted for analysis. Homologous series of isomeric pairs of dialkyl iso- and n-propylphosphonates, alkyl iso- and n-propylphosphonochloridates, and alkyl iso- and n-propylphosphonofluoridates, have been analysed by liquid chromatography-ion trap tandem mass spectrometry and/or by gas chromatography-Fourier transform infrared spectroscopy. The results show that P-propyl isomers can be reliably differentiated by collision induced dissociation (CID) of selected fragment ions and by their infrared P=O stretching and C-H deformation frequencies.

Derivatisation reactions in the chromatographic analysis of chemical warfare agents and their degradation products.

The analysis of chemical warfare agents and their degradation products is an important component of verification of compliance with the Chemical Weapons Convention. Gas and liquid chromatography, particularly combined with mass spectrometry, are the major techniques used to detect and identify chemicals of concern to the Convention. The more polar analytes, and some of the more reactive or highly volatile agents, are usually derivatised to facilitate chromatography, and to impart properties beneficial for detection. This review focuses on derivatisation reactions used in the chromatographic analysis of chemical warfare agents, their degradation products and metabolites.