Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information AbstractThe cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d 2 ), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra-and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.

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This paper reviews milestones in antidotal therapies for cyanide (CN) spanning early remedies, current antidotal systems and research towards next generation therapies. CN has been a part of plant defense mechanisms for millions of years. It became industrially important in the nineteenth century with the advent of CN assisted gold mining and the use of CN as a pest control agent. The biochemical basis of CN poisoning was actively studied and key mechanisms were understood as early as 1929. These fundamental studies led to a variety of antidotes, including indirect CN binders that generate methemoglobin, direct CN binders such as hydroxocobalamin, and sulfur donors that convert CN to the less toxic thiocyanate. Research on blood gases at the end of the twentieth century shed new light on the role of nitric oxide (NO) in the body. The discovery of NO's ability to compete with CN for enzymatic binding sites provided a previously missed explanation for the rapid efficacy of NO generating antidotes such as the nitrites. Presently used CN therapies include: methemoglobin/NO generators (e.g., sodium nitrite, amyl nitrite, and dimethyl aminophenol), sulfur donors (e.g., sodium thiosulfate and glutathione), and direct binding agents [(e.g., hydroxocobalamin and dicobalt salt of ethylenediaminetetraacetic acid (dicobalt edetate)]. A strong effort is being made to explore novel antidotal systems and to formulate them for rapid administration at the point of intoxication in mass casualty scenarios. New antidotes, formulations, and delivery systems are enhancing bioavailability and efficacy and hold promise for a new generation of improved CN countermeasures.

Cyanide causes toxic effects by inhibiting cytochrome c oxidase, resulting in cellular hypoxia and cytotoxic anoxia, and can eventually lead to death. Cyanide exposure can be verified by direct analysis of cyanide concentrations or analyzing its metabolites, including thiocyanate (SCN(-)) and 2-amino-2-thiazoline-4-carboxylic acid (ATCA) in blood. To determine the behavior of these markers following cyanide exposure, a toxicokinetics study was performed in three animal models: (i) rats (250-300 g), (ii) rabbits (3.5-4.2 kg) and (iii) swine (47-54 kg). Cyanide reached a maximum in blood and declined rapidly in each animal model as it was absorbed, distributed, metabolized and eliminated. Thiocyanate concentrations rose more slowly as cyanide was enzymatically converted to SCN(-). Concentrations of ATCA did not rise significantly above the baseline in the rat model, but rose quickly in rabbits (up to a 40-fold increase) and swine (up to a 3-fold increase) and then fell rapidly, generally following the relative behavior of cyanide. Rats were administered cyanide subcutaneously and the apparent half-life (t1/2) was determined to be 1,510 min. Rabbits were administered cyanide intravenously and the t1/2 was determined to be 177 min. Swine were administered cyanide intravenously and the t1/2 was determined to be 26.9 min. The SCN(-) t1/2 in rats was 3,010 min, but was not calculated in rabbits and swine because SCN(-) concentrations did not reach a maximum. The t1/2 of ATCA was 40.7 and 13.9 min in rabbits and swine, respectively, while it could not be determined in rats with confidence. The current study suggests that cyanide exposure may be verified shortly after exposure by determining significantly elevated cyanide and SCN(-) in each animal model and ATCA may be used when the ATCA detoxification pathway is significant.

An analytical method utilizing chemical ionization gas chromatography-mass spectrometry was developed for the simultaneous determination of cyanide and thiocyanate in plasma. Sample preparation for this analysis required essentially one-step by combining the reaction of cyanide and thiocyanate with pentafluorobenzyl bromide and simultaneous extraction of the product into ethyl acetate facilitated by a phase-transfer catalyst, tetrabutylammonium sulfate. The limits of detection for cyanide and thiocyanate were 1 μM and 50 nM, respectively. The linear dynamic range was from 10 μM to 20 mM for cyanide and from 500 nM to 200 μM for thiocyanate with correlation coefficients higher than 0.999 for both cyanide and thiocyanate. The precision, as measured by %RSD, was below 9 %, and the accuracy was within 15 % of the nominal concentration for all quality control standards analyzed. The gross recoveries of cyanide and thiocyanate from plasma were over 90 %. Using this method, the toxicokinetic behavior of cyanide and thiocyanate in swine plasma was assessed following cyanide exposure.