Multiple-technique analytical characterization of a mixture containing chemical-weapons simulant from a munition.

An amber yellow organic liquid was found in a munition shell at Dugway Proving Grounds, UT, USA, that was likely used as a simulant of chemical weapons. The primary analytical techniques to characterize the mixture were gas chromatography-infrared detection-mass spectral detection (GC-IR-MS); liquid chromatography-mass spectrometry (LC-MS); nuclear magnetic resonance (NMR) using the nuclei 1H, 13C and 31P; and gas chromatography-atomic emission detection (GC-AED). Six major phosphorus-containing components were identified and confirmed by at least three techniques, and several additional phosphorus-containing components of lower concentration have been identified by GC-IR-MS and LC-MS. Five major non-phosphorus components, including ethyl acetate, diethyl sulfide and dibutylamine, have been identified by multiple techniques. The major phosphorus compound (23.9+/-0.4 wt.%) was O,O,O-triethyl phosphorothioate (I) and the second most abundant (14.4+/-0.2 wt.%) was O,O,S-triethyl phosphorothioate (III). No VX, G-agent, or pesticide was observed in the sample, although III may be a cholinesterase inhibitor which produces delayed toxic response. III also produces a false hit for the pesticide cyanthoate when analyzed by GC-MS-EI. The mixture appears to have been formulated as a chemical warfare agent simulant, most likely as a challenge of agent detection techniques.

Detection of the chemical warfare agents bis-(2-chloroethyl)ethylamine (HN-1) and tris-(2-chloroethyl)amine (HN-3) in air.

This paper describes the method development and validation for detection of the chemical warfare agents HN-1 and HN-3 in air using C8 solid-phase extraction disks followed by liquid desorption and analysis by gas chromatography. The method is contrasted to the standard approach which uses solid sorbent tubes followed by thermal desorption and analysis by gas chromatography.

Analysis of chemical weapons degradation products by capillary electrophoresis with UV detection.

CE with UV detection is investigated for the separation and detection of 27 chemical weapons (CW) degradation products. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separation techniques are used with direct and indirect UV detection. Optimal buffer systems and instrumental conditions are established for a representative collection of chemical warfare degradation compounds. Anionic, cationic, zwitterionic, and neutral components are determined with either direct UV absorbance or indirect UV displacement. Sodium dodecyl sulfate is used as the micelle-forming surfactant, and phenylphosphonic acid is used as the background electrolyte in borate buffer solutions. This evaluation is designed to demonstrate the applicability of CE as an analytical method in CW detection.

Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta.

The phosphotriesterase from Pseudomonas diminuta was tested as a catalyst for the hydrolysis of phosphofluoridates. The purified enzyme has been shown to hydrolyze the phosphorus-fluorine bond of diisopropyl fluorophosphate, isopropyl methylphosphonofluoridate, and 1,2,2-trimethylpropylmethylphosphonofluoridate at pH 7.0, 25 degrees C, with turnover numbers of 41, 56, and 5 s-1, respectively. The enzymatic rate enhancement for the hydrolysis of sarin at pH 7.0 is 2.2 X 10(7). The turnover number for paraoxon hydrolysis is 2100 s-1. The enzyme does not hydrolyze methanesulfonyl fluoride, phenylmethylsulfonyl fluoride, or O-p-nitrophenyl phenylsulfonate nor do these compounds inactivate or inhibit the ability of the enzyme to hydrolyze diethyl p-nitrophenyl phosphate. The breadth of substrate utility and the efficiency of the hydrolytic reaction exceed the more limited abilities of other prokaryotic and eukaryotic enzymes that catalyze similar reactions. The substantial rate enhancement exhibited by this enzyme for the hydrolysis of a wide variety of organophosphorus nerve agents make this enzyme the prime candidate for the biological detoxification of insecticide and mammalian acetylcholinesterase inhibitors.

Seasonal frequency of ciguatoxic barracuda in southwest Puerto Rico.

Ciguatoxicity of barracuda (Sphyraena barracuda) head, viscera and flesh tissues has been determined in 219 specimens caught along the southwest coast of Puerto Rico from March 1985 through May 1987. Twenty-nine percent of these specimens were toxic. Monthly frequencies of ciguatoxic barracuda showed an apparent seasonal variability, with peak values (60-70% toxic fish) in the late winter-early spring (January-May) and fall (August-November). Minimal frequencies (0-10% toxic fish) were observed during June-July and December. The most frequently toxic tissues in poisonous animals were the viscera and head. Viscera tissue was the only toxic tissue found in 31% of the poisonous fish assayed, and this tissue was poisonous in all toxic fish. In no case was a poisonous specimen found to have toxic flesh alone. Marked temporal variation in frequency of ciguatoxicity suggests that ciguatera toxins, at least in their active form, are not accumulated in barracuda tissues for extended periods of time. Variability in barracuda ciguatoxicity may reflect fluctuations in the toxicity of smaller reef fish prey, seasonal fluctuations in toxic benthic dinoflagellates and/or changes in the ability of the barracuda to detoxify ingested poisons or their precursors.

Organofluorophosphate-hydrolyzing activity in an estuarine clam, Rangia cuneata.

1. The bivalve Rangia cuneata can enzymatically detoxify the organophosphorus acetylcholinesterase inhibitors DFP and soman. 2. Digestive gland homogenates contained Mazur-type DFPases based on response to Mn2+ ions, and relative rates of DFP: soman hydrolysis. Squid-type DFPase contributed little to the total organophosphate acid (OPA) anhydrase activity of these preparations. 3. The natural substrate(s) and physiological role(s) of OPA anhydrase in R. cuneata has yet to be determined; however, DFPase specific activity was pronounced in the digestive gland, the primary organ involved in bioconcentration and biotransformation of xenobiotics, and in the gills, which are in continuous contact with water-borne chemicals.

Discovery of multiple organofluorophosphate hydrolyzing activities in the protozoan Tetrahymena thermophila.

Recently it has been found that homogenates of Tetrahymena thermophila can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoridate (soman). Upon purification of the DFP hydrolyzing activity 10-fold it had been noted that the soman hydrolyzing activity increased only 2-3 fold. Treatment with manganous ion and comparison of the soman and DFP hydrolysis rates of the homogenate indicated that a mixture of the squid-type and Mazur-type DFPases may be present. Subsequent purification of the enzymatic activities within the Tetrahymena-homogenate demonstrated that there are at least five functioning proteins of molecular weights 67,000 to 96,000. None are directly homologous to the DFPases found in hog kidney or squid. The enzymatic activities are designated DFPase-1 through DFPase-5. A hypothesis is presented that the functions of DFPases are in the normal metabolism of organophosphates naturally synthesized by T. thermophila.

Lysis of red blood cells by extracts from benthic dinoflagellates.

Samples of the cultured benthic dinoflagellates Gambierdiscus toxicus and Ostreopsis lenticularis, both isolated from a shallow back reef habitat in southwestern Puerto Rico, were extracted in methanol, dried and resuspended in distilled water. After centrifugation, aliquots of the supernatant, or dilutions thereof, were added to suspensions of washed human and mouse red blood cells and incubated at different temperatures for different time periods. Further spectrophotometrical examinations of the samples showed a hemolytic activity against mouse and human red blood cells. The hemolytic activity of G. toxicus extract was 3 to 4 times greater than that of O. lenticularis and was less temperature-dependent. Such findings suggest that these two dinoflagellates produce chemically different hemolysins.

Liquid chromatographic separation and detection of nanogram quantities of biologically important dicarboxylic acids.

Phenacyl and benzyl derivatives of some biologically significant dicarboxylic acids have been prepared in a quantitative manner using a crown ether catalyst. They were separated using reversed-phase chromatography. The column used was packed with Corasil II to which a C9 phase (nonyl) was bonded. Water-methanol (68:32) was used as the mobile phase. Detection limits in the range of 5--15 ng have been obtained for a non-optimized system. The ease of preparation of these derivatives, coupled with their excellent chromatographic properties, makes this a very attractive procedure for the investigation of mixtures containing biologically significant acids. The implications and future of this technique are discussed.