Gas chromatography-tandem mass spectrometry-based detection of half nitrogen mustards in plasma as a new biomarker of nitrogen mustard exposure.

The development and optimization of an analytical method for the detection and identification of reactive metabolite of organochlorine chemical warfare agent nitrogen mustards (NMs), 2-[(2-chloroethyl)(alkyl)amino]ethanol (CEAAE), known as half nitrogen mustard, in blood samples is presented, herein. In this study, half nitrogen mustards in plasma are presented as a new and unambiguous biomarker of NM exposure since the fully hydrolyzed product, i.e., amino alcohols, are common industrial chemicals that can be present as such without getting exposed to NMs. Thus, the detection of half nitrogen mustard as a biomarker holds great significance for verification by the Chemical Weapon Convention (CWC) and will also be helpful in understanding the pharmacokinetics of NM-based chemotherapeutic pro-drugs. To the best of our knowledge, this is the first report on the detection of half nitrogen mustards in any matrice, including plasma. A very simple sample preparation protocol was developed for its extraction from plasma samples. Heptafluorobutyrylation and gas chromatography-tandem mass spectrometry in the positive chemical ionization mode were developed for the detection and identification of halfNMs. The developed method has shown excellent analytical figures of merits such as a wide range of linearity (1.0-50 ng mL-1), low limit of detection (0.3-0.5 ng mL-1), and low limit of quantification (1.0 ng mL-1). The interday and intraday reproducibilities were also less than 15%. The developed method was successfully applied to real-world samples; in vitro human plasma was spiked with ∼1 ng mL-1 of all the NMs and in vivo studies were done with rats intravenously exposed to 1 × LD50 of bis(2-chloroethyl)methylamine (HN2).

Quantitative Proteomic Changes after Organophosphorous Nerve Agent Exposure in the Rat Hippocampus.

The widespread use of organophosphorous (OP) compounds and recent misuse of nerve agents on civilians requires an urgent need to decode their complex biological response to develop effective drugs. Proteomic profiling of biological target tissues helps in identification of molecular toxicity mechanisms. Quantitative proteomics profiling of the rat hippocampus was studied in this study. Liquid chromatography mass spectrometry (LC-MS) analysis of tandem mass tag (TMT)-labeled lysates identified 6356 proteins. A total of 69, 61, and 77 proteins were upregulated, and 66, 35, and 70 proteins were downregulated at 30 min, 1 day, and 7 days after soman exposure. This is the first report on the soman-induced proteomic changes to the best of our knowledge. Bioinformatics analysis revealed soman-induced broad-range proteomic changes in key pathways related to glutamate, acetylcholine, GABA, 5-hydroxytryptamine, and adrenergic receptors, G-protein signaling, chemokine and cytokine-mediated inflammation, cytoskeleton, neurodegeneration (Parkinson's and Alzheimer's), Wnt signaling, synaptic vesicle trafficking, MAP kinases, proteosome degradation, metabolism, and cell death. Selected protein changes were verified by immunoblotting, and neuropathological findings indicated significant brain damage. Results demonstrate that persistent proteomic changes in the brain can cause multiple neurological effects through cholinergic and non-cholinergic pathways, and these mechanistic insights are useful in the development of novel drugs.

Mass spectral fragmentation of perfluoroacyl derivatives of half nitrogen mustards for their detection by gas chromatography/mass spectrometry.

RATIONALE: Analytical methods for the detection and identification of half nitrogen mustards (halfNMs), i.e., partially hydrolyzed products of nitrogen mustards (pHpNMs), using silyl derivatives are often associated with low sensitivity and selectivity. In order to overcome these limitations, the derivatization of halfNMs was performed using perfluoroacylation. METHODS: Two efficient derivatization techniques using trifluoroacetyl (TFA) and heptafluorobutyryl (HFB) groups were developed for the unambiguous identification of halfNMs. A mass spectral database was generated by performing gas chromatography/electron ionization mass spectrometry (GC/EI-MS) and gas chromatography/positive chemical ionization mass spectrometry (GC/PCI-MS). The fragmentation pathways were studied by tandem mass spectrometry (MS/MS) in both EI and PCI mode. RESULTS: The EI-MS spectra of the TFA and HFB derivatives of halfNMs contain intense molecular ions and fragment ions, thus making perfluoroacylation preferable to silylation. In addition, the background-free chromatogram obtained using these derivatives provides unambiguous identification of these compounds in blind samples. The structures of the fragment ions were postulated, and the sources of significant ions were traced by performing MS/MS precursor ion scans. In the PCI-MS spectra, along with the protonated molecule, significant peaks due to neutral losses of HF, HCl, CH3 Cl and CF3 COOH were observed. CONCLUSIONS: This is the first report of the elucidation of the fragmentation pathways of perfluoroacyl derivatives of halfNMs. The complementary GC/PCI-MS and GC/PCI-MS/MS data will be helpful in the identification of unknown metabolites in a fast and reliable fashion.

Mass spectral studies of silyl derivatives of partially hydrolyzed products of nitrogen mustards: Important markers of nitrogen mustard exposure.

RATIONALE: Nitrogen mustards (NMs) are vesicant class of chemical warfare agents. From the viewpoint of the Chemical Weapons Convention partially hydrolyzed products of nitrogen mustards (pHpNMs) are considered as important markers of nitrogen mustard exposure. The detection of pHpNMs from biological or environmental samples is highly useful for obtaining forensic evidence of exposure to NMs. METHODS: Gas chromatography interfaced with tandem mass spectrometry (GC/MS/MS) is a widely used tool for the identification and sensitive detection of metabolites of NMs in complex matrices. The pHpNMs were derivatized using silylating agents as they are highly polar and non-amenable to GC. The mass spectral studies of these silyl derivatives of pHpNMs were performed using GC/MS/MS in both electron ionization (EI) and chemical ionization (CI) mode. RESULTS: Various approaches have been proposed to assess the fragmentation pathways of the trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives of pHpNMs. All the proposed fragmentation pathways were based on the product and/or precursor ion scanning of corresponding ions in both EI and CI mode. In the case of EI, most of the fragmentation pathways involved either α-cleavage or inductive cleavage. CONCLUSIONS: This is the first report on the MS study of the silyl derivatives of pHpNMs. The study of the two different derivatives of pHpNMs using both EI- and CI-MS provides a reliable, unambiguous identification of pHpNMs in complex environmental and biomedical matrices (such as plasma and urine) during any verification activities.

Comparative evaluation of an improved test method for bioefficacy of insecticidal fabrics against dengue and malaria vectors.

BACKGROUND: Insecticidal fabrics are important personal protective measures against mosquitoes, ticks and other disease vectors. In the absence of internationally accepted guidelines, bioefficacy tests have been carried out using continuous exposure and three minutes exposure bioassay methods. Recently, we have reported an improved method for bioefficacy testing of insecticidal fabrics, which involves continuous exposure of mosquitoes to the test fabrics. The present paper reports the comparative evaluation of the outcomes of the continuous exposure bioassay and the three minutes bioassay on the same fabric samples. METHODS: Permethrin content in the treated fabric samples was determined through HPLC analysis and NMR studies were performed to establish the stability of the analyte. Bioefficacy tests were carried out against dengue vector Aedes aegypti and malaria vector Anopheles stephensi as per the improved test method and the three minutes bioassay method. RESULTS: The permethrin doses in the fabric samples ranged from 60 to 3000 mg/m2 and 36.2% of permethrin was retained after 10 washings. The extraction and chromatographic analysis were not found to affect the stability of permethrin. In continuous exposure, all fabric samples showed bioefficacy, as the mean complete knockdown time for both Ae. aegypti (10.5-34.5 min) and An. stephensi (14.5-36.8 min) was ≤ 71.5 min. The same samples were found to be not effective when tested using the three minutes bioassay method, since the knockdown and mortality percentages were well below the required bioefficacy values. The bioefficacy of the fabric samples in terms of complete knockdown time was significantly higher against Ae. aegypti in comparison to An. stephensi. The mean complete knockdown time of Ae. aegypti increased to 48.3 min after 10 washings indicating a significant reduction in bioefficacy. CONCLUSIONS: Bioefficacy testing of the insecticidal fabrics using the improved method resulted in outcomes, which could be correlated better with the permethrin content in the fabric samples. The improved method is more appropriate for the testing of insecticidal fabrics than the three minutes bioassay method. Further evaluation of the improved method using different test arthropods could help in the formulation of specific guidelines for the bioefficacy testing of insecticidal fabrics.

Epigenetic and autophagic changes after nerve agent exposure in the rat piriform cortex and hippocampus.

The detrimental effects of organophosphate (OP) nerve agents have been reported but the mechanisms mediating these multiple effects are not well understood. Recent use of nerve agents in Syria and the UK illustrate their continuous threat to the modern world. Epigenetic and autophagy studies are useful to address the issues related to regulation of gene and protein expression by which nerve agents could impact on human health. These studies help to understand molecular mechanisms underlying the multiple neurotoxic effects of nerve agents. In the present study, changes in epigenetic (global DNA methylation and histone acetylation) and autophagic marker proteins were studied in the nerve agent sensitive rat brain areas (piriform cortex and hippocampus) after soman (1xLD50) exposure. Global DNA methylation analysis revealed that nerve agent induced hypomethylation in the brain regions at 1 and 7 days post exposure. In contrast, DNA hypermethylation was observed at 30 days post soman exposure, demonstrating a possible compensatory mechanism. Western blot analysis showed significant increase in the histone acetylation levels after soman exposure in the piriform cortex and hippocampus. The present study observed the changes in autophagic proteins of nerve agent poisoning for the first time to the best of our knowledge. Immunoreactivity levels of autophagic proteins (LC3-II, ATG-5 and p62) were transiently increased in the rat piriform cortex and hippocampus after soman exposure. In conclusion, this study provides insight into the epigenetic and autophagic changes in the brain following soman exposure and their possible role in the neuronal damage and development of multiple neurological effects.

Triazine-Based Covalent Organic Framework: A Promising Sorbent for Efficient Elimination of the Hydrocarbon Backgrounds of Organic Sample for GC-MS and 1H NMR Analysis of Chemical Weapons Convention Related Compounds.

The strict monitoring and precise measurements of chemical warfare agents (CWAs) in environmental and other complex samples with high accuracy have great practical significance from the forensic and Chemical Weapons Convention (CWC) verification point of view. Therefore, this study was aimed to develop an efficient extraction and enrichment method for identification and quantification of toxic agents, especially with high sensitivity and multidetection ability in complex samples. It is the first study on solid-phase extraction (SPE) of CWAs and their related compounds from hydrocarbon backgrounds using covalent triazine-based frameworks (CTFs). This nitrogen-rich CTF sorbent has shown an excellent SPE performance toward sample cleanup by selective elimination of hydrocarbon backgrounds and enrich the CWC related analytes in comparison with the conventional and other reported methods. The best enrichment of the analytes was found with the washing solvent (1 mL of n-hexane) and the extraction solvent (1 mL of dichloromethane). Under the optimized conditions, the SPE method had good linearity in the concentration range of 0.050-10.0 µg mL-1 for organophosphorus esters, 0.040-20.0 µg mL-1 for nerve agents, and 0.200-20.0 µg mL-1 for mustards with correlation coefficients ( r2) between 0.9867 and 0.9998 for all analytes. Limits of detection ( S/ N = 3:1) in the SIM mode were found to be in the range of 0.015-0.050 µg mL-1 for organophosphorus esters, 0.010-0.030 µg mL-1 for nerve agents, and 0.050-0.100 µg mL-1 for blister agents. Limits of quantification ( S/ N = 10:1) were found in the range of 0.050-0.200 µg mL-1 for organophosphorus esters, 0.040-0.100 µg mL-1 for nerve agents, and 0.180-0.350 µg mL-1 for blister agents in the SIM mode. The recoveries of all analytes ranged from 87 to 100% with the relative standard deviations ranging from 1 to 8%. This method was also successfully applied for the sample preparation of 1H NMR analysis of sulfur and nitrogen mustards in the presence of hydrocarbon backgrounds. Therefore, this SPE method provides the single sample preparation for both NMR and GC-MS analyses.

Adductomics: a promising tool for the verification of chemical warfare agents' exposures in biological samples.

Humans are constantly exposed to a wide range of reactive and toxic chemicals from the different sources in everyday life. Identification of the exposed chemical helps in the detection and understanding the exposure associated adverse health effects. Covalent adducts of proteins and DNA formed after xenobiotics exposure may serve as readily measurable indicators of these exposures. Measuring the exposed chemicals with focus on adducts resulting from the nucleophilic interactions with blood proteins is useful in the development of diagnostic markers. Particularly, the most abundant proteins such as albumin and hemoglobin acts as dominant scavengers for many reactive chemicals in blood and can serve as excellent diagnostic candidates to determine the type of chemical exposure. This review focuses on the potential application of an adductomics approach for the screening of bimolecular adducts of chemical warfare agents (CWAs). Recent incidents of CWAs use in Syria, Malaysia, and the UK illustrate the continuing threat of chemical warfare agents in the modern world. Detection of CWAs and their metabolites in blood or in other body fluids of victims depends on immediate access to victims. Concentrations of intact CWAs in body fluids of surviving victims may decline rapidly within a few days. Certain CWAs, particularly nerve agents and vesicants, form covalent bonds with certain amino acids to form CWA-protein adducts. Proteins that are abundant in the blood, including albumin and hemoglobin, may carry these adducts longer after the original exposure. We searched MEDLINE and ISI Web of Science databases using the key terms "adductomics" "adducts of CWAs," "CWAs adducts detection in the biological samples," "protein adducts of CWAs," alone and in combination with the keywords "detection" "intoxication" "exposure" "adverse effects" and "toxicity." We also included non-peer-reviewed sources such as text books, relevant newspaper reports, and applicable Internet resources. We screened bibliographies of identified articles for additional relevant studies including non-indexed reports. These searches produced 1931 citations of which only relevant and nonduplicate citations were considered for this review. The analysis of biomedical samples has several purposes including detecting and identifying the type of chemical agent exposed, understanding the biological mechanism, assists in giving adequate treatment, determining the cause of death and providing evidence in a court of justice for forensic investigations. Rapid advances in the mass spectrometry to acquire high-quality data with greater resolution enabled the analysis of protein and DNA adducts of xenobiotics including CWAs and place the rapidly advancing 'adductomics' next to the other "-omics" technologies. Adductomics can serve as a powerful bioanalytical tool for the verification of CWAs exposure. This review mostly describes the protein adducts for nerve agents and vesicants, outlines the procedures for measuring adducts, and suggests the evolving (or future) use of adducts in the detection and verification of CWAs.

Neuroprotective Effects of Galantamine on Nerve Agent-Induced Neuroglial and Biochemical Changes.

Neuroprotection from nerve agent such as soman-induced neural damage is a major challenge for existing drugs. Nerve agent exposure can cause many neural effects in survivors arising mainly due to acetylcholinesterase (AChE) inhibition or death within minutes. Unraveling the mechanisms underlying the nerve agent-induced multiple neurological effects is useful to develop better and safe drugs. The present study aimed to understand the molecular response during soman exposure and to evaluate the neuroprotective efficacy of galantamine on nerve agent-induced neurotoxic changes. mRNA expression studies using quantitative real-time PCR revealed significant changes in S-100ß, Gfap, c-fos, and Bdnf in the hippocampus and piriform cortex after soman (90 µg/kg, s.c) exposure. Immunoblot analysis showed acute soman exposure significantly increased the protein levels of neuroglial markers (S100-ß and GFAP); c-Fos and protein oxidation in discrete rat brain areas indicate their role in nerve agent-induced neurotoxicity. Induction of BDNF levels during soman exposure may indicate the recovery mechanisms activation. AChE was inhibited in the blood and brain up to 82% after soman exposure. Antidotal treatment with galantamine alone (3 mg/kg) and galantamine plus atropine (10 mg/kg) has protected animals from nerve agent-induced intoxication, death, and soman-inhibited AChE up to 45% in the blood and brain. Animal received galantamine displayed increased levels of neuroprotective genes (nAChRα-7, Bcl-2, and Bdnf) in the brain suggest the neuroprotective value of galantamine. Neuroglial changes, c-Fos, and protein oxidation levels significantly reduced after galantamine and galantamine plus atropine treatment indicate their potential antidotal value in nerve agent treatment.

Time-dependent comparative evaluation of some important biomarkers of acute cyanide poisoning in rats: an aid in diagnosis.

OBJECTIVE: The study focuses on time-dependent comparative evaluation of various biomarkers of acute cyanide poisoning in rats. METHODS: Blood gas (analyzer), lactate, pyruvate, cyanide, thiocyanate (spectrophotometer) and 2-amino-2-thiazoline-4-carboxylic acid (ATCA; gas chromatography-mass spectrometry) in plasma or urine, and various physiological parameters (polygraph) were measured. RESULTS: Cyanide poisoning was characterized by elevated lactate, cyanide, thiocyanate and ATCA concentrations in plasma up to 15 min, 4, 16 and 24 h, respectively, while high urinary thiocyanate and ATCA levels were measured between 4 and 24 h. CONCLUSION: ATCA concentration in plasma and urine was found to be more reliable indicator of cyanide poisoning.

Gas chromatography-full scan mass spectrometry determination of traces of chemical warfare agents and their impurities in air samples by inlet based thermal desorption of sorbent tubes.

A sensitive gas chromatography-mass spectrometry (GC-MS) based analytical method was developed for detection of the chemical warfare agents (CWA) and related compounds in air/vapor samples. The method uses a Tenax TA packed GC liner as an air/vapor sampling tube and Programmable Temperature-Vaporization (PTV) GC inlet as the thermal desorber. This approach eliminates secondary focusing step and allows transfer of desorbed analytes as sharp bands directly to the head of GC column. Use of a Peltier element for rapid cooling eliminates need for an external coolant. Minimal logistic and hardware needs make the method relatively inexpensive and especially suitable for a mobile laboratory. The limits of detection (LODs) of 0.8-2.9ng on tube for selected nerve and blister agents were achieved in the full scan MS mode. Simple derivatization method applied for detection of Lewisites 1 and 2 did not affect simultaneous analysis of other agents. The method was extensively evaluated with authentic CWA during the field trainings of the inspectors from the Organization for the Prohibition of Chemical Weapons (OPCW). The environmental area and personal samples were collected for a semi-quantitative determination of averaged airborne CWA concentration levels.

Dispersive derivatization liquid-liquid extraction of degradation products/precursors of mustards and V-agents from aqueous samples.

A new derivatization and extraction technique termed as dispersive derivatization liquid-liquid extraction (DDLLE) speeds up the analysis process by removing the requirement for drying of the sample. The derivatization process takes place at the interface between the analyte containing aqueous phase and derivatization agent laden organic phase. The organic phase is highly dispersed using disperser solvent so that the total surface area is large. The derivatizing agent used is 1-(heptafluorobutyryl)imidazole and the resulting heptafluorobutyryl (HFB) derivatized analytes are partitioned into the organic phase. In addition to reduced sample preparation time, for some of the analytes, the HFB derivatives provide better spectral differentiation between isomers than conventional trimethylsilyl (TMS) derivatives. Method parameters for the DDLLE, such as extraction, and disperser solvent and their volume, type and amount of base, amount of heptafluorobutyrylimidazole and extraction time were optimized on diisopropylaminoethanol (DiPAE), ethyldiethanolamine (EDEA), triethanolamine (TEA) and thiodiglycol (TDG). The DDLLE was also used on various real world samples, which also includes few OPCW organized proficiency test and a spiked urine sample. The observed limit of detection (LOD) with 1mL of sample for DDLLE in full scan with AMDIS was 10ng/mL and with methane chemical ionization, multiple reaction monitoring (MRM) was 100pg/mL, i.e., 100fg on-column.

Thermally assisted methylation and subsequent silylation of scheduled acids of chemical weapon convention for on-site analysis and its comparison with the other methods of methylation.

On-site verification of the chemical weapon convention (CWC) requires provision for the detection and identification of alkyl phosphonic acids as well as some organic acids that are amenable to GC-MS only after derivatisation. Various derivatisation methods have been used for the identification of these acids and for many cases the methyl derivatives are less prone to artifacts possibly leading to false positive identification. Methylation with diazomethane is widely used but, especially for on-site analysis it has limitation due to the potential explosive and health hazards. Other methylation procedures like trimethylsilyldiazomethane (TMSD), thermally assisted methylation (TAM) by trimethylphenylammonium hydroxide (TMPAH) and trimethylsulfonium hydroxide (TMSH) are evaluated. Data for methylation for the alkyl alkylphosphonic acids, alkylphosphonic acids and benzilic acid are reported. In addition, TAM followed by the silylation in the same sample without any additional sample preparation is also reported. Several parameters such as solvent, temperature, amount of reagents, time, etc. were studied. The two commercially available reagents namely, TMPAH and TMSH for TAM and subsequent silylation were evaluated. The LOD with TMPAH was below 0.5 ng per injection since all of the acids were detected by GC-MS with the S/N of >3 in full scan mode by AMDIS and their inter day relative standard deviation was from 4.7% to 10.8%.

Fragmentation energy index for universalization of fragmentation energy in ion trap mass spectrometers for the analysis of chemical weapon convention related chemicals by atmospheric pressure ionization-tandem mass spectrometry analysis.

The use of mass spectra generated at 70 eV in electron ionization (EI) as a universal standard for EI has helped in the generation of searchable library databases and had a profound influence on the analytical applications of gas chromatography/mass spectrometry (GC/MS), similarly for liquid chromatography tandem mass spectrometry (LC-MS/MS), suggesting a novel method to normalize the collisional energy for the universalization of fragmentation energy for the analysis of Chemical Weapon Convention (CWC)-related chemicals by atmospheric pressure ionization-tandem mass spectrometry (API-MS(n)) using three-dimensional (3D) ion trap instruments. For normalizing fragmentation energy a "fragmentation energy index" (FEI) is proposed which is an arbitrary scale based on the fact of specific MS/MS fragmentation obtained at different collisional energies for the reference chemicals which are not CWC scheduled compounds. FEI 6 for the generation of an MS(n) library-searchable mass spectral database is recommended.

Electrospray ionization tandem mass spectral analysis of oxidation products of precursors of sulfur mustards.

Electrospray ionization tandem mass spectral (ESI-MSn) analysis of thiodiglycol, bis(2-hydroxyethylthio)alkanes (BHETAs) and their mono-, di-, tri-, and tetraoxygenated compounds was carried out to obtain their characteristic spectra for ESI-MS analysis. These compounds are important markers of chemical warfare agents, namely sulfur mustards. ESI-MSn (n > or = 3) analysis of a compound by collisionally induced dissociation in an ion trap gives rise to mass spectra that are somewhat similar to electron ionization mass spectra. These ESI-MSn spectra can be used for compound identification. Under ESI-MS and ESI-MS/MS the compounds mostly produced [M+NH4]+, [M+H]+ and [M+H--H2O]+ ions. Fragmentations of these even-electron precursors in the ion trap gave rise to characteristic product ions via neutral loss of O2, H2O, C2H4, HCHO, C2H4O, C2H4S, HSC2H4OH and C2H4SO. Fragmentation routes of these compounds are proposed that rationalize the formation of product ions in ESI-MSn analysis.

Clostridium schirmacherense sp. nov., an obligately anaerobic, proteolytic, psychrophilic bacterium isolated from lake sediment of Schirmacher Oasis, Antarctica.

A novel obligately anaerobic, proteolytic bacterium, designated AP15T, was isolated from lake sediments of Schirmacher Oasis, Antarctica. The bacterium produced maximum cell mass between 5 and 10 degrees C in an anaerobic basal medium containing 0.5 % tryptone and peptone. The strain grew optimally at a pH around 8.0 and tolerated NaCl up to a concentration of 7.5 %. It contained diphosphatidylglycerol as the major phospholipid and C(15 : 0), C(16 : 0) and C(17 : 0) as the major cellular fatty acids. Several amino acids, including arginine, leucine, isoleucine, cysteine, glutamate and serine, supported growth. Glutamate was degraded to acetate, propionate, CO2 and H2. In addition, the strain degraded carbohydrates including glucose, raffinose, adonitol, ribose and rhamnose. The main fermentation products during growth on glucose were H2, CO2, formate, acetate, propionate and isovalerate. The DNA G+C content of the bacterium was 24 mol%. On the basis of a phylogenetic analysis, strain AP15(T) is identified as a close relative of Clostridium subterminale ATCC 25774T, with which it shares 99.5 % similarity at the 16S rRNA gene sequence level; however, it exhibits a low DNA-DNA binding value (55 %) to this strain at the whole-genome level. In addition to showing other major differences with respect to C. subterminale and other members of the genus Clostridium, AP15T also exhibits phenotypic differences. On the basis of these differences, strain AP15T is identified as representing a novel species of the genus Clostridium, for which the name Clostridium schirmacherense sp. nov. is proposed. The type strain is AP15T (=DSM 17394T = JCM 13289T).

Microemulsion mediated in situ derivatization-extraction and gas chromatography-mass spectrometric analysis of alkylphosphonic acids.

Detection and identification of environmental signatures of chemical warfare agents is an important aspect of verification program of Chemical Weapons Convention (CWC). Alkylphosphonic acids (APAs) are ultimate and persistent degradation products of nerve agents. Their identification in a sample submitted for off-site analysis infers possible indication of contamination with nerve agents. This paper describes the development of a new sample preparation method which involves 'in situ derivatization and extraction' (INDEX) of acids from water. Derivatization is performed by alkylation of APAs with alkylbromides in surfactant less microemulsion (SLME). The derivatized analytes were analyzed by gas chromatography coupled with mass spectrometry. The developed method involves simultaneous derivatization (alkylation) and extraction of acidic analytes mediated by surfactant less microemulsion. Various derivatization-extraction parameters such as solvent, reaction time and temperature, base and alkyl bromides were optimized. Pentyl bromide in the presence of potassium carbonate and diisopropylamine at 100 degrees C derivatized the selected acids efficiently. Kinetic data for alkylation of methylphosphonic acids and some carboxylic acids were obtained to assess their relative susceptibility for alkylation in microemulsion. Methylphosphonic acid and isopropyl methylphosphonic acid took 140-150 min to reach completion while carboxylic acids took 100 min to complete the reaction. INDEX could be successfully performed even in the presence of interfering Ca(2+) and Mg(2+) ions.

Hollow fiber-mediated liquid-phase microextraction of chemical warfare agents from water.

Unambiguous detection and identification of chemical warfare agents (CWAs) and related compounds are of paramount importance from verification point of view of Chemical Weapons Convention (CWC). It requires development of fast, reliable, simple and reproducible sample preparation of CWAs from water which is likely to be contaminated during deliberate or inadvertent spread of CWAs. This work describes development of hollow fiber liquid-phase microextraction (HF-LPME) method for efficient extraction of CWAs (such as sarin, sulfur mustard and their analogues) from water followed by gas chromatography-mass spectrometric analysis. Extraction parameters, such as organic solvent, agitation, extraction time, and salt concentration were optimized. Best recoveries of target analytes were achieved using 1 microL trichloroethylene as extracting solvent, 1000 rpm stirring rate, 15 min extraction time, and 30% NaCl. Excellent precision was observed with less than 7.6% RSD. The limit of detection by HF-LPME was achieved up to 0.1 microg/L at 30% salt concentration.

Gas chromatography/mass spectrometric analysis of methyl esters of N,N-dialkylaminoethane-2-sulfonic acids for verification of the Chemical Weapons Convention.

This paper describes the synthesis and gas chromatography/electron ionization mass spectrometric (GC/EI-MS) analysis of methyl esters of N,N-dialkylaminoethane-2-sulfonic acids (DAESAs). These sulfonic acids are important environmental signatures of nerve agent VX and its toxic analogues, hence GC/EI-MS analysis of their methyl esters is of paramount importance for verification of the Chemical Weapons Convention. DAESAs were prepared by condensation of 2-bromoethane sulfonic acid with dialkylamines, and by condensation of dialkylaminoethyl chloride with sodium bisulfite. GC/EI-MS analysis of methyl esters of DAESAs yielded mass spectra; based on these spectra, generalized fragmentation routes are proposed that rationalize most of the characteristic ions.