Application of microcolumn liquid chromatography and capillary electrophoresis with flame photometric detection for the screening of degradation products of chemical warfare agents in water and soil.

Microcolumn liquid chromatography (microLC) and capillary electrophoresis (CE) coupled on-line with flame photometric detection (FPD) have been used for the screening of polar breakdown products of chemical warfare agents in water and soil samples, provided during Official Proficiency Tests organized by the Technical Secretariat of the Organization for the Prohibition of Chemical Weapons. CE-FPD is shown to be a powerful and rapid method for the determination of alkylphosphonic acids, which are the breakdown products of organophosphorus nerve agents. Gradient elution microLC-FPD is more sensitive and robust but less rapid in the determination of these compounds. In addition, microLC-FPD can be applied to screen for hydrolysis products of sulfur mustard and its analogues. Both methods can be applied without prior derivatization and are extremely selective. In order to unambiguously identify the relevant compounds, electrospray ionization (tandem) mass spectrometry, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry were applied.

Determination of alkylphosphonic acids by microcolumn liquid chromatography with gradient elution coupled on-line with flame photometric detection.

Microcolumn liquid chromatography with gradient elution and on-line flame photometric detection has been used for the selective and rapid determination of non-volatile alkylphosphonic acids, which are hydrolysis products of nerve agents, in aqueous samples. A make-up of water was used to maintain stable introduction of the eluent during a gradient run. The detection limits are strongly dependent on quenching, which is determined by the mass flow of methanol in the eluent. Large-volume injections of 100 microl of aqueous solutions of short- and long-chain alkylphosphonic acids resulted in detection limits in the range 6-800 ng/ml. The repeatability of the retention times and analyte response - peak area or peak height were 0.7-0.9% and 4-11%, respectively. The method was successfully applied to a local tap water sample and an aqueous soil extract.

Determination of the sulfur mustard hydrolysis product thiodiglycol by microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection using large-volume injections and peak compression.

A selective, direct and relatively rapid method has been developed for the determination of thiodiglycol (TDG) in aqueous samples. TDG is the main hydrolysis product of the chemical warfare agent sulfur mustard. The method of analysis is based on the on-line coupling of reversed-phase microcolumn liquid chromatography and sulfur-selective flame photometric detection. To improve sensitivity and efficiency, peak compression by displacement was used in combination with large-volume injections. A concentration of 1% n-propanol was added to the sample to obtain the best sensitivity and efficiency after a 10 microliters injection. Detection limits of 0.25 microgram/ml were achieved with efficiencies of 4.10(5) plates per meter. The method was successfully applied during the Fourth Official Proficiency Test organized by the Technical Secretariat of the Organization for Prohibition of Chemical Weapons for the determination of TDG in a soil sample.

Chromatography and mass spectrometry of chemical warfare agents, toxins and related compounds: state of the art and future prospects.

Methods for the identification of chemical warfare agents, toxins, bioregulators and related products are frequently reported in literature. These methods are often based on instrumental analysis using chromatography (gas and liquid) and mass spectrometry. Here, these instrumental techniques are discussed in several applications, new developments and trends based on a review of the literature published since 1990. Apart from new instrumental developments, it is shown that modern analytical chemistry can be successfully applied to perform identification in the broad field of analytes ranging from chemical to biological warfare agents.

Determination of staphylococcal enterotoxin B by on-line (micro) liquid chromatography-electrospray mass spectrometry.

The use of (micro) liquid chromatography electrospray mass spectrometry (LC-ES-MS) was investigated as potential technique for the determination of the high-molecular-mass protein toxin Staphylococcal Enterotoxin B (SEB). The molecular mass was determined (28,366.3 +/- 1.1) by flow injection analysis and micro-LC-MS with a TSK-gel Phenyl-5PW column packing. Both methods allowed molecular-mass determination of SEB at levels down to 3 pmol/ml. Additional evidence of identification was obtained by detecting the presence of a disulfide bridge by the addition of 2-mercaptoethanol and by tryptic digestion. Collision induced dissociation spectra were recorded from the major tryptic fragments resulting in a sufficient number of sequence ions to allow for the determination of the amino acid sequence. On the analysis of the tryptic digests with C18 reversed-phase columns the use of micro-LC-MS-MS resulted in an 30-40-fold increase in sensitivity as compared with conventional-size LC-MS-MS.

Capillary gas chromatographic analysis of nerve agents using large volume injections.

The use of large volume injections has been studied for the verification of intact organophosphorus chemical warfare agents in water samples. As the use of ethyl acetate caused severe detection problems new potential solvents were evaluated. With the developed procedure, the nerve agents sarin, tabun, soman, DFP and VX can be determined in freshly prepared water samples at ppt levels. Except for the nerve agent tabun all other agents added to the water samples were still present after 8 days at 20-60% levels, if the pH of the water sample is adjusted to ca. 5 shortly after sampling and adjusted to pH 7 for analysis.

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis.

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27 degrees C, maximum T-2 toxin yield (265 and 50 mug/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27 degrees C and slowly at 15 degrees C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15 degrees C (400 mug of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.

High-performance liquid chromatographic separation, isolation and identification of 1,2,3-thiadiazole-5-carboxaldoxime glucuronide in rabbit urine.

Reversed-phase high-performance liquid chromatography was used to separate and isolate the glucuronic acid conjugate of 1,2,3-thiadiazole-5-carboxaldoxime from urine of rabbits after intravenous injection of the oxime. The conjugate was identified by gas chromatography-mass spectrometry as its trimethylsilylated methyl ester and by nuclear magnetic resonance spectrometry. Additional information was obtained from thin-layer chromatography and high-voltage paper electrophoresis.