Liquid chromatography electrospray tandem mass spectrometric and desorption electrospray ionization tandem mass spectrometric analysis of chemical warfare agents in office media typically collected during a forensic investigation.

Most prior analytical studies have dealt with the determination of chemical warfare agents in environmental or biological matrices that would typically be collected following battlefield use or in support of the Chemical Weapons Convention. These methods may be useful for some investigations, but may not be practical for indoor forensic investigations where chemical warfare agent use is suspected. There is a need for analytical methods for chemical warfare agent identification in office media, including flooring, wall surfaces, office fabrics and paper products, which would typically be collected in an office environment during forensic investigations. During this study, typical office environment media were spiked at the 4-20microg/g level with either a complex munitions grade sample of tabun (GA) or with a standard containing the three nerve agents, sarin (GB), cyclohexyl methylphosphonofluoridate (GF), soman (GD) and the nerve agent simulant, triethyl phosphate (TEP), to evaluate the potentials of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for forensic purposes. An emerging technique, desorption electrospray ionization (DESI-MS/MS), was also investigated for the direct determination of TEP, GB and GD sampled onto solid phase microextraction (SPME) fibers exposed to spiked office media. The spiked chemical warfare agents were recovered with varying efficiencies during this study, but in all cases sufficient chemical warfare agent was recovered for mass spectrometric identification purposes. Full high resolution mass spectra were acquired for all the chemical warfare agents in the continuum mode, which typically resulted in mass measurement errors of 0.001Da or less.

Packed capillary liquid chromatography-electrospray ionization (tandem) mass spectrometry of mustard hydrolysis products in soil.

A packed capillary liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and applied to the identification of mustard hydrolysis products in aqueous extracts of soil. In the first application the LC-ESI-MS/MS method was used to identify thiodiglycol and nine longer chain diols in soil samples taken at different locations and depths from a former mustard storage site as part of an ongoing environmental assessment. Aqueous extracts of the soil samples were analysed by LC-ESI-MS/MS using a quadrupole/time-of-flight tandem mass spectrometer operating with a resolution of 9000. High resolution product mass spectra were acquired for thiodiglycol, the hydrolysis product of mustard and nine other sulfur containing diols, including five longer chain diols that could not be identified during prior LC-ESI-MS analyses. The high resolution LC-ESI-MS/MS method was also incorporated into an analytical approach designed to provide rapid chemical warfare agent identification in cases where the chemical and/or biological warfare agent content of a sample is unknown. A sample handling method involving aqueous extraction of the soil sample in biocontainment level 3 (BL-3), followed by autoclave sterilization of the aqueous extract was developed. Once sterilized, the container and aqueous extract can then be safely manipulated outside of BL-3 in the analytical laboratories and may be analysed for the presence or absence of chemical warfare agents, their hydrolysis products or related compounds by LC-ESI-MS/MS.

Packed capillary liquid chromatography-electrospray mass spectrometry of snow contaminated with sarin.

Packed capillary liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) was used for the analysis of a snow sample that was accidentally contaminated with an organophosphorus chemical warfare agent during the destruction of a chemical munition. Sarin, its hydrolysis products and a number of related compounds were identified on the basis of acquired LC-ESI-MS data. Full mass spectra were acquired for 14 compounds, with all exhibiting MH+, [MH+ACN]+ ions and/or protonated dimers that could be used to confirm molecular mass. Sampling cone voltages from 20 to 70 V were utilized with the higher sampling voltages enhancing formation of structurally important product ions in the ESI interface. All data were acquired with a time-of-flight mass spectrometer with a resolution of 5,000 (50% valley definition), a resolution that aided in the assignment of elemental composition of the observed ions. The application of LC-ESI-MS to snow analysis appears to be an attractive alternative to the GC-MS methods, since both chemical warfare agents and their hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.

Determination of sarin, soman and their hydrolysis products in soil by packed capillary liquid chromatography-electrospray mass spectrometry.

An analytical method based on aqueous ultrasonic extraction and packed capillary liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis was developed and compared to an existing gas chromatography(GC)-MS based method for the determination of sarin, soman and their hydrolysis products in contaminated soil. Three soils, a red clay, a tan sandy clay and a brown sandy clay loam, were spiked with sarin and soman and their initial hydrolysis products, isopropyl methylphosphonic acid and pinacolyl methylphosphonic acid, at the 10 microg/g level to assess recovery efficiency. Recovery of sarin and soman from the aqueous soil extracts was comparable to the existing analytical method, with a significant improvement in recovery being demonstrated for the chemical warfare agent hydrolysis products. Sarin and soman were recovered in the 20-90% range from the three soil types with aqueous extraction, while the hydrolysis products of these chemical warfare agents were extracted with recoveries in excess of 80%. The developed soil extraction and analysis method appears to be an attractive alternative to the GC-MS based method, since aqueous extracts containing chemical warfare agent hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.

Packed capillary liquid chromatography-electrospray mass spectrometry analysis of organophosphorus chemical warfare agents.

Packed capillary column liquid chromatography (LC)-electrospray mass spectrometry (ESI-MS) was used for the first time to detect and identify four common organophosphorus chemical warfare agents in aqueous samples. Aqueous samples containing the organophosphorus chemical warfare agents in the 0.01 to 0.1 mg/ml range were analyzed directly by packed capillary LC-ESI-MS with the chemical warfare agents and several minor related impurities being well resolved under acetonitrile-water gradient elution conditions. The ESI-MS data for isopropyl methylphosphonofluoridate (sarin or GB), O-ethyl N,N-dimethylphosphoramidocyanidate (tabun or GA), cyclohexyl methylphosphonofluoridate (GF) and pinacolyl methylphosphonofluoridate (soman or GD) were acquired with a sampling cone voltage setting that promoted collisionally activated dissociation, and resulted in the acquisition of informative mass spectra containing both molecular and product ion information. The developed method appears to be an attractive alternative to GC-MS for the analysis of aqueous samples containing organophosphorus chemical warfare agents and their hydrolysis products, since they may be analyzed directly without the need for additional sample handling.

Analysis of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and its degradation products by packed capillary liquid chromatography-electrospray mass spectrometry.

Packed capillary column liquid chromatography (LC)-electrospray mass spectrometry (ESI-MS) was used for the first time to detect and identify O-ethyl, S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and its degradation products, including compounds containing a P-CH3 bond, bis(diisopropylamino)thioalkanes and ureas commonly employed as VX stabilizers. The reported ESI-MS data were generally acquired with a higher sampling cone voltage, a setting that promoted collisionally activated dissociation, and resulted in the acquisition in informative mass spectra containing both molecular and product ion information. The developed method appears to be an attractive alternative to GC-MS for the analysis of aqueous sample containing the degradation products of VX, since they may be analysed directly with little risk of thermal decomposition and without the need for additional sample handling or derivatization. Application of this method to a degraded VX sample resulted in the detection of a number of novel polar and higher-molecular-mass degradation products, not previously associated with VX during GC-MS analysis.

l-thiocitrulline: A potent protective agent against the toxicity of sulphur mustard in vitro.

Previous studies in this laboratory have shown that the well-characterized arginine analogue nitric oxide synthase (NOS) inhibitor, l-nitroarginine methyl ester (l-NAME) is protective against the cytotoxicity of the vesicating agent bis (2-chloroethyl) sulphide (HD). Furthermore, these protective effects were not mediated through the inhibition of NOS. In studies designed to investigate the efficacy of additional arginine analogue NOS inhibitors as protective agents against this compound, l-thiocitrullline (l-TC) was identified as being extremely potent. In contrast to the protection conferred by l-NAME, however, l-TC was found to protect immature cultures of neurons against HD, as well as mature cultures. In addition, l-TC was found to exert its effects prophylactically, as opposed to the therapeutic characteristics of l-NAME. l-TC gave approximately 800% protection against HD with a one-h pretreatment compared to approximately 1500% protection with a 24-h pretreatment. The protection conferred by l-TC against HD was persistent, and did not require the continued presence of l-TC after the initial HD lesion was expressed. The protective characteristics of l-TC against HD are very different than those of l-NAME and suggest that these closely related arginine analogues act at as yet unidentified and different sites to exert their effects.

Liquid chromatographic-high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization.

Liquid chromatography-high-resolution electrospray mass spectrometry (LC-ESI-MS) was investigated for the identification of known and unknown synthetic peptides in a research effort designed to evaluate the applicability of this and complementary MS techniques for peptide characterization and identification. The monoisotopic molecular masses of five related peptides with molecular masses between 2000 and 2500 u were acquired with a resolution of 3000 (10% valley). Under narrow and wide mass range magnetic sector scanning conditions monoisotopic molecular mass errors were typically in the 10-20 and 30-40 ppm range, respectively. Tryptic maps were generated for each peptide following LC-ESI-MS analysis and collisionally activated dissociation (CAD) in the ESI interface resulted in the production of characteristic product ions that enabled amino acid sequencing of the tryptic fragments. Unknown identification was demonstrated during analysis of an incomplete synthetic peptide reaction mixture. The synthesis of an 18 amino acid peptide, LTTAVKKVLTTGLPALIS, was not successful. In its place were six unknown peptides that were identified on the basis of monoisotopic molecular mass and amino acid sequence data. The monoisotopic molecular masses of these unknowns were determined to within 10-20 ppm with a resolution of 3500 (10% valley). Amino acid sequences for the six peptides were generated during ESI-MS-MS analysis. Finally two synthetic peptides differing only by the incorporation of a 13C at leucine were analysed with a resolution of 6000 (10% valley) to confirm that the isotopic distributions were consistent with theoretical expectations.

Analysis of bioactive peptides by liquid chromatography-high-resolution electrospray mass spectrometry.

LC-high-resolution electrospray ionization (ESI)-MS data for a number of bioactive peptides, including substance P and bradykinins were acquired over a wide mass range by scanning the magnetic sector and calibrating externally with polyethylene glycol standards. Multiply charged ions were observed and errors between observed and theoretical monoisotopic molecular masses were typically in the 5 to 30 ppm range for the peptides during LC-ESI-MS and ESI-MS operation with magnetic sector resolutions between 2500 and 6000 (10% valley definition). Under collisionally activated dissociation conditions bn- and yn-series sequence ions were generally observed, enabling amino acid sequencing and the differentiation of lysine from glutamine, two amino acids differing in residue mass by only 0.0364 u. Mass accuracy was evaluated during an international round robin analytical exercise where the molecular masses of five unknown peptides were to be accurately determined. Isotopic clusters for charge states of up to +6 were fully resolved, facilitating the rapid and unambiguous assignment of charge states and calculation of monoisotopic molecular masses. Errors between theoretical and observed monoisotopic molecular masses were in the 2 to 18 ppm range for the five unknown peptides.

High resolution electrospray mass spectrometry with a magnetic sector instrument: accurate mass measurement and peptide sequencing.

The accurate molecular weights for a series of 37 unknown synthetic peptides, used in research studies involving synthetic vaccines, antibacterial peptides or the de novo design of helical peptides and proteins, were determined with a magnetic sector instrument. All data were obtained with external calibration over a wide mass range during magnetic scanning. Errors between observed and theoretical monoisotopic molecular weights were typically in the 5-60 ppm range for the unknowns at sector resolutions between 2500 and 9000 (10% valley). Isotopic clusters for charge states up to 10+ were resolved through the use of high resolution. Collisionally activated dissociation (CAD) in the electrospray interface resulted in product ions that enabled either full or partial sequencing of most unknown peptides of molecular weights below 2000 Da. The complete primary sequence for one peptide was determined and the importance of high resolution was demonstrated by the differentiation of lysine from glutamine, two amino acids differing in residue mass by only 0.0364 Da. Two other peptides, with identical monoisotopic masses, but different primary sequences, were differentiated based on CAD-MS data.

Bioassay of organophosphate nerve agents in soil using neuronal tissue cultures.

A soil sample originating from an area of suspected chemical warfare activity was subjected to chemical analysis and bioassay. Sarin and several related compounds were confirmed in the soil by capillary column gas chromatography-mass spectrometry (GC-MS); however, the binding of these compounds to the soil hindered quantitation. The chemical results were then compared to those obtained by bioassay in primary cultures of chick embryo forebrain neurons. By comparing the sample's anticholinesterase activity against those of purified standards in chick embryo neuron cultures, a reasonable agreement was found between the chemical and bioassay semi-quantitative estimates of sarin content in the soil extract. Furthermore, the in vitro system appears to offer a sensitive technique for the estimation of sarin remaining bound to the soil following solvent extraction as well as for an assessment of the potential toxicity of the contaminated soil in vivo.

Thermography in breast diagnosis.

The role of two types of thermography in the diagnosis of breast disease was studied in 502 women seen over a two-year period. Thirteen cancers were diagnosed in eleven women. The most significant finding was the large number of equivocal or abnormal thermograms in women with normal breasts of benign disease, while in patients with proven cancer, the thermogram was abnormal in less than half. Clinical diagnosis of breast cancer was not enhanced by either or both types of thermogram. Despite specific criteria, thermographic interpretation was inconsistent except in the thermograms reported as "normal." On the basis of the findings, the authors could not recommend that an abnormal thermogram be used as an indication for mammography, since this would result in an inordinate number of these studies, particularly in young women. The findings suggest that thermography is not a sufficiently precise modality for use in routine breast diagnosis.

Test sequences in screening for breast cancer.

Published data of Stark and Way on the screening of 2,684 women at high risk of breast cancer are analyzed to arrive at a preferred sequence of screening tests. In the practical situation where palpation first signals a problem, the analysis suggests thermography to follow. Women with positive thermograms should then be biopsied, and those with negative thermograms should be mammographed. A positive mammogram calls for biopsy, and a negative one calls for close follow-up. For high-risk women whose breasts appear normal on palpation, a subsequent negative thermogram is not definitive enough to terminate investigation, but a negative mammogram after a negative palpation is enough evidence to waive further investigation for some time. A positive mammogram calls for immediate biopsy in any circumstance.