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1.
Anal Bioanal Chem ; 406(21): 5195-202, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24633507

ABSTRACT

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.


Subject(s)
Chemical Warfare Agents/analysis , Organophosphorus Compounds/blood , Organothiophosphorus Compounds/blood , Sarin/blood , Soman/blood , Ammonium Compounds , Biotransformation , Buffers , Chemical Warfare Agents/metabolism , Chromatography, High Pressure Liquid/methods , Fluorides , Humans , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Limit of Detection , Quaternary Ammonium Compounds , Solid Phase Extraction , Tandem Mass Spectrometry
2.
J Anal Toxicol ; 32(1): 57-62, 2008.
Article in English | MEDLINE | ID: mdl-18269794

ABSTRACT

A method to detect fluoride ion generated O-ethyl methylphosphonofluoridate (VX-G) in Göttingen minipig red blood cells (RBC) following whole-body exposure to VX vapor utilizing a gas chromatograph-tandem mass spectrometer (GC-MS-MS) has been developed. Dose-response curves for VX exposure were generated after applying the fluoride ion reactivation assay to the RBC fraction of serially collected whole blood samples that were taken after whole-body exposures that varied in both duration and concentration. GC-MS-MS analysis of minipig RBC samples following 180-min exposures at two different concentrations was a more precise indicator for severity of exposure than the analysis of acetylcholinesterase (AChE) inhibition for the same samples. AChE enzyme activity recovered faster than indicated by the apparent elimination rate of VX-G. GC-MS-MS analyses of RBC samples following VX exposure demonstrate this technique has both adequate sensitivity and specificity to indicate the severity of exposure.


Subject(s)
Environmental Monitoring/methods , Erythrocytes/chemistry , Gas Chromatography-Mass Spectrometry/methods , Organothiophosphorus Compounds/blood , Tandem Mass Spectrometry/methods , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Administration, Inhalation , Animals , Biomarkers/blood , Calibration , Chemical Warfare Agents/analysis , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/administration & dosage , Cholinesterase Inhibitors/blood , Cholinesterase Inhibitors/metabolism , Environmental Exposure/analysis , Erythrocytes/enzymology , Fluorides/chemistry , Organothiophosphorus Compounds/administration & dosage , Organothiophosphorus Compounds/metabolism , Potassium Compounds/chemistry , Reproducibility of Results , Solid Phase Extraction/methods , Swine , Swine, Miniature
3.
J Anal Toxicol ; 32(1): 78-85, 2008.
Article in English | MEDLINE | ID: mdl-18269798

ABSTRACT

An analysis method for determining isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA), the metabolic hydrolysis products of toxic organophosphorus nerve agents isopropyl methylphosphonofluoridate (sarin, GB) and cyclohexyl methylphosphonofluoridate (cyclosarin, GF), respectively, has been developed and validated using high-performance liquid chromatography-mass spectrometry with negative ion electrospray ionization with time-of-flight detection (LC-ESI-MS-TOF). The linear range of quantitation was 5 to 125 ng/mL in plasma with a method detection limit of 2 ng/mL for each compound. This method was developed to determine the amount of metabolic hydrolysis that was formed during and after nerve agent exposure in minipigs to account for a major pathway of GB and GF elimination that had not been previously characterized in the bloodstream, particularly during low-level whole-body inhalation experiments. Metabolic hydrolysis accounted for 70% to 90% of the recoverable agent in the bloodstream during exposure, when compared to both unbound and cholinesterase bound agent recovered by fluoride ion reactivation analysis for the same samples. The estimated half-life of IMPA and CMPA in plasma was determined to be 44 and 61 min, respectively. The method utilizes the mass selectivity of LC-ESI-MS-TOF using a bench-top instrument to achieve a detection limit that is consistent with reported LC-MS-MS methods analyzing blood samples.


Subject(s)
Organophosphorus Compounds/blood , Organophosphorus Compounds/metabolism , Sarin/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Administration, Inhalation , Animals , Biomarkers/blood , Chemical Warfare Agents/analysis , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/administration & dosage , Cholinesterase Inhibitors/blood , Cholinesterase Inhibitors/metabolism , Chromatography, Liquid/methods , Environmental Exposure/analysis , Environmental Monitoring/methods , Half-Life , Organophosphorus Compounds/administration & dosage , Reproducibility of Results , Sarin/administration & dosage , Sarin/blood , Solid Phase Extraction/methods , Swine , Swine, Miniature
4.
Inhal Toxicol ; 20(2): 149-56, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18236229

ABSTRACT

The nerve agent O-pinacolyl methylphosphonofluoridate, also known as soman or by its military designation GD, is a highly toxic organophosphorous compound that exerts its effects through inhibition of the enzyme acetylcholinesterase (AChE). In the present study, a fluoride ion based regeneration assay was developed to quantify the level of soman present in the blood of rats following a low-level whole-body inhalation exposure. It was hypothesized that the amount of regenerated nerve agent in the blood would be dose dependent in rats subjected to a whole-body inhalation exposure to a low-level dose of soman vapor, and that the fluoride ion-based regeneration method would be more sensitive for the detection of a low-level exposure to soman vapor than the measurement of whole blood AChE activity. Regenerated soman was dose-dependently detected in both the red blood cells (RBCs) and plasma of exposed rats at all concentrations tested (0.033-0.280 mg/m(3) for a 240-min exposure). Significant inhibition of whole blood AChE activity did not occur below a concentration of 0.101 mg/m(3), and was only depressed by approximately 10-25% at concentrations ranging from 0.101 mg/m(3) to 0.280 mg/m(3). This study is the first to utilize a fluoride ion-based regeneration assay to demonstrate the dose-dependent increases in soman in the blood following whole-body inhalation exposure to low levels of vapor. Additionally, the results of the present study demonstrate that the fluoride ion based regeneration assay was approximately threefold more sensitive than the measurement of AChE activity in the blood for the detection of exposure to soman, and also that miosis is a more sensitive marker of soman exposure than inhibition of AChE activity.


Subject(s)
Biomarkers/blood , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/blood , Enzyme Reactivators/pharmacology , Sodium Fluoride/pharmacology , Soman/blood , Acetylcholinesterase/blood , Administration, Inhalation , Animals , Butyrylcholinesterase/blood , Chemical Warfare Agents/toxicity , Cholinesterase Inhibitors/toxicity , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Erythrocytes/metabolism , Female , Gas Chromatography-Mass Spectrometry , Inhalation Exposure , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Soman/toxicity , Volatilization
5.
Inhal Toxicol ; 18(14): 1091-9, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17050346

ABSTRACT

Male and female rats were whole-body exposed to VX vapor in a 1000-L single-pass exposure chamber. Estimated exposure dosages producing lethal (LCT50) effects in 50% of exposed male and female rats were established for 10, 60, and 240 min exposure durations. A potency comparison with GB and GF shows that VX becomes increasingly more potent than these G agents with increasing exposure duration. VX is approximately 4-30 times more potent than GB and 5-15 times more potent than GF. Gender differences in the estimated median dosages were not significant at the 10, 60, and 240 min exposure durations. An empirical toxic load model was developed and the toxic load exponent for lethality (n) in the equation Cn x T = k was determined to be n = 0.92. The VX-G regeneration assay was successfully used as a biomarker for the presence of VX in the blood plasma and RBC fractions of the blood 24 h postexposure.


Subject(s)
Cholinesterase Inhibitors/toxicity , Organothiophosphorus Compounds/toxicity , Animals , Cholinesterase Inhibitors/chemistry , Dose-Response Relationship, Drug , Female , Lethal Dose 50 , Male , Organophosphorus Compounds/toxicity , Organothiophosphorus Compounds/chemistry , Rats , Rats, Sprague-Dawley , Sarin/toxicity , Sex Characteristics , Volatilization
6.
J Anal Toxicol ; 28(5): 357-63, 2004.
Article in English | MEDLINE | ID: mdl-15239856

ABSTRACT

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.


Subject(s)
Chemical Warfare Agents/analysis , Erythrocytes/chemistry , Fluorides/analysis , Gas Chromatography-Mass Spectrometry/methods , Sarin/analysis , Swine, Miniature , Animals , Chemical Warfare Agents/pharmacokinetics , Chemical Warfare Agents/poisoning , Dose-Response Relationship, Drug , Guinea Pigs , Humans , Inhalation Exposure , Ion Exchange , Isotope Labeling , Rats , Rats, Sprague-Dawley , Sarin/pharmacokinetics , Sarin/poisoning , Spectrometry, Mass, Electrospray Ionization/instrumentation , Spectrometry, Mass, Electrospray Ionization/methods , Swine
7.
J Appl Toxicol ; 20 Suppl 1: S199-204, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11428637

ABSTRACT

Animal models are employed to investigate mechanisms of injury and to evaluate protective measures against sulfur mustard (HD) exposure. The ability to detect and quantify HD enables the researcher to follow safe procedures in handling skin samples. We designed an experimental procedure to measure HD offgassing from animal models. A Minicams--a portable gas chromatograph equipped with a flame photometric detector and on-line sorbent collection and desorption--was used to monitor the HD concentration. Confirming measurements were made using a two-step process that trapped HD on a Tenax sorbent off-line and then transferred the sample by means of an ACEM 900 to a gas chromatograph equipped with either a flame photometric detector or a mass spectrometer. Sulfur mustard offgassing data are presented from three experiments in which weanling pigs were exposed to saturated HD vapor via vapor caps containing 10 microl of HD. The HD concentration was measured in time-weighted-average (TWA) units at a specific HD application site. The current 8-h maximum exposure limit for HD is 3-ng l(-1), (1 TWA unit). The largest TWA value measured near a 3 h time point was a Minicams measurement of 0.48 TWA at 2 h and 53 min after removal of a vapor cap containing HD from a single exposure site on an animal that had 24 concurrent dorsal exposure sites. Gas chromatography/flame photometric detection and gas chromatography/mass spectrometry were used to confirm the Minicams data and to provide greater sensitivity and selectivity down to 0.1 TWA. The gas chromatography/mass spectrometry data confirmed that HD concentrations fell below 0.1 TWA in <5 h for a specific site. These measurements of HD concentrations provided information on the expeditious and safe handling of HD-exposed tissue.


Subject(s)
Dermatologic Agents/pharmacokinetics , Mustard Gas/pharmacokinetics , Animals , Chromatography, Gas , Dermatologic Agents/adverse effects , Dermatologic Agents/analysis , Inhalation Exposure , Male , Mustard Gas/adverse effects , Mustard Gas/analysis , Photometry , Reference Values , Safety , Sensitivity and Specificity , Specimen Handling , Swine , Toxicity Tests , Volatilization
9.
Experientia ; 38(4): 431-3, 1982 Apr 15.
Article in English | MEDLINE | ID: mdl-6806118

ABSTRACT

The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1,1-d2 was synthesized and intramolecular isotope effects determined. The preference of diamine oxidase for the unlabeled alpha position was about 4 times greater than for the deuterated methylene. This work shows that intramolecular deuterium isotope effects are observable in enzyme systems other than cytochrome P-450.


Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Putrescine/metabolism , Animals , Deuterium , Kidney/enzymology , Kinetics , Radioisotope Dilution Technique , Swine
10.
Biomed Mass Spectrom ; 7(11-12): 525-8, 1980 Nov.
Article in English | MEDLINE | ID: mdl-7194699

ABSTRACT

delta 1-Pyrroline is a putrescine metabolite that is biotransformed by rabbit liver preparations to 4-aminobutanoic acid and its lactam, 2-pyrrolidinone. Analysis of dilute aqueous solutions of delta 1-pyrroline by proton nuclear magnetic resonance indicated the the predominating species in the liver incubation preparations was delta 1-pyrroline monomer, although other species, such as 4-aminobutyraldehyde an delta 1-pyrroline timer, may exist in equilibrium with the monomer. [2H12]-delta 1-Pyrroline trimer was synthesized from [2H5]pyrrolidine by conversion to the N-chloro derivative followed by dehydrohalogenation. 4-Aminobutanoic acid was measured by a gas chromatographic mass spectrometric assay after derivatization with dimethylformamide dimethyl acetal. The 4-aminobutanoic acid homologue, 5-aminovaleric acid, served as internal standard. 2-Pyrrolidinone was hydrolyzed and measured as 4-aminobutanoic acid. A comparison of the amounts of product formed following incubation of labeled and unlabeled delta 1-pyrroline indicated a significant isotope effect in the formation of 2-pyrrolidinone. The influence of the label was much less on 4-aminobutanoic acid production. The results suggest that there are two separate pathways involved in the reaction.


Subject(s)
Pyrroles/metabolism , Pyrrolidinones/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Biotransformation , Deuterium , In Vitro Techniques , Liver/metabolism , Mass Spectrometry/methods , Rabbits
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