Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.

RATIONALE: Nitrogen mustards (NMs) are blistering chemical warfare agents. The ability to detect NMs in environmental samples is very important for obtaining forensic evidence. The most common analytical techniques for NM detection are gas chromatography-mass spectrometry, which detects NMs in their intact form but is disadvantaged by high limits of detection (LODs), and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) of their hydrolysis products, which do not provide robust evidence to support NM use. METHODS: We developed a novel approach to detect and identify NMs using LC/ESI-MS/MS after chemical derivatization. The method is based on ethoxide-promoted ethanolysis prior to analysis. The effects of reaction time, temperature, ethoxide concentration and chromatography behavior were studied and optimized. In the developed procedure, 0.1% (v/v) sodium ethoxide solution is added to the NMs in ethanol and agitated for 2 h at 50°C, followed by LC/ESI-MS/MS, without any other pretreatment. RESULTS: The ethanolysis reaction efficiencies were evaluated in ethanolic extracts from soil, asphalt, and ethanol contaminated with 0.5% (v/v) diesel fortified with NMs at a five-point calibration curve. The calibration curves showed good linearity in the range of 0.05-1 ng/mL, with an R2 value of 0.99, and were similar to those of LC/MS-grade ethanol, with almost no observable matrix effects. The derivatization products were stable at room temperature, with LODs of 10 pg/mL, in all investigated extracts. CONCLUSIONS: Through this newly developed strategy, the derivatization of active NMs by ethanolysis was achieved for the first time, and these derivatization products can serve as specific indicators for the use and presence of NMs. The methodology can also verify trace levels of NM chemical warfare agents collected in war or terror scenarios in forensic investigations.

Rapid and simple identification of trace amounts of sodium azide in beverages and bodily fluids followed by derivatization and liquid chromatography-electrospray ionization tandem mass spectrometry.

RATIONALE: Sodium azide (NaN3 ) is a toxic chemical agent to humans by ingestion and inhalation with a growing number of intentional exposures and accidental cases over the last few decades. Due to its low molecular weight and lack of any chromophore, its retention and detection by reverse-phase liquid chromatography-ultraviolet-mass spectrometry methods are a challenging task. METHODS: To be able to confirm azide exposure, we have developed a method to identify azide in both beverages and bodily fluids. The identification of azide (N3 - ) is based on derivatization with N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) at 25°C for 15 min followed by LC/ESI-MS/MS analysis, with no other sample preparation. RESULTS: The azide after derivatization (CAX-N3 ) was stable, retainable by LC and sensitively detected by selected reaction monitoring. The ESI-MS/MS fragmentation of the M+ precursor ion produced characteristic product ions at m/z 118, 100, 91 and 86. The calibration curves for CAX-N3 showed linearity over two orders of magnitude with R2 value of 0.99. Low limits of identification of 0.1-0.5 ng/mL were obtained in all investigated matrices (drinking water, tea, orange juice, plasma and urine). CONCLUSIONS: Compared with previously reported chromatography-based methods, this method that was based on derivatization and LC/ESI-MS/MS analysis was substantially more sensitive, simpler and faster. The method can be used for forensic investigation to confirm azide exposure from fatal use to much smaller intoxication dose.

Enhanced LC-ESI-MS/MS Sensitivity by Cationic Derivatization of Organophosphorus Acids.

The chemical derivatization to enhance the signal intensity and signal-to-noise (S/N) of several organophosphorus (OP) acids in liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) is illustrated. The OP class of compounds represents the environmental degradants of OP nerve agents and pesticides. N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) was utilized to derivatize a panel of eight acids consisting of five alkyl methylphosphonic acids (ethyl-, isopropyl-, isobutyl-, cyclohexyl-, and pinacolyl-methylphosphonic acid) along with three dialkylphosphate analogs (diethyl-, dibutyl-, and diethyl thio-phosphate). The derivatization reaction with CAX-B was conducted in acetonitrile in the presence of potassium carbonate at 70 °C for 1 h. The resulting acid derivatives were analyzed with an LC-Orbitrap-ESI-MS/MS, and their dissociation processes were investigated. It was found that the derivatization procedure increased the limits of identification (LOIs) by one to over two orders of magnitude from the range of 1 to 10 ng/mL for the intact OP-acids to the range of 0.02-0.2 ng/mL for the derivatized acids utilizing an LC-MS(QqQ) in MRM mode, regardless of the sample matrix (hair, concrete, or plant extracts). The interpretation of the corresponding ESI-MS/MS spectra for each type of derivatized sub-OP family revealed the formation of characteristic neutral losses and a characteristic ion for the organophosphorus core. This derivatization is beneficial and useful for screening and identifying target and "unknown" OP acids.

Oxidative derivatization of V-type nerve agents as a tool for their structural elucidation by liquid chromatography/tandem mass spectrometry.

RATIONALE: The identification of V-type nerve agents poses an analytical challenge. Their spectra obtained by electron ionization mass spectrometry (EI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) are dominated by ions originating from the N,N-dialkylaminoethyl moiety, while ions representative of the alkyl phosphonothiolate part are absent from the spectra or present at negligible abundance. Hence, analogs or isomers with the same amine residue exhibit similar mass spectral patterns, leading to unavoidable ambiguity in their identification. METHODS: Chemical derivatization was utilized for the structural elucidation of a series of five V-type nerve agents, including O-ethyl S-(2-diisopropylamino)ethyl methylalkyl phosphonothiolate (VX), O-isobutyl S-(2-diethylamino)ethyl methylalkyl phosphonothiolate (RVX) and O-ethyl S-(2-diethylamino)ethyl methylalkyl phosphonothiolate (VM). The procedure consisted of "in-vial" oxidation of the tertiary amine group with 3-chloroperbenzoic acid (m-CPBA) at ambient temperature followed by liquid chromatography (LC)/Orbitrap-ESI-MS/MS analysis with no other sample preparation. RESULTS: The generated N-oxide of the V-type nerve agents altered the charge distribution occurring during fragmentation and produced informative ESI-MS/MS spectra characteristic of the alkyl phosphonothiolate structure, enabling a higher degree of certainty in their identification. Moreover, two VX isomers possessing an identical tertiary amine moiety that coeluted at practically the same retention time and displayed high mass spectral similarity were easily differentiated, and their structures elucidated once derivatized. CONCLUSIONS: In contrast to the ESI-MS/MS spectra of the V-type nerve agents, which exhibited mostly/only information on the amine-containing residue, the ESI-MS/MS spectra of the V-type nerve agent N-oxides revealed ions indicative of both the alkyl phosphonothiolate and the amine parts, enabling their reliable structural elucidation.

Extended retrospective detection of regenerated sarin (GB) in rabbit blood and the IMPA metabolite in urine: a pharmacokinetics study.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70 days for 0.1 LD50 and 0.5 LD50 and 77 days for 0.9 LD50, where rapid elimination occurred in the first 8 days with an initial average half-life of 1.2 days, followed by a second, slower elimination, with a terminal average half-life of 8.4 days. The time frame for monitoring IMPA in urine was 7, 15 and 16 days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8 days on days 2-6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4 days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

A novel approach for the detection and identification of sulfur mustard using liquid chromatography-electrospray ionization-tandem mass spectrometry based on its selective oxidation to sulfur mustard monoxide with N-iodosuccinimide.

A new derivatization strategy for the detection and identification of sulfur mustard (HD) via liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is developed. The method incorporates selective oxidation of the sulfide group by the electrophilic iodine reagent N-iodosuccinimide (NIS) to produce sulfur mustard monoxide (HDSO). The derivatization reaction efficiencies were evaluated with acetonitrile extracts of soil, asphalt, cloth, Formica, and linoleum spiked with HD at concentrations of 50-5000 pg/ml and found to be similar to that with pure acetonitrile. The current derivatization approach is the first to preserve the identity of chloride groups and support HD regulation and evidentiary findings.

Structural elucidation of V-type nerve agents by liquid chromatography/electrospray ionization mass spectrometry.

V-nerve agents present information-poor spectra, both in GC-EI-MS and LC-ESI-MS/MS, with dominant fragments/product ions corresponding to the amine-containing residue. Hence, derivatives/isomers with the same amine residue exhibit similar mass spectral patterns, leading to ambiguity in the phosphonate structure. We present a simple approach for their structural elucidation based on two complementary experiments: ESI-MS/MS of the original compound, which provides information about the amine moiety, and ESI-MS/MS of the phosphonic acid hydrolysis products generated by N-iodosuccinimide, which provides ions' characteristic of the phosphonate structure. This approach enables the structural elucidation of the original V-agents with a higher degree of certainty.

Highly sensitive retrospective determination of organophosphorous nerve agent biomarkers in human urine implemented in vivo in rabbit.

Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO2 cartridge was accomplished with a 1% ammonium hydroxide (NH4OH) solution and was followed by analysis via liquid chromatography-mass spectrometry (LC-MS). The limits of quantitation (LOQs) were in the range of 10-100 pg/mL with recoveries of 64-71% (± 5-19%) after fast sample preparation and a total LC-MS analysis cycle time of 15 min and 13 min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD50 (7.5 µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15 days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC-MS/MS analysis.

Retrospective determination of regenerated nerve agent sarin in human blood by liquid chromatography-mass spectrometry and in vivo implementation in rabbit.

The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography-mass spectrometry (LC-MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC-ESI-MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5-1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC-MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC-MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC-MS/MS trace analysis of regenerated GB from biological matrices.

Identification of G-nerve agents at picogram levels from complex organic samples containing hydrocarbon interferences by aqueous extraction, followed by derivatization and liquid chromatography-mass spectrometry analysis.

The chromatograms obtained from the gas chromatography-electron ionization mass spectrometric (GC-EI-MS) analysis of extracts containing G-nerve agents in the presence of diesel, gasoline, etc., are dominated by hydrocarbon backgrounds that "mask" the G-nerve agents, leading to severe difficulties in identification. This paper presents a practical solution for this challenge by transferring the G-nerve agents from the organic phase into the aqueous phase using liquid-liquid extraction (LLE), followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP), allowing ultrasensitive LC-ESI-MS/MS analysis of the G-derivatives. The proposed approach enables rapid identification of trace amounts of G-nerve agents with limits of identification (LOIs) at the pg/mL scale.

Is rivastigmine safe as pretreatment against nerve agents poisoning? A pharmacological, physiological and cognitive assessment in healthy young adult volunteers.

Rivastigmine, a reversible cholinesterase inhibitor, approved as a remedy in Alzheimer's disease, was suggested as pretreatment against nerve agents poisoning. We evaluated the pharmacokinetic, pharmacodynamic, physiologic, cognitive and emotional effects of repeated rivastigmine in young healthy male adults, in a double blind, placebo controlled crossover trial. Three groups completed 3 treatment periods: 0, 1.5 and 3mg twice a day, for a total of 5 intakes. Parameters monitored were: vital signs, ECG, laboratory tests, sialometry, visual accommodation, inspiratory peak flow, and cognitive function tests. Adverse reactions were mild. Peak blood levels and peak cholinesterase inhibition increased with repeated intakes, and high variability and non-linear pharmacokinetics were demonstrated. In addition, two cognitive functions were affected (perceptual speed and dynamic tracking). The complicated pharmacological profile and the high inter-personal variability limit the potential use of rivastigmine as pretreatment for war fighters and first responders.