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).

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.

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.

Efficient Extraction of Sulfur and Nitrogen Mustards from Nonpolar Matrix and an Investigation on Their Sorption Behavior on Silica.

Extraction of vesicant class of chemical warfare agents (CWAs) such as sulfur mustard and nitrogen mustards from the environmental matrices is of prime importance, from a forensic and verification viewpoint of the Chemical Weapons Convention (CWC). For extraction of Convention Related Compounds from nonpolar organic medium, commercially available silica cartridges are used extensively, but silica cartridges exhibit limited efficiency toward vesicant classes of compounds. It is expected that sulfur mustard being nonpolar does not retain sufficiently on silica surface, and nitrogen mustards (being basic) are strongly adsorbed on acidic silica surface, resulting in their poor recoveries. Contrary to the expected higher recovery of sulfur mustard over nitrogen mustards, it was observed that the recovery of sulfur mustard was lower than that of nitrogen mustards with the silica based sorbent. The reason for this typical behavior of these agents on silica was investigated. This study was aimed to develop an analytical method for efficient extraction and enrichment of sulfur and nitrogen mustards from hydrophobic matrices. In this work, the polymeric sorbent was synthesized with polar methacrylic acid (MAA) as monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker and used for solid phase extraction (SPE) of sulfur mustard and nitrogen mustards. The extraction efficiency of the polymeric sorbent was optimized and compared with that of silica cartridges. Both classes of analytes were recovered in good amounts from the polymeric sorbent compared to silica. The extraction parameters were optimized for the proposed method which included extraction solvent ethyl acetate and washing solvent n-hexane (1 mL). The recoveries of the analytes ranged from 75 to 87% with relative standard deviations (RSDs) lower than 9%. The limit of detection (LOD) was found to be in the range of 0.075-0.150 µg mL-1, and limit of quantification (LOQ) was >0.25 µg mL-1. The linear dynamic range of optimized method was found to be 0.50-20 µg mL-1 ( r2 = 0.9994) for sulfur mustard and 0.25-20 µg mL-1 ( r2 = 0.9897-0.9987) for nitrogen mustards, respectively.

Polymeric Sorbent with Controlled Surface Polarity: An Alternate for Solid-Phase Extraction of Nerve Agents and Their Markers from Organic Matrix.

Extraction and identification of lethal nerve agents and their markers in complex organic background have a prime importance from the forensic and verification viewpoint of the Chemical Weapons Convention (CWC). Liquid-liquid extraction with acetonitrile and commercially available solid phase silica cartridges are extensively used for this purpose. Silica cartridges exhibit limited applicability for relatively polar analytes, and acetonitrile extraction shows limited efficacy toward relatively nonpolar analytes. The present study describes the synthesis of polymeric sorbents with tunable surface polarity, their application as a solid-phase extraction (SPE) material against nerve agents and their polar as well as nonpolar markers from nonpolar organic matrices. In comparison with the acetonitrile extraction and commercial silica cartridges, the new sorbent showed better extraction efficiency toward analytes of varying polarity. The extraction parameters were optimized for the proposed method, which included ethyl acetate as an extraction solvent and n-hexane as a washing solvent. Under optimized conditions, method linearity ranged from 0.10 to 10 µg mL-1 ( r2 = 0.9327-0.9988) for organophosphorus esters and 0.05-20 µg mL-1 ( r2 = 0.9976-0.9991) for nerve agents. Limits of detection (S:N = 3:1) in the SIM mode were found in the range of 0.03-0.075 µg mL-1 for organophosphorus esters and 0.015-0.025 µg mL-1 for nerve agents. Limits of quantification (S:N = 10:1) were found in the range of 0.100-0.25 µg mL-1 for organophosphorus esters and 0.05-0.100 µg mL-1 for nerve agents in the SIM mode. The recoveries of the nerve agents and their markers ranged from 90.0 to 98.0% and 75.0 to 95.0% respectively. The repeatability and reproducibility (with relative standard deviations (RSDs) %) for organophosphorus esters were found in the range of 1.35-8.61% and 2.30-9.25% respectively. For nerve agents, the repeatability range from 1.00 to 7.75% and reproducibility were found in the range of 2.17-6.90%.