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.

p-Hexafluoroisopropanol phenyl functionalized graphene for QCM based detection of dimethyl methylphosphonate, a simulant of the nerve agent sarin.

The detection of DMMP (dimethyl methylphosphonate, a simulant of nerve agent sarin) was performed by using p-hexafluoroisopropanol phenyl (HFIPP) functionalized graphene (GR) via hydrogen bond interactions. For this, the HFIPP moiety was covalently functionalized on the surface of GR by a diazo reaction. The HFIPP-GR film-modified QCM electrodes were fabricated and their sensing characteristics towards DMMP were investigated. The proposed sensor showed good response towards sensing DMMP vapor at room temperature. In order to see the effect of HFIPP derivatives on DMMP vapor sensing, a comparative study was also conducted with unfunctionalized graphene. The sensitivity and detection limit of the HFIPP-GR sensor against DMMP vapors were 12.24 Hz ppm-1 and 150 ppb respectively. The HFIPP-GR coated sensors showed good selectivity towards sensing DMMP vapors when compared with common organic vapors.

Electrochemical immunosensor for botulinum neurotoxin type-E using covalently ordered graphene nanosheets modified electrodes and gold nanoparticles-enzyme conjugate.

In this work, a novel electrochemical immunosensor was developed for the detection of botulinum neurotoxin-E (BoNT/E). This method relied on graphene nanosheets-aryldiazonium salt modified glassy carbon electrodes (GCE) as sensing platform and enzyme induced silver nanoparticles (AgNPs) deposited on gold nanoparticles (AuNPs) as signal amplifier. Herein, a GCE was electrografted with mixed monolayer of phenyl and aminophenyl (Ph-PhNH2/GCE) by diazotization reaction. Further, graphene nanosheets (GNS) were covalently attached on electrode surface (GNS/Ph-PhNH2/GCE). Field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize synthesized graphene oxide and modified electrode surfaces. In the sandwich immunoassay format, the sensitivity was amplified using rabbit anti-mouse IgG-alkaline phosphatase (RαMIgG-ALP) functionalized with gold nanoparticles (RαMIgG-ALP/AuNPs). In order to study the immunosensing performance of GNS/Ph-PhNH2/GCE, first the capturing antibody (rabbit-anti BoNT/E antibody) was covalently immobilized via EDC/NHS chemistry. Further, the electrode was sequentially subjected to sample containing spiked BoNT/E, revealing antibody (mouse-anti BoNT/E) followed by RαMIgG-ALP/AuNPs. 3-indoxyl phosphate (3-IP) was used as substrate which finally reduces the silver ions. The deposited AgNPs on electrode surface were determined by linear sweep voltammetry (LSV). The developed electrochemical immunosensor could detect BoNT/E with linear range from 10pg/ml to 10ng/ml with the minimum detection limit of 5.0pg/ml and total analysis time of 65min. In addition, the immunosensor was successfully evaluated against food samples (orange juice and milk).

Relative efficiency of zinc sulfide (ZnS) quantum dots (QDs) based electrochemical and fluorescence immunoassay for the detection of Staphylococcal enterotoxin B (SEB).

In this paper an attempt was made to detect Staphylococcal enterotoxin B (SEB) both by electrochemical and fluorescence immunoassay methods using zinc sulphide (ZnS) QDs. Wet-chemical method was adopted for the preparation of fluorescent ZnS QDs (diameter âˆ¼ 5-10 nm). These QDs were bioconjugated with monoclonal antibodies and then characterized by various method. A detection limit of 0.02 ng mL-1 by fluorescence assay and 1.0 ng mL-1 by electrochemical assay for SEB was achieved. While by sandwich ELISA it is possible to detect 0.24 ng mL-1 only. The sensitivity of all techniques is very good, since the LD50 of SEB is 20 ng kg-1. Electrochemical assay is faster, need low-cost instrument, independent to the size of QDs and found to be one of the best alternative methods as compared to the other existing methods studied herein. The presented method could be expanded to the development of electrochemical and fluorescence biosensors for various agents for field and laboratory use.