Unambiguous identification of γ-aminobutyric acid adducts as novel plant biomarkers and their ultra-sensitive detection by UPLC-MS/MS for retrospective analyses of nitrogen mustards exposure.

Plants are widely existing in the environments and have been considered as potential sentinel species of toxic chemicals' exposure. In this study, the deadly toxic chemicals of three nitrogen mustards (NMs, including NH1, NH2 and NH3) were selected as the investigated targets. First, the reactivities of common endogenous plant components with NMs were examined in vitro. Then, the model plant Nicotiana benthamiana Domin was exposed to NMs. Three γ-aminobutyric acid-nitrogen mustard adducts (GABA-NMs) were identified in the living plant by high resolution mass spectrometry and comparison with the synthesized references. A sensitive detection method with the limits of quantification of 0.0500 ng mL-1 was developed using ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry. The GABA-NMs could be detected after 120 days of the exposure and even in the dead leaves without obvious decrease. Furthermore, 20 different plant species grown in diverse climate zones were exposed to HN1, and the adduct of GABA-HN1 was identified in all the leaves. The results showed the good universality and specificity of GABA-NMs as plant biomarkers for NMs exposure. This work provides a new approach for the pollution investigation of toxic chemicals through analysing biomarkers in plant materials.

Qualitative Analysis of Novel Flavonoid Adducts from Nerve Agent Tabun-Exposed Arabidopsis thaliana (L.) Based on Quadrupole-Time of Flight Mass Spectrometry.

Flavonoids are a kind of secondary metabolite which widely exist in plants. They contain a lot of active hydroxyls, which can react with toxic chemicals to produce potential exposure biomarkers. In this article, the model plant Arabidopsis thaliana (L.) was exposed to the nerve agent O-Ethyl N,N-dimethyl phosphoramidocyanidate (Tabun). By comparing with the plant not exposed to Tabun, some characteristic ions were identified by quadrupole-time of flight mass spectrometry in the acetonitrile extract of the exposed leaves. These characteristic ions were selected as parent ions to produce product ion mass spectra (PIMS). Some interesting fragmentation pathways were revealed, including neutral loss of glucoside, rhamnose and ethylene. O-Ethyl N,N-dimethyl phosphoryl modified flavonoids were deduced from assignment of the PIMS. The element components and the accurate mass of the product ions from each parent ion matched well with those of the proposed fragmentation pathways. Through comparison with the PIMS of structurally closely related chemical of Isobutyl methylphosphonyl modified flavonoids, the structures and the fragmentation pathways of the O-Ethyl N,N-dimethyl phosphoryl modified flavonoids were finally confirmed. Successfully finding and identifying these three specific exposure biomarkers in plants provided a new strategy for the retrospective analysis of organophosphorus exposure and forensic analysis.

Identification of four novel flavonoid adducts in Arabidopsis thaliana (L.) exposed to isobutyl S-2-diethylaminoethyl methylphosphonothiolate as potential plant exposure biomarkers.

As vegetation is part of our lives, plants are good candidates as indicators of toxic chemicals. Numerous components in plants may react with toxic chemicals to produce exposure biomarkers. Plant biomarkers formed by the modification of endogenous plant components by chemical warfare agents have not been reported. In this article, the model plant Arabidopsis thaliana (L.) was exposed to the nerve agent isobutyl S-2-diethylaminoethyl methylphosphonothiolate (iBuVX). Some characteristic ions were identified by liquid chromatography-high resolution mass spectrometry and their product ion mass spectra were recorded and interpreted. Some interesting fragmentation pathways were revealed including neutral loss of glucoside, rhamnose and isobutylene. Isobutyl methylphosphonyl modified flavonoids were deduced from assignment of product ions. The element components and the accurate mass of the product ions matched well with those of the proposed fragmentation pathways. The binding site of the nerve agent on flavonoids was proved to be the hydroxyl group on the benzene ring of the flavonoids by density functional theory computation and by the synthesis of the reference chemical, which was confirmed by 1H-31P HMBC NMR. The phosphonyl-modified flavonoids were evaluated for specificity in different plants. Four new flavonoid adducts as potential biomarkers were identified in the leaves of the iBuVX-exposed plant, which provided a novel strategy for the retrospective analysis of organophosphorus exposure for chemical weapon verification and forensic analysis.

Sensitive and selective detections of mustard gas and its analogues by 4-mercaptocoumarins as fluorescent chemosensors in both solutions and gas phase.

Mustard gas has been used as a chemical warfare agent for a century, and is the most likely chemical weapon used in wars or by terrorists. Thus, it is important to develop a facile, rapid and highly selective method for the detection of mustard gas. In this paper, two fluorescent probe molecules, 4-mercaptocoumarins, have been developed for rapid and sensitive detections of SM and its analogues (CEES and NH1) in both solutions and gas phase. The sensing reaction is a nucleophilic addition at three-membered hetercyclic sulfonium/ammonium formed from SM, CEES/NH1 in ethanol. Two fluorescent probes (4-mercaptocoumarins, ArSH) in ethanol deprotonate to form thiophenol anions (ArS-) resulting from their low pKa values (3.2-3.4), and the nucleophilic addition of the anion ArS- generates the corresponding thioethers, giving a turn-on fluorescence response. The thiophenol anion can fast sense SM, CEES and NH1 (within 1-4 min) with high sensitivity (~nM level) at 60 °C, and high selectivity through adding a tertiary amine, and two probes exhibit excellent chemical and photostability in detection systems. Furthermore, a facile test strip with the sensor was fabricated for the detection of CEES vapor with rapid response (3 min), high sensitivity (9 ppb) and high selectivity.

Fluoride reactivation-enabled sensitive quantification of tabun adducts on human serum albumin by GC-MS/MS via isotope dilution.

Organophosphorus nerve agents inhibit the cholinesterase activity by phosphylation of the active site serine. The resulting phosphylated cholinesterase and adducts on human serum albumin (HSA) are appropriate biomarkers for nerve agents exposure. Several methods have been developed for the detection of nerve agents, including fluoride reactivation or alkaline cleavage. It was previously thought that some nerve agents adducts to HSA could not be detected via fluoride regeneration. In our study, the results showed that tabun (GA) adducts of HSA could be detected by fluoride regeneration. The sample preparation included acetone precipitation, washing and SPE. Deuterated tabun (d5-GA) was applied as the internal standard. The product of regenerated fluorotabun is detected with a good linearity (R2 > 0.997) in the concentration range from 0.02 to 100.0 ng/ml, small relative standard deviation (≤6.89%) and favorable recoveries between 94.8 and 106.3%. The established preparation confirmed the fluorotabun was regenerated from the GA-HSA adducts.