Retrospective Analyses Of Plant Samples Exposed To Typical Organophosphorus Agents

Organophosphorus nerve agents（OPNAs）are a kind of chemical agents with high toxicity,high efficiency and high lethality properties which are listed as Schedule IA toxic chemicals in the Chemical Weapons Convention（CWC）and have been controlled strictly by Organisation for the Prohibition of Chemical Weapons（OPCW）.In recent years,the OPCW and military chemists around the world have paid great attention to the search and discovery of plant biomarkers of chemical exposure.Through the detection of specific biomarkers which were produced by the reaction of specific plant components and OPNAs and through the monitoring of morphological changes of plants,retrospective analysis of organophosphorus exposure can be realized.This has become a new direction of investigation and research on alleged use of chemical weapons.In this paper,the well-known nerve agents of RVX、GA、GB were studied to search and identify specific plant biomarkers.The model plant of Arabidopsis thaliana（L.）and the widely cultivated Tagetes erecta Linn.were exposed to toxicants by matrix exposure as well as by living plant exposure.The general chemical components of flavonoids in plants were screened and 7 novel plant exposure biomarkers of OPNAs modified flavonoids were found.The structures of the flavonoid adducts were deduced by assignments of the HRMS PIMS and unambiguously identified through theoretical computation combining with the reference synthesis.The quantification approach was developed with isotope dilution UPLC-MS/MS（MRM）strategy for the biomarkers of phosphonyl/phosphoryl modified flavonoids in plants.In addition,the kinetics in RVX and GB exposed plants was studied by quantitively monitoring the phosphonyl-flavonoid and the free metabolite.The work could provide novel strategies and tools for retrospective analysis in the fields of CWC verification and chemical forensics.The main research contents and innovative findings are as follows:1.Finding and identifying of novel flavonoid adducts from OPNA exposed plantsThe model plants Arabidopsis thaliana（L.）and the widely distributed Tagetes erecta Linn were cultivated for the study.The exposure pathways were examined taking into account of exposure time and the pretreatment procedure of the plant samples was also determined.RVX and GA were selected as the typical OPNAs to expose the plants.Both exposed and non-exposed plants were analyzed by LC-HRMS.Seven novel flavonoid adducts were screened and the possible structures of the flavonoid adducts were deduced through the assignment of the PIMS.In the RVX exposed Arabidopsis thaliana（L.）,the endogenous flavonoid compounds kaempferol 3-O-rhamnose-glucose-7-O-rhamnoside（flavonoid compound（1））,kaempferol 3-O-glucose-7-O-rhamnoside（flavonoid compound（2））and kaempferol 3-O-rhamnose-7-O-rhamnoside（flavonoid compound（3））were isobutyl methylphosphonyl modified to form the corresponding RVX-flavonoid adducts（4）、（5）、（6）and the di-isobutyl methylphosphonyl modified RVX-flavonoid adduct（7）.In the RVX exposed Tagetes erecta Linn.RVX-flavonoid adduct（6）were detected.Using the same screening method,three GA-flavonoid adducts（8）,（9）,（10）were found in GA exposed Arabidopsis thaliana（L.）and GA-flavonoid adduct（10）was found in GA exposed Tagetes erecta Linn.Density functional theory was used to predict the phosphonyl modified sites.Reference chemicals were synthesized to confirm the structure of the seven novel flavonoid adducts.Interference was examined with leaf samples from different plants and no interference peak in the corresponding retention time windows of the flavonoid adducts,which indicated that the toxic chemical-flavonoid adducts could be used as plant biomarkers for the retrospective analysis of organophosphorus exposure.The successful discovery and accurate identification of RVX and GA exposure biomarkers in plants provide a new strategy for the retrospective analysis of organophosphorus exposure.2.Establishment of UPLC-MS/MS（MRM）method for plant biomarkersThe most abundant RVX-flavonoid adduct（6）and GA-flavonoid adduct（10）after exposure to RVX and GA were set as detection targets,and Arabidopsis thaliana（L.）was used as sample matrix to establish a quantitative analysis method.The isotope-labeled RVX-flavonoid adduct（11）and isotope-labeled GA-flavonoid adduct（12）were synthesized to be used as the internal standard for quantitative analysis.The targeted quantitative analysis conditions of MRM were optimized,and a quantitative method of isotope dilution mass spectrometry for toxic chemical-adduct was constructed.The recoveries and matrix effects of RVX-flavonoid adduct（6）in different matrix were investigated by using acetonitrile,acetone,methanol and water as extraction solvents.A simple sample preparation method based on acetonitrile extraction was established.At the same time,the specificity,sensitivity,accuracy,precision of the quantitative method and stability of RVX-flavonoid adduct（6）in plant substrates were systematically investigated.In the acetonitrile extract of Arabidopsis thaliana（L）,the linear ranges for RVX-flavonoid adduct（6）was 0.2～120 ng/m L,the detection limit of qualification and quantification is 0.02 ng/m L and 0.04 ng/m L,respectively,the accuracy of the method was 92.0～102.9%,the relative standard deviations of the intra-day and inter-day precision were 0.4～4.5%and 0.9～4.5%,respectively.The established quantitative analysis method of acetonitrile extraction combined with isotope dilution UPLC-MS/MS（MRM）is simple,sensitive and reliable,which provided an effective means for retrospective analysis of organophosphorus agents and kinetic study of organophosphorus agents.3.The kinetic study of typical organophosphorus agents exposed plantsThe extract solvent of plant and living plant were exposed to RVX with different concentrations.The kinetics of typical organophosphorus nerve agent exposed plants was studied.RVX-flavonoid adduct（6）was used as the exposure marker,and isotope-labeled biomarker（11）was used as the internal standard to quantitatively monitor the content of exposure marker at different time after exposure.Biomarkers could be detected in both the living plants and extract solvent of plants exposed with 200 ng RVX one day after the exposure.The content of biomarker in the extract solvent increased with time and the biomarker could still be detected in the exposed living plants after 31 days of exposure.The results showed that after exposure to the nerve agent RVX,plant markers accumulated in plants and retained for a long time window,which provided a possibility for the long-term retrospective analysis of organophosphorus agents based on plants.4.Study on the transformation and distribution of typical organophosphorus agents in plantsThe widely distributed Pisum sativum Linn.was exposed to isopropyl methylphosphonofluoridate（GB）by two different means,namely,one-time immersion and continuous exposure at different concentrations.The transformation and transfer of GB in Pisum sativum Linn.were studied by LC-MS/MS technique.Results shows:In Pisum sativum Linn.,GB existed in the form of the hydrolysate of isopropyl methylphosphonate（IMPA）.In the one-time immersed Pisum sativum Linn.,the IMPA accumulated in the roots and stems first,as time went by,the contents in the roots and stems decreased gradually while the contents in the leaves increased.In the continuous exposed Pisum sativum Linn.,the IMPA content in roots was higher than that in stems and leaves at the beginning,as time went by,IMPA content in stems and leaves increased gradually and the IMPA content in roots reached the highest on the 21^st day;the growth rate of IMPA in different parts:leaves>stems>roots.The results provide important reference for sampling time and sampling location of chemical weapon-related compounds exposure in plants.At the same time,the effect of GB on plant growth also provides the possibility for chemical exposure monitoring based on plant apparent responses.