The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1–1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3A^a,b,c,d, T12A^a, T15A^b, and T9A^c,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

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.

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.