Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics.

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

CHART: a novel system for detector evaluation against toxic chemical aerosols.

Concern over the possibility of deliberate dispersion of chemical warfare agents and highly toxic pharmaceutical based agents as persistent aerosols has raised the need for experimental assessment of current and future defensive capabilities of armed forces and law enforcement agencies. Therefor we herewith present the design, realization and validation of the Chemical Hot Aerosol Research Tool (CHART) as a validated and safe experimental set-up for performance evaluation of chemical detection and identification equipment against chemical warfare agents and other highly toxic compounds. In the CHART liquid and solid compounds in solution or suspension are being dispersed as aerosols in a nebulization chamber. A broad dynamic particle size range can be generated, including particles known to be able to reach the lower respiratory tract. The aerosol generated is presented to the detection system-under-test while being monitored and characterized in real-time, using an optical particle counter and a time-of-flight aerosol analyzer, respectively. Additionally, the chemical composition of the aerosol is ex situ measured by analytical chemical methods. Evidently, in the design of the CHART significant emphasis was placed on laboratory safety and containment of toxic chemicals. The CHART presented in this paper has proven to be an indispensable experimental tool to study detectors and fieldable identification equipment against toxic chemical aerosols.

Real-time characterization of chemical threat agent aerosols for improvement of inhalation studies.

OBJECTIVES: Deliberate or accidental release of chemical treat agents in the aerosol form can cause an inhalation hazard. Since the relationship between aerosol properties and health hazards is poorly understood, research into the toxicological consequences of exposure to aerosols is needed. The aim of the present study was to improve the characterization of particles for inhalation studies. METHODS: Several aerosol measurement technologies were compared for their potential to physically and chemically characterize particles in the inhalation size range in real-time. For that purpose, we compared the performance of an aerodynamic particle sizer (APS), a scanning mobility particle sizer (SMPS) and an electrical low-pressure impactor (ELPI) in an experimental set-up in which particles were generated by a Collison nebulizer and subsequently delivered into a nose-only inhalation exposure system. RESULTS: We found that more than 95% of the number of particles, equating to more than 83% of the mass generated by the 6-jet Collison nebulizer, were below 0.5 µm. To characterize the entire size range, the APS as single detector has only limited value, therefore the addition of supplementary instrumentation such as the SMPS or the ELPI is required. After real-time measurements in the size range of 30 nm to 10 µm, ex-situ chromatographic chemical analysis is essential for quantification of the delivered mass concentration. CONCLUSIONS: In summary, the present work demonstrates the utility of the ELPI technology, in combination with off-line analysis, for characterizing aerosols with various size, shape, charge, and composition. This makes the aerosol generation and analysis suite described a promising tool for quantitative inhalation exposure studies.

Multiple analysis techniques were applied for real-time aerosol characterizationAerosol size distributions are characterized for inhalation exposure studies.Analytical analysis following ELPI measurements is essential for mass quantification.

Verification of exposure to chemical warfare agents through analysis of persistent biomarkers in plants.

The continuing threats of military conflicts and terrorism may involve the misuse of chemical weapons. The present study aims to use environmental samples to find evidence of the release of such agents at an incident scene. A novel approach was developed for identifying protein adducts in plants. Basil (Ocimum basilicum), bay laurel leaf (Laurus nobilis) and stinging nettle (Urtica dioica) were exposed to 2.5 to 150 mg m-3 sulfur mustard, 2.5 to 250 mg m-3 sarin, and 0.5 to 25 g m-3 chlorine gas. The vapors of the selected chemicals were generated under controlled conditions in a dedicated set-up. After sample preparation and digestion, the samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS), respectively. In the case of chlorine exposure, it was found that 3-chloro- and 3,5-dichlorotyrosine adducts were formed. As a result of sarin exposure, the o-isopropyl methylphosphonic acid adduct to tyrosine could be analyzed, and after sulfur mustard exposure the N1- and N3-HETE-histidine adducts were identified. The lowest vapor exposure levels for which these plant adducts could be detected, were 2.5 mg m-3 for sarin, 50 mg m-3 for chlorine and 12.5 mg m-3 for sulfur mustard. Additionally, protein adducts following a liquid exposure of only 2 nmol Novichock A-234, 0.4 nmol sarin and 0.2 nmol sulfur mustard could still be observed. For both vapor and liquid exposure, the amount of adduct formed increased with the level of exposure. In all cases synthetic reference standards were used for unambiguous identification. The window of opportunity for investigation of agent exposure through the analysis of plant material was found to be remarkably long. Even three months after the actual exposure, the biomarkers could still be detected in the living plants, as well as in dried leaves. An important benefit of the current method is that a relatively simple and generic sample work-up procedure can be applied for all agents studied. In conclusion, the presented work clearly demonstrates the possibility of analyzing chemical warfare agent biomarkers in plants, which is useful for forensic reconstructions, including the investigation into alleged use in conflict areas.

Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure.

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Contact transfer risk from fentanyl-contaminated RSDL® Kit.

The RSDL® (Reactive Skin Decontamination Lotion) Kit contains a lotion-impregnated sponge extensively studied for the removal or neutralization of chemical warfare agents from skin. Pilot investigation of efficacy with industrial threat compounds noted that synthetic opioid fentanyl citrate was removed by the RSDL Kit but not chemically inactivated by the lotion. This implies that after use the RSDL Kit will contain intact fentanyl, which may pose a dermal health hazard if the fentanyl is then transferred to skin after use without proper handling. This in vitro investigation studied the contaminated RSDL Kit using three different concentrations of fentanyl with a skin contact time of 15 min under direct interaction from passive contact, light touch, and leaning with one hand. It was demonstrated that the expected transfer of fentanyl from contaminated RSDL depends on 1) the concentration of fentanyl and 2) the area of the exposed surface. From a toxicological perspective, the contact risk of fentanyl under the conditions tested can be considered low but not absent. The present study determined that a contaminated RSDL Kit, used for removal of fentanyl, should be handled with proper care. Use of protective gloves in operational use and washing skin afterwards is advised to prevent undesired contamination.