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.