Discovery of chlorine exposure signatures in plant material using targeted and comparative mass spectrometry methods†

Recent uses of chlorine gas in violation of the Chemical Weapons Convention are difficult to identify through chemical analysis as unique signatures of exposure have not been identified. We exposed living pine seedlings and English ivy to chlorine gas, extracted the pine needles, and analyzed the extracts by liquid chromatography-time of flight mass spectrometry (LC-qTOF) and comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-TOF). Data from exposed seedlings was compared to unexposed seedlings and bleach-treated seedlings using commercial and Battelle proprietary software to identify unique or elevated markers of exposure. Battelle also used targeted mass spectrometry to evaluate 3-chlorotyrosine and 3,5-chlorotyrosine as chlorine exposure biomarkers that were expected to be present in exposed pine needles. We discovered ten (10) chlorine exposure biomarkers in chlorine gas-exposed pine needle and ivy leaf extracts using survey mass spectrometry methods. Additional survey mass spectrometry analysis suggested additional biomarkers (chlorinated glycosylated flavonoid analogs) may be present but that sufficient levels were not generated for detection in extracts from the chlorine gas-exposed samples. Targeted analysis for 3-chlorotyrosine and 3,5-dichlorotyrosine indicated presence of 3-chlorotyrosine in extracts from exposed ivy.