Nasal Lavage Fluid as a Biomedical Sample for Verification of Chlorine Exposure

Chlorine is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography--tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.