determination of chlorine gas candidate biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in plasma samples

Abstract: chlorine gas (cl2) is a public health concern and potential threat due to its high reactivity, ease and scale of production, widespread industrial use, bulk transportation, massive stockpiles and history as a chemical weapon. after utilizing the chlorine, e.g. in chemical warfare, it is challenging to detect it in the environment, since it rapidly decomposes. toxicity is due to the formation of hydrochloric acid (hcl) and hypochlorous acid (hocl) through hydrolysis in the moist linings of the airways. chlorine or its decomposition products are known to form stable adducts with tyrosine (tyr) residues in proteins. this work describes a new and sensitive method for the retrospective detection and quantitation of two chlorotyrosine adduct biomarkers, 3-chlorotyrosine (cl-tyr) and 3,5-dichlorotyrosine (cl2-tyr), which were isolated after protein precipitation and pronase digestion of plasma sample spiked with sodium hypochlorite (naocl) solution. cl2-tyr adduct is distinct representative of exposure to chlorine gas and it is observed in the concentration of more than 50 ppm. digested samples were clean-up by solid-phase extraction (hlb) and identified by liquid chromatography tandem mass spectrometry (lc-ms/ms) [1-2]. analysis was performed in the positive ionization mode using multiple reaction monitoring (mrm) of the ion transitions at m/z 216.1 / 135, 170, 199 for cl-tyr [1]. afterwards, matrix effects, lod, loq, precision, calibration range and recovery range were evaluated for the proposed method. the calibration range was estimated at 0.1–50 mg/l (r2≥ 0.995) with lod and loq of 0.05 mg/l and 0.1 mg/l for cl-tyr, respectively. rsd for cl-tyr was obtained at 2.9%. matrix effects were also evaluated at 24.75%. spe recoveries for cl-tyr was 116%. for the animal phase study, 8 male rats (two groups each containing four rats) were intramuscularly treated with 5 and 50 mg/kg sodium hypochlorite (naocl) solution, respectively. the rats’ plasma were analyzed for the presence of cl-tyr through above mentioned method. the presence of cl-tyr in rats' plasma was confirmed in all the intramuscularly treated rats by lc-ms/ms. in conclusion, pronase digestion combined with lc-ms/ms (mrm) analysis of chlorinated tyrosine adduct could constitute a valuable technique for the forensic verification of exposure to chlorine gas.