LAUSR

Sulfur mustard (SM, bis(2-chloroethyl)-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins e.g., serum albumin (SA) are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant and re-dissolution analysis was performed by micro liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, μLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE]+ at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The 8-fold deuterated SM analogue (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C) and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at -30°C for at least 33 d and for three freeze-and-thaw cycles. At the body temperature of chicken (+40°C) Glu(-HETE) was degraded in serum (period of half-change 3 d) whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.