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We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a μLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9 weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)