Nontargeted High-Resolution Mass Spectrometric Workflow for the Detection of Butyrylcholinesterase-Derived Adducts with Organophosphorus Toxicants and Structural Characterization of Their Phosphyl Moiety after In-Source Fragmentation.

Organophosphorus (OP) nerve agents were used for chemical warfare, assassination, and attempted murder of individuals. Therefore, forensic methods are required to identify known and unknown incorporated OP poisons. Serum is tested for the presence of covalent reaction products (adducts) of the toxicant with, e.g., butyrylcholinesterase (BChE) typically by targeted analysis, thus only detecting known OP adducts. We herein present a nontargeted two-step mass spectrometry (MS)-based workflow taking advantage of a high-resolution (HR) Orbitrap mass spectrometer and its option for in-source collision-induced dissociation (IS-CID) highly valuable for the detection of unknown agents. BChE adducts are extracted by immunomagnetic separation and proteolyzed with pepsin yielding a phosphylated nonapeptide (NP) biomarker NP(OP). In step 1, the sample is separated by micro liquid chromatography (μLC) detecting the NP(OP) by nontargeted HR MS followed by data-dependent tandem-MS (ddMS2). Extracted ion chromatograms of diagnostic product ions at