Paper Report for: John_2022_Anal.Chem__

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 (microLC) detecting the NP(OP) by nontargeted HR MS followed by data-dependent tandem-MS (ddMS2). Extracted ion chromatograms of diagnostic product ions at m/z 778.33661, 673.29402, and 602.25690 reveal the accurate mass of the NP(OP) precursor ion as well as the elemental composition of the adducted phosphyl moiety. Considering this information, a second microLC run is performed (step 2) for nonselective IS-CID of NP(OP) yielding the cleaved charged phosphyl moiety. This fragment ion is immediately subjected to targeted CID in parallel reaction monitoring (PRM). The accurate mass of its product ions allows the determination of their elemental composition and thus supports its structural elucidation. The described workflow was exemplarily applied to NP(OP) of three Tamelin esters and VX providing highly appropriate abilities for the detection of adducts even of unknown OP poisons like Novichok agents.