Verification of organophosphorus pesticide poisoning: Detection of phosphorylated tyrosines and a cysteine-proline disulfide-adduct from human serum albumin after intoxication with dimethoate/omethoate

A method is described allowing forensic analysis of plasma samples to prove human poisoning with the organophosphorus pesticides omethoate (OM) and dimethoate (DIM). Upon incubation of human serum albumin (HSA) with both pesticides tyrosine residues were phosphorylated. In addition, a novel disulfide-adduct between the identical thiol-containing leaving group of OM and DIM (2-mercapto-N-methylacetamide, MNMA) and the only free cysteine residue in HSA (Cys34) was formed. Following pronase-catalyzed proteolysis either O,O-dimethyl phosphotyrosine (Tyr-dmp) or O,O-dimethyl thiophosphotyrosine (Tyr-dmsp) as well as the cysteine-proline dipeptide disulfide-adduct (MNMA-CysPro) were produced. All biomarkers were simultaneously detected using modern microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). Corresponding limits of identification (LOI) for tyrosine-adducts (LOIOM: 30 μM, LOIDIM: 120 μM) and disulfide-adducts (LOIOM: 1.2 μM, LOIDIM: 30 μM) demonstrated that MNMA-CysPro allowed a considerably more sensitive detection. Finally, this novel method was applied to a plasma sample of an 87-year-old man, who had unintentionally ingested the pesticide Roxion® containing DIM as active ingredient. Unambiguous proof of poisoning demonstrated suitability of the novel biomarkers for sensitive verification analysis.