verification of nerve agents exposure by identification of free metabolites and protein adduct biomarkers in human plasma samples in the 7th international official opcw biomedical proficiency test

Abstract: the purpose of this study was analysis of human plasma samples for protein adducts and free metabolites with different analytical techniques in order to investigate the presences or absence of nerve agents. organophosphorus nerve agents (opnas) target the enzyme butyrylcholinesterase (buche) and albumin to cause acute toxicity [1-2]. in this biomedical proficiency test, plasma samples (7×5 ml vials with codes of p071-p077) were received (1 march, 2022) from opcw laboratory (rijiswijk, the netherlands) and analyzed for verification of human exposure to nerve agents. buche activity was measured spectrophotometrically using an ellman assay as a preliminary test to diagnose exposure to opnas. fluoride regeneration method and clean-up with hlb solid phase extraction applied to release the agents again. buche from plasma purified by sepharose q fast flow gel (anion exchange)-procainamide affinity gel chromatography, digested with pepsin, cleaned-up by c18 solid phase extraction and the modified nonapeptide adducts were identified by lc-ms/ms. totally protein precipitated, digested with pronase e enzyme, cleaned-up by c18 solid phase extraction, then the tyrosine adducts were identified using lc-ms/ms. cleaning up by lc-si cartridge, derivatization with pentafluorobenzyl bromide followed by gc-ms/ms analysis was applied to identify the free metabolites of agents in plasma samples. all reported chemicals were in accordance with preliminary report of the seventh official opcw biomedical proficiency test. cyclosarin was identified in plasma samples with codes of p073 and p074 with real concentration of 20 ng/ml according to opcw reports. vx and cvx with concentrations of 10 and 20 ng/ml were identified in the plasma samples with codes of p075 and p077, respectively. reference chemicals (own synthesis) were used for comparison of the obtained data with the test samples. the samples with codes of p071, p072 and p076 were blank samples. the defense chemical research laboratory (dcrl) correctly identified all the spiked chemicals in the concentration range of 10-20 ng/ml in plasma samples with sufficient analytical data. in conclusion, presented methods could constitute valuable techniques for the forensic verification of exposure to nerve agents.