Unambiguous identification and determination of A234-Novichok nerve agent biomarkers in biological fluids using...

The present study was intended to develop suitable methods for unambiguous identification and determination of ethyl (1-(diethylamino)ethylidene) phosphoramidofluoridate (known as A234-Novichok) biomarkers in urine and plasma samples. Multiple biomarkers were investigated for the first time, to verify intoxication by the A234-Novichok agent, using sensitive and accurate techniques including gas and liquid chromatography-tandem mass spectrometry (GC-MS/MS and LC-MS/MS). Like other nerve agents, in biological matrices, the A234-Novichok agent reacts with several proteins to form related adducts. Considering this, two different protein adduct biomarkers in blood samples were analyzed, and the regenerated A234 was determined. Two-dimensional chromatography and solid-phase extraction techniques were employed for blood sample preparation. Limits of detection for butyrylcholinesterase (BChE) adduct, the regenerated A234, and albumin covalent adduct were determined and reported as 1, 1, and 10 ng mL^-1, while the related calibration curves were linear within the range of 2-100, 2-100, and 15-100 ng mL^-1, respectively. The detection limit and linear range for the intact agent in the urine sample were determined as 0.1 and 1-100 ng mL^-1, respectively. Since A234 and some other Novichok chemicals have been added to the Schedule 1 of the Chemical Weapons Convention (CWC), Annex on Chemicals, after UK incidents, the analytical methods developed in this work might be used for verification purposes, as well as OPCW Biomedical Proficiency Tests.