Identification of 2-(diethylamino)ethylthiol dipeptide (Cys-Pro) adduct as biomarker of nerve agents VR and CVX in human plasma using liquid chromatography-high-resolution tandem mass spectrometry.

Abstract

Organophosphorus nerve agents pose a significant threat to human health. The most toxic compounds in this class include V-type poisonous substances such as VX, CVX, and VR. Although all stockpiles of this type of substance are subject to destruction under the Chemical Weapons Convention (CWC), there is still a risk that they could be used for criminal and terrorist purposes. The latter determines the relevance of studies aimed at identification of biomarkers that may indicate the exposure of these group substances to the organism. A liquid chromatography mass spectrometry/high-resolution mass spectrometry (LC-MS/HR MS) method for determination of trace amounts of nerve agents such as VR and CVX in human plasma was proposed. The method is based on enzymatic plasma hydrolysis with the use of pronase to form a stable adduct of 2-(diethylamino)ethylthiol with dipeptide cysteine-proline (DEAET-CP) with its subsequent determination by LC-MS/HR MS. Synthesis of DEAET-CP as reference compound was conducted using non-toxic precursors. Sample preparation of human blood plasma samples exposed to VR was carried out with the use of solid-phase extraction (SPE). Liquid chromatography (LC) separation on the reversed-phase column and mass spectrometric detection (selection of optimal transitions and detection modes) were performed. The achieved limit of detection (LOD) of VR (in the form of DEAET-CP) in human blood plasma was 0.05ngmL-1. The proposed approach was developed using plasma samples exposed to VR and CVX obtained in the frame of the Fifth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons (OPCW) and showed good specificity of detection.